in vitro and in vivo activity of dinb: role in ......for the students with me and after: belen,...
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IN VITRO AND IN VIVO ACTIVITY OF DINB: ROLE IN MUCOID CONVERSION
OF PSEUDOMONAS AERUGINOSA
BY
ANDREA BETH ROCKEL
A Dissertation Submitted to the Graduate Faculty of
WAKE FOREST UNIVERSITY GRADUATE SCHOOL OF ARTS AND SCIENCES
in Partial Fulfillment of the Requirements
for the Degree of
DOCTOR OF PHILOSOPHY
Microbiology and Immunology
December, 2008
Winston Salem, North Carolina
Approved By:
Daniel J. Wozniak, Ph.D., Advisor ____________________________________
Examining Committee:
Fred W. Perrino, Ph.D., Chairman ____________________________________
Karin D. Scarpinato, Ph.D. ____________________________________
David A. Ornelles, Ph.D. ____________________________________
Sean D. Reid, Ph.D. ____________________________________
ACKNOWLEDGEMENTS
I would like to first thank my parents, Mike and Peg Rockel, for not only raising
me in a wonderful home, but also for being my first teachers; for inspiring in me a love of
learning, a curiosity about the world around me, and sense of awe about the beauty of the
creation. I want to thank them for always believing in me, for always encouraging me to
pursue my dreams, and for the guidance they have always offered along the way. And I
would like to thank my brother Matthew, for listening to my ramblings when frustrated,
and for his ability to always cheer me up when I am down. I am truly blessed.
I also want to express my whole-hearted appreciation for the guidance of my
advisor, Dan Wozniak. He is truly a model of what a scientist should be, and the best
mentor I could have imagined. Almost all of my important lessons in graduate school
came from Dan, and I doubt that I would have survived parts of it without his guidance. I
also want to thank the members of my thesis committee: Fred Perrino, David Ornelles,
Karin Scarpinato and Sean Reid. They have been a source of direction for the project, and
an invaluable resource for me and my work.
I would also likely not have survived (or enjoyed) parts of graduate school
without the support of my classmates: Leslie, Belen, Shayla, Jerry and Patrick. They have
been a constant source of laughter and fun, but have also been there through the tough
times when laughter was not easy. I love them all very much, and am excited to see and
hear the amazing things that they will be accomplishing over the next several years.
I also worked with some very wonderful people during my time in graduate
school. I want to thank April Sprinkle for keeping everything in the lab running
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smoothly, and also for her support and help throughout my time in the lab. I would also
like to thank Haiping Lu for the many ways he helped me move my project along. For the
students before me; Debbie, Kara, and Anne were wonderful resources and role models.
For the students with me and after: Belen, Elizabeth, Cynthia and Matt were great
colleagues and friends. And for Dr. Stephen Richardson, for his advice and
encouragement throughout my time in graduate school.
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TABLE OF CONTENTS
Acknowledgements ……………………………………………………………. ii
Table of Contents ……………………………………………………………. iv
Illustrations ……………………………………………………………. vii
Abstract ……………………………………………………………. ix
Chapter 1: Introduction ……………………………………………………. 1
Pseudomonas aeruginosa lifestyle and virulence ……………………. 1
Pseudomonas aeruginosa as a human pathogen in cystic fibrosis ……. 3
Mucoidy as a virulence factor ……………………………………. 4
DNA damage response and repair mechanisms in bacteria ……………. 7
DinB as a Y-family DNA polymerase: activity, regulation and fidelity … 10
Overall objective of thesis ……………………………………………. 15
Chapter 2: Materials and Methods ……………………………………………. 16
Bacterial strains, plasmids, chemicals and growth conditions …...... 16
Construction of bacterial strains …………………………………….. 16
Lac+ reversion assay …………………………………………………….. 18
Protein expression and purification …………………………………….. 18
Primer extension assays …………………………………………….. 19
Electrophoretic mobility shift assays …………………………………….. 21
Immunoblot analysis …………………………………………………….. 22
RNA harvest and real-time RT-PCR …………………………………….. 22
Mucoid conversion assay …………………………………………….. 23
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PCR sequencing and complementation …………………………….. 24
Chapter 3: Activity and kinetics of DinB on mucA sequences …………………. 25
DinBPa is a DNA polymerase …………………………………….. 25
DinBPa can use both magnesium and manganese as co-factors for activity …………………………………………….. 28
Metal ion choice affects both the kinetics and fidelity of DinB on a mucA-derived template …………………………………….. 32
DinB creates -1 frameshifts on a mismatched mucA-derived template …... 44
The -1 frameshifting ability of DinB on mismatched templates appears to be due to slippage of the polyG tract …………………….. 55
Chapter Summary …………………………………………………….. 59
Chapter 4: Role of DinB in mucoid conversion …………………………….. 60
Expression of DinB is DNA damage regulated …………………….. 60
LexA regulates dinB by binding upstream of the dinB coding sequence .... 63
Expression of DinB increases the frequency of ΔG-dependent Lac+ reversion …………………………………….. 72
Development of a selection strategy for isolation of mucoid P. aeruginosa …………………………………………….. 76
Hydrogen peroxide treatment increases the frequency of mucoid conversion ………………………………………………….. 76
Deletion of lexA increases the frequency of mucoid conversion in response to hydrogen peroxide …………………………………….. 81
Deletion of dinB decreases both spontaneous and induced mucoid conversion …………………………………….. 86
Mucoid conversion is increased when mutS is deleted, while deletion of dinB in a ΔmutS background eliminates mucoid conversion ………… 86
Determination of the frequency of mucA-dependent vs mucA-independent mucoid algD-cat variants …………………….. 89
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Chapter Summary …………………………………………………….. 97
Chapter 5: Conclusions and discussion …………………………………….. 98
References …………………………………………………………………….. 108
Appendix …………………………………………………………………….. 116
Scholastic vitae …………………………………………………………….. 122
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ILLUSTRATIONS
Fig. 1. Schematic overview of alginate control in Pseudomonas aeruginosa …... 5
Fig. 2. Representative structure diagram of a Y family DNA polymerase ……… 12
Fig. 3. DinB is a DNA polymerase ……………………………………………… 26
Fig. 4. DinB is active with both magnesium and manganese co-factors on a polyG tract mucA sequence ..………………………………………………. 30
Fig. 5. Time course of DinB activity with magnesium and manganese on a polyG tract mucA sequence ………………………………………………… 33
Fig. 6. dGTP incorporation with magnesium and manganese on a polyG tract mucA sequence ………………………………………………… 36
Fig. 7. Kinetic analysis of dGTP incorporation with magnesium and manganese on a polyG tract mucA sequence ………………………………... 38
Fig. 8. dCTP mis-incorporation with magnesium and manganese on a polyG tract mucA sequence …………………………………………………. 40
Fig. 9. Kinetic analysis of dCTP mis-incorporation with manganese on a polyG tract mucA sequence …………………………………………………. 42
Fig. 10. dGTP incorporation with magnesium and manganese on a non-polyG tract mucA sequence …………………………………………….. 45
Fig. 11. Kinetic analysis of dGTP incorporation with magnesium and manganese on a non-polyG tract mucA sequence …………………………..... 47
Fig. 12. DinB creates -1 frameshifts on the polyG tract sequence of the mucA gene with a G:G mismatch ………………………………………….. 50
Fig. 13. DinB creates -1 frameshifts on a polyG tract non-mucA sequence with a G:G mismatch ……………………………………….. 53
Fig. 14. DinB does not extend a G:G mismatch on a non-polyG tract sequence …………………………………………………… 57
Fig. 15. Real-time RT-PCR analysis reveals dinB mRNA levels are elevated in a lexA- mutant ……………………………………………………. 61
Fig. 16. Real-time RT-PCR analysis reveals elevated dinB mRNA levels with DNA damage ………………………………………………… 64
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Fig. 17. DinB protein levels are elevated by deleting lexA or treating with MMC ……………………………………………………………. 66
Fig. 18. E. coli LexA does not bind P. aeruginosa pdinB ……………………….. 68
Fig. 19. P. aeruginosa LexA binds P. aeruginosa pdinB ………………………... 70
Fig. 20. DinB increases the frequency of DG mutations ……………………….... 74
Fig. 21. Schematic of palgD-cat construct and mucoid conversion selection ..…. 77
Fig. 22. Hydrogen peroxide treatment increases the frequency of mucoid conversion ……………………………………………... …………….. 79 Fig. 23. Hydrogen peroxide treatment increases the frequency of mucoid conversion of a ΔlexA mutant ………………………………………… 82
Fig. 24. Deletion of dinB decreases the frequency of both spontaneous and peroxide-induced mucoid conversion …………………. 84
Fig. 25. Deletion of mutS increases the frequency of both spontaneous and peroxide-induced mucoid conversion …………………. 87
Fig. 26. Deletion of dinB in a ΔmutS background eliminates mucoid conversion ……………………………………………………. 90
Fig. 27. Results of mucA sequencing show crucial role for mutS and dinB in mucA mutagenesis …………………………………………. 93
Fig. 28. Results of mucA sequencing show a role for mutS and dinB in altering the spectrum of mucA mutations ……………………………………… 95
Fig. 29. Proposed model for mucA mutagenesis in the CF lung …………………. 106
Fig. A1. Effect of P. aeruginosa dinB overexpression on the frequency of rifampicin resistance in E. coli ……………………………… 118
Fig. A2. Ability of plasmid-expressed DinB to influence frequencies of Lac+ reversion of different lacZ alleles ………………………………………... 120
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ABSTRACT
The opportunistic pathogen Pseudomonas aeruginosa undergoes a phenotypic
change called mucoid conversion in the lungs of chronically infected cystic fibrosis (CF)
patients. This phenotypic change leads to the production of the exopolysaccharide
alginate, which gives the organism a selective advantage in the CF lung, as alginate
protects against reactive oxygen species, antibiotics and host immune cells. The majority
of the mucoid variants that emerge from CF patients have mutations in mucA, which
encodes an anti-sigma factor and this mutation allows for the biosynthesis of alginate.
The mechanisms leading to the mutagenesis of mucA have yet to be discovered, and are
the main focus of this work.
We first characterized the protein DinB of P. aeruginosa, which we hypothesized
would be involved in the response to DNA damage. We have shown that DinB is a DNA
polymerase with no exonuclease activity, and that it is able to utilize both magnesium and
manganese as metal ion cofactors for catalysis. The Km for dNTP is over 600-fold lower
when manganese is present compared with magnesium, indicating a role for the cofactor
in changing the affinity of the enzyme for dNTP. Given the E. coli DinB’s preference for
creating -1 frameshifts in poly-guanine runs, we also assayed the activity of P.
aeruginosa DinB on the poly-guanine run of a mucA-derived template. When there was a
G:G mismatch as the terminal base pair of the template/primer, DinB was able to extend
the primer but caused -1 frameshifts. We have determined that this effect is most likely
due to DNA slippage of the template and primer strands during replication, based on
DinB’s lack of frameshifting on a template without the homopolymeric run. In the
context of a chronic CF respiratory infection, where a high frequency of hypermutable
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mutants emerge, this ability of DinB to extend a mismatch at this site in mucA with a -1
frameshift could be a partial explanation for mucoid conversion in vivo.
Having examined the activity of DinB, we have also shown that dinB is directly
repressed by LexA as part of an SOS-type response, and that DinB is up-regulated during
response to DNA damage by mitomycin C and hydrogen peroxide. Using a Lac+
reversion assay, plasmid-expressed DinB increased the frequency of -1 frameshift
mutations in a poly-guanine run. Given the similarity of this sequence with the mutation
hotspot in mucA, we next considered DinB’s role in mucoid conversion. After developing
a selection system for detecting mucoid variants, we determined the effect of both
hydrogen peroxide treatment as well as the role of DNA repair. Following sub-lethal
hydrogen peroxide treatment, mucoid conversion frequency increased in both the wild-
type and isogenic ΔlexA mutant. When a strain has lost mismatch repair function by
deletion of mutS, the mucoid conversion frequency was drastically increased. Both with
the single deletion ΔdinB as well as the double deletion ΔmutSΔdinB, mucoid conversion
is essentially abolished. This indicates a crucial role for both DinB and MutS in mucoid
conversion. Given this data, we conclude that there is a DinB-dependent pathway for
mucoid conversion, and that impairment of MMR serves to exacerbate this phenomenon.
CHAPTER 1: Introduction
Pseudomonas aeruginosa lifestyle and virulence
Pseudomonas aeruginosa is a ubiquitous Gram-negative bacteria commonly
isolated from many environmental niches. It is a motile, rod-shaped obligate aerobe
capable of surviving harsh environments due to its limited requirements for nutrients
(61). It can be cultured from many aquatic sources, such as swimming pools, sinks and
drains, and from the soil, its natural habitat, especially in association with the roots of
plants (8). P. aeruginosa is inherently resistant to many antibiotics, due to several factors:
outer membrane with low permeability, multiple enzymes to degrade antibiotics, and the
production of several efflux pumps, in addition to its capacity to create and obtain new
methods for resistance. This ability to enhance its antibiotic resistance profile is thought
to be due at least in part to the large size of the genome [6.2 megabases]. It has also been
partly attributed to the aquatic habitat where P. aeruginosa is frequently found, which
often contain bacteria with multiple resistance genes that can be acquired through genetic
transfer (46).
P. aeruginosa has a host of virulence factors that contribute to its pathogenesis
and survival, including but not limited to: elastase, exotoxin A, type III secreted proteins,
flagella, type IV pili, pyocyanin, proteases, lipopolysaccharide and alginate (42). Elastase
is an especially crucial virulence factor for burn infections, as it has been shown to break
down collagen and other host proteins, and disturb the basement membrane (2, 42).
Exotoxin A is considered to be the most toxic virulence factor secreted by P. aeruginosa,
and is known to strongly inhibit eukaryotic protein synthesis and contribute to host cell
1
death (8). Many of the adhesins produced by P. aeruginosa are important for virulence,
including type IV pili, which is crucial for dissemination during an infection, particularly
burn infections (42). Pyocyanin, the greenish-blue pigment that was first studied in the
late nineteenth century, decreases the normal ciliary movement of the nasal epithelium
and thus impairs clearance of the bacteria (8). Alginate, an exopolysaccharide which will
be addressed more fully in a later section, is an especially important virulence factor in
the respiratory infections of cystic fibrosis patients, where a phenotypic change occurs
known as mucoid conversion (23).
The first scientific study on P. aeruginosa was published in 1882, examining the
blue and green discoloration of infected wound bandages, later found to be caused by the
bacterially-produced pigment pyocyanin (8). Currently, P. aeruginosa is a significant
source of nosocomial infections, responsible for 11-14% of hospital-acquired infections
that have an isolated microbiological cause. This percentage goes up even higher for
intensive care units (ICUs), where P. aeruginosa infections account for 13-22% of the
hospital-acquired infections (16). With regard to the types of infections commonly found,
P. aeruginosa is second only to Staphylococcus aureus as the cause for hospital-
associated pneumonia, healthcare-associated pneumonia, and ventilator-associated
pneumonia. P. aeruginosa is also the leading cause of hospital-acquired pneumonias in
pediatric ICUs. P. aeruginosa is the major cause of the skin infections of burn patients, as
well as being associated with cases of bacteremia, nosocomial urinary tract infections,
and infection of surgical sites (16).
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Pseudomonas aeruginosa as a human pathogen in cystic fibrosis
Cystic fibrosis (CF) is a genetic disorder caused by mutations in a gene on the
long arm of chromosome 7. This gene encodes the CF transmembrane conductance
regulator (CFTR), which is responsible for maintenance of chloride ion concentrations in
epithelial cells (35). When mutations arise in the gene for this channel protein, ion
transport across the cell membrane is impaired and the milieu of the lung environment is
changed, along with other changes in the gastrointestinal and reproductive tracts. As the
most common fatal genetic disorder in the Caucasian population, CF affects
approximately 1 in every 2500 live births (22, 23). Among the nearly 1000 mutations that
have been identified, the most common alteration observed in the CFTR gene leads to the
deletion of a phenylalanine residue at position 508 of the CFTR protein; this mutation is
present in approximately 70% of CF patients (15). By far the most severe problems are
associated with the respiratory tract, with chronic bacterial infections of the lungs leading
to respiratory failure and death of 80 to 95% of CF patients by respiratory failure (23).
While early colonization of the lungs may involve several bacterial pathogens
including Haemophilus influenzae and Staphylococcus aureus, by the time CF patients
reach their teen years, 70 to 80% of them are chronically colonized by P. aeruginosa.
Using more advanced testing techniques, by the time they are 3 years old, approximately
98% of CF patients have some history of infection with P. aeruginosa (24). Aggressive
use of antibiotics can help to control the non-Pseudomonas infections; however, P.
aeruginosa is rarely completely eliminated, even with the most aggressive antibiotic
therapy (23). This chronic colonization with P. aeruginosa can be correlated with a
decline in prognosis for the infected patient, such that P. aeruginosa infection is
3
considered to be the cause of an infected patient’s deterioration. The chronic P.
aeruginosa infection that is established in a CF patient’s lungs is most often the terminal
infection leading to respiratory failure and death; therefore research into the pathogenesis
of P. aeruginosa infection is extremely important to the health of CF patients.
Mucoidy as a virulence factor
The mucoid phenotype, as stated earlier, is an important virulence factor for P.
aeruginosa, especially in the chronic respiratory infections of CF patients. This
phenotype results from the over-production of the exopolysaccharide alginate, which
creates a capsule-like protective slime on the bacteria and gives the characteristic sticky
mucoid morphology (17). The induction of the alginate biosynthetic operon is dependent
on the alternative sigma factor σ22 which is encoded by the algT gene (also referred to as
algU) (42). AlgT exerts positive control over its own transcription, as well as being
responsible for the up-regulation of AlgB, AlgR and AmrZ (Fig. 1). These DNA binding
proteins lead to the transcription of algD, which encodes a GDPmannose dehydrogenase
and is the first gene in the alginate biosynthetic operon. AlgD converts the precursor
GDP-mannose into GDP-mannuronate as the first committed step of alginate
biosynthesis. The mature alginate product is a linear copolymer of (1 4)-linked β-D-
mannuronic acid and α-L-guluronic acid (23).
Wild-type, non-mucoid bacteria have an anti-sigma factor, MucA, which acts to
sequester and repress the activity of the sigma factor AlgT, thereby effectively
eliminating alginate production (13) (Fig. 1). When MucA is mutated, it loses its ability
to repress the activity of AlgT, which is therefore able to activate alginate production.
4
Figure 1. Schematic overview of alginate control. Diagram of the major cellular factors involved in the control of alginate production. Anti-sigma factor MucA represses sigma factor AlgT and therefore all downstream targets of AlgT in a non-mucoid bacterium. Mutations in MucA lead to release of AlgT and transcription of downstream targets (notably AlgB, AlgR and AmrZ). This results in transcription of algD and production of alginate.
5
MucA
Alginate
OM
IM
AlgT
AlgB AlgR AmrZ
AlgD
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This conversion to mucoidy is seen at a high frequency with CF patients who have had
chronic lung infections and often indicates a decline in pulmonary function (14). In the
majority (~84%) of mucoid P. aeruginosa isolates from CF patient sputa, there is a
mutation in the mucA gene, leading to alginate production (4). Among these mucoid
isolates, the most common mutation that occurs in the mucA gene is a deletion of a
guanine residue in a stretch of 5 guanines, leading to a -1 frameshift mutation and early
truncation of the protein. This mutation is referred to as mucA22 and accounts for
approximately 25% of the mucA-dependent mucoid variants (3). There is also a minority
of mucoid isolates that don’t conform to this pattern and have a wild-type mucA gene,
while still producing excessive amounts of alginate, which are referred to as mucA-
independent variants. The mucoidy of these variants has been postulated to be due to
another sigma factor, RpoN (σ54), and these variants are traditionally referred to as
muc23 (28). Although the nature of many of the mucA mutations in CF patients has been
described, there is still little known about the molecular mechanisms underlying mucA
mutagenesis.
DNA damage response and repair mechanisms in bacteria
Maintaining the integrity of the genome during normal growth and stressful
situations is crucial for the survival of bacteria. Bacterial species have several
mechanisms to respond to DNA damage, and two of these mechanisms will be discussed
as relevant to this thesis: the mismatch repair (MMR) system, and the SOS response to
DNA damage.
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DNA mismatch repair (MMR) is a highly conserved process for protecting the
genome of almost all organisms from bacteria to mammals (57). MMR is responsible for
finding and repairing base-base mispairing as well as insertion/deletion loops (IDLs). The
repair pathway has been extensively researched in E. coli, so while little is known about
the pathway in P. aeruginosa, the MMR of E. coli will be used as a model system. This
process begins with the binding of MutS to mismatched DNA; MutS binds to the β-clamp
of the replication complex and that may be a mechanism for recruiting MutS to the site of
mispairing (39). The next step requires both MutL and ATP with the mismatch-bound
MutS, and this activates a third protein, MutH. MutH is an endonuclease that creates a
nick in the newly synthesized DNA strand that is still unmethylated for a short time after
replication (26). This nick is not necessarily at the point of the mismatch, but is typically
within about 1000 bases. Once the nick is created, MutL can assist with loading DNA
helicase II and single-stranded binding protein (SSB), which results in single-stranded
DNA that can then be digested by exonucleases. DNA polymerase III is then able to
resynthesize the missing strand to fill in the gap before DNA ligase seals everything
together (51).
While there are many similarities between the MMR system in E. coli and
P. aeruginosa, there are also several differences that exist between the two systems.
There has been no MutH homologue identified in P. aeruginosa, though its genome does
contain homologues of the mutS, mutL and uvrD genes. This, in combination with the
fact that there is no Dam homologue in P. aeruginosa, indicates that P. aeruginosa likely
uses a different method than E. coli for distinguishing between strands when repairing
mismatches. In addition to this, P. aeruginosa MutS is incapable of complementing an E.
8
coli MutS mutation; in fact, the P. aeruginosa MutS creates a dominant-negative effect
when introduced into a wild-type E. coli strain (68). It has been shown recently, however,
that P. aeruginosa MutL can complement an E. coli MutL mutation (67). P. aeruginosa
MutS has also been shown to vary in its oligomerization state, with the tetramer
appearing most active in vitro (68).
In P. putida, a closely-related organism to P. aeruginosa, inactivation of the
MMR system leads to an increase in the accumulation of mutations during starvation, and
the types of mutations found in wild-type versus MMR-deficient strains were distinct.
This could be important for the P. aeruginosa infection of the cystic fibrosis lung,
considering the harsh conditions and possible reduction in access to nutrients in the
inflamed respiratory tract during chronic infections (69).
The SOS response of Escherichia coli causes the induction of approximately 30
genes that assist with tolerance to damage and damage repair; these genes include lexA,
recA, polB, sulA and dinB. LexA is a repressor protein of many of the damage-responsive
genes, and binds to the ‘SOS box’ site upstream of these genes and prevents the RNA
polymerase from gaining access (58). Following DNA damage, RecA is activated to form
a complex with the ssDNA that is a result of the failed replication attempts on damaged
DNA. This complex acts as a co-protease to facilitate the auto-cleavage of LexA, which
relieves LexA binding and repression of the SOS-induced genes (27). Once the
repression is relieved, transcription of those genes takes place and the cell can attempt to
recover from the damage to its genome with the DNA repair proteins that are the
products of those genes. Among the genes regulated by the SOS system in E. coli are
dinB, which encodes DNA Polymerase IV, and umuDC, which encodes DNA Polymerase
9
V (58). While P. aeruginosa does not have a homolog of UmuDC, it does have a DinB
homolog (PA0923), which has not been characterized until now.
DinB as a Y-family DNA polymerase: activity, regulation and fidelity
DNA polymerases are grouped into families based on sequence similarity. There
are currently 6 families identified: A, B, C, D, X and Y. Families A, B, C and D are
involved in replication during the normal cell cycle and are considered of relatively high
fidelity with error rates of 10-5 to 10-6 . Family X contains polymerases that are mainly
involved in the gap-filling polymerization during base excision repair, with error rates
slightly higher than the replicative counterparts at 10-4 to 10-5 (65).
The Y family of DNA polymerases is another mechanism of the cell to maintain
the integrity of its genome during times of DNA damage. The ability of these
polymerases to bypass DNA lesions sets them apart from the A, B, C, D and X
polymerase families, as well as their relatively low fidelity on non-damaged DNA
templates. The many members of this family display great diversity in terms of the types
of lesions which they will most faithfully bypass, from bulky adduct lesions to thymine-
thymine dimers (47, 56, 62). These enzymes are not significantly similar in sequence
with the A, B or X polymerases, and while there is considerable variation in the size of
the Y family members, they all maintain 5 conserved motifs in the N-terminal sequence
of each enzyme (38).
To date, all of the crystal structures of the Y family polymerases have shown a
similar arrangement of the finger, palm and thumb domains, fitting the right hand-like
arrangement of most DNA polymerases (Fig. 2). Part of what sets the Y family
10
polymerases apart, however, is the open nature of the active site in the palm domain.
Small ‘finger’ and ‘thumb’ domains create a more solvent-accessible active site, and less
contact between the active site and the replicating DNA. This apparent loosening of the
‘grip’ of the enzyme on the DNA has been proposed as a possible structural explanation
for the low fidelity of DNA replication by the Y family polymerases (40).
Another significant difference between the replicative DNA polymerase and the Y
family is that the Y family member enzymes lack the ‘induced-fit’ conformational change
that occurs with other polymerases to ensure that incorrect nucleotides are excluded from
the active site. For the replicative enzymes, the correct Watson-Crick pairing of the
incoming deoxynucleoside triphosphate with the template base creates a conformational
‘induced-fit’ change and the active site is then said to be closed. However, the active sites
of the Y family members appear to already be in a closed conformation and ready to
catalyze the nucleotidyl transfer reaction, regardless of the correctness or condition of the
incoming deoxynucleoside triphosphate or template base (66).
Another difference between the replicative polymerases and the Y family
polymerases is the presence of a ‘little finger’ domain in the C-terminal region of the
enzyme (also referred to as the polymerase-associated domain (PAD) or the wrist
domain). This flexible domain has a role in maintaining the DNA duplex across from the
thumb domain, and it is only moved into position when in close association with the
DNA (53, 60). This domain also has a dramatic effect on the activity and mutation
spectra of the Y family enzymes, based on the ‘little finger’ domain’s ability to alter the
strength of the association between the polymerase and the DNA (5).
11
Figure 2. Representative structure diagram of a Y family DNA polymerase. Crystal structure of Sulfolobus sulfataricus Dpo4 complexed with DNA; a view of the ternary complex in ribbon diagram. The palm domain is in red, finger domain in blue, thumb domain in green, and little finger domain in purple. DNA is in gold, and is shown in the plane of the page. Reproduced with permission from Yang, Current Opinion in Structural Biology 2003, 13:23-30 (License number 2085380231836)
12
13
In E. coli, DNA polymerase IV (DinB) and DNA polymerase V (UmuDC) are the
two identified Y family polymerases. DinB, encoded by the dinB gene, was originally
found in a screen attempting to identify genes that are up-regulated in response to UV
light and mitomycin C exposure (32). In vivo, deleting dinB does not cause a dramatic
phenotype change in response to DNA damaging agents, as is seen when umuDC is
deleted (29). dinB has been implicated in λ untargeted mutagenesis (in combination with
recA), as well as adaptive mutagenesis (6, 7, 44). Over-expressing DinB also leads to an
increase in mutagenesis, with a preference for causing -1 frameshifts (33).
DinB exists at a high cellular concentration, relative to other polymerases,
approximately 250 copies per cell in E. coli under normal growth conditions (34). This is
in contrast to Pol V, for example, of which there are only 15 copies per cell in an
uninduced state (31). In E. coli, dinB is directly repressed by LexA, though not
stringently (18). It has also more recently been shown to be positively regulated by RpoS,
the stationary phase sigma factor σ38, though this may be an indirect effect caused by
another RpoS-regulated gene product (31).
While many of these pathways have been elucidated for E. coli, there is still very
little known about the mechanisms of DNA repair in P. aeruginosa, from SOS induction
to the activity and regulation of DNA polymerases. Given the implications of DinB
activity on mutagenesis in other organisms, we believe it is crucial to develop a more
complete picture of the DNA damage response in P. aeruginosa, especially as it relates to
mucA mutagenesis and mucoid conversion.
14
Overall objective of thesis
The goal of this work is to gain an understanding of the regulation and activity of
DinB, and also to ascertain whether or not DinB plays a role in the mucoid conversion of
P. aeruginosa. We addressed this in two major sections: 1) The in vitro activity of DinB,
and 2) The regulation of DinB and the role of DinB in mucoid conversion. We set about
to study a previously uncharacterized DNA polymerase in P. aeruginosa, DinB; to
describe how it is regulated in the cell, its activity on relevant substrates, and its
mutagenic potential. We also sought to determine if there is a link between this
polymerase and mucoid conversion. Understanding the factors that lead to mucoid
conversion is crucial for developing new strategies for therapeutic treatment of CF
patients, and this work is an attempt to isolate some of those factors.
15
CHAPTER 2: Materials and Methods
Bacterial strains, plasmids, chemicals and growth conditions
All strains were routinely grown in Luria-Bertani medium (LB: 10 g tryptone, 5 g
yeast extract, 10 g NaCl, 1 L water) or LB with no salt added (LBNS). M9 media was
used for the Lac+ Reversion Assay with either glucose or lactose added. The following
antibiotics were used at the indicated concentrations with E. coli strains: ampicillin, 150
μg/ml; kanamycin, 60 μg/ml; and rifampin (Rif), 100 μg/ml. Gentamicin and rifampin
were each used at 100 μg/ml with P. aeruginosa strains, while tetracycline was used at 60
μg/ml, and carbenicillin was used at 250 μg/ml.
Construction of bacterial strains
The dinB gene (including its native promoter) of P. aeruginosa (DinBPa) was
PCR amplified from P. aeruginosa PAO1 genomic DNA using primers dinB1 (5’-CGG
GAT CCG AGT TCC ATC CGG TTC ACG-3’) and dinB2 (5’-CCC AAG CTT GGG
ACG CCG TGC TGA AGG CC-3’). The amplified DNA fragment was digested with
BamHI and HindIII and cloned into pWSK29, resulting in pHL6. For the expression of
DinBPa, a PCR amplification of plasmid pHL6 with primers dinB4 (5’-GGA ATT CCA
TAT GCG GAA AAT CAT CCA TAT AGA CTG-3’) and dinB5 (5’-CCC AAG CTT
GAA CAA CCT GAG TTG TTC GT-3’) was performed. The fragment was digested
with NdeI and HindIII and cloned into pET29a (Novagen), resulting in plasmid pHL8,
which was used for the overproduction of the hexahistidine-tagged, wild-type DinBPa.
Three individual dinB missense mutations (D8A [aspartic acid position 8 changed to
16
alanine], R49A, and D103A) were constructed in both pHL6 and pHL8 by site-directed
mutagenesis using the QuikChange mutagenesis kit (Stratagene).
The lexA gene was PCR amplified from P. aeruginosa PAO1 genomic DNA
using primers lexA5 (5’-CCC AAG CTT GCG CCG GAT CAC GC-3’) and lexA6 (5’-
CGG GAT CCA GGC GAC GAC ATG CA-3’). The resulting product was digested with
HindIII and BamHI and cloned into the vector pET29a, resulting in pAR88, which was
used for expression with a C-terminally hexahistidine-tagged P. aeruginosa LexA protein
(LexAPa).
Standard reverse genetic techniques were used for the construction of P.
aeruginosa dinB- and lexA-null mutants (25, 52). For the ΔdinB mutant, plasmid pHL6
was digested with BamHI and HindIII and the dinB fragment was subcloned into
pEX18Ap, resulting in pHL13. An ~200-bp fragment within the dinB coding sequence
was removed by SalI cleavage of pHL13 and replaced with a gentamicin resistance
cassette (aacC1) derived from SalI cleavage of pMS266, generating pHL16. P.
aeruginosa WFPA334 (ΔdinB::aacC1) was generated using standard mating and sucrose
selection gene replacement strategies of wild-type dinB in P. aeruginosa PAO1 with
ΔdinB::aacC1 from pHL16. A similar strategy was used to generate the lexA::aacC1
mutant, WFPA340. The lexA gene was amplified from PAO1 genomic DNA using
primers lexA1 (5’-CGG GAT CCG CAG GAG GTC CTC CAG GGT-3’) and lexA2 (5’-
CCC AAG CTT TAT TCA GGC TCT GTG CTT GGC CC-3’), digested with BamHI
and HindIII, and cloned into similarly digested pEX18Ap, resulting in pHL10. A
gentamicin resistance cassette derived from SphI cleavage of pMS266 was cloned into
the SphI site of pHL10, within the lexA coding sequence, generating pHL11. P.
17
aeruginosa WFPA340 (ΔlexA::aacC1) was generated using standard mating and sucrose
selection gene replacement strategies of wild-type lexA in P. aeruginosa PAO1 with
ΔlexA::aacC1 from pHL11. The sulA gene is located immediately downstream of lexA;
the expression of sulA may be affected by the lexA::aacC1 allele, as we did not have to
disrupt sulA prior to constructing the ΔlexA strain.
Lac+ reversion assay
The E. coli strains harboring the empty vector pWSK29, the wild-type DinB
pHL6 or the mutant R49A DinB pHL6R49A were grown in LB containing kanamycin,
ampicillin and IPTG overnight at 37°C. Serial dilutions were made of the cultures, and 4
X 100 μl of the 10-6 dilution were plated on M9 Salts + glucose plates, and 4 X 100 μl of
the undiluted culture were plated on M9 Salts + lactose plates. These plates were then
incubated at 38°C for 48 hours. The colonies on each plate were then enumerated and
averaged for the 4 replicate plates in each group. A ratio of the number of colonies
growing on the lactose-containing plates divided by the number of colonies growing on
the glucose-containing plates was generated. These ratios were then compared to generate
a fold-change of either of the two DinB-containing strains over the empty vector strain.
Protein expression and purification
The P. aeruginosa wild-type DinB protein was overproduced using E. coli
BL21(DE3) harboring pHL8 grown at 30°C in LB supplemented with ampicillin. When
the culture reached an optical density at 595 nm (OD595) of 0.8, IPTG (isopropyl-β-D
thiogalactopyranoside) (Promega) was added to 50 μM and growth was continued for
18
another 3 h. Cells were harvested by centrifugation, resuspended in buffer H (50 mM
NaPO4 [pH 6.8], 500 mM NaCl, 10% glycerol, 20 mM 2-mercaptoethanol, and 1 mM
imidazole), and lysed by double passage through a chilled French press. The soluble
fraction was clarified by centrifugation at 4°C prior to chromatography. The extract
containing the C-terminally hexahistidinetagged DinBPa protein was first applied to a 5-
ml Ni-nitrilotriacetic acid (NTA) agarose column (QIAGEN). Bound protein was eluted
with buffer H containing 50 mM EDTA. Fractions containing DinBPa, identified by
sodium dodecyl sulfate- polyacrylamide gel electrophoresis (SDS-PAGE), were pooled
and dialyzed against buffer A (25 mM Tris-HCl [pH 7.0], 10% glycerol, and 20 mM 2-
mercaptoethanol) containing 25 mM NaCl prior to chromatography using a 1-ml HiTrap
heparin column (GE Healthcare) equilibrated in the same buffer. Bound protein was
eluted from the heparin column by using a linear gradient of 2.5% to 100% buffer B (25
mM Tris-HCl [pH 7.0], 10% glycerol, 20 mM 2-mercaptoethanol, and 1 M NaCl).
Fractions containing DinBPa were pooled, dialyzed, and applied to a Mono S HR 5/5
column (GE Healthcare) equilibrated in buffer A containing 25 mM NaCl. Bound protein
was eluted using a linear gradient of 2.5% to 100% buffer B. The purity of the final
material was judged to be ~95% based on densitometric scanning of Coomassie-stained
SDS-PAGE gels.
Primer extension assays
There were two types of primer extension (or replication assays) performed. The
first replication assay shown (Fig. 3) uses the following method: Replication assays (10
μl) consisted of a 5’-[32P]ATP radiolabeled (using T4 polynucleotide kinase; Promega)
19
synthetic 20-mer (5’-AC GCC TGT AGC ATT CCA CAG-3’)/100-mer (5’-AA TCC
CAT ACA GAA AAT TCA TTT ACT AAC GTC TGG AAA CTC GAC AAA ACT
TTA GAT CGA AAC GCT AAC TAT GAG GGG TGT CTG TGG AAT GCT ACA
GGC GT-3’) DNA template (gel purified) at a final concentration of 5 nM in replication
buffer (20 mM Tris-HCl [pH 7.5], 8 mM MgCl2, 4% glycerol, 50 mM NaCl, 5 mM
dithiothreitol, 40 μg/ml bovine serum albumin) containing 133 μM of either each
individual or all four ultrapure deoxynucleoside triphosphates (dNTPs) (GE Healthcare).
Reactions were initiated by the addition of DinBPa or the E. coli DinB protein (DinBEc)
at the indicated concentrations for 10 or 20 min at 37°C, as noted. Reactions were
quenched with EDTA, heat denatured, and electrophoresed through 10% denaturing
polyacrylamide gels. Radiolabeled products were visualized using a phosphorimager with
a signal-intensifying phosphorimaging screen (Bio-Rad).
All remaining replication assays consisted of fluorescently-labeled synthetic DNA
primers annealed to non-labeled synthetic DNA templates at a final concentration of 5 or
10 nM in each reaction (20 mM Tris-HCl [pH 7.5], 5 mM dithiothreitol, 40 μg/ml bovine
serum albumin). Each reaction contained none, individual or all four ultrapure
deoxynucleoside triphosphates (dNTPs) (GE Healthcare), as indicated for each figure.
Reactions were initiated by the addition of DinB or at the indicated concentrations for 12
min at 37°C, as noted. Reactions were quenched with EDTA, heat denatured, and
electrophoresed through 23% denaturing polyacrylamide gels. Fluorescently-labeled
products were visualized using a phosphorimager to detect the fluorescein tag.
20
Electrophoretic mobility shift assays
Electrophoretic mobility shift assay (EMSA) experiments were performed
generally as described previously (18). DNA fragments for dinBPa EMSA were labeled
by PCR amplification of P. aeruginosa PAO1 genomic DNA with primers dinB1 (above)
and dinB3 (5’-CCC AAG CTT CGA GGG CGG CAT AGA AAC AGT-3’) and
[α32P]dCTP following previously published protocols (Baynham 1999). Primers
umuprom1 (5’-GCG TCG TCG CCA GAA GG-3’) and umuprom2 (5’-CGT AAT CTG
CTG CCG GTG-3’) were used to amplify the 5’ regulatory sequences of E coli umuC
using E. coli MG1655 genomic DNA. Cell extracts, purified LexAEc, or LexAPa was
incubated with the DNA fragments in a 5-μl final volume for 30 min in binding buffer
(10 mM HEPES-NaOH [pH 8.0], 10 mM Tris-HCl [pH 8.0], 5% glycerol, 50 mM NaCl,
1 mM EDTA, and 1 mM dithiothreitol). A 6% nondenaturing polyacrylamide gel was
prerun for 1 h at 50 V at 4°C with electrophoresis buffer (25 mM Tris-HCl [pH 8.0], 250
mM glycine, 1 mM EDTA). Samples were loaded and resolved overnight at 50 V at 4°C.
The gel was dried and visualized using a phosphorimager with a signal-intensifying
phosphorimaging screen (Bio-Rad). LexAEc was kindly provided by John Little
(University of Arizona). Hexahistidine-tagged LexAPa protein derived from
JM109(DE3)(pAR88) was purified using Ni-NTA agarose resin (QIAGEN). Bound
protein was eluted from Ni-NTA agarose using imidazole. LexAPa purified in this
fashion contained two distinct bands of approximately 26 and 14 kDa, which likely
represent mature and cleaved LexA, respectively.
21
Immunoblot analysis
Purified hexahistidine-tagged DinBPa derived from pHL8 was used to elicit
antibodies from New Zealand White rabbits (Covance Research Products). Anti-DinBPa
serum was adsorbed against extracts derived from WFPA334 (ΔdinB::aacC1) and
subsequently used at a dilution of 1/10,000 as the primary antibody for the detection of
DinBPa by Western blotting. Horseradish peroxidase-conjugated goat anti-rabbit
immunoglobulin (Pierce) was used as the secondary antibody at a dilution of 1/10,000.
Western blotting was performed using either whole-cell extracts or cell extracts, and
purified hexahistidine-tagged DinBPa protein as a standard. Whole-cell extracts were
prepared as described previously (Sutton 2005). For the preparation of cell extracts, P.
aeruginosa strains were grown to an OD600 of 0.6, 10 ml of each culture was centrifuged,
and the pellets were resuspended in 1.0 ml of FBG (100 mM NaCl, 10 mM Tris-HCl [pH
8.0], 1 mM MgCl2, 5% glycerol). Samples were subjected to sonication and centrifuged
for 15 min at 14,000 rpm. Protein concentration in the supernatant fraction was quantified
by the bicinchoninic acid assay. Equivalent amounts of protein from extracts were
separated by 12% SDS-PAGE and analyzed by Western blotting.
RNA harvest and real-time RT-PCR
Total RNA was harvested from P. aeruginosa strains PAO1 (lexA+) and
WFPA340 (ΔlexA::aacC1) using the Ambion Ribo-Pure kit according to the
manufacturer’s instructions. For experiments investigating DNA damage-induced
expression of dinB, cultures were grown to an OD600 of 0.6 and then treated with
mitomycin C (MMC; 1 μg/ml) for 2 h. RNA was then harvested and quantified for real-
22
time reverse transcription-PCR (RT-PCR) analysis. Following purity confirmation by gel
electrophoresis, 100 ng of this RNA was used as a template in a one-step real-time RT-
PCR (TaqMan one-step RT-PCR kit) with primers (dinBF [5’-GGC GGT GTC GAA
GCA GAT-3’] and dinBR [5’-CGA AAG CGG CTC GAT CAG-3’]) and a fluorescent
probe specific for dinB mRNA (dinBP [5’-CAT GCG ATC TTC CGC GAT TAT ACC
GA-3’]). Reactions were performed and analyzed using an ABI Prism 7000 sequence
detection system. For each RNA sample, dinB mRNA levels were normalized against
rpoD mRNA levels, which were determined using primers rpoDF (5’-CCT GCC GGA
GGA TAT TTC C-3’) and rpoDR (5’-GAT CCC CAT GTC GTT GAT CAT-3’) and a
fluorescent probe specific for rpoD mRNA (rpoDP, 5’-ATC CGG AAC AGG TGG
AAG ACA TCA TCC-3’). Once normalized against rpoD, data were presented as a
several-fold increase over wild-type (PAO1) levels of dinBPa mRNA, which were set
equal to 1.0.
Mucoid conversion assay
The algD-cat construct was created by first digesting the algD promoter region
fused to the chloramphenicol resistance gene cat (6.8 kilobases; palgD-cat) from plasmid
pDJW145 (palgD-cat in pACYC177 vector) with BamHI and HindIII. The vector pmini-
CTX was cut with BamHI and HindIII as well. The palgD-cat fragment was then ligated
to pmini-CTX with T4 DNA ligase. This plasmid was then transformed into E. coli strain
SM10 for mating with P. aeruginosa strains. Following mating, the construct was then
integrated into the neutral attB site of the wild-type and mutant P. aeruginosa strains of
interest (ΔdinB, ΔlexA, ΔmutS, ΔmutsΔdinB) (Fig. 21). These strains were then used in
23
the mucoid conversion assay as follows. Strains were grown in overnight cultures and
then diluted into fresh media with or without hydrogen peroxide at 0.3% (from stock 30%
solution) and allowed to grow for 24 hours at 37°C. The cultures were then serially
diluted for plating on non-selective Pseudomonas isolation agar (PIA) for colony counts
after 24 hours of plate growth at 37°C. Cultures were also plated straight onto PIA
containing chloramphenicol to determine colony counts after 24 hours at 37°C. The
mucoid conversion frequency was then determined by dividing the number of mucoid
variants by the total number of CFU.
PCR sequencing and complementation
Genomic DNA was harvested from the mucoid variants isolated in the mucoid
conversion assay. Single colonies of each strain were suspended in 12 μl TE buffer + 50
μg/ml proteinase K, then heated to 55°C for 15 minutes, then to 85°C for 15 minutes,
then cooled on ice for 1 minutes before centrifugation for 3 minutes. 1.5-3 μl of this was
removed for PCR amplification with mucA-specific primers mucAS1 and mucAS3
(mucAS1, 5’- GGA ATT CCG GAT CTT CCG CGC TCG TGA -3’; mucAS3, 5’- GGA
ATT CCG CCT GAG TGG CGG GAA CC -3’). After verification of PCR product by
agarose gel electrophoresis, the PCR products were sequenced by MWG Biotech with the
mucAS1 and mucAS3 primers. The sequence data produced were then aligned with the
wild-type PAO1 mucA gene sequence to determine if and/or where mutations occurred in
the mucA gene of the variants.
24
CHAPTER 3: Activity and kinetic analyses of P. aeruginosa DinB
DinBPa is a DNA polymerase
Based on the Cluster of Orthologous Groups of the National Center for
Biotechnology Information, the P. aeruginosa genome contains a single ortholog of E.
coli umuC and dinB designated PA0923 (55). As PA0923 resembled E. coli dinB more
closely than it did E. coli umuC, it was suggested to be a dinB (DNA Pol IV) ortholog.
The predicted P. aeruginosa DinB protein (DinBPa) is 342 amino acids (aa) and exhibits
49% identity with DinBEc (340 aa). Moreover, residues D8, R49, and D103, which are
essential for DinBEc activity, are completely conserved in DinBPa (62). In order to
determine whether DinBPa possessed an intrinsic nucleotidyl transferase activity, we
purified the C-terminally hexahistidine-tagged protein to apparent homogeneity and
assayed its activity in an in vitro primer extension assay. Using a synthetic 20-mer primer
annealed to a 100-mer as a template (20/100 template) (Fig. 3), DinBPa was able to
incorporate dNTPs with efficiencies comparable to that observed with DinBEc (Fig. 3,
compare lanes 1 to 3 and 9 to 11). The absence of primer degradation in the reaction
containing only DinBPa and the 20/100 DNA substrate (Fig. 3, lane 12) suggests that
DinBPa, like DinBEc (Fig. 3, lane 4), is devoid of an intrinsic 3’-to-5’ exonuclease
(proofreading) activity. We investigated the accuracy of nucleotide incorporation by
DinBPa in vitro on an undamaged template qualitatively by assessing the efficiency of
the template-directed incorporation of each single dNTP. Using the 20/100 as a template,
DinBPa efficiently catalyzed the incorporation of dATP opposite template dTMP (Fig. 3,
lane 13). In contrast, DinBPa did not incorporate dTTP, dGTP, or dCTP opposite
25
Figure 3. DinB is a DNA polymerase. In vitro primer extension assay was performed as described in Materials and Methods using the template/primer as pictured. DinBEc, DinBPa, and DinBPaR49A were used at 5, 25, or 125 nM, and DinBPaD8A was used at 250 nM. Reaction times were 10 or 20 minutes, as indicated. dNTPs were added individually or together, as indicated. Published in collaboration: Sanders et al, Journal of Bacteriology, 188:8573-8585.
26
27
template dTMP (Fig. 3, lanes 14 to 16). In the presence of all four nucleotides, DinBPa
extended the primer in what appeared to be a largely distributive manner (Fig. 3, lane 11).
Similar results were observed for DinBEc (Fig. 3, lanes 3 and 5 to 8), consistent with
previous reports (62).
Amino acid substitutions at residues D8, R49, and D103 of DinBEc severely
impair its catalytic activity both in vitro and in vivo (62). Using site-directed mutagenesis,
we replaced these corresponding residues in DinBPa with alanine. The DinB(D8A)Pa
and DinB(R49A)Pa mutant proteins purified in a manner that was indistinguishable from
that of the wild-type DinBPa. In contrast, the DinB(D103A)Pa mutant protein became
insoluble during its purification and was therefore not evaluated for in vitro activity.
Although no polymerase activity was detected with the DinB(D8A)Pa mutant protein in
vitro (Fig. 3, lanes 17 to 18), the DinB(R49A)Pa mutant protein retained detectable,
albeit significantly reduced compared with wild-type, polymerase activity (Fig. 3, lanes
19 to 21).
DinBPa can use both magnesium and manganese as co-factors for activity
Having shown that P. aeruginosa DinB (hereafter referred to as DinB) is indeed a
DNA polymerase, we wished to address the activity of DinB on a physiologically
relevant DNA template. As shown previously (33, 63) and in our work (Fig. 20 and
Appendix Fig. A2), DinB shows a strong preference for creating -1 frameshifts,
especially in homopolymeric purine runs. Based on this, we chose for our replication
template an internal sequence of the mucA gene, with the 5-guanine run in the center of
the template. A -1 frameshift at this homopolymeric run (mucA22) is the most common
28
mucA mutation to occur in CF clinical isolates (3). We therefore chose a 36-mer template
from the internal region of the mucA sequence, with the 5-guanine run centered in the
sequence. We also designed 17-mer fluorescently-labeled primers that were
complementary to the template such that the 3’ end of each primer annealed across from
the homopolymeric run (Fig. 4A).
DinB was able to replicate the mucA-derived template, as evidenced by its ability
to extend the primers (Fig. 4B, left side). We next turned to the role of the metal ion
cofactor used by the enzyme during catalysis. It has recently been shown for a Y family
polymerase (Pol ι) that metal ion choice (comparing magnesium and manganese) affects
the activity as well as the fidelity of the enzyme (19). Therefore, we first determined
whether DinB can utilize manganese as a cofactor, and if so what the optimal
concentration of the metal would be, as we already have evidence that it will use
magnesium (Fig. 3). To address this, we performed a replication assay with our mucA-
derived template system and varied the magnesium chloride or manganese chloride
concentrations under saturating dNTP conditions and high enzyme concentration. We
found that the magnesium and manganese concentrations that gave optimal replication
activity were very similar, approximately 2.5-4 mM (Fig. 4B). This is in contrast to what
was found for polymerase ι, where a very low manganese concentration (0.075 mM)
gave the optimal activity, and concentrations over 0.5 mM inhibited the replicative ability
of the enzyme (19). Thus, DinB can utilize manganese as a metal ion cofactor for its
enzyme activity, and optimal activity was found using 2.5 mM MnCl2 or 4 mM MgCl2.
These metal ion conditions will be used for the remainder of the assays testing DinB
activity and fidelity.
29
Figure 4. DinB is active with both magnesium and manganese co-factors on a polyG tract mucA sequence. A. Schematic of DNA template/primer used for this assay. B. In vitro primer extension assay was performed as described in Materials and Methods using a primer/template pair that cover an internal region of the mucA gene, with the 5G tract in the center. The primer was used at a concentration of 50 nM, the dNTPs were used at 200 μM (each), and the concentration of DinB was 100 nM. The concentrations shown in the figure are the metal ion concentrations used for each reaction lane.
30
A. 5’ ----ACGCCGCGGGGGACGAGA---- 3’
3’ CCCCCTGCTCT---- 5’
B.
μM μM μM mM mM mM mM mM mM μM μM μM μM μM μM mM mM mM
0 10 100 500 1 2 4 6 8 10 0 1 5 10 50 100 500 1 2.5 5 [Metal]:
Manganese Magnesium
31
Metal ion choice affects both the kinetics and fidelity of DinB on a mucA-derived
template
Based on previously published work with polymerase eta as a representative of
the Y family polymerases, we hypothesized that manganese, as the cofactor for enzyme
activity, would alter the kinetics and fidelity of DinB (19). To test this, we set out to
determine the kinetics of nucleotide incorporation on our mucA-derived template system.
To achieve the correct parameters for kinetic measurements, we first set up single
completed hit reactions in which less than 20% of the labeled primer would be extended
(10). Using 10 nM DinB, we set up a time course set of reactions from 0-16 minutes
under both metal ion conditions (MnCl2 and MgCl2). Under these conditions and the
correct nucleotide (dGTP), approximately 20% of the primer was extended after 12
minute at 37°C, and the remaining assays were performed under these parameters (Fig.
5). In the presence of manganese, there were clearly more replication products than just
the product that would result from a single insertion of dGTP onto the primer (Fig. 5,
right side). This indicates a change in the fidelity of the enzyme depending on the metal
ion condition of the reaction mixture, with the presence of manganese decreasing the
fidelity of DinB such that it will incorporate non-template-directed bases.
Using the parameters obtained from the above experiments, we next determined
the kinetics of dNTP incorporation by DinB on the mucA-derived template system. To do
this, we kept the enzyme concentration and reaction time constant, and varied the dNTP
concentration. We first used a broad dGTP concentration range to approximate the Km of
32
Figure 5. Time course of DinB activity with magnesium and manganese on a polyG tract mucA sequence. In vitro primer extension assay was performed as described in Materials and Methods using a primer/template pair that cover an internal region of the mucA gene, with the 5G tract in the center. The primer was used at a concentration of 100 nM, dGTP was used at 200 μM, the metal ion concentrations were 4 mM for MgCl2 and 2.5 mM for MnCl2, and the concentration of DinB was 10 nM. A time course was generated to determine the optimal reaction time for extending approximately 20% of the starting primers.
33
Time (min): 0 1 2 4 6 8 10 12 14 16 0 1 2 4 6 8 10 12 14 16
Magnesium Manganese
34
nucleotide incorporation. This was done using Sigma Plot software by quantitating the
amount of product formed by pixel density and then plotting this on the Y axis against the
dGTP concentration on the X axis. A hyperbolic line of best fit was generated following
Michaelis-Menten kinetics, and based on the slope of the line, we extracted the Vmax and
Km of incorporation. Using this approximation of the Km, we set up reactions such that
the dGTP concentrations ranged from 0.2 X – 5 X Km (Fig. 6). By repeating the
quantitation and plotting of the data using Sigma Plot, we calculated the Km of dGTP
incorporation under both metal ion conditions: 80 μM for magnesium and 130 nM for
manganese (Fig. 7). It is clear from the Km values for magnesium and manganese that
there is a dramatic effect of the metal ion cofactor on the kinetics of incorporation, with
an over 600-fold difference between the two conditions and manganese having the much
lower Km value (Fig. 7). This indicates that under manganese conditions, the amount of
dGTP necessary to extend a certain amount of the primer is lower than under magnesium
conditions.
We next wished to determine if the metal ion cofactor affected the fidelity of
DinB on the mucA-derived template system. We repeated the above experiment, with
each of the remaining dNTPs individually. However, the only condition under which we
saw extension was with dCTP as the nucleotide and manganese as the cofactor (Fig. 8).
We calculated a Km value for dCTP misincorporation with manganese of 14 μM, which
is >100 times the Km value for dGTP incorporation with manganese (Fig. 9). There is
therefore some effect of manganese on the fidelity of DinB, but overall, it is surprising
that there is so little misincorporation in general, as the Y family polymerases are
typically thought of as low-fidelity enzymes (65).
35
Figure 6. dGTP incorporation with magnesium and manganese on a polyG tract mucA sequence. In vitro primer extension assay was performed as described in Materials and Methods using a primer/template pair that cover an internal region of the mucA gene, with the 5G tract in the center. The primer was used at a concentration of 100 nM, the metal ion concentrations were 4 mM for MgCl2 and 2.5 mM for MnCl2, the reaction time was 12 minutes, and the concentration of DinB was 10 nM. The concentrations shown in the figure represent the dGTP concentrations tested in the reactions. The concentrations were designed in a range of 0.2X-5X, with X being the apparent Km, based on previous reactions.
36
0 12 24 36 48 60 120 180 240 300 0 80 160 240 360 400 800 1200 1600 2000
nM dGTP μM dGTP
Magnesium Manganese
37
Figure 7. Kinetic analysis of dGTP incorporation with magnesium and manganese on a polyG tract mucA sequence. Sigmaplot was used to analyze the data from the proceeding figure, graphing the amount of product created per minute per milligram of enzyme against the concentration of dGTP in the reaction. A hyperbolic line was fit to the data points, and Vmax and Km were then calculated.
38
nM dGTP
0 200 400 600 800 1000 1200
nmol
/min
/mgE
nz
0.0
0.2
0.4
0.6
0.8
1.0
0.0
0.2
0.4
0.6
0.8
1.0
μM dGTP
nmol
/min
/mgE
nz
0 20 40 60 80 100 120 140 160
Magnesium Manganese Km= 130±72 nM Km= 80±60 μM
39
Figure 8. dCTP mis-incorporation with magnesium and manganese on a polyG tract mucA sequence. In vitro primer extension assay was performed as described in Materials and Methods using a primer/template pair that cover an internal region of the mucA gene, with the 5G tract in the center. The primer was used at a concentration of 100 nM, the metal ion concentrations were 4 mM for MgCl2 and 2.5 mM for MnCl2, the reaction time was 12 minutes, and the concentration of DinB was 10 nM. The concentrations shown in the figure represent the dCTP concentrations tested in the reactions. The concentrations for the MnCl2 reactions were designed in a range of 0.2X-5X, with X being the apparent Km, based on previous reactions; the concentrations for the MgCl2 reactions were essentially at the maximum level.
40
0 37.5 30 22.5 15 7.5 6 4.5 3 1.5 0 25 20 15 10 5 4 3 2 1
mM dCTP μM dCTP
Magnesium Manganese
41
Figure 9. Kinetic analysis of dCTP mis-incorporation with manganese on a polyG tract mucA sequence. Sigmaplot was used to analyze the data from the proceeding figure, graphing the amount of product created per minute per milligram of enzyme against the concentration of dCTP in the reaction. A hyperbolic line was fit to the data points, and Vmax and Km were then calculated.
42
0.0
0.2
0.4
0.6
0.8
1.0
μM dCTP
nmol
/min
/mgE
nz
Manganese
Km= 14±20 μM
0 10 20 30
43
To ensure that our kinetic data is reproducible on another relevant template, we
created a mucA-derived template/primer system in which the primer annealed with the
template downstream of the 5-guanine run (Fig. 10A). The first correct nucleotide to be
incorporated would still be dGTP, as with the original template system, but there is no
homopolymeric run at the replication site. We then repeated the kinetic experiments on
this new template, beginning with correct dGTP incorporation, which appeared similar in
nature to the previous template system (Fig. 10B). The Km values for dGTP incorporation
with the new template under both metal ion conditions were similar to those obtained
with the original template (Fig. 11). We then evaluated misincorporation of the other 3
nucleotides (dATP, dTTP, dCTP). Unlike with the original template, there was no
extension of the primer with any of the incorrect nucleotides, regardless of metal ion
cofactor conditions. This, again, is surprising, given the reputation of the Y family
polymerases as low-fidelity enzymes, though it has been proposed that at least on
undamaged DNA under some conditions, DinB’s activity may be mostly anti-mutagenic
(29).
DinB creates -1 frameshifts on a mismatched mucA-derived template
As we have already shown that DinB is active on the physiologically relevant
mucA-derived templates we designed, we also wanted to address DinB’s activity on
another relevant template system. To do this, we re-examined the role of hypermutability
and mismatch repair in the CF respiratory tract. It has been found that a significant
proportion of the P. aeruginosa population in CF patient sputum has a hypermutability
phenotype, indicating an increase in the global mutation frequency across the bacterial
44
Figure 10. dGTP incorporation with magnesium and manganese on a non-polyG tract mucA sequence. A. Schematic of DNA template/primer used for this assay. B. In vitro primer extension assay was performed as described in Materials and Methods using a primer/template pair that covers an internal region of the mucA gene, downstream of the polyG tract. The primer was used at a concentration of 100 nM, the metal ion concentrations were 4 mM for MgCl2 and 2.5 mM for MnCl2, the reaction time was 12 minutes, and the concentration of DinB was 10 nM. The concentrations shown in the figure represent the dGTP concentrations tested in the reactions. The concentrations were designed in a range of 0.2X-5X, with X being the apparent Km, based on previous reactions.
45
5’ ----CGGGGGACGAGAAGCGAC---- 3’ 3’ CTCTTCGCTG---- 5’
B.
A.
0 6 12 18 24 30 60 90 120 150 0 40 80 120 160 200 400 600 800 1000
nM dGTP μM dGTP
Manganese Magnesium
46
Figure 11. Kinetic analysis of dGTP incorporation with magnesium and manganese on a non-polyG tract mucA sequence. Sigmaplot was used to analyze the data from the proceeding figure, graphing the amount of product created per minute per milligram of enzyme against the concentration of dGTP in the reaction. A hyperbolic line was fit to the data points, and Vmax and Km were then calculated.
47
μM dGTP
0 20 40 60 80 100 120 140 160
0.0
0.2
0.4
0.6
0.8
1.0
0.0
0.1
0.2
0.3
0.4
0.5
0.6
0.7
nmol
/min
/mgE
nz
nM dGTP
nmol
/min
/mgE
nz
0 200 400 600
Magnesium Manganese Km= 288±575 nM Km= 57±60 μM
48
genome (49). This phenotype was later discovered to be primarily due to mutations in the
mismatch repair pathway of DNA damage repair, with mutS being the predominant
location for the mutations (48). We therefore wanted to determine the ability of DinB to
replicate a mismatched template/primer pair, as this seemed to be a relevant substrate
which DinB could encounter in vivo.
Our new template system was again using the internal region of mucA, with the 5-
guanine run in the center of the template. We created two labeled primers, one which
annealed perfectly with the template just up through the 5-guanine run, and one which
contained a G:G mismatch at the 3’ end of the primer across from the 5-guanine run, such
that the template and primer were not perfectly matched (Fig. 12A). Using these
template/primer pairs, we were then able to determine DinB’s activity on a mismatched
substrate. As shown in Fig. 12B, DinB was able to extend a mismatched template/primer
pair as seen by the extension products visible when all 4 dNTPs are present in the
reaction (right half of figure). We also performed reactions with dGTP alone (the next
correct nucleotide to be added), dCTP alone (the second nucleotide to be added) and
dGTP + dCTP together. While dGTP is added correctly with the non-mismatched
template/primer, dCTP was incorporated with the mismatched template/primer (Fig.
12B). When dGTP + dCTP were present together in the reaction, the extension products
were different sizes for the non-mismatched and mismatched template/primer pairs. For
the non-mismatched template/primer, we observed an extension product of 6 nucleotides,
which is in agreement with the sequence of the template, in which there are 6 guanine or
cytosine residues immediately following the start of replication. For the mismatched
template/primer, however, we saw
49
Figure 12. DinB creates -1 frameshifts on the polyG tract sequence of the mucA gene with a G:G mismatch. A. Schematic of DNA template/primer used in this assay. B. In vitro primer extension assay was performed as described in Materials and Methods using a primer/template pair that cover an internal region of the mucA gene, with the 5G tract in the center. The primer was used at a concentration of 100 nM, the metal ion concentrations were 5 mM for MgCl2 and 5 mM for MnCl2, the reaction time was 10 minutes, and the concentration of DinB was 100 nM. The concentrations shown in the figure represent the dNTP concentrations tested in the reactions.
50
A. 5’ ----ACGCCGCGGGGGACGAGA---- 3’ 3’ CCCCCTGCTCT---- 5’ 3’ GCCCCTGCTCT---- 5’
B.
- 4 G C G/C 4 G C G/C - 4 G C G/C 4 G C G/C
100 μM 1 μM 100 μM 1 μMdNTP:
G:C G:G
Magnesium Manganese
51
an extension product indicative of 5 nucleotides, indicating -1 frameshift in the product
(Fig. 12B).
We next wished to ascertain if the -1 frameshift effect was dependent on any of
the flanking sequence context of the surrounding mucA gene. To do this, we generated a
random DNA sequence for the flanking area surrounding the 5-guanine run plus the
adjacent cytosine for the template, and two labeled primers, one correctly paired with the
template and one with a G:G mismatched at the end of the 5-guanine run. DinB was
active on this mismatched template/primer (Fig. 13). We also performed reactions with
dGTP alone (the next correct nucleotide to be added), dTTP alone (the second nucleotide
to be added), dATP alone (the third nucleotide to be added) and dGTP + dTTP + dATP
together. While dGTP was added correctly with the non-mismatched template/primer,
dTTP was incorporated with the mismatched template/primer (Fig. 13). As we saw with
the previous template/primer pair, we also see that with the non-mismatched
template/primer and dGTP + dTTP + dATP, there was an extension product that indicates
incorporation of 3 nucleotides. And with the mismatched template/primer and dGTP +
dTTP + dATP, we observed an extension product that indicates the incorporation of only
2 nucleotides, indicating a likely -1 frameshift (Fig. 13). The mechanism of this -1
frameshift is not discernable from this experiment, as it could result from the bulging out
of one of the guanine bases to allow the primer G to pair with the adjacent C, or it could
be due to slippage in the DNA as the DNA undergoes the normal ‘breathing’ of
separating and re-annealing during replication. We will address this question in the next
section.
52
Figure 13. DinB creates -1 frameshifts on a polyG tract non-mucA sequence with a G:G mismatch. In vitro primer extension assay was performed as described in Materials and Methods using a primer/template pair that was randomly generated in the flanking sequence, but contains the polyG tract and adjacent C. The primer was used at a concentration of 100 nM, the metal ion concentrations were 5 mM for MgCl2 and 5 mM for MnCl2, the reaction time was 10 minutes, the dNTPs were used at a concentration of 10 μM, and the concentration of DinB was 100 nM.
53
- 4 G T A G/T/A - 4 G T A G/T/A 4 G T A G/T/A 4 G T A G/T/A
G:C G:G G:C G:G
Manganese Magnesium
54
One other point to mention as regards these frameshift replication assays. It is
striking that with these assays at relatively high enzyme concentration (100 nM), the
presence of manganese does have an effect on the apparent fidelity of DinB. This can be
seen most clearly in the manganese lanes of Fig. 13 where misincorporation occurs with
all of the nucleotides tested, regardless of whether the template/primer as mismatched or
not (see: multiple bands for dGTP and dTTP, and incorporation of dATP to a small extent
with the G:C template/primer). This would seem to indicate that manganese does have a
potential role to play in changing the fidelity of DinB.
The -1 frameshifting ability of DinB on mismatched templates appears to be due to
slippage of the polyG tract
In order to determine a possible mechanism for the -1 frameshifting of DinB on
the mismatched template/primers, we designed a set of template/primer pairs that are still
internal to mucA but in which the primers anneal downstream of the 5-guanine run. One
primer was perfectly matched to the template, and one contained a G:G mismatch at the
3’ end of the primer, with an adjacent cytosine being the next nucleotide in the template
(Fig. 14A). This template/primer system was designed to eliminate the possibility of
‘slippage’ of the DNA due to the homopolymeric run in the earlier template/primer
system. These new template/primer pairs were then used to perform a replication assay
similar to those described in the previous section, with manganese as the metal ion
cofactor. As seen in Fig. 13, the left-hand side of the figure shows the -1 frameshift with
the mismatched template/primer pair with the 5-guanine (PolyG) run, discussed in the
previous section. The right-hand side of the figure shows replication of the new
template/primer pairs, with the non-mismatched pair on the left and the mismatched pair
55
on the right. It is clear that DinB is active on this template, as shown by the extension
seen when all 4 dNTPs were present in the reaction. When dGTP alone was added to the
reaction, we also saw extension indicative of a single addition onto the primer, which is
expected as cytosine is the first nucleotide of the template past where the primer is
annealed. However, using the mismatched template/primer, we saw little extension of the
primer, even with all 4 dNTPs in the reaction (Fig. 14B). There were also no extension
products for any of the reactions with individual nucleotides (Fig. 14B). This would seem
to indicate that the reason for the -1 frameshifting with the earlier mismatched
template/primer (Fig. 12 & 13) is due to DNA ‘slippage’, as the homopolymeric run of
the previous template/primer makes it likely to ‘slip’ and this new template/primer pair,
even mismatched, would be not likely to do so.
56
Figure 14. DinB does not extend a G:G mismatch on a non-polyG tract sequence. A. Schematic of the DNA templates/primers used in this assay. B. In vitro primer extension assay was performed as described in Materials and Methods using 2 primer/template pairs, one that covers an internal region of the mucA gene, with the primer annealing up through the polyG tract, and one in which the primer anneals downstream of the polyG tract. The primer was used at a concentration of 100 nM, the metal ion concentration was 5 mM for MnCl2, the reaction time was 10 minutes, the dNTPs were used at a concentration of 10 mM, and the concentration of DinB was 100 nM.
57
A. 5’ ----ACGCCGCGGGGGACGAGA---- 3’ 3’ CCCCCTGCTCT---- 5’
5’ ----CGGGGGACGAGAAGCGAC---- 3’ 3’ CTCTTCGCTG---- 5’
B.
0 4 G C G/C 0 4 G C G/C 0 4 G C G/C 0 4 G C dNTP:
C:G G:G C:G G:G
Non-PolyG PolyG
58
Chapter Summary
In this chapter, we have presented data in support of the hypothesis that DinB is a
DNA polymerase, as well as addressing the specific activity and fidelity of DinB on
various templates. We have shown that DinB does have DNA polymerase activity, both
on randomly-generated DNA templates as well as mucA-specific templates. We can also
conclude from our data that choice of metal ion used as a cofactor is important to not
only the activity of DinB, but also to its fidelity under some conditions. We have also
seen that DinB causes -1 frameshift while replicating past mismatches in a poly-guanine
template sequence. This frameshifting effect, however, seems to be due to the nature of
the poly-guanine run, as the same G:G mismatch at another site in the mucA sequence
cannot be extended by DinB. We conclude that the -1 frameshift is likely generated as the
DNA duplex re-aligns (or slips) during replication, given the sequence context of the
homopolymeric run and its potential for slipping. In the context of a chronic CF
respiratory infection, where MMR-impaired P. aeruginosa mutants are prevalent and
mismatches would go un-repaired, this ability of DinB to extend a mismatch at this site in
mucA with a -1 frameshift could be a partial explanation for mucoid conversion in vivo.
59
CHAPTER 4: Role of DinB in mucoid conversion
Expression of DinB is DNA damage regulated
As DinB is able to contribute to mutagenesis in P. aeruginosa when over-
expressed (Appendix Fig. A1), we were interested in investigating the regulation of dinB.
In E. coli and related bacteria, translesion synthesis (TLS) is largely regulated by the SOS
response (20, 21, 59). As part of this response, LexA represses the transcription of more
than 40 different genes by binding to DNA sequences overlapping their promoter regions
(9, 41). Following replication-blocking DNA damage, the LexA protein undergoes a
RecA-facilitated autodigestion that serves to inactivate its DNA binding activity, leading
to the transcriptional derepression of the SOS-regulated genes, including the three E. coli
DNA Pols involved in TLS (Pol II, Pol IV, and Pol V). In an attempt to understand the
regulation of dinB, we constructed a PAO1 ΔlexA::aacC1 mutant (WFPA340) by
inserting a gentamicin resistance cassette (aacC1) within lexA coding sequences. Real-
time RT-PCR analysis was then performed with RNA derived from the PAO1 (lexA+)
and the WFPA340 (ΔlexA::aacC1) strains using a dinB-specific probe and primers. Real-
time RT-PCR experiments showed a 5.5-fold increase in dinB mRNA levels in
WFPA340 compared with PAO1 dinB mRNA levels (Fig. 15).
We also tested whether DNA damage caused a similar up-regulation of dinB
mRNA. To do this, we treated wild-type PAO1 cultures with either mitomycin C (MMC)
or hydrogen peroxide to create a situation where DNA damage would occur. Real-time
RT-PCR analysis of MMC-treated PAO1 cells showed a 6.5-fold increase in dinB mRNA
60
Figure 15. Real-time RT-PCR analysis reveals dinB mRNA levels are elevated in a lexA- mutant. RNA was harvested from wild-type (PAO1) and lexA- strains. The RNA was then analyzed with real-time RT-PCR with probes and primers designed to detect dinB mRNA. The dinB levels are normalized against the housekeeping gene rpoD. Wild-type levels are set at 1 to express the fold increase over wild-type.
61
0
1
2
3
4
5
6
PAO1 lexA-
Fold
Incr
ease
Ove
r Wild
-Typ
e
62
levels, while hydrogen peroxide-treated PAO1 cells showed just over a 2-fold increase
(Fig. 16).
We next addressed the effect of the ΔlexA::aacC1 allele on DinB protein levels.
Cell-free protein extracts of PAO1 and WFPA340 were prepared, separated by SDS-
PAGE, and processed by western blotting using anti-DinB antibodies. A significant
increase in DinB protein levels was observed in WFPA340 compared with PAO1 (Fig.
17). When extracts were made from cultures of PAO1 treated with MMC, an increase in
the steady-state levels of DinB was observed. These data support the hypothesis that
LexA acts to repress dinB transcription and suggest that, like in E. coli, the transcription
of dinB in P. aeruginosa is regulated as part of an SOS-like response to DNA damage.
LexA regulates dinB by binding upstream of the dinB coding sequence
We next wished to determine whether LexA-dependent repression of dinB was
due to a direct interaction of LexA with dinB. Electrophoretic mobility shift assays
(EMSAs) were used in all DNA binding studies. First, we examined whether purified
LexA from E. coli would bind radiolabeled dinB promoter DNA from P. aeruginosa. A
dinB fragment extending 450 bp upstream of the coding sequence and containing a
putative LexA binding site (5’-CTGT-N8-ACAG-3’) located ~350 bp upstream of the
predicted dinB start codon was used in these studies (pdinB). At all concentrations tested,
LexA from E. coli was unable to bind to dinB sequences from P. aeruginosa (Fig. 18),
despite the fact that the same LexA preparation readily recognized umuDC target DNA
from E. coli (Fig. 18).
63
Figure 16. Real-time RT-PCR analysis reveals elevated dinB mRNA levels with DNA damage. RNA was harvested from PAO1 treated with mitomycin C (MMC) or hydrogen peroxide (H2O2). The RNA was then analyzed with real-time RT-PCR with probes and primers designed to detect dinB mRNA. The dinB levels are normalized against the housekeeping gene rpoD. Wild-type untreated levels are set at 1 to express the fold increase over wild-type.
64
0
1
2
3
4
5
6
7
PAO1 PAO1+MMC PAO1+Hydrogenperoxide
Fold
incr
ease
ove
r wild
-type
65
Figure 17. DinB protein levels are elevated by deleting lexA or treating with MMC. Western blot showing DinB levels in PAO1 (lexA+), WFPA340 (lexA-), and WFPA334 (dinB-). Cell extracts were prepared and separated by SDSPAGE, and blotting was performed using anti-DinB antibodies as described in Materials and Methods. Upper panel, DinB; lower panel, nonspecific cross-reactive band used for a normalizing control. Lane 1, purified His-tagged DinB; lane 2, PAO1 extract; lane 3, WFPA340 extract; lane 4, PAO1+MMC extract; lane 5, WFPA334 extract.
66
67
Figure 18. E. coli LexA does not bind P. aeruginosa pdinB. A. Radiolabeled dinB promoter DNA fragment (pdinB) was left untreated (lane 1) or incubated with increasing amounts of purified LexAEc (2.5 nM, 10 nM, 50 nM, 100 nM; lanes 2 to 5, respectively). B. Radiolabeled umuDCEc promoter DNA (pumuDCEc) was incubated with the following extracts or purified LexAEc samples: lane 1, no protein; lane 2, 50 nM; lane 3, 100 nM; lane 4, 10 g extract; lane 5, 10 g extract.
68
69
Figure 19. P. aeruginosa LexA binds P. aeruginosa pdinB. A radiolabeled pdinB fragment was incubated with the following: lane 1, no protein; lane 2 to 4, 5.0 μg extract; lane 5 to 7, 10 μg extract; lane 8, no protein; lane 9 to 14, increasing amounts of LexA (100 ng, 500 ng, 750 ng, 1,000 ng, 1,500 ng, and 2,000 ng, respectively).
70
71
However, a protein(s) present in extracts derived from P. aeruginosa PAO1
bound to the umuDC fragment from E. coli (Fig. 18). Data supporting the hypothesis that
this protein is LexA are (i) that no protein-DNA complex was formed when WFPA340
(ΔlexA) extracts were tested (Fig. 19), while protein-DNA complexes were readily
observed when wild-type P. aeruginosa extracts were tested with a radiolabeled dinB
fragment (Fig. 19), and (ii) that no binding was observed when the extracts were prepared
from PAO1 cells treated with MMC, under which conditions we would expect to have
eliminated LexA repression (Fig. 19).
To verify the above findings, P. aeruginosa lexA was cloned and a hexahistidine-
tagged form of LexA (LexAPa) was generated. When LexAPa was incubated with the
32P-radiolabeled dinB promoter DNA, a protein-DNA complex with a mobility similar to
that seen with P. aeruginosa extracts was observed (Fig. 19), indicating that purified
LexA from P. aeruginosa binds to dinB promoter sequences. This finding, when
combined with the real time RT-PCR and DinB western blot analysis (Fig. 15-17 ),
strongly implies a role for LexA in directly repressing dinB, likely as part of an SOS-like
response.
Expression of DinB increases the frequency of ΔG-dependent Lac+ reversion
In order to determine the spectrum of mutations made by DinBPa, we utilized a
lacZ Lac+ reversion assay developed by Cupples and Miller (11, 12). This assay takes
advantage of a series of isogenic strains bearing different mutant lacZ alleles. The ability
of these strains to grow on minimal medium containing lactose as a sole carbon source
requires that the lacZ allele (either a base substitution or a frameshift mutation) undergo a
72
“true” reversion, as no other mutation will restore the Lac+ phenotype. We specifically
focused on the lacZ allele with which we see the restoration of the Lac+ phenotype only
with a ΔG mutation, deleting a single guanine residue from a homo-polymeric of 6
guanines (CC108). Into the CC108 E. coli strain we introduced either the empty plasmid
(pWSK29), wild-type DinBPa (pHL6), or the mutant R49ADinBPa (pHL6-R49A).
These strains were then plated on glucose+ and lactose+ media to determine the relative
frequency of Lac+ reversion. When comparing the ratio of colonies able to grow on
lactose+ media with those grown on lactose- media, we saw a 15-fold increase in the
frequency of Lac+ reversion when wild-type DinB was introduced into the system when
compared with empty vector (Fig. 20). This effect was not seen with the introduction of
the mutant R49ADinB, where less than a 2-fold increase in reversion frequency was seen
compared with empty vector (Fig. 20). This indicates that the expression of wild-type
DinB increases the frequency of Lac+ reversion by causing -1 frameshifts at a homo-
polymeric run of guanine residues. Also, this effect of DinB was dependent on its
replication ability, as the mutant R49ADinB, which is highly impaired in its replicative
activity (Fig. 3), was not able to create the same frequency of -1 frameshifts as seen with
wild-type DinB. This -1 frameshift effect at poly-guanine runs is reminiscent of the
mucA22 mutation commonly found in the mucA gene of P. aeruginosa isolate from CF
patients, and thus we hypothesize that this mutagenic potential of DinB is related to mucA
mutagenesis and mucoid conversion.
73
Figure 20. DinB increases the frequency of ΔG mutations . lacZ Lac+ reversion frequencies were measured using cultures inoculated with single colonies and grown overnight as described in Materials and Methods. Strains either contained empty vector, wild-type dinB, or dinBR49A mutant. Wild-type frequency is set at 1.
74
0
2
4
6
8
10
12
14
16
Vector DinB DinBR49A
Fold
incr
ease
ove
r wild
-typ
e
75
Development of a selection strategy for isolation of mucoid P. aeruginosa
The study of mucoid conversion in the laboratory has been hampered partly by
the difficulty in isolating the relatively rare mucoid variants that arise in a population. In
an attempt to circumvent this problem, we developed a system for selecting mucoid
variants.
We developed a construct that places the chloramphenicol-resistance gene cat under the
control of the algD promoter and utilized the integrating vector mini-CTX to move the
construct into the genome at the neutral attB site (Fig. 21). Under this system, mucoid
bacteria that are producing alginate and therefore expressing algD will be
chloramphenicol-resistant, allowing for selection of mucoid variants from a population
by growth on chloramphenicol-containing media. In order to study the effects of specific
genes on mucoid conversion, we moved the algD-cat construct into wild-type PAO1 and
its isogenic mutants: ΔdinB, ΔlexA, ΔmutS, ΔmutSΔdinB. By comparing the numbers of
colonies on non-selective versus chloramphenicol media we can determine a relative
frequency of mucoid conversion.
Hydrogen peroxide treatment increases the frequency of mucoid conversion
Previously published data shows that mucoid conversion frequencies are
increased in response to treatment of wild-type, non-mucoid P. aeruginosa with
hydrogen peroxide (43, 45). To determine if this same effect was seen with our selection
strategy, PAO1algD-cat was grown in broth culture and treated with a non-lethal dose of
hydrogen peroxide for 24 hours. The culture was then plated on chloramphenicol media
and serially diluted for plating on non-selective media. The colonies were counted to
76
Figure 21. Schematic of palgD-cat construct and mucoid conversion selection. The promoter region of algD was cloned upstream of the cat gene (confers chloramphenicol resistance). This palgD-cat construct was created in a vector capable of integrating, and was then integrated into the neutral attB site of the wild-type PAO1 and specific gene mutants. The flowchart illustrates the general scheme for the mucoid conversion assay and the calculation of mucoid conversion frequency.
77
palgD
cat
attB
Strains grown +/- H2O2 24h
Dilutions plated on non-selective
media
Cmr mucoid CFU
counted
Non-diluted culture plated on chloramphenicol
Cmr mucoid CFU ÷
Total CFU =
Mucoid conversion frequency Total CFU
counted
78
Figure 22. Hydrogen peroxide treatment increases the frequency of mucoid conversion. Strains containing the algD:cat construct were grown 24 hours with or without hydrogen peroxide at a sublethal dose, and then plated on chloramphenicol and serially diluted and plated on non-selective media. Mucoid colonies were counted on the chloramphenicol plates and then determined the mucoid conversion frequency by dividing the number of mucoid variants by the total number of CFU.
79
0
10
20
30
40
50
60
PAO1 untreated PAO1 H2O2
Con
vers
ion
freq
uenc
y (X
10^1
0)
80
determine the mucoid conversion frequency by dividing the number of mucoid variants
by the total number of CFU. Our results show a 7-fold increase in the frequency of
mucoid conversion in response to hydrogen peroxide treatment (Fig. 22).
Deletion of lexA increases the frequency of mucoid conversion in response to
hydrogen peroxide
As we have shown previously with real-time RT-PCR, immunoblot and EMSA
analysis, LexA directly represses the expression of dinB (Fig. 15-19). These data are in
agreement with what is known about the SOS response in other organisms, where LexA
directly regulates multiple genes in response to DNA damage or environmental stress
(58). We therefore hypothesized that the frequency of mucoid conversion in response to
hydrogen peroxide would be different in a ΔlexA mutant when compared with wild-type
P. aeruginosa. Using the PAO1algD-cat strain and its isogenic ΔlexA mutant, we
compared the mucoid conversion frequencies with and without exposure of hydrogen
peroxide. While there appeared to be no change in the basal level of mucoid conversion
comparing the untreated PAO1 and ΔlexA mutant, the mucoid conversion frequency was
increased 18-fold in the ΔlexA mutant in response to hydrogen peroxide treatment (Fig.
23). This indicates that elevated DinB levels, which are the result of mutating lexA, lead
to increased mucoid conversion.
81
Figure 23. Hydrogen peroxide treatment increases the frequency of mucoid conversion of a lexA- mutant. Strains containing the algD:cat construct were grown 24 hours with or without hydrogen peroxide at a sublethal dose, and then plated on chloramphenicol and serially diluted and plated on non-selective media. Mucoid colonies were counted on the chloramphenicol plates and then determined the mucoid conversion frequency by dividing the number of mucoid variants by the total number of CFU.
82
0
10
20
30
40
50
60
70
80
90
100
lexA- untreated lexA- H2O2
Con
vers
ion
freq
uenc
y (X
10^1
0)
83
Figure 24. Deletion of dinB decreases the frequency of both spontaneous and peroxide-induced mucoid conversion. Strains containing the algD:cat construct were grown 24 hours with or without hydrogen peroxide at a sublethal dose, and then plated on chloramphenicol and serially diluted and plated on non-selective media. Mucoid colonies were counted on the chloramphenicol plates and then determined the mucoid conversion frequency by dividing the number of mucoid variants by the total number of CFU.
84
0
10
20
30
40
50
60
PAO1 untreated PAO1 H2O2 dinB- untreated dinB- H2O2
Con
vers
ion
freq
uenc
y (X
10^1
0)
85
Deletion of dinB decreases both non-induced and induced mucoid conversion
The above data supports a DinB-dependent strategy for mucoid conversion. To
further investigate this hypothesis, we created a ΔdinB mutant in our PAO1algD-cat
strain. The non-induced mucoid conversion frequency for this strain was almost 10-fold
less than for PAO1 and there was no increase in that frequency in response to hydrogen
peroxide treatment (Fig. 24). In combination with our previous data, this indicates that
DinB is responsible for the majority of mucoid conversion, both non-induced and those
induced by hydrogen peroxide treatment. This argues for a physiological role for DinB in
response to DNA damage: from the perspective of regulation, LexA and DNA damage
both have a role in DinB expression (Fig. 15-19), and from the perspective of mucoid
conversion, LexA, DinB and DNA damage all have a role in changing the frequency of
mucoid conversion (Fig. 22-24).
Mucoid conversion is increased when mutS is deleted, while deletion of dinB in a
ΔmutS background eliminates mucoid conversion
Previous studies with P. aeruginosa isolates from CF patients have shown that the
hypermutable phenotype is common among these isolates, and that this phenotype is
most often associated with mutations of the mutS, mutL, and uvrD genes, which are
involved in MMR (Oliver 2000, Oliver 2002). ΔmutS mutants in P. aeruginosa have also
been shown to have an increased mucoid conversion frequency (45). We therefore tested
a ΔmutS mutant in our mucoid conversion assay, and found that it had a higher frequency
of non-induced mucoid conversion in the ΔmutS mutant as compared with the wild-type
PAO1algD-cat strain (Fig. 25). There was also an increase in mucoid
86
Figure 25. Deletion of mutS increases the frequency of both spontaneous and peroxide-induced mucoid conversion. Strains containing the algD:cat construct were grown 24 hours with or without hydrogen peroxide at a sublethal dose, and then plated on chloramphenicol and serially diluted and plated on non-selective media. Mucoid colonies were counted on the chloramphenicol plates and then determined the mucoid conversion frequency by dividing the number of mucoid variants by the total number of CFU.
87
0
200
400
600
800
1000
1200
1400
PAO1 untreated PAO1 H2O2 mutS- untreated mutS- H2O2
Con
vers
ion
freq
uenc
y (X
10^1
0)
88
conversion in response to hydrogen peroxide treatment of the ΔmutS mutant (Fig. 25).
These data make a connection between MMR and mucoid conversion that is likely
physiologically relevant in the CF lung, where both hypermutable and mucoid bacteria
emerge.
To test the role of dinB in the high frequency of mucoid conversion in the ΔmutS
strain, we created a ΔmutSΔdinB strain in the PAO1algD-cat background. The high
frequency of mucoid conversion of the single ΔmutS strain was completely eliminated by
deleting dinB (Fig. 26). We were unable to recover any mucoid variants from this double
mutant strain, even following hydrogen peroxide treatment. We have already shown that
deleting dinB alone almost completely eliminates both non-induced and induced mucoid
conversion (Fig. 24). This double mutant data strongly suggests a very important role for
dinB in the mucoid conversion when MMR is interrupted.
Determination of the frequency of mucA-dependent vs mucA-independent mucoid
algD-cat variants
In order to determine the classes of mucA mutants present in the mucoid variants
that arose in our mucoid conversion assay, we sequenced the mucA allele from
representative variants of each population. Given the frequency of the mucA22 allele in
isolates from CF patients (25-40%), we determined that we would need to sequence at
least 15 alleles from each group to ensure our results were statistically significant (3). We
harvested genomic DNA from each of the variants, amplified the mucA open reading
frame by PCR and had the PCR products sequenced. These sequence data were then
89
Figure 26. Deletion of dinB in a ΔmutS background eliminates mucoid conversion. Strains containing the algD:cat construct were grown 24 hours with or without hydrogen peroxide at a sublethal dose, and then plated on chloramphenicol and serially diluted and plated on non-selective media. Mucoid colonies were counted on the chloramphenicol plates and then determined the mucoid conversion frequency by dividing the number of mucoid variants by the total number of CFU.
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91
aligned with the wild-type PAO1 mucA sequence to determine if or where any mutations
occurred in the mucA gene of the variants.
The variants were first grouped into one of two categories: mucA-independent or
mucA-dependent. This was determined based on the variant sequences being either the
same as wild-type (mucA-independent) or having a mutation in the gene (mucA-
dependent). What is most striking about the sequence data for these groups is the drastic
decrease in the number of mucA-dependent mucoid conversion in the ΔmutS strain and
the ΔdinB strain (Fig. 27). The ΔmutS strain (both treated and untreated with hydrogen
peroxide) shows a trend reversed from that of the wild-type and ΔlexA strains, in which
the majority of the variants are mucA-independent. This same trend was also seen with
the ΔdinB strain, but to an even greater extent, where all of the variants from this strain
were mucA-independent (Fig. 27). Also, there does not appear to be any change in the
ratio of these groups upon treatment with hydrogen peroxide, as the ratio of mucA-
dependent to mucA-independent variants does not change with treatment (Fig. 27). This
indicates that hydrogen peroxide treatment, while changing the frequency of mucoid
conversion, does not alter the mucA-dependence or mucA-independence of the mucoidy
of the resulting variants. We also conclude that mutS and dinB are crucial for mucA
mutagenesis, from the mucoid conversion data, as well as the sequencing of the mucA
alleles.
We also grouped the variant sequence data by the types of mutations found:
mucA-independent (wild-type sequence), mucA22 (ΔG at the 5-guanine run), transitions,
transversions and other mutations (deletions, insertions). As seen in Fig. 28, it is clear
that mutS again plays a role in determining the diversity of mucA mutations, as the
92
Figure 27. Results of mucA sequencing show crucial role for mutS and dinB in mucA mutagenesis. The mucA gene of the mucoid isolates obtained from the experiments represented by the proceeding figures was PCR amplified and sent for direct nucleotide sequencing. The results are shown here as mucA-independent vs mucA-dependent, represented as percentages of the whole mucoid population. mucA-independent isolates were those with wild-type mucA sequences. 1: PAO1 untreated, 2: PAO1 treated, 3: ΔlexA untreated, 4: ΔlexA treated, 5: ΔmutS untreated, 6: ΔmutS treated, 7: ΔdinB treated.
93
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rcen
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otal
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94
Figure 28. Results of mucA sequencing show a role for mutS and dinB in altering the spectrum of mucA mutations. The results of the mucA gene sequencing are shown, with the different categories of mutations. Specifically shown are mucA22 mutations (ΔG at the 5G tract), transversion, transitions, mucA-independent mutations, and other (including frameshifts due to insertions and deletions). 1: PAO1 untreated, 2: PAO1 treated, 3: ΔlexA untreated, 4: ΔlexA treated, 5: ΔmutS untreated, 6: ΔmutS treated, 7: ΔdinB treated.
95
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mucA22mutationsmucA-independent
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96
diversity of the mucA mutations is decreased in the ΔmutS strain, with almost all mucA-
dependent variants having the mucA22 mutation. All of the mucoid variants from the
ΔdinB strain were mucA-independent, indicating a clear role for dinB in mucA
mutagenesis.
Chapter Summary
In this chapter, we have presented data that connects DinB (as well as other
factors) with mucoid conversion. We first developed a system to reproducibly isolate
mucoid variants in the lab using selection rather than traditional screening. Using this, we
showed that hydrogen peroxide increases the frequency of mucoid conversion, in
agreement with previous findings (43). The role of mutS was also examined in this
system, and we found that deletion of mutS significantly increased the frequency of
mucoid conversion, both spontaneously and in response to hydrogen peroxide treatment.
DinB was also shown to be essential for this process, as deletion of dinB in the ΔmutS
background completely eliminated mucoid conversion in this high-converting strain.
Deletion of dinB on its own also decreased mucoid conversion, both spontaneously and
with hydrogen peroxide treatment. MutS and DinB were also shown to have a crucial role
in determining mucA mutagenesis, based on the spectrum of mucA mutations from the
mucoid variants of the ΔmutS, ΔdinB and ΔmutSΔdinB strains. This shows an important
role for DinB is mucoid conversion, especially in the context of the hyptermutable MMR-
impaired P. aeruginosa that emerge from the CF lung.
97
CHAPTER 5: Conclusions and Discussion
The conversion of P. aeruginosa to the mucoid phenotype has long been an
important area of ongoing research, as it has become clear that the production of alginate
is one of the factors associated with a poor prognosis for the CF patient (23). The
presence of alginate contributes to resistance to phagocytosis, protection from reactive
oxygen species, and resistance to antibiotics (23, 36, 54). While the molecular
mechanism for mucoid conversion was accelerated with the discovery of the role of
mucA mutagenesis, there is still much to be learned about the physiological factors that
produce an environment that ultimately leads to mucoid conversion. Therefore, a better
understanding of these factors that lead to mucoid conversion is essential to developing
more effective treatment and prevention therapies for the P. aeruginosa respiratory
infections of CF.
In beginning to study DinB as one of the potential factors involved in mucoid
conversion, we first assayed the enzyme for polymerase activity. The results clearly show
that DinB is a DNA polymerase, with no exonuclease (proofreading) activity and activity
similar to that seen for the E. coli homolog (Fig. 3). In order to analyze DinB’s activity
on a relevant DNA template, we created several mucA-derived template/primer pairs and
completed replication assays to determine several important biochemical characteristics
of the enzyme. This included showing that DinB is active in replicating mucA sequences,
and that DinB can utilize both magnesium and manganese as metal ion cofactors for
enzymatic activity (Fig. 4). As manganese has long been known to have a mutagenic
effect on DNA replication, and as it has recently been shown to have an effect on the
98
activity and fidelity of a Y family polymerase (1, 19), we also chose to consider the effect
of metal ion cofactor on DinB activity and fidelity. We showed that there is an over 600-
fold decrease in the Km for the correct nucleotide with magnesium versus manganese,
indicating an increase in DinB’s apparent affinity for dNTP in the presence of manganese
(Fig. 7). This was also shown to be an effect independent of the specific poly-guanine run
of the template/primer, as we repeated the Km for dNTP determination with another
mucA-derived template but with the primer annealed downstream of the poly-guanine
run, and the kinetics were similar to those observed with the original template/primer
(Fig. 10 & 11). To examine the role of cofactor in fidelity, we also determined the ability
of DinB to misincorporate incorrect nucleotides and found that the only nucleotide
misincorporated was dCTP across from the template cytosine, and this only in the
presence of manganese as the metal ion cofactor (Fig. 8 & 9). This shows i) that overall,
DinB is more faithful on this template than we originally hypothesized, and ii) that
manganese does appear to decrease the fidelity of DinB under specific circumstances. It
has been proposed that manganese is more loosely coordinated in the enzyme structure
than magnesium, and that this difference in coordination may result in a more open or
accessible conformation of the active site, which could result in both the increased
activity and decreased fidelity of DinB with manganese (19).
Also in an attempt to understand the nature of DinB activity on another relevant
template, we used a mismatched G:G in the DNA sequences for our template/primer,
with the G:G mismatch at the 3’ terminus on the primer and at the end of the poly-
guanine run of the mucA sequence. Using this template, we have shown that DinB is
capable of both extending the mismatched template/primer and also of creating -1
99
frameshifts as a result of that extension (Fig. 12). This effect extends to a random, non-
mucA-derived sequence flanking the same core poly-guanine run with adjacent cytosine,
indicating that there is nothing specific about the flanking sequence context of mucA that
allows for this effect (Fig. 13). It is also worth noting at this point that under the
conditions we used for these assays, manganese does have an effect on the fidelity of
DinB, as seen by the increased number of presumably incorrect extension products we
see with manganese present in the reactions.
To address the mechanism for the -1 frameshifting we see with the mismatch at
the poly-guanine run, we have also showed that this frameshifting effect is not seen with
a G:G mismatch in the context of a non-homopolymeric run (Fig. 14). We therefore
conclude that it is likely that the frameshifting we see with the poly-guanine run is due to
slippage of the DNA strands, as the duplex ‘breathes’ during replication and comes back
together annealed one nucleotide position off from where it started. This would result in
the G:G mismatch becoming a G:C match of 3’ guanine of the primer annealed to the
cytosine adjacent to the poly-guanine run of the template. This would mean that DinB is
not actually extending a mismatched template/primer per se, as what the enzyme is
‘seeing’ in its active site is actually a correctly-paired template/primer sequence. Thus,
this activity of DinB does not really address the enzyme’s fidelity, but it is still a
potentially relevant physiological phenomenon that could be happening in the
hypermutable P. aeruginosa undergoing mucoid conversion in the lungs of CF patients.
In this study, we have made several intriguing discoveries that relate to the
connection between the DNA damaging nature of the inflammatory environment of the
CF lung and the conversion to the mucoid phenotype. First, we have shown that the P.
100
aeruginosa Y family DNA polymerase DinB is repressed by the SOS factor LexA, at the
mRNA and protein levels (Fig. 15 & 17). As LexA is known to respond to DNA damage
in the cell, this argues that the regulation of DinB is thus DNA damage-responsive. This
hypothesis is confirmed with our data showing that both hydrogen peroxide and
mitomycin C cause an up-regulation of DinB mRNA levels (Fig. 16). It was also
important to determine whether or not this repression of DinB exerted by LexA is direct
or indirect, and we addressed that question with DNA binding assays that showed that
LexA does bind directly to the upstream region of the dinB open reading frame (Fig. 19).
This data argues for a model where DinB levels are elevated following exposure of a cell
to DNA damaging agents through LexA de-repression in an SOS-type response,
presumably in an attempt to rescue the cell from otherwise lethal mutations in the
genome by translesion synthesis.
We have also shown that DinB has a preference for creating -1 frameshifts in
homopolymeric guanine runs, as measured by introducing a plasmid-born wild-type copy
of dinB into the Lac+ reversion assay (Fig. 20). This ability of DinB is linked to its
polymerase activity, as the several impaired R49A mutant DinB shows almost no
increase in the frequency of Lac+ reversion with the same sequence (Fig. 20). We have
also shown that there are other mutations that DinB seems prone to making (Appendix
Fig.), but the -1 frameshift in the poly-guanine run is by far the most significantly
affected by elevating cellular DinB levels. Given this sequence’s similarity to the
mutational hotspot 5-guanine run in the mucA gene, it was intriguing to think of a
connection between the mutational specificity of DinB and the mutational spectrum of
mucA. When taken together with the modest mutator phenotype we see when DinB is
101
over-expressed (Appendix Fig. A1), this fits well with the hypothesis that DinB has a role
in inactivating mucA and therefore in mucoid conversion.
The fact that the process of mucoid conversion has been difficult to model in the
lab has been an issue slowing down the studying of the factors that lead to mucoid
conversion. To address this issue, we created a novel panel of strains that would allow us
to select for the expression of alginate, using a fusion of the algD promoter with the gene
for chloramphenicol resistance (Fig. 21). This method allows us to select for the
relatively infrequent mucoid conversion by plating on chloramphenicol, rather than
visually screening for the mucoid variants among the rest of the non-mucoid population.
One of our goals with this strain was to test whether hydrogen peroxide treatment would
alter the frequency of mucoid conversion, and we found that it does indeed increase the
frequency 7-fold over the non-induced conversion that occurs without treatment in the
parental strain (Fig. 22). This is in agreement with previous work that observed a
noticeable increase in mucoid conversion using a different growth system upon treatment
with hydrogen peroxide (43).
Another goal with this system was to determine if any of our genes of interest had
a role in mucoid conversion, so we created mutants in our mucoid-selectable strain:
ΔlexA, ΔdinB, ΔmutS, and ΔmutSΔdinB. We determined that while there is no change in
the non-induced frequency of mucoid conversion between the parental and ΔlexA strains,
there is a much higher frequency of conversion upon treatment of the ΔlexA strain with
hydrogen peroxide, compared with treatment of the parental strain (Fig. 23). As we have
shown that LexA represses DinB, this data indicates that bacteria with higher levels of
DinB have higher frequencies of mucoid conversion than those with repressed DinB.
102
Looking at this from the opposite side, deleting dinB in this system meant almost no
mucoid conversion occurred, either non-induced or treatment-induced (Fig. 24). Again,
this argues for an important role to be played by DinB in mucoid conversion, shown by
both elevating DinB levels and eliminating DinB expression.
Given the significance of the hypermutable phenotype and mismatch repair in the
P. aeruginosa isolates from CF patients, we also examined the role of mutS in our
mucoid-selectable system (45, 48). Deletion of mutS dramatically increased the frequency
of both non-induced and treatment-induced mucoid conversion (Fig. 25). We believe that
this is likely occurring because the replication errors being introduced into the genome by
DinB are going unnoticed due to the lack of mismatch repair. This also fits with the fact
that we see complete elimination of mucoid conversion with the double mutant
ΔmutSΔdinB (Fig. 26). Even missing the function of mismatch repair, without DinB
around to create errors, mucoid conversion is nonexistent.
It is also of interest to note at this point that when the mucA alleles of these
variants were sequenced, the most striking effects were seen with the ΔmutS and the
ΔdinB strains. Deletion of mutS shifted the majority of the variants to the mucA-
independent category, versus the parental and ΔlexA strains, where the majority of the
variants were of the mucA-dependent category (Fig. 27). Deletion of dinB also led to this
same trend, with all of the sequenced variants falling in the mucA-independent category
(Fig. 27). The mucA alleles from the ΔmutS variants that were mutated also had less
diversity in the kinds of mutations found (compared with the parental and ΔlexA), with
most of them having the mucA22 mutation (ΔG) (Fig. 28). This strongly indicates an
103
important role for both mutS and dinB in determining mucA mutagenesis and therefore
mucoid conversion.
This data, taken together and in the context of the recent work in the field
especially regarding hyptermutable isolates and biofilms, leads us to propose the
following model for mucoid conversion (Fig. 29). We have shown here that DinB is
regulated in an SOS-type response, being directly repressed by LexA and being induced
by agents of DNA damage. Once DinB is active in a cell that has undergone DNA
damage, it would be recruited to replicate damaged DNA. This could include lesions or
adducts in the DNA, and if any of those occur in or near the mucA, we have shown that
DinB is able to replicate at least part of the internal mucA sequence. At this point, DinB
may introduce errors as it is replicating, either due to incorrect incorporation bypassing a
lesion or possibly creating the -1 frameshift that we have seen as it is copying past a
mismatch. It is also clear that there is a strong selection for hypermutators in the CF lung,
and that this phenotype has so far been found to be caused by mutations in the MMR
pathway, especially in mutS (48, 49). This impairment of the MMR system would lead to
a lack of mutational oversight of the genome, and any errors introduced by DinB would
not be caught by the normal DNA repair pathway intended to fix them. Given the strong
selective pressure towards mucoid conversion in the CF lung, mutations that inactivate
MucA would be highly advantageous in combating host defenses and therapeutic
regimens. We believe that MutS and DinB both play pivotal roles in mucoid conversion,
confirmed by our mucoid conversion assays in which both genes were shown be
important for mucA mutagenesis.
104
Given the emerging view in the field that mutator bacteria are critical for many
chronic infections, we believe that the data represented in this thesis can lead to better
understanding of these chronic infections. Mutator bacteria have been found not only
with P. aeruginosa in cystic fibrosis infections but also in meningitis (Neisseria
meningitidis serogroup A), peptic ulcers and gastric cancer (Helicobacter pylori), and
cystic fibrosis (Streptococcus pneumoniae). Most of the characterized mutators have been
found to have mutations in the DNA repair genes, especially those involved in mismatch
repair. These mutators have a selective advantage in harsh, often inflammatory
environments, and have been linked in some cases to phase variation, which can aid in
pathogenesis. This phenotype is also often associated with a high resistance to antibiotics,
which is in itself a complication during infections.
Our work shows the important role of DNA repair mechanisms and the mutator
phenotype in mucoid conversion during a modeled chronic infection. Our results indicate
that the judicious use of anti-inflammatory treatment could lead to better outcomes for
patients with cystic fibrosis-related P. aeruginosa infections, especially early in the
disease progression, before the chronic mucoid infection has been established. We also
believe that this work may shed some light on the impact of mutators in persistent
bacterial infections in general.
105
Figure 29. Proposed model for mucA mutagenesis in the CF lung. Our model for mucoid conversion of P. aeruginosa due to chronic respiratory infection. Initially non-mucoid bacteria are exposed to years of chronic inflammation, leading to DNA damage and induction of an SOS response. DinB is up-regulated, mutS is mutated, and these two factors lead to mucA mutagenesis and alginate production.
106
S. aureus, H. influenzae Nonmucoid P. aeruginosa
Mucous layer
Inflammation, ROS, DNA damage, DinB induction,
LexA
Mucoid P. aeruginosa
DinB
CF airway epithelium
Alginate AlgT
mucA mutagenesis
mutS
Chronic infection
107
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APPENDIX
Elevated expression of DinBPa confers a mutator phenotype in vivo
Overexpressed levels of DinBEc from a plasmid were reported to increase the
frequency of mutagenesis in E. coli as measured using a Rifampicin resistance (Rifr)
assay (30, 37, 63, 64). We therefore hypothesized that if DinBPa was similarly error
prone, then plasmid-expressed DinBPa would likewise increase the frequency of Rifr in
E. coli. The expression of dinB of P. aeruginosa from a low-copy-number plasmid
(pHL6) conferred a 4.7-fold increase in the frequency of Rifr relative to the same strain
bearing the pWSK29 control vector (Appendix Fig. A1). In contrast, neither plasmid-
expressed DinB(D8A)Pa nor DinB(R49A)Pa enhanced the frequency of Rifr (Appendix
Fig. A1). Given that the D8A and R49A mutants were expressed at steady-state levels
comparable to that of the wild-type protein (50), these findings indicate that the modest
Rifr mutator phenotype conferred by ectopically expressed DinBPa was attributable to its
intrinsic DNA polymerase activity.
Ability of DinB to influence frequency of Lac+ reversion
In order to determine the spectrum of mutations made by DinBPa, we utilized a
lacZ Lac+ reversion assay developed by Cupples and Miller (11, 12, 50). This assay
takes advantage of a series of isogenic strains bearing different mutant lacZ alleles. The
ability of these strains to grow on minimal medium containing lactose as a sole carbon
source requires that the lacZ allele (either a base substitution or a frameshift mutation)
undergo a “true” reversion, as no other mutation will restore the Lac+ phenotype (11, 12,
50). By comparing frequencies of lacZ Lac+ reversion for transformants of these
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reporter strains bearing either the pWSK29 control vector or the pHL6, which expresses
DinBPa, we were able to determine the spectrum of mutations generated by ectopic
expression of DinBPa. As summarized in Appendix Figure A2, DinBPa clearly favored -
1 frameshift mutations: the frequency of Lac+ reversion was increased 339- and 3-fold in
the CC108 (lacZ[6G 5G]) and the CC111 (lacZ[7A 6A]) strains, respectively, relative
to the pWSK29 control (P values of 0.001 and 0.036, respectively). Furthermore,
DinBPa also increased by 117-fold the frequency of Lac+ reversion in the CC104
(lacZ[GCG GAG]) strain (P value 0.056). Frequencies of Lac+ reversion observed with
the other reporter strains bearing the DinBPa-expressing plasmid (pHL6) were
comparable to those of the respective strains bearing pWSK29 (Appendix Figure A2).
We next asked whether the DNA polymerase activity of DinBPa was necessary
for increased lacZ Lac+ reversion. Due to the fact that DinBPa enhanced lacZ Lac+
reversion in the CC104 and CC108 strains more strongly than in the other strains
examined (Appendix Figure A2), we focused on these two strains for this experiment. As
summarized in Appendix Figure A2, the D8A mutant was unable to significantly alter the
frequency of lacZ Lac+ reversion in both the CC104 and the CC108 strains. Despite its
inability to confer a mutator phenotype in E. coli as measured by Rifr (Appendix Figure
A1), the R49A mutant nonetheless enhanced the frequency of lacZ Lac+ reversion in
both the CC104 and CC108 strains by factors of 53- and 19-fold, respectively: these
increases were statistically significant (P values of 0.012 and 0.005, respectively) and are
consistent with the R49A mutant retaining partial catalytic activity (Fig. 3, lanes 19 to
21). Taken together, these findings indicate that DinBPa is capable of catalyzing both
misinsertion and frameshift mutations.
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Appendix Figure A1. Effect of P. aeruginosa dinB overexpression on the frequency of rifampicin resistance in E. coli. Each plasmid was transformed into E. coli strain AB1157. Aliquots of appropriate serial dilutions of cultures started from single colonies inoculated into 5 ml of LB-Ap and grown 16 h were plated on LB to determine survival and on LB-Rif to assess spontaneous mutation rate. Values shown represent averages of 4 to 16 independent experiments the standard error of the means. Published in collaboration: Sanders et al, Journal of Bacteriology, 188:8573-8585.
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Appendix Figure A2. Ability of plasmid-expressed DinB to influence frequencies of Lac+ reversion of different lacZ alleles. lacZ Lac+ reversion frequencies were measured using cultures inoculated with single colonies and grown overnight as described in Materials and Methods. Strains either contained empty vector, wild-type DinB, DinBR49A mutant, or DinBD8A mutant. Values shown represent the averages of at least three independent determinations the standard error of the means. Parenthetical values indicate P values (Student’s t test) for the difference between the frequencies of lacZ Lac+ reversion for the indicated plasmid-bearing strain relative to the same strain bearing the pWSK29 control plasmid. Published in collaboration: Sanders et al, Journal of Bacteriology, 188:8573-8585.
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SCHOLASTIC VITAE
Andrea Beth Rockel School Home Department of Microbiology and Immunology 2425 Lyndhurst Ave Wake Forest University Winston Salem, NC 27103 Medical Center Blvd 336.407.4100 Winston Salem, NC 27157 [email protected] 336.716.9333 Education Wake Forest University, Winston Salem, North Carolina, Summer 2008 Ph.D. candidate in Microbiology and Immunology Thesis advisor: Daniel J. Wozniak, Ph.D.
Area of specialization: Bacterial pathogenesis Mars Hill College, Mars Hill, North Carolina, 2002 B.S. in Biology, summa cum laude Awards and Honors Biofilms Summer Workshop Student Travel Grant, 2006 Center for Biofilm Engineering, Bozeman, Montana Dean’s Fellowship, Wake Forest University, 2002 – present Awarded based on academic record Teaching Experience Teaching Assistant, Fundamentals of Bacteriology, Wake Forest University, 2006
• Organized and delivered lecture in special topics to first year graduate students
Research Mentor, Wake Forest University, 2005 – 2006
• Served as mentor for graduate and undergraduate students: o Scott Hardison (Wake Forest University, 2006) o Doug Grunwald (Davidson College, 2005) o Cynthia Ryder (Wake Forest University, 2005)
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• Helped to direct short-term student research projects Advanced Topics Lectures, Wake Forest University, 2003-2005
• Researched and organized lectures on advances topics in microbiology and
immunology, including bacterial pathogensis, advanced laboratory techniques and vaccine development.
Teaching Assistant, Mars Hill College, 1999 – 2002
• Prepared for undergraduate laboratory courses, including microbiology, organismal biology, ecology and genetics
• Ran the above laboratory sessions, with class sizes of 15-30 • Graded exams and laboratory papers from the above courses
Research experience Doctoral Research: Department of Microbiology and Immunology, Wake Forest University Health Sciences, 2002 – present (research advisor: Dr. Daniel J. Wozniak)
• Cloned a novel, previously uncharacterized gene, dinB • Showed that DinB is an error prone DNA polymerase that is essential for the
response to stress and DNA damage • Developed novel gene replacement strategies • Developed new DNA replication assays
Publications Rockel, A., Sanders, L.H., Sutton, M.D., and D.J. Wozniak. 2008. Role of DinB in the mucoid conversion of Pseudomonas aeruginosa. Manuscript in preparation. Rockel, A., Pence, M., Perrino, F.W. and D.J. Wozniak. 2008. Activity and kinetics of Pseudomonas aeruginosa DinB. Manuscript in preparation. Sanders, L.H., Rockel, A., Lu, H., Wozniak, D.J. and M.D. Sutton. 2006. Role of Pseudomonas aeruginosa dinB-Encoded DNA Polymerase IV in Mutagenesis. Journal of Bacteriology 188:8573-8585. West-Barnette, S., Rockel, A., and W.E. Swords. 2006. Biofilm growth increases phosphorylcholine content and decreases potency of nontypeable Haemophilus influenzae endotoxins. Infection and Immunity. 74:1828-36.
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Brzoza, K.L., Rockel, A.B., and E.M. Hiltbold. 2004. Cytoplasmic entry of Listeria monocytogenes enhances dendritic cell maturation and T cell differentiation and function. Journal of Immunology. 173:2641-51. Scientific presentations Rockel, A.B., Sanders, L.H., Sutton, M.D., Lu, H., and D.J. Wozniak. Identification of DinB, a novel DNA polymerase of Pseudomonas aeruginosa. Midatlantic Microbial Pathogenesis Meeting, Wintergreen Resort, VA. Poster Presentation. February 2007. Rockel, A.B., Sanders, L., Sutton, M.D., Lu, H. and D.J. Wozniak. Identification of DinB, a novel DNA polymerase of Pseudomonas aeruginosa. North Carolina Branch of American Society of Microbiology, North Carolina State University. Poster Presentation. October 2006. Rockel, A.B., Sanders, L., Sutton, M.D., Lu, H. and D.J. Wozniak. Identification of DinB, a novel DNA polymerase of Pseudomonas aeruginosa. North Carolina Branch of American Society of Microbiology, North Carolina State University. Oral Presentation. October 2005. Rockel, A.B., Sutton, M.D., Lu, H., and D.J. Wozniak. Identification of DinB, a novel DNA polymerase of Pseudomonas aeruginosa. Midatlantic Microbial Pathogenesis Meeting, Wintergreen Resort, VA. Poster Presentation. February 2005.