Department Of BiologyIndependent Study Thesis

Progress toward the Investigation of the role of Arginine Kinase in the bacterium Myxococcus xanthus

Shelby Kratt

Adviser: Dr. Dean Fraga

Submitted in Partial Fulfillment of the Requirement for

Independent Study Thesis in Biology at the

COLLEGE OF WOOSTER 2015-2016

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TABLE OF CONTENTS

I. ABSTRACT …………...……………………………………………………………..3

II. INTRODUCTION ...………………………………………………………………...4

a. Phosphagen Kinases

b. Background Information on a Function of Phosphagen Kinases

i. General Information about the Arginine Kinase Lineage

c. Bacterial Arginine Kinases

i. Bacterial Acquisition of AK Feasible via Horizontal Gene Transfer

ii. Fitness Advantage of Organisms with Novel Arginine Kinase Function

iii. Bacteria with Functional Arginine Kinases

d. Function of Arginine Kinase in Myxococcus xanthus

i. Effect of AK deletion on M. xanthus Stress Response

ii. AK deletion Affects M. xanthus Development

e. Intention of this Study

III. MATERIALS AND METHODS ………………………………………………..17

a. Strains and Growth Conditions

b. Midiprep DNA Purification of the Four Plasmids

c. Transformations of Deletion Strain with Various Plasmids

i. Procedure for transforming Myxococcus

d. GalK Counter-Selection of Transformants

e. Glycerol Preservation of Strains

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TABLE OF CONTENTS CONTINUED

IV. RESULTS ………………………………………………………………………......22

a. Strains and Growth Conditions

b. Midiprep DNA Purification of the Four Plasmids

c. Transformations of Deletion Strain with Various Plasmids

d. GalK Counter-Selection of Transformants

e. Glycerol Preservation of Strains

V. DISCUSSION ……………………………………………………………………....33

VI. ACKNOWLEDGEMENTS ...……………………………………………………..41

VII. LITERATURE CITED …………………………………………………………....42

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ABSRACT

Recent studies have characterized novel arginine kinase (AK) genes in bacteria, extending the

phylogenetic realm in which they were previously thought to occur. Elimination of the AK gene

in the bacteria Myxococcus xanthus, gave rise to increased salt and pH sensitivity. Surprisingly,

the AK gene was additionally found to be required for normal development of M. xanthus during

times of decreased nutrient availability, inciting the need for further research on the enzyme in

this bacterium. The research described here was begun in hopes of furthering the investigation of

the role of AK in the bacterium Myxococcus xanthus. As such, the M. xanthus Δark mutant was

transformed with the original M. xanthus ark gene (MyxoAK), both with and without a His-tag,

as well as with the horseshoe crab AK homolog (HcAK). Inserting the HcAK gene in isolated M.

xanthus cultures alongside insertions of the bacterium’s original AK gene in others was

performed so that the effects these homologs have on the bacterium can be evaluated in future

studies. Successful transformation of various pBJ114 plasmids containing the different AK genes

was confirmed with selection for kanamycin resistance and homologous recombination of the

plasmid AK insert was suggested by galK counter-selection. Continuation of this work may then

include constructing M. xanthus Δark mutant strains with HcAK-His and verifying Δark of the

transformants was replaced with an AK homolog, as well as assessing the transformants’

responses to non-starvation and starvation stress, as compared to the wild-type (DK1622) and the

M. xanthus Δark (MS2252) strains.

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INTRODUCTION

Phosphagen Kinases

Background Information on a Function of Phosphagen Kinases

To better understand one of the various roles phosphagen kinases (PKs) play in cellular

systems, it is first imperative to understand the importance of cellular adenosine triphosphate

(ATP). This molecule is just one of the substrates for this family of enzymes and is used as a

"high-energy" exchange medium with which a cell can perform many different cellular

processes. ATP is primarily generated via oxidative phosphorylation, which occurs in the

mitochondrial membrane of eukaryotic cells and the cytoplasmic membrane of aerobic

prokaryotic cells. As this metabolic pathway passes electrons through electron transport chains in

a series of reduction-oxidation reactions, the energy released by electron flow is used to transport

protons across the inner membrane of either the mitochondria or the bacterial cell. This electron

transport thus generates potential energy in the form of a pH gradient and an electrical potential

across this membrane. ATP synthase now uses the energy from allowing protons to flow back

across the membrane and down these gradients to generate ATP from adenosine diphosphate

(ADP) and cellular phosphate (Pi), “storing” the energy in the third (ɣ) phosphate’s bond. Energy

from cleaving the phosphate bond from ATP can then be used to power activities such as

metabolism and signal transduction, which influence various cellular functions.

While ATP acts as a substrate itself in many cellular pathways, kinases such as

phosphagen (guanidino) kinases, which specifically transfer the phosphorus-containing groups

(phosphotransferases) with a nitrogenous group as acceptor, can additionally transfer phosphate

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from these energy-providing molecules to proteins and lipids. Figure 1 displays the reversible

transfer of the high-energy ɣ phosphate group between adenosine triphosphate (ATP) and a

guanidino molecule, catalyzed by arginine kinase, a member of the phosphagen kinase family.

Through this transference, the signal transduction and metabolic processes that use ATP as an

energy source convert it back into its precursors (ADP and Pi), which can then be used to again

generate ATP as needed.

Figure 1: The reversible transfer of a high-energy ɣ phosphate group between adenosine triphosphate (ATP) and a

guanidino molecule catalyzed by arginine kinase, referred to as oxidative phosphorylation.

As the majority of ATP is routinely hydrolyzed to form ADP and Pi, then re-

phosphorylated, and is not generally synthesized anew, the total amount of ATP and ADP

remains fairly constant at any given time. However, at any given moment, a cell may require

more available ATP than ADP in response to certain intracellular and extracellular conditions. In

these situations, PKs help maintain energy homeostasis within cells by buffering the

concentration of stored ATP, (Ratto, Shapiro, and Christen 1989; Uda et al. 2006; Newsholme et

al. 1978). This can include spatial buffering; the stabilization of ATP levels throughout a cell, as

well as temporal buffering; the stabilization of ATP levels in response to fluctuations in ATP

usage within a cell. PKs are able to maintain spatial ATP buffering as the phosphorylated

guanidino-containing molecules are smaller than ATP, and can thus diffuse more quickly

through a cell to where ATP is being utilized (Ellington 2001). These phosphorylated enzymes

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can then transfer their phosphates to the consumed ADP, thus generating more ATP to satisfy the

need. Additionally, the temporal buffering of ATP in a cell is sustained as these enzymes can

rapidly produce more of this high-energy molecule in times when there is an increased cellular

demand for energy, relative to ATP/ADP systems that lack these proteins to catalyze the

phosphate transfer (Ellington 2001). In these ways, PKs can support a cell’s rapidly changing

regional or comprehensive ATP demands.

Given their significance in sustaining energy homeostasis within cells, it is not surprising

that phosphagen kinases are conserved across a wide array of organisms - such as animals, some

protozoa, and a few species of bacteria. The two most well-studied phosphagen kinases are

arginine kinase (AK) and creatine kinase (CK), but other PKs include glycocyamine (GK),

lombricine (LK), taurocyamine (TK), hypotaurocyamine (HTK), opheline (OK), and

thalassemine (ThK) kinases. As seen in Figure 2, and supported by Ellington (2001) and Uda, et

al. (2006), these PKs can be phylogenetically clustered into two main evolutionary families: the

AKs and CKs. The AK lineage is thought to consist of HTK and AK only; the CK lineage

consists of CK, GK, LK, and TK kinases. AKs appear to be largely coded for expression in the

cytoplasm of cells, whereas CKs comprise flagellar, mitochondrial, and cytoplasmic genes that,

when transcribed, are targeted to diverse cellular components (M. Suzuki et al. 2004). Though

there are cases in which PKs from both of these distinctive groups have been found in the same

organism (Ratto, Shapiro, and Christen 1989), creatine kinases typically occur in vertebrates and

are rarely observed in invertebrates, whereas arginine kinases so far have only been observed in

invertebrates, protozoa, and bacteria (Ratto, Shapiro, and Christen 1989; T. Suzuki and

Furukohri 1994; Andrews et al. 2008; Uda et al. 2010; Bragg et al. 2012).

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Figure 2: Phylogenetic lineage of phosphagen kinases determined by Neighbor Joining (NJ) method. Sequence

data used in tree assembly were from DNA databases at the time the figure was created (adapted from Vial, 2006).

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General Information about Arginine Kinase Lineage

While the physical and enzymatic properties of CK have been extensively studied, there

is still much to learn about the evolution, function, and differentiation of AKs. As opposed to

CKs, AK genes vary more in the number and organization of introns and exons, and as multiple

duplication/fission events of the gene have occurred throughout evolution, there also exists 2-

domain and dimeric AKs (Uda et al. 2006). This variation in AK genomic sequences and

structures, as well as their extensive distribution across phylogenetic branches (Uda et al. 2006;

Andrews et al. 2008; Bragg et al. 2012) suggest an ancient origin of these proteins. The relative

lack of genetic conservation in the coding of AKs is in sharp contrast to the maintenance of the

more evolutionarily recent CK gene in vertebrates, and its sequence variability may be a factor

contributing to the considerable amount of knowledge yet to be discovered about specific

activities of the AK family.

Notwithstanding the differences in the coding sequences for AKs, comparisons between

the gene for this enzyme from Limulus polyphemus and other organisms has identified 12 highly

conserved residues of the molecule that are presumed necessary for AKs’ function (Uda et al.

2006; 2010; Zhou et al. 1998). All known AK enzymes specifically catalyze the reversible

transfer of a phosphate group from MgATP to arginine and from phosphoarginine back to

MgADP ( ), similarly represented

in Figure 1). In a monomeric AK protein, these 12 residues include seven that interact with the

arginine substrate and five that recognize the ADP substrate. In conjunction with the crystal

structure of the L. polyphemus AK, analysis of sequence alignments has also suggested that a

loop arrangement in the molecule may be an additional factor in governing the enzyme’s

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substrate specificity (Zhou et al. 1998). Via the phosphate exchange and interactions at each end

of the protein, the AK loop is stimulated to make the conformational changes required for the

enzyme’s activity. The explicit maintenance of the 12 residues central to substrate binding in

addition to the flexibility of this molecular feature could be the reason for the conservation of

AK function despite the general sequence and structural variability of the gene across

phylogenetic branches.

Bacterial Arginine Kinases

Bacterial Acquisition of AK Feasible via Horizontal Gene Transfer

Though PKs are widely distributed and conserved among metazoan and some protozoan

organisms, they are rarely found in bacteria. Nonetheless, a recent study by Andrews et al.

(2008) was the first to describe a bacterial AK in Desulfotalea psychrophila and subsequent

Blast searches revealed several other purported bacterial AK gene homologs amongst organisms

such as Sulfurovum sp. NBC37-1, Moritella sp. PE36, and Myxococcus xanthus. Interestingly,

the sequences of these putative genes are not as similar among themselves as they are to

divergent eukaryotic AKs, as represented in neighbor-joining and maximum likelihood

phylogenetic trees such as in Figure 3, (Andrews et al. 2008; Bragg et al. 2012). Rather than this

event occurring as a result of wide-spread loss-of-function mutations across a broad spectrum of

organisms, it is more probable the nonlinear relationship of AK homologs among distally related

organisms occurred as a result of horizontal gene transfer (HGT). This concept is not a novel

assumption, as the reputed ability of AKs to be acquired by HGT is supported in other research

characterizing the novel occurrence of this enzyme in invertebrates (Pereira et al. 2000; Uda et

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al. 2006). In fact, operational genes for “housekeeping” molecules such as AK are believed to be

transferred by this mechanism far more frequently than informational genes, particularly in

archaea and nonpathogenic bacteria (Jain, Rivera, and Lake 1999; Garcia-Vallvé, Romeu, and

Palau 2000).

Figure 3: The phylogenetic relationships of selected phosphagen kinases determined by maximum likelihood

analysis. This illustration highlights the relationships between identified bacterial PKs and those of various

eukaryotic species, with an emphasis on protozoan species. Microbial species are shown in red, and protozoan

species are shown in blue. Only clades with bootstrap values of >50% are shown. The sequences used

(GenBank accession numbers) can be viewed in original source: Bragg, et al., 2012.

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Fitness Advantage of Organisms with Novel Arginine Kinase Function

As PKs improve cellular ATP buffering and energy homeostasis through the catalyzed

transfer of phosphates to or from ATP, either storing energy in the formation of a phosphagen

bond or releasing it in the cleavage of the bond (Sauer and Schlattner 2004), they are accordingly

observed in cells that experience large energy demands or fluxes. Cells with these systems can

thus drive greater endergonic processes with more speed and efficiency without exhausting their

ATP reserves. Therefore, it stands to reason that in cells that lack PKs, cellular ATP energy

stores should deteriorate when ATP energy production is impaired, such as during transient

stress conditions, because ATP synthesis cannot match the increased rate of utilization. It is not

surprising then, why PK functionality has been conserved throughout evolution.

However, as not all organisms encode and/or express PKs, or more specifically AKs,

studies such as those done by Canonaco et al., (2003; 2002), have been performed to determine

whether AKs provide an appreciable selective advantage when expressed as novel proteins in

organisms. Several experiments revealed that the addition of AKs to heterologous systems

resulted in improved recovery and tolerance of stress in Saccharomyces cerevisiae, and

Escherichia coli as opposed to strains that did not contain this enzyme (Canonaco et al. 2002;

2003). The 2002 study found that AK-expressing yeast were able maintain around 10% more

biomass during temporary low nutrient stress (Canonaco et al. 2002). In confirming that AK

activity supported the greater biomass concentrations of the transformed strain during the periods

of starvation, the researchers also observed the intracellular ATP and ADP levels of the two

cultures. The temporal buffering function of AK was found to enable AK-expressing yeast to

sustain stable ATP concentrations, whereas ATP concentrations of the wild type dropped

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significantly (approx. 50%) during that time. Conversely, ADP levels remained relatively

consistent in both strains throughout all of the conditions.

Additionally, though AK expression neither significantly improved intracellular ATP

levels nor growth rates during transient pH stress in transformed E. coli strains as compared to

the wild-type, the post-stress growth rate of the AK-expressing differed considerably (Canonaco

et al. 2003). The transformed bacteria grew 131% faster than the wild type controls after

temporary exposure to low pH, and similar results were observed for the post-stress growth rates

in transformed yeast (Canonaco et al. 2002). The outcomes from these and the other related

studies therefore recognize that the addition of a functional AK system to a genome instantly

imparts a selective advantage to the species in the event of recovery from a transient stress.

Function of Arginine Kinase in Myxococcus xanthus

Effect of AK deletion on M. xanthus Stress Response

Given the functionality of a homologous AK gene characterized in D. psychrophila, the

strong sequence similarities Myxococcus xanthus shares with the Limulus polyphemus AK gene

(Andrews et al. 2008), and the selective advantage novel AK function conveys in organisms

(Canonaco et al. 2002; 2003; Sauer and Schlattner 2004), Bragg et al. (2012) investigated the

role of the putative gene in the M. xanthus bacteria. To do so, a knock-out strain for the AK gene

(ark) was created from M. xanthus DK1622. As previous studies found that novel AK expression

had protective effects against stressors such as salt and pH in yeast and E. coli (Canonaco et al.

2002; 2003), M. xanthus was subjected to analogous conditions (oxidative, osmotic, pH, and salt

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stress) to determine whether its AK expression afforded any selective advantage. When grown

in the presence of the ionic stressors NaCl (0.2M) or KCl (0.2M) in CTTYE, the ∆ark mutant

strain displayed a decreased doubling time when compared to the wild-type, though the strains

did not display any significant difference in doubling time when grown in the nonionic 0.2 M

sucrose solution. Additionally, the mutants recovered at a slower rate upon exposure to a more

acidic environment, comparable to results observed by Canonaco et al. that documented reduced

recovery time from pH stress in yeast and E. coli expressing AKs (2002; 2003). When studied in

the presence of transient H2O2, however, no significant differences between the survivability of

the M. xanthus wild-type and ∆ark mutant strains were observed. These results therefore

revealed that, in the presence of certain non-starvation stressors (salt and pH), AK function is

beneficial for the doubling time and rate of recovery of the M. xanthus bacteria (Bragg et al.

2012).

AK deletion Affects M. xanthus Development

While effects of non-starvation stressors on M. xanthus were not unexpected, the

observation that the AK gene is necessary for normal development of the bacteria in the event of

nutrient depletion is surprising and warrants further research (Bragg et al. 2012). Additional

investigation of this finding is necessary in part because the effect of non-starvation stressors on

multicellular group dynamics was not specifically addressed in the studies by Canonaco et al.,

(2002; 2003). For instance, a single cell of this gram-negative soil bacteria typically glides only

when in direct contact with another, resulting in swarms or "wolf-packs" of M. xanthus. A swarm

of M. xanthus reaching up to several inches wide, contains millions of individuals that

communicate among themselves in a non-centralized fashion in response to environmental cues,

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such as stress conditions, (Zusman et al. 2007; MacNeil, Mouzeyan, and Hartzell 1994). Indeed,

even the bacteria’s response to starvation conditions is collective in nature; a pack undergoes

colonial transformation involving the alteration of multiple biosynthetic processes and

morphology of specific cells, dependent on the position of those individuals in the bacterial

cluster. Thus, while elimination of AK expression from M. xanthus would principally affect the

cellular activities of an individual, the collective behavior of this bacterial may also experience

changes related to this deletion.

This process in which members of the myxobacteria family cluster and individually alter

multiple biosynthetic processes as a result of starvation conditions is termed “development”.

When nutrients are plentiful, members M. xanthus form small colonies of aligned cells a few

cell-layers thick and individuals independently hunt for food, i.e. other bacteria (Sliusarenko,

Zusman, and Oster 2007; Igoshin, Kaiser, and Oster 2004). This behavior is advantageous to

members of the group, as it increases the concentration of extracellular digestive enzymes

secreted by the bacteria, thus facilitating predatory feeding. Conversely, when threatened with

nutrient restriction, groups of roughly 109 individual rod-shaped cells cooperatively mobilize

and aggregate over the course of several hours to form multicellular fruiting bodies and sporulate

for survival, (Tzeng, Ellis, and Singer 2006; Kaiser and Welch 2004; Sliusarenko, Zusman, and

Oster 2007; Igoshin, Kaiser, and Oster 2004). The cells on the interior of the fruiting body

undergo changes in protein synthases as well as morphological alterations in their cell walls to

differentiate into spherical, thick-walled spores, thus inciting the morphological changes of the

entire fruiting body (Kuspa, Kroos, and Kaiser 1986; Hagen, Bretscher, and Kaiser 1978a;

Zusman et al. 2007). The morphological changes that occur specifically during M. xanthus

development are displayed in Figure 4, (Kaiser, 2003).

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Figure 4: The developmental cycle of Myxococcus xanthus under normal growth conditions and when nutritionally

stressed. The figure can be viewed in its original source: Kaiser, 2003.

The various biosynthetic processes triggered during M. xanthus development are initiated

by different regulators, including ATP and its precursors (ADP and Pi), the cellular

concentrations of which AKs influences. This enzyme could therefore ultimately affect the

regulation of genes and other molecules crucial to this process and spore formation, such as

motility (Kaiser and Welch 2004; Sliusarenko, Zusman, and Oster 2007). Genetic manipulations

have identified more than 40 loci involved in individual gliding of M. xanthus cells and reveal

the existence of two independent motility systems controlling separate behavioral phenotypes

(MacNeil, Mouzeyan, and Hartzell 1994). These differ between social (S) and adventurous (A)

motility. S motility refers to the actions of a group of cells, whereas A motility refers to the

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movement of isolated cells. (A third group of genes have been found to be involved in M.

xanthus motility in addition to the A and S motility systems, though these are more involved in

controlling a specific feature of gliding motility (Blackhart and Zusman 1985)). If any of these

genetic regions are influenced by signaling pathways regulated by AKs, similar to other kinases

that play a role in signal amplification and transduction, then its expression in M. xanthus could

very well impact the cooperative behavior among colony members in addition to the overall

growth and recovery of these colonies. However, this is highly speculative as there are currently

no known signaling pathways regulated by AKs.

Intention of this Study

The results observed by Bragg et al., in which the M. xanthus Δark mutant was unable to

form fruiting bodies under starvation conditions (2012), led the researchers to suggested two

possible hypotheses. The work proposed the knock-out mutants either were unable to store

enough ATP and therefore easily-available energy was limited under more stringent starvation

conditions, or that the phosphoarginine normally encoded for by the gene is also uniquely acting

as a signal molecule and the development defects were due to a mis-regulation of this AK-

dependent pathway in its absence (2012). To further the exploration of the role of AK, I

proposed to transform M. xanthus Δark with the original M. xanthus ark gene (MyxoAK), both

with and without a His-tag, as well as with the horseshoe crab AK homolog (HcAK), both with

and without a His-tag. Knocking-in MyxoAK in place of Δark should rescue WT responses to

non-starvation and starvation stress, whereas the results of instead inserting HcAK are unknown.

Future works may then attempt to further research the conditions of the hypotheses proposed by

Bragg et. al.

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MATERIALS AND METHODS

Strains and Growth Conditions

M. xanthus DK1622 was used as the wild-type strain and M. xanthus MS2252 (Bragg et

al. 2012), a derivative of this strain with an in-frame deletion of the ark gene (MXAN2252) with

the first and last 6 codons remaining, was used as the parent strain for all subsequent

recombinant strains. Both strains were grown at 32°C in CTTYE broth (1.0% Casitone, 10 mM

Tris-HCl [pH 7.6], 1 mM KH2PO4, 8 mM MgSO4, 0.5% yeast extract) with vigorous aeration

(250rpm) or on CTTYE plates containing 1.5% agar.

Additionally, standard growth curves of the M. xanthus wild-type (WT) and in-frame ark

deletion (Δark) strains were observed. Both strains were grown at 32°C in CTTYE broth with

vigorous aeration (250rpm) for two separate 48 hr trials. Klett readings were recorded every 3

hrs throughout the first 12 hrs, then every 6 hrs the following 36 hrs. M. xanthus WT and Δark

cultures were also streaked onto CTTYE hard agar (CTTYE HA) plates as to ensure the

cultureswere free of contamination at the time of the readings.

Midiprep DNA Purification of the Four Plasmids

Previously, Gentry Kerwood and Scott Bingman (Class of 2015, The College of Wooster,

Wooster Ohio) designed four pMTP vectors containing the AK gene analogs MyxoAK,

MyxoAK-His, HcAK and HcAK-His (hereafter these names will be used to refer to each pMTP

vector with these genes). Each vector was transformed into separate DH5α E. coli colonies,

following the Addgene Bacterial Transformation protocol (Addgene. Cambridge, MA, USA).

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The chemically competent cells transformed with the plasmids were plated onto Luria-Bertani

(LB) plates supplemented with kanamycin (LB broth, 1% agar, 40 µg/ml kanamycin). Colonies

were then selected and grown up for plasmid DNA purification using Qiagen Midiprep Kit

(Qiagen. Valencia, CA, USA). DNA concentrations of the plasmids were measured as well as

subjected to diagnostic restriction digest and analyzed by gel electrophoresis (83-120V). The

digest was conducted using enzymes EcoRI, HindiIII, and NdeI, with restriction sites at 872 bp,

923 bp, and 2038 bp in pMTP, respectively. Each vector type was exposed to separate mixtures

of all three possible combinations of two of the restriction digest enzymes in unison.

Transformations of Deletion Strain with Various Plasmids

Transformations of the M. xanthus ark gene deletion strain (MS2252) with any of the

MyxoAK, MyxoAK-His, HcAK and HcAK-His pMTP vectors followed a previously described

method of galK positive/negative selection described by Shi et al. (2008). Though, instead of

constructing a knock-out strain, cultures of the pre-existing MS2252 knock-out strain was

transformed with one of the four described plasmids to construct a “knock-in” strain.

Procedure for transforming Myxococcus

DNA from each purified plasmid was introduced into MXAN2252 by electroporation as

similarly described by the Myxococcus Electroporation Protocol accessible on the laboratory

website of Dr. Mitch Singer in association with University of California’s Department of

Microbiology and Molecular Genetics (http://microbiology.ucdavis.edu/singer/). As such, Δark

cultures were grown in 25 mL CTTYE media to 80-120 Klett units, at 32ºC and 250 rpm. Cells

were then pelleted at 8 Krpm for 8 min at 15ºC in 30 mL centrifuge tubes. After discarding the

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supernatant, pellets were re-suspend in 1.5 mL distilled water (dH2O) and transferred to 2 mL

centrifuge tubes. The resuspensions were pelleted again for 3 min at 8 Krpm, and, again after

discarding the supernatant, washed with 1.5 mL dH2O. The pelleting and wash was repeated

three more times, with a final resuspension in 1.5 mL dH2O. Each resuspension of Δark culture

was individually mixed with 11-20 µL (~1-3 µg) of DNA from one of the plasmids in a

centrifuge tube and transferred to a 0.1cm cuvette. Electroporation of an individual sample

occurred at room temperature with 400 W, 25 mFD, and 0.65 kV, (ideal time constant values at

9.0 or higher). Immediately following electroporation, 1 mL of CTTYE media was added to the

cuvette, then the contents were transferred by pipette to a 125 mL Erlenmeyer flask containing

1.5 mL of CTTYE media. The cultures were grown for 6 hours at 32ºC and 250 rpm to allow for

phenotypic lag. After transferring the contents of the flask to 30 mL centrifuge tubes, 5 mL of

cool-to-touch molten CTTYE soft agar supplemented with kanamycin (1.0% Casitone, 10 mM

Tris-HCl [pH 7.6], 1 mM KH2PO4, 8 mM MgSO4, 0.5% yeast extract, 1.% agar, 40 µg/ml

kanamycin) was slowly added to each sample and briefly Vortexed to mix. The mixtures were

then promptly poured onto CTTYE hard agar plates supplemented with 40% kanamycin

(CTTYE HA+ Kan), cautious to minimize bubbles. M. xanthus Δark transformants were grown

for 6-9 days at 33ºC and resulting colonies were re-streaked onto CTTYE HA+ Kan plates.

GalK Counter-Selection of Transformants

Whereas transformation of the vectors into the M. xanthus Δark strain was confirmed

with kanamycin resistance, homologous recombination of the AK homologs with the deletion

region (effectively replacing Δark with a vector’s AK homolog while removing the remainder of

the inserted plasmid) was verified through galK counter-selection. As the pBJ114 vector cannot

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replicate in M. xanthus but can be accepted for its kanamycin resistance, the transformed

bacterium could possess a recombination plasmid insertion likely up- or downstream of the

target region of DNA. The galK (E. coli galactokinase) gene counter-selectable marker is also

present in the pBJ114 plasmid and is crucial to select for the recombination of the target gene

carried by the plasmid with the homologous promoter and terminator regions in the bacteria’s

DNA. When expressed, the galK gene converts galactose into its phosphorylated form, galactose

phosphate. Yet because M. xanthus cannot metabolize this compound, it accumulates to toxic

levels when the cells are grown in galactose-containing media. Only cells that have undergone a

second recombination to excise the plasmid then are viable. Therefore, this two-step selection

procedure described by Shi et al. (2008) allows M. xanthus DNA to be modified without

introducing unwanted plasmid fragments at the modification site.

To isolate clones containing the in-frame insertion, colonies of the re-streaked M. xanthus

Δark + plasmid transformants confirmed by kanamycin resistance were first grown under normal

growth conditions in 25 mL CTTYE broth until they had grown to 40-80 Klett units. Aliquots of

200mL of each culture were then transferred sterile 10 mL glass tubes and 3mL of molten

CTTYE soft agar (CTTYE SA) containing 2.5% D-galactose was slowly added to each sample

and briefly Vortexed. The mixtures were then promptly poured on CTTYE +2.5% D-galactose

(CTTYE HA+ Gal) plates, careful to minimize bubbles. Positive colonies developed after 3-5

days. As some plates were more densely populated with GalR colonies than others, unreliable

“distinct” colonies were steaked. Discernibly single colonies were streaked firstly onto CTTYE

HA+ Gal plates, then immediately onto CTTYE HA+ Kan plates. Galactose-resistant (GalR) and

kanamycin-sensitive (KanS) M. xanthus Δark + plasmid colonies were identified accordingly.

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*Some GalR/KanS colonies were also obtained by simply streaking the cultures onto

CTTYE HA+ Gal plates, although individual colonies were more difficult to distinguish.

Glycerol Preservation of Strains

Glycerol stocks of the M. xanthus DK1622 and MS2252 strains used in this study as well

as each of the KanR cultures and all and KanS M. xanthus Δark + plasmid cultures were made as

similarly described by the Addgene protocol for Creating Bacterial Glycerol Stocks for Long-

term Storage of Plasmids (Addgene. Cambridge, MA, USA). However, cultures were grown 39-

42 hrs rather than overnight, and 0.5 mL of 60% glycerol was used instead of 0.5 mL of 50%

glycerol. Additionally, the cultures from which the aliquots were obtained for the glycerol stocks

were streaked onto CTTYE, CTTYE+ Kan, and CTTYE+ Gal plates as necessary to ensure the

preserved cultures were free of contamination.

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RESULTS

Strains and Growth Conditions

Standard growth curves of the M. xanthus WT and Δark strains were observed for two

cultures of each strain during two separate 48 hr growth trials in CTTYE media incubated at

32°C with vigorous aeration (250 rpm). Klett readings were recorded every 3 hrs throughout the

first 12 hrs of observation, then every 6 hrs the remaining 36 hrs. As recorded in Tables 1 and 2,

both WT and Δark M. xanthus strains grew to mid-exponential phase (80-120 Klett units) 36 to

42 hrs after cultures were started. Figures 5 was also included as plotted example of the general

growth curves observed for the four cultures each of the WT and Δark strains to display the

similarities in their growth rates.

Table 1: First 48 hr Growth Curve Trail of Two Cultures Each of M. xanthus WT and Δark Strains

WT5.1 WT5.2 ΔAK1.1 ΔAK1.2Hours Kletts Hours Kletts Hours Kletts Hours Kletts

0 0.00 0 0.00 0 0.00 0 0.003 3.00 3 2.00 3 3.00 3 2.006 4.50 6 4.00 6 5.50 6 4.009 5.50 9 5.75 9 6.00 9 5.75

12 6.75 12 7.50 12 8.00 12 7.5018 8.50 18 11.25 18 13.50 18 11.2524 13.00 24 16.00 24 19.00 24 15.0030 28.00 30 37.00 30 38.00 30 27.0036 63.00 36 69.00 36 72.00 36 62.0042 125.00 42 130.00 42 135.00 42 123.0048 182.00 48 184.00 48 201.00 48 179.00

23

Table 2: Second 48 hr Growth Curve Trail of Two Cultures Each of M. xanthus WT and Δark Strains

WT5.1 WT5.2 ΔAK1.1 ΔAK1.2Hours Kletts Hours Kletts Hours Kletts Hours Kletts

0 0.00 0 0.00 0 0.00 0 0.003 3.50 3 2.50 3 3.00 3 3.506 4.50 6 3.00 6 4.50 6 4.509 7.50 9 5.00 9 8.00 9 7.00

12 13.00 12 11.50 12 14.00 12 12.5018 17.50 18 15.25 18 17.50 18 17.7524 22.00 24 19.00 24 21.00 24 23.0030 43.00 30 34.00 30 40.00 30 45.0036 78.00 36 69.00 36 75.00 36 81.0042 142.00 42 132.00 42 136.00 42 145.0048 208.00 48 189.00 48 199.00 48 211.00

0 6 12 18 24 30 36 42 480.00

25.00

50.00

75.00

100.00

125.00

150.00

175.00

200.00

Growth Curve of WT5.1 and ΔAK1.2 in Trial 1

WT5.2Exponential (WT5.2)ΔAK1.2Exponential (ΔAK1.2)Exponential (ΔAK1.2)

Time (Hours)

Klett

Uni

ts

Figure 5: Growth curve of a culture of M. xanthus WT and Δark strains recorded in the first 48 hour trial period, incubated at 32°C with vigorous aeration (250 rpm) and measured in Klett units. Used as an example to display the typical growth curves observed for the four M. xanthus WT and Δark cultures each.

24

Doubling time for each culture of M. xanthus WT and Δark strains was also calculated

for the mid-exponential phase (80-120 Klett units) of growth. Doubling or generation time is

used to express the rate of exponential growth of a bacterial culture, defined as the time (t) per

generation (n = number of generations). The calculated generation times of the cultures during

mid-exponential phase (80-120 Klett units) occurring 36 to 42 hrs are presented in Table 3.

Table 3: Doubling Times (Dt) of Each of the M. xanthus WT and Δark Growth Curve Cultures

Trial 1 Doubling time (hours) Trial 2 Doubling time (hours)WT5.1 6.070 WT5.1 6.942WT5.2 6.566 WT5.2 6.411ΔAK1.1

6.616 ΔAK1.1 6.988

ΔAK1.2

6.071 ΔAK1.2 7.142

*No significant difference between doubling times of M. xanthus WT and Δark strains was found

(P= 0.38979), consistent with the findings of Bragg et al. when these strains were not exposed to pH,

salt, or starvation stress (2012).

Midiprep DNA Purification of the Four Plasmids

Midiprep purification for each of the four pMTP vectors containing the AK gene analogs

MyxoAK, MyxoAK-His, HcAK and HcAK-His was performed until an ample concentration of

DNA from each of the vectors was attained. The greatest DNA concentrations acquired for each

of the vectors from a single Midiprep procedure go as follows; [MyxoAK] = 424.5 ng/µl,

[MyxoAK-His] = 152.4 ng/µl, [HcAK] = 578.3ng/µl, and [HcAK-His] = 101.4 ng/µl.

Interestingly, Midiprep purifications of the pMTP vectors containing the AK gene analogs with

the His-tags repeatedly yielded lower DNA concentrations than those of their counterparts

without His-tags. Samples from each purified Midiprep DNA product were then analyzed using

25

gel electrophoresis, after first applying a diagnostic restriction digest. As the length of the pMTP

vector itself is approximately 5 Kb and the ark gene without His-tag is approximately 1.1 Kb, the

total uncut base pair length of the vectors should be circa 6.1 Kb. Separate mixtures of all three

possible combinations for two of the restriction digest enzymes in unison (EcoRI, HindiIII, and

NdeI, with restriction sites in pMTP at 872 bp, 923 bp, and 2038 bp, respectively) produced

bands at the anticipated positions. The combination of EcoRI and HindiIII generated bands of

approximately 51 bp, HindiIII, and NdeI had bands 1115, and NdeI and EcoRI 1166 base pairs.

Transformations of Deletion Strain with Various Plasmids

Initial transformations attempts of M. xanthus Δark with the plasmids using the M.

xanthus Electroporation Protocol available on Dr. Mitch Singer’s laboratory website were

unsuccessful. The procedure indicated cultures could, after electroporation, be grown 4-8 hours

until phenotypic lag was reached or incubated overnight. Consequently, several transformation

attempts were made allowing the cells to recover for 8, 12, and 16 hrs, the assumption being that

as more time was allowed for growth, the possibility positive colonies of M. xanthus Δark

transformed with the plasmids would also increase. However, the resulting cultures were very

dense, sometimes to the point that the bacteria had begun to mass together in the flask, even with

agitation. The congregating of the cells in the cultures made the resultant CTTYE SA+ Kan

plating exceptionally irregular. Patches of cells from the liquid cultures as well as the bubbles

that were created when it and the CTTYE SA+ Kan were Vortexed made discerning any

resulting KanR transformant colonies exceedingly difficult.

26

After expounding on the protocol available on Dr. Mitch Singer’s website, KanR

transformants of the M. xanthus ark gene deletion strain (MS2252) with one of the four pMTP

vectors were ultimately generated for all but the HcAK-His plasmid. In fact, the electroporation

of M. xanthus Δark cultures with the various plasmids yielded three separate colonies of

MyxoAK, three colonies of MyxoAK-His, and a single colony of HcAK. Each of the different

cultures cultivated from the three colonies of MyxoAK and MyxoAK-His were identified as A,

B, or C accordingly. These positive colonies resulted from allowing cultures recovering from

electroporation to grow for exactly 6 hrs. Both the cultures and the CTTYE SA+ Kan plating of

these cultures were free of cellular aggregation and individual positive colonies could be easily

1identified on the CTTYE SA/HA+ Kan plates.

GalK Counter-Selection of Transformants

A two-step selection procedure was used to knock-in the AK homologs carried by the

pBJ114 vectors into M. xanthus Δark in place of the deletion region. Plasmid recombination into

the genome was confirmed by kanamycin resistance, as the vectors are not maintainable as free

plasmids in this bacterium and must be incorporated into a cell’s DNA to impart this selective

advantage. Homologous recombination of the plasmid out of the genome was then verified

through galK counter-selection. Upon multiple CTTYE SA+ Gal and culture plating trials, 16 of

the 28 CTTYE SA/HA+ Gal plates resulted in positive GalR colonies. Of these plates, five

originated from the MS2252+ MyxoAK transformants, seven were from MS2252+ MyxoAK-

His transformants, and four arose from MS2252+ HcAK transformants. Examples of the cultures

plated on the CTTYE SA/HA+ Gal plates can be seen in Figure 6 parts A and B. From these

27

cultures, 32 colonies were identified (10 MS2252+ MyxoAK, 10 MS2252+ MyxoAK-His, and

12 MS2252+ HcAK) and re-streaked first onto CTTYE HA+ Gal plates, then immediately onto

CTTYE HA+ Kan plates. Colonies that underwent homologous recombination with the vectors

could then be identified as GalR and KanS.if they were able to proliferate on the CTTYE HA+

Gal plates and not the CTTYE HA+ Kan plates. Successful results of this second selection are

represented in Figures 7 and 8, and cases of the 12 ambiguous results that appeared to be both

galactose and kanamycin resistant are presented in Figure 9.

Glycerol Preservation of Strains

All glycerol stocks created and kept at -80ºC were verified as free of contamination and

competent before final long-term storage. Three aliquots of M. xanthus DK1622 and five of

MS2252 strains used in this study were preserved. At least two glycerol stocks of each of the

three subsets of MyxoAK and MyxoAK+His KanR transformants and the HcAK transformant

were stored Lastly, six GalR and kanamycin-sensitive (KanS) M. xanthus Δark + plasmid cultures

that were free of post-plating contamination were suspended in glycerol as well.

28

BA

DC

Figure 6A: Examples of the GalR M. xanthus Δark, transformants with the pBJ114 vectors on CTTYE HA/SA +Gal. A) Culture streaked directly from CTTYE media, individual colonies relatively difficult to distinguish; B) Culture plated with only 3 mL of CTTYE SA +Gal SA, distinct colonies rather challenging to recognize; C) Large, singular colonies of the transformant; D) Numerous colonies with some joining and marginally challenging to differentiate.

29

Figure 6B: Continued examples of the GalR M. xanthus Δark, transformants with the pBJ114 vectors on CTTYE HA/SA +Gal. E) Countless colonies, fairly challenging to discern individuals; F) Again, many single colonies moderately difficult to discriminate; G) Culture was contaminated prior to plating, resulting in many contaminant colonies and very few M. xanthus Δark, transformant colonies; H) Discrete singular colonies of the transformant

HG

FE

30

Figure 7: Illustrations of M. xanthus transformant colonies that were identified as GalR and KanS when streaked first onto CTTYE HA+ Gal plates (identified on top with a green circle), then immediately onto CTTYE HA+ Kan plates (identified on bottom with a red circle). Of the samples plated in column A), only the colony plated on side 2 appears to have undergone homologous recombination with a vector. Of the samples plated in column B), only the colony plated on side 1 appears to have undergone homologous recombination with a vector (some contamination arose on the second plate, side 1).

A

1

B

21

1 21

31

Figure 8: Illustrations of M. xanthus transformant colonies that were identified as GalR and KanS when streaked first onto CTTYE HA+ Gal plates (identified on top with a green circle), then immediately onto CTTYE HA+ Kan plates (identified on bottom with a red circle). Both colonies on the plates in column A) appear to have undergone homologous recombination with a vector (some contamination arose on the second plate, side 2). Both colonies on the plates in column B), also appear to have undergone homologous recombination with a vector.

A B

1

11

32

Figure 9: Illustrations of M. xanthus transformant colonies that were ambiguous as to if they were truly GalR and KanS when streaked first onto CTTYE HA+ Gal plates (identified on top with a green circle), then immediately onto CTTYE HA+ Kan plates (identified on bottom with a red circle). Both colonies on either of the plates in column A) and B) appear to have equal resistance to galactose as well as kanamycin.

A

11

B

1

33

DISCUSSION

As recent studies have characterized novel arginine kinase (AK) genes in bacteria

(Andrews et al. 2008; Bragg et al. 2012; T. Suzuki et al. 2013), reverse genetics techniques have

been utilized to better elucidate the role these genes play in a bacterial system. As previously

described, earlier research has revealed that establishing novel AK systems can impart selective

advantages in the organisms, including the enhanced ability to handle non-starvation stress

conditions, (Canonaco et al. 2002; 2003). The consequences of knocking out existing AK

expression in M. xanthus was equally observed to decrease tolerance of non-starvation stress

(Bragg et al. 2012). Moreover, this study discovered the absence of the AK gene resulted in an

unexpected developmental phenotype. While the effects from non-starvation stressors were not

unanticipated in this deletion event, the observation that the AK gene was required for normal

development of the bacterium during times of decreased nutrient availability was unforeseen and

prompted the researchers to advocate for further research on this finding. Bragg et al. (2012)

speculated that the function of the M. xanthus ark gene may either be enabling the cells to store

reserves of high-energy phosphoarginine that can be quickly converted to ATP for sudden high

energy demands, or it may be that phosphoarginine has a novel signaling function that influences

other proteins or gene expression in the bacterium.

This research intended to further the exploration of the role of AK in the bacterium and

provide data relating to one of the proposed hypotheses by knocking-in the original M. xanthus

ark gene (MyxoAK), both with and without a His-tag into the Δark mutant by homologous

recombination and assess the bacteria’s responses to non-starvation and starvation stress. These

34

phenotypic assays would be implemented alongside identical experiments with the wild-type

strain (DK1622), the M. xanthus Δark strain, and M. xanthus Δark mutant strains transformed

through homologous recombination with the horseshoe crab AK homolog (HcAK), both with

and without a His-tag. MyxoAK knocked-in in place of Δark should rescue WT responses to

non-starvation and starvation stress, whereas the results of instead inserting HcAK are unknown.

Performing the experiments simultaneously would allow the effect the original gene conveys to

the bacterium to be assessed in comparison to a novel, homologous AK gene. If the integration

of either the ark gene or the horseshoe crab AK homolog can rescue both non-starvation and

starvation stress responses in the bacteria, it may be determined that the M. xanthus AK gene

simply catalyzes the reversible transfer of a phosphate group between adenosine triphosphate

(ATP) and a guanidino molecule as do other PKs. If, however, only the re-integration of the ark

gene rescues both non-starvation and starvation stress responses in the bacteria, it would suggest

that the gene has developed a novel function in M. xanthus. Additionally, if the presence of the

His-tag on the MyxoAK and HcAK homologs has no significant effect on development or

starvation and non-starvation stress, it may be possible to purify the proteins from the bacterium

via immunoprecipitation and identify the substrate binding partners of these homologous

molecules. Future works could then analyze these enzymes in vitro to determine if their

enzymatic activities differ and potentially challenge the hypothesis that the M. xanthus AK gene

solely functions as a PK.

However, this anticipated stage of the study -testing the non-starvation and starvation

stress responses of the WT and knock-in constructs- was not reached due to several experimental

limitations. The first concern of this study involved establishing viable M. xanthus WT and Δark

35

cultures (DK1622 and MS2252, respectively), on CTTYE HA plates from glycerol stocks that

had been preserved for over six years at the time this research commenced. Initial plating of

these cultures took 6-9 days of incubation at 32°C to display signs of growth, whereas plating of

later M. xanthus cultures exhibited signs of growth only after 3 days of incubation. This slow

recovery from -80°C may be due to any prior improper handling of the glycerol stocks, such as

allowing them to thaw completely or thawing and re-freezing the solutions too many times.

Glycerol is commonly used to prevent freezing damage such as ice crystal formation in the event

cells are frozen, though the AddGene protocol for Creating Bacterial Glycerol Stocks (Addgene.

Cambridge, MA, USA) repeatedly states that multiple thawing and re-freezing events will reduce

the effectiveness of this technique, thus diminishing the viability of the preserved bacteria.

Nevertheless, after the viable M. xanthus WT and Δark cultures were re-streaked or grown in

media, their doubling times normalized as shown in Tables 1-3 and Figures 5 and 6. These

growth rates mirror those found in Mason and Powelson’s work (D. J. Mason and Dorothy

Powelson) and indicate that both strains were growing as expected after their revival from

glycerol preservation.

Another limitation this study encountered was the use of an outdated and undescriptive

protocol for M. xanthus electroporation for the preliminary transformations attempts of M.

xanthus Δark with the plasmids. Direct correspondence with a member of Dr. Mitch Singer’s

laboratory group revealed that techniques described in the protocol on the website

(http://microbiology.ucdavis.edu/singer/) had been amended in practice, though the posted

procedure had not been revised (J. Liang, personal communication January 9, 2016).

Clarification of the available protocol revealed that each sample to be electroplated ought to be

36

mixed with 11-20 µL (~1-3 µg) of DNA from the desired plasmid for optimal transformation

results. Furthermore, the addition of CTTYE media should occur in direct succession of the act

of electroporation for each individual sample so that recovery can begin immediately after the

plasmid DNA is introduced into the bacteria. Lastly, it was discovered that the optimal time to

allow for recovery growth was observed to be six hours, rather than in the range of four to eight

hours or overnight. Upon implementing these suggestions for the Myxococcus Electroporation

Protocol, the plated cultures were remarkably free of cellular aggregation, permitting positive

KanR colonies to be easily identified. Transformants of the M. xanthus ark gene deletion strain

(MS2252) with one of the four pMTP vectors were ultimately generated for all but the HcAK-

His plasmid and confirmed upon re-plating KanR colonies from the CTTYE HA/SA+ Kan plates

onto CTTYE HA+ Kan plates.

Upon transformation, the M. xanthus Δark cultures could possess a recombination

plasmid insertion of the pBJ114 vector likely up- or downstream of the target region of DNA, as

exhibited in Figure 10. While this vector cannot replicate in the bacterium, it can be recombined

into the genome for its kanamycin resistance and thus can be used to confirm transformation.

Nonetheless, KanR colonies only confirmed that the plasmid had been accepted by M. xanthus

Δark, not that the deletion region was replaced by an AK homolog carried by the MyxoAK,

MyxoAK-His, or HcAK vectors. This homologous recombination was suggested then through

galK counter-selection. The galK (E. coli galactokinase) gene pBJ114 also contains converts

galactose into its phosphorylated form, galactose phosphate. As M. xanthus cannot metabolize

this compound, it accumulates to toxic levels when the cells are grown in galactose-containing

media and to survive these conditions, cells may effect a second recombination to excise the

37

plasmid fragment containing the galK gene. Surviving cells are then GalR and KanS, as the

kanamycin-resistance would similarly be lost upon excising the remaining pBJ114 vector. This

two-step selection procedure therefore allows M. xanthus DNA to be modified without

introducing unwanted plasmid fragments.

2A

2B

Figure 10: Illustration of first recombination event for M. xanthus Δark, transformation with pBJ114. 1) Displays the vector and the deletion region of the bacterium’s DNA with the possible recombination areas between the promotor and terminator expanses of both pBJ114 and M. xanthus Δark DNA. 2A) Recombination result as though plasmid insertion occurred downstream of Δark, and conversely 2B) is as though plasmid insertion occurred upstream of Δark.

**

**

**

38

To assess the galactose-resistance and kanamycin-sensitivity of the transformants,

colonies of each KanR transformant strain were first grown in CTTYE media, then separately

mixed with CTTYE SA+ Gal and plated onto CTTYE HA+ Gal plates. Growth in unselective

media prior to plating onto galactose-rich CTTYE agar was to increase the opportunity for

homologous recombination to take place. Had the KanR transformant strains been transferred

directly from CTTYE HA+ Kan plates, which selected for the inclusion of the pBJ114 vector in

M. xanthus, to the CTTYE HA+ Gal plates, which would select for the removal of the vector,

homologous recombination would have needed to occur instantaneously upon plating. Lone GalR

colonies that developed on the CTTYE HA/SA + Gal plates were first streaked onto CTTYE

HA+ Gal plates, then immediately onto CTTYE HA+ Kan plates. This method, outlined in

Figure 11, was used to evaluate the galactose-resistance and kanamycin-sensitivity of a specific

colony. If a colony was able to grow on CTTYE HA+ Gal plates but not CTTYE HA+ Kan

plates as seen in Figures 8 and 9, it was deemed GalR and KanS. As Figure 10 illustrates though,

not all cultures conveyed this pattern. Some transformed M. xanthus colonies appeared to be both

GalR and KanR. This outcome may have arisen if more than one colony had been isolated from

the original CTTYE HA/SA+ Gal plates and at least one of those colonies was GalR while at

least one other was KanR. Contamination that transpired in the process of plating might also have

given the appearance of M. xanthus growth on both CTTYE HA+ Gal and CTTYE HA+ Kan

plates. Additionally, mutation of the galK gene that would have disrupted its function may have

allowed colonies to forgo the second homologous recombination while still permitting them to

appear GalR.

39

Figure 11: Diagram of basic process used to isolate transformants having the in-frame insertion of a pBJ114 vector containing one of the AK homologs. M. xanthus Δark + plasmid transformants confirmed as KanR (first red box) were grown under normal growth conditions in CTTYE media (blue box) and checked for contamination on CTTYE HA and CTTYE HA +Kan plate (blue and red diagonal boxes). They were then transferred to CTTYE HA/SA+ Gal plates (first green box). Densely populated CTTYE HA/SA+ Gal plates were steaked (backwards arrow). Positive GalR colonies were then streaked firstly onto CTTYE HA+ Gal plates (second green box), then immediately onto CTTYE HA+ Kan plates (second red box).

Nevertheless, while noticeably GalR and KanS colonies can be identified with this double-

selection method, it does not ensure that the transformed bacteria will contain the desired AK

homologs in place of the ark deletion region. This is due to its expectation that the organism will

retain a non-essential AK homolog while the elimination of the plasmid fragment that introduced

that gene is encouraged. Statistically, around half of the M. xanthus mutants growing on the

selective galactose plates that appear to be GalR and KanS will have only lost the galK-containing

expanse of the insertion while retaining the new genomic region, and the remaining 50% will

have restored the original genomic situation upon excluding the transformed region. To confirm

that the GalR and KanS M. xanthus colonies obtained are indeed knock-in mutants with the AK

homologs in replace of Δark, DNA should be extracted and evaluated with PCR. The primers

40

that flanked the deletion region in the bacterium are the same as those used for the inserts in the

vectors and can be used to either amplify Δark or the AK homologs with this technique. The size

of the fragments could then be analyzed using gel electrophoresis to determine which colonies

only eliminated the portion of the plasmid fragment containing galK and maintained the AK

gene to grow on the CTTYE HA+ Gal plates, and which surviving cells became GalR and KanS

by rejecting the entirety of the plasmid insert (thus producing bands equivalent to the length of

Δark). Sequencing of the DNA samples may additionally be performed to exhibit homologous

recombination between the deletion region and the inserts was achieved.

In addition to ensuring that transformation and galK counter-selection of M. xanthus Δark

mutants was successful, future studies might continue this work by first constructing M. xanthus

Δark mutant strains with the HcAK-His insert and, secondly, assessing each of the

transformants’ responses to non-starvation and starvation stress, as compared to the wild-type

(DK1622) and M. xanthus Δark (MS2252) strains. The enactment of this proposition would

include duplicating the pH, salt, and osmotic non-starvation stress assays that the Bragg et. al.

paper described, as well as the starvation stress/development assays (2012). These are only a

few suggestions in which the exploration of the role of AK in M. xanthus may be continued and

it may be discovered whether the knock-out mutants either are simply unable to store enough

ATP as they lack the AK enzyme, or that the phosphoarginine normally encoded for by the gene

is now also uniquely acting as a signal molecule (2012). If the ark gene is suspected to be acting

as a signaling molecule, the substrate may be isolated along with the expressed MyxoAK+His

gene through protein purification methods.

41

ACKNOWLEDGEMENTS

Though this research process is identified as an ‘Independent Study’, it is in no way completed

alone and unaided. Friends encourage you during your progress and setbacks, peers lament with

you over the struggles of this academic exploration, and professors recognize your endeavors to

expand the information available on the topic you pursue. While there were numerous friends,

colleagues, and acquaintances that abetted me throughout the course of this research, I would

like to identify a few specific individuals who were particularly supportive and accommodating

with respect to the progress of my “Independent” Study.

Firstly, I must recognize my advisor for inspiring me to enhance my critical thinking skills and

develop my knowledge and expertise in the laboratory for the advancement my project.

Copeland Funding is also to thank for the progress of my study as it provided the funds that

made visiting Dr. Mitchel Singer and his Myxococcus-centered research laboratory possible.

Dr. Mitchel Singer was more than willing to open his laboratory to me and Jennifer Liang, a

member of his research group, was an immeasurable help in providing general information about

the bacterium itself, as well as recommendations for transformation and counter-selection.

Gilian Lee was an invaluable aid for the mechanical details of this thesis, as well as a prominent

source of emotional support and encouragement while completing this work.

Daniel Boyce served as an infinite basis of emotional support and encouragement as well, in

addition to participating in this work, assisting as a Lab Helper/Safety Buddy .

And finally, my parents deserve more gratitude than I can express. Without their constant,

unconditional support and encouragement throughout not just this research, but also my life, I

would never have become the person I am or achieved all that I have.

42

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