CrdA regulates endogenous beta-lactamase activity in Myxococcus xanthus

University of IowaIowa Research Online

Theses and Dissertations

Fall 2009

CrdA regulates endogenous beta-lactamase activityin Myxococcus xanthusDi LiUniversity of Iowa

Copyright 2009 Di Li

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Recommended CitationLi, Di. "CrdA regulates endogenous beta-lactamase activity in Myxococcus xanthus." MS (Master of Science) thesis, University ofIowa, 2009.https://doi.org/10.17077/etd.wq35fgd0

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CRDA REGULATES ENDOGENOUS BETA-LACTAMASE ACTIVITY IN

MYXOCOCCUS XANTHUS

by

Di Li

A thesis submitted in partial fulfillment of the requirements for the Master of

Science degree in Microbiology in the Graduate College of

The University of Iowa

December 2009

Thesis Supervisor: Associate Professor John R. Kirby

Graduate College The University of Iowa

Iowa City, Iowa

CERTIFICATE OF APPROVAL

_______________________

MASTER'S THESIS

_______________

This is to certify that the Master's thesis of

Di Li

has been approved by the Examining Committee for the thesis requirement for the Master of Science degree in Microbiology at the December 2009 graduation.

Thesis Committee: ___________________________________ John R. Kirby, Thesis Supervisor

___________________________________ Alexander R. Horswill

___________________________________ Craig D. Ellermeier

TABLE OF CONTENTS

LIST OF TABLES............................................................................................................. iii

LIST OF FIGURES ........................................................................................................... iv

LIST OF ABBREVATIONS ............................................................................................. vi

CHAPTER

I. INTRODUCTION ............................................................................................1 Myxococcus xanthus and Two-component Signal Transduction......................1 NtrC Family Regulators....................................................................................2 Sporulation of M. xanthus and Beta-lactamase Activity ..................................4 Beta-lactam Resistance Mechanism .................................................................5 Chemosensory Systems in M. xanthus .............................................................7

II. MATERIALS AND METHODS ...................................................................17 Bacterial Strains, Plasmids and Media ...........................................................17 Construction of Mutants .................................................................................17 Growth and Development Phenotypes Analysis ............................................18 Assay for Beta-galactosidase and Beta-lactamase Activity............................18 Sonication and Heat Resistance Assay ...........................................................20 Protein Over-expression, Purification and Dialysis........................................20 Gel Shift Assay ...............................................................................................21

III. RESULTS.......................................................................................................24

In silico Analyis..............................................................................................24 crdA .........................................................................................................24 Beta-lactamases in M. xanthus ................................................................26

Affect of Ampicillin During Aggregation, Sporulation and Growth .............27 Developmental Phenotype Analysis........................................................27 Starvation-independent Sporulation Analysis .........................................28 Ampicillin Resistance Assay...................................................................29

crdA Regulates Beta-lactamase Activity ........................................................29 Specific Induction of Beta-lactamases ....................................................29 Non-specific Induction of Beta-lactamases.............................................30

Regulation of the che3 Cluster, crdS and crdA...............................................31 lacZ Expression from che3, cheB3,crdS and crdA Promters...................31 DNA-binding Assay of CrdA..................................................................33

IV. CONCLUSIONS AND FUTURE DIRECTIONS .........................................57

General Discussion .........................................................................................57 Model for CrdA ..............................................................................................59

REFERENCES ..................................................................................................................63

ii

LIST OF TABLES

Table

II.1. Strains and Plasmids Used in This Study ...............................................................22

II.2. Primers Used in This Study….................................................................................23

III.1. Numbers of Beta-lactamases in Different Organisms ............................................34

III.2. Sonication and Heat Resistance of Glycerol-induced Spores …............................35

III.3. Sonication and Heat Resistance of Ampicillin-induced DZ4513 cells...................36

iii

LIST OF FIGURES

Figure

I.1. Life Cycle of Myxococcus xanthus ...........................................................................9

I.2. The Classical Model of a Two-component Signal Transduction System...............10

I.3. Regulation of Transcription by NtrC......................................................................11

I.4. Promoters of the glnALG operon ............................................................................12

I.5. Positively and Negatively Regulated NtrCs ...........................................................13

I.6. Induction of Beta-lactamase ...................................................................................14

I.7. Chemotaxis and Chemosensory Systems ..............................................................15

I.8. Eight Chemosensory Gene Clusters in M. xanthus................................................16

III.1. Predicted Domains in crdA. ...................................................................................37

III.2. Alignment of CrdA and Its Homologs...................................................................38

III.3. Prediction of Coiled Coils in crdA.........................................................................39

III.4. Comparison of crdA-crdB gene neighborhoods in M. xanthus and Anaeromyxobacter Fw109-5.....................................................................................40

III.5. Structures of Beta-lactamases in M. xanthus .........................................................41

III.6. Beta-lactamase Expression Patterns during Development ....................................44

III.7. Affect of ampicillin on development of M. xanthus cells......................................45

III.8. Affect of ampicillin and EDTA on growth and development of M. xanthus cells ...........................................................................................................................46

III.9. Affect of ampicillin on growth of M. xanthus .......................................................47

III.10 Ampicillin-induced Sporulation ............................................................................48

III.11 Glycerol-induced Sporulation................................................................................49

III.12. Beta-lactamase Activity Induced by Ampicillin....................................................50

III.13. Induction of Beta-lactamase Activity by Glycerol and EDTA..............................51

III.14 lacZ Expression of the Promoters in WT and the crdA Mutant Background .......52

III.15 Solubility and Purity Analysis of CrdA................................................................55

iv

III.16 CrdA and crdAB Promoter-binding Assay ...........................................................56

IV.1. Model for Chemosensory Regulation of CrdA to Affect Cell Wall Stability .....62

v

vi

LIST OF ABBREVIATION

BLAST............................................................................. Basic local alignment search tool

BP…………………………………………………………………………………Base pair

BSA………………………………………………………………..bovine serium albumin

CF....................................................................................................................Clone-fruiting

CYE.................................................................................................... Casitone yeast extract

DMSO...........................................................................................……..Dimethyl sulfoxide

HK………………………………………………………………………....Histidine kinase

KU…….………………………………………..…………………………………Klett unit

PBP…….. ……………………………………………………....Penicillin-binding protein

PCR............................................................................................. Polymerase chain reaction

RR…………………………………………………………………..….Response regulator

TCST............................................................................Two-component signal transduction

1

CHAPTER I

INTRODUCTION

Myxococcus xanthus and Two-component Signal Transduction

Myxococcus xanthus is a rod-shaped, gram-negative bacterium, which has a life

cycle requiring interactions between individual cells. It is a model organism for studying

the genetic, biochemical, and mechanistic basis in prokaryotic multicellular structures.

When there are enough nutrients in the environment, cells grow vegetatively and swarm

as groups using type IV pili (TFP), which is referred to as S-motility, or as individuals,

which is called A-motility and explained by a slime extrusion model (Wall and Kaiser,

1999, Wolgemuth, 2002). When M. xanthus cells encounter prey bacteria, they move in

a coordinated manner and consume other bacteria efficiently (Berleman and Kirby, 2007,

Berleman et al., 2006, Berleman et al., 2008). When the prey or nutrients are not enough

to support the population, the M. xanthus cells begin to aggregate and form fruiting

bodies that contain environmentally resistant myxospores (Dworkin, 1963, Shimkets,

1999). Some reagents, such as glycerol, DMSO and antibiotics that interrupt

peptidoglycan synthesis, are able to induce starvation-independent sporulation even when

there are enough nutrients to support growth (Dworkin and Gibson, 1964, O'Connor and

Zusman, 1997). During starvation-independent sporulation, individual cells differentiate

into spores directly without aggregation. When the environment is appropriate,

myxospores germinate and the life cycle restarts (Fig I.1).

M. xanthus has a large number of signal transduction proteins, which enable it to

respond rapidly to all kinds of stimuli and survive in changing environmental conditions.

One class of the regulators is the two-component signal transduction (TCS) system. It

classically is comprised of a membrane-bound sensor histidine kinase (HK) and a cognate

response regulator (RR), which are usually located in the same operon. The HK is able to

sense an environmental stimulus such as osmolarity, sugars, or nitrogen, via its input

2

domain, and the resulting protein conformational change leads to auto-phosphorylation at

a conserved histidine residue in its transmitter domain (Mascher, 2006). This phosphoryl

group is then transferred to the conserved aspartate-containing receiver domain of the RR

(Fig I.2). Phosphorylation of the RR receiver domain usually modifies the activity of the

output domain, which is most frequently a helix–turn–helix DNA-binding domain

(Gooderham, 2008). The RR may control gene transcription, chemotaxis, methylation, or

other aspects of physiology (Stock, 2000).

NtrC Family Regulators

NtrC, which is also called NRI or GlnG, is a sigma54-dependent response

regulator. In Escherichia coli, NtrC and its cognate HK NtrB compose a TCS system,

which regulates nitrogen status of the cell. NtrB phosphorylates NtrC under nitrogen

limiting conditions and dephosphorylates NtrC under nitrogen-excess conditions (Jiang,

2000). The phosphorylated form of NtrC activates transcription of glnALG, nifA, and

other operons involved in nitrogen fixation and assimilation (Reitzer, 1985, Zhang,

2005).

NtrC family regulators are composed of receiver, AAA ATPase, and sequence-

specific DNA-binding domains. The receiver domain has a conserved aspartate which

can be phosphorylated by NtrB. The ATPase domain is able to interact with the sigma54

polymerase-DNA complex. The DNA-binding domain usually binds 100-150 bp

upstream of the promoter sequence. Some sequences have one binding site, whereas

others have two. Generally, when NtrC is unphosphorylated, two NtrC molecules form a

dimer and bind to a single binding site on the DNA. If there are two binding sites, two

dimers can interact with each other cooperatively, which appears to strengthen the

binding (Yang, 2004). When NtrC is phosphorylated, central domain-mediated

oligomerization is stimulated so that the structure of the ATPase active site becomes

complete (Lee, 2003). The hydrolysis of ATP is required for remodeling the sigma54

3

polymerase-DNA complex and initiates the transcription (Weiss, 1991, De, 2006) (Fig

I.3).

NtrC can also function as a repressor of transcription. It has been found that some

genes have both sigma54 and other sigma factor-dependent promoters, which are induced

under different conditions (Reitzer, 1985, Shiau, 1993, Kiupakis and Reitzer, 2002,

Zhang and Rainey, 2007, Schwab, 2007). For example, the ast operon in E.coli is

transcribed from sigma54-dependent promoter during exponential phase in glucose-

arginine minimal medium, and from the sigmaS-dependent promoter during stationary

phase in Luria-Bertani (LB) medium. These two types of transcription inhibit each other

and do not happen at the same time (Kiupakis and Reitzer, 2002). NtrC binding DNA

prevents start of transcription from the other promoter (Fig I.3) (Shiau, 1993, Yang, 2004,

Zhang and Rainey, 2007). The central domain of NtrC is not required for the repressing

function (Yang, 2004).

Auto-regulation of transcription of ntrC has been studied (Reitzer, 1985,

Magasanik, 1989). In E. coli, glnA, which encodes glutamine synthetase, ntrB (glnL) and

ntrC (glnG) compose a glnALG operon (Fig I.4). There are two promoters in front of

glnA, which are glnAp1 and glnAp2. Another promoter, which is glnLp, is between glnA

and ntrB. Both glnAp1 and glnLp promote transcription by the sigma70 form of RNA

polymerase, which provides a basal level of production of GlnA, NtrB and NtrC. They

are inhibited by unphosphorylated NtrC under nitrogen excess condition. There are two

NtrC binding sites in front of glnA and one in front of ntrB. When nitrogen is limited,

NtrC is phosphorylated by NtrB, and then activate transcription of glnALG from glnAp2

and glnLp, which enhances the protein level of GlnA, NtrB and NtrC.

NtrC family regulators can be divided into two groups: positively regulated and

negatively regulated (Fig I.5), considering the function of the receiver domain. If

phosphorylation of the receiver domain facilitates ATPase assembly, the group is

positively regulated by the receiver domain, such as NtrC from Salmonella enterica

4

Serovar Typhimurium (Lee, 2000). If the unphosphorylated status represses ATPase

assembly, the NtrC family is negatively regulated by the receiver domain, such as NtrC1

from Aquifex aeolicus (Doucleff, 2005, Lee, 2003). In general, there are three differences

between the structures of these two groups of proteins. Negatively regulated proteins

have (1) a GHG motif (2) a coiled-coil linker between the receiver domain and the central

domain, and (3) a shorter linker between the central domain and DNA-binding domain

(C-D linker) compared to that in positively regulated ones (Doucleff, 2005).

Sporulation of M. xanthus and Beta-lactamase Activity

As mentioned above, there are two types of sporulation in M. xanthus: starvation-

dependent and starvation-independent. In the laboratory, starvation-dependent

sporulation is induced by spotting cell suspension on low nutrient plates (CF plates).

Starvation-independent sporulation is induced by incubating cells in rich media

containing reagents, such as glycerol, DMSO, etc. Antibiotics which affect peptidoglycan

synthesis or assembly (ampicillin and cephalosporins), are all able to induce the

starvation-independent sporulation, while those that may inhibit translation and DNA

replication do not (neomycin and novobiocin) (O’Connor, 1997). It suggests that damage

to the peptidoglycan induces starvation-independent sporulation. The peptidoglycan layer

is degraded during the transition from vegetative cells to glycerol-induced myxospores.

Newly constructed spherical coat contains no muropeptides, and has different protein

expression patterns compared to that in fruiting bodies (Downard, 1985, McCleary, 1991,

Bui, 2009), which may be the reason why glycerol spores are less resistant to sonication

and heat.

Some M. xanthus mutants are only defective in one type of sporulation. For

example, asgA and csgA mutants can form spores induced by glycerol but not starvation

(LaRossa, 1983). An early study isolated glc mutants, which cannot form glycerol spores.

Some of them still sporulate well during starvation (Burchard, 1975). The Omega7536

5

mutant, a transposon insertion in a gene encoding a chain length determinant protein

homolog, cannot change from rod to sphere under both conditions (Licking, 2000), which

suggests that these two types of sporulation use different pathways yet share some

common steps.

Sporulation occurring through starvation-dependent and independent pathways is

accompanied by an increase in endogenous beta-lactamase activities. Agents that induce

glycerol spores also induce beta-lactamase. Beta-lactamase activity peaks when the cells

change from rod to sphere (O’Connor, 1997). However, the role of beta-lactamases in

sporulation remains unclear. It is possible that beta-lactamase plays a role in restructuring

the cell wall during morphogenesis, or it is induced by signals which also induce

sporulation.

Beta-lactam Resistance Mechanism

Beta-lactam antibiotics can bind penicillin-binding proteins (PBP) to prevent

them from cross-linking the nascent peptidoglycan layer, an important part of cell wall

structure (Zapun, 2008). Beta-lactamases are enzymes which are able to deactivate beta-

lactam antibiotics like penicillins, cephalosporins, etc. There are currently more than 300

known beta-lactamases, which have been subdivided into four classes: A, B, C and D.

Class A, C and D have a serine active site. Class B is a metallo-enzyme which uses zinc

to catalyze hydrolysis of the beta-lactam ring.

Two beta-lactam resistance mechanisms have been developed by bacteria. Some

strains have evolved PBPs with low affinity for beta-lactams while some have

chromosomally-encoded beta-lactamases which are induced in the presence of certain

beta-lactams (Poole, 2004). The latter mechanism is dominant in gram-negative rod-

shaped bacteria (Livermore, 1995) (Fig I.6). Among gram-negative rods, the most widely

distributed beta-lactamases are AmpC homologs, which belong to class C beta-

lactamases. Generally, ampC is divergently transcribed from ampR, which encodes a

6

LysR-type global transcriptional regulator that activates the transcription of ampC (Kong,

2005). The activity of AmpR is regulated by muropeptides. ampC is negatively regulated

by AmpD, a N-acetyl-anhydromuramyl-L-alaline amidase, in the cytoplasm ((Lindberg,

1987, Langaee, 1998, Lee, 2009). During the bacterial life cycle, peptidoglycan is

consistently degraded. Degraded peptidoglycan is transported into the cytoplasm by a

permease AmpG, and then digested by AmpD and some other enzymes, such as Mpl,

LdcA, etc. Peptides, sugars, amino acids generated are used to construct new cell wall or

just as nutrients (Park and Uehara, 2008) It is believed that recycling intermediates, such

as UDP-MurNAc-pentapeptide, repress activation of ampC by inhibiting the activity of

AmpR. Recently, inactivation of PBP4 in Pseudomonas aeruginosa has been found to

activate the production of ampC, which is parallel from AmpD pathway (a defect in one

of them can be complemented by increasing the amount of the other). Both of them are

AmpR-dependent (Moya et al, 2009). It is considered that inactivation of PBP4 causes

accumulation of some kind of muropeptides, which activate AmpR. This study also found

that a CreBC/BlrAB two component system, which regulates ampC in Aeromonas

hydrophila (Niumsup, 2003, Avison, 2004), does not affect AmpC expression in P.

aeruginosa.

Compared to P. aeruginosa or A. hydrophila, which only has one ampC gene in

the chromosome, M. xanthus has 9 AmpC variants and 20 metallo-beta-lactamases, but

none of the ampC homologs are encoded next to an ampR homolog. Why beta-lactamase

activity is up-regulated during sporulation has not been studied yet. One possible

mechanism is that old peptidoglycan layer is degraded and new cell wall is constructed

during sporulation, so that the change of the amount of intermediate peptides may induce

the transcription of beta-lactamases. Why M. xanthus has such a large number of beta-

lactamases is also a mystery. Considering that beta-lactamases have similar structures as

penicillin-binding proteins, they may be able to interact with some kind of muropeptides

and play a role in sporulation. It is also possible that M. xanthus employs beta lactam

7

antibiotics to digest other prey bacteria so that it needs a large amount of beta-lactamase

to protect itself.

Chemosensory Systems in M. xanthus

Chemotaxis systems are used by bacteria to perceive environmental stimuli and

generate responses. The most well known chemotaxis system is that in E. coli and

regulates motility via flagellar rotation (Khan, 2000). The chemotaxis pathway makes use

of a phosphorelay to pass information through the methyl-accepting chemotaxis proteins

(MCPs) that serve as sensors and are coupled to the histidine kinase, CheA, through the

coupling protein CheW. The response regulator, CheY, interacts with the flagellar

switch, altering the direction of motor rotation. CheA also phosphorylates CheB, a

methylesterase, which is then activated and able to demethylate the MCPs. The system

contains a methyltransferase, CheR, which serves to adapt the system to the presence of

the stimulus in conjunction with CheB (Fig I.7). The steady state level of methylation

provides the cell with a primitive memory and is the hallmark feature of chemosensory

regulation. Without stimulus the cell executes a random walk by alternating runs and

tumbles. When a stimulus is provided, the walk becomes biased through the suppression

of tumbling. In order to remain in an area of optimal stimulus concentration the system

adapts through the actions of CheB and CheR on the MCP. This returns the system to the

pre-stimulus state and the cell begins a random walk even in the presence of a stimulus.

Some other chemotaxis genes are found in the genomes of other organisms, such as cheC

and cheD in Bacillus subtilis. CheC is a CheY-P phosphotase (Kirby, 2001) and CheD is

a chemoreceptor modification enzyme (Kristich, 2002).

The adaption module is not only employed to regulate motility, such as in E.coli,

Bacillus subtilis and Vibrio cholerae (Rao, 2008, Hyakutake, 2005), but also used to

regulate a variety of cell functions first discovered in M. xanthus (Kirby, 2003) (Fig I.7).

The latter system is called a chemosensory system, which differs from a chemotaxis

8

system in that genes other than the che genes are found within the operons. For example,

Rhodospirillum centenum has a chemosensory system that regulates timing of

differentiation (Berleman, 2005) and Pseudomonas aeruginosa has Wsp chemosensory

cluster, which is involved in biofilm formation (Hickman, 2005).

There are eight chemosensory systems in M. xanthus (Fig I.8). The Frz system

regulates reversal frequency of the cells while the Che4 system couples velocity and

reversal frequency when the cell is using type IV pilus (TFP) based motility (Blackhart,

1985, Vlamakis, 2004). The Dif system regulates exo-polysaccharide production, which

is required to stimulate TFP retraction (Yang, 2000). The Che3 system regulates

developmental gene expression (Kirby, 2003) and response to cell wall stress (Muller and

Kirby, unpublished data). The mRNA level of cheA3(the cheA kinase gene within the

che3 cluster) is up-regulated during development (Jakobsen, 2004), and the mcp3B (the

MCP gene within the che3 cluster) mutant is premature in development (Kirby, 2003).

The che3 operon does not have a CheY homolog. CrdA, a NtrC homolog, is known to be

the output of the Che3 system, because the crdA mutant and crdAmcp3B double mutant

show a delayed phenotype in development (Kirby, 2003). The Che5, Che6, Che7 and

Che8 systems have not yet been fully characterized, but are predicted to regulate

alternative cellular functions (Kirby, 2009).

9

Fig I.1. Life Cycle of Myxococcus xanthus. When there are enough nutrients, the cells are able to grow and swarm on a solid surface. They form rippling patterns while preying on other bacteria. If there are not enough nutrients, the cells aggregate and form fruiting bodies that contain myxospores. Fruiting bodies are also developed during predation. Cell wall stress can induce individual cells to sporulate without aggregation, even in rich media. The heat and sonication resistant spores are able to germinate and restart the life cycle when nutrients become available.

10

HK

RR

P

P

stimulus

RR inactive

active

Fig I.2. The Classical Model of a Two-component Signal Transduction System. The histidine kinase (HK) senses the external stimulus and autophosphorylates a histidine residue, the phosphoryl group (P) is then transferred to an aspartate residue on the response regulator (RR). Phosphorylated RR may control gene transcription, chemotaxis, methylation, or other aspects of physiology.

11

A

Transcriptional start site

Sigma54 binding sequenceNtrC Binding Sites

P PP

Complex of Sigma54

and RNA polymerase

B

Transcriptional start site

Complex of Sigma70 and RNA polymerase

Sigma70 binding site

Sigma54 binding sequenceNtrC Binding Sites

Fig I.3. Regulation of Transcription by NtrC. (A)Unphosphorylated NtrC forms dimers and binds to binding sites on the DNA. When NtrC is phosphorylated, central domain-mediated oligomerization is stimulated. NtrC oligomers interact with the sigma54 polymerase-DNA complex and initiate transcription. (B) When NtrC binding sites overlap with a sigma70 binding site, interaction between unphosphorylated NtrC and DNA represses transcription from sigma70.

12

glnAp1 glnAp2 glnLp1glnA

ntrB(glnL)

ntrC(glnG)

Sigma70 binding site NtrC binding site

Fig I.4. Promoters of the glnALG operon. Two promoters (glnAp1 and glnAp2) are in front of glnA. glnAp1 contains a sigma70 binding site. glnAp2 contains two NtrC binding sites. Another promoter, which is glnLp, is between glnA and ntrB. It has both sigma70 and NtrC binding sites. Under nitrogen excess condition, transcription is started from glnAp1 and glnLp by the sigma70 form of RNA polymerase to provide a basal level of production of GlnA, NtrB and NtrC. When nitrogen is limited, transcription is activated by phosphorylated NtrC from glnAp2 and glnLp.

13

PP

P P

Positive

Negative

GHG

Fig I.5. Positively and Negatively regulated NtrCs Positively regulated NtrCs form dimers via their DNA-binding domains. After being phosphorylated, the receiver domain interacts with the central domain, which helps form oligomers. The oligomeric form interacts RNA polymerase complex and initiates the transcription. Negatively regulated NtrCs form dimers tightly via their receiver domains, which contain a GHG motif, in the absence of phosphorylation. Binding inhibits the formation of oligomers. After being phosphorylated, the receiver domains induce a conformational change, which allows the interaction between central domains, so that oligomers can be formed, which results in activation of transcription.

14

Beta‐lactam drug

PBP

OM

IM

ampCAmpR

beta‐lactamase

peptidoglycanrecycling

AmpD

ampR

transcription regulator

energy

AmpG

muropeptides

Fig I.6. Induction of Beta-lactamase. ampC is divergently transcribed from ampR, which is a LysR-type global transcriptional regulator that activates transcription of ampC. When beta-lactam drugs penetrate into the periplasm, they bind penicillin-binding protein (PBP) to prevent synthesis of the peptidoglycan layer. Inactivation of PBPs up-regulates the transcription of ampC, which is AmpR-dependent, which may due to accumulation of some kind of muropeptides. AmpC is secreted into periplasm and deactivates beta lactam drugs. Components of peptidoglycan are transported into cytoplasm by a permease AmpG, and digested by a N-acetyl-anhydromuramyl-L-alaline amidase AmpD. The products are used to re-construct peptidoglycan or are consumed in other metabolic pathways. The muropeptide intermediates bind AmpR, which activates or inhibits the transcription of ampC.

15

Chemotaxis System

PP

CH3CH3

MCP

WCheR CheBCheA

motility

P

stimulus

CheY

PP

CH3CH3

MCP

WCheR CheBCheA

stimulus

Chemosensory System

motility other

RR RRP P

Fig I.7. Chemotaxis and Chemosensory Systems. In a typical chemotaxis system, the MCP senses the stimulus and interacts with the histidine kinase CheA and the coupling protein CheW. Conformational changes within the MCP induced by ligand binding leads to autophosphorylation of CheA. Phosphorylated CheA can phosphorylate the response regulators CheY and CheB. Phosphorylated CheY interacts with the flagellar motor, therefore affects the cell motility. CheR constitutively methylates the MCP while phosphorylated CheB demethylates the MCP. The methylation state of the MCP controls its ability to respond to attractants and repellents. The chemosensory system employs a similar pathway using other response regulators to control motility and other cell functions, such as gene expression.

16

Fig I.8. Eight Chemosensory Gene Clusters in M. xanthus. The color filled arrows represent chemotaxis homologs as annotated above the frz and dif clusters. The white arrows correspond to the non-chemotaxis homologs in the cluster. The small black arrows indicate the promoters.

17

CHAPTER II

MATERIALS AND METHODS

Bacterial Strains, Plasmids and Media

Bacterial strains and plasmids are listed in Table II.1. strains were grown in

Luria-Bertani broth at 37°C. . strains were grown at 32°C in Casitone Yeast

Extract (CYE) media (1% casitone, 0.5% yeast extract, 4 mM MgSO4, 10 mM Mops, pH

7.6) or agar (1.5%) plates. 100 ug/ml ampicillin (Amp), 100 ug/ml kanamycin (Km) or 10

ug/ml oxytetracycline (Tc) were used for selection. Development was assayed at 32°C

on Clone-fruiting (CF) (0.015% casitone, 1 mM potassium phosphate, 8 mM MgSO4, 1.5

mM (NH4)2SO4, 6.8 mM sodium citrate, 9.1 mM sodium pyruvate, 10 mM Mops, pH

7.6) agar (1.5%) plates.

Construction of Mutants

For construction of CrdA-expressing plasmids: the BamHI-HindIII fragment of

full-length was PCR amplified from the chromosome of wild-type strain

DZ2 and cloned into pUC18 and pQE80L to construct pDL162 and pDL180. pDL180 is

used in overexpression in . pDL162 served as the DNA template in mutagenesis. A

QuikChange site-directed mutagenesis kit (Stratagene) was used to mutate aspartate 53 in

to alanine or glutamate. Primers containing the mutation were used to amplify the

whole plasmid. Then the methylated parental DNA was digested by DpnI and the non-

methylated dsDNA containing the mutation was transformed into XL1-Blue competent

cells. Plasmids isolated from colonies on resulting plates were sequenced by the Nevada

Genomics Center to confirm the mutation. c fragments with or without the point

mutation were digested by BamHI and HindIII, and subcloned into pET28a. Then a

XbaI-BamHI fragment of promoter from bp -217 to 3 relative to its translational start

site, generated by PCR, was cloned into these plasmids. After that, the promoter and

fragments were digested from pET28a derivatives using XbaI and HindIII, and

E.coli

M xanthus

crdA M. xanthus

E. coli

crdA

rdA

pilA

pilA

crdA

18

subcloned into pWB300 to construct pDL336, pDL337 and pDL338.

For the construction of promoter-lacZ fusion plasmids: trpA-lacZ fragment was

digeste

transformed into M. xanthus strains

by elec

Growth and Development Phenotypes Analysis

For growth s cells were

harvest it

and

maging

Assay for Beta-galactosidase and Beta-lactamase activity

Sample ssay were

the sam

washed

d from pSM63 using EcoRI and HindIII, and inserted into pWB300 to construct

pDL323. Then different promoters were PCR amplified and cloned into pDL323, which

was digested by XbaI and EcoRI, or PvuI and XbaI.

Plasmids which are pWB300 derivatives were

troporation in a 0.2 cm cuvette at 25mF, 400Ω, 0.65kV (Xu, 2005). The pWB300

plasmid contains integrase and phage Mx8 attP attachment site. It is integrated at attB site

in the genome of M. xanthus by a mechanism of site-specific recombination. (Magrini,

1999) The integration was confirmed by PCR. All the primers used for construction were

synthesized by Integrated DNA Technologies. The sequences are listed in table II.2.

and development phenotypes observation, M. xanthu

ed from liquid CYE culture when the density was between 100 – 150 Klett Un

(KU). Cells were washed by water twice, and concentrated to 200 KU. 10 ul of the

resulting cell suspension was spotted on CYE or CF plates with appropriate reagents

allowed to dry at room temperature. Plates were incubated at 32°C for 48 hours.

Photographs were taken using a Nikon SMZ1500 dissecting microscope with Q-I

camera and software.

preparation for beta-galactosidase assay and beta-lactamase a

e. M. xanthus cells were harvested from liquid culture and concentrated to 500

KU. 200 ul of the resulting cell suspension was plated on the entire plate. After

incubation at 32oC for the time indicated, cells were scraped from the plates and

by water once, then re-suspended in the working buffer to be assayed later (Kirby, 2003).

Alternatively cells were harvested from liquid culture and concentrated to 200 KU. 500 ul

19

of the resulting cells was centrifuged and resuspended in the working buffer.

Cells in the working buffer were then sonicated using a Branson sonifier 150 set

at inten

ant was

sample supernatant was measured using the Bio-Rad

protein

or the beta-galactosidase activity assay, the working buffer was Z buffer (0.85%

Na2HP

on

amase activity assay, the working buffer was 0.1 M phosphate

buffer. in

x

sity 5 for 20 seconds or until the solution was clear. Sonicated cells were

centrifuged at 13,000 g for 5 min and the supernatant was collected. The supernat

used to determine the protein level and for the subsequent beta-galactosidase activity

assay or beta-lactamase assay.

The protein level of the

assay (Bio-Rad). 2-20 ul sample was added into 900 ul 1x dye reagent and

incubated for 5 minutes. Then the protein concentration was calculated by measured

OD585 using an Eppendorf Biophotometer (BSA was used to calculate the standard

curve).

F

O4, 0.55% NaH2PO4•H2O, 1 mM MgSO4, 10 mM KCl, pH 7.0). 10-100 ul sample

supernatant, 300 ul Z/BME buffer (Z buffer plus 0.05 M beta-mercaptoethanol), and 100

ul ONPG (4 mg/ml ONPG in Z buffer) were mixed together and incubated at 37oC. After

the solution became yellow, 500 ul of 1 M Na2CO3 was added into the reaction and the

time of incubation was recorded (Kirby, 2003). Then the OD420 was measured using

Nanodrop ND-1000 spectrophotometer. The beta-galactosidase specific activity was

calculated using the formula: 213 x OD420 / (sample volume (ml) x protein concentrati

(mg/ml) x time (min)).

For the beta-lact

20 ul sample was mixed with 1 ul nitrocefin stock solution (10 mg/ml nitrocefin

dimethylsulfoxide (DMSO) ), 1 ul EDTA (200 mM EDTA, pH 8.0), and 178 ul 0.1 M

phosphate buffer. The beta-lactamase activity was calculated using the formula: 228000

OD486 / (protein concentration (mg/ml) x time (min)) (King, 1980).

20

Sonication and Heat Resistance Assay

500 ul of 150 KU M. xanthus cells were incubated with reagents, such as EDTA

and/or ampicillin, for a period of time as indicated. Spores were collected and re-

suspended in 500 ul water. To test sonication resistance, samples were sonicated using a

Branson sonifier 150 set at intensity 4 for 30 seconds as previously described (Licking,

2000). To test heat resistance, samples were incubated in waterbath at 50oC for 2 h. After

sonication and heat treatment, spores were diluted 105 times and then mixed with 4 ml

CYE 0.7% agar, then plated on CYE plates. Seven days later, germinated colonies were

counted using an aCOLyte colony counter.

Protein over-expression, purification and dialysis

The E.coli strain containing plasmid pDL180 was grown in 25 ml LB broth at

37oC until the OD600 reached 0.4-0.6, then IPTG was added into the culture as the

inducer. After expressing at 32oC for 4 h, cells were lysed by adding Cell-Lytic Express

(Sigma) with shaking at room temperature for 20 minutes.

The transparent lysed cell solution was loaded to a polypropylene column (Pierce)

containing HIS-Select Nickel Affinity Gel (Sigma), which was pre-equilibrated with

equilibration buffer (50 mM Tris HCl, 0.3 M sodium chloride, 10 mM imidazole, pH

8.0), then washed with wash buffer (50 mM Tris HCl, 0.3 M sodium chloride, 20 mM

imidazole, pH 8.0) until the amount of the protein in the material eluting from the column

was very low and steady, which was measured using BioRad protein assay as mentioned

above. After that, the protein was eluted with elution buffer (50 mM Tris HCl, 0.3 M

sodium chloride, 250 mM imidazole, pH 8.0). Fractions were collected from the flow, run

on 12% polyacrylamide gel and stained with Coomassie Brilliant Blue to verify the size

and purity of the protein. The fraction having the highest protein amount was used for the

subsequent experiments.

1 ml purified CrdA was dialyzed in a dialysis cassette (BioRad ) in 1 L dialysis

21

buffer (50 mM Tris HCl pH 7.5, 50 mM KCl, 10 mM MgCl2, 1 mM CaCl2, 0.2 mM DTT,

50% Glycerol) at 40C overnight. The concentration of dialyzed CrdA was measured

using BioRad protein assay. To assay for the solubility of purified CrdA, 200 ul of

undialyzed and dialyzed CrdA were centrifuged at 100,000 g for 15 min. The supernatant

was removed and the pellet was resuspended in 200 ul elution buffer. After that, SDS-

PAGE was performed to detect the amount of CrdA in each fraction.

Gel Shift Assay

The gel shift assay was performed using the LightShift Chemiluminescent EMSA

kit (Pierce). DNA samples were generated by PCR using biotin 5’end-labeled primers

and cleaned by the PCR product purification kit (Qiagen). Each 20 ul reaction contains 2

ul 10x Binding Buffer (100 mM Tris, 500 mM KCl, 10 mM DTT; pH 7.5), 1 ul 50%

glycerol, 1 ul 2 mg/ml BSA, dialyzed CrdA , crdB promoter and DNA competitors. All

the reagents were mixed together and incubated at room temperature for 20 minutes, then

loaded to 5% native polyacrylamide gel in 0.5x TBE which had been pre-electrophoresed

for 30 minutes. Samples were run at 100 V until the bromophenol blue dye had migrated

to the bottom of the gel. After electrophoresis, samples were transferred to Nylon

membrane (Milipore) at 20 V for 30 minutes using a BioRad TRANS-BLOT SD semi-

dry transfer cell. The transferred DNA was cross-linked to membrane at 120 mJ/cm2

using a UV-light cross-linker (use the auto cross-link function twice). Biotin-labeled

DNA on the membrane was then detected by chemiluminescence according to

manufacturer’s instructions.

22

Table II.1. Strains and Plasmids used in This Study Strain or plasmid Relevant genotype Ref. M. xanthus strains DZ2 Wild Type Campos, 1975 DZ4513 DZ2 crdA::pJK412, KmR Kirby, 2003 JK1746 DZ4513 attB::pDL336, KmR, TcR This work JK1747 DZ4513 attB::pDL337, KmR, TcR This work JK1748 DZ4513 attB::pDL338, KmR, TcR This work JK1749 DZ2 attB::pWB300, TcR This work JK1750 DZ4513 attB::pWB300, KmR, TcR This work JK1751 DZ2 attB::pDL323, TcR This work JK1752 DZ2 attB::pDL418, TcR This work JK1753 DZ2 attB::pDL378, TcR This work JK1754 DZ2 attB::pDL381, TcR This work JK1755 DZ2 attB::pDL366, TcR This work JK1756 DZ4513 attB::pDL418, KmR, TcR This work JK1757 DZ4513 attB::pDL378, KmR, TcR This work JK1758 DZ4513 attB::pDL381, KmR, TcR This work JK1759 DZ4513 attB::pDL366, KmR, TcR This work E.coli strains

DH5a F- φ80lacZΔM15 Δ(lacZYA-argF)U169 recA1 endA1 hsdR17(rk-, mk+) phoAsupE44 thi-1 gyrA96 relA1 λ-

Invitrogen

XL1Blue recA1 endA1 gyrA96 thi-1 hsdR17 supE44 relA1 lac [F´ proAB lacIqZΔM15 Tn10 , TetR

Stratagene

BL21(DE3) F-ompT hsdSB dcm gal l(DE3) Stratagene Plasmid pUC18 Used for mutagenesis, AmpR Invitrogen pDL162 pUC18 with full-length crdA, AmpR This work pDL308 pUC18 with D to A mutation at bp 53 in crdA, AmpR This work pDL319 pUC18 with D to E mutation at bp 53 in crdA, AmpR This work pET28a Used for plasmid construction, KanR This work pDL313 pET28a with full-length crdA, KanR This work pDL320 pET28a with crdAD53A, KanR This work pDL321 pET28a with crdAD53E, KanR This work pDL330 pET28a with pilA promoter and crdA, KanR This work pDL331 pET28a with pilA promoter and crdAD53A, KanR This work pDL332 pET28a with pilA promoter and crdAD53E, KanR This work pWB300 Used for expression in M. xanthus, TcR Xu, 2005 pDL336 pWB300 with pilA promoter and crdA, TcR This work pDL337 pWB300 with pilA promoter and crdAD53A, TcR This work pDL338 pWB300 with pilA promoter and crdAD53E, TcR This work pQE80L Vector for protein expression in E.coli Qiagen pDL180 pQE80L with full-length crdA, AmpR This work pSM63 Plasmid offering trpA-lacZ fragment Mueller, 2006 pDL323 pWB300 with trpA-lacZ fragment, TcR This work pDL418 pWB300 with crdA promoter and trpA-lacZ fragment, TcR This work pDL378 pWB300 with crdS promoter and trpA-lacZ fragment, TcR This work pDL381 pWB300 with che3 promoter and trpA-lacZ fragment, TcR This work pDL366 pWB300 with cheB3 promoter and trpA-lacZ fragment, TcR This work

23

Table II. 2. Primers Used in This Study

Primers Sequence (5' - 3')

crdA fwd GGAAGGATCCATGCCCGCCGCCTCTGTCCTC

crdA rev GGAAAAGCTTTTACGACTCGGAGCTGCC

crdA D53A fwd CGCGGTGCTGATGGCCGTGAAGATGCCGG

crdA D53A rev CCGGCATCTTCACGGCCATCAGCACCGCG

crdA D53E fwd CGCGGTGCTGATGGAGGTGAAGATGCCGGAC

crdA D53E rev GTCCGGCATCTTCACCTCCATCAGCACCGCG

PpilA fwd GGAATCTAGAGGGAGCGCTTCGGA

PpilA rev GGAACATATGCATGGGGGTCCTCAGAGAAG

PcrdA fwd GGAACGATCGTCGGATAGGAAGGGGTGCAG

PcrdA rev GGAATCTAGACATCGACGATGAGGACAGAG

PcrdS fwd GGAATCTAGACTCAACAACCTGGGCCTCAC

PcrdS rev GGAAGAATTCTGATGAGCAGCCGCTTCGTC

Pche3 fwd GGAACGATCGTCTGATACCCCGCCAGTTGG

Pche3 rev GGAATCTAGAAGGTGTTCTCGGGAACCTGG

PcheB3 fwd GGAATCTAGACGTTCCAATCGCCATCGAAG

PcheB3 rev GGAAGAATTCAGATGAGGGAGTCATCGACC

attR fwd AAAAAAGCTTCCGGGCGGCCTTGCGGAATGAT

attR rev TCAGCGCTTCAGGTCCGGGACTGGGAC

PcrdAB fwd TCTGATACCCCGCCAGTTGG

PcrdAB rev TGTAGTCGTTGGCGTGTAGTC

asgB fwd GGAAGGTACCGCGCCATCCTGAAAATCTTCC

asgB rev GGAATCTAGACTGCTTCACTTCCGACATGTC

24

CHAPTER III

RESULTS

In silico analysis

crdA

A database search using NCBI-BLAST predicted the domains in CrdA and

identified homologs (Altschul, 1990, Altschul, 1997). As shown in Fig III.1, CrdA is a

NtrC homolog, composed of a receiver domain, an AAA ATPase domain and a helix-

turn-helix DNA-binding domain. The receiver domain has the conserved aspartate (D53)

for phosphorylation.

To determine whether CrdA is positively or negatively regulated by the receiver

domain, the sequence was aligned with twenty NtrCs randomly selected from other

organisms. Ten of them have a long C-D linker and don’t have a GHG motif, including

NtrC from E. coli and Salmonella enterica Serovar Typhimurium, which are positively

regulated. Ten of them have a short C-D linker and a GHG motif, including NtrC1 from

Aquifex aeolicus and DctD from Sinorhizobium meliloti, both of which are negatively

regulated. COILS (Lupas, 1991) was used to predict the coiled-coil motifs. CrdA has a

GHG motif (Fig III.2) and a coiled-coil motif (Fig III.3) between the receiver domain and

the central domain, which exist in negatively regulated NtrCs, such as NtrC1 and DctD,

but not in positively regulated NtrCs, such as NtrC from E. coli and S. enterica. The

receiver domain in negatively regulated NtrCs form dimers when it is unphosphorylated,

which inhibits the ATPase assembly. The receiver domain in CrdA may play the same

function. Usually, negatively regulated NtrCs have shorter C-D linkers than the positively

regulated ones do. However, CrdA has a C-D linker, which is about 30 residues longer

than that in positively regulated NtrCs (Fig III.2). It implies that the mechanism of DNA-

binding by CrdA may be more similar to positively regulated NtrCs.

25

Sequence analysis of the M. xanthus genome shows that crdA is not in an operon

with other genes. It is divergently transcribed from crdB, which encodes a lipoprotein

located in the cell membrane and is required for EDTA-induced cell wall stress

responses. CrdB is an input to the Che3 system, which is involved in the regulation of

development (Kirby, 2003, Muller and Kirby, unpublished data). We tried to predict the

functional partners of CrdA using SMART database (Jensen et al, 2009) and found that

co-occurrence of crdA, crdB, cheA3 and MXAN_5184, designated crdS, have high

confidence scores, meaning that these genes co-exist in many organisms. CrdS is

predicted to be a histidine kinase and encode a NtrB homolog, which is about 37kb away

from crdA in M. xanthus. It is in an operon including four other genes. One of them

encodes a penicillin-binding protein (PBP) homolog. PBPs synthesize the peptidoglycan

layer in the cell wall and they are the targets of beta-lactam antibiotics. We studied the

position of crdA homologs in the genome of other organisms and found that a similar

crdA-crdB fragment exists in other organisms, such as Anaeromyxobacter sp, Stigmatella

aurantiaca, Geobacter sulfurreducens. Alignment of the similar operons in M. xanthus

and Anaeromyxobacter sp Fw109-5 is shown in Fig III.4. Anaeromyxobacter sp has very

similar arrangement of crdA, crdB, crdF, crdG, crdH genes except that it doesn’t have

the che genes. The che3 cluster, with the exception of crdB, was either inserted into M.

xanthus or deleted from Anaeromyxobacter species. It implies that besides functioning in

a signal transduction pathway, CrdB may have other functions related to those genes

present in neighboring clusters, including synthesis of peptidoglycan. In

Anaeromyxobacter sp, the crdA-like gene and the crdS homolog are in the same operon,

which implies that CrdS may be the cognate histidine kinase of CrdA.

An in vitro assay was performed and the histidine kinase CrdS proved to be able

to phosphorylate CrdA (Willett and Kirby, unpublished data). Purified CrdS and/or CrdA

were incubated with 32P-labeled ATP for a certain amount of time and fractionated by

SDS-PAGE. CrdS can autophosphorylate and is stable in the buffer for several days.

26

Phosphorylated CrdS can transfer the 32P to CrdA within seconds. The phosphorylation

is inhibited by CheA3. As CrdA proved to be downstream of CheA3 by epistasis assay, it

might be the output of both the Che3 system and the CrdS pathway.

Considering the function of CrdB and the PBP are both related to peptidoglycan

layer and cell wall stability, CrdA is predicted to function in cell wall stress regulation.

Beta-lactamases in M. xanthus

Compared to other model organisms, M. xanthus has a large number of beta-

lactamases (Table III.1). There are twenty-nine beta-lactamase homologs in the

chromosome of M. xanthus. Their sequences and predicted domains were obtained from

the MiST2 database (Ulrich and Zhulin, 2007). Whether the gene contains a signal

peptide for secretion was predicted by PrediSi software (Hiller, 2004) (Fig III.5).

Twenty of these beta-lactamases belong to the metallo-enzyme class, which

utilize a catalytic zinc center and the other nine are serine-hydrolases. Beta-lactamase

expression patterns during the first 24 hours of development are shown in Fig III.6, along

with the evolutionary tree (Cullen and Kirby, unpublished data). The data is from

previous microarray experiment (Shi, 2008). RNA was isolated from M. xanthus cells

grown in liquid 1% CTT medium (reference RNA) or MC7 buffer (developmental RNA)

over a 24h period of time. cDNA of the reference probe was labeled with Cy5 and cDNA

of the developmental probe with Cy3. The M. xanthus DNA microarrays covered 88% of

all protein-coding genes in the M. xanthus genome. The data produced was filtered by the

Genepix Pro 6.0 spot-finding algorithm and signal-to-noise ratio. Therefore, not all the

beta-lactamases are included in the microarray data. Among metallo-enzyme class or

serine-enzyme class, some beta-lactamases are predicted to be secreted and some are not;

some are up-regulated during development and some are down-regulated. We found no

corresponding relationship between their structures, locations and functions.

27

Affect of Ampicillin during Aggregation, Sporulation and Growth

Affect of Ampicillin on Growth and Development

Based on previous studies and in silico analyses, the function of CrdA is an output

of the Che3 system, which is involved in regulation of development and responses to

cell wall stress. Therefore, developmental phenotype analysis was performed using

ampicillin as cell wall stress inducer (Fig III.7). The wild-type strain and the crdA mutant

containing the empty expression vectors respectively were assayed on CF media

containing 10ug/ml ampicillin. The crdA mutant cells formed smaller aggregation centers

compared to wildtype cells. Both of them grew well on CYE plates, which suggests that

the smaller aggregation is not due to growth defect. Wildtype crdA and the point mutants

crdAD53A and crdAD53E were fused to the promoter of pilA and the expression plasmids

were transformed into the crdA mutant. CrdAD53A doesn’t have the aspartate

phosphorylation site such that the mutant protein functions as the unphosphorylated form

of the response regulator. Glutamate is used to mimic the phosphorylated aspartate such

that CrdAD53E is likely to be constitutively active (Nohaile, 1997). Overexpression of

wildtype CrdA complemented the crdA null mutant phenotype, while overexpression of

CrdAD53A did not. The size of the aggregation centers formed by crdA/pCrdAD53E was

between the crdA mutant and crdA/pCrdA, indicating that CrdA-P is the active form of

CrdA, which is required for aggregation under stress. CrdAD53E is able to mimic the

phosphorylated form of CrdA, but may be not as functional as the wildtype CrdA-P.

In order to determine if more stress would cause more severe defects in

development, 5ug/ml ampicillin and 0.5 mM EDTA were added to the CF plates together

or separately (Fig III.8). EDTA chelates Mg2+ disrupting LPS and increases the fluidity of

cell membranes so that ampicillin can penetrate into the periplasm more easily. It also

chelates Zn2+, which is required for proper function of metallo-beta-lactamases, so that

the cells would be more sensitive to ampicillin. All strains looked similar on CF plates

28

containing only 5 ug/ml ampicillin or 0.5 mM EDTA (data not shown). However, when

both 5 ug/ml ampicillin and 0.5 mM EDTA were used, the crdA mutant displayed a

severe defect in aggregation. In contrast, at 48 h, the wildtype strain had already formed

large aggregates, which started to darken (indicating sporulation). Overexpression of

wildtype CrdA and CrdAD53E complemented the crdA mutant defect while overexpression

of CrdAD53A did not, indicating that CrdA is phosphorylated under this condition.

Furthermore, when these cells were plated on CYE containing the same amount of EDTA

and ampicillin, the crdA mutant and crdA/pCrdAD53A did not grow well. These results

suggest that the severe aggregation defect during development may due to growth defect.

Higher amounts of ampicillin were tested and found to cause a similar phenotype (Fig

III.9). The crdA mutant and crdA/pCrdAD53A didn’t grow on CYE plates containing

50ug/ml ampicillin, while the wildtype, crdA/pCrdA and crdA/pCrdAD53E grew well.

These results suggest that phosphorylation of CrdA is required for ampicillin resistance

during both growth and development.

Affect of Ampicillin on Cell Wall

The results above show that the crdA mutant cells are more sensitive to ampicillin

compared to the wildtype strain. Whether ampicillin kills the crdA mutant cells was

tested by incubating M. xanthus cells in CYE broth with 1 mg/ml ampicillin for 12 h.

After that, cells were observed by microscopy and photographed (Fig III.10). The

wildtype cells were still rod-shaped, while all the crdA mutant and crdA/pCrdAD53A cells

became spherical. crdA/pCrdA and crdA/pCrdAD53E had both rod-shaped and spherical

cells. The spherical crdA mutant cells were collected, resuspended in water, and plated on

CYE plates with or without heat and sonication treatment. The cells in water are viable,

but not resistant to sonication and heat (Table III.2), which suggests that the crdA mutant

spherical cells treated by ampicillin are neither pure spheroplasts nor mature spores. The

29

spherical cells of crdA/pCrdA, crdA/pCrdAD53A and crdA/pCrdAD53E also need to be

assayed in the future to identify the role of CrdA in cell wall synthesis and sporulation.

Peptidoglycan is the shape-determining structure in the cell envelope of M.

xanthus. It is degraded during sporulation when the cells differentiate from rod-shaped

vegetative cells to spherical spores. Ampicillin inhibits the synthesis and assembly of

peptidoglycan. Without the support of peptidoglycan, rod-shaped cells become spherical.

The wildtype cells are still rod-shaped after being incubated with high amounts of

ampicillin for 12 h, which suggests that M. xanthus employs a mechanism that makes

cells strongly resistant to ampicillin. The crdA mutant data indicates that CrdA must be

involved in this regulatory pathway.

Glycerol-induced Sporulation Analysis

Starvation-independent sporulation is thought to occur by the interruption of cell

wall synthesis. As the crdA mutant cells can not be induced to mature spores by

ampicillin, whether they have normal glycerol-induced sporulation was tested. After 12 h

of incubation with 0.5 M glycerol, the cells of all the strains become spherical (Fig

III.11). Spores formed by the crdA mutant cells are resistant to sonication and heat as

well as those formed by wild-type cells (Table III.3). It suggests that crdA is not required

for starvation-independent sporulation. Spores formed by crdA/pCrdA, crdA/pCrdAD53A

and crdA/pCrdAD53E cells will be assayed in the future to confirm this conclustion.

crdA Regulates Beta-lactamase Activity

Specific Induction of Beta-lactamase

Ampicillin is a beta-lactam antibiotic targeting cell wall synthesis. In some

bacteria, it can specifically induce the production of beta-lactamases, which inactivate

ampicillin to protect the cells. The crdA mutant shows defects in sporulation and growth

30

when the medium contains ampicillin. Therefore, the beta-lactamase activity assays were

performed to test whether crdA is required for induction of beta-lactamases. The wild-

type strain and the crdA mutant cells were assayed after incubation with 100 ug/ml

ampicillin overnight. After ampicillin treatment, the beta-lactamase activity in wild-type

is up-regulated about four fold, whereas beta-lactamase activity in the crdA mutant

remains unchanged. Overexpressing CrdA in the crdA mutant background complements

this defect (Fig III.12). The results suggest that CrdA regulates beta-lactamase

production and that decreased beta-lactamase activity may be the reason for the crdA

mutant’s sensitivity to ampicillin being not resistant to ampicillin during development

and growth.

Non-specific Induction of Beta-lactamase

Both starvation-dependent and independent sporulation are accompanied by the

induction of beta-lactamases (O’Connor, 1997, O’Connor, 1999). The crdA mutant is not

able to induce beta-lactamases when grown with ampicillin, and it has a defect in

ampicillin-induced sporulation. However, it can undergo glycerol-induced sporulation

normally. We then assayed whether beta-lactamase activity would be stimulated by

glycerol or other reagents in the crdA mutant. Beta-lactamase activity was assayed in the

presence of glycerol and EDTA as inducers. Cells were grown with different amounts of

glycerol or EDTA overnight, and then collected for the assay. Beta-lactamase activity of

the crdA mutant cells is lower than the wild-type cells under all conditions. However,

higher concentrations of glycerol and EDTA are also able to enhance beta-lactamase

activity in the crdA mutant cells (Fig III.13), which may explain why the crdA mutant

produces normal spores when induced by glycerol. The results also indicate that there is

more than one pathway in addition to Che3-CrdA, capable of regulating beta-lactamase

gene expression.

31

Regulation of the che3 Cluster, crdS and crdA

lacZ Expression from che3, cheB3, crdS and crdA Promoters

Because NtrC performs auto-regulation when it responds to nitrogen

concentration in E. coli, we tested various promoters in the Che3 system to see if they are

under the control of CrdA. Transcription levels of the crdA, crdS, cheB3 and che3

operon were assayed when cell wall stress is induced. Promoter regions from ~200 to

500 bp upstream of the transcriptional start site for each of these genes were fused to lacZ

and the plasmids were transformed into the wild-type strain and the crdA mutant. Beta-

galactosidase activity was measured under both growth and development conditions with

or without stress inducer. A pilA promoter was used as a positive control and lacZ gene

only was used as a negative control (Fig III.14). All the strains were cultured in CYE

until the cell density was approximately 150 KU when cells were harvested. Some of the

cells were directly used for beta-galactosidase assays and are labeled as “CYE” (Fig

III.14). Some of the cells were subsequently incubated in fresh CYE broth with 200

ug/ml ampicillin for 4 more hours before being collected, labeled as “CYEAmp” in Fig

III.14. In addition, some of the cells were plated on CF plates with or without 5 ug/ml

ampicillin and 0.5 mM EDTA, and harvested at 48 h. These samples are labeled as “CF”

or “CFEA” (Fig III.14).

The results show that the promoters of crdS, crdA and cheB3 are up-regulated

when wildtype cells enter development (CF plates) (Fig III.14B). However, the

transcription level of crdB doesn’t change, which was unexpected based on previous

results that showed the che3 genes upregulated during development (Jakobsen, 2004).

One possible explanation is that the promoter of crdB is not long enough so that some

activator binding sites are missing. The upregulation of crdA and cheB3 in the crdA

mutant is not to the level of wild type, which suggests that CrdA stimulates the

transcription of crdA and cheB3 during development as predicted, similar to other NtrC

32

homologs that are known to be autoregulatory (Reitzer, 1985, Schwab, 2007). Increasing

the level of CheB3 and CheR3 would allow the Che3 system to adapt to the signals more

quickly, which may be important during development because multiple signals play roles

in different stages during starvation-dependent sporulation.

The results in Fig III.14(C) shows that in both wildtype and the crdA mutant

background, the promoters of crdB and cheB3 are down-regulated by ampicillin during

growth. The down-regulation is not CrdA-dependent. As CheA3 inhibits the

phosphorylation of CrdA, down-regulation of crdB and cheB3 would lead to an increase

in phosphorylated CrdA, which is required for ampicillin-resistance mechanism.

The results in Fig III.14(D) shows that under developmental conditions, all the

promoters tested are down-regulated by ampicillin and EDTA in the wild-type

background, but not in the crdA mutant. This result was surprising because we expected

that crdS and crdA would be up-regulated during stress response. One possible

explanation is that cells initiate the starvation-independent sporulation process

responding to damage of the cell wall. In contrast, during sporulation, peptidoglycan is

degraded such that the amount of CrdA, which helps to stabilize the peptidoglycan layer,

needs to be decreased.

To access whether CrdA functions to regulate these promoters, the ratio of the

promoter activities for the crdA mutant relative to the wild-type is presented in Fig

III.14E. Numbers smaller than 1 suggest that the genes are activated by CrdA under

those conditions. Numbers bigger than 1 suggest repression by CrdA. The results indicate

that when cells are grown in rich liquid media (CYE), crdB is activated by CrdA. This

induction is attenuated after incubation with ampicillin. During development (CF), crdA

and cheB3 are upregulated by CrdA. However, inducing stress during development did

not lead to CrdA dependent upregulation of crdA, crdB and cheB3. Lastly, cell wall

induced stress during development (CFEA) revealed that the transcription of crdA is

33

repressed by CrdA itself. Together, the results imply that without severe stress, CrdA

functions as an activator, and with severe stress, CrdA functions as a repressor.

DNA-binding Assay of CrdA

Whether CrdA directly binds promoter sequences was further tested using a gel

shift assay. CrdA was over-expressed in E.coli, then purified and dialyzed (see Materials

and Methods part). Dialyzed and undialyzed solution was centrifuged at 100,000 g for 15

min. CrdA was present in the soluble fraction indicating the purified protein was soluble

(Fig III.14). Binding between CrdA and PcrdAB (the 255 bp fragment between crdA and

crdB) was assayed. The protein and DNA form a large complex and a shift was observed

at the top of the gel. The shift disappears partly or completely when adding competitor

DNA such as polydIdC or a M. xanthus asgB fragment into the reaction. The results

suggest that binding between CrdA and PcrdAB may be non-specific, which can be

inhibited by other DNA fragments. Further analysis of larger promoter regions is required

to determine if CrdA regulates a promoter in this region.

34

Table III. 1. Numbers of Beta-lactamases in Different Organisms

Organism Number of Beta-lactamases

E.coli K12 1

Staphylococcus aureus subsp. aureus JH1 14

Pseudomonas aeruginosa PAO1 1

Helicobacter pylori Shi470 0

Bacillus subtilis subsp. subtilis str. 168 2

Mycobacterium tuberculosis CDC1551 7

Streptococcus pneumoniae TCH8431/19A 5

Haemophilus influenzae 86-028NP 1

Klebsiella pneumoniae 342 10

Myxococcus xanthus DK 1622 29

35

Table III.2 Sonication and Heat Resistance of Ampicillin-induced crdA mutant Cells

Treatment Germinated spores (per ml)

None ( 7.35±0.64 ) x 106

Sonicate for 30 s and heat at 50oC for 2 h 0*

Note: * - Sonicated and heated spores were diluted 105 times and plated, no colonies grow.

36

Table III.3 Sonication and Heat Resistance of Glycerol-induced Spores

Strain Germinated spores (per ml)

WT ( 9.4±0.85 ) x 106

crdA ( 9.7±0.42 ) x 106

Note: P = 0.698 > 0.05. P is the probability associated with a Student’s t-Test, which determines whether two samples are likey to have come from the same two underlying population that have the same mean.

37

REC AAA ATPase HTH

Fig III.1. Predicted Domains in crdA, crdA contains a signal receiver (REC) domain, an AAA ATPase domain and a helix-turn-helix (HTH) DNA-binding domain. The REC domain is a CheY-homologous receiver domain, which receives the signal from the sensor partner in a two-component system and contains a phosphor-acceptor site that is phosphorylated by histidine kinase homologs. The AAA ATPase domain is characterized by a conserved ATP-binding motif, which is involved in manipulation of protein structure via nucleotide-dependent conformational changes

38

A

GHG motif

B

C-D linker

Fig III.2. Alignment of CrdA and Its Homologs. NtrC family regulators randomly selected from different organisms are aligned. Ten of them have long C-D linker, without GHG motif , which are the signs of positively regulated NtrCs. Ten of them have GHG motif and short C-D linker, which are the signs of negatively regulated NtrCs. CrdA has a both GHG motif and a long C-D linker, suggesting it may regulate gene expression using elements of both mechanisms. (A) The red-line square represents the GHG motif. (B) The blue-line square shows the C-D linker.

378 390 400 410 420 430 440 450 460 470 4(378)TVMAPGQTIEVADLPPEMRDR------PERE-----------------------------MPVAWVDGLAVEADRLIATSPGEVFDRLTREFERTLIRRALTANtrC A. aromaticum(366)TVMAPGQTVEIKDLPQDLVEERVHAPQPAQNRGCSGIGGSPSDGLYRACPGVASASADGAAGASWITLLETEAAQMLASEQPEVMDILGRQFEAALIKVALKHNtrC H. seropedicae(364)TVMAAGQEVLIQDLPGELFESTVAES------------------------------TSQMQPDSWATLLAQWADRALRSGHQNLLSEAQPELERTLLTTALRHNtrC E. coli(368)TVMASGREVHIDDLPPELLTQPQDS----------------------------------APAANWEQALRQWADQALGRGQSNLLDSAVPAFERIMIETALKHNtrC P. aeruginosa(369)TVMASSREVLIGDLPPELLNLPHDA----------------------------------APVTNWEQALRQWADQALARGQTSLLDSAVPSFERIMIETALKHNtrc P. putida(369)TVMASGQEILPQDLPPELLKEPTSIN------------------------------PMAKGSQDWQSALTEWIDQKLSEGNSDLLTEVQPAFERILLETALRHNtrC S. oneidensis(370)TVMASSQEVLPSDLPPELFSTPIPQ--------------------------------QQHQITDWQEQLSSWVENQLNKGEEDILNKVIPNVERILLDKALHHNtrC V. fischeri(370)AALYPQDVITASVIDGELA---PPSVSPGAAVQQGV---------------------DNLGGAVEAYLSSHFQGFPNGVPPPGLYHRILKEIEVPLLTAALAANtrC B. japonicum(367)TALYPQDVITREIIENELRSEIPDSPIEKAAARSGS---------------------LSISQAVEENMRQYFASFGDALPPSGLYDRVLAEMEYPLILAALTANtrC S. melilot i(366)MVTSAEAEITRAEVEAVLGN--QPAMEPLKGGGEG----------------------EKLSSSVARHLRRYFDLHGGALPPPGVYQRILREVEAPLIEIALDANtrC R. sphaeroides(340)AILCEGPIVTRTDALELLPRGRNVPPPAPVETPASPLPPPSPE------AAVVLASSTPAPAIAAPVATEPPAPVGFRPRADRTFREQVEDAEREIIQHVLSHCrdA M. xanthus(367)VLFSEGKFIDRGELSCLVNS-----------------------------------------------------------KGIKNKHKSIKEIEKEEIIKVLKENtrC1 A. aeolicus(367)LILSDGPTITARDVQRYVHPGSTSGSG----------------------------------------------SLQELIERYPTFAEFRDAAEKLFLEHKLRENtrC R. marinus(373)LILVEGDVIEGDDIDQFVQPGGN-GDG----------------------------------------------PTQELIEAYNDFSDARDQFEKHFIQHKLHENtrC S. ruber(372)LIMYAGDEVGPEHLPGDVEAPMAGGHERG-------------------------------------------------VGWDDDFKNARARFEKDFLTQKLEQNtrC D. retbaense(370)VIMTPGKVITPDQVPDTIGSAAGEAHR------------------------------------------------PGAPLELNSLREAREGFEREFILQKLEENtrC G. metallireducens(368)VIMTPGRTITVNQIPDYIGAGEATREMGG--------------------------------------------SKPGSALELSSLREAREEFEKEFIIQKLEENtrC G. uraniireducens(368)VIMSPSQTISSADLPSSVLAAGPSAQP----------------------------------------------TKFDLPASDLSLRQAREEFEKEFILQKLQENtrC P. carbinolicus(367)ALGVEGNLGVPAAAPASSG---------------------------------------------------------------ATLPERLERYEADILKQALTADctd S. melilot i(373)VIMTPGQRIGPRDLPLDFLNRLPKPPE------------------------------------------------EAGPYQCATLREARSVFERAYLLRKLDENtrC S. fumaroxidans(368)AILCDGPEIGADDVVAMLPGAR--------------------------------------------------RPRGDRLRAGAAFHELVEEAEREIVLAALEANtrC A. dehalogenans(368)

1 10 20 30 40 50 60 70 80 90(1)---MNTVWIVDDDRSIRWVIEKALSRENISHRSFASAGEALTALETAPHPPKVLVSDIRMPGESGLGLLQRVKTLHPHLPVIIMTAYSDLESAVSAFQGNtrC A. aromaticum (1)---MKPIWIVDDDESIRWVLEKALARENLATQSFSSARDAIAALQNG--TPQVLVSDIRMPGASGLELLQTVKAKHPGIPVIIITAFSDLDSAVSAFQGNtrC H. seropedicae (1)-MQRGIVWVVDDDSSIRWVLERALAGAGLTCTTFENGAEVLEALASK--TPDVLLSDIRMPGMDGLALLKQIKQRHPMLPVIIMTAHSDLDAAVSAYQQNtrC E. coli (1)MSRSETVWIVDDDRSIRWVLEKALQQEGMTTVSFDSADSVIGRLGRQ--QPDVIISDIRMPGASGLDLLAQIRELHPRLPVIIMTAHSDLDSAVASYQGNtrC P. aeruginosa (1)MSRSETVWIVDDDRSIRWVLEKALQQEGMTTQSFDSADGVMGRLARQ--QPDVIISDIRMPGTSGLDLLAQIREQHPRLPVIIMTAHSDLDSAVASYQGNtrc P. put ida (1)MRISEQVWILDDDSSIRWVLEKALQGAKLSTASFAAAESLWQALEIS--QPRVIVSDIRMPGTDGLTLLERLQIHYPHIPVIIMTAHSDLDSAVSAYQANtrC S. oneidensis (1)-MSKGFIWVVDDDSSIRWVLEKTLTSTNMMCESFGDAESVIQALERN--VPDVIISDIRMPGMDGLSLLHHIQENYPELPVIIMTAHSDLDAAVSAYQKNtrC V. fischeri (1)-MPAGSILVADDDTAIRTVLNQALSRAGYEVRLTGNAATLWRWVSQG--EGDLVITDVVMPDENAFDLLPRIKKMRPNLPVIVMSAQNTFMTAIRASERNtrC B. japonicum (1)-MTGATILVADDDAAIRTVLNQALSRAGYDVRITSNAATLWRWIAAG--DGDLVVTDVVMPDENAFDLLPRIKKARPDLPVLVMSAQNTFMTAIKASEKNtrC S. melilot i (1)--MDGTVLVADDDRTIRTVLTQALTRAGCKVHATSSLMTLMRWVEEG--KGDLVISDVVMPDGNGLEALPRISKLRPGLPVIVISAQNTIMTAIQAAEANtrC R. sphaeroides (1)--MPASVLIVDDEKNILLTLSQSLQLAGYQTHLANSGQVALDVVSAR--PVDAVLMDVKMPDMDGLTALAKLTELKPELPVIMMSGHGTIDTAVKATQLCrdA M. xanthus (1)----MNVLVIEDDKVFRGLLEEYLSMKGIKVESAERGKEAYKLLSEK--HFNVVLLDLLLPDVNGLEILKWIKERSPETEVIVITGHGTIKTAVEAMKMNtrC1 A. aeolicus (1)--MAATILVVDDERSIRRTLREILEYEGYAVEEAADGDEALEKLREG--RYDLVLLDIKMPRRDGMEVLRTLAAEQPELPVVMISGHGTIETAVEATRLNtrC R. marinus (1)---MPTILVVDDEASIRRTLREILEYEDFGVEEAVDGEEALVALREN--AYDLVILDVKMPKMDGMEVLETIADEGYEVPVLMISGHGTIETAVESTKLNtrC S. ruber (1)--MQARILVVDDELDIRVSLSGILEDEGHTVMEADSGEAGLTAMAGK--EIDLVFLDIWLPGMDGLAVLERLRQEWPDIPVIMISGHGTIETAVSAIKNNtrC D. retbaense (1)--MNETILVVDDEQNIRTALAGILEDEGYRPVFAKDGLEALDMAKKE--NPDLVLLDIWMPRLDGLETLQALKEFHPLLTVVMMSGHGTIETAVKSTKLNtrC G. metallireducens (1)--MSATILVVDDEESIRTSLAGILEDEGYRTLFAVDGVEALSVVQQE--MPALVLLDIWMPRMDGMETLQKLKELYPVLTVIMMSGHGTIETAVKSTKMNtrC G. uraniireducens (1)---MKTILIVDDEQSIRESLDGILQDEGFRTLSAETGEDALTLLCGE--NPDLILLDIWLPGMDGLETLRRIRDNDPEQIVIMMSGHGTIETAVKATKLNtrC P. carbinolicus (1)MSAAPSVFLIDDDRDLRKAMQQTLELAGFTVSSFASATEALAGLSAD--FAGIVISDIRMPGMDGLALFRKILALDPDLPMILVTGHGDIPMAVQAIQDDctd S. melilot i (1)--MKPKILVVDDEISILQSLRGVLQDEGYRIGVAASGEEALEELRRD--TPDLMLLDIWMPGMDGLAVLEEIKKSHAHLPVIIISGHGNIETAVKATRMNtrC S. fumaroxidans (1)--MPATVLVVDDERNIQLTLSRALSMEGYAVETASGGREALEKLAAL--PVDVVVMDVRMPDLDGLAVLQKARETRPELPVVIMSGHGSIDTVRSAFKLNtrC A. dehalogenans (1)

39

Fig III.3 Predition of Coiled coils in CrdA. The sequence of CrdA is analyzed by

software COILS to predict coiled coils. The height of peaks represents the possibility of a coiled-coil structure. Scanning windows of 14, 21 and 28 residues are all tested. Each algorithm predicts a high possibility of coiled-coil structure between about 110 ~ 150 residues, which corresponds to the region between the receiver domain and the ATPase domain of CrdA.

40

Fig III.4. Comparison of crdA-crdB gene neighborhoods in M. xanthus and Anaeromyxobacter Fw109-5. The genome of Anaeromyxobacter has a crdA - crdB fragment similar to that in M. xanthus. The crdA homolog is in an operon including other four genes, one of which is an ntrB homolog. These four genes also exist in the genome of M. xanthus. The crdB homolog is next to crdF, crdG and crdH homologs. The chemosensory genes of the M. xanthus Che3 system are missing in Anaeromyxobacter

41

Fig III.5. Structures of Beta-lactamases in M. xanthus. The domains are predicted by MiST2 database. Each predicted domain is labeled in a white, bordered box. “Lactamase B” domain represents Metallo-beta-lactamase superfamily. “Beta-lactamase” domain represents the serine beta-lactamase-like superfamily. “RMMBL” represents RNA metabolising metallo-beta-lactamase. “Rhodanese” represents a single copy of a duplicated domain in rhodanese. It is also found in phosphatases and ubiquitin C-terminal hydrolases. “TRP_2” represents the tetratrico peptide repeat domain, which mediates protein-protein interactions and the assembly of multiprotein complexes. Blue vertical boxes ( ) represent transmembrane regions.

42

Gene Structure Signal Peptide

MXAN_0061 Lactamase B

No


No


Yes


Yes

MXAN_1479 Lactamase B RMMBL

No


No


No


No


No


No


No

MXAN_5359 Lactamase B No

MXAN_5428 LactamaseB Rhodanese

No


Yes

MXAN_5721 Lactamase B No


No


No


No

MXAN_6901 LactamaseB

No

MXAN_1911 LactamaseB No

MXAN_7313 Lactamase B Yes

MXAN_2136 Beta‐lactamase Yes


MXAN_2321 Beta‐lactamase

No

MXAN_2364 Beta‐lactamase TPR_2 No

MXAN_5519 Beta‐lactamase No

43

Figure III.5 - Continued

MXAN_6409 Beta‐lactamase TPR_2 Yes



44

A

0 2 4 6 9 12 15 18 24 h

serin

e be

ta-la

ctam

ase

met

allo

-bet

a-la

ctam

ase

B

0 2 4 6 9 12 15 18 24 h

Fig III.6. Beta-lactamases Expression Pattern during Development and Evolutionary Tree. (A) Left part of the pictures show the evolutionary tree of the betalactamases, which can be divided into metallo-beta-lactamase family and serine beta-lactamase family. Right part of the picture is a heat map indicating beta-lactamase expression patterns during the first 24 hours of development. Green squares represent down-regulation and red ones represent up-regulation. (B) The same date from above is rearrayed based on expression (Cullen and Kirby, unpublished data).

45

CF Amp10

CYE Amp10

WT

crdA

crdA pCrdA

crdA pCrdA D53A

crdA pCrdA D53E

Fig III.7. Affect of ampicillin on growth and development of M. xanthus. 7.5x photographs of cells were taken on CYE or CF plates with 10 ug/ml ampicillin at 48 h. The wildtype strain, the crdA mutant, and strains over-expressing wildtype CrdA, CrdAD53A, and CrdAD53E in the crdA mutant background were assayed.

46

CF

Amp5 EDTA0.5 CYE

Amp5 EDTA0.5

WT

crdA

crdA pCrdA

crdA pCrdA D53A

crdA pCrdA D53E

Fig III.8. Affect of Ampicillin and EDTA on Growth and Developmental of M. xanthus cells. 7.5x photographs of cells were taken on CYE or CF plates with 0.5 mM EDTA and 5 ug/ml ampicillin at 48 h. The wildtype strain, the crdA mutant, and strains over-expressing wildtype CrdA, CrdAD53A, and CrdAD53E in the crdA mutant background were assayed.

47

CYE Amp50

WT

crdA

crdA pCrdA

crdA pCrdA D53A

crdA pCrdA D53E

Fig III.9. Affect of Ampicillin on growth of M. xanthus cells. 7.5x photographs of cells were taken on CYE plates with 50 ug/ml ampicillin at 48 h. The wildtype strain, the crdA mutant, and strains over-expressing wildtype CrdA, CrdAD53A, and CrdAD53E in the crdA mutant background were assayed.

48

CYE

Amp 1000

WT

crdA

crdA pCrdA

crdA pCrdA D53A

crdA pCrdA D53E

Fig III.10. Ampicillin-induced Sporulation. Cells were incubated in CYE broth with 1 mg/ml ampicillin for 12 h. The wildtype strain, the crdA mutant, and strains over-expressing wildtype CrdA, CrdAD53A, and CrdAD53E in the crdA mutant background were assayed. 400x photographs were taken by microscopy.

49

CYE

Glycerol 0.5

WT

crdA

crdA pCrdA

crdA pCrdA D53A

crdA pCrdA D53E

Fig III.11. Glycerol-induced Sporulation. Cells were incubated in CYE broth with 0.5 M glycerol for 12 h. The wildtype strain, the crdA mutant, and strains over-expressing wildtype CrdA, CrdAD53A, and CrdAD53E in the crdA mutant background were assayed. 400x photographs were taken by microscopy.

50

0

500

1000

1500

2000

2500

3000

3500

4000

4500

5000

CYE

CYEAmp100

Beta‐lactam

ase activity

(mmol x m

in‐1 x m

g‐1).

WT crdA crdA/pCrdA

Fig III.12. Beta-lactamase Activity Induced by Ampicillin. Cells were incubated in CYE broth at 32oC with or without 100 ug/ml ampicillin overnight. The wildtype strain, the crdA mutant, and strains over-expressing wildtype CrdA in the crdA mutant background were assayed.

51

0

2000

4000

6000

8000

0.1 M 0.2 M

Beta‐lactam

ase

(mmol /g/min)

Glycerol

WT

crdA

0

500

1000

1500

2000

1 mM 3 mM

Beta‐lactam

ase

(mmol /g/min)

EDTA

WT

crdA

A B

Fig III.13. Induction of Beta-lactamase Activity by Glycerol and EDTA. (A) Cells were incubated in CYE broth at 32oC with 0.1 M or 0.2 M glycerol overnight. (B) Cells were incubated in CYE broth at 32oC with 1 mM or 3 mM EDTA overnight.

52

Fig III.14. lacZ Expression of the Promoters in WT and the crdA Mutant Background. (A) Strains containing a lacZ gene without a promoter or with the M. xanthus pilA promoter in the wildtype background were assayed as negative and

density

ter

positive controls. (B) Cells were harvested from liquid CYE when the cell was 100-150KU, or CF plates after 48 h of incubation. (C) Cells were harvested before and after incubation in liquid CYE with 200 ug/ml ampicillin for 4 h. (D)Cells were harvested from CF plates with or without 0.5 mM EDTA and 5 ug/ml ampicillin (CFEA) after 48 h of incubation. (E). Under each condition, the promoactivities in the crdA mutant background are divided by those in the wildtype background.

53

A

B

C

D

54

Figure III.14 - Continued

E

0.00

0.50

1.00

1.50

2.00

2.50

PcrdS PcrdA PcrdB PcheB3

CYE

CYEAmp

CF

CFEA

crdA / W

T

55

dialyzed undialyzed S P S P

132 kD

78

46

33

18

Fig III.15. Solubility and Purity Analysis of CrdA. 200 ul of dialyzed (~1mM) and undialyzed (~4mM) CrdA were centrifuged at 100,000 g for 15 min, 5 ul of the samples from the soluble fraction (S) and the pellet fraction (P) were loaded into the 12% polyacrylamide gel.

56

Positive control 1 2 3 4 5 6 + + + + CrdA - + + + PcrdAB - - asgB polydIdC Competitor

Fig III.16. CrdA and crdAB Promoter-binding Assay. Lane 1 and 2 are positive controls using Epstein-Barr Nuclear Antigen (EBNA) Extract and biotin EBNA control DNA. Lane 1 is DNA only. Lane 2 is DNA and EBNA extract. Lane 3 to 6 all have 0.25 nM 257 bp crdA and crdB promoter fragment, which is from -304 to -48 from the translational start site of crdB. Lane 3 has 200 nM CrdA only. Lane 4 is PcrdAB and 200 nM CrdA. Lane 5 is PcrdAB, 200 nM CrdA, and 980 nM asgB fragment. Lane 6 is PcrdAB, 200 nM CrdA, and 250 ng polydIdC.

CHAPTER IV

57

CONCLUSIONS AND FUTURE DIRECTIONS

General Discussion

The focus of this dissertation was to study the function of CrdA. CrdA is a

sigma54-dependent transcriptional regulator homolog, which belongs to the NtrC family.

The alignment of CrdA and other NtrC proteins shows that the structure of CrdA is

different from those that have been well studied. CrdA has a long linker between the

central domain and the DNA-binding domain compared to those NtrC homologs which

have been crystallized. It implies that the function and structure of CrdA may be different

from what is known for the NtrC homologs in other organisms.

Previous studies from our laboratory demonstrated that crdA is downstream of the

che3 pathway and may be involved in regulation of developmental genes (Kirby, 2003).

However, the developmental defect was not as apparent after our laboratory moved to the

University of Iowa. The change of this phenotype may be due to the change of water we

used. It implies that CrdA may function in a signal transduction pathway, which is able to

sense subtle changes in the environment.

Previous data, in silico analysis and new findings about CrdB’s function suggest

that CrdA may function in the regulation of cell wall stress. Therefore, we hypothesize

that CrdA is involved in a signal transduction pathway, which monitors the integrity of

peptidoglycan envelop. We used ampicillin and EDTA as stress inducers because

ampicillin is a beta-lactam drug, which affects peptidoglycan synthesis and has proved to

induce starvation-independent sporulation, and the crdB mutant has defect in response to

EDTA-induced stress. Experiments in this dissertation have demonstrated that under both

growth and developmental conditions, the crdA mutant is more sensitive to ampicillin

than the wild-type strain. In liquid CYE with high ampicillin concentrations, the wild-

type strain keeps growing, whereas the crdA mutant becomes spherical. Whether these

spherical cells are immature spores or are more similar to spheroplasts remains to be

58

elucidated. Under starvation conditions with ampicillin and EDTA, the crdA mutant

forms fewer and smaller aggregates compared to the wild type. When we added

ampicillin or EDTA separately into the plates, the crdA mutant didn’t show any obvious

defect (data not shown). One possible explanation for the synergistic effect is that EDTA

affects a large number of proteins and induces fluidization and destabilization of the cell

membrane, which helps ampicillin penetrate the periplasm more easily and therefore

causes much more damage to the cell wall. Under the conditions we have tested, the crdA

mutant shows normal glycerol-induced sporulation, which suggests that CrdA responds

to ampicillin specifically.

In many gram-negative rod-shaped bacteria, production of chromosomally-

encoded beta-lactamases is induced by beta-lactams to protect the cell wall. As the crdA

mutant is very sensitive to ampicillin, which is a beta-lactam, we assayed for beta-

lactamase activity. As we expected, the crdA mutant is not able to produce beta-

lactamases as much as the wild-type strain when ampicillin is present. Adding glycerol

and EDTA can still enhance beta-lactamase activity in the crdA mutant, but not to the

level of the wild type. It suggests that some beta-lactamases are not regulated by CrdA

and may explain why the crdA mutant shows a normal glycerol sporulation phenotype.

qPCR is being performed to study which beta-lactamases are under the control of CrdA.

NtrC proteins typically regulate the ntrBC operon (Shiau,1993). In order to

determine if CrdA regulates itself, the che3 operon or the crdS gene was studied by

measuring lacZ expression from these promoters. The results show that CrdA positively

regulates the che3 operon in CYE liquid, cheB3 and itself during starvation-dependent

development, and negatively regulates the che3 operon, cheB3, and itself during

starvation under cell wall stress. As CrdA can switch between phosphorylated and

unphosphorylated states, it is possible that in one state, CrdA functions as an activator

and in the other state, it functions as a repressor. We also noticed that, under the

conditions tested, all the promoters have a certain level of expression in the crdA mutant

59

background. This observation suggests that without CrdA, these genes are expressed at

basal level. It is possible that other transcriptional mechanisms are involved in regulation

of these genes.

Whether CrdA directly binds the promoter sequence between crdA and crdB was

tested using a gel shift assay. Under all the conditions we have tried, CrdA binds the

DNA but may be non-specific. It may due to the inappropriate in vitro condition, or other

proteins may be required for transcription.

Model for CrdA

We hypothesize that CrdA is regulated by both the CrdS pathway and the Che3

system. CrdS, the NtrB homolog, may phosphorylate CrdA corresponding to input from

the PBP (Mxan_5181). Phosphorylated CrdA activates the transcription of genes

necessary for resistance to envelope stress, including beta-lactamases. CrdB may sense

changes in the peptidoglycan layer and transfer the signal to the che3 system. CheA3 then

inhibits the phosphorylation of CrdA by an unknown mechanism. Dephosphorylated

CrdA can then act as a repressor. Therefore, the ratio between phosphorylated and

unphosphorylated CrdA determines whether the transcription is activated or repressed. It

may vary for different promoters.

When ampicillin is added into the CYE culture of the wild-type strain, it

inactivates the PBP, which is sensed by CrdS. CrdA is then phosphorylated and activates

the transcription of genes which stabilize the cell wall. At this time, the crdB and cheB3

genes are down regulated by an unknown CrdA-independent mechanism. Therefore, the

inhibition of phosphorylation of CrdA by CheA3 is relieved, which increases the amount

of phosphorylated CrdA. In the crdA mutant, genes that stabilize the cell wall are not

induced and synthesis of the peptidoglycan layer is quickly interrupted. Overexpression

of wildtype CrdA or CrdAD53E (constitutively active form of CrdA) is able to complement

60

this defect. CrdAD53A can not be phosphorylated, and therefore, overexpression of this

protein can not activate transcription of down-stream genes.

If there is damage to the cell wall, it may be better for the M. xanthus cells to

enter a starvation-independent sporulation program for protection and energy

consumption. Therefore, we hypothesize that when peptidoglycan damage occurs

quickly, the majority of CrdA will be dephosphorylated by CheA3 and the protein will

function as a repressor. This proposal is supported by the transcriptional results on the

crdA, crdB and cheB3 genes. One interesting question here is that why M. xanthus cells

want to employ a chemosensory system to monitor the cell wall change instead of a

prototypical one/two-component system. The biggest difference between a chemosensory

system and a two-component system is that a chemosensory system is able to adapt the

cells to the existing signal and a two-component system is not. Therefore, a

chemosensory system can sense the changing rate of the signal level, instead of the

absolute value of the signal level. Our hypothesis is that M. xanthus cells always need to

sense signals, such as cell wall integrity, etc, to determine whether they want to keep

replicating or start the sporulation process. How fast the cell wall is degraded may be

more important than how much cell wall is degraded, because if the cell wall damage is

severe, but peptidoglycan is no longer being degraded, then the cell would be able to fix

any damage by producing new peptidoglycan. Thus the Che3 system provides an

advantage over a simple switch-like mechanism.

This model is based on previous data, and results presented herein. To further

explore the mechanism of this system and confirm aspects of the model, many

experiments will need to be performed in the future. First, characterization of the crdS

operon is required. Whether ampicillin is able to bind the PBP and whether binding

triggers the phosphorylation of CrdA by CrdS needs to be assayed. Secondly, the target

genes of CrdA need to be determined. Whether CrdA directly regulates the transcription

of beta-lactamase genes is currently being tested by qPCR. If targets are confirmed,

61

further experimentation will determine if CrdA directly binds to the promoter sequences.

It is also unknown if CrdA directly regulates other cell-wall related genes to help stabilize

the peptidoglycan layer. The overexpression of beta-lactamases to complement the crdA

mutant’s phenotype would be further evidence that CrdA is needed for the transcription

of those genes. Third, the mechanism for Che3 regulation of CrdA remains unclear. More

biochemical experiments are needed to elucidate the relationship between CheA3 and

CrdA.

62

P CrdC

CH3CH3

PCrdA

CrdS

CrdB

CheR3

PCheB3

CheA3

W

Mcp3A

Pbp1A

ATP

Mcp3B

genes which help stabililize peptidoglycan layer

CrdA

Fig IV.1 Model for Chemosensory Regulation of CrdA to Affect Cell Wall Stability. CrdA is regulated by both the CrdS pathway and the Che3 system. CrdS senses the condition of PBP and phosphorylates CrdA corresponding to the signal. CrdB senses the condition of peptydoglycan layer and transfers the signal to the Che3 system. CheA3 inhibits the phosphorylation of CrdA. When CrdA is phosphorylated, it activates the transcription of genes which help stabilize the peptidoglycan layer. When CrdA is dephosphorylated, it functions as a repressor.

63

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CrdA regulates endogenous beta-lactamase activity in Myxococcus xanthus