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INVESTIGATING THE ROLE OF RNA DEGRADOSOME COMPONENT RNASE J2 IN
THE COMPETITIVE FITNESS OF BACILLUS SUBTILIS UNDER HYPOBARIA
By
HOANG VINH NGUYEN
A THESIS PRESENTED TO THE GRADUATE SCHOOL OF THE UNIVERSITY OF
FLORIDA IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF
MASTER OF SCIENCE
UNIVERSITY OF FLORIDA
2017
© 2017 Hoang Vinh Nguyen
3
ACKNOWLEDGMENTS
Most importantly, I would like to thank my family for supporting me throughout
whatever endeavors I have ever had. Mom and Dad thank you for letting me make my own
decisions, you have never pushed me into doing anything I did not want. You have always
allowed me to chart my own path and make my own mistakes. That has been the most important
aspect of my life. I would also like to thank my best friends, Ian Finley and Derek Milligan. We
have all known each other for the better part of a decade now. You guys have been there
supporting me in my happiest moments and my darkest moments. It has been a very tumultuous
several years for me but with your support I have pushed forward through the darkness. You
guys are my brothers. Thank you for always being there for me. I promise I will always be there
for you guys. I would also like to give my sincere thanks to Dr. Wayne Nicholson, I have been a
big trouble maker in his lab. Regardless, he has supported, guided, and encouraged me to be a
better scientist. I would also like to thank my lab mate, Michael Morrison from Texas, thank you
for being a friend as well as helping me throughout the analysis of my data. I don’t think I would
have been able to complete everything on time without your help. I would also like to thank Dr.
Kelly Rice and her lab, I appreciate that you could provide lab space, equipment, and reagents
for me to continue working on the main Microbiology and Cell Science Building. I don’t think I
would be able to complete this thesis without your help.
4
TABLE OF CONTENTS
page
ACKNOWLEDGMENTS ...............................................................................................................3
LIST OF TABLES ...........................................................................................................................6
LIST OF FIGURES .........................................................................................................................7
ABSTRACT .....................................................................................................................................8
CHAPTER
1 INTRODUCTION ..................................................................................................................10
Thermodynamic Effects of Pressure .......................................................................................10 Effects of Pressure on Cells and Macromolecular Structure ..................................................11
Pressure Response and Adaptation by Terrestrial Microorganisms .......................................12 High Pressure Adaptations by Piezophiles ......................................................................12
High Pressure Response by Surface Dwelling Mesophilic Organisms ...........................14 Low Pressure Response by Terrestrial Organisms ..........................................................16
Hypothesis and Specific Aims ................................................................................................18
2 MATERIAL AND METHODS ..............................................................................................22
Bacterial Strains, Media and Growth Conditions ...................................................................22 Plasmid Constructions ............................................................................................................22 B. subtilis Strain Constructions...............................................................................................23
Competitive Fitness Assays ....................................................................................................24 Growth Rate Measurements ...................................................................................................26
Statistical Analyses .................................................................................................................26 Total RNA Preparation ...........................................................................................................27
RNA Sequencing and Data Analysis ......................................................................................27
3 RESULTS ...............................................................................................................................31
Detection and Validation of Mixed Fluorescent Populations of B. Subtilis ...........................31 Effects of the rnjBΔ9 Mutation on the Fitness of B. subtilis at Various T-P Regimes...........33 Growth Rates of w.t. and rnjBΔ9 Strains at 25°C ..................................................................33 RNA-seq Analysis of WN1589 at LP and SP ........................................................................34 Regulons Affected by rnjBΔ9 Under LP ................................................................................35
GO Term Enrichment of DEGs in WN1589 ..........................................................................35 KEGG Pathway Enrichment of DEGs in LP Dataset .............................................................36
4 DISCUSSION AND CONCLUSIONS ..................................................................................49
APPENDIX: DIFFERENTIAL GENE ANALYSIS AND REGULON TABLES .......................62
5
LIST OF REFERENCES .............................................................................................................109
BIOGRAPHICAL SKETCH .......................................................................................................120
6
LIST OF TABLES
Table page
1-1 Stability of chemical interactions under pressure. .............................................................20
1-2 Cellular processes impaired by high pressures in E.coli ....................................................20
1-3 Mutations identified by whole genome sequencing of WN1106.......................................21
2-1 Bacterial strains and plasmids used in this study ...............................................................29
2-2 Oligonucleotides used in this study ...................................................................................30
3-1 Number of differentially expressed genes in rnjBΔ9 mutant strain WN1589
compared to w.t. strain WN1591 at ~101.3 kPa (SP) and 5 kPa (LP). ..............................46
3-2 Summary of regulons with at least 5 genes affected. .........................................................47
3-3 Upregulated GO terms for WN1589:WN1591 at LP ........................................................48
A-1 Differential gene expression data for WN1589 at SP and LP. ..........................................62
A-2 Regulons of DEGs found in WN1589 at 5 kPa .................................................................77
7
LIST OF FIGURES
Figure page
3-1 Two-parameter flow cytometric analysis of B. subtilis at 30˚C and normal
atmospheric pressure (~101.3 kPa) under the FL1-A (GFP) vs FL2-A (RFP) filters. ......38
3-2 Flow cytometric analysis of B. subtilis grown at 30˚C at LP (5 kPa). ...............................39
3-3 Restoration of RFP fluorescence in WN1591 after exposure to 5 kPa. .............................40
3-4 Competition experiments to compare the fitness of the rnjBΔ9 mutation by viable
counts and flow cytometric counts. ...................................................................................41
3-5 Relative fitness conferred on the rnjBΔ9 mutant at different T-P combinations. ..............42
3-6 Exponential growth rates of w.t. strains WN1590 (GFP), WN1591(RFP), and mutant
strain WN1589 (rnjb∆9, RFP). ..........................................................................................43
3-7 GO-term Enrichment analysis for downregulated genes in WN1589 under LP. ..............44
3-8 KEGG Pathway analsysis of downregulated genes in WN1589 under LP. ......................45
4-1 SWISS-MODEL diagrams of: the predicted structure of the RNase J1/J2
heterotetramer. ...................................................................................................................60
4-2 KEGG Pathway analysis for purine metabolism. ..............................................................61
8
Abstract of Thesis Presented to the Graduate School
of the University of Florida in Partial Fulfillment of the
Requirements for the Degree of Master of Science
INVESTIGATING THE ROLE OF RNA DEGRADOSOME COMPONENT RNASE J2 IN
THE COMPETITIVE FITNESS OF BACILLUS SUBTILIS UNDER HYPOBARIA
By
Hoang Vinh Nguyen
May 2017
Chair: Wayne L. Nicholson
Major: Microbiology and Cell Science
To understand how terrestrial microbes are affected by, and adapt to, low pressure (LP),
Bacillus subtilis strain WN624 was evolved for 1,000 generations at 5 kPa to produce the LP
evolved strain. WN1106. Whole-genome sequence revealed an in-frame 9-nucleotide deletion in
the rnjB gene (rnjBΔ9) that encodes the RNase J2 subunit of the RNA degradosome. To
understand the role of rnjB in LP fitness, congenic strains were constructed carrying the rnjBΔ9
or rnjB+ allele and tagged with a red fluorescent or green fluorescent protein marker,
respectively. The strains were competed under a range of temperatures (T) (20-30°C) and at LP
(5 kPa) or at standard atmospheric pressure (SP) of 101.3 kPa to assess temperature effects on
fitness. rnjBΔ9 conferred an LP fitness advantage to B. subtilis at 25°C, and growth rate
measurements revealed that the mutant had a significantly faster growth rate than the w.t. at
25°C, at LP and SP. RNA-seq was performed on the two strains grown at 25˚C and LP and SP.
At LP, the rnjBΔ9 strain exhibited 225 up-regulated and 276 down-regulated transcripts (P <
0.05). At, SP the rnjBΔ9 strain exhibited 1 up-regulated and 1 down-regulated transcript (P <
0.05). rnjBΔ9 altered RNA turnover at LP for many genes, but had small effects on transcripts at
SP. Transcriptomic analysis suggests that under LP the rnjBΔ9 strain may enhanced fitness by
9
triggering the stringent response. At SP, we were unable to explain how the differential
expression in sspH and purA might contribute to the reduced fitness.
10
CHAPTER 1
INTRODUCTION
Thermodynamic Effects of Pressure
Pressure (P) is a thermodynamic parameter that is defined as the force per unit area
applied on a surface perpendicularly:
P = F/A
Where P is the pressure, F is the normal force applied to the surface, and A is the area. In
general, the point of influence for pressure is that it affects reaction volumes. As pressure
increases, more compact structural forms are favored and chemical equilibria are disrupted1. This
can be mathematically described by a derivation of Le Chatelier’s principle:
(𝛿(∆𝐺)/𝛿𝑃)) = −∆𝑉/𝑅𝑇
This equation states that the Gibb’s free energy (ΔG) for a reaction at constant
temperature (T) favors the state resulting in the smallest volume (V) with increasing pressure (P).
Reactions that proceed with a volume increase will be inhibited by increasing pressure, and those
that proceed with a volume decrease will be promoted by increasing pressure- the larger the
volume change, the greater the effect2. For instance, a reaction that is accompanied by a ∆V
value of 10 mL mol-1 at 0.1 MPa can be enhanced over 1000 fold if pressure were to be increased
to 100 MPa. The forces that are involved in biochemical reactions and maintaining the structure
of biomolecules (e.g. proteins and DNA) are affected by pressure. Electrostatic and hydrophobic
interactions proceeding with the most negative ΔV (greatest increase in volume) are destabilized
with increasing pressure, while hydrogen and covalent bonds are stabilized3. These effects are
summarized in Table 1-1. The majority of studies conducted on pressure have been done within
the range of 0.1 MPa- 200 MPa, which are commonly encountered by Earth organisms4. At
these pressures, only intermolecular distances are affected, not covalent bond distances or angles;
11
as a result, pressure predominantly affects the conformation of biomolecules, thus their
functionality within cellular systems1,4. Different pressures on various enzymes, have been
shown to affect ligand binding, enzymatic reaction rate, and structural state3. A pressure
decrease or increase can have sizeable effects on reaction rate or biomolecular structure.
Effects of Pressure on Cells and Macromolecular Structure
As stated before, pressure affects (bio)chemical equilibria and reaction rates through the
volumes involved in the reaction. It shifts chemical equilibria and reaction processes towards
states that result in smaller volumes with increasing pressure. This single point of influence
exerts effects on many cellular processes in cells. Pressure is thought to primarily act upon the
weak interactions (hydrogen bonds, electrostatic, and hydrophobic) that define macromolecular
structure, thereby function5. High pressure (>101.3 kPa) effects have been widely described in
nucleic acids, lipid membranes, and proteins. Under increasing pressures, DNA hydrogen bonds
and base stacking interactions are stabilized increasing the melting temperature (Tm) of the
transition from duplex to single strand; this may make transcription, translation, and replication
more difficult6. Lipid membranes are among the most pressure sensitive structures; under high
pressure membranes become tightly packed which increases rigidity and decreases permeability
to small molecules as well as inhibits protein-lipid interactions 7. High pressure disrupts weak
interactions that are important for protein structure and function. Proteins will adapt to the
constriction in volume by changing their conformation, which can have an effect on substrate
binding, oligomeric association, and catalytic activity5. Prominent examples of this can be seen
in ribosomal assembly and the enzyme chymotrypsin. The E.coli 70S ribosome has been shown
to dissociate with increasing pressure, which is thought to be one of the major causes of cell
death for pressure-exposed bacterial cells8. Chymotrypsin loses its enzymatic activity when
exposed to 1000 MPa due to the destabilization of the salt bridges in its active site3.
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On the other side of the spectrum, nothing is currently known about low pressure effects
(<101.3 kPa) on macromolecular structure. However, based on the effects of hyperbaria one
could easily postulate that low pressures would have opposite but similarly destabilizing effects
on macromolecular structure. Overall, the numerous effects on cellular systems suggests that
pressure acts as a selective force corresponding to other fundamental parameters such as pH,
osmolarity, and temperature. This selection can be seen in the evolution of pressure-loving
organisms (piezophiles) and their distribution in high pressure environments >10 MPa9.
Physiological systems have a minimal, optimal, and maximal operating pressure. For
instance, B. subtilis maintains a turgor pressure of 2 MPa to drive expansion during growth 10. In
addition, all enzymes appear to have an optimal pressure for their activity11. This suggests that in
order to acquire the ability to survive and grow under a pressure-stressed condition it is thought
that a population must meet three criteria: 1) Tune its gene expression to compensate for lower
biological activity 2) Express pressure specific genes 3) Modify the structure of biomolecules to
be tolerant to pressure induced conformational changes 12–14.
Pressure Response and Adaptation by Terrestrial Microorganisms
High Pressure Adaptations by Piezophiles
Piezophiles are organisms that have optimal growth rates at pressures above atmospheric
pressure (~101.3 kPa). These organisms’ habitats span the “deep biosphere” which is composed
of the deep sea and subterranean habitats 15. Over the course of their evolutionary history, they
have developed several adaptations to survive and proliferate at pressures lethal to surface-
dwelling organisms. Lipid membranes are among the most sensitive components to pressure due
to their high compressibility 7. The combination of high pressures and cold temperatures
experienced by deep-sea microbes have the effects of reducing membrane fluidity by increasing
the packing of fatty acyl chains7. To increase membrane fluidity, mono-unsaturated fatty acids
13
levels are increased in the membrane. In addition, many deep-sea bacterial membranes also
contain poly-unsaturated fatty acids (PUFAs) such as eicosapentaenoic acid (20:5) or
docosahexaenoic acid (22:6)16. The “kinks” generated in the fatty acyl backbone by the double
bonds prevent tight packing in the membrane as well as lower the melting point, both of which
lead to enhanced fluidity. Studies of the piezophiles Photobacterium profundum strain SS9,
Shewanella sp. strain DB21MT-2, and Moritella sp. strain DB21MT-5 have revealed an increase
in the proportions of mono and poly-unsaturated fatty acids which were correlated with
increasing pressures 1,16–18.
The evidence is less clear for the adaptations to changes in the compression by protein
and nucleic acid structures that allows optimal function under high pressure. Under pressure,
DNA is thought to exist in a highly supercoiled state which is inhibitory for transcription,
translation, and replication19. In a transposon library of the model piezophile, Photobacterium
profundum SS9, a mutation in recD (DNA helicase) created a pressure sensitive mutant, and
insertion of the wild-type (w.t.) P. profundum recD gene into Escherichia coli allowed it to
replicate normally at high pressure (3 MPa)20. In Shewanella benthica, the catalytic activity of its
dihydrofolate reductase increases with pressure as compared to pressure sensitive species21. As
one of the most important component of all organisms, the ribosome would be expected to have
been adapted to pressure. Lauro et al. (2007) demonstrated that piezophiles have extended loops
8, 11, and 49 in their 16S rRNA; they proposed that these loops may play a role in ribosomal
structure stability22. Comparison of a group of Shewanella spp. containing piezo-sensitive, piezo-
tolerant, and obligate piezophiles demonstrated a trend towards proteins containing lower
proportions of proline and glycine; it is hypothesized that decreasing flexibility enhanced protein
resistance to pressure stress14. The advent of -omics profiling has given further insight to major
14
piezophilic adaptations. Genomic analysis of piezophilic organisms reveal an abundance of non-
novel pathways involved in the synthesis of mono- and poly-unsaturated fatty acids, high
intragenomic variation among rRNA operon (possibly due to functionality at different pressures),
a large number of chemotaxis proteins, and expectedly the lack of DNA repair genes23.
Proteomic analyses of Desulfovibrio piezophilus and Shewanella violacea exposed to different
pressures demonstrated the impact pressure has on the abundance of key respiratory chain
components24. This suggests that respiratory chain component modulations may represent a key
adaptation to changes in pressure. Transcriptome analysis of Desulfovibrio hydrothermalis and
Pyrococcus yayanosii in response to different hydrostatic pressure revealed a broad array cellular
targets – translation, replication, respiratory chain elements, chemotaxis, transport, and energy
metabolism. An interesting response shown in the transcriptomic data was the accumulation of
“piezolytes” like glutamate and β-hydroxybutyrate. These compounds are protein stabilizing
solutes that offset the effects of hydrostatic pressure on conformation, packing, and
intermolecular interactions of macromolecules25. Even with the recent influx of -omics data, no
novel genes, gene sets, or pathways have been discovered that define growth under high
pressure, suggesting that life does not require novel functions to grow at high pressure15.
High Pressure Response by Surface Dwelling Mesophilic Organisms
Piezophiles are well adapted to pressure eliciting specific responses to changes in
pressure. Surface-dwelling mesophililes are not usually exposed to high pressure, and high
pressure exposure elicits a very broad stress response26. High pressure affects many cellular
processes from macromolecular structure to metabolic function, thus it is difficult to determine
the key mechanism affected by pressure. Studies into pressure effects on mesophilic organisms
have revealed key processes affected by pressure27 (Table 1-2). As one of the most compressible
components of a cell, the lipid membrane is one of the first sites to look for pressure effects. The
15
lipid membrane decreases in fluidity with increasing pressure which eventually leads it to
becoming impermeable to the movement of small molecules as well as dysfunction of lipid-
protein interactions7. Studies in Lactobacillus rhamnosus exposed to high pressure correlated cell
death to the inactivation of ATPase mediated dye exclusion28. Measurement of ATP
concentration and membrane potential of Salmonella typhimurium and Listeria monocytogenes
when stressed with increasing pressure showed a decrease in membrane potential and
intracellular ATP29. These studies suggest that membranes in un-adapted cells will be impaired
by pressure eventually leading to loss of homeostasis. To prevent this, mesophilic cells undergo a
response similar to piezophiles. E.coli and Saccharomyces cerevisiae respond to high pressure by
increasing the ratio of unsaturated fatty acid in their membranes30,31. Upon exposure to 200 MPa
for 30 minutes, S. cerevisiae upregulates the gene expression of OLE1, a desaturase, which is
commonly induced during cold stress30. Deletion of FabF from E. coli, an 3-ketoacyl-acyl carrier
protein synthase responsible for the accumulation of the unsaturated cis-vaccenic fatty acid,
caused null mutants to display diminished capacity to survive at increasing pressures31. A variety
of other cellular components are also targets of pressure. In E.coli, ribosomes are dissociated by
pressure and this will inhibit protein synthesis8. Recent evidence has linked protein misfolding
and turnover with pressure resistance. Disruption of cold shock protein CspA, heat shock protein
DnaK, heat shock protein DnaS, and IbpAB decreased the resistance of E.coli to increasing
pressures32,33. In addition, high pressures have been shown to induce oxidative stress; exposure
of E.coli to 250 MPa for 15 minutes increased the production of endogenous reactive oxygen
species and decreased viability of cells when exposed to plumbagin, a superoxide generator34.
Studies into this oxidative stress response, have shown that pressure disrupts proteins containing
iron-sulfur clusters, and the accumulation of free iron in the cytoplasm catalyzes the formation of
16
oxidative species33,34. As a result, proteins that prevent oxidative stress have been linked to
pressure resistance such as Dps, an iron-sequestering protein (ferritin) that protects against DNA
damage, and the KatE catalase.35. Transcriptomics in E. coli and S. cerevisiae demonstrated a
broad high-pressure stress response with up-regulation of transcripts involved in carbohydrate
metabolism, oxidative stress, membrane stress, transport, and molecular chaperones36,37. There
was also noted down-regulation in genes responsible for cell proliferation and protein synthesis.
The response to high pressure is akin to stresses that typically cause alteration of protein function
or lipid bilayer phase transition37. This generalized stress response is likely a consequence of the
fact that neither organism needed to develop a specific response to pressure throughout their
evolutionary history. The importance of a general stress response to high pressure has been
shown in E.coli, as the disruption of the rpoE and rpoS genes encoding stress-response sigma
factors resulted in greatly reduced viability at high pressures.32,33.
Low Pressure Response by Terrestrial Organisms
Because no natural low-pressure environments exist on Earth, our understanding of how
low pressure affects microorganisms is much less advanced. Most of the knowledge to date has
been gained through astrobiological studies driven by interest in understanding whether Earth
microbes could survive and proliferate in the surface environment of Mars, on which pressure
ranges from ~0.1-1.0 kPa38–41. Early experiments indicated that the growth of most terrestrial
bacteria is inhibited at pressures lower than ~2.5 kPa40. However, a few species of the genera
Bacillus, Carnobacterium, Clostridium, Cryobacterium, Exiguobacterium, Paenibacillus,
Rhodococcus, Serratia, Streptomyces, and Trichococcus have recently been isolated from a
variety of soils (arctic, Antarctic, alpine, permafrost), and have been shown to be capable of
growth under simulated Mars atmospheric conditions of 0˚C, anoxic CO2-dominated atmosphere,
and 0.7 kPa pressure40–42.
17
Low pressure has been shown to alter metabolism in plants and microbes. Methane
production from methanogenic microbes greatly decreased as pressure was reduced from 40 kPa
to 5 kPa43. The cyanobacterium Microcytis aeruginosa exposed to low pressure (50 kPa)
increased production of exopolysaccharides, which is a typical environmental stress response by
this organism when exposed to high salt, heavy metals, or desiccation. In wheat, a low pressure
of 50 kPa promoted growth but at 2.5 kPa prevented reproductive organ development44.
Microarray analysis of gene expression of Arabidopsis thaliana reveal a complex set of
adaptations unique to hypobaria with broad differential expression of stress genes related to heat-
shock, cold-shock, drought stress, and hypoxia45. In B. subtilis, a broad array of stress related
regulons are differentially expressed when the organism is placed under low pressure; genes
belonging to the regulons AbrB, CcpA, CodY, Fur, IolR, ResD, Rok, SigH, Spo0A, and the
general stress response (GSR) SigB regulon were induced46, which mirrors the non-specific
responses by E.coli and S. cerevisiae when placed under high pressure stress33,37.
To better understand the cellular components targeted by exposure to LP, and the
molecular mechanisms of the LP response, our lab performed a laboratory evolution experiment
using LP as a selective condition. We chose as the model organism B. subtilis, a Gram-positive
spore-forming bacterium that: (i) is a model organism both for molecular and astrobiological
studies, and (ii) represents a variety of spore-forming Bacillus spp. which are common
contaminants of spacecraft and their assembly facilities. In the LP evolution experiment, B.
subtilis strain WN624, a 168 derived strain containing an SpcR cassette, was cultured for 1,000
generations at the near-inhibitory pressure of 5 kPa, during which the population evolved an
enhanced ability to grow at LP. Strain WN1106 was then isolated from the 1,000-generation
culture, and shown to exhibit increased fitness at 5 kPa pressure when competed with the
18
ancestral strain WN62447. To identify mutations which contribute to its enhanced LP growth
phenotype, ancestral strain WN624 and LP-evolved strain WN1106 were both subjected to
whole-genome sequencing, which revealed amino acid-changing mutations in eight candidate
genes (Table 1-3). The most interesting mutation observed was a 9-bp in-frame deletion within
the rnjB gene encoding RNase J2, dubbed rnjBΔ9. Kinetic analysis of the appearance of
mutations during 1000 generations of evolution at LP revealed that the rnjB mutation was among
the earliest arising and most persistent48. The RNA degradosome in Bacteria and Archaea is a
complex of RNA-degrading enzymes used for efficient RNA turnover49. RNase J2 is a
phylogenetically widespread but poorly understood component of the RNA degradosome among
Firmicute bacteria50. These observations suggest that the rnjBΔ9 mutation may be important in
conferring LP fitness to B. subtilis.
Hypothesis and Specific Aims
Pressure (P) and temperature (T) are two fundamental and synergizing parameters that
govern all biological systems. The interplay between P and T has been shown to induce a variety
of effects on macromolecular structure and function, as well as drive the evolution of specifically
adapted proteins4,13,27. Our understanding of how cells sense and respond to low T, high T, and
high P is relatively well established from studies of terrestrial extremophiles. However, there is
currently a dearth of knowledge about the molecular mechanisms and adaptations by microbial
systems towards low pressure (LP), as there exist essentially no natural, and few human-made,
LP environments on Earth's surface.
The primary goal of this project is to understand the molecular mechanisms and cellular
targets of LP on microbes, using B. subtilis as the model system. Based on the preliminary
results presented above, I have formulated the working hypotheses that:
19
1. RNase J2 is an important component in the evolution of B. subtilis to increased fitness to
growth at LP, and
2. RNase J2 exerts its enhanced fitness through global modulation of RNA turnover under
LP conditions.
Specific Aims (SA): By competition experiments, LP-evolved strain WN1106 was demonstrated
to be more fit at LP than ancestral strain WN62447; however, strain WN1106 carries all 8
mutations listed in Table 1-3. In order to test the contribution of only the rnjBΔ9 mutation to LP
fitness:
SA1-1: Congenic strains differing only at the rnjB locus were constructed, and furthermore
marked with different fluorescent tags to distinguish them in mixed culture.
SA1-2: Pairwise competition experiments were performed to measure the relative fitness of
strain carrying the rnjBΔ9 mutation to the w.t. strain under several different T-P
combinations.
SA2-1: Both the w.t. and the rnjBΔ9-bearing strain were cultivated in triplicate at either standard
pressure or LP, and total RNA was isolated from each culture. These RNA samples were
analyzed by high-throughput RNA sequencing (RNA-seq) to identify genes whose
transcript levels were altered by (i) LP exposure and (ii) the rnjBΔ9 mutation.
20
Table 1-1. Stability of chemical interactions under pressure. Adapted from Rivlain et al. 201351.
Interaction
ΔV Effect of
Pressure
Covalent +10 +
Ionic -10 -
Hydrophobic +3 to -1 +/-
Hydrogen -20 to -10 -
Table 1-2. Cellular processes impaired by high pressures in E.coli
Cell Process Pressure abolished ( MPa) Reference
Motility 80 Nishiyama et. 201252
Substrate Transport 26 Schwarz and Landau 196753
Cell Division 30-50 Welch et al. 1993, Ishii et al. 200454,55
Growth 50 Zobell and Cobet 196456
DNA synthesis 50 Yayanos and Pollard 196957
RNA synthesis 77 Welch et al. 199354
Translation 60-67 Schwarz and Landau 1972, Gross et al.
199353,58
Transcription 80 Erijman and Clegg 199859
Viability >150 Manas and Mackey 2004, Moussa et al.
200760,61
21
Table 1-3. Mutations identified by whole genome sequencing of WN1106. Adapted from Waters
et al. 201548.
Gene Essential Annotated function(s) Mutation Amino acid
change
Codon
Change
fliI No Flagellum-specific ATP
synthase; motility and
chemotaxis
C→A P35T CCA →
ACA
rnjB No RNase J2; RNA
processing and
degradation
ΔAGATCGCCA Δ183-AKI-
185
parC Yes Subunit of DNA
topoisomerase IV;
chromosome
segregation and
compaction
G→C D205H GAC →
CAC
resD No Two-component
response regulator;
regulation of anaerobic
respiration
C→T P110L CCG →
CTG
ytoI No Unknown G→T V77F GTT → TTT
yvlD No Unknown T→A Stop120K TAA →
AAA
bacD No Alanine-anticapsin
ligase; bacilysin
biosynthesis
G→A E97K GAA →
AAA
walK Yes Two-component sensor
kinase; control of cell
wall metabolism; co-
ordinates cell walls
remodelling with cell
division by controlling
the transcription of
genes for autolysins and
their inhibitors
C→T T195M ACG →
ATG
22
CHAPTER 2
MATERIAL AND METHODS
Bacterial Strains, Media and Growth Conditions
Bacillus subtilis and Escherichia coli strains used are listed in Table 2-1. For routine
growth and plating, strains were grown at 37°C in liquid Miller LB broth (10 g/L tryptone, 5 g/L
yeast extract, 5 g/L sodium chloride) or on LB plates solidified with 1.5% (w/v) agar62.
Appropriate antibiotics were supplemented to the medium when required: 100 µg/mL ampicillin
for Escherichia coli and 5 µg/mL chloramphenicol, 5 µg/mL kanamycin, or 5 µg/mL
erythromycin for Bacillus subtilis. Glycerol stocks for each strain were prepared by mixing equal
volumes of overnight culture with sterile 50% (v/v) glycerol in cryogenic tubes and storing at
-80oC.
Plasmid Constructions
All plasmids and oligonucleotides used in this study are listed in Tables 2-1 and 2-2. To
construct the promoterless – dsRed fusion plasmid pRFP_star, the dsRed.T3 construct63 was
amplified from plasmid pCR-RFP, generously donated from Kelly Rice Lab, by polymerase
chain reaction (PCR) using the oligonucleotide primers XmaILIC-RFPF and HindIII-RFPR.
GFP_star and the dsRed.T3 product were then digested with restriction enzymes XmaI and
HindIII, ligated, and introduced into E.coli strain DH5α by standard heat shock tranformation64.
Transformants were screened by colony PCR, and proper insert integration and orientation were
confirmed by DNA sequencing using primers XFP5629F and pGFP_StarAmy6810R.
To construct the veg promoter – GFP fusion plasmid pGFP_veg, the veg promoter region
(Pveg) from B.subtilis 168 genomic DNA was amplified by PCR using primers pLIC-249F and
pLICveg-13R. To insert the PCR product into the ligation independent cloning site (LIC),
pGFP_Star was digested with SmaI, 600 ng of GFP_star was treated with 1.2 U of T4 DNA
23
polymerase in presence of 2.5 mM dATP and 400 ng of the Pveg PCR product (250 bp) was
treated with 1.2 U of T4 DNA polymerase in presence of 2.5 mM dTTP. Samples were incubated
for 30 min at 22°C and inactivated for 20 min at 75°C. After T4 DNA polymerase treatment, the
insert and vector were ligated by combining at a molar ratio of 1:3 at room temperature and
introduced into E.coli DH5α by standard heat shock transformation64. Transformants were
confirmed by colony PCR and sequenced using primers XFP5629F and GFP5983R. The
insertion of the veg promoter into pRFP_star to create pRFP_veg followed the same procedure.
B. subtilis Strain Constructions
Description of strain and construction methods are listed in Table 2-1. All B.subtilis
transformations were performed using the standard two-stage competence method65. The w.t.
(i.e., rnjB+) strain WN1561 was constructed by transformation of laboratory strain B. subtilis 168
with plasmid pDG364, an integration vector that inserts a chloramphenical-resistance (CmR) into
the chromosomal amyE locus66. The congenic w.t. strain WN1574 was created by swapping out
the CmR cassette of WN1561 with an ErmR cassette from plasmid pECE7267. WN1573
containing the rnjBΔ9 mutation was constructed by amplifying the rnjBΔ9 gene from strain
WN110647 using primers rnjB-556F and rnjB+2180R which are 556 bp upstream and 2180 bp
downstream from the rnjB translational start site. Two-marker transformation (i.e.,
congression)68 was used in order to create a strain carrying both the rnjBΔ9 mutation and an EmR
marker. Recipient strain WN1561 was transformed simultaneously with plasmid pECE72 and the
2.8 kb rnjB∆9 PCR product amplified from WN1106 at a 1:167 molar ratio. EmR transformants
were screened by colony PCR using the rnjB+493F and rnjB+573R primers and separation of the
PCR products by electrophoresis through a 20% polyacrylamide gel. PCR products carrying the
rnjB∆9 deletion mutation were identified by their faster mobility, and further confirmed by DNA
sequencing. One of the transformants was designated strain WN1573 (Table 2-1).
24
To construct fluorescent versions of the w.t. strain and mutants, strains WN1574 and
WN1573 were transformed with plasmids pGFP_veg and/or pRFP_veg as described above to
create the fluorescent strains WN1590 and WN1589 respectively. Strains were confirmed by
antibiotic screening and presence of fluorescence performed on the BioTek Cytation™ 3
Imaging Multimode Reader (Biotek Instruments, Inc, USA) at excitation:emission of 488:518
(GFP) and 560:587 (RFP).
Competitive Fitness Assays
Growth competition assays were performed between the w.t. strain and mutant in the
following pairwise comparison: WN1590:WN1589 and WN1590:WN1591 at standard pressure
(~101.3 kPa) or LP (5 kPa). For LP competition assays, the pressure inside a vacuum jar was
maintained by a programmable vacuum pump system (KNF Neuberger, Trenton, NJ) fitted with
0.2-μm in-line air filters, as previously described47. Prior to the assay, strains were freshly
streaked from frozen glycerol stocks onto LB agar containing the appropriate antibiotics. Strains
were then acclimated to the testing conditions by growing them overnight with shaking (300
rpm) in LB broth containing the appropriate antibiotics at the temperature (ranging from 20-
30˚C) and pressure (~101.3 or 5 kPa) to be tested. An equivalent volume of cells from the two
overnight cultures were mixed and inoculated at a dilution of 1:100 into 400 µL of LB broth in
triplicate wells within a sterile deep 96-well microtiter plates (AB0061, ThermoFisher Scientific,
Waltham, MA, USA). Initial ratios of (RFP) mutant to (GFP) w.t. (M:W) were determined using
flow cytometry using a BD Accuri C6 Flow Cytometer (BD Accuri, USA). To measure initial
cell ratios, culture samples were diluted in 0.22 uM-filtered phosphate-buffered saline (PBS)
until there were <1000 events/second, to limit doublets. Events were measured at a flow rate of
35 µL per min, FSC-H threshold of 80,000, core size: 16 µm, until 50,000 events were collected.
Cells were excited with a 488 nm laser, and separation was performed with the
25
excitation/emission filter sets 510/15 (for GFP) and 585/40 (for RFP). After initial counting, the
plates were sealed with a gas- permeable film (AB0718, ThermoFisher Scientific, Waltham, MA,
USA). The 96-square deep well plate were placed at either ~101.3 kPa or 5 kPa. At 24-h
intervals, the plates were removed, samples taken for fluorescent counts as described above, co-
cultures were diluted 1:100 into 400 µL fresh LB, and returned to the incubation chamber. Each
competition experiment was conducted for 3 consecutive days.
Subsequent analyses of the fluorescent events were performed using FlowJo version
10.07 (FlowJo LLC, USA). Single colored w.t. strain WN1590 (GFP), WN1591 (RFP), and
uncolored w.t. strain WN1574 were used to automatically compensate for fluorescent spillover
with FlowJo as well as standards for gating populations. Relative fitness values (S) were
calculated using the formula:
Relative Fitness (S) = [ln(RT / RT-1)/6.6]69, where
RT is the Ratio of M:W on Day T,
RT-1 is the Ratio of M:W on the previous day,
6.6 is the number of generations cultures undergo per day, using a 1:100 dilution.
If S > 0 the mutant has greater fitness than the w.t. strain, if S < 0 the mutant has lower
fitness than the w.t. strain, and if S = 0 both the mutant and and the w.t. strain are equally fit.
Obtained (S) values from WN1591:WN1590 competition were subtracted from the
WN1590:WN1589 competition to normalize for fitness differences attributed to the fluorescent
markers.
To validate that fitness measurements by flow cytometry corresponded to fitness
measurements previously obtained by viable counts, competition experiments using viable
counts were also performed with non-fluorescent strains of WN1561(CmR) vs WN1573(rnjBΔ9,
26
ErmR) as previously described47,48,70. To account for fitness differences caused by the antibiotic
cassette, congenic w.t. strains were also competed: WN1561 (CmR) vs WN1574 (ErmR).
Competitions were performed at 30oC at both standard pressure and LP and fitnesses were
measured as described above. At daily intervals, an aliquot was removed from each culture,
diluted serially tenfold in PBS, and plated on LB+Cm or LB+Erm to obtain viable counts of each
subpopulation. Obtained (S) values were subtracted from the WN1561 vs WN1573 dataset to
normalize data for any fitness effects attributed to the antibiotic cassettes.
Growth Rate Measurements
Strains were acclimated to the specified temperature and pressure growth condition as
described above. Cultures were diluted 1:100 into 125 mL flasks containing 10 mL LB broth and
incubated at 25°C with shaking (300 rpm). For normal atmospheric pressure (~101.3 kPa),
growth was measured using pathlength-corrected OD600 on a Biotek Synergy HT plate reader
(BioTek Instruments, Inc., Winooski, VT). For growth at low pressure (5 kPa), cultures were
grown in 125-mL nephelometer (Klett) flasks with 0.2-μm-pore in-line air filters, connected
directly to the vacuum pumping system. Growth was measured using a Klett-Summerson
colorimeter fitted with the no. 66 (red, 660-nm) filter. (For purposes of comparison, 100 Klett
units = 1 OD660). Growth rates were calculated from the slopes of the exponential portion of the
growth curves and are reported as doubings hr-1.
Statistical Analyses
Basic statistical parameters and analyses of variance (ANOVA) were performed using
commercial statistical software (Graphpad Prism, version 6.0.1 ; Graphpad Software, Inc., La
Jolla, CA). Differences with P values of ≤ 0.05 were considered statistically significant.
27
Total RNA Preparation
Triplicate 10-mL cultures of strains WN1591 and WN1589 were grown in liquid LB
medium at 25°C and either ~101.3 kPa or 5 kPa to mid-logarithmic phase and harvested by
centrifugation at 4800 rpm for 5 minutes at 0°C to prepare total RNA. Cell pellets were
resuspended in RNALater (Ambion, Naugatuck, CT, USA) and stored at -80oC. Total RNA was
extracted using the RiboPure™-Bacteria Kit (Ambion, Naugatuck, CT, USA) according to the
manufacturer's instructions. The extracted RNA was incubated with DNase to remove any
contaminating DNA. RNA concentrations were measured using a Qubit fluorometer and the
Quant-iT RNA high sensitivity assay kit (Life Technologies, Grand Island, NY, USA) according
to the manufacturer's guidelines. The isolated total RNA was further evaluated using RNA
electropherograms generated with an Agilent 2100 Bioanalyzer (Santa Clara, CA, USA) to
assess the quantity, quality, and RNA integrity number(RIN). RIN scores of samples ranged
from 8.0 to 9.0.
RNA Sequencing and Data Analysis
Total RNA samples were shipped on dry ice to the Hudson Alpha Institute for
Biotechnology (http://hudsonalpha.org/), where preparation of cDNA libraries, depletion of
rRNA, and RNA-seq were performed. RNA-seq was performed on the Illumina HiSeq 2500
instrument to generate non-directional, single-ended, 50-base pair reads. RNA-seq data
processing was conducted through available software packages on the open-source Galaxy
bioinformatics cluster (galaxy.psu.edu) through the University of Florida Research Computing
Center (http://wiki.hpc.ufl.edu/doc/Galaxy). Single-end reads were mapped to the Bacillus
subtilis subsp. subtilis str. 168 chromosome reference sequence (NCBI accession number
NC_000964.3) using Bowtie271. The number of reads mapped to each gene was counted by
using HTSeq-count. Only genes with mapped counts >10 were considered for further
28
analysis.The DEseq2 v 1.14.072 and LIMMA v 3.30.473 packages from R/BioConductor were
used to normalize the mapped count data and for differential gene expression analysis. For both
programs a corrected P-value of 0.05 was set as a threshold for significantly differential
expression. Furthermore, only genes that were deemed significantly differentiated by both
software packages were selected for further analysis. Gene Ontology (GO) enrichment was
performed using the Gene Ontology Consortium Database74 (http://geneontology.org) with GO
terms considered significant if the threshold of a corrected P < 0.05 was matched. The KEGG
pathway enrichment analysis was performed using String-DB v1075 (http://string-db.org). Only
terms and pathways that were less than the false discovery rate (FDR) threshold of 0.05 were
considered significant.
29
Table 2-1. Bacterial strains and plasmids used in this study
Strain/Plasmid Genotype Reference
Escherichia coli
DH5α
F– endA1 glnV44 thi-
1 recA1 relA1 gyrA96 deoR nupG purB20 φ80dlacZΔM1
5 Δ(lacZYA-argF)U169, hsdR17(rK–mK
+), λ–
Taylor76
pDG364 amyE::cat, integrates CmR cassette at amyE locus BGSCA
pECE72 cat::erm, antibiotic switching cassette BGSC
pECE73 cat::neo, antibiotic switching cassette BGSC
pGFP_Star ‘amyE cat, TygrA LIC promoterless gfpmut3 amyE’ bla
ColE1 origin
BGSC, 77
pGFP_veg ‘amyE cat TygrA LIC pVeg -gfpmut3 amyE’ bla ColE1
origin
This Study
pRFP_promoterles
s
‘amyE cat TygrA LIC promoterless DsRed.T3 amyE’ bla
ColE1 origin
This Study
pRFP_veg ‘amyE cat TygrA LIC pVeg-DsRed.T3 amyE’ bla ColE1
origin
This Study
Bacillus subtilis
168 trpC2 Laboratory
Stock
(Spizizen78)
WN1106 trpC2 amyE::spc, evolved for enhanced growth at 5 kPa Nicholson
et al. 2010 47
WN1561 trpC2, amyE::cat pDG364 >
168 (tf)
WN1573 trpC2, amyE::erm, rnjb∆9 pECE72 +
rnjb∆9 PCR
product >
WN1561
(tf)
WN1574 trpC2, amyE::erm pECE72 >
WN1561(tf
)
WN1589 trpC2, amyE::[cat, TygrA Pveg-dsRed.T3], rnjB∆9 pRFP_veg
> WN1573
(tf)
WN1590 trpC2, amyE::[cat TygrA Pveg-gfpmut3]
pGFP_veg
> WN1574
(tf)
WN1591 trpC2, amyE::[cat TygrA Pveg-dsRed.T3]
pRFP_veg
> WN1574
(tf) ABGSC, Bacillus Genetic Stock Center; tf, transformation.
30
Table 2-2. Oligonucleotides used in this study
Primer Name Sequence (5' to 3') Purpose
rnjB-556F TTAAAGAAGCGAGCGG
AGAC
Primer for amplification and
sequencing of rnjB
rnjB+2180R AAGATACAGCAATCGG
AACG
Primer for amplification and
sequencing of rnjB
rnjB+493F GCACTTAATCAGACGTG
CGAC
Primer for screening of rnjBΔ9
containing transformants
rnjB+573R GAGCACGCCGCTATTG Primer for screening of rnjBΔ9
containing transformants
pLICveg-249F CCGCGGGCTTTCCCGGA
GTTCTGAGAATTGGTAT
GC
Primer to add LIC site to veg
promoter) to insert into pXFP_Star
pLICveg-13R GTTCCTCCTTCCCACTA
CATTTATTGTACAACAC
GAGC
Primer to add LIC site to veg
promoter to insert into pXFP_Star
XFP5629F GTGAATTTAGGAGGCTT
ACTTGTCT
Sequencing primer for GFP_Star
starts at position 5629
GFP5983R TCACCTTCACCCTCTCC
ACT
Sequencing primer for GFP_Star
promoter insertion, starts at position
5983
XmaILIC-RFPF CCCCCCGGGAAGGAGG
AACTTGATTAACTTTAT
AAGGAGGAAAAACATA
TGGA
Adds XmaI site to dsRed.T3 from
pCR-RFP and adds LIC site
HindIII-RFPR CCCAAGCTTTTATAAAA
ACAAATGATGACGACC
TTCTGTAC
Adds HindIII site to dsRed.T3 from
pCR-RFP
pGFP_StarAmy6810R CCAATGAGGTTAAGAG
TATTCCAAAC
Sequencing primer for GFP_star ,
starts at position 6810.
31
CHAPTER 3
RESULTS
Detection and Validation of Mixed Fluorescent Populations of B. Subtilis
B. subtilis cells are rod shaped, about 4-10 µm in length and 0.25-1.0 µm width, making
them amenable flow cytometric measurements (FCM), as the typical limit for measureable
particle size is 1 µm79. In order to assess the ability of FCM to separate and count mixed
populations for competitive fitness measurements, B. subtilis 168-based strains were transformed
with vectors containing green fluorescent protein (GFP) or red fluorescent protein (RFP)
controlled by the strong constitutive Pveg promoter integrated at the amyE locus.
Non-fluorescent, GFP-, and RFP-expressing w.t. strains WN1574 (trpC2, amyE::erm),
WN1590 (trpC2, amyE::[cat TygrA Pveg-gfpmut3]), and WN1591 (trpC2, amyE:: [cat TygrA
pVeg-dsRed.T3]) were subjected to FCM after growth at 30°C and standard atmospheric pressure
(101.3 kPa, SP). [Note: 30oC was arbitrarily chosen as the validation temperature due to
availability and accessibility to an incubator that could maintain this temperature.] A threshold of
80,000 forward scatter height (FSC-H) was set in order to eliminate the majority of the electronic
noise and small particles within the phosphate-buffered solution(PBS) used to suspend the cells.
Based on the measurements of each single strain, quadrant gates were drawn upon an FL1-A vs
FL2-A plot with FL1-A and FL2-A representing GFP and RFP fluorescence, respectively (Fig 3-
1). Using the above settings, populations were able to be well separated and defined by the
gating, with gates for each population containing >98% of the desired population (Fig 3-1, panels
A-C). These gates could further be applied to a co-culture of strains WN1590 and WN1591 to
easily separate and measure the ratio of each population in the culture (Fig 3-1, panel D).
The above settings were also used for analysis of cell populations grown at LP (5 kPa). It
was observed that GFP-expressing strain WN1590 retained its fluorescence (Fig 3-2, panel B),
32
however RFP-expressing strain WN1591 lost its fluorescence, comparable to non-fluorescing
strain WN1574 (compare Fig 3-2, panels A and C). Although low pressure effects on
fluorescence proteins have not been investigated, high pressures have been shown to have a
reversible effect on the structure which in turns affects the activity and fluorescent intensity of
GFP80. It was suspected that a similar effect on protein structure might be at play that quenched
RFP fluorescence at low-P. To confirm that the lack of RFP fluorescence was not the result of
LP inhibiting the synthesis of RFP, but merely its folding or activity, overnight cultures of the
RFP w.t. strain WN1591 grown at 30°C and LP were resuspended in sterile PBS. These
resuspended cultures were allowed to shake under standard pressure for 24 hr at 4°C to prevent
the synthesis of more RFP while allowing its renaturation. After 24 hrs, FCM measurements
demonstrated that WN1591 had regained its fluorescence (Fig 3-3). A possible explanation for
this is that while both GFP and the dsRed RFP used in this study are dependent on oxygen to
create a functional chromophore, the GFP in this study exists as a monomer with a single
chromophore whereas dsRed demonstrates a greater dependence on oxygen as it requires the
tetramerization of four chromophore containing monomeric subunits in order to fully fluoresce81–
83. At 5 kPa, only 5% of the oxygen at SP is available. The combination of a low-oxygen
environment and low pressure on oligermization could be responsible for the lack of RFP
fluorescence.
Because it was observed that RFP did not fluoresce in vivo after cultivation at LP, to
simplify the gating the GFP and RFP populations were gated based on fluorescence on the FL1
(GFP) filters for analysis of mixed populations (Fig 3-2, panels E-H).
Using the above parameters, a competition experiment was performed between strains
WN1590 (w.t., GFP) vs. WN1589 (rnjB∆9, RFP) grown in co-culture at 30°C under either SP or
33
LP and quantification by FCM. For purposes of comparison, a parallel competition experiment
using viable plate counts was performed between congenic background strains WN1561(trpC2,
amyE::cat) vs. WN1573(trpC2, amyE::erm, rnjB∆9). The data were normalized to account for
the fitness cost of the different reporter cassettes between the two groups, and relative fitnesses
were determined by computing the selection coefficient (S) for each competition. No significant
differences were observed between measurement of fitness by viable counts vs. FCM (Fig 3-4).
This experiment demonstrated that flow cytometry is a rapid, quantitative, and real-time method
to measure competitive fitness in B. subtilis; thus flow cytometry was used for subsequent fitness
measurements.
Effects of the rnjBΔ9 Mutation on the Fitness of B. subtilis at Various T-P Regimes
Because T and P are inseparable physical parameters, they must both be taken into
account in fitness determinations. In the original LP evolution experiment resulting in
identification of the rnjB∆9 mutation, the culture was evolved for 1000 generations at 5 kPa and
27°C47. Therefore, to assess the fitness effects of the rnjBΔ9 mutation as a function of T and P,
growth competition assays were performed between w.t. strain WN1591 (GFP) and mutant strain
WN1589 (rnjb∆9, RFP) at T's of 20, 25, and 30°C and at either ~101.3 kPa (SP) and 5 kPa (LP),
and selection coefficients measured under each condition. A distinct T effect was noted in the
fitness of strain WN1589 carrying the rnjB∆9 mutation (Fig 3-5). At 20°C the rnjB∆9 mutant
was less fit than w.t. regardless of P, and at 30°C the rnjB∆9 mutant was more fit than w.t.
regardless of P. Only at 25°C, did the rnjB∆9 mutant show a differential fitness with respect to P,
being less fit at SP (S = -0.06 + 0.006) and more fit (S = +0.05 + 0.002) at LP (Fig. 3-5).
Growth Rates of w.t. and rnjBΔ9 Strains at 25°C
In co-culture, the higher fitness of one strain over another can be due to a faster
exponential growth rate, among other reasons84,85. In order to test whether the increased fitness
34
of rnjBΔ9-bearing strain WN1589 was due to an increased growth rate, the growth rates at 25˚C
in LB of w.t. strains WN1590 (GFP) and WN1591 (RFP) were compared to the growth rate of
strain WN1589 (rnjB∆9, RFP) at both SP and LP. Strain WN1589 was observed to grow
significantly faster than either w.t. strain both at SP and at LP (Fig 3-6). Therefore, the
differential competitive fitness observed at 25°C (Fig. 3-5) is likely not due to differences in
exponential growth rate. Other possibilities will be discussed below.
Collectively the above data indicate that the rnjBΔ9 mutation specifically confers an LP
fitness advantage to B. subtilis at 25°C, close to the temperature at which it was isolated
(27°C)47. Because the rnjB gene encodes the RNase J2 component of the B. subtilis RNA
degradosome, we reasoned that the rnjBΔ9 mutation could lead to alterations in the global
transcriptome of B. subtilis involved in growth at low pressure (LP).
RNA-seq Analysis of WN1589 at LP and SP
To assess how the rnjBΔ9 mutation could contribute to the LP fitness of B. subtilis, RNA-
seq analysis was conducted on strains WN1589 and WN1591 grown under conditions
demonstrating maximum fitness differences (Fig 3-4), i.e. in liquid LB at 25˚C and at either SP
or LP (Table 3-1). Surprisingly, at SP only 2 genes found to be significantly up-regulated (sspH;
log-2 fold change (L2FC) = 1.09) or down-regulated (purA; L2FC = -0.71) in the rnjBΔ9 mutant
(Table 3-1). In stark contrast, at LP a total of 501 genes were found to be differentially
expressed; 225 (45%) were up-regulated and 276 (55%) were down-regulated (Table 3-1). On
the basis of the criteria (P < 0.05) applied to differentially expressed gene (DEG) sets generated
by LIMMA and DEseq2 on rnjBΔ9 mutant strain WN1589 compared to w.t. strain WN1591, a
merged list containing DEG sets discovered by both algorithms was created and is presented
(Table A-1).
35
Regulons Affected by rnjBΔ9 Under LP
Exposure of WN1589 to LP caused differential expression in several regulons (Table A-2). A
summary (Table 3-2) of the affected regulators revealed that the majority of the expression
changes were largely concentrated in the stringent response86, transition state regulators
AbrB/Abh87, and purine metabolism XptR (G-box)/PurR88. Expression changes in T-box89,
CcpA90, SigB91, Rok92, Spo0A93, LexA94, SigD95, ResD96, and SigG97 regulatory networks
occurred to a lesser degree. Interestingly, the majority of transcripts (272) were unclassified to
any regulatory network.
GO Term Enrichment of DEGs in WN1589
Gene ontology (GO) category enrichment is a commonly used technique to highlight
interesting biological processes. Genes are assembled into biological terms and then tested to
find terms that are over represented among the differentially expressed genes98. To highlight
important biological processes and molecular functions affected by pressure in WN1589, all
DEGs were mapped to terms in the biological processes and molecular function GO database and
compared to whole genomic background of B. subtilis strain 168 from the Gene Ontology
Consortium Database. GO terms with corrected P-value < 0.05 were considered significantly
enriched. The GO terms table generated for upregulated and downregulated genes are described
in Table 3-3 and Fig 3-7 respectively. Of the up-regulated 225 genes, 107 genes are unclassified
to any biological processes and 114 are unclassified to any molecular function term ID. Up-
regulated genes that were enriched were placed into two biological process categories: single
organism cellular processes and small molecule metabolic processes. In terms of molecular
function, transcripts were categorized into categories of catalytic activity and/or binding. This
suggests that the up-regulatory expressional changes significantly affected the activity in
WN1589 of genes involved in cellular processes and small molecule processes at LP.
36
For the down-regulated gene set, 9 biological process GO terms were enriched (Table 3-
3). Of the 276 down-regulated genes, 35 were unclassified to any biological process GO term
and 32 were unclassified to any molecular function GO term. GO biological process term
enrichment revealed that a large set of cellular processes were down-regulated in WN1589: de
novo inosoine monosphosphate (IMP) biosynthesis, aminoacylation of tRNA for protein
translation, ribosomal biogenesis, fatty acid biosynthesis, nucleotide phosphorylation, pyruvate
metabolism, coenzyme metabolism, oxidiation-reduction reactions, transmembrane transport,
and glycolytic processes. The most significantly enriched term was de novo IMP biosynthesis
which is associated with purine biosynthesis. Categorization under GO molecular function terms
indicated that the down-regulatory expressional changes in WN1589 caused significant effects
on the binding and activity of many gene products (Fig 3-7). The most frequent and significantly
enriched terms occurred in molecular activities involved in GO terms associated with protein
synthesis. This includes rRNA binding, ribosomal structure, tRNA binding, and aminoacyl-
tRNA ligase activity. ATP binding was also significantly enriched, indicating that the
downregulation of genes within the de novo IMP (purine) biosynthesis pathway had significant
effects on the activity of gene products that require ATP. Ligase, oxidoreductase, transferase,
and transporter activities were also affected in WN1589 indicating significant effects on cellular
reactions. Overall, the most interesting results for GO biological processes and molecular
functions indicated that pathways involved in protein and purine synthesis were particularly
down-regulated in WN1589 at LP.
KEGG Pathway Enrichment of DEGs in LP Dataset
To understand the biochemical pathways involved in the LP response of WN1589,
KEGG pathway enrichment was performed using String-DB with a false discovery rate (FDR)
cutoff of 0.05 on the 501 differentially expressed genes. Surprisingly, no KEGG pathways were
37
found to be enriched in the 225 up-regulated genes. For the 276 down-regulated genes, 17
pathways were significantly enriched (Fig 3-8). Metabolic pathways that were down-regulated
included energy metabolism, carbohydrate and lipid metabolism, amino acid metabolism, and
secondary metabolism. The most significantly enriched non-metabolic pathway was
“Ribosome”, which is associated with the biogenesis of ribosomal proteins and RNAs.
Interestingly, there was also enrichment of pathways involved in DNA repair.
38
Figure 3-1. Two-parameter flow cytometric analysis of B. subtilis at 30˚C and normal
atmospheric pressure (~101.3 kPa) under the FL1-A (GFP) vs FL2-A (RFP) filters.
Non-fluorescent B. subtilis w.t. strain WN1574 A), GFP-fluorescing B. subtilis w.t.
strain WN1590 (amyE::cat TygrA Pveg-gfpmut3) B), RFP fluorescing w.t. B. subtilis
strain WN1591 (trpC2 amyE::cat TygrA Pveg-dsRed.T3) C), example of separation in
co-culture of fluorescent B. subtilis w.t. strains WN1590 and WN1591 D).
A
C
B
D
39
A B
C D
E F
G H
Figure 3-2. Flow cytometric analysis of B. subtilis grown at 30˚C at LP (5 kPa). Panels A-D are
two parameter plots of FL1-A (GFP) vs FL2-A (RFP) fluorescence. Panels E-H are
histograms of relative fluorescence in the FL1-A (GFP) channel corresponding to
Panels A-D. Non-fluorescent w.t. strain WN1574 (A and E), GFP-fluorescing w.t.
strain WN1590 (B and F), RFP-fluorescing w.t. strain WN1591 became non-fluorescent
under low pressure (C and G). Panels D and H show an example of separation of strains
WN1590 and WN1591 grown in co-culture at LP (5 kPa).
40
Figure 3-3. Restoration of RFP fluorescence in WN1591 after exposure to 5 kPa. WN1591 RFP
population at Day 0 after overnight growth at 5 kPA A). RFP population from panel
A after 24 hrs shaking at 4°C in PBS at 101.3 kPa B).
41
Figure 3-4. Competition experiments to compare the fitness of the rnjBΔ9 mutation by viable
counts and flow cytometric counts. Viable counts: strain WN1573 ( rnjBΔ9,
amyE::erm) was competed against congenic w.t. strain WN1561 (amyE::cat). Flow
cyometric counts - strain WN1589 (amyE::cat TygrA Pveg-dsRed.T3), rnjBΔ9) was
competed against w.t. strain WN1590 (amyE::cat TygrA Pveg-gfpmut3).
Competitions were performed in liquid LB medium at 30˚C and either ∼101.3 kPa
(black bars) or 5 kPa (gray bars). Data are averages ± standard error, derived from
triplicate timepoints of triplicate independent experiments. Selection coefficients have
been normalized to the fitness differences caused by the antibiotic or fluorescent
cassettes obtained through competition of WN1561(amyE::cat) vs.
WN1574(amyE::erm) or WN1590 (amyE::cat TygrA Pveg-gfpmut3) vs.
WN1591(amyE::cat TygrA Pveg-dsRed.T3).
42
Se
lec
tio
n C
oe
ffic
ien
t
(s)
20o C
25o C
30o C
20o C
25o C
30o C
-0 .1 0
-0 .0 5
0 .0 0
0 .0 5
0 .1 0
0 .1 5
1 0 1 .3 k P a
5 k P a*
*
n s
Figure 3-5. Relative fitness conferred on the rnjBΔ9 mutant at different T-P combinations. Strain
WN1589 (rnjb∆9, RFP) was competed against congenic w.t. strain WN1590 (w.t.,
GFP) at the indicated P and T combinations. Competitions were performed in LB
medium and the data are averages ± standard errors of triplicate time points from
triplicate independent experiments. Data are averages ± standard error from triplicate
independent experiments. Asterisks denote significant differences (ANOVA, P <
0.05). Selection coefficients have been normalized to the fitness differences caused
by fluorescent cassettes obtained through competition of WN1590 (amyE::cat TygrA
Pveg-gfpmut3) vs. WN1591(amyE::cat TygrA Pveg-dsRed.T3) for all P-T
combinations.
43
Figure 3-6. Exponential growth rates of w.t. strains WN1590 (GFP), WN1591(RFP), and mutant
strain WN1589 (rnjb∆9, RFP). Cultures were grown at 25˚C and the indicated
pressure. Data are averages ± standard deviations from triplicate independent
experiments. Asterisks denote significant differences (ANOVA, P < 0.05).
Do
ub
lin
gs
hr
-1
5 k
Pa
101 k
Pa
0 .0
0 .5
1 .0
1 .5
W N 1 5 9 0
W N 1 5 9 1
W N 1 5 8 9
*
*
*
*
44
Figure 3-7. GO-term Enrichment analysis for downregulated genes in WN1589 under LP. The
GO-term enrichment analysis was performed using Gene Ontology Consortium.
Significant terms were P < 0.05. Fold enrichment values were represented as the
minus base 10 log of their corresponding p-values.
45
Figure 3-8. KEGG Pathway analsysis of downregulated genes in WN1589 under LP. KEGG Pathway
analysis was conducted using String-DB with a cutoff for significance of False Discovery Rate
(FDR) < 0.05. Fold enrichment values were represented as minus base 10 log of their
corresponding p-value.
46
Table 3-1. Number of differentially expressed genes in rnjBΔ9 mutant strain WN1589 compared
to w.t. strain WN1591 at ~101.3 kPa (SP) and 5 kPa (LP).
WN1589SP
WN1591SP
WN1589LP
WN1591LP
Upregulated 1 225
Downregulated 1 276
47
Table 3-2. Summary of regulons with at least 5 genes affected. Regulators No of genes: Response
Upregulated Downregulated Total
Unknown 129 143 272 Unknown
Stringent response 1 50 51 Coordinate response
to a variety of
stresses
Abh/AbrB 37 0 37 Transition from
active growth to
stationary phase
G-box (XptR)/PurR 0 25 25 Purine de novo
synthesis, purine
salvage, and
transport
T-Box 0 13 13 Regulation of
aminoacyl-tRNA
synthetase genes and
genes involved in
amino acid
biosynthesis and
uptake expression
CcpA 7 5 12 Carbon catabolite
repression
SigB 5 6 11 Transcription of
general stress
response genes
Rok 10 0 10 Regulation of
genetic competence
Spo0A 6 4 10 Initiation of
sporulation
LexA 4 4 8 SOS response to
DNA damage
SigD 6 0 6 Regulation of
flagella, motility,
chemotaxis and
autolysis
ResD 3 2 5 Regulation of
anaerobic/aerobic
respiration
SigG 2 3 5 Transcription of
sporulation genes
48
Table 3-3. Upregulated GO terms for WN1589:WN1591 at LP
Functional Categories
# Genes % of
Genes
corrected
p-value
Biological Processes
single-organism cellular process 31 2.8 3.44E-03
small molecule metabolic process 15 2.3 4.75E-02
Molecular Function
catalytic activity 67 3.7 1.18E-02
ion binding 28 2.7 4.53E-03
49
CHAPTER 4
DISCUSSION AND CONCLUSIONS
Temperature (T) and pressure (P) exert thermodynamic effects on biological systems that
affect biochemical processes within these systems; T affects molecular motion, and P affects the
apparent volume for reactions. The ability to adapt to different P-T conditions has influenced the
distribution and evolution of life1. Our understanding of how cells sense and respond to high P at
low or high T combinations have been established from studies of piezophiles, mesophiles and
thermophiles. In order to adapt to these P-T conditions, it has been proposed that piezophiles
have evolved the ability to finely tune overall gene expression to different P-T conditions, the
development of high pressure-responsive genes and pressure-adapted biomolecules27. However,
the adaptations by biological systems towards (LP) remains a mystery as there are a limited
number of man-made or natural LP environments on Earth. B. subtilis is a Gram-positive spore-
forming bacterium that is both a model organism for astrobiological studies and a common
contaminant of space craft assembly facilities and spacecraft. B. subtilis strain WN624 grows
well at normal laboratory pressure (~101.3 kPa), but its growth is completely inhibited at ~2.5
kPa 40. To better understand the cellular targets and molecular mechanisms of low pressure, a
laboratory evolution experiment was performed using low pressure as a selective condition. In
the experiment, strain WN624 was cultured for 1,000 generations at the near-inhibitory pressure
of 5 kPa, during which the population evolved an enhanced ability to grow at LP. Strain
WN1106 was then isolated from the 1,000-generation culture, and shown to exhibit increased
fitness at 5 kPa pressure when competed with the ancestral strain WN62447.
Genomic sequencing of the LP-evolved B. subtilis strain WN1106 revealed the presence
of 8 mutations all occurring in coding regions48(Table 1-3). A 9-bp in-frame deletion (rnjBΔ9),
occured within the rnjB gene which encodes RNase J2. The function of RNase J2 is currently
50
unknown in B. subtilis, as a knockout mutation in this gene showed no mutant phenotype despite
the alteration of 44 transcripts99. In vitro assays have shown that paralogs RNase J1 and RNase
J2 both exhibited endonucleolytic activity, but RNase J2 lacked the 5’ to 3’ exonucleolytic
activity exhibited by RNase J1100. The current model for RNase J2 is that it dimerizes with its
paralog RNase J1 which exonucleolytically degrades mono-phosphorylated RNA101. Current
evidence suggests that RNase J2 may serve an unknown regulatory role on RNase J1 activity.
The RNase J1/J2 complex has altered endonucleolytic properties compared to the individual
proteins100. In Stapylococcus aureus, inactivation of the RNase J2 catalytic site elicited no
phenotype, but an RNase J2 deletion mutant exhibited a narrowed range of temperature and
media for growth102. It has been reported that the RNase J1/J2 dimer self-oligeromizes to form a
heterotetramer, but there are conflicting reports as to which oligomeric species dominates in vivo
and the property difference of each state is currently unknown 100,103. The predicted protein
structures of the w.t.- and rnjBΔ9-encoded RNase J2 proteins were generated using the SWISS-
MODEL program and compared. The loss of three amino acids (AKI) in the RnjBΔ9 protein was
shown to occur at α4 within the highly conserved β-lactamase domain, causing the loss of a
helical turn (Fig 4-1). This area is located at the putative RNase J1/J2 dimerization interface for
tetramerization, thus may affect the oligomeric state of the RNase J1/J2 complex103. This in turn
might exert effects on the ability of RNase J1 and J2 to interact at LP. Although the effects on
protein structure and oligomerization at LP remains an unknown topic, high pressures have been
demonstrated to cause notable effects on these processes. High pressures have been shown to
causes subunit dissociation and unfolding in a variety of protein structures, such as dimeric beta
lactogobulin, ribosomes, and viral protein coat polymers8,104,105.
51
Because the normal habitat of B. subtilis is soil, the rhizosphere, and the gastrointestinal
tracts of animals, it most likely has not developed a specific response to low pressure. As such,
its response would be similar to those of other stresses it normally encounters – temperature,
dessication, pH, and salinity. A T stress response would be the most likely predicted response.
While B. subtilis may not have developed a specific response to P, T and P always operate
simultaneously on any thermodynamic system, and it is impossible to study one parameter
without taking the other into account. For example, membrane fluidity responds oppositely to
pressure than it does to temperature—increasing temperature makes membranes more fluid,
while increasing pressure makes them more rigid. Bacteria possess homeoviscous mechanisms to
maintain their membrane fluidity within an optimal range in response to changes in T and P1. As
such, our hypothesis is that the mutation in RNase J2 (rnjBΔ9) contributes to the LP fitness by
impacting the temperature response by B. subtilis. To test this, a competition experiment was
conducted between the rnjBΔ9 mutant (WN1589) and the congenic w.t. strain WN1590 within a
temperature range of 20-30oC at SP or LP (Fig 3-5). It was observed that the fitness contribution
by rnjBΔ9 was dependent on temperature. Only at 25˚C was fitness of strain WN1589 observed
to be decreased at SP and increased at LP.
Exponential growth rate is an important contributor to fitness of haploid organisms106.
Due to the opposing fitness effects observed SP and LP at 25˚C, growth rates were then
measured for each strain at this T. Interestingly, at both SP and LP, strain WN1589 carrying the
rnjBΔ9 mutation had a faster doubling time than its wild-type counterpart, suggesting that a
component of growth other than exponential growth rate may be the determinant of LP fitness,
such as lag time, final cell density, survival in stationary phase, or co-culture effects on growth
rates that we were unable to measure in our study. It has previously been shown that B. subtilis
52
cells indeed shorten their lag times and increase their final cell densities during evolution in
batch culture84,85. Although we did not perform an extensive growth kinetic analysis, we
postulate that the observed effects on competitive fitness most likely stem from changes in gene
expression that the rnjBΔ9 mutation exerts on the B. subtilis response to T and P.
To ascertain the global gene expression changes that occurred in WN1589 that may have
enhanced its fitness over w.t. strain WN1591 at LP, RNA-seq was conducted on mid-exponential
phase cells grown at 25oC and either SP or LP (Tables 3-1 and 3-2). Strain WN1589 grown
under SP resulted in only slight upregulation and downregulation of two genes: sspH (L2FC =
1.09) and purA (L2FC = -0.71). The sspH gene encodes a minor small, acid-soluble protein
(SASP) and is normally expressed in the developing forespore compartment during Stage III of
sporulation107, or during anaerobic growth (data from Subtiwiki Expression Browser,
http://www.subtiwiki.uni-goettingen.de). Little is currently known about the function of this
SASP as mutants have not shown any discernible phenotype. The lowered fitness seen in
WN1589 at SP might be due to the slight downregulation in purA-encoded adenylosuccinate
synthetase, which catalyzes the rate limiting and committed step of IMP conversion to AMP.
purA plays an important role in controlling metabolic flux within the purine biosynthesis
pathway. The expression of purA controls the de novo biosynthesis of AMP and therefore the
balance between GTP and ATP pools. As such, a decrease in its activity has been shown to
decrease growth rate in B. subtilis108. However, growth rate data of single strains at SP shows
that strain WN1589 grew slightly faster than its congenic w.t. background strains, WN1590 and
WN1591 (Fig 3-6). We are unable to explain how the upregulation of a SASP and the slight
downregulation of purA would lead to lowered fitness, especially as Luria-bertani broth (LB)
53
contains an abundance of the AMP intermediates. It is interesting to note that a purA knockout in
B. cereus grew similarly to w.t. in rich LB medium but not in minimal medium109.
Exposing mutant strain WN1589 to LP caused differential gene expression of many
transcripts when compared to w.t. strain WN1591 (Table A-1). To analyze this large dataset,
genes were assigned to their corresponding regulons. In addition, gene ontology (GO)
enrichment analysis and KEGG pathway enrichment analyses were performed. Strain WN1589
displayed differential expression in a large number of regulons (Table A-2). A summary of the
most dramatically altered regulons are seen in Table 3-2. The rnjBΔ9 mutation affected all of the
regulons summarized in previous microarray data on B. subtilis exposed to LP46, indicating that
RNase J2 plays a prominent role in global transcriptomics at LP. The top three most strongly
affected regulons were the stringent response, growth-to-stationary phase transition regulators
AbrH/AbH, and purine metabolism regulators XptR(G-box)/PurR. The prominence of the
stringent response and purine metabolism not seen in the previous microarray data46 suggests
that these are important regulatory pathways affected by the rnjBΔ9 mutation.
GO term enrichment was conducted on up-regulated and down-regulated datasets for LP-
exposed strain WN1589. The up-regulated dataset was found to be enriched for two very broad
GO biological processes – "single organism cellular processes" and "small molecule metabolic
processes". Single organism cellular processes include processes such as amino acid or
nucleotide biosynthesis; however, there was no strong enrichment for any specific process. Small
molecule metabolic process is a GO term that denotes chemical reactions or pathways that
involve small molecules, any non-coding, low-molecular-weight, monomeric molecules. GO
molecular function enrichment for “catalytic activity” and “ion binding” illustrates that the up-
regulated expression of enzymes involved in the enriched biological processes significantly
54
change their activity. Enrichment of the the down-regulated dataset reveals effects on growth
related processes. This includes terms involved in processes to generate important
macromolecular building blocks for growing cells, including protein synthesis (ribosomal
biogenesis, tRNA-aminoacylation for translation), fatty acid biosynthesis, glycolytic processes,
and coenzyme synthesis. In addition, processes involved in energy flux are also affected:
pyruvate metabolism, de novo IMP biosynthetic processes, nucleotide phosphorylation, and
oxidation-reduction processes. In summary, GO term analysis was not very effective at
identifying specific possible clues to the increased fitness of rnjBΔ9-bearing strains at LP.
KEGG pathway analysis of the up-regulated dataset yielded no enriched pathways.
However, KEGG analysis of the down-regulated datasets revealed several metabolic pathways
important for growth (Fig 3-7). Down-regulation of processes involved in energy metabolism
were once again seen, such as the TCA cycle and Pyruvate metabolism. The biosynthesis of
amino acids and fatty acids, components required for the growing cell were also down-regulated.
In addition, the pathway “Ribosome” was highly significantly enriched, as well as the associated
pathway "tRNA-aminoacylation". The purine metabolism pathway was also strongly enriched.
Purines are essential metabolites involved in many structural components of DNA and RNA,
energy carriers ATP and GTP, cofactors like NAD+, biosynthesis of amino acids, and folate
based compounds like riboflavin110. As such, downregulated pathways associated with purine
metabolism are also enriched (Fig 3-8). This includes biosynthesis of amino acids, one-carbon
pathways mediated by folate, and the pentose phosphate pathway. The GO term enrichment and
KEGG pathway analysis suggested that the reduced transcription or altered stability of mRNAs
contributing to growth-related metabolic processes would lower the growth rate of WN1589 at
55
LP. Paradoxically, however, we found that this organism had a slightly faster growth rate than
the w.t. strains WN1590 and WN1591 at both SP and LP.
The comparative analysis of the downregulated genes in w.t. WN1591 to the rnjBΔ9
mutant strain WN1589 suggests that the mutant mounted the stringent response at LP. In nature
B. subtilis and other bacteria are often faced with a variety of stresses such as nutrient and
physicochemical stresses. Whereas the general stress response prepares cells to resist multiple
incoming stresses, the widely conserved stringent response is thought to prevent waste of
nutrients during starvation86. In B. subtilis, it is mediated by the “alarmones” guanosine-5′-
triphosphate-3′-diphosphate and guanosine-5′-diphosphate-3′-diphosphate, collectively known as
(p)ppGpp, which in B. subtilis are synthesized by RelA, YwaC, and YjbM from ATP and
GTP111. The alarmones (p)ppGpp trigger broad reprogramming of genes related to biosynthetic
activities and the activation of survival responses112. A hallmark of the stringent response is the
down-regulation of components of the translational machinery that include tRNAs, rRNA,
ribosomal proteins, initiation factors, elongation factors, and trigger factors86. In Table A-2, 26
of the 52 ribosomal proteins in B. subtilis were significantly down-regulated in WN1589 grown
at LP. In addition, other components of the translational machinery, such as the initiation factors
(infA and infB) and elongation factor (fusA) are also significantly down-regulated. T-box-
regulated Aminoacyl-tRNA synthetases for alanine, isoleucine, leucine, lysine, phenyalanine,
threonine, tyrosine, and valine also demonstrated significant down-regulation in WN1589 at LP.
Reducing the aminoacylation of the tRNAs for the above amino acids could explain the
activation of the stringent response in WN1589 by mimicking amino acid starvation113. RNase J2
in a heterotetramer with its paralog RNase J1 has been implicated in regulatory
processing/maturation of the T-box family members of RNA114. How the rnjBΔ9 mutation
56
affects the activity of the RNase J1/J2 complex is speculative at present, but its occurance at an
interface for tetramerization of the RNase J1/J2 heterodimer could affect its catalytic activity or
RNA binding through changes in its oligomeric state.
Aside from the translational machinery, genes encoding for the RNA and DNA
machinery are also downregulated in WN1589, including polC, nusA, rpoA, and rpoC (Table A-
2). The polC gene encodes the DNA polymerase III alpha subunit. nusA encodes a transcription
termination factor for RNA polymerase, rpoA encodes the alpha subunit of RNA polymerase,
and rpoC encodes the β' subunit of RNA polymerase. Down-regulation of these genes was
expected, as they are located within operons encoding the ribosomal proteins and thus are subject
to the stringent response.
Other genes typical of growing cells are downregulated (nucleotide biosynthesis, energy
metabolism, lipid metabolism, and coenzyme production), as shown in the GO term enrichment
and KEGG pathway analyses. This expression pattern was typical of B. subtilis cells undergoing
a stringent response112. A prominent biosynthetic pathway that was enriched from both analyses
was the purine pathway. Mapping the expressional changes on KEGG demonstrated that
enzymes required for the synthesis of IMP ( purCDFHLMNQRS), ATP(adk, purA), and GTP
(guaB and guaC) were significantly downregulated (Fig 4-2). The figure also shows the
downregulation of DNA and RNA synthesis which was expected with the downregulation of the
DNA and RNA machinery described above. Aside from growth rate, the availability of NTPs
have been shown to affect the transcriptional initiation of rRNA genes and other stringent
promoters115. This regulation seems to be limited to stringent response promoters as B. subtilis
initiates the RNA chains with ATP or GTP116.
57
For the up-regulated genes dataset of WN1589 exposed to LP, little was gained from
transcriptomic analysis from the data as no specific terms were enriched by GO term or KEGG
pathway analysis; in addition, the majority of genes identified were unclassified to any regulons.
As such the data had to manually analyzed (Table A-1). If strain WN1589 were undergoing a
stringent response, in B. subtilis and E. coli the expected response would be the induction of
stress and survival related genes86,117. Previously described up-regulated stringent response genes
in B. subtilis were genes prominently involved sporulation, amino acid acquisition, and ilv-leu
operon of branched chain amino acid biosynthesis. Several sporulation genes are shown to be
upregulated at LP in strain WN1589. They include genes that induce sporulation initiation (rapA-
J and ftsX ), SASPs (sspHL), anti-sigma factor (csfB) for sporulation specific SigG, the anti-
toxin (spoIISB) required for efficient sporulation, the spbC-encoded toxin against non-
sporulating cells, and the SpbC immunity protein encoded by spbI. Sporulation in LB is highly
inefficient, sporulation occurs at the ~21 hr mark upon nutrient exhaustion with sporulating cells
accounting for <0.5% of the total cell population118. In addition, the differential expression of
such a small number and varied the stage of sporulation would imply that sporulation is not an
important contributor to fitness. However, the upregulation of the SpbC toxin and the SpbI
immunity protein are promising candidates for enhanced fitness at LP by contributing to the
killing of the w.t. strain in co-culture. No branched chain amino acid genes with the ilv-leu
operon were found to be up-regulated. This is usually a marker for stringent response in B.
subtilis as the CodY protein is a GTP-binding repressor of several operons, including ilv-leu, that
are inactive when cells are growing in abundant nutrients119. The lowered levels of GTP relieves
several operons of CodY regulation. The downregulated and upregulated genes presented here
correspond with microarray data published previously in which the B. subtilis stringent response
58
was induced by simulation of an amino acid starvation through the addition of D-norvaline to
minimal media by Eymann et al. 200286.
Furthermore, in Gram-positive bacteria, the stringent response has been shown to
influence antibiotic production120. In B. subtilis Marburg, the stringent response was shown to
enhance the production of antibiotics121. In our dataset, we found significant changes in the
Gram-positive targeting antimicrobial peptide sublancin 168; its synthesis and transport genes
(sunS, sunT, and sunA) exhibited a >1 L2FC. This would enhance WN1589’s fitness under LP
through further killing of the w.t. population in co-culture.
In summary, the rnjBΔ9 mutation discovered in the LP-evolved strain WN1106 is
important in the LP fitness of B. subtilis. The fitness advantage or disadvantage conferred by this
mutation is dependent on the T and P of exposure. At 25oC, this mutation confers a fitness
disadvantage at SP whereas it confers a fitness advantage at LP. Growth measurements were
taken and the rnjBΔ9 mutant, WN1589, demonstrated a higher growth rate than w.t. at both SP
and LP. While growth rate is an important determinant of fitness, this result reveals that more
complex mechanisms are at play. RNA-seq was conducted on exponentially growing WN1589
and its congenic w.t. strain WN1591 at 25˚C and SP or LP. Differential gene analysis at SP
revealed differences in only two genes: the upregulated sspH and downregulated purA. How
these may contribute to the fitness disadvantage experienced by WN1589 at SP is unclear. One
possible mechanism is that by downregulating purA, WN1589 may be less competitive in a co-
culture as it is unable to produce as much AMP as the w.t. strain WN1590 under the stress of
competitive nutrient sequestration. Exposure of WN1589 to LP revealed a major effect on the
global gene expression pattern. Transcriptomic analysis suggests that WN1589 is experiencing a
stringent response. There was a down-regulation of major pathways and genes involved in
59
growth such as protein synthesis, energy metabolism, energy flux (purine biosynthesis), and lipid
biosynthesis. In addition, genes previously demonstrated to be induced under a stringent
response by B. subtilis were also present like sporulation and antibiotic production. This
stringent response might enhance the viability of WN1589 after overnight co-culture with the
w.t. WN1590 at LP. However, to truly say for sure whether WN1589 experienced a stringent
response at LP, future experiments will need to be conducted to measure the stringent response
alarmone (p)ppGpp and the signal molecules of metabolic flux GTP and ATP. How exactly the
rnjBΔ9 mutation affects RNase J2 activity to trigger the stringent response is not understood and
is currently beyond the scope of this study. Future experiments would involve confirming the
RNA-seq data with RT-PCR, purifying the mutant enzyme and comparing its activity across a
variety of targets like those identified here that are important for stringent response activation
such as T-box regulated aminoacyl tRNA synthetases.
60
Figure 4-1. SWISS-MODEL diagrams of: the predicted structure of the RNase J1/J2
heterotetramer (left); the predicted structure helix 4 in w.t. RNase J2 (center); and
the predicted structure RNase J2 helix 4 resulting from the rnjBΔ9 mutation.
61
Figure 4-2. KEGG Pathway analysis for purine metabolism. Differentially expressed genes are highlighted in green
according to the gradient (legend, upper right) representing L2FC of the gene expression rnjBΔ9 mutant strain
WN1589 compared to w.t. strain WN1591 at LP.
62
APPENDIX
DIFFERENTIAL GENE ANALYSIS AND REGULON TABLES
Table A-1. Differential gene expression data for WN1589 at SP and LP. NS = Not significantly
different. Log-2 Fold Change
Locus tag Gene
Name
Protein name WN1589:WN1591
at 101.3 kPa
WN1589:WN1591
at 5kPa
BSU_misc_RNA26 S26 Integernic to yabD and yabE NS 0.89
BSU_misc_RNA27 S27 5’ UTR of rnmV NS 1.09
BSU_misc_RNA4 S4 Lies within DnaA transcript NS -0.61
BSU_misc_RNA43 S43 5’ UTR of rrnJ-16s NS -0.33
BSU00090 guaB inosine-5\'-monophosphate
dehydrogenase
NS -0.56
BSU00100 dacA D-alanyl-D-alanine
carboxypeptidase DacA
NS 0.94
BSU00240 csfB sporulation protein CsfB NS 1.42
BSU00250 xpaC 5-bromo 4-chloroindolyl phosphate
hydrolysis protein XpaC
NS 0.53
BSU00390 yabD deoxyribonuclease YabD NS -0.54
BSU00500 glmU bifunctional N-acetylglucosamine-
1-phosphate
uridyltransferase/glucosamine-1-
phosphate acetyltransferase
NS -0.44
BSU00510 prs ribose-phosphate
pyrophosphokinase
NS -0.65
BSU00550 mfd transcription-repair-coupling factor NS -0.48
BSU00630 yabR hypothetical protein BSU00630 NS 0.57
BSU00700 coaX type III pantothenate kinase NS -0.47
BSU00820 lysS lysine--tRNA ligase NS -0.42
BSU00930 cysE serine acetyltransferase NS -0.64
BSU00960 rlmB tRNA/rRNA methyltransferase
TrmH
NS -0.64
BSU00970 yacP hypothetical protein BSU00970 NS -0.58
BSU00990 rpmG 50S ribosomal protein L33 2 NS 0.67
BSU01000 secE preprotein translocase subunit SecE NS 0.81
BSU01020 rplK 50S ribosomal protein L11 NS 0.52
BSU01060 ybxB hypothetical protein BSU01060 NS -1.11
BSU01080 rpoC DNA-directed RNA polymerase
subunit beta\'
NS -0.46
BSU01090 ybxF ribosome-associated protein L7Ae-
like
NS -0.44
BSU01100 rpsL 30S ribosomal protein S12 NS -0.73
BSU01110 rpsG 30S ribosomal protein S7 NS -0.73
BSU01120 fusA elongation factor G NS -0.65
BSU01140 ybaC aminopeptidase YbaC NS -0.67
BSU01170 rplD 50S ribosomal protein L4 NS -0.61
BSU01180 rplW 50S ribosomal protein L23 NS -0.69
63
Table A-1. Continued
Log-2 Fold Change
Locus tag Gene
Name
Protein name WN1589:WN1591
at 101.3 kPa
WN1589:WN1591
at 5kPa
BSU01190 rplB 50S ribosomal protein L2 NS -0.73
BSU01200 rpsS 30S ribosomal protein S19 NS -0.94
BSU01210 rplV 50S ribosomal protein L22 NS -0.92
BSU01220 rpsC 30S ribosomal protein S3 NS -0.98
BSU01230 rplP 50S ribosomal protein L16 NS -1.03
BSU01240 rpmC 50S ribosomal protein L29 NS -1.00
BSU01250 rpsQ 30S ribosomal protein S17 NS -0.96
BSU01260 rplN 50S ribosomal protein L14 NS -0.99
BSU01270 rplX 50S ribosomal protein L24 NS -0.97
BSU01280 rplE 50S ribosomal protein L5 NS -1.00
BSU01290 rpsN 30S ribosomal protein S14 NS -1.03
BSU01300 rpsH 30S ribosomal protein S8 NS -0.99
BSU01310 rplF 50S ribosomal protein L6 NS -1.00
BSU01320 rplR 50S ribosomal protein L18 NS -1.00
BSU01330 rpsE 30S ribosomal protein S5 NS -0.98
BSU01340 rpmD 50S ribosomal protein L30 NS -0.96
BSU01350 rplO 50S ribosomal protein L15 NS -0.88
BSU01360 secY protein translocase subunit SecY NS -0.65
BSU01370 adk adenylate kinase NS -0.78
BSU01380 mapA methionine aminopeptidase 1 NS -0.77
BSU01389 ybzG ribosome-binding protein YbzG NS -0.81
BSU01390 infA translation initiation factor IF-1 NS -0.83
BSU01400 rpmJ 50S ribosomal protein L36 NS -0.83
BSU01410 rpsM 30S ribosomal protein S13 NS -0.77
BSU01420 rpsK 30S ribosomal protein S11 NS -0.71
BSU01430 rpoA DNA-directed RNA polymerase
subunit alpha
NS -0.65
BSU01450 cbiO energy-coupling factor transporter
ATP-binding protein EcfA1
NS -0.71
BSU01460 cbiO energy-coupling factor transporter
ATP-binding protein EcfA2
NS -0.67
BSU01470 ybaF energy-coupling factor transporter
transmembrane protein EcfT
NS -0.48
BSU01480 truA tRNA pseudouridine synthase A NS -0.97
BSU01490 rplM 50S ribosomal protein L13 NS -0.56
BSU01820 adaB methylated-dna--protein-cysteine
methyltransferase
NS 0.78
BSU02290 psd phosphatidylserine decarboxylase
proenzyme
NS 0.42
BSU02520 ycbJ hypothetical protein BSU02520 NS 0.76
BSU02580 ycbO hypothetical protein BSU02580 NS 0.67
BSU02670 lmrB lincomycin resistance protein LmrB NS 0.77
64
Table A-1. Continued
Log-2 Fold Change
Locus tag Gene
Name
Protein name WN1589:WN1591
at 101.3 kPa
WN1589:WN1591
at 5kPa
BSU02770 yccK oxidoreductase NS -0.59
BSU02820 rapJ response regulator aspartate
phosphatase J
NS 0.54
BSU02890 yceC stress response protein SCP2 NS -0.58
BSU02990 opuAB glycine betaine transport system
permease protein OpuAB
NS -0.58
BSU03140 tmrB tunicamycin resistance protein NS -0.49
BSU03450 hxlB 3-hexulose-6-phosphate isomerase NS -0.71
BSU03460 hxlA 3-hexulose-6-phosphate synthase NS -0.69
BSU03540 ycxB hypothetical protein BSU03540 NS 0.90
BSU03600 tcyB L-cystine transport system
permease protein TcyB
NS -0.74
BSU03610 tcyA L-cystine-binding protein TcyA NS -0.47
BSU03860 ycnD FMN reductase NS -0.81
BSU03870 ycnE monooxygenase YcnE NS -1.06
BSU03880 yczG ArsR family transcriptional
regulator
NS 0.81
BSU04280 ydaK hypothetical protein BSU04280 NS -0.69
BSU04290 ydaL hypothetical protein BSU04290 NS -0.65
BSU04300 ydaM glycosyltransferase YdaM NS -0.55
BSU04310 ydaN hypothetical protein BSU04310 NS -0.50
BSU04359 ydzK hypothetical protein BSU04359 NS 0.83
BSU04510 ydbL hypothetical protein BSU04510 NS 0.90
BSU04610 ydcA rhomboid protease YdcA NS 0.39
BSU04680 rsbS RsbT antagonist protein RsbS NS -0.57
BSU04690 rsbT serine/threonine protein kinase NS -0.64
BSU04700 rsbU phosphoserine phosphatase RsbU NS -0.59
BSU04750 ydcF hypothetical protein BSU04750 NS -1.20
BSU04780 ydcI hypothetical protein BSU04780 NS -0.67
BSU04850 ydcP hypothetical protein BSU04850 NS 0.95
BSU04860 ydcQ Ftsk domain-containing protein
YdcQ
NS 0.95
BSU05000 yddK hypothetical protein BSU05000 NS 0.77
BSU05010 rapI response regulator aspartate
phosphatase I
NS 0.71
BSU05020 phrI secreted regulator of the activity of
phosphatase RapI
NS 0.63
BSU05050 lrpA AsnC family transcriptional
regulator
NS 0.85
BSU05110 ydeA protease YdeA NS 0.73
BSU05200 ydeH hypothetical protein BSU05200 NS 0.74
BSU05220 ydeJ Lipoprotein NS 1.00
65
Table A-1. Continued
Log-2 Fold Change
Locus tag Gene
Name
Protein name WN1589:WN1591
at 101.3 kPa
WN1589:WN1591
at 5kPa
BSU05390 ydfF ArsR family transcriptional
regulator
NS 0.75
BSU05440 nap carboxylesterase nap NS 0.66
BSU05610 vmlR nucleotide-binding protein ExpZ NS -0.53
BSU05930 rimI ribosomal-protein-alanine
acetyltransferase
NS -0.58
BSU06110 ydjA type-2 restriction enzyme BsuMI
component YdjA
NS 0.72
BSU06140 gutR transcription activator GutR NS -0.48
BSU06250 ydjM hypothetical protein BSU06250 NS 1.14
BSU06320 yeaB Transporter NS 0.54
BSU06450 purC phosphoribosylaminoimidazole-
succinocarboxamide synthase
NS -1.67
BSU06460 purS hypothetical protein BSU06460 NS -1.62
BSU06470 purQ phosphoribosylformylglycinamidine
synthase
NS -1.54
BSU06480 purL phosphoribosylformylglycinamidine
synthase
NS -1.56
BSU06490 purF amidophosphoribosyltransferase NS -1.38
BSU06500 purM phosphoribosylformylglycinamidine
cyclo-ligase
NS -1.31
BSU06510 purN phosphoribosylglycinamide
formyltransferase
NS -1.29
BSU06520 purH bifunctional purine biosynthesis
protein PurH
NS -1.24
BSU06530 purD phosphoribosylamine--glycine
ligase
NS -0.97
BSU06580 yerC hypothetical protein BSU06580 NS 0.65
BSU06610 pcrA ATP-dependent DNA helicase PcrA NS -0.51
BSU06620 ligA DNA ligase NS -0.48
BSU07230 yetM Oxidoreductase NS -0.75
BSU07240 yetN hypothetical protein BSU07240 NS -0.46
BSU07330 yfnB HAD-hydrolase YfnB NS 0.70
BSU07340 yfnA amino acid permease YfnA NS 0.99
BSU07440 yfmK N-acetyltransferase YfmK NS 0.95
BSU08029 yfzA hypothetical protein BSU08029 NS 1.01
BSU08100 acoR acetoin dehydrogenase operon
transcriptional activator AcoR
NS 0.79
BSU08110 sspH small acid-soluble spore protein H 1.09 0.88
BSU08290 yfiJ sensor histidine kinase NS 0.54
BSU08300 yfiK transcriptional regulator NS 0.69
BSU08350 estB extracellular esterase EstB NS -0.52
BSU08470 yfhB isomerase NS -1.00
BSU08630 yfhQ A/G-specific adenine glycosylase
YfhQ
NS -0.58
66
Table A-1. Continued
Log-2 Fold Change
Locus tag Gene
Name
Protein name WN1589:WN1591
at 101.3 kPa
WN1589:WN1591
at 5kPa
BSU08640 yfhS hypothetical protein BSU08640 NS -0.61
BSU08650 fabL enoyl NS -0.52
BSU08740 ygzB hypothetical protein BSU08740 NS 0.65
BSU08820 katA vegetative catalase NS -0.60
BSU08990 yhbI MarR family transcriptional
regulator
NS -0.66
BSU09000 yhbJ efflux system component YhbJ NS -0.75
BSU09010 yhcA MFS transporter NS -0.91
BSU09020 yhcB hypothetical protein BSU09020 NS -0.81
BSU09030 yhcC hypothetical protein BSU09030 NS -0.62
BSU09270 glpP glycerol uptake operon
antiterminator regulatory protein
NS 0.36
BSU09340 yhdA FMN-dependent NADPH-
azoreductase
NS 0.48
BSU09440 citA citrate synthase NS -0.46
BSU09570 yhdR aspartate aminotransferase NS -0.51
BSU09590 yhdT hypothetical protein BSU09590 NS 0.41
BSU09910 yhaO metallophosphoesterase NS -0.56
BSU09930 yhaM 3\'-5\' exoribonuclease YhaM NS 0.49
BSU10150 yhgD TetR family transcriptional
regulator
NS -0.49
BSU10440 yhjA hypothetical protein BSU10440 NS 0.88
BSU10530 ntdC NTD biosynthesis operon putative
oxidoreductase NtdC
NS 0.71
BSU10550 ntdA NTD biosynthesis operon protein
NtdA
NS 0.88
BSU10560 yhjM NTD biosynthesis operon regulator
NtdR
NS 0.83
BSU10800 yizA hypothetical protein BSU10800 NS 0.63
BSU11020 yitK hypothetical protein BSU11020 NS 0.46
BSU11330 fabHA 3-oxoacyl NS -0.47
BSU11550 yjbH hypothetical protein BSU11550 NS 0.54
BSU11910 yjcM hypothetical protein BSU11910 NS 1.19
BSU11940 yjcP hypothetical protein BSU11940 NS 1.13
BSU11950 yjcQ hypothetical protein BSU11950 NS 1.04
BSU12040 yjdG N-acetyltransferase YjdG NS 0.79
BSU12069 yjzH hypothetical protein BSU12069 NS 0.65
BSU12220 yjiC UDP-glucosyltransferase YjiC NS 0.70
BSU12430 rapA response regulator aspartate
phosphatase A
NS 0.91
BSU12450 yjpA hypothetical protein BSU12450 NS 0.63
BSU12490 yjqC hypothetical protein BSU12490 NS 0.77
BSU12820 spoIISB stage II sporulation protein SB NS 0.60
67
Table A-1. Continued
Log-2 Fold Change
Locus tag Gene
Name
Protein name WN1589:WN1591
at 101.3 kPa
WN1589:WN1591
at 5kPa
BSU13010 ykgB 6-phosphogluconolactonase NS -0.59
BSU13030 ykhA acyl-CoA thioester hydrolase YkhA NS 0.59
BSU13310 tnrA MerR family transcriptional
regulator
NS 0.52
BSU13550 mtnA methylthioribose-1-phosphate
isomerase
NS -0.62
BSU13630 ykvA hypothetical protein BSU13630 NS 0.60
BSU13670 mhqR MarR family transcriptional
regulator
NS 0.70
BSU13680 motB motility protein B NS 0.65
BSU13880 glcT BglG family transcription
antiterminator
NS -0.44
BSU13890 ptsG PTS system-glucose-specific
transporter subunit EIICBA
NS -0.65
BSU13910 ptsI phosphoenolpyruvate-protein
phosphotransferase
NS -0.63
BSU13920 splA transcriptional regulator SplA NS -0.82
BSU14010 cheV chemotaxis protein CheV NS 0.82
BSU14060 ykuF 2,4-dienoyl-CoA reductase NS -0.91
BSU14072 ykzU hypothetical protein BSU14072 NS 1.16
BSU14410 sipT signal peptidase I T NS 0.93
BSU14420 ykoA hypothetical protein BSU14420 NS 0.65
BSU14570 ykyA lipoprotein NS 0.75
BSU14580 pdhA pyruvate dehydrogenase E1
component subunit alpha
NS -0.80
BSU14590 pdhB pyruvate dehydrogenase E1
component subunit beta
NS -0.80
BSU14600 pdhC dihydrolipoyllysine-residue
acetyltransferase component of
pyruvate dehydrogenase complex
NS -0.52
BSU14640 yktA hypothetical protein BSU14640 NS 0.82
BSU14840 ylaN hypothetical protein BSU14840 NS 0.54
BSU14890 ctaC cytochrome c oxidase subunit 2 NS 0.82
BSU14920 ctaF cytochrome c oxidase subunit 4B NS 0.74
BSU14930 ctaG cytochrome c oxidase assembly
factor CtaG
NS 0.80
BSU15000 ylbG hypothetical protein BSU15000 NS 0.57
BSU15069 ylzH hypothetical protein BSU15069 NS 0.69
BSU15290 ftsZ cell division protein FtsZ NS 0.49
BSU15410 ylmH RNA-binding protein YlmH NS 0.44
BSU15420 divIVA septum site-determining protein
DivIVA
NS 0.89
BSU15430 ileS isoleucine--tRNA ligase NS -0.41
BSU15450 lspA lipoprotein signal peptidase NS -0.39
68
Table A-1. Continued
Log-2 Fold Change
Locus tag Gene
Name
Protein name WN1589:WN1591
at 101.3 kPa
WN1589:WN1591
at 5kPa
BSU15470 pyrR bifunctional protein PyrR NS 1.12
BSU15570 cysH phosphoadenosine phosphosulfate
reductase
NS -1.24
BSU15700 coaBC coenzyme A biosynthesis
bifunctional protein CoaBC
NS -0.51
BSU15740 rsmB ribosomal RNA small subunit
methyltransferase B
NS -0.47
BSU15760 prpC protein phosphatase PrpC NS -0.58
BSU15770 prkC serine/threonine protein kinase NS -0.60
BSU15870 recG ATP-dependent DNA helicase
RecG
NS -0.42
BSU15890 plsX phosphate acyltransferase NS -0.63
BSU15900 fabD malonyl CoA-acyl carrier protein
transacylase
NS -0.69
BSU15910 fabG 3-oxoacyl NS -0.63
BSU15940 smc chromosome partition protein Smc NS -0.47
BSU15960 ylqB hypothetical protein BSU15960 NS 1.14
BSU16010 ylqD hypothetical protein BSU16010 NS -0.52
BSU16020 rimM ribosome maturation factor RimM NS -0.50
BSU16030 trmD tRNA (guanine-N(1)-)-
methyltransferase
NS -0.66
BSU16090 sucC succinyl-CoA ligase NS -0.57
BSU16130 gid methylenetetrahydrofolate--tRNA-
(uracil-5-)-methyltransferase
TrmFO
NS -0.69
BSU16530 uppS undecaprenyl pyrophosphate
synthase
NS -0.52
BSU16540 cdsA phosphatidate cytidylyltransferase NS -0.44
BSU16550 dxr 1-deoxy-D-xylulose 5-phosphate
reductoisomerase
NS -0.43
BSU16560 rseP zinc metalloprotease RasP NS -0.40
BSU16580 polC DNA polymerase III PolC-type NS -0.53
BSU16600 nusA transcription
termination/antitermination protein
NusA
NS -0.60
BSU16610 ylxR hypothetical protein BSU16610 NS -0.76
BSU16620 ylxQ ribosomal protein YlxQ NS -0.71
BSU16630 infB translation initiation factor IF-2 NS -0.50
BSU16660 truB tRNA pseudouridine synthase B NS -0.77
BSU16700 ylxY hypothetical protein BSU16700 NS -0.71
BSU16710 mlpA zinc protease YmxG NS -0.58
BSU16870 fabG oxidoreductase NS -0.61
BSU16910 ymfM hypothetical protein BSU16910 NS 0.75
BSU16950 pbpX penicillin-binding protein PbpX NS -0.64
69
Table A-1. Continued
Log-2 Fold Change
Locus tag Gene
Name
Protein name WN1589:WN1591
at 101.3 kPa
WN1589:WN1591
at 5kPa
BSU16970 ymdB hypothetical protein BSU16970 NS -0.90
BSU17040 mutS DNA mismatch repair protein MutS NS -0.54
BSU17060 ymzD hypothetical protein BSU17060 NS 0.62
BSU17080 pksA TetR family transcriptional
regulator
NS 0.95
BSU17110 pksD polyketide biosynthesis
acyltransferase homolog PksD
NS 0.51
BSU17200 pksM polyketide synthase PksM NS -0.52
BSU17210 pksN polyketide synthase PksN NS -0.52
BSU17220 pksR polyketide synthase PksR NS -0.51
BSU17380 nrdE ribonucleoside-diphosphate
reductase subunit alpha
NS -0.72
BSU17390 nrdF ribonucleoside-diphosphate
reductase subunit beta
NS -0.43
BSU17430 ynbA GTPase HflX NS -0.54
BSU17440 ynbB hypothetical protein BSU17440 NS -0.67
BSU17500 ynaB hypothetical protein BSU17500 NS 0.78
BSU17510 ynaC hypothetical protein BSU17510 NS 0.82
BSU17530 ynaE hypothetical protein BSU17530 NS 0.88
BSU17540 ynaF hypothetical protein BSU17540 NS 0.77
BSU17650 yncE hypothetical protein BSU17650 NS 0.55
BSU17660 yncF deoxyuridine 5\'-triphosphate
nucleotidohydrolase YncF
NS 0.62
BSU18060 yneR hypothetical protein BSU18060 NS 0.62
BSU18470 proJ glutamate 5-kinase NS -0.85
BSU18500 fabG oxidoreductase NS -0.77
BSU18660 yoaM hypothetical protein BSU18660 NS 0.45
BSU18780 yoaW hypothetical protein BSU18780 NS 0.92
BSU18830 pps phosphoenolpyruvate synthase NS -0.62
BSU18850 yobD XRE family transcriptional
regulator
NS 0.64
BSU18860 yozH hypothetical protein BSU18860 NS 0.73
BSU18870 yozI hypothetical protein BSU18870 NS 0.74
BSU18890 yobF hypothetical protein BSU18890 NS 0.61
BSU18990 yobK hypothetical protein BSU18990 NS 0.91
BSU19000 yobL hypothetical protein BSU19000 NS 0.65
BSU19060 yobR acetyltransferase NS -0.57
BSU19120 czrA ArsR family transcriptional
regulator
NS 0.63
BSU19230 yocJ FMN-dependent NADH-
azoreductase 1
NS -0.88
BSU19290 yozO hypothetical protein BSU19290 NS 0.81
70
Table A-1. Continued
Log-2 Fold Change
Locus tag Gene
Name
Protein name WN1589:WN1591
at 101.3 kPa
WN1589:WN1591
at 5kPa
BSU19370 sucA 2-oxoglutarate dehydrogenase E1
component
NS -0.61
BSU19380 yojO hypothetical protein BSU19380 NS -0.60
BSU19540 yodB ArsR family transcriptional
regulator
NS 0.61
BSU19550 yodC NAD(P)H nitroreductase NS -0.72
BSU19570 yodE ring-cleaving dioxygenase MhqE NS -0.56
BSU20010 yosT transcriptional regulator YosT NS 0.70
BSU20420 yorD stress response protein SCP1 NS 1.32
BSU20430 yorC hypothetical protein BSU20430 NS 1.02
BSU20440 yorB hypothetical protein BSU20440 NS 1.19
BSU20860 yopK hypothetical protein BSU20860 NS 0.89
BSU20870 yopJ hypothetical protein BSU20870 NS 1.00
BSU20900 yopG hypothetical protein BSU20900 NS 0.79
BSU20910 yopF hypothetical protein BSU20910 NS 0.69
BSU20929 yoyI hypothetical protein BSU20929 NS 0.84
BSU20930 yopD hypothetical protein BSU20930 NS 0.81
BSU20940 yopC hypothetical protein BSU20940 NS 0.82
BSU20960 yopA hypothetical protein BSU20960 NS 0.87
BSU20970 yonX hypothetical protein BSU20970 NS 1.28
BSU20999 yoyJ hypothetical protein BSU20999 NS 1.00
BSU21010 yonS Lipoprotein NS 0.87
BSU21060 yonK hypothetical protein BSU21060 NS 1.68
BSU21080 yonI hypothetical protein BSU21080 NS 0.71
BSU21100 yonG hypothetical protein BSU21100 NS 1.51
BSU21350 yomI transglycosylase YomI NS 0.74
BSU21410 blyA N-acetylmuramoyl-L-alanine
amidase BlyA
NS 0.77
BSU21430 bhlB holin-like bacteriophage SPbeta
protein BhlB
NS 1.13
BSU21440 bdbB disulfide bond formation protein B NS 1.35
BSU21450 yolJ glycosyltransferase SunS NS 1.33
BSU21470 sunT sublancin-168-processing and
transport ATP-binding protein SunT
NS 1.22
BSU21480 sunA bacteriocin sublancin-168 NS 1.24
BSU21520 yolC hypothetical protein BSU21520 NS 0.95
BSU21530 yolB hypothetical protein BSU21530 NS 1.37
BSU21540 yolA hypothetical protein BSU21540 NS 1.38
BSU21550 yokL N-acetyltransferase YokL NS 0.82
BSU21560 yokK hypothetical protein BSU21560 NS 0.75
BSU21570 yokJ hypothetical protein BSU21570 NS 0.64
71
Table A-1. Continued
Log-2 Fold Change
Locus tag Gene
Name
Protein name WN1589:WN1591
at 101.3 kPa
WN1589:WN1591
at 5kPa
BSU21590 yokH hypothetical protein BSU21590 NS 0.70
BSU21630 yokD aminoglycoside N(3\')-
acetyltransferase-like protein YokD
NS 0.81
BSU21650 yokB Lipoprotein NS 1.33
BSU21700 ypoP MarR family transcriptional
regulator
NS 1.02
BSU21770 ilvA threonine dehydratase NS -0.67
BSU22000 sspL small acid-soluble spore protein L NS 0.51
BSU22010 exoA 5\'-3\' exonuclease NS 0.45
BSU22020 ypbS hypothetical protein BSU22020 NS 0.68
BSU22030 ypbR hypothetical protein BSU22030 NS -0.69
BSU22080 ypwA metalloprotease YpwA NS -0.58
BSU22150 ypvA ATP-dependent helicase YpvA NS -0.57
BSU22170 ypsC RNA methyltransferase YpsC NS -0.53
BSU22220 yprA ATP-dependent helicase YprA NS -0.52
BSU22300 yppC hypothetical protein BSU22300 NS -0.64
BSU22350 dnaD DNA replication protein DnaD NS -0.50
BSU22410 panD aspartate 1-decarboxylase NS -0.38
BSU22420 panC pantothenate synthetase NS -0.72
BSU22430 panB 3-methyl-2-oxobutanoate
hydroxymethyltransferase
NS -0.79
BSU22440 birA bifunctional protein BirA NS -0.68
BSU22450 cca CCA-adding enzyme NS -0.66
BSU22460 ypjH glycosyltransferase YpjH NS -0.61
BSU22480 mgsA methylglyoxal synthase NS -0.65
BSU22490 dapB 4-hydroxy-tetrahydrodipicolinate
reductase
NS -0.63
BSU22500 ypjD hypothetical protein BSU22500 NS -0.54
BSU22590 ypiA TPR repeat-containing protein
YpiA
NS -0.70
BSU22600 aroE 3-phosphoshikimate 1-
carboxyvinyltransferase
NS 0.60
BSU22680 trpE anthranilate synthase component 1 NS 0.96
BSU22750 ubiE demethylmenaquinone
methyltransferase
NS -0.46
BSU22830 gpsA glycerol-3-phosphate
dehydrogenase
NS -0.51
BSU22840 engA GTPase Der NS -0.59
BSU22940 prsW protease PrsW NS 0.92
BSU22960 gudB cryptic catabolic NAD-specific
glutamate dehydrogenase GudB
NS -0.55
BSU23040 fer ferredoxin NS 0.66
BSU23050 fmnP riboflavin transporter FmnP NS 0.47
72
Table A-1. Continued
Log-2 Fold Change
Locus tag Gene
Name
Protein name WN1589:WN1591
at 101.3 kPa
WN1589:WN1591
at 5kPa
BSU23240 ypzK riboflavin biosynthesis protein RibT NS 0.70
BSU23310 sipS signal peptidase I S NS 0.66
BSU23590 ansR XRE family transcriptional
regulator
NS 0.61
BSU23740 yqjU hypothetical protein BSU23740 NS 0.51
BSU23750 yqjT hypothetical protein BSU23750 NS 0.38
BSU23820 yqjM NADPH dehydrogenase NS -0.95
BSU24010 bmr multidrug resistance protein NS 0.58
BSU24210 yqiG NADH-dependent flavin
oxidoreductase YqiG
NS -0.73
BSU24280 ispA farnesyl diphosphate synthase NS -0.59
BSU24290 xseB exodeoxyribonuclease 7 small
subunit
NS -0.83
BSU24300 xseA exodeoxyribonuclease 7 large
subunit
NS -0.76
BSU24310 folD bifunctional protein FolD NS -0.70
BSU24320 nusB N utilization substance protein B NS -0.67
BSU24340 accC biotin carboxylase 1 NS -0.58
BSU24350 accB biotin carboxyl carrier protein of
acetyl-CoA carboxylase
NS -0.47
BSU24450 efp elongation factor P NS 0.47
BSU24530 yqhM octanoyltransferase LipM NS -0.72
BSU24540 yqhL hypothetical protein BSU24540 NS 0.70
BSU24620 tasA spore coat protein N NS 1.37
BSU24630 sipW signal peptidase I W NS 0.84
BSU24780 yqgY hypothetical protein BSU24780 NS 0.91
BSU24800 yqgW hypothetical protein BSU24800 NS 0.69
BSU24880 yqgO hypothetical protein BSU24880 NS -0.56
BSU24890 yqgN hypothetical protein BSU24890 NS -0.83
BSU24990 pstS phosphate-binding protein PstS NS 0.99
BSU25050 yqgA cell wall-binding protein YqgA NS 0.65
BSU25090 yqfW nucleotidase YqfW NS 0.69
BSU25250 ccpN transcriptional repressor CcpN NS -0.51
BSU25500 hemN oxygen-independent
coproporphyrinogen-III oxidase 1
NS -0.63
BSU25780 arsC arsenate reductase ArsC NS 0.70
BSU25790 arsB arsenite resistance protein ArsB NS 0.66
BSU25800 yqcK hypothetical protein BSU25800 NS 0.78
BSU25830 rapE response regulator aspartate
phosphatase E
NS 0.85
BSU25870 yqcF hypothetical protein BSU25870 NS 0.73
BSU26160 yqbC hypothetical protein BSU26160 NS 0.91
73
Table A-1. Continued
Log-2 Fold Change
Locus tag Gene
Name
Protein name WN1589:WN1591
at 101.3 kPa
WN1589:WN1591
at 5kPa
BSU26210 yqaR hypothetical protein BSU26210 NS 0.99
BSU26230 yqaP hypothetical protein BSU26230 NS 0.78
BSU26740 cypA cytochrome P450 NS -0.82
BSU26850 yrpG oxidoreductase NS -0.94
BSU26910 yraK hydrolase YraK NS -0.83
BSU27190 yrzI hypothetical protein BSU27190 NS 1.09
BSU27340 yrrO protease YrrO NS -0.48
BSU27350 yrrN protease YrrN NS -0.52
BSU27360 yrrM O-methyltransferase YrrM NS -0.50
BSU27410 alaS alanine--tRNA ligase NS -0.49
BSU27540 yrvM hypothetical protein BSU27540 NS 0.46
BSU27620 recJ single-stranded-dna-specific
exonuclease RecJ
NS -0.36
BSU27700 yajC preprotein translocase subunit YajC NS 0.85
BSU27960 rplU 50S ribosomal protein L21 NS -0.29
BSU28080 folC folylpolyglutamate synthase NS -0.59
BSU28090 valS valine--tRNA ligase NS -0.70
BSU28360 ysnA non-canonical purine NTP
pyrophosphatase
NS -0.60
BSU28370 rph ribonuclease PH NS -0.78
BSU28490 uvrC UvrABC system protein C NS -0.60
BSU28620 rnhC ribonuclease HIII NS -0.41
BSU28640 pheS phenylalanine--tRNA ligase alpha
subunit
NS -0.61
BSU28670 ysfB hypothetical protein BSU28670 NS -0.55
BSU28700 ysfE hypothetical protein BSU28700 NS 0.75
BSU28820 ysdC aminopeptidase NS -0.55
BSU28840 ysdA hypothetical protein BSU28840 NS -0.45
BSU28900 ysbB antiholin-like protein LrgB NS 0.58
BSU28950 thrS threonine--tRNA ligase 1 NS -0.38
BSU29070 ytaF hypothetical protein BSU29070 NS -0.55
BSU29130 icd isocitrate dehydrogenase NS -0.69
BSU29200 accA acetyl-coenzyme A carboxylase
carboxyl transferase subunit alpha
NS -0.40
BSU29470 ackA acetate kinase NS -0.62
BSU29590 iscS cysteine desulfurase IscS 2 NS -0.37
BSU29670 tyrS tyrosine--tRNA ligase 1 NS -0.55
BSU29760 ytxJ hypothetical protein BSU29760 NS 0.60
BSU29805 ytpS DNA translocase SftA NS -0.54
BSU29830 ytpQ hypothetical protein BSU29830 NS -0.56
BSU29840 ytpP thioredoxin NS -0.67
74
Table A-1. Continued
Log-2 Fold Change
Locus tag Gene
Name
Protein name WN1589:WN1591
at 101.3 kPa
WN1589:WN1591
at 5kPa
BSU29990 pbuO guanine/hypoxanthine permease
PbuO
NS -0.55
BSU30320 leuS leucine--tRNA ligase NS -0.63
BSU30420 ytrE ABC transporter ATP-binding
protein
NS -0.65
BSU30990 thiT thiamine transporter ThiT NS 0.57
BSU31350 pgi glucose-6-phosphate isomerase NS -0.54
BSU31360 yugK NADH-dependent butanol
dehydrogenase 2
NS -0.96
BSU31370 yugJ NADH-dependent butanol
dehydrogenase 1
NS -1.00
BSU31380 yuzA hypothetical protein BSU31380 NS -0.76
BSU31440 patB cystathionine beta-lyase NS -0.44
BSU31450 kinB sporulation kinase B NS -0.66
BSU31760 pncA isochorismatase NS -0.54
BSU32040 yuiF amino acid transporter YuiF NS 1.19
BSU32110 yumC ferredoxin--NADP reductase 2 NS -0.52
BSU32130 guaC GMP reductase NS -0.92
BSU32310 yutD hypothetical protein BSU32310 NS -0.47
BSU32420 pucR purine catabolism regulatory protein NS -0.80
BSU32680 iscU NifU-like protein NS -0.43
BSU32690 sufS cysteine desulfurase NS -0.57
BSU32700 sufD FeS cluster assembly protein SufD NS -0.44
BSU32710 sufC vegetative protein 296 NS -0.69
BSU32719 yuzK hypothetical protein BSU32719 NS -0.95
BSU32770 yusE thioredoxin NS -0.64
BSU32780 yusF hypothetical protein BSU32780 NS -0.74
BSU32810 yusI hypothetical protein BSU32810 NS -0.73
BSU32940 yusV siderophore transport system ATP-
binding protein YusV
NS 0.40
BSU33210 yvrG sensor histidine kinase NS -0.48
BSU33260 yvrN ABC transporter permease NS 0.88
BSU33400 yvgN glyoxal reductase NS -0.81
BSU33430 yvgQ sulfite reductase NS 0.53
BSU33560 yvaD hypothetical protein BSU33560 NS 0.69
BSU33570 yvaE hypothetical protein BSU33570 NS 0.74
BSU33610 rnr ribonuclease R NS -0.37
BSU33620 yvaK carboxylesterase NS -0.66
BSU33770 spbC killing factor SdpC NS 0.71
BSU33780 sdpI immunity protein SdpI NS 0.89
BSU33840 yvbF HTH-type transcriptional regulator
YvbF
NS 0.73
75
Table A-1. Continued
Log-2 Fold Change
Locus tag Gene
Name
Protein name WN1589:WN1591
at 101.3 kPa
WN1589:WN1591
at 5kPa
BSU33910 pgm 2,3-bisphosphoglycerate-
independent phosphoglycerate
mutase
NS -0.92
BSU33920 tpiA triosephosphate isomerase NS -0.91
BSU33930 pgk phosphoglycerate kinase NS -0.93
BSU33950 cggR central glycolytic genes regulator NS -0.86
BSU33970 araR transcriptional repressor NS -0.51
BSU34640 yvdD LOG family protein YvdD NS -0.70
BSU34880 hisA 1-(5-phosphoribosyl)-5 NS 0.85
BSU35050 yvnA HTH-type transcriptional regulator
YvnA
NS 0.83
BSU35250 ftsX cell division protein FtsX NS 0.58
BSU35490 degU transcriptional regulatory protein
DegU
NS 0.49
BSU35530 tagO undecaprenyl-phosphate N-
acetylglucosaminyl 1-phosphate
transferase
NS 0.69
BSU35740 tagD glycerol-3-phosphate
cytidylyltransferase
NS 1.02
BSU35750 tagA N-acetylmannosaminyltransferase NS 0.75
BSU35990 ywrO general stress protein 14 NS -0.55
BSU36900 glyA serine hydroxymethyltransferase NS -0.84
BSU36980 ywlA hypothetical protein BSU36980 NS 0.64
BSU37000 prmC release factor glutamine
methyltransferase
NS -0.55
BSU37020 ywkD hypothetical protein BSU37020 NS -0.75
BSU37030 racA chromosome-anchoring protein
RacA
NS -0.49
BSU37050 ywkA NAD-dependent malic enzyme 2 NS -0.58
BSU37080 rho transcription termination factor Rho NS -0.41
BSU37110 ywjH transaldolase NS -0.48
BSU37330 argS arginine--tRNA ligase NS 0.31
BSU37460 rapF response regulator aspartate
phosphatase F
NS 0.94
BSU37610 ywzC hypothetical protein BSU37610 NS 0.86
BSU38020 thiD pyridoxine kinase NS -0.59
BSU38120 rodA rod shape-determining protein
RodA
NS 0.74
BSU38190 galT galactose-1-phosphate
uridylyltransferase
NS 0.40
BSU38460 tyrZ tyrosine--tRNA ligase 2 NS -0.64
BSU38850 yxkC hypothetical protein BSU38850 NS 0.86
BSU38930 yxjJ hypothetical protein BSU38930 NS 0.74
BSU38940 yxjI hypothetical protein BSU38940 NS 0.60
76
Table A-1. Continued
Log-2 Fold Change
Locus tag Gene
Name
Protein name WN1589:WN1591
at 101.3 kPa
WN1589:WN1591
at 5kPa
BSU39040 yxiS hypothetical protein BSU39040 NS 0.82
BSU39120 yxiM esterase YxiM NS -0.64
BSU39280 yxxE hypothetical protein BSU39280 NS 0.81
BSU39290 yxxD hypothetical protein BSU39290 NS 0.87
BSU39300 yxiD hypothetical protein BSU39300 NS 0.66
BSU39420 deoC deoxyribose-phosphate aldolase NS -0.60
BSU39580 yxeE hypothetical protein BSU39580 NS 0.61
BSU39830 yxcA hypothetical protein BSU39830 NS 0.65
BSU39940 yxaL hypothetical protein BSU39940 NS 0.96
BSU40140 yydJ peptide export permease protein
YydJ
NS 1.11
BSU40150 yydI peptide export ATP-binding protein
YydI
NS 0.97
BSU40170 yydG peptide biosynthesis protein YydG NS 0.77
BSU40200 yydD hypothetical protein BSU40200 NS 0.89
BSU40210 yydC hypothetical protein BSU40210 NS 0.81
BSU40220 yydB metallophosphoesterase NS 0.74
BSU40240 yycS hypothetical protein BSU40240 NS 0.69
BSU40350 rocR arginine utilization regulatory
protein RocR
NS -0.44
BSU40390 walH two-component system YycF/YycG
regulatory protein YycH
NS -0.53
BSU40400 walK sensor histidine kinase NS -0.59
BSU40420 purA adenylosuccinate synthetase -0.71 -0.83
BSU40520 yybS hypothetical protein BSU40520 NS -0.39
BSU40540 yybR HTH-type transcriptional regulator
YybR
NS 1.00
BSU40580 yybN hypothetical protein BSU40580 NS 1.26
BSU40610 yybK hypothetical protein BSU40610 NS 0.67
BSU40620 yybJ ABC transporter ATP-binding
protein
NS 0.68
BSU40970 parA sporulation initiation inhibitor
protein Soj
NS -0.39
77
Table A-2. Regulons of DEGs found in WN1589 at 5 kPa
Locus tag L2FC Gene
Name Protein name Regulon Regulator Action
BSU21440 1.35 bdbB disulfide bond formation
protein B Abh Abh activation
BSU21450 1.33 yolJ glycosyltransferase SunS Abh Abh activation
BSU21480 1.24 sunA bacteriocin sublancin-168 Abh Abh activation
BSU21470 1.22 sunT
sublancin-168-processing
and transport ATP-binding
protein SunT
Abh Abh activation
BSU14890 0.82 ctaC cytochrome c oxidase
subunit 2 Abh Abh repression
BSU14930 0.80 ctaG cytochrome c oxidase
assembly factor CtaG Abh Abh repression
BSU14920 0.74 ctaF cytochrome c oxidase
subunit 4B Abh Abh repression
BSU21540 1.38 yolA hypothetical protein
BSU21540 AbrB AbrB repression
BSU24620 1.37 tasA spore coat protein N AbrB AbrB repression
BSU21530 1.37 yolB hypothetical protein
BSU21530 AbrB AbrB repression
BSU21440 1.35 bdbB disulfide bond formation
protein B AbrB AbrB repression
BSU21450 1.33 yolJ glycosyltransferase SunS AbrB AbrB repression
BSU20420 1.32 yorD stress response protein SCP1 AbrB AbrB repression
BSU21480 1.24 sunA bacteriocin sublancin-168 AbrB AbrB repression
BSU21470 1.22 sunT
sublancin-168-processing
and transport ATP-binding
protein SunT
AbrB AbrB repression
BSU11910 1.19 yjcM hypothetical protein
BSU11910 AbrB AbrB repression
BSU15960 1.14 ylqB hypothetical protein
BSU15960 AbrB AbrB repression
BSU40140 1.11 yydJ peptide export permease
protein YydJ AbrB AbrB repression
BSU27190 1.09 yrzI hypothetical protein
BSU27190 AbrB AbrB repression
BSU05220 1.00 ydeJ lipoprotein AbrB AbrB repression
BSU40150 0.97 yydI peptide export ATP-binding
protein YydI AbrB AbrB repression
BSU39940 0.96 yxaL hypothetical protein
BSU39940 AbrB AbrB repression
BSU21520 0.95 yolC hypothetical protein
BSU21520 AbrB AbrB repression
BSU18780 0.92 yoaW hypothetical protein
BSU18780 AbrB AbrB repression
BSU33780 0.89 sdpI immunity protein SdpI AbrB AbrB repression
BSU33260 0.88 yvrN ABC transporter permease AbrB AbrB repression
78
Table A-2. Continued
Locus tag L2FC Gene
Name Protein name Regulon Regulator Action
BSU24630 0.84 sipW signal peptidase I W AbrB AbrB repression
BSU35050 0.83 yvnA HTH-type transcriptional
regulator YvnA AbrB AbrB repression
BSU10560 0.83 yhjM NTD biosynthesis operon
regulator NtdR AbrB AbrB repression
BSU14890 0.82 ctaC cytochrome c oxidase
subunit 2 AbrB AbrB repression
BSU21550 0.82 yokL N-acetyltransferase YokL AbrB AbrB repression
BSU14930 0.80 ctaG cytochrome c oxidase
assembly factor CtaG AbrB AbrB repression
BSU12040 0.79 yjdG N-acetyltransferase YjdG AbrB AbrB repression
BSU26230 0.78 yqaP hypothetical protein
BSU26230 AbrB AbrB repression
BSU40170 0.77 yydG peptide biosynthesis protein
YydG AbrB AbrB repression
BSU02520 0.76 ycbJ hypothetical protein
BSU02520 AbrB AbrB repression
BSU21560 0.75 yokK hypothetical protein
BSU21560 AbrB AbrB repression
BSU14920 0.74 ctaF cytochrome c oxidase
subunit 4B AbrB AbrB repression
BSU05200 0.74 ydeH hypothetical protein
BSU05200 AbrB AbrB repression
BSU33770 0.71 spbC killing factor SdpC AbrB AbrB repression
BSU21570 0.64 yokJ hypothetical protein
BSU21570 AbrB AbrB repression
BSU01820 0.78 adaB methylated-dna--protein-
cysteine methyltransferase AdaA AdaA
positive
regulation
BSU23590 0.61 ansR XRE family transcriptional
regulator AnsR AnsR repression
BSU33970 -0.51 araR transcriptional repressor AraR AraR repression
BSU25800 0.78 yqcK hypothetical protein
BSU25800 ArsR ArsR repression
BSU25780 0.70 arsC arsenate reductase ArsC ArsR ArsR repression
BSU25790 0.66 arsB arsenite resistance protein
ArsB ArsR ArsR repression
BSU24010 0.58 bmr multidrug resistance protein BmrR BmrR activation
BSU37460 0.94 rapF response regulator aspartate
phosphatase F CcpA CcpA repression
BSU08110 0.88 sspH small acid-soluble spore
protein H CcpA CcpA repression
BSU35050 0.83 yvnA HTH-type transcriptional
regulator YvnA CcpA CcpA repression
BSU14890 0.82 ctaC cytochrome c oxidase
subunit 2 CcpA CcpA repression
79
Table A-2. Continued
Locus tag L2FC Gene
Name Protein name Regulon Regulator Action
BSU08100 0.79 acoR
acetoin dehydrogenase
operon transcriptional
activator AcoR
CcpA CcpA repression
BSU35490 0.49 degU transcriptional regulatory
protein DegU CcpA CcpA activation
BSU38190 0.40 galT galactose-1-phosphate
uridylyltransferase CcpA CcpA repression
BSU16090 -0.57 sucC succinyl-CoA ligase CcpA CcpA repression
BSU39420 -0.60 deoC deoxyribose-phosphate
aldolase CcpA CcpA repression
BSU19370 -0.61 sucA
2-oxoglutarate
dehydrogenase E1
component
CcpA CcpA repression
BSU29470 -0.62 ackA acetate kinase CcpA CcpA activation
BSU29130 -0.69 icd isocitrate dehydrogenase CcpA CcpA repression
BSU29130 -0.69 icd isocitrate dehydrogenase CcpC CcpC repression
BSU33950 -0.86 cggR central glycolytic genes
regulator CggR CggR repression
BSU33920 -0.91 tpiA triosephosphate isomerase CggR CggR repression
BSU33910 -0.92 pgm
2,3-bisphosphoglycerate-
independent
phosphoglycerate mutase
CggR CggR repression
BSU33930 -0.93 pgk phosphoglycerate kinase CggR CggR repression
BSU09440 -0.46 citA citrate synthase CitR CitR repression
BSU00090 -0.56 guaB inosine-5\'-monophosphate
dehydrogenase CodY CodY activation
BSU29470 -0.62 ackA acetate kinase CodY CodY activation
BSU32130 -0.92 guaC GMP reductase CodY CodY activation
BSU37460 0.94 rapF response regulator aspartate
phosphatase F ComA ComA activation
BSU12430 0.91 rapA response regulator aspartate
phosphatase A ComA ComA activation
BSU25830 0.85 rapE response regulator aspartate
phosphatase E ComA ComA activation
BSU15890 -0.63 plsX phosphate acyltransferase ComA ComA activation
BSU33260 0.88 yvrN ABC transporter permease ComK ComK activation
BSU15570 -1.24 cysH phosphoadenosine
phosphosulfate reductase CymR CymR repression
BSU33430 0.53 yvgQ sulfite reductase CysL CysL activation
BSU14410 0.93 sipT signal peptidase I T DegU DegU activation
BSU38930 0.74 yxjJ hypothetical protein
BSU38930 DegU DegU
Repression
80
Table A-2. Continued
Locus tag L2FC Gene
Name Protein name Regulon Regulator Action
BSU38940 0.60 yxjI hypothetical protein
BSU38940 DegU DegU repression
BSU35490 0.49 degU transcriptional regulatory
protein DegU DegU DegU activation
BSU39420 -0.60 deoC deoxyribose-phosphate
aldolase DeoR DeoR repression
BSU14060 -0.91 ykuF 2,4-dienoyl-CoA reductase FadR FadR repression
BSU11330 -0.47 fabHA 3-oxoacyl FapR FapR repression
BSU15910 -0.63 fabG 3-oxoacyl FapR FapR repression
BSU15890 -0.63 plsX phosphate acyltransferase FapR FapR repression
BSU15900 -0.69 fabD malonyl CoA-acyl carrier
protein transacylase FapR FapR repression
BSU23240 0.70 ypzK riboflavin biosynthesis
protein RibT FMN-box FMN-box termination
BSU23050 0.47 fmnP riboflavin transporter FmnP FMN-box FMN-box termination
BSU32940 0.40 yusV siderophore transport system
ATP-binding protein YusV Fur Fur repression
BSU06530 -0.97 purD phosphoribosylamine--
glycine ligase G-box G-box termination
BSU06520 -1.24 purH bifunctional purine
biosynthesis protein PurH G-box G-box termination
BSU06510 -1.29 purN phosphoribosylglycinamide
formyltransferase G-box G-box termination
BSU06500 -1.31 purM phosphoribosylformylglycina
midine cyclo-ligase G-box G-box termination
BSU06490 -1.38 purF amidophosphoribosyltransfer
ase G-box G-box termination
BSU06470 -1.54 purQ phosphoribosylformylglycina
midine synthase G-box G-box termination
BSU06480 -1.56 purL phosphoribosylformylglycina
midine synthase G-box G-box termination
BSU06460 -1.62 purS hypothetical protein
BSU06460 G-box G-box termination
BSU06450 -1.67 purC
phosphoribosylaminoimidazo
le-succinocarboxamide
synthase
G-box G-box termination
BSU39580 0.61 yxeE hypothetical protein
BSU39580 GerE GerE activation
BSU13910 -0.63 ptsI phosphoenolpyruvate-protein
phosphotransferase GlcT GlcT
Antiterminat
ion
BSU13890 -0.65 ptsG PTS system-glucose-specific
transporter subunit EIICBA GlcT GlcT
Antiterminat
ion
81
Table A-2. Continued
Locus tag L2FC Gene
Name Protein name Regulon Regulator Action
BSU13310 0.52 tnrA MerR family transcriptional
regulator GlnR GlnR repression
BSU03460 -0.69 hxlA 3-hexulose-6-phosphate
synthase HxlR HxlR activation
BSU03450 -0.71 hxlB 3-hexulose-6-phosphate
isomerase HxlR HxlR activation
BSU20440 1.19 yorB hypothetical protein
BSU20440 LexA LexA repression
BSU20430 1.02 yorC hypothetical protein
BSU20430 LexA LexA repression
BSU21520 0.95 yolC hypothetical protein
BSU21520 LexA LexA repression
BSU09930 0.49 yhaM 3\'-5\' exoribonuclease YhaM LexA LexA repression
BSU06620 -0.48 ligA DNA ligase LexA LexA repression
BSU06610 -0.51 pcrA ATP-dependent DNA
helicase PcrA LexA LexA repression
BSU09910 -0.56 yhaO metallophosphoesterase LexA LexA repression
BSU28490 -0.60 uvrC UvrABC system protein C LexA LexA repression
BSU09340 0.48 yhdA FMN-dependent NADPH-
azoreductase LiaR LiaR activation
BSU02670 0.77 lmrB lincomycin resistance protein
LmrB LmrA LmrA repression
BSU37050 -0.58 ywkA NAD-dependent malic
enzyme 2 MalR MalR activation
BSU19570 -0.56 yodE ring-cleaving dioxygenase
MhqE MhqR MhqR repression
BSU24010 0.58 bmr multidrug resistance protein Mta Mta activation
BSU10550 0.88 ntdA NTD biosynthesis operon
protein NtdA NtdR NtdR activation
BSU10530 0.71 ntdC
NTD biosynthesis operon
putative oxidoreductase
NtdC
NtdR NtdR activation
BSU08820 -0.60 katA vegetative catalase PerR PerR repression
BSU06250 1.14 ydjM hypothetical protein
BSU06250 PhoP PhoP activation
BSU35740 1.02 tagD glycerol-3-phosphate
cytidylyltransferase PhoP PhoP repression
BSU24990 0.99 pstS phosphate-binding protein
PstS PhoP PhoP activation
BSU35750 0.75 tagA
N-
acetylmannosaminyltransfera
se
PhoP PhoP repression
BSU32420 -0.80 pucR purine catabolism regulatory
protein PucR PucR repression
82
Table A-2. Continued
Locus tag L2FC Gene
Name Protein name Regulon Regulator Action
BSU29990 -0.55 pbuO guanine/hypoxanthine
permease PbuO PurR PurR repression
BSU24320 -0.67 nusB N utilization substance
protein B PurR PurR repression
BSU24310 -0.70 folD bifunctional protein FolD PurR PurR repression
BSU40420 -0.83 purA adenylosuccinate synthetase PurR PurR repression
BSU36900 -0.84 glyA serine
hydroxymethyltransferase PurR PurR repression
BSU32130 -0.92 guaC GMP reductase PurR PurR repression
BSU06530 -0.97 purD phosphoribosylamine--
glycine ligase PurR PurR repression
BSU06520 -1.24 purH bifunctional purine
biosynthesis protein PurH PurR PurR repression
BSU06510 -1.29 purN phosphoribosylglycinamide
formyltransferase PurR PurR repression
BSU06500 -1.31 purM phosphoribosylformylglycina
midine cyclo-ligase PurR PurR repression
BSU06490 -1.38 purF amidophosphoribosyltransfer
ase PurR PurR repression
BSU06470 -1.54 purQ phosphoribosylformylglycina
midine synthase PurR PurR repression
BSU06480 -1.56 purL phosphoribosylformylglycina
midine synthase PurR PurR repression
BSU06460 -1.62 purS hypothetical protein
BSU06460 PurR PurR repression
BSU06450 -1.67 purC
phosphoribosylaminoimidazo
le-succinocarboxamide
synthase
PurR PurR repression
BSU15470 1.12 pyrR bifunctional protein PyrR PyrR PyrR antiterminati
on
BSU14890 0.82 ctaC cytochrome c oxidase
subunit 2 ResD ResD activation
BSU14930 0.80 ctaG cytochrome c oxidase
assembly factor CtaG ResD ResD activation
BSU14920 0.74 ctaF cytochrome c oxidase
subunit 4B ResD ResD activation
BSU17390 -0.43 nrdF ribonucleoside-diphosphate
reductase subunit beta ResD ResD activation
BSU17380 -0.72 nrdE ribonucleoside-diphosphate
reductase subunit alpha ResD ResD activation
BSU05610 -0.53 vmlR nucleotide-binding protein
ExpZ
RNA
switch/
other
RNA switch unknown
mechanism
83
Table A-2. Continued
Locus tag L2FC Gene
Name Protein name Regulon Regulator Action
BSU21440 1.35 bdbB disulfide bond formation
protein B Rok Rok repression
BSU21450 1.33 yolJ glycosyltransferase SunS Rok Rok repression
BSU40580 1.26 yybN hypothetical protein
BSU40580 Rok Rok repression
BSU21480 1.24 sunA bacteriocin sublancin-168 Rok Rok Repression
BSU21470 1.22 sunT
sublancin-168-processing
and transport ATP-binding
protein SunT
Rok Rok Repression
BSU40140 1.11 yydJ peptide export permease
protein YydJ Rok Rok Repression
BSU40150 0.97 yydI peptide export ATP-binding
protein YydI Rok Rok Repression
BSU39940 0.96 yxaL hypothetical protein
BSU39940 Rok Rok Repression
BSU33770 0.71 spbC killing factor SdpC Rok Rok Repression
BSU40610 0.67 yybK hypothetical protein
BSU40610 Rok Rok Repression
BSU28840 -0.45 ysdA hypothetical protein
BSU28840 RplT RplT Termination
BSU13550 -0.62 mtnA methylthioribose-1-
phosphate isomerase S-box S-box Termination
BSU15570 -1.24 cysH phosphoadenosine
phosphosulfate reductase S-box S-box Termination
BSU33780 0.89 sdpI immunity protein SdpI SdpR SdpR Repression
BSU39040 0.82 yxiS hypothetical protein
BSU39040 SigB SigB Unknown
BSU38930 0.74 yxjJ hypothetical protein
BSU38930 SigB SigB Unknown
BSU38940 0.60 yxjI hypothetical protein
BSU38940 SigB SigB Unknown
BSU29760 0.60 ytxJ hypothetical protein
BSU29760 SigB SigB Unknown
BSU24010 0.58 bmr multidrug resistance protein SigB SigB Unknown
BSU33610 -0.37 rnr ribonuclease R SigB SigB Unknown
BSU02890 -0.58 yceC stress response protein SCP2 SigB SigB Unknown
BSU13010 -0.59 ykgB 6-phosphogluconolactonase SigB SigB Unknown
BSU33620 -0.66 yvaK carboxylesterase SigB SigB Unknown
BSU31380 -0.76 yuzA hypothetical protein
BSU31380 SigB SigB Unknown
BSU33400 -0.81 yvgN glyoxal reductase SigB SigB Unknown
BSU15960 1.14 ylqB hypothetical protein
BSU15960 SigD SigD Unknown
84
Table A-2. Continued
Locus tag L2FC Gene
Name Protein name Regulon Regulator Action
BSU11940 1.13 yjcP hypothetical protein
BSU11940 SigD SigD Unknown
BSU11950 1.04 yjcQ hypothetical protein
BSU11950 SigD SigD Unknown
BSU38850 0.86 yxkC hypothetical protein
BSU38850 SigD SigD Unknown
BSU14010 0.82 cheV chemotaxis protein CheV SigD SigD Unknown
BSU13680 0.65 motB motility protein B SigD SigD Unknown
BSU18780 0.92 yoaW hypothetical protein
BSU18780 SigE SigE Unknown
BSU00630 0.57 yabR hypothetical protein
BSU00630 SigE SigE Unknown
BSU04610 0.39 ydcA rhomboid protease YdcA SigE SigE Unknown
BSU29070 -0.55 ytaF hypothetical protein
BSU29070 SigE SigE Unknown
BSU00240 1.42 csfB sporulation protein CsfB SigF SigF Unknown
BSU25800 0.78 yqcK hypothetical protein
BSU25800 SigF SigF Unknown
BSU25780 0.70 arsC arsenate reductase ArsC SigF SigF Unknown
BSU25790 0.66 arsB arsenite resistance protein
ArsB SigF SigF Unknown
BSU08110 0.88 sspH small acid-soluble spore
protein H SigG SigG Unknown
BSU22000 0.51 sspL small acid-soluble spore
protein L SigG SigG Unknown
BSU08650 -0.52 fabL enoyl SigG SigG Unknown
BSU31380 -0.76 yuzA hypothetical protein
BSU31380 SigG SigG Unknown
BSU13920 -0.82 splA transcriptional regulator
SplA SigG SigG Unknown
BSU05020 0.63 phrI secreted regulator of the
activity of phosphatase RapI SigH SigH Unknown
BSU29760 0.60 ytxJ hypothetical protein
BSU29760 SigH SigH Unknown
BSU15290 0.49 ftsZ cell division protein FtsZ SigH SigH Unknown
BSU37030 -0.49 racA chromosome-anchoring
protein RacA SigH SigH Unknown
BSU39580 0.61 yxeE hypothetical protein
BSU39580 SigK SigK Unknown
BSU38120 0.74 rodA rod shape-determining
protein RodA SigM SigM Unknown
BSU00700 -0.47 coaX type III pantothenate kinase SigM SigM Unknown
85
Table A-2. Continued
Locus tag L2FC Gene
Name Protein name Regulon Regulator Action
BSU02890 -0.58 yceC stress response protein SCP2 SigM SigM Unknown
BSU16950 -0.64 pbpX penicillin-binding protein
PbpX SigM SigM Unknown
BSU19290 0.81 yozO hypothetical protein
BSU19290 SigW SigW Unknown
BSU38940 0.60 yxjI hypothetical protein
BSU38940 SigW SigW Unknown
BSU00250 0.53 xpaC
5-bromo 4-chloroindolyl
phosphate hydrolysis protein
XpaC
SigW SigW Unknown
BSU02890 -0.58 yceC stress response protein SCP2 SigW SigW Unknown
BSU02290 0.42 psd phosphatidylserine
decarboxylase proenzyme SigX SigX Unknown
BSU02890 -0.58 yceC stress response protein SCP2 SigX SigX Unknown
BSU16950 -0.64 pbpX penicillin-binding protein
PbpX SigX SigX Unknown
BSU24620 1.37 tasA spore coat protein N SinR SinR Repression
BSU24630 0.84 sipW signal peptidase I W SinR SinR Repression
BSU33400 -0.81 yvgN glyoxal reductase SinR SinR Repression
BSU12430 0.91 rapA response regulator aspartate
phosphatase A Spo0A Spo0A Repression
BSU15420 0.89 divIV
A
septum site-determining
protein DivIVA Spo0A Spo0A Repression
BSU25870 0.73 yqcF hypothetical protein
BSU25870 Spo0A Spo0A Activation
BSU33770 0.71 spbC killing factor SdpC Spo0A Spo0A Activation
BSU35250 0.58 ftsX cell division protein FtsX Spo0A Spo0A Repression
BSU15410 0.44 ylmH RNA-binding protein YlmH Spo0A Spo0A Repression
BSU40970 -0.39 parA sporulation initiation
inhibitor protein Soj Spo0A Spo0A Repression
BSU29200 -0.40 accA
acetyl-coenzyme A
carboxylase carboxyl
transferase subunit alpha
Spo0A Spo0A Activation
BSU37030 -0.49 racA chromosome-anchoring
protein RacA Spo0A Spo0A Activation
BSU32770 -0.64 yusE thioredoxin Spo0A Spo0A Activation
BSU08650 -0.52 fabL enoyl SpoVT SpoVT Repression
BSU29830 -0.56 ytpQ hypothetical protein
BSU29830 Spx Spx Activation
BSU29840 -0.67 ytpP thioredoxin Spx Spx Activation
BSU31370 -1.00 yugJ NADH-dependent butanol
dehydrogenase 1 Spx Spx Activation
86
Table A-2. Continued
Locus tag L2FC Gene
Name Protein name Regulon Regulator Action
BSU01020 0.52 rplK 50S ribosomal protein L11 stringent
response
stringent
response
negative
regulation
BSU27960 -0.29 rplU 50S ribosomal protein L21 stringent
response
stringent
response
negative
regulation
BSU27620 -0.36 recJ single-stranded-dna-specific
exonuclease RecJ
stringent
response
stringent
response
negative
regulation
BSU00500 -0.44 glmU
bifunctional N-
acetylglucosamine-1-
phosphate
uridyltransferase/glucosamin
e-1-phosphate
acetyltransferase
stringent
response
stringent
response
negative
regulation
BSU01090 -0.44 ybxF ribosome-associated protein
L7Ae-like
stringent
response
stringent
response
negative
regulation
BSU01080 -0.46 rpoC DNA-directed RNA
polymerase subunit beta\'
stringent
response
stringent
response
negative
regulation
BSU16630 -0.50 infB translation initiation factor
IF-2
stringent
response
stringent
response
negative
regulation
BSU14600 -0.52 pdhC
dihydrolipoyllysine-residue
acetyltransferase component
of pyruvate dehydrogenase
complex
stringent
response
stringent
response
negative
regulation
BSU01490 -0.56 rplM 50S ribosomal protein L13 stringent
response
stringent
response
negative
regulation
BSU16600 -0.60 nusA
transcription
termination/antitermination
protein NusA
stringent
response
stringent
response
negative
regulation
BSU01170 -0.61 rplD 50S ribosomal protein L4 stringent
response
stringent
response
negative
regulation
BSU13910 -0.63 ptsI phosphoenolpyruvate-protein
phosphotransferase
stringent
response
stringent
response
negative
regulation
BSU01430 -0.65 rpoA DNA-directed RNA
polymerase subunit alpha
stringent
response
stringent
response
negative
regulation
BSU13890 -0.65 ptsG PTS system-glucose-specific
transporter subunit EIICBA
stringent
response
stringent
response
negative
regulation
BSU01360 -0.65 secY protein translocase subunit
SecY
stringent
response
stringent
response
negative
regulation
BSU00510 -0.65 prs ribose-phosphate
pyrophosphokinase
stringent
response
stringent
response
negative
regulation
BSU01120 -0.65 fusA elongation factor G stringent
response
stringent
response
negative
regulation
BSU31450 -0.66 kinB sporulation kinase B stringent
response
stringent
response
positive
regulation
BSU24320 -0.67 nusB N utilization substance
protein B
stringent
response
stringent
response
negative
regulation
87
Table A-2. Continued
Locus tag L2FC Gene
Name Protein name Regulon Regulator Action
BSU01180 -0.69 rplW 50S ribosomal protein L23 stringent
response
stringent
response
negative
regulation
BSU01420 -0.71 rpsK 30S ribosomal protein S11 stringent
response
stringent
response
negative
regulation
BSU16620 -0.71 ylxQ ribosomal protein YlxQ stringent
response
stringent
response
negative
regulation
BSU01100 -0.73 rpsL 30S ribosomal protein S12 stringent
response
stringent
response
negative
regulation
BSU01190 -0.73 rplB 50S ribosomal protein L2 stringent
response
stringent
response
negative
regulation
BSU01110 -0.73 rpsG 30S ribosomal protein S7 stringent
response
stringent
response
negative
regulation
BSU16610 -0.76 ylxR hypothetical protein
BSU16610
stringent
response
stringent
response
negative
regulation
BSU01380 -0.77 mapA methionine aminopeptidase 1 stringent
response
stringent
response
negative
regulation
BSU01410 -0.77 rpsM 30S ribosomal protein S13 stringent
response
stringent
response
negative
regulation
BSU01370 -0.78 adk adenylate kinase stringent
response
stringent
response
negative
regulation
BSU14580 -0.80 pdhA pyruvate dehydrogenase E1
component subunit alpha
stringent
response
stringent
response
negative
regulation
BSU14590 -0.80 pdhB pyruvate dehydrogenase E1
component subunit beta
stringent
response
stringent
response
negative
regulation
BSU01390 -0.83 infA translation initiation factor
IF-1
stringent
response
stringent
response
negative
regulation
BSU01400 -0.83 rpmJ 50S ribosomal protein L36 stringent
response
stringent
response
negative
regulation
BSU01350 -0.88 rplO 50S ribosomal protein L15 stringent
response
stringent
response
negative
regulation
BSU01210 -0.92 rplV 50S ribosomal protein L22 stringent
response
stringent
response
negative
regulation
BSU01200 -0.94 rpsS 30S ribosomal protein S19 stringent
response
stringent
response
negative
regulation
BSU01340 -0.96 rpmD 50S ribosomal protein L30 stringent
response
stringent
response
negative
regulation
BSU01250 -0.96 rpsQ 30S ribosomal protein S17 stringent
response
stringent
response
negative
regulation
BSU01480 -0.97 truA tRNA pseudouridine
synthase A
stringent
response
stringent
response
negative
regulation
BSU01270 -0.97 rplX 50S ribosomal protein L24 stringent
response
stringent
response
negative
regulation
BSU01220 -0.98 rpsC 30S ribosomal protein S3 stringent
response
stringent
response
negative
regulation
88
Table A-2. Continued
Locus tag L2FC Gene
Name Protein name Regulon Regulator Action
BSU01330 -0.98 rpsE 30S ribosomal protein S5 stringent
response
stringent
response
negative
regulation
BSU01300 -0.99 rpsH 30S ribosomal protein S8 stringent
response
stringent
response
negative
regulation
BSU01260 -0.99 rplN 50S ribosomal protein L14 stringent
response
stringent
response
negative
regulation
BSU01240 -1.00 rpmC 50S ribosomal protein L29 stringent
response
stringent
response
negative
regulation
BSU01280 -1.00 rplE 50S ribosomal protein L5 stringent
response
stringent
response
negative
regulation
BSU01310 -1.00 rplF 50S ribosomal protein L6 stringent
response
stringent
response
negative
regulation
BSU01320 -1.00 rplR 50S ribosomal protein L18 stringent
response
stringent
response
negative
regulation
BSU01290 -1.03 rpsN 30S ribosomal protein S14 stringent
response
stringent
response
negative
regulation
BSU01230 -1.03 rplP 50S ribosomal protein L16 stringent
response
stringent
response
negative
regulation
BSU01060 -1.11 ybxB hypothetical protein
BSU01060
stringent
response
stringent
response
negative
regulation
BSU28950 -0.38 thrS threonine--tRNA ligase 1 T-box T-box Antiterminat
ion
BSU15430 -0.41 ileS isoleucine--tRNA ligase T-box T-box Antiterminat
ion
BSU27410 -0.49 alaS alanine--tRNA ligase T-box T-box Antiterminat
ion
BSU29670 -0.55 tyrS tyrosine--tRNA ligase 1 T-box T-box Antiterminat
ion
BSU00970 -0.58 yacP hypothetical protein
BSU00970 T-box T-box
Antiterminat
ion
BSU28080 -0.59 folC folylpolyglutamate synthase T-box T-box Antiterminat
ion
BSU28640 -0.61 pheS phenylalanine--tRNA ligase
alpha subunit T-box T-box
Antiterminat
ion
BSU30320 -0.63 leuS leucine--tRNA ligase T-box T-box Antiterminat
ion
BSU00960 -0.64 rlmB tRNA/rRNA
methyltransferase TrmH T-box T-box
Antiterminat
ion
BSU38460 -0.64 tyrZ tyrosine--tRNA ligase 2 T-box T-box Antiterminat
ion
BSU00930 -0.64 cysE serine acetyltransferase T-box T-box Antiterminat
ion
BSU28090 -0.70 valS valine--tRNA ligase T-box T-box Antiterminat
ion
BSU36900 -0.84 glyA serine
hydroxymethyltransferase T-box T-box
Antiterminat
ion
89
Table A-2. Continued
Locus tag L2FC Gene
Name Protein name Regulon Regulator Action
BSU30990 0.57 thiT thiamine transporter ThiT Thi-box Thi-box Termination
BSU38850 0.86 yxkC hypothetical protein
BSU38850 TnrA TnrA Activation
BSU13310 0.52 tnrA MerR family transcriptional
regulator TnrA TnrA Activation
BSU35490 0.49 degU transcriptional regulatory
protein DegU TnrA TnrA Repression
BSU22680 0.96 trpE anthranilate synthase
component 1 TRAP TRAP Termination
BSU22600 0.60 aroE 3-phosphoshikimate 1-
carboxyvinyltransferase TRAP TRAP Termination
BSU06250 1.14 ydjM hypothetical protein
BSU06250 WalR WalR Activation
BSU35740 1.02 tagD glycerol-3-phosphate
cytidylyltransferase WalR WalR Activation
BSU35750 0.75 tagA
N-
acetylmannosaminyltransfera
se
WalR WalR Activation
BSU15290 0.49 ftsZ cell division protein FtsZ WalR WalR Activation
BSU02520 0.76 ycbJ hypothetical protein
BSU02520 YcbG YcbG Repression
BSU07230 -0.75 yetM oxidoreductase YetL YetL Repression
BSU08650 -0.52 fabL enoyl YfhP YfhP Repression
BSU08630 -0.58 yfhQ A/G-specific adenine
glycosylase YfhQ YfhP YfhP Repression
BSU19540 0.61 yodB ArsR family transcriptional
regulator YodB YodB Repression
BSU19550 -0.72 yodC NAD(P)H nitroreductase YodB YodB Repression
BSU19230 -0.88 yocJ FMN-dependent NADH-
azoreductase 1 YodB YodB Repression
BSU30420 -0.65 ytrE ABC transporter ATP-
binding protein YtrA YtrA Repression
BSU21440 1.35 bdbB disulfide bond formation
protein B YvrHb YvrHb Activation
BSU21450 1.33 yolJ glycosyltransferase SunS YvrHb YvrHb Activation
BSU21480 1.24 sunA bacteriocin sublancin-168 YvrHb YvrHb Activation
BSU21470 1.22 sunT
sublancin-168-processing
and transport ATP-binding
protein SunT
YvrHb YvrHb Activation
BSU21060 1.68 yonK hypothetical protein
BSU21060 Unknown Unknown Unknown
90
Table A-2. Continued
Locus tag L2FC Gene
Name Protein name Regulon Regulator Action
BSU21100 1.51 yonG hypothetical protein
BSU21100 Unknown Unknown Unknown
BSU21650 1.33 yokB lipoprotein Unknown Unknown Unknown
BSU20970 1.28 yonX hypothetical protein
BSU20970 Unknown Unknown Unknown
BSU32040 1.19 yuiF amino acid transporter YuiF Unknown Unknown Unknown
BSU14072 1.16 ykzU hypothetical protein
BSU14072 Unknown Unknown Unknown
BSU21430 1.13 bhlB holin-like bacteriophage
SPbeta protein BhlB Unknown Unknown Unknown
BSU_misc_
RNA_27 1.09 S27 NA Unknown Unknown Unknown
BSU21700 1.02 ypoP MarR family transcriptional
regulator Unknown Unknown Unknown
BSU08029 1.01 yfzA hypothetical protein
BSU08029 Unknown Unknown Unknown
BSU40540 1.00 yybR HTH-type transcriptional
regulator YybR Unknown Unknown Unknown
BSU20870 1.00 yopJ hypothetical protein
BSU20870 Unknown Unknown Unknown
BSU20999 1.00 yoyJ hypothetical protein
BSU20999 Unknown Unknown Unknown
BSU26210 0.99 yqaR hypothetical protein
BSU26210 Unknown Unknown Unknown
BSU07340 0.99 yfnA amino acid permease YfnA Unknown Unknown Unknown
BSU17080 0.95 pksA TetR family transcriptional
regulator Unknown Unknown Unknown
91
Table A-2. Continued
Locus tag L2FC Gene
Name Protein name Regulon Regulator Action
BSU04850 0.95 ydcP hypothetical protein
BSU04850 Unknown Unknown Unknown
BSU07440 0.95 yfmK N-acetyltransferase YfmK Unknown Unknown Unknown
BSU04860 0.95 ydcQ Ftsk domain-containing
protein YdcQ Unknown Unknown Unknown
BSU00100 0.94 dacA D-alanyl-D-alanine
carboxypeptidase DacA Unknown Unknown Unknown
BSU22940 0.92 prsW protease PrsW Unknown Unknown Unknown
BSU26160 0.91 yqbC hypothetical protein
BSU26160 Unknown Unknown Unknown
BSU18990 0.91 yobK hypothetical protein
BSU18990 Unknown Unknown Unknown
BSU24780 0.91 yqgY hypothetical protein
BSU24780 Unknown Unknown Unknown
BSU04510 0.90 ydbL hypothetical protein
BSU04510 Unknown Unknown Unknown
BSU03540 0.90 ycxB hypothetical protein
BSU03540 Unknown Unknown Unknown
BSU20860 0.89 yopK hypothetical protein
BSU20860 Unknown Unknown Unknown
BSU40200 0.89 yydD hypothetical protein
BSU40200 Unknown Unknown Unknown
BSU_misc_
RNA_26 0.89 S26 NA Unknown Unknown Unknown
BSU17530 0.88 ynaE hypothetical protein
BSU17530 Unknown Unknown Unknown
BSU10440 0.88 yhjA hypothetical protein
BSU10440 Unknown Unknown Unknown
92
Table A-2. Continued
Locus tag L2FC Gene
Name Protein name Regulon Regulator Action
BSU20960 0.87 yopA hypothetical protein
BSU20960 Unknown Unknown Unknown
BSU21010 0.87 yonS lipoprotein Unknown Unknown Unknown
BSU39290 0.87 yxxD hypothetical protein
BSU39290 Unknown Unknown Unknown
BSU37610 0.86 ywzC hypothetical protein
BSU37610 Unknown Unknown Unknown
BSU05050 0.85 lrpA AsnC family transcriptional
regulator Unknown Unknown Unknown
BSU27700 0.85 yajC preprotein translocase
subunit YajC Unknown Unknown Unknown
BSU34880 0.85 hisA 1-(5-phosphoribosyl)-5 Unknown Unknown Unknown
BSU20929 0.84 yoyI hypothetical protein
BSU20929 Unknown Unknown Unknown
BSU04359 0.83 ydzK hypothetical protein
BSU04359 Unknown Unknown Unknown
BSU20940 0.82 yopC hypothetical protein
BSU20940 Unknown Unknown Unknown
BSU14640 0.82 yktA hypothetical protein
BSU14640 Unknown Unknown Unknown
BSU17510 0.82 ynaC hypothetical protein
BSU17510 Unknown Unknown Unknown
BSU40210 0.81 yydC hypothetical protein
BSU40210 Unknown Unknown Unknown
BSU39280 0.81 yxxE hypothetical protein
BSU39280 Unknown Unknown Unknown
BSU20930 0.81 yopD hypothetical protein
BSU20930 Unknown Unknown Unknown
93
Table A-2. Continued
Locus tag L2FC Gene
Name Protein name Regulon Regulator Action
BSU01000 0.81 secE preprotein translocase
subunit SecE Unknown Unknown Unknown
BSU21630 0.81 yokD
aminoglycoside N(3\')-
acetyltransferase-like protein
YokD
Unknown Unknown Unknown
BSU03880 0.81 yczG ArsR family transcriptional
regulator Unknown Unknown Unknown
BSU20900 0.79 yopG hypothetical protein
BSU20900 Unknown Unknown Unknown
BSU17500 0.78 ynaB hypothetical protein
BSU17500 Unknown Unknown Unknown
BSU05000 0.77 yddK hypothetical protein
BSU05000 Unknown Unknown Unknown
BSU21410 0.77 blyA N-acetylmuramoyl-L-alanine
amidase BlyA Unknown Unknown Unknown
BSU17540 0.77 ynaF hypothetical protein
BSU17540 Unknown Unknown Unknown
BSU12490 0.77 yjqC hypothetical protein
BSU12490 Unknown Unknown Unknown
BSU05390 0.75 ydfF ArsR family transcriptional
regulator Unknown Unknown Unknown
BSU16910 0.75 ymfM hypothetical protein
BSU16910 Unknown Unknown Unknown
BSU28700 0.75 ysfE hypothetical protein
BSU28700 Unknown Unknown Unknown
BSU14570 0.75 ykyA lipoprotein Unknown Unknown Unknown
BSU40220 0.74 yydB metallophosphoesterase Unknown Unknown Unknown
BSU18870 0.74 yozI hypothetical protein
BSU18870 Unknown Unknown Unknown
94
Table A-2. Continued
Locus tag L2FC Gene
Name Protein name Regulon Regulator Action
BSU33570 0.74 yvaE hypothetical protein
BSU33570 Unknown Unknown Unknown
BSU21350 0.74 yomI transglycosylase YomI Unknown Unknown Unknown
BSU33840 0.73 yvbF HTH-type transcriptional
regulator YvbF Unknown Unknown Unknown
BSU18860 0.73 yozH hypothetical protein
BSU18860 Unknown Unknown Unknown
BSU05110 0.73 ydeA protease YdeA Unknown Unknown Unknown
BSU06110 0.72 ydjA type-2 restriction enzyme
BsuMI component YdjA Unknown Unknown Unknown
BSU21080 0.71 yonI hypothetical protein
BSU21080 Unknown Unknown Unknown
BSU05010 0.71 rapI response regulator aspartate
phosphatase I Unknown Unknown Unknown
BSU20010 0.70 yosT transcriptional regulator
YosT Unknown Unknown Unknown
BSU12220 0.70 yjiC UDP-glucosyltransferase
YjiC Unknown Unknown Unknown
BSU07330 0.70 yfnB HAD-hydrolase YfnB Unknown Unknown Unknown
BSU13670 0.70 mhqR MarR family transcriptional
regulator Unknown Unknown Unknown
BSU24540 0.70 yqhL hypothetical protein
BSU24540 Unknown Unknown Unknown
BSU21590 0.70 yokH hypothetical protein
BSU21590 Unknown Unknown Unknown
BSU08300 0.69 yfiK transcriptional regulator Unknown Unknown Unknown
95
Table A-2. Continued
Locus tag L2FC Gene
Name Protein name Regulon Regulator Action
BSU35530 0.69 tagO
undecaprenyl-phosphate N-
acetylglucosaminyl 1-
phosphate transferase
Unknown Unknown Unknown
BSU40240 0.69 yycS hypothetical protein
BSU40240 Unknown Unknown Unknown
BSU24800 0.69 yqgW hypothetical protein
BSU24800 Unknown Unknown Unknown
BSU25090 0.69 yqfW nucleotidase YqfW Unknown Unknown Unknown
BSU33560 0.69 yvaD hypothetical protein
BSU33560 Unknown Unknown Unknown
BSU20910 0.69 yopF hypothetical protein
BSU20910 Unknown Unknown Unknown
BSU15069 0.69 ylzH hypothetical protein
BSU15069 Unknown Unknown Unknown
BSU22020 0.68 ypbS hypothetical protein
BSU22020 Unknown Unknown Unknown
BSU40620 0.68 yybJ ABC transporter ATP-
binding protein Unknown Unknown Unknown
BSU02580 0.67 ycbO hypothetical protein
BSU02580 Unknown Unknown Unknown
BSU00990 0.67 rpmG 50S ribosomal protein L33 2 Unknown Unknown Unknown
BSU39300 0.66 yxiD hypothetical protein
BSU39300 Unknown Unknown Unknown
BSU23310 0.66 sipS signal peptidase I S Unknown Unknown Unknown
BSU05440 0.66 nap carboxylesterase nap Unknown Unknown Unknown
BSU23040 0.66 fer ferredoxin Unknown Unknown Unknown
96
Table A-2. Continued
Locus tag L2FC Gene
Name Protein name Regulon Regulator Action
BSU08740 0.65 ygzB hypothetical protein
BSU08740 Unknown Unknown Unknown
BSU12069 0.65 yjzH hypothetical protein
BSU12069 Unknown Unknown Unknown
BSU39830 0.65 yxcA hypothetical protein
BSU39830 Unknown Unknown Unknown
BSU25050 0.65 yqgA cell wall-binding protein
YqgA Unknown Unknown Unknown
BSU06580 0.65 yerC hypothetical protein
BSU06580 Unknown Unknown Unknown
BSU19000 0.65 yobL hypothetical protein
BSU19000 Unknown Unknown Unknown
BSU14420 0.65 ykoA hypothetical protein
BSU14420 Unknown Unknown Unknown
BSU18850 0.64 yobD XRE family transcriptional
regulator Unknown Unknown Unknown
BSU36980 0.64 ywlA hypothetical protein
BSU36980 Unknown Unknown Unknown
BSU10800 0.63 yizA hypothetical protein
BSU10800 Unknown Unknown Unknown
BSU12450 0.63 yjpA hypothetical protein
BSU12450 Unknown Unknown Unknown
BSU19120 0.63 czrA ArsR family transcriptional
regulator Unknown Unknown Unknown
BSU17660 0.62 yncF deoxyuridine 5\'-triphosphate
nucleotidohydrolase YncF Unknown Unknown Unknown
BSU18060 0.62 yneR hypothetical protein
BSU18060 Unknown Unknown Unknown
BSU17060 0.62 ymzD hypothetical protein
BSU17060 Unknown Unknown Unknown
97
Table A-2. Continued
Locus tag L2FC Gene
Name Protein name Regulon Regulator Action
BSU18890 0.61 yobF hypothetical protein
BSU18890 Unknown Unknown Unknown
BSU12820 0.60 spoIIS
B
stage II sporulation protein
SB Unknown Unknown Unknown
BSU13630 0.60 ykvA hypothetical protein
BSU13630 Unknown Unknown Unknown
BSU13030 0.59 ykhA acyl-CoA thioester hydrolase
YkhA Unknown Unknown Unknown
BSU28900 0.58 ysbB antiholin-like protein LrgB Unknown Unknown Unknown
BSU15000 0.57 ylbG hypothetical protein
BSU15000 Unknown Unknown Unknown
BSU17650 0.55 yncE hypothetical protein
BSU17650 Unknown Unknown Unknown
BSU11550 0.54 yjbH hypothetical protein
BSU11550 Unknown Unknown Unknown
BSU02820 0.54 rapJ response regulator aspartate
phosphatase J Unknown Unknown Unknown
BSU14840 0.54 ylaN hypothetical protein
BSU14840 Unknown Unknown Unknown
BSU08290 0.54 yfiJ sensor histidine kinase Unknown Unknown Unknown
BSU06320 0.54 yeaB transporter Unknown Unknown Unknown
BSU23740 0.51 yqjU hypothetical protein
BSU23740 Unknown Unknown Unknown
BSU17110 0.51 pksD
polyketide biosynthesis
acyltransferase homolog
PksD
Unknown Unknown Unknown
BSU24450 0.47 efp elongation factor P Unknown Unknown Unknown
98
Table A-2. Continued
Locus tag L2FC Gene
Name Protein name Regulon Regulator Action
BSU27540 0.46 yrvM hypothetical protein
BSU27540 Unknown Unknown Unknown
BSU11020 0.46 yitK hypothetical protein
BSU11020 Unknown Unknown Unknown
BSU18660 0.45 yoaM hypothetical protein
BSU18660 Unknown Unknown Unknown
BSU22010 0.45 exoA 5\'-3\' exonuclease Unknown Unknown Unknown
BSU09590 0.41 yhdT hypothetical protein
BSU09590 Unknown Unknown Unknown
BSU23750 0.38 yqjT hypothetical protein
BSU23750 Unknown Unknown Unknown
BSU09270 0.36 glpP
glycerol uptake operon
antiterminator regulatory
protein
Unknown Unknown Unknown
BSU37330 0.31 argS arginine--tRNA ligase Unknown Unknown Unknown
BSU_misc_
RNA_43 -0.33 S43 NA Unknown Unknown Unknown
BSU29590 -0.37 iscS cysteine desulfurase IscS 2 Unknown Unknown Unknown
BSU22410 -0.38 panD aspartate 1-decarboxylase Unknown Unknown Unknown
BSU40520 -0.39 yybS hypothetical protein
BSU40520 Unknown Unknown Unknown
BSU15450 -0.39 lspA lipoprotein signal peptidase Unknown Unknown Unknown
BSU16560 -0.40 rseP zinc metalloprotease RasP Unknown Unknown Unknown
BSU37080 -0.41 rho transcription termination
factor Rho Unknown Unknown Unknown
99
Table A-2. Continued
Locus tag L2FC Gene
Name Protein name Regulon Regulator Action
BSU28620 -0.41 rnhC ribonuclease HIII Unknown Unknown Unknown
BSU00820 -0.42 lysS lysine--tRNA ligase Unknown Unknown Unknown
BSU15870 -0.42 recG ATP-dependent DNA
helicase RecG Unknown Unknown Unknown
BSU16550 -0.43 dxr 1-deoxy-D-xylulose 5-
phosphate reductoisomerase Unknown Unknown Unknown
BSU32680 -0.43 iscU NifU-like protein Unknown Unknown Unknown
BSU40350 -0.44 rocR arginine utilization
regulatory protein RocR Unknown Unknown Unknown
BSU32700 -0.44 sufD FeS cluster assembly protein
SufD Unknown Unknown Unknown
BSU31440 -0.44 patB cystathionine beta-lyase Unknown Unknown Unknown
BSU16540 -0.44 cdsA phosphatidate
cytidylyltransferase Unknown Unknown Unknown
BSU13880 -0.44 glcT BglG family transcription
antiterminator Unknown Unknown Unknown
BSU22750 -0.46 ubiE demethylmenaquinone
methyltransferase Unknown Unknown Unknown
BSU07240 -0.46 yetN hypothetical protein
BSU07240 Unknown Unknown Unknown
BSU03610 -0.47 tcyA L-cystine-binding protein
TcyA Unknown Unknown Unknown
BSU15940 -0.47 smc chromosome partition protein
Smc Unknown Unknown Unknown
BSU32310 -0.47 yutD hypothetical protein
BSU32310 Unknown Unknown Unknown
100
Table A-2. Continued
Locus tag L2FC Gene
Name Protein name Regulon Regulator Action
BSU15740 -0.47 rsmB ribosomal RNA small
subunit methyltransferase B Unknown Unknown Unknown
BSU24350 -0.47 accB
biotin carboxyl carrier
protein of acetyl-CoA
carboxylase
Unknown Unknown Unknown
BSU06140 -0.48 gutR transcription activator GutR Unknown Unknown Unknown
BSU27340 -0.48 yrrO protease YrrO Unknown Unknown Unknown
BSU37110 -0.48 ywjH transaldolase Unknown Unknown Unknown
BSU00550 -0.48 mfd transcription-repair-coupling
factor Unknown Unknown Unknown
BSU33210 -0.48 yvrG sensor histidine kinase Unknown Unknown Unknown
BSU01470 -0.48 ybaF
energy-coupling factor
transporter transmembrane
protein EcfT
Unknown Unknown Unknown
BSU03140 -0.49 tmrB tunicamycin resistance
protein Unknown Unknown Unknown
BSU10150 -0.49 yhgD TetR family transcriptional
regulator Unknown Unknown Unknown
BSU22350 -0.50 dnaD DNA replication protein
DnaD Unknown Unknown Unknown
BSU16020 -0.50 rimM ribosome maturation factor
RimM Unknown Unknown Unknown
BSU27360 -0.50 yrrM O-methyltransferase YrrM Unknown Unknown Unknown
BSU04310 -0.50 ydaN hypothetical protein
BSU04310 Unknown Unknown Unknown
BSU25250 -0.51 ccpN transcriptional repressor
CcpN Unknown Unknown Unknown
101
Table A-2. Continued
Locus tag L2FC Gene
Name Protein name Regulon Regulator Action
BSU15700 -0.51 coaB
C
coenzyme A biosynthesis
bifunctional protein CoaBC Unknown Unknown Unknown
BSU17220 -0.51 pksR polyketide synthase PksR Unknown Unknown Unknown
BSU22830 -0.51 gpsA glycerol-3-phosphate
dehydrogenase Unknown Unknown Unknown
BSU09570 -0.51 yhdR aspartate aminotransferase Unknown Unknown Unknown
BSU16010 -0.52 ylqD hypothetical protein
BSU16010 Unknown Unknown Unknown
BSU22220 -0.52 yprA ATP-dependent helicase
YprA Unknown Unknown Unknown
BSU27350 -0.52 yrrN protease YrrN Unknown Unknown Unknown
BSU17210 -0.52 pksN polyketide synthase PksN Unknown Unknown Unknown
BSU32110 -0.52 yumC ferredoxin--NADP reductase
2 Unknown Unknown Unknown
BSU08350 -0.52 estB extracellular esterase EstB Unknown Unknown Unknown
BSU16530 -0.52 uppS undecaprenyl pyrophosphate
synthase Unknown Unknown Unknown
BSU17200 -0.52 pksM polyketide synthase PksM Unknown Unknown Unknown
BSU16580 -0.53 polC DNA polymerase III PolC-
type Unknown Unknown Unknown
BSU22170 -0.53 ypsC RNA methyltransferase
YpsC Unknown Unknown Unknown
BSU40390 -0.53 walH
two-component system
YycF/YycG regulatory
protein YycH
Unknown Unknown Unknown
102
Table A-2. Continued
Locus tag L2FC Gene
Name Protein name Regulon Regulator Action
BSU22500 -0.54 ypjD hypothetical protein
BSU22500 Unknown Unknown Unknown
BSU17040 -0.54 mutS DNA mismatch repair
protein MutS Unknown Unknown Unknown
BSU31760 -0.54 pncA isochorismatase Unknown Unknown Unknown
BSU29805 -0.54 ytpS DNA translocase SftA Unknown Unknown Unknown
BSU31350 -0.54 pgi glucose-6-phosphate
isomerase Unknown Unknown Unknown
BSU00390 -0.54 yabD deoxyribonuclease YabD Unknown Unknown Unknown
BSU17430 -0.54 ynbA GTPase HflX Unknown Unknown Unknown
BSU28820 -0.55 ysdC aminopeptidase Unknown Unknown Unknown
BSU37000 -0.55 prmC release factor glutamine
methyltransferase Unknown Unknown Unknown
BSU28670 -0.55 ysfB hypothetical protein
BSU28670 Unknown Unknown Unknown
BSU35990 -0.55 ywrO general stress protein 14 Unknown Unknown Unknown
BSU04300 -0.55 ydaM glycosyltransferase YdaM Unknown Unknown Unknown
BSU22960 -0.55 gudB
cryptic catabolic NAD-
specific glutamate
dehydrogenase GudB
Unknown Unknown Unknown
BSU24880 -0.56 yqgO hypothetical protein
BSU24880 Unknown Unknown Unknown
BSU32690 -0.57 sufS cysteine desulfurase Unknown Unknown Unknown
103
Table A-2. Continued
Locus tag L2FC Gene
Name Protein name Regulon Regulator Action
BSU22150 -0.57 ypvA ATP-dependent helicase
YpvA Unknown Unknown Unknown
BSU04680 -0.57 rsbS RsbT antagonist protein
RsbS Unknown Unknown Unknown
BSU19060 -0.57 yobR acetyltransferase Unknown Unknown Unknown
BSU24340 -0.58 accC biotin carboxylase 1 Unknown Unknown Unknown
BSU15760 -0.58 prpC protein phosphatase PrpC Unknown Unknown Unknown
BSU02990 -0.58 opuA
B
glycine betaine transport
system permease protein
OpuAB
Unknown Unknown Unknown
BSU05930 -0.58 rimI ribosomal-protein-alanine
acetyltransferase Unknown Unknown Unknown
BSU16710 -0.58 mlpA zinc protease YmxG Unknown Unknown Unknown
BSU22080 -0.58 ypwA metalloprotease YpwA Unknown Unknown Unknown
BSU38020 -0.59 thiD pyridoxine kinase Unknown Unknown Unknown
BSU04700 -0.59 rsbU phosphoserine phosphatase
RsbU Unknown Unknown Unknown
BSU22840 -0.59 engA GTPase Der Unknown Unknown Unknown
BSU02770 -0.59 yccK oxidoreductase Unknown Unknown Unknown
BSU24280 -0.59 ispA farnesyl diphosphate
synthase Unknown Unknown Unknown
BSU40400 -0.59 walK sensor histidine kinase Unknown Unknown Unknown
104
Table A-2. Continued
Locus tag L2FC Gene
Name Protein name Regulon Regulator Action
BSU19380 -0.60 yojO hypothetical protein
BSU19380 Unknown Unknown Unknown
BSU15770 -0.60 prkC serine/threonine protein
kinase Unknown Unknown Unknown
BSU28360 -0.60 ysnA non-canonical purine NTP
pyrophosphatase Unknown Unknown Unknown
BSU08640 -0.61 yfhS hypothetical protein
BSU08640 Unknown Unknown Unknown
BSU16870 -0.61 fabG oxidoreductase Unknown Unknown Unknown
BSU22460 -0.61 ypjH glycosyltransferase YpjH Unknown Unknown Unknown
BSU_misc_
RNA_4 -0.61 S4 NA Unknown Unknown Unknown
BSU18830 -0.62 pps phosphoenolpyruvate
synthase Unknown Unknown Unknown
BSU09030 -0.62 yhcC hypothetical protein
BSU09030 Unknown Unknown Unknown
BSU25500 -0.63 hemN
oxygen-independent
coproporphyrinogen-III
oxidase 1
Unknown Unknown Unknown
BSU22490 -0.63 dapB
4-hydroxy-
tetrahydrodipicolinate
reductase
Unknown Unknown Unknown
BSU39120 -0.64 yxiM esterase YxiM Unknown Unknown Unknown
BSU22300 -0.64 yppC hypothetical protein
BSU22300 Unknown Unknown Unknown
BSU04690 -0.64 rsbT serine/threonine protein
kinase Unknown Unknown Unknown
BSU04290 -0.65 ydaL hypothetical protein
BSU04290 Unknown Unknown Unknown
105
Table A-2. Continued
Locus tag L2FC Gene
Name Protein name Regulon Regulator Action
BSU22480 -0.65 mgsA methylglyoxal synthase Unknown Unknown Unknown
BSU22450 -0.66 cca CCA-adding enzyme Unknown Unknown Unknown
BSU16030 -0.66 trmD tRNA (guanine-N(1)-)-
methyltransferase Unknown Unknown Unknown
BSU08990 -0.66 yhbI MarR family transcriptional
regulator Unknown Unknown Unknown
BSU01460 -0.67 cbiO
energy-coupling factor
transporter ATP-binding
protein EcfA2
Unknown Unknown Unknown
BSU17440 -0.67 ynbB hypothetical protein
BSU17440 Unknown Unknown Unknown
BSU01140 -0.67 ybaC aminopeptidase YbaC Unknown Unknown Unknown
BSU04780 -0.67 ydcI hypothetical protein
BSU04780 Unknown Unknown Unknown
BSU22440 -0.68 birA bifunctional protein BirA Unknown Unknown Unknown
BSU04280 -0.69 ydaK hypothetical protein
BSU04280 Unknown Unknown Unknown
BSU22030 -0.69 ypbR hypothetical protein
BSU22030 Unknown Unknown Unknown
BSU32710 -0.69 sufC vegetative protein 296 Unknown Unknown Unknown
BSU16130 -0.69 gid
methylenetetrahydrofolate--
tRNA-(uracil-5-)-
methyltransferase TrmFO
Unknown Unknown Unknown
BSU22590 -0.70 ypiA TPR repeat-containing
protein YpiA Unknown Unknown Unknown
BSU34640 -0.70 yvdD LOG family protein YvdD Unknown Unknown Unknown
106
Table A-2. Continued
Locus tag L2FC Gene
Name Protein name Regulon Regulator Action
BSU16700 -0.71 ylxY hypothetical protein
BSU16700 Unknown Unknown Unknown
BSU01450 -0.71 cbiO
energy-coupling factor
transporter ATP-binding
protein EcfA1
Unknown Unknown Unknown
BSU24530 -0.72 yqhM octanoyltransferase LipM Unknown Unknown Unknown
BSU22420 -0.72 panC pantothenate synthetase Unknown Unknown Unknown
BSU32810 -0.73 yusI hypothetical protein
BSU32810 Unknown Unknown Unknown
BSU24210 -0.73 yqiG NADH-dependent flavin
oxidoreductase YqiG Unknown Unknown Unknown
BSU32780 -0.74 yusF hypothetical protein
BSU32780 Unknown Unknown Unknown
BSU03600 -0.74 tcyB L-cystine transport system
permease protein TcyB Unknown Unknown Unknown
BSU09000 -0.75 yhbJ efflux system component
YhbJ Unknown Unknown Unknown
BSU37020 -0.75 ywkD hypothetical protein
BSU37020 Unknown Unknown Unknown
BSU24300 -0.76 xseA exodeoxyribonuclease 7
large subunit Unknown Unknown Unknown
BSU18500 -0.77 fabG oxidoreductase Unknown Unknown Unknown
BSU16660 -0.77 truB tRNA pseudouridine
synthase B Unknown Unknown Unknown
BSU28370 -0.78 rph ribonuclease PH Unknown Unknown Unknown
BSU22430 -0.79 panB 3-methyl-2-oxobutanoate
hydroxymethyltransferase Unknown Unknown Unknown
107
Table A-2. Continued
Locus tag L2FC Gene
Name Protein name Regulon Regulator Action
BSU09020 -0.81 yhcB hypothetical protein
BSU09020 Unknown Unknown Unknown
BSU03860 -0.81 ycnD FMN reductase Unknown Unknown Unknown
BSU01389 -0.81 ybzG ribosome-binding protein
YbzG Unknown Unknown Unknown
BSU26740 -0.82 cypA cytochrome P450 Unknown Unknown Unknown
BSU24290 -0.83 xseB exodeoxyribonuclease 7
small subunit Unknown Unknown Unknown
BSU24890 -0.83 yqgN hypothetical protein
BSU24890 Unknown Unknown Unknown
BSU26910 -0.83 yraK hydrolase YraK Unknown Unknown Unknown
BSU18470 -0.85 proJ glutamate 5-kinase Unknown Unknown Unknown
BSU16970 -0.90 ymdB hypothetical protein
BSU16970 Unknown Unknown Unknown
BSU09010 -0.91 yhcA MFS transporter Unknown Unknown Unknown
BSU26850 -0.94 yrpG oxidoreductase Unknown Unknown Unknown
BSU32719 -0.95 yuzK hypothetical protein
BSU32719 Unknown Unknown Unknown
BSU23820 -0.95 yqjM NADPH dehydrogenase Unknown Unknown Unknown
BSU31360 -0.96 yugK NADH-dependent butanol
dehydrogenase 2 Unknown Unknown Unknown
BSU08470 -1.00 yfhB isomerase Unknown Unknown Unknown
108
Table A-2. Continued
Locus tag L2FC Gene
Name Protein name Regulon Regulator Action
BSU03870 -1.06 ycnE monooxygenase YcnE Unknown Unknown Unknown
BSU04750 -1.20 ydcF hypothetical protein
BSU04750 Unknown Unknown Unknown
109
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BIOGRAPHICAL SKETCH
Hoang Nguyen is an Oklahoma City, Oklahoma native who graduated from Westmoore
Highschool in 2008. From there he proceeded to attend the University of Oklahoma-Norman,
where he graduated with a Bachelor of Science degree in microbiology with a minor in
Chemistry in 2013. He then pursued his Master of Science at the Department of Microbology
and Cell Science where he graduated in May 2017.
After graduation, Hoang will be applying for Microbiology related jobs throughout the
United States. He hopes to get a job working as a research scientist within an industrial
biotechnology company.