IDENTIFICATION OF A SPECIFIC TARGET SEQUENCE

OF A POTENTIAL REGULATORY PROTEIN

IN Pseudomonas aemginosa

by

ANN MARIE LUNA, B.S.

A THESIS

IN

MICROBIOLOGY

Submitted to the Graduate Faculty

of Texas Tech University in Partial Fulfillment of the Requirements for

the Degree of

MASTER OF SCIENCE

Approved

December, 2001

ACKNOWLEDGMENTS

The work presented here could not have been possible without the love, support,

and encouragement from several people in my life. I would first like to thank my family.

My wonderful parents Armando and Mary Lou Luna who at an early age taught me the

importance of an education. Their unconditional love gave me the space I needed to

grow into my own person. I thank my sisters Amanda and Rosanna, and my brother

Alex, who have always encouraged me to pursue my goals while giving me the

confidence to know that I could do it.

I would like to thank my committee members Dr. Jeter, Dr. San Francisco, and

Dr. Hamood for their much appreciated guidance throughout my graduate career. I

particularly want to express my gratitude to Dr. Hamood who taught me the basis of a

good work ethic by example, through his passion for research. I would also like to thank

Dr. Jane Colmer-Hamood for her ability to help anyone with any type of problem, while

doing the many other jobs that keep our lab functional, along with a special thanks to Dr.

Britta Swanson, who helped answer many questions throughout my research. I would

also like to thank Landon Westfall for his contribution to the protein purification aspect

of my project.

I would like to mention the wonderful friendships that I have made over the years

David, Ravi, Florencia, Naomi, Landon, Marisela, and Trevor have made the stresses of

being a graduate student much more tolerable. I would like to make a special mention of

my friends Missy Smith and Eric Graham who have seen me through many good times as

11

well as bad. All of which will always be memorable. Hours and hours of laughter with

them have taught me not to take life so seriously.

My friends Mehnda Lackey and Nancy Carty have been a major influence in my

life over the past two years. We have shared numerous times of frustration, anger,

sadness, laughter, and joy. They are my sisters, and I hope they know how much they

mean to me. I have the utmost respect and admiration for them as research scientists and

friends.

Lastiy, I would like to thank Nathan Simmons, to whom I dedicate this work.

Together we have endured though many difficult times but have always managed to

focus on what is most important and that is each other. The past six and a half years with

him have represented the most wonderful years of my life.

Ill

TABLE OF CONTENTS

ACKNOWLEDGEMENTS ii

ABSTRACT vii

LIST OF TABLES ix

LIST OF FIGURES x

CHAPTER

L INTRODUCTION 1

General characteristics 1

P. aeruginosa as a human pathogen 1

P. aeruginosa in healthy patients 2

Pathogenesis in cystic fibrosis patients 3

Pathogenesis in bum victims 4

Pathogenesis in immunocompromised

hosts 4

Antibiotic Resistance 5

Stages of a P. aeruginosa infection 5

Virulence factors produced by P. aeruginosa 6

Flagellum 6

Pih 6

Exoenzyme S 7

Alginate 7

IV

Proteases 7

Exotoxin A 8

Regulation of Exotoxin A 8

Environmental regulation of exotoxin A production 9

Genetic regulation of exotoxin A production 9

Purpose of this study 12

IL MATERIALS AND METHODS 13

Bacterial strains, plasmids, media and growth conditions 13

Construction of recombinant plasmids carrying the ptxRIptxS intergenic region 13

Isolation of DNA fragments for the Electrophoretic Mobility Shift Assays 14

Preparation of the DNA probes labeled with [y-^^P]-

ATP 15

Preparation of the P. aeruginosa lysate 15

Electrophoretic Mobility Shift Assay 16

Protein Purification 16

Heparin-Sepharose column 17

DNase I Footprinting Analysis 19

Preparation of DNA probe 19

DNase I Reactions 20

Sequencing Reactions 21

T7 Expression System 22

Using the T7 Expressions Experiments to examine the translational product of the P. aeruginosa MvaT homologue 22

T7 Expression experiments 23

m. RESULTS 28

Identification of the ptxR/ptxS Intergenic Region 28

Localization of the Binding Region within the 201-bp Fragment 30

Binding Activity of the 150-bp fragment that overlaps the junction between the 102-bp and the 98-bp fragments 30

Further Experiments to Localize the Binding to the 150-bp

fragment 31

Purification of the Potential DNA-binding proteins 32

Preparation of the PAOl lysate 32

Enrichment of the DNA binding proteins using the Heparin-Sepharose Column 32

Identification of the Potential Binding Proteins 33

Identification of the Binding Sequence for the 16-kDa and 9-kDa proteins within the ptxRIptxS intergenic regions 34

T7 Expression of the P. aeruginosa MvaT protein 35

Analysis of the Putative P. aeruginosa MvaT homologue 36

IV. DISCUSSION 53

REFERENCES 59

VI

ABSTRACT

Exotoxin A is one of the most toxic virulence factor produced by Pseudomonas

aeruginosa. Exotoxin A production by P. aeruginosa is regulated by several genes in

response to different environmental conditions. We have previously characterized the P.

aeruginosa genes, ptxR and ptxS that are divergently transcribed. However, the

mechanism that regulates the expression of either gene in not known. Preliminary

analysis suggested that presence of several potential regulatory proteins that specifically

bind to the ptxRIptxS intergenic region. The present study was conducted to characterize

one of these proteins and to identify the specific nucleotide sequence to which it binds.

Initial DNA gel shift experiments revealed the presence of a specific gel shift band when

the lysate of P. aeruginosa was incubated with a 201-bp fragment within iht ptxRIptxS

intergenic region. Additional experiments, using several overlapping probes within the

201-bp fragment, localized the binding to a 150-bp fragment. The potential binding

protein was identified using the Heparin-Sepharose columns. One eluted column

fraction, which produced the gel shift band, contained only two proteins, which were 9-

kDa and 16-kDa in molecular weight. The amino acid sequence of the first 15 amino

acids of each protein was determined. Computer analysis of the sequences confirmed

that one protein is the previously characterized P. aeruginosa HU protein, while the other

is an uncharacterized protein that carries an 82% homology to the Pseudomonas

mevalonii transcriptional activator, MvaT. DNase I protection experiments using the

Heparin-Sepharose fraction that contains both proteins, produces a 25-bp protected

Vll

region. Analysis of this region revealed the presence of an 8-bp palindromic sequence.

Beside the MvaT homologue. P. aeruginosa carries a hypothetical protein that has 647r

homology to the MvaT. However, P. aeruginosa does not contain a homologue of the

mvaAB operon, which is the target of the MvaT protein. Instead, a hypothetical protein

that has limited homology to the mvaB was detected. This suggests that the HU protein,

the P. aeruginosa MvaT homologue, or both regulate ptxS expression by specifically

binding to the ptxS upstream region.

Vl l l

LIST OF TABLES

2.1 Bacterial strains and plasmids 24

2.2 Oligonucleotides used in PCR synthesis of DNA fragments from the ptxRIptxS intergenic region for Electrophoretic mobility assay 25

IX

LIST OF FIGURES

2.1 Diagram of DNA fragments generated by PCR synthesis, with specific oligonucleotides primers and specific orientation in relationship to ptxR and ptxS 26

2.2 Diagram of DNA manipulations for DNase I Footprinting Analysis 27

3.1 Diagram of the different probes (within the ptxR-ptxS intergenic region) used in the Electrophoretic mobility gel shift assay 37

3.2 Electrophoretic mobility shift assay of 201-bp fragment and PAO1 lysate 38

3.3 Electrophoretic mobility shift assay of the 102-bp and 98-bp fragments with PAOl lysate that was French pressed and concentrated by dialysis 39

3.4 Electrophoretic mobility shift assay of the 150-bp fragment with PAOl lysate 40

3.5 Electrophoretic mobility shift assay of the 75( R) bp fragment with PAOl lysate 41

3.6 Electrophoretic mobility shift assay of the 75(L)bp fragment with PAOl lysate 42

3.7 SDS-PAGE of the 0.6 M KCl Heparin-sepharose colunrn fraction... 43

3.8 Amino acid sequence of first 20 amino acids of the 9-kDa and thel6-kDa proteins 44

3.9 Identification of the open reading frame (ORE) that codes for the P. aeruginosa MvaT homologue 45

3.10 Competitive Electrophoretic Mobility Shift Assay of the 150-bp probe with excessl5-0bp fragment 48

3.11 Determining the specific nucleotide sequence within the ptxS/ptxR intergenic region to which the putative regulatory protein binds 49

3.12 Expression experiment to detect the production of the P. aeruginosa MvaT homologue 51

3.13 Homology comparison between the P. mevalonii MvaT protein and the P. aeruginosa homologues 52

XI

CHAPTER I

INTRODUCTION

General Characteristics

Pseudomonas aeruginosa is a Gram negative, opportunistic, bacterial pathogen. A

typical cell of P. aeruginosa, is rod shaped, and has a single motile flagellum.

Pseudomonas aeruginosa can normally be found in soil and water (Vasil et al., 1986;

Wick et al., 1990b). Because the bacteria have few nutritional requirements, they can

utilize a variety of natural and artificial compounds as carbon energy sources, allowing

easy survival in hot tubs, mop water, in dilute disinfectants as well as on hospital

instruments such as catheters and bedpans (Rolston and Bodey, 1992). Most importantly,

because of its ubiquitous nature, the bacterium can be found in the normal human flora

where it survives against the human immune responses (Wood, 1976).

Pseudomonas aeruginosa can be both invasive and toxinogenic. The pathogenic

nature of Pseudomonas infections makes it an important specimen to investigate and to

understand. The pathogenicity of the bacterium is controlled by numerous environmental

and genetic components that play important roles in the establishment of severe

infections and life threatening diseases.

P. aeruginosa as a human pathogen

Pseudomonas aeruginosa is a gram-negative opportunistic pathogen that causes

various infections such as: nosocomial infections, wound infections, and lung infections

in cystic fibrosis patients (Wood, 1976). An opportunistic pathogen is a pathogen that is

unable to cause disease in healthy, immunocompetent people but can infect a person

whose specific or nonspecific immune defenses have been impaired (Doggett, 1979).

What specifically defines P. aeruginosa as an opportunistic pathogen is that the normal

nonspecific immune defenses of a healthy person can easily guard against initial

attachment of the bacterium. It is when a serious breakdown of this defense occurs that

P. aeruginosa has the ability to attach, colonize and invade the body, and inevitably

progress into fatal systemic diseases (Salyers, 1994). Thus, the probability of establishing

a serious P. aeruginosa infection is greatly increased for persons that are

immunocompromised.

P. aeruginosa in healthy patients

P. aeruginosa is the predominant pathogen in most cases of external otitis,

including "swimmer's ear." The bacteria can be found in the normal ear, but often

inhabits the external auditory canal in response to injury, maceration, inflammation or

merely wet and humid conditions (Bodey et al., 1983). Pseudomonas aeruginosa can

also cause severe infections in the human eye. It is one of the most common causes of

bacterial keratitis and has been isolated as the etiological agent of neonatal ophthalmia

(Bodey et al., 1983; Pollack 1995). If the environment within the eye region is physically

compromised in any way, the bacteria can rapidly proliferate and produce numerous

toxins that can lead to the loss of the entire eye (Bodey et al., 1983; Pollack, 1995). P.

aeruginosa can also be acquired in hospitals and nursing homes, and it accounts for 25%

of all hospital-acquired Gram-negative bacteremias (Salyers, 1994). Urinary tract

infections (UTI) can be caused by Pseudomonas aeruginosa in a hospital setting by the

onset of urinary tract catheterization, instrumentation, or surgery (Salyers, 1994). The

bacterium also appears to be among the most adherent of common urinary pathogens

(including E. coli) to the bladder uroepithelium (Doggett, 1979; Bodey et al., 1983).

Pathogenesis in cystic fibrosis patients

Cystic fibrosis is a genetic disease associated with a defect in chloride secretion

(Govan, 1992; Campa et al., 1993). Cystic fibrosis patients are highly susceptible to

infection since the disease provides an optimal environment for the bacterial cells to

easily attach and colonize (Wood, 1976). This defect causes an increase in the

production of unusually thick mucin in the lungs. Thick mucin not only allows bacterial

cells to attach and colonize the inner lining of the lung; but also hinders the movement of

important phagocytes, neutrophils, and macrophages into the area (Patrick and Larkin,

1995; Hillman, 1997). After a P. aeruginosa infection is established, patients undergo

several cycles of antibiotic treatment until the bacterium eventually become resistant

(Brown, 1975; Bryan, 1975). Because of this resistance, the patient soon dies as control

of the infection by antibiotic therapy diminishes and the patient can no longer tolerate the

infection (Govan et al., 1992, 1996). Statistics have shown that P. aeruginosa infections

cause 90% of deaths in cystic fibrosis patients (Salyers, 1994).

Pathogenesis in bum patients

Pseudomonas aeruginosa causes the most severe and life-threatening infections in

bum patients (Campa et al., 1993). Bum victims are highly susceptible to infection

followed by septicemia due to the loss of epithelial layers. The skin provides a primary

defense mechanism to prevent invasion of bacteria into the body. Once the skin barrier is

breached, the progression from infection of burned skin tissue followed by systemic

invasion of the bacteria can quickly become fatal due to multiple organ failure (Patrick

and Larkin, 1995). It is critical for a bum victim that has survived the severity of a

thermal injury to undergo extensive precautions in preventing the establishment of a P.

aeruginosa infection.

Pathogenesis in immunocompromised hosts

Immunocompromised people typically include patients undergoing

chemotherapy, intravenous drug users, bum victims, people with cystic fibrosis (a genetic

disorder), and AIDS patients (Rolston and Bodey, 1992; Gupta and Griscelli, 1993).

Other diseases and infections associated with Pseudomonal infections include

endocarditis, bacteremia, and infections of the ears, eyes, bones and joints, urinary tracts

and gastrointestinal (Doggett, 1979; Bodey et al., 1983; Gupta, 1993). Infections become

fatal once the bacteria have established direct invasion into the bloodstream. Bacteremia

is common among immunocompromised patients, particularly in AIDS patients and

people that have suffered severe bums (Wood, 1976).

Antibiotic Resistance

The infection cannot be eUminated by antibiotic treatment (Brown, 1975; Bryan,

1989). Such systemic infections are associated with cystic fibrosis patients, bum patients

and cancer patients undergoing chemotherapy. The primary antibiotics that have been

shown to be effective against P. aeruginosa infections belong to the p-lactam and

aminoglycoside families (Brown, 1975). Flouroquinolones such as ciprofloxacin have

also been shown to be useful agents against P. aeruginosa infection (Bryan, 1989;

Campa, 1993).

Stages of a P. aeruginosa infection

A serious Pseudomonas infection undergoes three stages: (1) bacterial attachment

and colonization, (2) local tissue invasion, and (3) disseminated systemic disease

(Pollack, 1995). In the bacterial attachment and colonization stage, the bacteria utilize

cell surface-associated components to attach and to colonize. Local invasion involves the

spread of the bacteria throughout the tissue in search of nutrients after colonization.

Treating the infection during local invasion is critical, since hindrance of the third stage is

extremely difficult. The third stage is systemic dissemination, meaning the infection has

been incorporated into the blood stream. Once sepsis has been established, death is

inevitable due to multiple organ failure (Lory et al, 1983; Roth et al., 1995).

Vimlence factors produced by P. aeruginosa

Pseudomonas has numerous vimlent factors that play important roles in the

development of each stage of infection. Some vimlence factors are merely outer

structural components of the bacterium itself that establish and develop a strong defense

mechanism from the host response. Other stmctures are critical components that initiate

epithelial cell injury of the host organism (Liu. 1973; Nicas and Iglewski, 1985; Holder,

1985).

Flagellum

P. aeruginosa contains a single, polar flagella, which rapidly moves the bacterium

through its surroundings (Montie, 1998). The role of the flagellum in vimlence is,

therefore, associated with directed motility. Directed motility is essential for the

progression of systemic P. aeruginosa infections, as well as evasion of host cell defenses.

Pih

Pili are cell-associated virulence factors that are involved in the initial attachment

and colonization of the bacterial cell. These small, hair-like projections protmde from

the periphery of the cell and bind to receptors on epithelial cells (Woods, 1980). The

retractable nature of the structure allowing other cell-associated factors to produce their

effect by bringing the bacterial cell and the eukaryotic cell in close proximity (Hahn,

1997).

Exoenzvme S

Exoenzyme S is an ADP-ribosylating enzyme that causes cell death in numerous

eukaryotic cells (Baker et al, 1991). Exoenzyme S has been shown to play a role in

adherence to epithelial cells in conjunction with the pili of P. aeruginosa (Baker et al,

1991). Localization to the cell membrane allows exoenzyme S to be exposed to the

external environment where the protein behaves as ligand to buccal cells (Hillman, 1997).

Alginate

One of the most important cell-associated vimlence factors of P. aeruginosa is the

alginate capsule or outer slime layer (Martin et al, 1993). The alginate capsule is an

exopolysaccharide that consists of repeating polymers of glucuronic and mannuronic acid

(Gacesa, 1998). The slime layer provides a means of adhesion as well as protection from

host defenses (Gacesa, 1998). Alginate production is most commonly seen in strains that

have been isolated from the lungs of cystic fibrosis patients (Govan et al, 1992; Govan et

al , 1996).

Proteases

The extracellular virulence factors produce by Pseudomonas aeruginosa include

elastase A and B and alkaline protease (Ohman and Iglewski, 1980a; Blackwood et al,

1983; Bergen, 1985; Gambello and Iglewski, 1991). These virulence factors are

associated with the physical breakdown of host cellular membranes. Elastase has been

shown to be an important virulence factor in bum wound infections (Rumbaugh et al.

1999). Elastase contributes directly in the invasiveness of P. aeruginosa by the

degradation of elastin (Campa et al, 1993). Mutants defective in elastase production were

not capable of spreading effectively through the burned skin tissue and therefore did not

disseminate into the bloodstream (Ohman and Iglewski, 1980a; Rumbaugh et al, 1999).

Alkaline protease interferes with fibrin formation, allowing the bacterium to spread

effectively through tissue (Campa et al, 1993).

Exotoxin A

Pseudomonas aeruginosa production of exotoxin A is thought to be the most

toxic of the secreted factors (Iglewski and Kabat, 1975). Exotoxin A is an ADP-ribosyl

transferase enzyme that catalyzes the transfer of the ADP-ribosyl moiety of the NAD"

onto elongation factor-2 of the host cells, thus causing the termination of the host protein

synthesis process and ultimately cell death (Iglewski and Kabat, 1975). Exotoxin A acts

at the local level, allowing the bacteria to spread within the surrounding tissue and to

disseminate at the systemic level (Chen et al, 1987; Montie, 1998). Because of its

apparent importance in the infection process, the biology of exotoxin A was investigated

further at the molecular level

Regulation of exotoxin A

The production of exotoxin A is regulated by multiple factors, including

environmental and genetic elements (Cross et al, 1980; Nakazawa, 1996). Certain

factors allow maximal expression of toxA, while others suppress its expression. Thus,

8

this multiple level of control on toxin expression is vital to the pathogenicity of

Pseudomonas aeruginosa (Nakazawa, 1996).

Environmental regulation of exotoxin A production

During initial attempts in the purification of the exotoxin A protein. se\eral

environmental parameters have been determined to maximize the yield (Liu, 1973). For

maximal production of exotoxin A in Pseudomonas aeruginosa PAOl, cultures are

grown to stationary phase at 32°C with rigorous aeration in TSB-DC medium (Liu,

1973). TSB-DC is dialyzed trypticase soy broth that has been treated with Chelex to

remove iron and that is supplemented with glycerol and glutamic acid (Ohman et al.

1980a). Specific cations in the culture medium are known to influence the production of

exotoxin A. The addition of calcium and glycerol increases ToxA. while minimal

amounts of cobalt, copper, or magnesium decrease the amounts of ToxA produced (Liu.

1973; Ohman and Iglewski. 1980a). The amount of ToxA is significantiy reduced by the

presence of iron (Bjom et al, 1978: Frank et al, 1989; Oschner and Vasil. 1995).

Genetic regulation of exotoxin A production

The genetic regulation of exotoxin A is a highly complex and multifaceted

process that involves numerous positive and negative regulators. Although many of these

regulator}' genes ha\'e been identified, the mechanisms b\ which many of these genes

contribute to pathogenicity are still unknown. The known positive regulators include

regA, regB, vfr, lasR, pvdS, Siud ptxR (Hedstrom et al. 1986; Wick et al, 1990a; Storey et

al, 1991; West et al, 1994b; Hamood et al, 1992, 1996; Oschner et al, 1996; Colmer et

al , 1997). The known negative regulators art fur and ptxS (Prince et al, 1991; Colmer

and Hamood, 1998; Swanson, 2000a).

fur

The ferric uptake regulator, r , is an important negative regulator of exotoxin A

production in Pseudomonas aeruginosa (Prince et al, 1991, 1993). The Fur protein

regulates the expression of siderophores (iron chelators) (Gensburg et al, 1992; Prince et

al , 1993, 1991). When iron is available. Fur binds to ferrous iron (Fe " ) (Bagg and

Neilands, 1987). This complex then binds within the siderophore upstream region and

represses transcription (Bagg and Neilands, 1987). When iron is low. Fur does not form

a complex with Fe " and transcription of siderophore genes are active (Bagg and

Neilands, 1987). Exotoxin A production is negatively regulated by iron through the fur

gene (Prince et al , 1991, 1993).

pvdS

The pvdS gene is a positive regulator of the production of siderophore pyoverdine

(Miyazaki et al, 1995). Transcription of pvdS is negatively regulated by the presence of

iron and the Fur protein (Miyazaki et al, 1995; Oschner et al, 1996; Stintzi et al, 1999).

PvdS is thought to enhance exotoxin A expression, possibly through the gene ptxR (Vasil

et al , 1998).

10

vfr

P. aeruginosa positive regulator Vfr enhances toxA transcription through regA

(West et al , 1994a). The regA gene is essential in activating the transcription of the toxA

gene.

lasR

LasR is one of the major components in the Quomm sensing system. It was

originally identified as an elastase regulator, promoting both lasA and the lasB expression

(Gambello and Iglewski, 1991). Further studies have found that LasR regulates alkaline

protease, alginate, and exotoxin A synthesis (Gambello et al, 1993; Storey et al, 1998).

The effect of lasR was thought to have a direct affect on the toxA upstream region

(Gambello et al, 1993). However, LasR does not affect the expression of regA

(Gambello et al , 1993).

regAB

Two of the most studied regulatory genes for the production of exotoxin A are

regAB. The regAB genes are required for maximal production of exotoxin A (Hamood

and Iglewski, 1990; Wick et al, 1990a). Neither toxA mRNA nor exotoxin A probe were

detected in a P. aeruginosa regA mutant (Walker et al, 1994, 1995; West et al, 1994).

However, the exact mechanism of toxA regulation by regA is not completely understood.

11

ptxR and ptxS

Our lab has isolated two toxA regulatory genes, ptxR and ptxS. Through the regA

gene, ptxR enhances the production of exotoxin A at the transcriptional level (Hamood et

al. 1996; Colmer. 1997: Colmer and Hamood. 2001). The ptxR gene encodes a 34-kDa

protein, PtxR. uhich belongs to the LysR family of transcriptional activators (Weickert

and Adhya, 1992; Colmer, 1997). The other regulatory gene, ptxS, interferes w ith the

effect of ptxR on toxA transcription and negatively autoregulates its ov\n synthesis

(Swanson and Hamood, 1999; Swanson, 2000). The ptxS gene encodes a 37-kDa protein,

PtxS, \\ hich belongs to the GalR family of transcriptional repressors (Weickert and

Adhya, 1992; Swanson, 2000a). The ptxS gene is located 5' of the ptxR gene and is

transcribed in the opposite orientation of the ptxR gene.

Purpose of this study

The aim of this study is to determine if P. aeruginosa contains a potential

regulatory protein(s) that binds specifically to the ptxRIptxS upstream region. Using the

electrophoretic mobility shift assay, we found that an intense gel shift band was detected

when a 201-bp probe taken from the ptxRIptxS intergenic region was incubated with the

lysate of P. aeruginosa PAOl. The two main goals of this research were:

1. To purify and characterize the potential DNA binding regulatory

protein(s).

2. To determine the target nucleotide sequence to w hich these protein(s)

bind.

12

CHAPTER II

MATERIALS AND METHODS

Bacterial strains, plasmids media and growth conditions

Bacterial strains and plasmids used in this work are listed in Table 2.1.

Escherichia coli strains were routinely grown in Luria Bertani (LB) medium (1% bacto-

tryptone [Difco Laboratories, Detroit, MI], 0.5% yeast extract, and 1% NaCl). Cultures

were incubated with maximum aeration at 37°C in the presence of the antibiotics. P.

aeruginosa strains were grown in Chelexed trypticase soy broth dialysate (BBL

Microbiology Systems, Cockeysville, MD) to which 1% glycerol and 0.05 M

monosodium glutamate were added (TSB-DC) (Ohman et al, 1980b) except where

otherwise noted. P. aeruginosa strains were grown at 32°C with vigorous aeration and

with the addition of appropriate antibiotics. Growth conditions for specific assays are

described in the text. Antibiotics were used in the following concentrations: E. coli:

ampicillin, 75pg/ml; carbenicillin lOOpg/ml; nalidixic acid, 20pg/ml; streptomycin,

lOOpg/ml; kanamycin, 50pg/ml P. aeruginosa: carbenicillin, 300pg/ml; rifampicin,

80pg/ml.

Construction of recombinant plasmids carrying the ptxRIptxS intergenic region

The 553-hp ptxRIptxS intergenic region was originally obtained from pSW205.

Using BamHl and Kpnl, vector restriction sites, the 720-bp fragment was generated that

included the 553-hp ptxRIptxS intergenic region and 167 bp of the ptxR structural gene.

13

The 720-bp fragment was cloned into pSK cloning vector, generating the plasmid

pBS720. To examine the potential proteins that bind to either p a P or to ptxS, the 720-bp

fragment was divided into three regions. The Kpnl-BamHl fragment was digested with

Dpnl restriction enzyme, generating three fragments; a 304-bp Dpnl-Kpnl fragment

containing the putative ptxS upstream region, a 157-bp Dpnl-Dpnl intergenic fragment,

and a 257-bp BamHl-Dpnl putative prjcP upstream region (Swanson, 2000a). The 304-bp

fragment was originally was cloned into the Hincll-Kpnl sites of pUC18, producing

pBS300 (Figure 3.1).

A previous analysis has shown that PtxS specifically binds to 103-bp region

within the 304-bp fragment (Swanson, 2000a). Other cellular proteins are bound to the

201-bp region (Figure 3.1). It is in this region that the present study is concerned with.

The regions were isolated as a Dpnl-Hincll fragment and cloned into pSK generating

plasmid pBS201. Additional fragments, within the 201-bp region, were generated from

pBS201.

Isolation of DNA fragments for the Electrophoretic Mobility Shift Assays

Different DNA fragments were synthesized by Polymerase Chain Reaction (PCR)

using pBS201 as a template. Ohgonucleotides used in the PCR synthesis of each DNA

fragment are shown in Table 2.2. Figure 2.1 contains a schematic illustration of the

different DNA fragments, the orientation of the DNA fragments, and oligonucleotides

that were used to generate the fragments.

14

Preparation of the DNA probes labeled with ly-^^PI-ATP

The PCR fragments were treated with Calf-Intestinal Alkaline Phosphatase to

remove the free phosphate groups from the 5' ends. Phosphatase treated DNA was

extracted with phenol chloroform and precipitated in 95% ethanol Radioactive

phosphates were incorporated to the free 5' ends by incubating the DNA fragment with

[Y- ^P]-ATP and T4 Polynucleotide Kinase for l-!/2 hours at 37°C. Unincorporated [y-

32

P]-ATP was removed by passing the reaction through a size exclusion (Sephadex G50)

column.

Preparation of the P. aeruginosa lysate

All the experiments were conducted using the P. aeruginosa strain PAOl. Initial

experiments were conducted using lysate from PAOl that was grown in Luria- Bertani

(LB) medium (1% bacto-tryptone, 0.5% yeast extract, and 1% NaCl) for 16-18 hours at

37°C with rigorous aeration. We also tried to determine if the potential regulatory

protein(s) is regulated by iron. The lysate was prepared from PAOl that was grown in

either iron deficient medium (TSB-DC) or iron sufficient medium (TSB-DC -i- iron).

TSB-DC is Chelexed trypticase soy broth dialysate (BBL Microbiology Systems,

Cockeysville, MD) to which 1% glycerol and 0.05M monosodium glutamate were added

(Ohman et al , 1980b). TSB-DC was made iron sufficient by the addition of 5pi of FeCb

(10 mg/ml). Regardless of the growth medium, cells were grown for 14-16 hours at 32°C

with vigorous aeration. A 1ml aliquot was isolated, centrifuged (23,708 x g) and the

pellet was resuspended in lOOpl of sterile H2O. The suspension was then sonicated for 2

15

minutes to lyse the cells and centrifuged again. The superaatants (lysate fractions) from

several samples were pooled and stored at -80°C in 50pl aliquots.

Electrophoretic Mobility Shift Assay

The electrophoretic mobility shift assay experiments were performed using

approx. 1x10^ cpm of end-labeled ly-^^Pl-ATP DNA fragment. The reaction mixture

consisted of 15-20pg of PAOl cell lystate, l-2pl of labeled fragment, and 20-30pl of

DNA binding buffer. The DNA binding buffer contains dithiothreitol, bovine semm

albumin, poly dl-dC and a base solution of lOmM Tris-HCl (pH 7.4), lOmM KCl, ImM

EDTA and 5% glycerol. All binding reactions contain a negative control, which

consisted of the probe, DNA binding buffer, and Nuclease Free H2O. The reaction

mixtures were incubated at 32°C for 30 minutes. Three pi of DNA tracking dye (50%

sucrose, 0.6% bromophenol blue) was added to each reaction. The mixtures were mn on

an 8% polyacrylamide gel for 2 and V2 hours at 180V. The gels were dried at 62°C for

45 minutes and exposed to X-ray film.

Protein Purification

Different preliminary experiments showed that the potential DNA binding protein

is produced when PAOl is grown in LB medium. Therefore, further experiments for the

purification of potential DNA binding proteins were conducted using lysate that was

obtained from PAOl grown in LB medium. The P. aeruginosa strain PAOl was grown

in 10 ml of LB medium for 16-18 hours at 37°C with rigorous aeration. The PAOl cells

16

were then subcultured into 1 L of LB and grown for 16-18 hours at 37°C with rigorous

aeration. The cultures were centrifuged at 1,453 (x g) for 10 minutes at 4°C. The pellet

was resuspended in 10 ml of sterile H2O, and lysed by passing them twice through a

French pressure cell at 1000 Ib/inl Lysed cells were ultra-centrifuged at 97,252 (x g) for

45 minutes at 4°C. The supernatant fraction (lysate) was collected, divided into several

aliquots, and stored at -80°C. The same procedure was repeated until the lysate from 10

liters of culture was collected. The proteins within the lysate were concentrated further

using ammonium sulfate. Solid ammonium sulfate was added to 65 ml of the lysate

fraction to a concentration of 50%. The mixture was centrifuged at 1,453 (x g) for 30

minutes at 4°C. The supernatant was discarded and the pellets were resuspended in a

total volume of 4 ml of sterile H2O. Through these procedures, the proteins within the

cell lysate were concentrated approximately lOOOX. To remove residual ammonium

sulfate salt, 10 ml of precipitated lysate was injected into a 12 ml dialysis cassette

(Pierce, Rockford, IL) and dialyzed in IL of DNA binding buffer (pH 7.4) for 1 hour at

4°C. The lysate was dialyzed every hour with IL of fresh DNA binding buffer (without

the addition of DTT, poly dl-dC, or BSA) for four hours and 2 L of fresh DNA binding

buffer ovemight. The amount of protein was determined by the Bradford assay (Pierce,

Rockford, IL). The concentrated lysate was stored at -80°C.

Heparin-Sepharose column

The heparin-sepharose column is an ionic exchange chromatography column in

which positively charged DNA binding proteins can be excluded from other proteins

17

within the PAOl whole-cell lysate. Heparin acts as a cation exchanger. The presence of

anionic sulphate groups in the heparin column eliminates any negatively charged proteins

by binding to, positively charged and putative DNA-binding proteins. DNA binding

proteins within the concentrated lysate were enriched using a Hi-Trap Heparin Affinity

colunrn (Pharmacia, Piscataway, NJ) (Carlsson, 1997). The column was first washed and

equilibrated with 5 column volumes of column buffer (DNA binding buffer without

dithiothreitol DTT, bovines semm albumin BSA, or poly dl-dC). Five milliliters of

concentrated lysate were passed through the column. The unbound proteins were washed

from the column with 3 column volumes of column buffer. The bound proteins were

eluted from the column using a step-wise KCl gradient (0.1-1.0 M KCl). Each of these

fractions was desalted using a Microcon-100 spin column (Amicon, Beverly, MA). A

total of 10 ml of the fractions were placed in the Microcon-100 spin columns and

centrifuged at 24,434 (x g) for 20 minutes. The solution that passed through the filter

column contained the salt buffer and was discarded. The proteins that were retained on

the filter were washed 3 times with column buffer, decanting the lower residual salt

solution each time. The column filter was then inverted and placed in a new collection

tube, where about 250pl of column buffer was added and the columns were centrifuged at

24,434 (x g) for 20 minutes to collect the elution fractions. Each elution fraction was

measured using the Bradford assay. The proteins within each fraction were separated

using SDS-PAGE and the gels were stained with silver stain. In addition, samples from

each fraction were examined for DNA binding to the 150-bp fragment. The proteins were

separated on a 15% SDS polyacrylamide gel to obtain an accurate estimation of their

18

molecular masses. Fractions eluted with 0.6 M KCl produced a significant gel shift band

and contained only two proteins. Therefore, further analyses, including the DNase I

protection assay, were conducted using this fraction (0.6 M KCl fraction).

DNase I Footprinting Analysis

DNase I protection experiments were conducted using a commercially available

kit (Promega, Madison, WI). Based on the manufacturer" s recommendations and our

previous experiments with these protocols, we needed a 100-600-bp fragment with the

potential binding site no closer than 25-bp from the labeled end. This would provide the

optimal conditions for the DNase I protection experiments. Based on these parameters

and because the location of the binding site within the 201-bp region is not known, we

have utilized the 304-bp fragment (Figure 3.13).

Preparation of the DNA probe

The DNase I Protection assay was conducted using the 304 bp fragment within

the ptxRIptxS intergenic region. The fragment was obtained from pBS300 by restriction

enzyme digestion with £coRI and HiruJUl. Figure 2.3 illustrates the preparation of the

304-bp probe for the DNase I footprinting analysis. The 304-bp fragment was incubated

with Calf-Intestinal Alkaline Phosphatase enzyme for 1 and Vi hours at 37°C in order to

remove the 5' phosphate groups. Radioactive phosphates were incorporated to the free 5'

ends by incubating the 304-bp fragment with 1Y-^"P]-ATP and T4 Polynucleotide Kinase

for 1 and Vi hours at 37°C. The probe was separated from uiuncorporated [y-^^PJ-ATP by

19

passing the labeled reaction through a size exclusion colunrn (Sephadex G-50). The

DNA probe was then digested with P^rl to create a singly end-labeled probe (Figure 2.2).

Again, the 304-bp fragment was passed through the size exclusion Sephadex column to

remove the small Pstl-Hindlll fragment.

DNase I Reactions

Two DNA binding reactions were set up. The first reaction contained 25pl of the

304-bp probe, 25pl of DNA binding buffer and 35pl of sterile H2O. The second reaction

contained 25pl of the 304-bp probe, 25pl of DNA binding buffer, 5pl of sterileH20, and

20pl (6pg) of 0.6 M KCl fraction. Both reactions were incubated for 30 minutes at 32°C.

Fifty pi of Ca" /Mg " solution (Promega, Madison, WI) that was warmed to room

temperature were added to both DNA binding reaction mixtures and incubated at room

temperature for 1 minute. Nine microhters of previously dilute RQl RNase-Free DNase

(5pl of RQl RNase-Free DNase were added to lOOpl of cold Tris-HCl [pH 8.0]) were

added to both reactions and incubated at room temperature for 1 minute. The reaction

was terminated by adding 90pl of Stop Solution, which was provided by the Promega

Core Footprinting kit. The reactions were phenol extracted and ethanol precipitated at -

20°C for 1 hour. The reaction mixture was then centrifuged at 23,708 (x g) for 5

minutes, washed with 70% ethanol and dried under a vacuum. Each reaction pellet was

resuspended in lOpl of Loading Solution (Promega, Madison, WI). Resuspended

material was vortexed and heated at 95°C for 2 minutes.

20

Sequencing Reactions

The nucleotide sequence termination reaction was done in conjunction with the

DNase I protection experiments to define the protected nucleotides. The DNA

sequencing reaction was performed by the di-deoxy chain termination method using a

sequencing kit (Amersham Pharmacia, Cleveland, OH). The double-stranded DNA

template used for the sequencing reaction was pBS300. Preparation of the double

stranded plasmid DNA template for sequencing reactions was done by the alkaline lysis

method (Ausubel et al, 1988). Approximately 7pg of pBS300 was denatured by adding

1/25 volume of 5M NaOH at 37°C for 5 minutes. The denatured DNA was precipitated

at room temperature for 30 minutes by adding 2 volumes of premixed

potassium/acetate/isopropanol The DNA was then ethanol precipitated and resuspended

in 20pl TE Buffer (pH 8). For the annealing reaction lOpl (3.5pg) pBS300 was added to

3pl (3.5-5 pmol) seq300 primer, and 2.6 pi of 1 X reaction buffer. The reaction was

incubated at 70°C for 2 minutes and cooled to 37°C over 20 minutes. The primer seq300

5' -GCTCGAGCGGTACCTCTTGGTGCT- 3'was constmcted to anneal to the

complementary strand of the denature pBS300 DNA template beginning at the EcoRl

region of the 304bp region which corresponds to the labeled-end of the 304-bp probe

used in the DNase I Footprinting reactions.

The four di-deoxy chain termination reactions were heated to 95°C for 5 minutes

and loaded with the DNase I Footprinting reactions on an 8% polyacrylamide sequencing

gel and run for 4 and Vi hours at 1500 Volts. The sequencing gel was soaked in fixative

solution (20%methanol, 10%acetic acid, 70% H2O), air dried for 6 hours and exposed to

21

X-ray film witii an intensifying screen for 17-19 hours at -SOX. The complementary

nucleotide sequence was determined by double stranded sequencing using the

oligonucleotide primer (seq300), which did not contain the P^rl restriction site.

T7 Expression System

The T7 expression system is usually used for the detection of the protein encoded

by a gene that is selecti\ ely expressed by tiie bacteriophage T7-RNA polymerase from a

T7 promoter (Tabor and Richardson. 1985). An E. coli strain containing a T7-RNA

polymerase plasmid and a recombinant plasmid in which the T7 promoter expresses the

gene of interest is subjected to different experimental conditions. Under these conditions,

the T7 promoter is selectively expressed by the T7-RNA polymerase and the protein

encoded by the gene is selectively labeled.

Using the T7 Expressions Experiments to examine the Translational product of the P. aeruginosa MvaT homologue

Analysis of the P. aeruginosa genome revealed the presence of an open reading

frame that codes for a 16-kDa protein. The protein is highly homologous to the MvaT

protein of Pseudomonas mevalonii. Therefore, we tried to isolate the region that codes for

this gene and determine the translational products. A 750-bp fragment that carries the

entire ORE was synthesized from the P. aeruginosa chromosome by PCR. The 750-bp

fragment was cloned into pCR® 2.1 TOPO® clotting vector (Invitrogene, Carlsbad, CA).

The recombinant plasmid pCR® 2.1 TOPO®-wvar was transformed into TOPIO

chemically competent cells (Invitrogene, Carlsbad, CA). Recombinant plasmid DNA

11

was isolated and the insertion of the 750-bp fragment was confirmed by nucleotide

sequence analysis. The production of the P. aeruginosa MvaT homologue was examined

using two approaches. In one approach, the 750-bp fragment was synthesized by PCR

(using PAOl chromosomal DNA) and cloned into pCR® 2.1 TOPO® cloning vector and

pCR'*T7/CT-T0P0® cloning vector (Invitrogene, Carlsbad, CA). The recombinant

plasmids were then transformed into either One Shot® BL21 (DE3) chemically competent

E. coli (Invitrogene, Carisbad, CA) or E. coli chemically competent K38 pGPl-2 cells

(Tabor and Richardson, 1985).

T7 Expression experiments

The mvaT gene was over expressed in the E. coli strain K38 using the T7

expression system as previously described (Tabor and Richardson, 1985; Colmer and

Hamood, 1998). E. coli K38 (pGPl-2) carrying T7 recombinant plasmids were grown to

an OD590 of >0.4 to<0.6. A 200pl sample of cells was harvested, washed, and

resuspended in 1 ml of M-9 minimal medium containing all amino acids minus

methionine and cysteine. Following incubation at 32°C for 3 hours, the cultures were

grown at 42°C for 20 minutes. Rifampicin (200pg/nti) was then added and the cells were

incubated at 42°C an additional 15 minutes. The cultures were shifted back to 32°C for

30 ntinutes and were pulse-labeled with 10 pCi of Tran [^^S]-methionine/cysteine (NEN

Biochemicals) for five minutes. The proteins in each sample were separated on a 12%

SDS polyacrylamide gel. The gels were dried for 45 minutes at 62°C and exposed to X-

ray film.

23

Table 2.1: Bacterial strains and plasmids.

Strains

Pseudomonas aeruginosa PAOl PAOl:: ptxR

Escherichia coli DH5a K38

Plasmids

pUC18 pSK pT7-5, pT7-6

Recombinant Plasmids pJAC17

pBS720

pBS300

pBS201

pCR'2.1-TOPO® mvaT

pCR®T7/CT-TOPO' mvaT

Description

Prototroph A ptxR strain

5/<pE44, thil, recAl, gyrA, Nal'* HfrC, host for pT7 expression system

CbVAp" ColEl, general cloning vector* pBluescript cloning vector Cloning vectors for the T7 expression system

pT7-5, which contains the HincU-HindUl fragment containing the intact ptxS open reading frame pSK containing the BamiU-Kpnl fragment which carries the ptxRJptxS intergenic region pUClS containing the 304bp Kpnl-Dpnl ptxRJptxS intergenic fragment digested from pBS720 pSK containing the 201bp HincU-HindUl intergenic fragment Topoisomerase cloning vector from Invitrogene cloning kit, containing the 750bp PCR product that carries the structural mvar gene" Topoisomerase T7 cloning vector from Invitrogene cloning kit, containing the 750bp PCR product that carries the structural mvaT gene"*"

Source/Reference

(HoUoway et al.. 1979) (Colmer and Hamood. 1997)

(Ausubel, 1988) (Tabor and Richardson, 1 1985)

Strategene Strategene (Tabor and Richardson, 1985)

i

(Colmer. 1997)

1

(Swanson, 2000)

(Swanson, 2001)

(Swanson. 2001)

Invitrogene This study

Invitrogene This study

* Abbreviations: Cb, carbeiucilhn; Ap, Ampicillin; Nal, nalidixic acid; ColEl, ColEl origin of replication; A, deletion; r, resistant • pCR^2.1-T0P0^ mvaT was transformed into TOPIO competent cells •'pCR®T7/CT-TOPO® mvaT^as transformed into BL21(DE3) and K38 competent cells

24

Table 2.2: List of oligonucleotides selected from the ptxR/ptxS intergenic region. These oligonucleotides were used for the PCR synthesis of DNA fragments to be used in the Electrophoretic Mobility Assays. The PCR template used was pBS201.

PCR fragment size

98bp

102bp

150bp

50bp

75Lbp

75Rbp

750bp

(mvaTgene)

Oligonucleotides

forward: "hcf1"

reverse: "hcr1"

forward: "dpf2"

reverse: "dpr2"

forward: "dpf150"

reverse: "dpr2"

forward: "dpflSO"

reverse: "hcrSO"

forward: "dpflSO"

reverse: "dpr75"

fonward: "F75/102"

reverse: "dpr2"

forward: "mvatP"

reverse: "mvatR"

Sequence

5' -GTC AAC GGG AAA AAA ACG CCT- 3'

5' -TGC AAA CCA TCA TTC GAA ACG- 3'

5' -ATT ATT AAT GTC GAA TGG AAT G- 3'

5" -TCG CTT TCT ACC GAC AAA A- 3'

5' -GTT CAT GAA AAC CGA ATA A- 3"

5" -TCG CTT TCT ACC GAC AAA A- 3'

5' -GTT CAT GAA AAC CGA ATA A- 3'

5' -TAA TAA TTG CAA ACC ATC A- 3'

5* -GTT CAT GAA AAC CGA ATA A- 3"

5' -ATG GCA CAT TCA TTC CAT T- 3'

5' -CCA TAA ATA AAA AGG AAG T- 3'

5' -TCG CTT TCT ACC GAC AAA A- 3'

5' -AGA AAC ATC GCG AAA GGC CGC CAT TCT A- 3"

5' -TAC GCC GCG GAA CTG CGT CAA CGC TAT T- 3'

* The PCR template used for the mvaT gene was chromosomal DNA from PAOl.

25

Dpnl HincU

ptxR 201bp

ptxS

102bp dpH 98bp hcfl

dpr2 hcrl

ISObp dpflSO

dpr2

5(tbp f l p f l 5 0

he 1-5(1

^

75Lbp f75/102 ^5khp cIpllSO

dpr2 (Ipr75

Figure 2.1: Diagram of DNA fragments generated by PCR synthesis. The different oligonucleotides that were used to generate the fragments are indicated: dpr2, dpf2, hcrl, hcfl, dpfl50, hcr50, f75/l02, and dpr75. The 75L is the left half of the 150-bp fragment and the 75R is the right half

26

P04-5' Kpnl

EcoRl

P - 5 ' Kpnl

EcoRl

P-5 ' Kpnl

EcoRl

pBS300

Hindlll pUClSCb^

Double digestion with £coRl and HindlW

Hindi

304bp Pstl

5'-P04 Hindlll

Incubation with Calf-Intestinal Alkaline Phosphatase followed by end-labeling with [y- "P]-.-\TP and PoKnucIeotide

• Kinase Hindi

Pstl 304bp

5 - P Hindlll

Restriction enzyme digestion with Pstl

Hindi

300bp

5^ Pstl

Figure 2.2: Diagram illustrating manipulations of the 304-bp fragment from pBS300 to generate the single, end-labeled 300-bp fragment for the DNase I footprinting analysis.

27

CHAPTER III

RESULTS

Considering that ptxR and ptxS are transcribed on opposite DNA strands in

opposite orientations relative to each other, the intergenic region containing the ptxR and

the ptxS upstream regions are likely to overlap. Computer analysis indicated that the ptxR

and the ptxS upstream regions have specific sequences that may represent binding sites

for several potential regulatory proteins (Colmer and Hamood, 1998).

Identification of the ptxR/ptxS Intergenic Region

Previous studies have identified at least four potential P. aeruginosa regulatory

proteins that bind specifically to the 720-bp fragment that contains the ptxRIptxS

upstream regions. In addition, Swanson, 2000a have shown that PtxS binds to a specific

sequence within the 103-bp fragment, which is directiy upstream of the ptxS gene (data

not shown). This sequence appears to be involved in the autoregulation of PtxS synthesis

(Swanson, 2000).

Another potential protein(s) bind to a 201-bp fragment, which is also within the

ptxRIptxS upstream region (Figure 3.2). In this study, we have tried to identify the

potential protein(s) that bind to the 201-bp fragment. In addition, we have attempted to

identify the nucleotide sequence within the ptxS upstream region to which the potential

protein(s) bind.

The 201-bp DpnllHindi fragment, which is located within the 720-bp intergenic

fragment, was used as a source for other small DNA fragments and as a template for the

28

synthesis of specific DNA fragments (Figure 3.1). Plasmid pBS201, which is a pUC18

recombinant plasmid carrying the 201-bp fragment was used as a template for the

synthesis of smaller DNA fragments by PCR. Different oligonucleotides that were used

in the PCR synthesis of the DNA fragments are described in Table 2.1.

Our initial approach was to confirm the presence of potential DNA binding

proteins within the lysate of P. aeruginosa, strain PAOl that specifically bind to the 201-

bp fragment. The fragment was synthesized by PCR and end-labeled with ty-^^P]- ATP

as described in Materials and Methods. The source of the potential DNA binding protein

was the lysate of PAOl that was grown in LB medium. As shown in Figure 3.2, a large

gel shift band was detected when the P. aeruginosa cell lysate was incubated with the

201-bp fragment. The binding was specific. When the binding reaction was conducted

in the presence of excess iron lysates and the 201-bp fragment, the intensity of the gel

shift band was reduced (data not shown).

In an attempt to determine if the binding of the potential protein(s) is regulated by

iron, we repeated the gel shift assay using PAOl lysate that was grown in either iron

deficient medium TSB-DC, or iron sufficient medium (TSB-DC + iron). A gel shift band

was detected with the lysate that was obtained from PAOl that was grown in both iron-

deficient and iron-sufficient media (data not shown). We also examined the possibility

that the binding protein may be PtxR. The lysate for the binding reaction was prepared

from PAOl and from the PAOl isogenic mutant ?AO::ptxR (Colmer and Hamood,

1998). The gel shift band was detected with the lysate from both strains, indicating that

the protein is not PtxR.

29

Localization of the Binding Region within the 201-bp fragment

In our initial attempt to localize the binding region, we divided the 201-bp

fragment into two fragments, the 102-bp fragment and the 98-bp fragment (Figure 3.1).

The fragments were synthesized by PCR using the plasmid pBS201 as a template. As

shown in Figure 3.3, no gel shift band was detected when the lysate was incubated with

the 98-bp probe. However, a clean gel shift band was detected with the 102-bp probe

(Figure 3.3). This gel shift band was detected with the lOOX, but not the lOX

concentrated lysate fraction (data not shown). In addition, the gel shift band that was

detected with the 102-bp probe was less intense than that produced with the 201-bp probe

(Figure 3.3). These results suggest that the gel shift band of the 102-bp fragment may

represent a single protein, while the gel shift band with the 201-bp fragment may

represent multiple proteins. We have conducted additional experiments that exclude the

possibility that the binding to the 102-bp fragment is caused by the PtxR protein. The

lysates were obtained from PAOl cultures that were grown in LB medium for either 6

hours or 18 hours (these are two possible time points for the maximal expression of the

PtxR protein) (Colmer and Hamood, 1998). However, no difference in the binding

activity was detected (data not shown).

Binding Activity of the 150-bp fragment that overlaps the junction between the 102-bp and the 98-bp fragments

Based on the above described results, we attempted to determine if the difference

between the intensity of the binding bands within the 201-bp and the 102-bp probes is

due to additional proteins that may bind to the region that overlaps the junction between

30

the 102-bp and the 98-bp fragments. A 150-bp fragment that overiaps the junction

between the 102-bp and the 98-bp fragments was synthesized by PCR (Figure 3.1).

Binding experiments were conducted using the lOOX concentrated lysate of PAOl that

was grown in LB medium. In addition, the lysates of PAOl that was grown in iron

deficient and iron sufficient media were examined. As shown in Figure 3.4, a strong gel

shift band was detected when the 150-bp probe was incubated with the PAOl grown in

iron-deficient medium. A less intense band was detected with the lysate of PAOl that

was grown in iron sufficient medium (Figure 3.4). However, a strong gel shift band was

detected with PAOl that was grown in LB medium (Figure 3.4). Thus, the presence of

iron is less likely to play a role in the binding activity. In addition, these results suggest

that at least one of the potential regulatory protein(s) binds to the 150-bp fragment. The

possible effect of the growth medium (TSB-DC vs. LB) on the binding activity is not

within the focus of my study and was not considered further.

Further Experiments to Locahze the Binding to the 150-bp fragment

We first divided the 150-bp fragment was divided into two 75 bp fragments, the

left 75-bp fragment (75L) and the right 75-bp fragment (75R) (Figure 3.1). These

fragments were synthesized by PCR using specific oligonucleotides as primers (Table

2.1). A faint gel shift band was detected with the 75L probe and the lysate of PAOl

(Figure 3.5). No gel-shift binding band was detected with the 75R fragment (Figure 3.6).

This band is clearly different from the strong gel shift band usually detected with the 150-

bp fragment. We have also examined the binding activity to a 50-bp fragment within the

31

75L fragment (Figure 3.1). However, no gel shift band was detected with tiie 50-bp

fragment (data not shown). Therefore, due to these variations, we have decided to limit

the binding activit) of the 150-bp fragment and concentrate our effort on identifying the

specific nucleotide sequence to which tiie potential protein(s) bind.

Purification of the Potential DNA-binding Proteins

Preparation of the PAOl Ivsate

The PAOl cells were grown in a lliter culture of LB medium at 37 C for (14-16)

hours. Prior to processing of the pellet of the lliter culture, a Iml sample was isolated

and pelleted. The lysate from the 1ml sample was examined for an\ binding acti\ ity to

the 150-bp fragment. Using this application, the lysate from 10 liters (from which a 1ml

sample provided a gel shift band) was prepared. The cells were lysed and the proteins

within the lysate were concentrated to about lOOOX as described in Materials and

Methods. Samples of concentrated proteins were examined for DNA binding activity. A

specific gel shift band was detected (data not shown).

Enrichment of DNA binding Proteins using Heparin-Sepharose Column

Heparin is a cationic exchanger. The presence of anionic sulfate groups in the

colunrn bind positively charged proteins. Most DNA-binding proteins are positively

charged. Concentrated proteins from PAOl lysate were further concentrated by passing

them through a 5ml Hi-Trap Heparin-Sepharose Column (Pharmacia, Piscataway, NJ).

Five milliliters of the concentrated proteins were passed through the column. The

32

unbound proteins were washed and bound proteins were eluted from the column using a

step-wise KCl gradient (0.1-1.0 M KCl). Eluted fractions were desalted and

concentrated using a Microcon-100 spin column (Amicon, Beveriy, MA).

Samples of the concentrated proteins were examined for their binding activity to

the 150-bp fragment (data not shown). In addition, the proteins in the other samples were

separated on SDS polyacrylamide gels and the gels were stained with silver stain to

examine the separation of the proteins. Proteins from the samples that were eluted with

the 0.3 M-0.6 M KCl, produced specific gel shift bands (data not shown). Silver stained

PAGE gels showed several proteins within the 0.3 M-0.5 M KCl eluted fractions.

However, the 0.6 M KCl fraction produced only (2) small proteins (Figure 3.7). These

proteins were also detected in the 0.3-0.5 M KCl fractions. Based on these results, we

decided to examine the two proteins within the 0.6 M KCl fraction. The proteins were

separated on a 15% SDS polyacrylamide gel to obtain an accurate estimation of their

molecular masses. Position indicated that the molecular weight of the proteins to be 16-

kDa and 9-kDa (Figure 3.7).

Identification of the Potential Binding Proteins

To identify the two proteins described above, we tried to determine the amino

acid sequence of the first 15-20 amino acids of each protein. Sufficient amounts of the

proteins were separated by SDS-PAGE and transferred to a PVDF (polyvynildine

diflouride) protein membrane (BIO-RAD, Hercules, CA). Regions of the membrane that

contained each protein were isolated. The sequences of the first 20 amino acids of both

33

proteins were determined by sequencing analysis at the Biotechnolog\ Core Facility Lab.

Texas Tech Uni\ersity (Figure 3.8).

Using the amino acid sequence, we searched the Genbank database to determine

if either one or both of the proteins have been previousl) characterized. The amino acid

sequence of the 9-kDa protein showed a 100% homology to the first 20 amino acids of

the previously characterized HU protein, which belongs to the family of transcriptional

regulatory proteins in E. coli (Aki. 1997). A similar analysis re\ ealed that the amino acid

sequence of the 16-kDa protein is 100% homologous to the first 20 amino acids of the

MvaT protein, which was identified as a transcriptional activator found in Pseudomonas

mevalonii (Rosenthall. 1998). We have also searched tiie P. aeruginosa Genbank

database to determine if a P. aeruginosa homologue of the MvaT protein exists. We

have identified an open reading frame (ORE) that codes for the 16-kDa protein (Figure

3.9).

Identification of the Binding Sequence for the 16-kDa and 9-kDa proteins within the ptxRJptxS intergenic region

It is possible that the gel shift band obser\ ed with the 0.6 M KCl fraction is

caused by binding of the 9-kDa and/or the 16-kDa protein to the 150-bp fragment. A

competitive binding assay was conducted to determine the specificity of the binding of

the potential proteins to the 150-bp probe in the presence of excess uitiabeled 150-bp

fragment. The gel shift band was either reduced or eliminated (Figure 3.10). Thus

confirming that the binding of either on or both proteins is specific (Figure 3.10).

Therefore, determining the specific nucleotide sequence to which this binding occurs is

34

important. This analysis was determined by the DNase I protection assay. The probe for

the DNase I protection experiment was a 304-bp fragment from the ptxRIptxS intergenic

region. This fragment was obtained as an EcoRUHindlW digestion from pBS300. The

fragment was end-labeled and purified as described in Materials and Methods. Reactions

from the DNase I protection assay were separated on an 8% urea sequencing gel along

with a nucleotide sequencing reaction of the 300-bp fragment. As shown in Figure

3.11 A, the DNase I protection experiment revealed the presence of a 25-bp-protected

region. This region is located -145-bp upstream from the ptxS structural gene (Figure

3.1 IB). Further analysis showed the presence of a potential 8-bp palindromic sequence

within the 25-bp-protected region (Figure 3.1 IB). Computer analysis showed no

potential region within the P. aeruginosa chromosome that contains most of the

nucleotide within the palindromic sequence. This palindromic sequence may represent a

potential binding site for either the 9-kDa or the 16-kDa protein or both.

T7 Expression of the P. aeruginosa MvaT homologue

Using the pCR®T7/CT-T0P0®-mvar, we were not able to detect the translational

product of the ORF (data not shown). Similarly, when we utilized the K38 pGPl-2 pT7-

mvaT, no translational product was detected (Figure 3.12). There were faint bands that

migrated at about 35-kDa, 33-kDa, 30-kDa, 16-kDa, and 9-kDa (Figure 3.12). These

bands are likely to be background or insufficient incubation of the E. coli RNA

polymerase. As a positive control, we have utilized the plasmid pJAC17 (in which PtxS

is expressed from tiie T7 promoter). An expected 38-kDa protein was detected (Figure

35

3.12). At this time, we do not know the reason for our failure to detect the product.

However, we have not utilized pCR^T7/CT-T0P0® system in the selective labeling of

proteins. As an alternative, we have isolated the 750-bp fragment from the pCR®T7/CT-

TOPO®-mvaT plasmid (utilizing the EcoRW and Hindlll sites of the vector). The

fragment will be cloned into (by the Hindi and Hindlll sites) of the pT7-6 expression

vector. This directional cloning will allow the ORF to be in the correct orientation

resulting in expression by the T7 promoter.

Analysis of the Putative P. aeruginosa MvaT homologue

The computer program BLASTp 2.2.1 was utilized to examine the regions of

homology between Pseudomonas mevalonii MvaT and the Pseudomonas aeruginosa

MvaT homologue. As shown in Figure 3.13, the two proteins share several identical

amino acids. In addition, two regions of amino acids identity(13 and 19 amino acids) are

located toward the carboxy terminus end of the proteins. We have searched the P.

aeruginosa genome for other proteins that contain MvaT homologous sequences and

have identified a hypothetical protein that carries a 64% homology. Comparison of the

hypothetical protein with that of the P. aeruginosa MvaT homologue also revealed

regions of identity (Figure 3.13). One region (12 amino acids) contains all but one of the

consensus amino acids in the MvaT and the P. aeruginosa MvaT homologue

(GEVXETKGGNHK) (Figure 3.16).

36

BamHl

ptxR 4 720bp Kpnl

Dpnl Kpnl 304bp

Dpnl HificU 201bp

102bp 98bp

150bp

'5bp(L)

50bp

"5bp(R)

Figure 3.1: Diagram, which illustrates, the \arious DNA fragments that were s\nthesized by PCR for the electrophoresis mobilit) shift assays and their onentation in relationship to the ptxR SLudptxS genes. The 304-bp and the 201-bp fragments were generated by restnction enz\nie digestion. .AJTOWS indicate the direction of transcnption of ptxS and ptxR.

37

Figure 3.2: Electrophoretic mobility shift assay of 201-bp fragment and PAOl lysate. Lanes: (I) 201-bp fragment probe alone. (2) 201-bp probe + PAOl lysate. (3) 201-bp probe + PAOl ::ptxR lysate.

38

1 2 3 4 5 6

Figure 3.3: Electrophoretic mobility shift assay of the 102-bp and 98-bp fragments with PAOl lysate that was French pressed and concentrated by dialysis. Lanes: (I) 98-bp fragment probe alone. (2) 98-bp probe + PAOl lysate (lOOx). (3) 102-bp fragment probe alone. (4) l02-bp probe +PA01 lysate (lOOx). (5) 201-bp fragment probe alone. (6) 201-bp probe + PAOl lysate (lOOx).

39

1 2 3 4 5 6

I

m

Figure 3.4: Electrophoretic mobility shift assay of the 150-bp fragment with PAOl lysate. Lanes: (1) 150-bp fragment probe alone. (2) 150-bp probe + PAOl lysate (low iron). (3) 150-bp probe + PAOl lysate (high iron). (4) 150-bp probe + PAOl lysate (LB). (5) 201-bp fragment probe alone. (6) 201-bp fragment + PAOl lysate (low iron).

40

I 2 3

m*^

<. » v 9

Figure 3.5: Electrophoretic mobility shift assay of the 75(L) fragment with PAOl lysate. Lanes: (1) 75(L) fragment probe alone. (2) 75(L) probe + PAOl lysate grown in LB media. (3) 75(L) probe + ?AOl::ptxR lysate.

41

Figure 3.6: Electrophoretic mobility shift assa\ of the ~5(R) fragment with P.AOl lysate. Lanes: (1) 75(R) probe alone. (2) 75(R) probe with lOOx PAO llysate. (3) 150-bp probe alone. (4) 150-bp probe - lOOx PAOl lysate.

42

97kDa B 66 kOa ^2 -^b'ijbff^

45 kDa

31 kOa

21 S kOa

14 5kna

Figure 3.7: Heparin-Sepharose 0.6 M KCl fraction. The proteins obtained from fractionation of the PAOl lysate through the heparin column and eluted with 0.6 M KCl were electrophoresed on a 15% SDS polyacrylaimde gel. The gel was silver stained for enhanced sensitivity.

43

16-kDa protein - MSLINEYRATEEAIKELQER.

9-kDa protein - MNKSELIDAIAASAD.

Figure 3.8: Amino acid sequence of first 20 amino acids of the 16-kDa protein and the first 15 amino acids of the 9-kDa proteins.

44

purUl mvaT ) <r sbcB

formyltetrahydrofolate transcriptional exodeoxyribonuclease I deformylase regulator

951bp 374bp 1442bp

14 kDa 53 kDa

B

1 GTCCTCGTCGCCGAGGGCGTCGATGTCGTAACGGCTTGTGCAACCCTCGCCGAGGGTCGG 60

61 TGGCGGGCTGGCGGTGCCCGCCGACCTGGGATCGCTCATGTTCGTTCCTCCGGGTGCCGG 120

121 CCCCGGCCGGCATTCCAGGGGAGCCGGCTCAGTCGAACACCACCGTCTTGTTGCCGTGTA 180

181 CCAGTACGCGGTCTTCCAGGTGATAGCGCAGTCCGCGCGCGAGCACCAGTTTTTCCACAT 240

241 CCTTGCCCAGCCGCACCATGTCTTCGACGTTGTCGCGGTGAGTGACCCGCACCACGTCCT 300 T V V F D

301 GCTCGATGATCGGACCGGCATCCAGTTCCTCGGTCACGTAATGGCTGGTCGCGCCGATCA 360 K N H Y R L G R A L V L K E V D K G L R

3 61 GCTTGACCCCGCGCTTCGACGCCTGGTGATACGGCTTGGCGCCGATGAAGGACGGCAGGA 420 V M D E V N D R H T V R V V D Q E I I P

421 AGCTGTGGTGGATGTTGATCACCTGGTGGGCGTACTTGCGGCACAGGTCCGGCGGCAGGA 480 G A D L E E T V Y H S K S A Q H Y P K A

481 TCTGCATGTAGCGAGCCAGGACGATGCAGTCGGCGCCATGTTCGTCGATCAGCCGGGAAA 540 G I F S P L F S H H I N I V Q H A Y K R

541 CTTCATCGAAGGCCGGCTGCTTGTCCTGCGGATCGACGGGAACGTGGAAATACGGGATGC 600 C L D P P L I Q M Y R A L V I C D A G H

601 CGTGCCACTCGACCATGCTGCGCAGGTCGTCGTGGTTGGCGATCACGCAGGGAATCTCGC 660 F A P Q K D Q P D V P V H F Y P I G H W

661 AGTCCAGTTCGCCGCTGTGCCAGCGGTGCAGCAGATCGGCCAGGCAATGGGACTCCTTGC 720 E V M S R L D D H N A I V C P I E C D L

Figure 3.9: Identification of the open reading frame (ORF) that codes for the P. aeruginosa MvaT homologue. A. Location of the putative genes within the P A O l chromosome. B. Nucleotide and amino acid sequence of the ORF.Putative - 1 0 and - 3 5 sites, based on homologies with E. coli a^° promoters, are underlined by dashed lines.

45

721 TGGCCATCAGCACCACGCGCTTCTTCACCTCGGAATCGGTGATTCGCCATTCCATGGAGA 780 E G S H W R H L L A M L V V R K K V E S

7 81 ATTCGCGGGCGATCGGGGCGAAAGCCTGGCGGAAGCCATCCAGGTCGAACGGCAGAGAGT 84 0 D T I R W E M S F E R A I P A F A Q R F

841 CGGCACGGATCTCGTGGCGCATGAAGAACCAGCCGTTATCGTTGTCGGAATGATGGCTGG 900 G D L D F P L S D A R I E H R M F F S A

901 CTTCGGTGATCCAGCCATTGTAGGTGGCCAGGAAGTTACTCACCTTGGCAACGATCCCGA 960 E T I W G N Y T A L F N S V K A V I G V

961 CCCCGTCGGGACAGGCGATCACCAGCCGAAAAGTGCGCATGGGTATACTCCAGAAACATC 102 0 G D P C A I V L R F T R M <—purUl

1021 GCGAAAGGCCGCCATTCTAGCGGCATGGCGGGAAAAGTTCAGCCTCGGCGAAGGTCTTTC 1080

1081 GACAGGCTGTTGCGCGAGAGAATAAACCCGCACAACAGGGCAATCCCGATGCGGAATTAA 1140

1141 TTACTTTGAATTTCATCGAGAAAAGTTTTCATCGCGGTTTACTTACAGTTTCGCCCTGAC 1200

1201 TATTATTGAATCTACTTACATGCCCGCCACCGCCACTCAGCACAGACAAGGTACCTGACA 1260 mvaT •

1261 TGTCCCTGATCAACGAATATCGCGCCACGGAAGAAGCCATCAAGGAACTTCAGGAGCGCC 1320 M S L I N E Y R A T E E A I K E L Q E R

1321 TGAAGTCCCTGGAACAAGACGACAAACTGAAAAAAGAACTGGAATTCGAAGAGAAGCTGC 1380 L K S L E Q D D K L K K E L E G T Y Q K

1381 GCACGCTGATGGGCACTTACCAGAAGTCCCTGCGTGACGTGATTTCCCTGCTCGATCCGG 1440 S L R D V I S L L D P D A K I G K S T R

1441 ACGCCAAGATCGGCAAGAGCACCCGCACCGCCAAGGCACCTGCCGGCAAGCGCGCGCGCA 1500 T A K A P A G K R A R K V K Q Y K N P H

1501 AGGTCAAGCAGTACAAGAACCCGCACACCGGCGAAGTCATCGAGACCAAGGGCGGCAACC 1560 T G E V T L K E W K A K W G P E A V E S

1561 ACAAGACTTTGAAAGAGTGGAAAGCCAAGTGGGGCCCCGAGGCCGTCGAGAGCTGGGCCA 1620 W A T L L G

1621 CCCTGCTCGGCTAAACCAGTCAGTTCCACGAAGAACGCCAGCCCAGTGCTGGCGTTTTTC 1680 STOP

1681 ATTTCCGCCGGATTGTATCGGCTCCTCTGCCTGCGCCCTCAAGTCTCCAGCGAATAGCGT 1740

1741 TGACGCAGTTCCGCGGCGTAATCGCGCCAGGCCGCAAGTACCGCCGGCAGTGTGCCGCCC 1800

1801 TGCTCTTGCCGACAAGCCTCCAGCGCCGCCTCGAAGGCCGGCAAAGTATTCGGTGCGCCG 1860

1861 TATTCTTCATGGGACAAACGATTGCGGCAGAAGCTTTCCCATTGTTGGCGCTCGGCCACA 1920

1921 TTTAAAGTTTCCGGGAAATTTCGCGCACGATAGCGAAAGAACAACTCCTGCAAACGGGCA 1980 T E L S Y R Q R L E A A Y D

1981 TCGTCGAAGGGCCATTGTTCTTTGGCCAATTGCTCCGGTTCGGCAAGGCGCAGCTGTTCA 2040 R W A A L V A P L T G G Q E Q R P L T N

2041 CAAAGGCGTCGATCCCGATCGCCAATAAATCCGTCATACAACTGTTGCTCGGGGTCGTCG 2100 P A G Y E E H S L R N R C F S E W Q Q R

2101 CTGGCGGAGAAACTTTCTTCGCCATATAGCGCTGTCAGTTTGTCCGCCCAGACGGTTTGA 2160 E A V N L T E P F N R A R Y R F F L E Q

2161 TGCTGGCGCAACAGCTCGGCTTTTTGCTGGCACTCATCGAGTTCGATGCCGGTTCGCTGG 2220 I R A D D E P E A L R L Q E C L R R D R

Fig. 3.9 continued

46

2221 CGATCCTCGGCGCGCAATACCGACAGCGGCGCCACCACCGGGCAACGGTTGACCTGGATC 2280 D G I F G D Y L Q Q E P D D S A S F S E

2281 TGCTTCAGCGGCACCGGCAACTCGCCCTCGGCCAGTTCGTCGCGACGGGTATACAGGCGC 2340 E G Y L A T L K D A W V T Q Q C E D L E

2341 CTGCGCAAGTCTTCGGCCGACAACTCCAGCAGCGGCGCGGGATCGGCGCAGAGGTCGCAG 2400 I G T R Q R D E A R L V S L P A V V P C

2401 ACGATCAGAGCATTGCGGTTGCGCGGGTGCCAGGCCAGCGGCAGGACCACCGAGAGGAAA 2460 R N V Q I Q K L P V P L E G E A L E D R

2461 TGGCGCTCCGCGGAAAAGCGTCCGGAGACATGCACCAGCGGCTGCAGCAGGCGCACCTGG 2 520 R T Y L E L L P A P D A C L D C V I L A

2521 TCGAGCACCTTGTGCTTGCTGCGCAACTGGTAGAGGTAATCGTACAGGCGCGGCTGGCGC 2580 N R N R P H Q A L P L V V S L F H R E A

2581 TGACGGATCAGGCGGGCCAGGGCAATGGTGGCGCGGACATCGGAAAGCGCGTCGTGGGCC 2640 S F R G S V H V L P Q L K S R L Q Y L Y

2641 TGCCCATGCTCCAGGCCGTTCGCCGCGGTGAGCATTTCCAGCTTCAGCGACAGCCGGCCG 2700 D Y L R P Q R Q R I L R A L A I T A R V

2701 TCCAGCTGCGGCCACTGGATGCCCTCCGGGCGCAGGGCATAGGCGGTACGGACCATGTCG 27 60 D S L A D H A Q G H E L G N A A T L M E

27 61 ATCAGGTCCCAGCGGCTATTGCCGCCTTGCCACTCGCGGGCGTAGGGATCGAAGAAGTTG 2820 L P W Q I G E P R L A Y A T R V M D I L

2821 CGGTACAGGCTGTAGCGCGTCACTTCGTCGTCGAAGCGCAGGGAGTTGTAGCCGGCCACG 2880 D W R S N G G Q W E R A Y P D F F N R Y

2881 CAGGTCGCCGGCTGCGCCAGCTGGGCGTGTACCCGGGTCATGAAGTCGGCCTCGGACAGC 2940 L S Y R T V E D D F T A P Q A L Q A H V

2941 CCCCGCTCGGCCAGCCGCTGGGGAGTGATGCCGGTGATCAGGCAAGCCATAGGGTGGGGC 3000 R T M F D A E S L G R E A L R Q P T I G

3001 AGGATATCGTCGCTGGGACGGCAATACAGGTTCATCGGCTCGCCGATCTCGTTCAGCGCC 3060 T I L C A M P H P L I D D S P R C Y L A

3061 TCGTCCGTGCGGATCCCGGCGATCTGCAACGGCCGGTCGCGGCGAGGGTCGATGCCGGTG 3120 E D T R I G A I Q L P R D R R P D I G T

3121 GTTTCGTAGTCGTACCAAAAAATACTCGCGTTCACGGTCATTCCCTTTCTGGCGGCACCG 3180 T E Y D Y W F I S A N V T M . sbcB

3181 GGCAGTCTACCAGTGACTGCCGCGAAGCTCGCGACCCACTTCGGGGATTTCCAGGTTTTC 3240

3241 CAACGAACTCACGGAAAGCGCCGTAGGCACGGCCCGCCAGGGCGCGTCAGTGCGCATATC 3300

3301 TGCCATCCGAACCTTGCGTTGTCCCCTTCGCCCTCGCTAGCATTCCGCTCGCACGGATGC 3360

3361 CCGTTTCACTGACAAGGACAGCACCATGAACGACACCAGCAATCCCTACGCCACCCCCGC 3420

Figure 3.9 continued

47

1 2 3

Figure 3.10: Competitive Electrophoretic Mobility Shift Assay of the 150-bp probe with 150-bp fragment. Lanes: (I) 150-bp fragment probe alone. (2) 150-bp probe + 0.6 M KCl heparin-sepharose fraction. (3) 150-bp probe + 0.6 M KCl heparin-Sepharose fraction + lOX unlabeled 150-bp fragment

48

49

Figure 3.11: Determining the specific nucleotide sequence within fheptxS/ptxR intergenic region to which the putative regulatory protem binds.

(A) DNase I protection analysis of the 0.6 M KCl heparin-Sepharose fraction binding to the ptxS upstream region. The 300 bp fragment of the ptxS upstream region was single end-labeled with [y- ^P]-ATP and incubated with the 0.6 M KCl elution fraction from the heparin-Sepharose column. Lanes: (1) 300-bp probe alone. (2) 300-bp probe + 6pg of 0.6 M KCl protein fraction lysate. All reactions were treated with DNase I, The complementary nucleotide sequence of the fragment is shown. In addition, specific nucleotides that constitute the DNase I protected region are indicated on the side. (B) The nucleotide sequence of the ptxS upstream region. The solid line mdicates the 25-bp DNase I protected region. The 8-bp palindrome is shown by opposing arrows. The potential -10 and -35 sites, based on homologies with E. coli a ^ promoters, are underlined by dashed lines.

A

< •

V

V

B

c A A C A A G T A C T T T T G G C T T A T T A G C

TCGCTTTCTACCGACAAAACCTTCTTAAAGTAGTTTCTCAAGTATTGGACA

AACTTCCTTTTTATTTATGGAGATATGGCACATTCATTCCATTCGACATTAA

TAATTGCAAACCATCATTCGAAACGATTATTCGGTTTTCATGAACAACGT

-35 CTGGCCTCATGCCTGGCTCGGCCCCGGTAGGCGTTTTTTTCCCGTTGACGG

. . . . I P . . . . GCATCGGCCTTCTGGTATTTTCTGAAACCGGTTTCAACTCCTGGCATCCGCT

M N GGCGAAGACCAGCCGACACCAATAAGAACAGCACCAAGAGGTGAAT

PtxS

50

112 kDa

«1 kDa

4QQkna

36.2 kDa

29.9 kDa

21.3 kDa

Figure 3.12: Expression experiment to detect the production of the P. aeruginosa MvaT homologue. Expression experiments were conducted using the E. coli T7 expression system as described in the Materials and Methods. Lanes: (I) Molecular weight standards in kiloDaltons. (2) K38 pGPl-2 containing plasmid vector pT7-5 (negative control). (3) K38 pGPl-2 containing recombinant plasmid pJAC17 (which contains theptxS ORF) (positive control) (4&5) K38 pGPl-2 containing recombinant plasmid pCR®T7/CT-T0P0-wvar),which carries the intact OR of the P. aeruginosa MvaT homologuevectors carrying the 750 bp mvaT gene fragment.

51

p. mevalonii MvaT

P.aeruginosa MvaT (82%) Homologue

1 YRATEqAIKELQARLANLSQDDKLKKELEFEGKLRALMJGEYSKSLRDVIALLDP 54 YRATE^AIKELQ R R LNp Y KSLRDVI+LLDP YRATEgAIKEijQERX2LX2C}gC)OC2LX2CXXXX2^

55ESKLSKAPRGAVKAVATKRARKVKQYKNPHN3EVIETKGGNHKTLEWKATWGGDLLO ++K+ K-F- R A KA A KRARKVKQYKNPH '3EVIETKGGNHKTLEWKA WG DAKIGKSTRTA-KAPAGKRARKVKQYKNPHTPEVIETKGGNHKITLEWKAKWG-

l l lWGDWESWATLL 124 + VESWATLL

--PEAVESWATLL

B

P.aeruginosa MvaT (82%) Homologue

P. aeruginosa hypothetical protein (64%)

1 MSLINEYRATEBAI MS + E+R E MSKLAEFREAEF

I KELQREXXXXXXXXXXXXXXXXXXXXRTLNJPTYQKSLRDV 54

++E + Ll^ Y +L + + KLQEQLALLEKLKSDSSLKQELEFKDKLQALNIDKYGMTLHNI

55ISLLDPDAKIGKSTRTAKSPAGKRARKVKQYKNPHlGEVIETKGGNHP I-t-+LDP A + lAILDPKAPV-

lllKAKWGPEAVESW 119 K ++G E VESW KEQYGSETVESW

+RAR +K YKNP-i-- - TVS AAPQRRARALKVYKNPNl GE WETKGGNHIVLKAWK

GEV+ETKGGNHP; LK W TLKEWK 110

Figure 3.13 Homology comparison between the P. mevalonii MvaT protein and the P. aeruginosa homologues. (A) Specific regions of homology between the Pseudomonas mevalonii MvaT protein (red) and the P. aeruginosa MvaT homologue (blue) showing an 82% homology. (B) Specific regions of homology between the P. aeruginosa MvaT homologue (blue) and the P. aeruginosa hypothetical protein (green) showing a 64% homology.

Closed boxes represent identical amino acid homology. Dashed boxes represent putative helix-tum-helix motif

52

CHAPTER IV

DISCUSSION

In this study we have identified two particular P. aeruginosa regulatory proteins

that specifically bind to a 150-bp fragment within the ptxRIptxS intergenic region. The

150-bp fragment is localized closer to the ptxS upstream region and the potential proteins

are likely to represent ptxS regulatory proteins. One of the identified proteins is the HU

protein, which was previously characterized in E. coli and P. aeruginosa (Delic-Attree et

al., 1995; Aki et al., 1996, 1997). The other protein carries a significant (82%) homology

to the MvaT protein, which is a transcriptional activator of the mevalonate utilization

operon mvaAB in Pseudomonas mevalonii (Rosenthal and Rodwell, 1998). The region to

which the binding of either one or both proteins was localized to a 25 bp-protected region

identified from the DNase I analysis. This protected region contains a typical 8-bp

palindromic sequence 5' -TTCATGAA- 3'.

The binding of individual proteins to the target sequence has not yet been

determined. The binding band in the gel shift experiments was detected in the presence

of both proteins. Passing the PAOl lysate through the heparin-sepharose column, which

enriched for all DNA binding proteins, purified the two proteins. Therefore, at this time

three possibilities can be suggested to explain the gel shift band. The band could be due

to the binding of the HU protein, the MvaT protein, or both. In an attempt to determine

the binding of the MvaT homologue, we are currentiy trying to selectively overexpress

the gene that codes for the protein using the E. coli T7 expression system (Tabor and

53

Richardson, 1985). The binding of the lysate fraction of the E. coli T7 expression system

to the 150-bp fragment and to the 25-bp fragment will then be examined. This will be

done in comparison with the lysate from E. coli carrying the T7 expression vector. If

both proteins bind to the 150-bp fragment, they can either bind together as a complex or

they may bind separately to different regions within the fragment. Careful examination of

the gel shift band, with the 150-bp fragment (Figure 3.4) shows a broad band, which

suggests that the binding is a complex of more than one protein. Previous studies have

shown that the binding of more than one protein to the same DNA fragment usually

results in a diffused or broad gel shift band (Swanson et al., 1999).

We will first consider the possibility that the binding band represents the binding

of the HU protein only. The HU protein from E. coli is predominantly a heterodimer of a

and P subunits, each with a molecular weight of 9-kDa. It is an abundant DNA-binding

protein associated with the bacterial nucleoid (Aki et al., 1996, 1997). HU is not known

to show DNA sequence specificity and has a tendency to bind to distorted regions of

DNA, such as kinks bends, single-stranded gaps and cruciforms (Aki et al, 1996, 1997).

Although the HU protein binds non-specifically to DNA, recent studies have shown that

bind affects the expression of several genes. For example, previous studies have shown

that the HU protein plays a role or is involved in the regulation of the E. coli GalR

protein (Aki et al., 1996). We have previously shown that the regulation of ptxS in P.

aeruginosa has several similarities to the regulation of GalR in E. coli (Swanson, 2000b).

For example, both proteins autoregulate their own synthesis; by binding to the upstream

regions of their respective genes. This binding was localized to a specific palindromic

54

sequence. However, the HU protein is unlikely to affect the specific binding of the PtxS

protein to the ptxS upstream region. We have already shown that PtxS binds to a 103-bp

region but not to the 201-bp fragment, with which current binding is detected.

The second possibility is that the specific binding of the MvaT homologue to the

150-bp fragment produces the gel shift band. The MvaT protein is a 35-kDa heteromeric

protein made up of a 15-kDa subunit called P15 and a 16kDa subunit called PI6. The

MvaT protein is a transcriptional activator that regulates the expression of the mvaAB

operon (Wang et al., 1991). The mvaAB operon consists of two genes, the mvaA and the

mvaB. The mvaA and the mvaB genes of the catabolic mvaAB operon encode HMG-

CoA reductase and HMG-CoA lyase respectively (Rosenthal and Rodwell, 1998;

Anderson and Rodwell, 1989). These enzymes catalyze the oxidative acylation of

mevalonate to HMG-CoA and its subsequent cleavage to acetoacetate and acetyl-CoA

(Rodwell et al., 1990). The microorganism, Pseudomonas mevalonii was isolated from

soil by the growth on specific media that contained mevalonate. In order to enrich for

potential DNA binding proteins, P. mevalonii lysates were passed through a heparin-

sepharose column (Rosenthall and Rodwell, 1998). Purification enrichment of the MvaT

protein resulted in the identification of two polypeptides, the PI5 and the PI6.

Comparison of at least the first 10 amino acids of P16 and PI5 showed that the two

proteins are not related, (i.e., P15 is not a proteolytic product of P16) (Rosenthall and

Rodwell, 1998). At this time, other than the sequence of its first 10 amino acids, no other

information is available regarding P15 protein. Analysis of the MvaT protein revealed

that it is a DNA binding protein that regulates the expression of the mvaAB operon by

55

binding to the operator region that contains three 10-bp direct repeats with the consensus

sequence TGGGTACAGT (Wang and Rodwell, 1991). The regulation of the mvaAB

operon is affected by the presence of mevalonate in the growth medium. However,

mevalonate does not affect the binding of MvaT to the target sequence within the mvaAB

upstream region. Rather, mevalonate affects the level of mvaAB expression (Wang et al.,

1989). Similar to the mvaAB operon, regulation of the kgu operon is affected by the

presence of 2-ketogluconate in the growth medium (Swanson, 2000b). These results

show that along with ptxS, four other genes (kguE, kguK, kguT, and kguD) constitute a 2-

ketogluconate utilization operon in P. aeruginosa. PtxS regulates the expression of the

kgu operon by binding to two operators within the operon, while 2-ketogluconate behaves

as the molecular inducer of the kgu operon or the molecular effector of PtxS (Swanson,

2000b). However, unlike the MvaT, ketogluconate directly affect the binding of PtxS to

its target sequence (Swanson, 2000b). PtxS binding to the target sequence was

eliminated when P. aeruginosa is grown in the presence of lOmM of 2-ketogluconate

(Swanson, 2000b).

Results of the present study raised several questions that need to be addressed.

For example, the homology between the MvaT protein and the P. aeruginosa MvaT

homologue is localized to two or three specific regions (Figure 3.13). These regions may

play an essential role in the function of these proteins. However, at this time, the amount

of information regarding the MvaT function is very limited. Whether this consensus

region represents a DNA binding motif is unknown. GCG PepPlot and Peptide Structure

software programs predict that PI6 is predominantiy a-helical, and may contain a helix-

56

tum-helix DNA binding motif between residues 7 and 38 (Rosenthal and Rodwell, 1998).

It is also not known if mevalonate plays a role in the metabolism of P. aeruginosa or

affects its growth. Furthermore, we do not know at this time if the ptxS upstream region

is the only target for the MvaT homologue. We have searched the P. aeruginosa genome

for any possible proteins that contain the same homology to the target of the MvaT

proteins (mvaA and mvaB). Our results revealed no homology to the mvaA gene.

However, a hypothetical protein that carries a 64% homology to mvaB was identified.

Whether the protein or its gene is regulated by the MvaT homologue is not knovs n at this

time.

With respect to their target sequences, MvaT and the P. aeruginosa MvaT

homologue appear to differ. As stated above, the known target sequence for MvaT is a

direct repeat of the 10-bp consensus sequence TGGGTACAGT. However, no such

sequence exists within the 150-bp fragment. Instead, the 150-bp fragment contains a 25

bp-protected region, in which exists an 8-bp palindromic sequence. Whether the two

proteins contain different domains to bind to the target sequence is not known at this

time. We have searched the P. aeruginosa genome for a possible sequence that may

contain all or most of the conserved nucleotides within the 10-bp MvaT target sequence.

One sequence that contains all the consensus nucleotides was detected. Further analysis

revealed that this region is located 3' of the P. aeruginosa lasR gene, which is one of the

components of the Quomm sensing system, and also 3' of rsaL gene, which is located

downstream form lasR and is transcribed antisense relative to lasR (Kievit et al., 1999).

The rsaL gene encodes an 11-kDa protein that when overexpressed reduces the

57

expression of lasB and elastase activity; RsaL represses transcription of the PAI-1

autoinducer synthase gene, lasl, which also a component of the Quomm sensing system

(Kievit et al., 1999). Whether the P. aeruginosa MvaT homologue binds to this region is

yet to be determined. Other regions that contain 3-9 of the consensus nucleotides were

also detected throughout the P. aeruginosa chromosome.

58

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