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REGULATION OF THE TYPE III SECRETION SYSTEM IN Pseudomonas aeruginosa
A DISSERTATION PRESENTED TO THE GRADUATE SCHOOL
OF THE UNIVERSITY OF FLORIDA IN PARTIAL FULFILLMENT
OF THE REQUIREMENTS FOR THE DEGREE OF
DOCTOR OF PHILOSOPHY
UNIVERSITY OF FLORIDA
This dissertation is dedicated to my parents, Liuting Wu, Hong Wang and my wife,
This work was carried out at the Department of Molecular Genetics and
Microbiology, College of Medicine, University of Florida, during the years 2001-2006.
It is my great pleasure to thank the following persons who have taken part in this work
and thus made it possible.
I owe my deepest thanks to my mentor, Dr. Shouguang Jin. His encouragement,
support, and enthusiastic attitude towards research and life in general have been inspiring
and have guided me during these years and have been more than I could have ever asked
for. I greatly appreciate the opportunity to be part of his research team.
I would like to sincerely thank my committee, Dr. Shouguang Jin, Dr. Ann
Progulske-Fox, Dr. Paul A. Gulig and Dr. Reuben Ramphal, whose insightful advice in
the last 4 years has made a great difference in my research progress and in my view of
being a serious scientist.
Far too many people to mention individually have assisted in so many ways during
my work. They all have my sincere gratitude. In particular, I would like to thank the
past and present members of the Jin laboratory, Dr. Unhwan Ha, Dr. Mounia Alaoue-El-
Azher, Dr. Li Liu, Dr. Jae Wha Kim, Xiaoling Wang, Dr. Hongjiang Yang and Dan Li,
for their help and advice. Especially, many thanks go to Dr. Lin Zeng and Dr. Jinghua
Jia, who have given me tremendous help in my life and research since I came to America.
I am also grateful to Wei Lian, M.D., for her help and suggestions these years.
I would like to thank Dr. William W. Metcalf of the University of Illinois at
Urbana-Champaign for providing the transposon plasmid and related E. coli strains used
in my work. I would like to thank Dr. Shiwani Aurora from Dr. Reuben Ramphal's lab
and Dr. Hassan Badrane from Dr. Henry V. Baker's lab, who have contributed to my
research and offered valuable technical support and discussions.
My final, and most heartfelt, acknowledgments must go to my family. I want to
express my earnest gratitude to my parents, Liuting Wu and Hong Wang, for their
unconditional love, encouragement and for always being there when I needed them most.
My wife, Chang Xu, deserves my warmest thanks. She is the source of my strength. Her
support, encouragement, and companionship have turned my journey through graduate
school into a pleasure. For all that, she has my everlasting love.
TABLE OF CONTENTS
A C K N O W L E D G M E N T S ................................................................................................. iv
LIST OF TA BLES .................................................................... ............ .. ix
LIST OF FIGURES ............................... ... ...... ... ................. .x
ABSTRACT .............. ..................... .......... .............. xii
1 INTRODUCTION AND BACKGROUND ..................................... ...............
Pseudomonas aeruginosa .............................................................................. 1
B asic B acteriology ................................................... ....... .............. .
Infections ................................. .................................. ...............1
CF Airway Infection by P. aeruginosa ...................................... ............... 2
Antibiotic Resistance ............................................ .............. ..... ......... 2
A acquired R resistance .................... ............................ .. ......... ............ .. .. .
Intrinsic R esistance.......... ..... ...................................................... ............... .3
M ultidrug Efflux System s .................................. ...................... ...............3
V irulence F actors ................................................................................................. 4
F lag ellu m .................................................................................. . 4
P ilu s .................................................................................................. . 5
E xtracellular T oxin s ................................................... .. ........ .......... .. .. ....
Q uorum Sensing .................................................................... 6
Iron M etab olism ....................................................... 6
A lginate ............................................................. . 7
B iofilm ................................................... 7
Type III Secretion System ....................................................... 7
Function of TTSS Structure G enes.................................................. 8
Needle structure genes ................. ................................. 8
Pore form ing com ponents .......................................................8
Polarization of type III translocation .......................................................9
Effector proteins ....................................................................... 9
R regulation of TTSS ................................................................................ 10
O their TTSS R elated G enes ................................ ........................ ...............11
2 MucA-MEDIATED COORDINATION OF TYPE III SECRETION AND
ALGINATE SYNTHESIS IN Pseudomonas aeruginosa .........................................13
In tro d u ctio n ...................................... ................................................ 13
M material and M ethods ........................................................... ........................ 14
Bacterial Strains and Growth Conditions............. ............................................14
Construction of Tn insertional M utant Bank.....................................................16
Determination of Tn Insertion Sites .............. ..................................... ......... 17
Generation of Knockout M utants ................................. ............. .................. 17
Plasmid Constructs for Complementation and Overexpression.......................18
W western Blotting ............... .. ................ ......... ....... ........ ...............19
RNA Isolation and M icroarray Analysis............................................... 20
R results .................... ...................... ..... .. ........... .. .............. ..... ........20
Activation of the TTSS Requires a Functional mucA Gene.............................20
Microarray Analysis of Gene Expression in the mucA Mutant.........................25
TTSS Repression in the mucA Mutant is AlgU Dependent. ............................31
AlgR has a Negative Regulatory Function on the TTSS..................................33
D discussion and Future D directions ...................... ............... ....... .....................34
The Expression of exsA in the mucA Mutant ................................. ...............34
The Regulatory Pathway of AlgU Regulon.................................. ................. 35
The TTSS Activity in P. aeruginosa CF Isolates................................ ..........36
Genes Differently Expressed in the mucA Mutant and Isogenic Wild-type
P A K ........................................................................... 3 8
3 PtrB OF Pseudomonas aeruginosa SUPPRESSES THE TYPE III SECRETION
SYSTEM UNDER THE STRESS OF DNA DAMAGE ...................... ...............42
Introdu action ...................................... ................................................. 42
M material and M ethords................................................................... .....................43
Bacterial Strains and Growth Conditions...................................................43
RT-PCR and Quantitative Real-time PCR ...................................................46
C ytotoxicity A ssay ............... .... ................ ..................... 47
Application of BacterioMatch Two-hybrid System ........................................48
Other M methods ............... ...... .. ................ .......... 48
TTSS Is Repressed in a prtR M utant .................................................................. 48
Identification of the PrtR-regulated Repressor of the TTSS ...............................49
PA0612 and PA0613 Form an Operon Which Is Under the Control of PrtR .....52
PA0612 Is Required for the Repression of the TTSS In the prtR Mutant...........53
The Expression of exsA Is Repressed by PtrB in prtR mutants...........................57
PtrB M ight Not Directly Interact with ExsA .................................................... 57
Mitomycin C-mediated Suppression of the TTSS Genes Requires PtrB ..........59
Twitching Motility Was Not Affected by the prtR mutation .............................61
D isc u ssio n .............................................. ........................ ................ 6 2
4 DISCUSSION AND FUTURE DIRECTIONS....................................66
The TTSS and Environm mental Stresses ........................................... ............... 66
Repression of the TTSS under Environmental Stresses..................... ...........66
Indication for the Control of P. aeruginosa Infection...................................67
Regulation of the TTSS under Environmental Stresses ...................................68
Expression of ExsA .................................. .......... .......... .............. 68
Transcriptional control ........................................ ........................... 69
Post-transcriptional control ........................................ ....... ............... 69
Transposon M utagenesis ................................................. ............................... 71
M utagenesis E efficiency ............................................... ............................ 71
C characteristics of the T n ........................................................................ ... ..... 72
Screen Sensitivity ........................................ ................... ..... .... 72
L IST O F R E F E R E N C E S ....................................................................... ... ................... 75
B IO G R A PH IC A L SK E TCH ..................................................................... ..................90
LIST OF TABLES
1-1 Regulation and substrates of multidrug efflux systems ...........................................4
2-1 Strains and plasmids used in this study .......................................... ...............15
2-2 Expression of AlgU regulon genes in PAKmucA22 .............................................25
2-3 Expression of TTSS-related genes in PAKmucA22 .............................................27
2-4 Genes up regulated in PAKmucA22.................... ... ........................... 28
2-5 Genes down regulated in PAKmucA22 ...................................................... 30
3-1 Strains and plasmids used in this study ............... .......................... .... ............. 43
3-2 PCR prim ers used in this study ........................................... ......................... 46
LIST OF FIGURES
1-1. A m odel of the regulation of ExsA ..................................................... ........... 10
1-2. TTSS related regulatory netw ork.. ....................................................... .....................12
2-1. Expression of type III secretion genes in Tn insertional mutants of mucA................22
2-2. Expression and secretion of ExoS protein......... .......................................24
2-3. Expression of exsA::lacZ (A) and exoS::lacZ (B)................... .................................32
2-4. Expression of exsA::lacZ (A) and exoS::lacZ (B) in algR mutants .........................34
2-5. Proposed model of MucA-mediated coordination of alginate production and
T T S S expression .. ......................................................................37
3-1. Expression and secretion of E xoS.. ................................................. .....................50
3-2. Genetic organization and putative promoter regions ofprtN, prtR, PA0612-3. ........52
3-3. Expression of PA0612 is repressed by prtR .............................................................54
3-4. Expression of PA0612::lacZ................................................... ...............55
3-5. Characterization of ExoS expression and cytotoxicity ...........................................56
3-6. Expression of exsA operon inprtR mutants ...................... ............... ........... 57
3-7. Monitoring of protein-protein interactions by the BacterioMatch two-hybrid
system ............... ..... ......... ......................... ...... ................. .. 58
3-8. Effect of mitomycin C on bacteria growth and TTSS activity................................60
3-9. Twitching motility ofprtR, ptrB and PA0613 mutants.........................................61
3-10. Proposed model ofPtrB-mediated TTSS repression.............................................64
4-1. Structure of the exsCEBA operon. ........................................ ......................... 69
4-2. The secondary structure of exsA mRNA 5' terminus. The sequence was analyzed
b y m fo ld .. .................................................................................7 0
Abstract of Dissertation Presented to the Graduate School
of the University of Florida in Partial Fulfillment of the
Requirements for the Degree of Doctor of Philosophy
REGULATION OF THE TYPE III SECRETION SYSTEM IN Pseudomonas aeruginosa
Chair: Shouguang Jin
Major Department: Molecular Genetics and Microbiology
Pseudomonas aeruginosa is an opportunistic bacterial pathogen which primarily
infects patients with cystic fibrosis (CF), severe bums, or immunosuppression. P.
aeruginosa possesses a type III secretion system (TTSS) which injects effector proteins
into host cells, resulting in cell rounding, lifting, and death by necrosis or apoptosis. By
screening a transposon insertional mutant library of a wild-type strain PAK, mutation in
the mucA orprtR gene was found to cause repression of the TTSS.
Mutation in the mucA gene causes alginate overproduction, resulting in a mucoid
phenotype. Comparison of global gene expression profiles of the mucA mutant and wild-
type PAK under TTSS inducing condition confirmed the down regulation of TTSS genes
and up regulation of genes involved in the alginate biosynthesis. Further analysis
indicated that the repression of the TTSS in the mucA mutant was AlgU and AlgR
dependent. Overexpression of the algR gene inhibited type III gene expression.
PrtR is an inhibitor ofprtN, which encodes a transcriptional activator for pyocin
synthesis genes. In P. aeruginosa, pyocin synthesis is activated when PrtR is degraded
during the SOS response. Treatment of a wild-type P. aeruginosa strain with mitomycin
C, a DNA-damaging agent resulted in the inhibition of TTSS activation. AprtR/prtN
double mutant had the same TTSS defect as theprtR mutant, and complementation by a
prtR gene but not by aprtNgene restored the TTSS function. Also, overexpression of the
prtN gene in wild-type PAK had no effect on the TTSS; thus PrtN is not involved in the
repression of the TTSS. To identify the PrtR-regulated TTSS repressor, another round of
Tn mutagenesis was performed in the background of aprtR/prtN double mutant.
Insertion in a small gene, designatedptrB, restored the normal TTSS activity. Expression
ofptrB is specifically repressed by PrtR, and mitomycin C-mediated suppression of the
TTSS is abolished in aptrB mutant strain. Therefore, PtrB is a newly discovered TTSS
repressor that regulates the TTSS under the stress of DNA damage.
My study revealed new regulatory relationship between MucA, PrtR and the TTSS,
and indicated that the TTSS might be repressed under environmental stresses.
INTRODUCTION AND BACKGROUND
Pseudomonas aeruginosa is a versatile bacterium that is present in soil, marshes,
tap water, and coastal marine habitats. It is a straight or slightly curved, gram negative
bacillus (0.5-1.0 x 3-4 [m), belonging to the y-subdivision of the Proteobacteria. The
bacterium is defined as an obligate aerobe; however, anaerobic growth can occur when
nitrate or arginine is used as an alternate electron acceptor.
The genome sequence of this microorganism was completed several years ago and
is freely available to the public (www.pseudomonas.com) (124). The complete sequence
of this genome was one of the largest bacterial genomes sequenced to date, with 6.3-Mbp
in size encoding 5570 predicted genes (124). Most interesting is the fact that as high as
8% of the genome encodes transcriptional regulators, which is consistent with the
observed bacterial adaptability to various growth environments.
P. aeruginosa causes a wide range of infections, from minor skin infections to
serious and sometimes life-threatening complications. P. aeruginosa is also a causative
agent of systemic infections in immunocompromised patients, such as those receiving
chemotherapy, elderly patients, and burn victims (105, 109). Chronic bronchopulmonary
infection of P. aeruginosa is the major cause of morbidity and mortality in cystic fibrosis
(CF) patients (57).
CF Airway Infection by P. aeruginosa
Today, CF is one of the most common genetic disorders in Caucasian populations.
Approximately 30,000 individuals are affected in the United States. CF patients bear a
defect in the cystic fibrosis transmembrane conductance regulator (CFTR) gene located
on the human chromosome 7q31.2 (41, 103). CFTR functions as an apical membrane
chloride channel. Due to the mutation in CFTR, little or no C1- is transported across the
apical surface of secretary cells, which leads to an unopposed reabsorbtion ofNa+, Cl-,
and water. This results in thick mucus in a CF patient's airway. The thickened mucus
provides a favorable environment for opportunistic pathogens including P. aeruginosa,
Staphylococcus aureus, Haemophilus influenza, and Burkholderia cepacia (51). During
progression of the infection, P. aeruginosa predominates and grows as a biofilm, which is
highly resistant to antibiotics and cannot be eradicated. Most clinical isolates from CF
patients overproduce an extracellular polysaccharide called alginate, resulting in a
It is believed that the recurring infections that culminate with chronic P. aeruginosa
colonization cause the respiratory damage in CF patients, the progressive deterioration of
respiratory function, and eventually the mortality of the patient. The clinical treatment
typically includes antibiotics, anti-inflammatory drugs, bronchodilators, and physical
therapy (96, 99).
P. aeruginosa exhibits a remarkable ability to develop resistance to multiple
antibiotics. The resistance arises through an acquired and/or intrinsic mechanism.
Acquired resistance is developed from a mutation or an acquisition of an antibiotic
modification enzyme by horizontal transfer, such as p-lactamase (76, 88) and acetyl-
transferases (resistance to aminoglycosides) (91, 117).
The target gene will avoid recognition of the antibiotic if mutation occurs. For
example, mutations causing lipopolysaccharide changes reduce the uptake of
aminoglycosides (14). Mutations in GyrA (a DNA gyrase) result in the resistance to
fluoroquinolone (94). Other mutations will cause the decrease of membrane permeability
(134) or up regulation of intrinsic resistant genes/systems (110).
P. aeruginosa is intrinsically resistant to many antibiotics. The mechanisms
include chromosomally encoded P-lactamase (76), low permeability of outer membrane
and multidrug efflux systems (100). Besides these mechanisms, the biofilm mode of
growth also leads to an increased antibiotic resistance (58). More of the biofilm will be
discussed in the next section.
Multidrug Efflux Systems
The multidrug efflux system is a three-component channel through the inner and
outer membrane which pumps out antimicrobial agents in an energy dependent manner.
It contributes to the reduced susceptibility or resistance to many antibiotics such as P-
lactams, aminoglycosides, tetracycline, quinolones, chloramphenicol, sulphonamides,
macrolides and trimethoprim (110). Six multidrug efflux systems have been identified in
P. aeruginosa, including MexAB-OprM, MexCD-OprJ, MexEF-OprN, MexXY-OprM,
MexJK-OprM and MexGHI-OpmD. Each of them has a different substrate specificity.
MexJK-OprM and MexGHI-OpmD were found to provide resistance against triclosan
(18) and vanadium (1), respectively. MexAB-OprM is constitutively expressed at a low
level. In wild-type strains, the expression of MexAB-OprM, MexCD-OprJ, MexEF-
OprN and MexXY-OprM is repressed. Mutation in their respective regulator will lead to
derepression and increased antibiotic resistance. The substrates and regulation of these
efflux systems are summarized in Table 1-1.
Table 1-1 Regulation and substrates of multidrug efflux systems
Multidrug efflux Regulator Substrates
MexAB-OprM MexR (-) p-lactams, quinolones, chloramphenicol,
tetracycline, trimethoprim, sulphonamides
MexCD-OprJ NfxB (-) Cefpirome, quinolones, chloramphenicol,
erythromycin, tetracycline (118)
MexEF-OprN MexT (+) Imipenem, quinolones, tetracycline (68)
MexXY-OprM MexZ (-) Aminoglycosides, tetracycline, erythromycin
-, negative regulator; +, positive regulator
P. aeruginosa harbors an arsenal of virulence factors, which enable it to establish
localized, chronic colonization or systemic infection. The virulence factors include
flagella, pili, extracellular toxins, quorum sensing systems, iron metabolism factors,
alginate production, and a type III secretion system (5, 19, 21, 25, 58, 64, 65, 67, 87, 97,
104, 111, 121).
P. aeruginosa possesses a single polar flagellum which serves as a motive
organelle on the bacterial surface. The flagellum consists of a basal body, hook, flagellar
filament and motor. The basal body anchors the flagellum on the surface of the
bacterium, the hook functions as a joint connecting the filament to the basal body, the
filament functions as a propeller, and the rotation of the flagellum is generated by the
motor. By rotating the flagellum, the bacterium can move in the surrounding
environment. Two types of movement depend on flagella, swimming and swarming.
Swimming is a movement of bacteria in the surrounding liquid, and swarming is a
surface translocation by groups of bacteria (17, 50). During the infection, flagella
mediate the adhesion of P. aeruginosa to mucin in human airways (5, 104).
Besides flagella, P. aeruginosa produces another motive organelle, the type IV
pilus. A pilus is a polar filament structure, mediating attachment to host epithelial cells
and a type of surface translocation called twitching motility (141). The pilus is composed
of a small subunit (pilin). Pilin is synthesized in the cytoplasm as pre-pilin and
translocated through the inner membrane, cell wall, and outer membrane to the surface of
bacterium. During translocation, pre-pilin is cleaved to pilin and made ready to be
assembled into a pilus. The pilus is able to extend and retract, resulting in surface
translocation (twitching motility) (25, 87).
P. aeruginosa produces a variety of extracellular virulence determinants and
secondary metabolites, which could cause extensive tissue damage, inflammation, and
disruption of host defense mechanisms. The extracellular toxins include exotoxin A,
alkaline protease, phospholipase C, elastase, hydrogen cyanide, pyocyanin, phenazine
and rhamnolipid. Exotoxin A and alkaline protease are under the control of the iron
metabolism system and are expressed at a much higher level under iron limited
environments (19, 97). The regulation of phospholipase C is regulated by inorganic
phosphate (Pi) (121). The remaining virulence factors are under the control of a quorum
sensing system (65).
P. aeruginosa possesses a signaling system for cell-cell communication, called
quorum sensing. P. aeruginosa possesses three quorum sensing systems, known as las,
rhl and PQS (pseudomonas quinolone signal). Each system contains a small molecule
involved in signal communication. The las and rhl systems use acyl-homoserine
lactones, C4-HSL and 30C12-HSL, as signal molecules, respectively. The signal
molecule of the PQS system is quinolone. The signal molecules are secreted into the
surrounding environment, and when their concentrations reach a threshold (usually at the
mid or late log phase), they can interact with their respective receptors and modulate gene
expression in the population. The three quorum sensing systems can interact with each
other. When the quorum sensing systems are activated, the expression of many virulence
genes is up regulated, as reported previously (65, 133). Besides functioning as signal
molecules, C4-HSL and 30C12-HSL can directly modulate the host immune system.
30C12-HSL is able to promote induction of apoptosis in macrophages and neutrophils
(128). Quorum sensing is required for biofilm formation (119, 145). Therefore, quorum
sensing can be a drug target for the treatment and eradication ofP. aeruginosa infection
Iron is essential for the metabolism and survival ofP. aeruginosa. To acquire iron
from the surrounding environment, P. aeruginosa produces and secretes iron-chelating
compounds called siderophores. Two types of siderophores, pyoverdine and pyochelin,
are produced by P. aeruginosa, with the former having much higher affinity than the
latter in binding iron (III). The pyoverdine and pyochelin synthesis genes and receptors
are under the negative control of a regulator, Fur. Under iron depleted environments, the
expression of these genes is derepressed (97, 132).
Alginate is an exopolysaccharide synthesised by P. aeruginosa. Alginate
production is known to be activated by environmental stress such as high osmolarity,
nitrogen limitation, and membrane perturbation induced by ethanol (10). Over
production of alginate renders mucoidy to the bacterium. Most P. aeruginosa clinical
isolates from CF patients display a mucoid phenotype (111). The function and regulation
of alginate are described in the introduction section of Chapter 3.
During chronic infection of CF airways, P. aeruginosa forms a biofilm on the
respiratory epithelial surface. The biofilm consists of microcolonies surrounded by
alginate (58), although alginate is not essential for the biofilm formation. The formation
ofbiofilm requires flagella, pili, and quorum sensing systems (28, 51, 58). Bacteria
growing in a biofilm are much more resistant to antibiotics than when growing in
planktonic mode. It is believed that the slow, anaerobic growth inside the biofilm
increases the antibiotic resistance. The surrounding negatively charged alginate may
function as a barrier against antibiotics, especially positively charged aminoglycosides
(58). Due to the biofilm mode of growth, antibiotic treatment usually fails to eradicate
the bacteria (58).
Type III Secretion System
Type III secretion systems (TTSSs) are complex protein secretion and delivery
machineries existing in many animal and plant pathogens. The TTSS directly
translocates bacterial effector molecules into the host cell cytoplasm, causing disruption
of intracellular signaling or even cell death (35). Components of TTSSs from a variety of
gram-negative bacteria display sequence and structural similarity. Most TTSS apparatus
are composed of two sets of protein rings embedded in the bacterial inner and outer
membranes and a needle-like structure (102). According to the current working model,
the needle forms a pole in the host cell membrane, and effector proteins are delivered
through the hollow needle (49, 95, 127).
The TTSS is an important virulence factor ofP. aeruginosa: it inhibits host defense
systems by inducing apoptosis in macrophages, polymorphonuclear phagocytes, and
epithelial cells (21, 64, 67). The loss of the TTSS resulted in an avirulent phenotype in a
burned mouse model (59).
Function of TTSS Structural Genes
The P. aeruginosa TTSS machinery is encoded by 31 genes arranged in four
operons on the chromosome. Several genes have been well studied for their functions
Needle structure genes
The P. aeruginosa TTSS needle is primarily composed of a 9-kDa protein named
PscF. Partially purified needles measured about 7 nm in width and 60-80 nm in length
(98). PscF has been shown to have two intracellular partners, PscE and PscG, which
prevent it from polymerizing prematurely in the cytoplasm and keep it in a secretion-
prone conformation (102).
Pore forming components
In order for TTSS-containing bacteria to directly deliver effector proteins into the
eukaryotic cytoplasm, a mechanism is required for the TTSS to penetrate the double
phospholipid cell membrane. The pore-forming activity possessed by the TTSS is
dependent on thepcrGVHpopBD operon (24). Upon contact with the host cell
membrane, PopB and PopD polymerize and form a ring-like structure in the membrane,
through which effector proteins are tanslocated. PopB and PopD have a common
cytoplasmic chaperon, PcrH, which prevents their premature aggregation (114). Another
gene product of this operon, PcrV, is required for the assembly and insertion of the
PopB/PopD ring into the host cell membrane (44). However, no direct interaction has
been detected between PcrV and PopB/PopD (44). PcrG was found to interact with PcrV
Polarization of type III translocation
When cultured mammalian cells were infected with wild type P. aeruginosa, TTSS
effector proteins could be detected only in the eukaryotic cytoplasm, but not in the tissue
culture medium (131). This phenomenon is called polarized translocation, during which
PopN, PcrG and PcrV are all required. Mutation in eitherpopN orpcrG does not affect
the TTSS-related cytotoxicity against HeLa cells; however, it results in high levels of
ExoS in the tissue culture medium (126).
Four different effector proteins have been found in P. aeruginosa, ExoS, ExoT,
ExoY and ExoU. However, no natural P. aeruginosa isolates harbor both ExoS and
ExoU simultaneously. ExoS and ExoT share significant sequence homology and
structural similarity, with both bearing an ADP-ribosyltransferase activity and a GTPase-
activating protein activity. ExoU and ExoY have lipase and adenylate cyclase activities,
respectively (6, 39, 81, 112, 125). The ADP-ribosyltransferase activity of ExoS has been
shown to cause programmed cell death in various types of tissue culture cells (64, 67).
Regulation of TTSS
Expression of the TTSS regulon can be stimulated by direct contact with the host
cell or by growth under a low Ca2+ environment (61, 131). The expression of type III-
related genes is coordinately regulated by a transcriptional activator, ExsA (60). ExsA is
an AraC-type DNA binding protein that recognizes a consensus sequence, TXAAAXA,
located upstream of the transcriptional start site of type III secretion genes, including the
exsA gene itself (60). Three proteins, ExsD, ExsC and ExsE, directly regulate the activity
of ExsA. ExsD represses ExsA activity by directly interacting with it (89). ExsC on the
other hand has the ability to interact with both ExsD and ExsE (106, 130). Under TTSS
non-inducing conditions, ExsC binds to ExsE; however, when the TTSS is induced, ExsE
is secreted outside of the cell by TTSS machinery. This leads to the increased level of
free ExsC, which in turn binds to ExsD and releases ExsA, allowing the transcriptional
activation of the TTSS (27, 106, 130). The regulation cascade of the TTSS through ExsA
is summarized in Fig. 1-1.
TTSS non-inducing conditions TTSS inducing conditions
Basal level TTSS Activated TTSS
Figure 1-1. A model of the regulation of ExsA. See text for detail. *, derepressed ExsA.
Other TTSS Related Genes
In addition to genes described thus far, a number of other genes have been shown
to affect the expression of type III genes, although the regulatory mechanisms are not
known. Under TTSS-inducing conditions (low Ca2+), the cyclic AMP level increases and
a CRP homologue, Vfr, is required for TTSS activation (140). Vfr is a global regulator
which mediates the activation of quorum sensing (3), twitching motility (8), type II
secretion (140), and repression of flagellum synthesis (26). A novel gene,fimL, is also
required for both TTSS and twitching motility (116, 137). Transcription of vfr is reduced
in afimL mutant, and over expression of Vfr restores both the TTSS and twitching
motility, which suggests that the regulatory role of Vfr is downstream of FimL (137).
Mutation in a hybrid sensor kinase/response regulator (RtsM or RetS) results in a defect
in the TTSS and hyperbiofilm phenotype (42). Over expression of either Vfr or ExsA in
a ArtsM mutant restores the TTSS activity (72). Furthermore, a three-component
regulatory system (SadARS) is also required for both TTSS and biofilm formation in P.
Some enzymes and metabolic pathways in P. aeruginosa are also found to be
essential for the activation of TTSS. These include a periplasmic thiol:disulfide
oxidoreductase (DsbA) (48), a tRNA pseudouridine synthase (TruA) (2), pyruvate
dehydrogenases (AceAB) (23), and a normal histidine metabolism pathway (107).
Additionally, the TTSS in P. aeruginosa is under the negative control of the rhl quorum-
sensing system and the stationary-phase sigma factor RpoS (12, 56). Over expression of
MexCD-OprJ or MexEF-OprN also cause the repression of the TTSS (75). Recently, our
lab has demonstrated that a gene highly inducible during infection of the bum mouse
model, designated ptrA, encodes a small protein which inhibits TTSS through direct
binding to ExsA and thus functions as an anti-ExsA factor. Expression of this gene is
specifically inducible by high copper signal in vitro through a CopR/S two-component
regulatory system (47). Fig. 1-2 summarizes the knowledge of the TTSS-related
regulatory network in P. aeruginosa. The regulatory roles played by AlgR and PtrB in
TTSS regulation were discovered during my doctoral research period and will be
described in Chapter 2 and 3, respectively.
trLt -00 TTSS
FimL + Twitching
+ Flagellum +
Vfr + Quorum + Biofilm
\+ aRpoS +
4- + SadARS
/ 1 +\
Normal histidine TruA AceAB DsbA
Figure 1-2. TTSS related regulatory network. See text for detail. +, positive
regulation/relationship; -, negative regulation. 1, direct protein-DNA binding
has been proved. 2, direct protein-protein interaction has been proved. 3, 4, this
relationship was newly discovered from the work during my Ph.D. program.
MucA-MEDIATED COORDINATION OF TYPE III SECRETION AND ALGINATE
SYNTHESIS IN Pseudomonas aeruginosa
Among CF patients, P. aeruginosa colonizes inside the thick mucus layer of the
airway. In this anaerobic environment, P. aeruginosa overproduces the
exopolysaccharide alginate and forms a biofilm which protects the bacterium from
reactive oxygen intermediates and inhibits phagocytosis (51). More than 90% of P.
aeruginosa strains isolated from CF patients show the mucoid phenotype, due to the
overproduction of alginate (111). Clearly, alginate overproduction is a strategy to
overcome environmental stresses. A number of stress signals trigger the overproduction
of alginate, converting the bacterium to the mucoid phenotype (84).
The genes encoding enzymes for alginate synthesis form an operon (algD operon),
and the expression of this operon is under the tight control of several regulators. The key
regulatory gene of this operon is the algU gene (also called algT), included in an algU
operon which consists of algU-mucA-mucB-mucC-mucD. The algU gene encodes a
sigma factor, 22, which autoregulates its own promoter and activates many other genes,
including those for alginate biosynthesis (85). The second gene in the algU operon, the
mucA gene, encodes a transmembrane protein with a cytoplasmic portion binding to and
inactivating AlgU (85). The third gene of the algU operon, the mucB gene, encodes a
periplasmic protein, possibly sensing certain environmental signals. Upon sensing
certain environmental signals, MucB transduces the signal to MucA, which in turn
releases the bound form of AlgU, resulting in activation of alginate production (85). The
majority of P. aeruginosa isolates from the lungs of older CF patients carry mutations in
the mucA or mucB gene and display a mucoid phenotype (82). In the AlgU regulon, two-
component regulatory systems AlgB-FimS (78) and AlgR-AlgZ (146) and regulators
AlgP (29) and AlgQ (73) are required for alginate synthesis. Among them, AlgR was
also shown to be essential for P. aeruginosa pathogenesis (77). An algR mutant is less
virulent than a wild-type strain in an acute septicemia infection mouse model (77). AlgR
is also required for twitching motility (136, 138). Proteomic analysis of the algR mutant
suggested that AlgR is a global regulator, affecting the expression of multiple genes (77).
In this chapter, a transposon (Tn) insertional mutant bank of a wild type P.
aeruginosa strain, PAK, was screened for mutants that are defective in TTSS expression.
I found that mutation in the mucA gene suppresses the expression of TTSS genes, greatly
reducing the response of the TTSS to low Ca2+. Furthermore, the suppression is
dependent on the AlgU and AlgR functions. Comparison of global gene expression of
the mucA mutant and wild type PAK under type Ill-inducing conditions confirmed the
above observation. Several groups of genes have been found to be differently expressed
in the mucA mutant and PAK, and their possible roles in TTSS expression are discussed.
Material and Methods
Bacterial Strains and Growth Conditions
Plasmids and bacterial strains used in this study are listed in Table 2-1. Bacteria
were gown in Luria broth (LB) at 370C. Antibiotics were used at the following
concentrations: for Escherichia coli, ampicillin at 100 [tg/ml, gentamicin at 10 [tg/ml,
tetracycline at 10 [g/ml, and kanamycin at 50 ig/ml; for P. aeruginosa, carbenicillin at
150 ig/ml, gentamicin at 150 ig/ml, tetracycline at 100 ig/ml, spectinomycin at 200
ig/ml, streptomycin at 200 ig/ml, and neomycin at 400 ig/ml. For 8-galactosidase
assays, three single colonies of each strain were used. The overnight cultures were diluted
100-fold with fresh LB or 30-fold with LB containing 5 mM EGTA. Bacteria were
grown to an optical density at 600 nm (OD600) between 1.0 and 2.0 before 8-galactosidase
assays (92). The data were subjected to t-test and P <0.05 was considered as statistically
Table 2-1. Strains and plasmids used in this study
Strain or plasmid Description Source or
E. coli strains
P. aeruginosa strains
RP4-2-Tc::Mu-1 Kan::Tn7 integrant leu-
63::ISO1 recAl zbf-5 creB510 hsdR17 endAl
thi uidA (AMluI::pir)/pRL27
#80dlacZAM15 &(lacZYA-argF)U169 recAl
hsdR1 7 deoR thi-1 supE44 gyrA96 relA 1/pir
Wild-type P. aeruginosa strain
PAK with exsA disrupted by insertion of V.
PAK mucA 1: :Tn5 mutant isolate; Neor
PAK mucA2::Tn5 mutant isolate; Neor
Point mutation (AG440) in mucA gene of PAK
mucA22 with algU disrupted by insertion of
Gm cassette; Gmr
mucA22 with algR disrupted by insertion of
Gm cassette; Gmr
PAK with algU disrupted by insertion of Gm
Cloning vector for the PCR products
exoS promoter of PAK fused to promoterless
lacZ on pDN191acZQ; Spr Smr Tcr
exoT promoter of PAK fused to promoterless
lacZ on pDN191acZL; Spr Smr Tcr
pscN promoter of PAK fused to promoterless
lacZ on pDN191acZQ; Spr Smr Tcr
exsA promoter of PAK fused to promoterless
lacZ on pDN191acZQ; Spr Smr Tcr
Shuttle vector between E. coli and P.
Table 2-1. Continued
Strain or plasmid Description Source or
pWW020 mucA gene on pUCP19 driven by algU This study
pWW021 mucA gene on pUCP19 driven by lac This study
pWW025 algU gene on pUCP19 driven by lac This study
pMMB67EH Low-copy-number broad-host-range cloning (38)
pWW022 algR gene on pMMB67EH driven by lac This study
pEX18Tc Gene replacement vector; Tcr oriF sacB+ (55)
pEX18Ap Gene replacement vector; Apr ori7 sacB (55)
pPS856 Source of Gm' cassette; Ap' Gm' (55)
algU::Gm- algU disrupted by insertion of Gmr cassette This study
pEX18Tc on pEX18Tc; Gmr Tc' oriT sacB
algR::Gm- algR disrupted by insertion of Gmr cassette This study
pEX18Ap on pEX18Ap; Gm' Ap' oriT sacB+
PAK algR::Gm PAK with algR disrupted by insertion of Gm This study
Construction of Tn insertional Mutant Bank
The P. aeruginosa PAK strain containing the exoT: :lacZ fusion plasmid
(pHW0006) was grown overnight at 420C, while E. coli donor strain BW20767/pRL27
was cultured to mid-log phase at 370C. Cells of the two types of bacteria were washed
with LB once to remove antibiotics in the culture medium. About 5 x 108
PAK/pHW0006 cells were mixed with 109 donor E. coli cells, and the mixture was
filtered onto a sterile nitrocellulose membrane (pore size, 0.22 .im). The membrane was
laid on top of nutrient agar and incubated at 370C for 7 to 9 h before washing off the
bacterial mixture from the membrane with LB. The bacterial suspension was serially
diluted with LB and spread on L-agar plates containing spectinomycin at 100 [tg/ml,
streptomycin at 100 [tg/ml, tetracycline at 50 [tg/ml, neomycin at 400 [tg/ml, and 20 tg
of 5-bromo-4-chloro-3-indolyl-B-L-thiogalactopyranoside (X-Gal) per ml, with or
without 2.5 mM EGTA for colony counting as well as mutant screening.
Determination of Tn Insertion Sites
To locate the Tn insertion sites of the isolated mutants, the Tn with flanking DNA
was rescued as a plasmid from the mutant chromosome. Plasmid rescue was carried out
as previously described (71). Briefly, genomic DNA of the Tn insertion mutants was
isolated with the Wizard genomic DNA purification kit (Promega) and digested with PstI.
The digested DNA was subjected to self-ligation with T4 DNA ligase and electroporated
into DH5a/pir. Plasmids were isolated from the transformants and sequenced with
primers tpnRL17-1 (5'-AAC AAG CCA GGG ATG TAA CG-3') and tpnRL13-2 (5'-
CAG CAA CAC CTT CTT CAC GA-3') for the DNA flanking the two ends of the Tn.
The DNA sequences were then compared with the P. aeruginosa genomic sequence by
using BLASTN (124).
Generation of Knockout Mutants
Chromosome gene knockout mutants were generated as previously described (55).
The target genes were amplified by PCR and cloned into pCR-TOPO2.1 (Invitrogen).
After subcloning the PCR product into pEX18Tc or pEX18Ap, the target gene was
disrupted by insertion of a gentamicin resistance cassette, leaving about 1 kb upstream
and downstream of the insertion-mutation site. The plasmids were electroporated into
wild-type PAK and single-crossover mutants were selected on LB plates containing
gentamicin at 150 [tg/ml, and tetracycline at 100 [tg/ml or carbenicillin at 150 [tg/ml.
Double-crossover mutants were selected by plating single-crossover mutants on LB
plates containing 5% sucrose and gentamicin at 150 [g/ml. In the case of the mucA22
mutant, a 1.8-kb fragment of the mucA gene region was amplified from FRD1 mucoidd
strain) (78) genomic DNA, and the fragment was cloned into the HindIII site of
pEX18Gm. The plasmid was transformed into P. aeruginosa to select for single
crossover mutants on LB agar plates containing 150 [g/ml gentamicin. Single-crossover
mutants were plated on L-agar plates containing 5% sucrose to select for double-
crossover mutants. The double-crossover mutants were mucoid, and the introduction of
the mucA22 mutation was confirmed by sequencing of the mucA gene.
Plasmid Constructs for Complementation and Overexpression
Reporter fusions between the exsA, exoT, exoS, and pscN genes and promoterless
lacZ on pDN191acZ were generated by Ha et al. (47, 48). For mucA gene
complementation, the mucA gene was amplified from PAK genomic DNA by PCR with
primers MucA-1 (5'-CGG ATC CTC CGC GCT CGT GAA GCA ATC G-3') and MucA-
2 (5'-TAC TGC GGC GCA CGG TCT CGA CCC ATA C-3'). The PCR product was
cloned into pCR-TOPO2.1 and transformed into E. coli TOP 1OF'. The obtained plasmid
was digested with HindII-XmnI and cloned into the HindIIl-Smal sites of pUCP19. The
mucA gene in the resulting plasmid, pWWO21, is driven by a lac promoter on the vector.
To generate a mucA gene driven by the algU promoter, the mucA gene on the pCR-
TOPO2.1 plasmid was subcloned into the BamHI and XmnI sites of pEX18Tc, resulting
in mucA-pEX18Tc. To obtain the algU gene promoter, an 800-bp DNA fragment
upstream of the algUgene open reading frame (ORF) was amplified by PCR with
primers AlgT1 (5'-CCT TCG CGG GTC AGG TGG TAT TCG AAG C-3') and AlgT2
(5'-TTG GAT CCG CGC TGT ACC CGT TCA ACC A-3') and cloned into pCR-
TOPO2.1. Then, this fragment was ligated into the EcoRI and BamHI sites upstream of
the mucA gene on the plasmid mucA-pEX18Tc. The obtained plasmid was digested with
EcoRI-XmnI, and the algU promoter and mucA gene ORF fragment were cloned into the
EcoRI-Smal sites of pUCP19. On the resulting plasmid (pWW020), the mucA gene is
driven by the algU promoter, and the transcriptional direction is opposite to that of the
lac promoter on the vector. For algR complementation, the algR gene was amplified
from PAK genomic DNA by PCR with primers algR1 (5'-GGT CTA GAG GCC GAG
CCC CTC GGG AAA G-3') and algR2 (5'-GTG GAT CCT ACT GCT CTC GGC GGC
GCT G-3'). The PCR product was initially cloned into pCR-TOPO2.1. The resulting
plasmid was digested with Clal, blunted ended with Klenow enzyme, and digested with
Xbal. The algR gene-containing fragment was ligated into XbaI-Smal sites of plasmid
pMMB67EH, resulting in pWW022, on which the algR gene is driven by the tac
promoter on the vector. For algU gene over expression, the algU gene ORF was
amplified from PAK genomic DNA by PCR with primers algUl (5'-GGG AAA GCT
TTT GCA AGA AGC CCG AGT C-3') and algU2 (5'-GCT TCG TTA TCC ATC ACA
GCG GAC AGA G-3'). The algUgene was cloned into Hindll-EcoRI sites of pUCP19,
where the expression of the algU gene in the resulting plasmid pWW025 was driven by
lac promoter on the vector.
P. aeruginosa strains were cultured overnight in LB at 370C. Bacterial cells were
diluted 100-fold with fresh LB or 30-fold with LB containing 5 mM EGTA and cultured
for 3.5 h. Supernatant and pellet were separated by centrifugation and mixed with
sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) loading buffer.
Equal loading of the protein samples was based on the same number of bacterial cells.
The proteins were transferred onto polyvinylidene difluoride membrane and probed with
rabbit polyclonal antibody against ExoS (self-developed). The signal was detected by
enhanced chemiluminescence following the protocol provided by the manufacturer
RNA Isolation and Microarray Analysis
For RNA isolation, three single colonies of PAK and the isogenic mutant
PAKmucA22 were each inoculated into 3 ml of LB and grown overnight. PAK and
PAKmucA22 were subcultured into LB containing 5 mM EGTA. PAK started with an
OD600 of 0.03, and the mucA22 mutant started with an OD600 of 0.06. After 3 to 4 h of
culture, bacteria were harvested at an OD600 of 1.0 to 1.2. Total RNA was isolated using
an RNeasy mini kit (QIAGEN) according to the manufacturer's instructions. The purity
and quantity were determined by spectrometry and electrophoresis. Fifteen micrograms
of RNA of each sample was used for cDNA synthesis. cDNA fragmentation and biotin
terminal labeling were carried out as instructed (Affymetrix). The experiments were
performed in triplicate. Microarray analysis was performed with the Affymetrix
GeneChip P. aeruginosa genome array. The experimental procedure followed the
manufacturer's instructions. Data were acquired and analyzed with Microarray Suite
version 5.0 (Affymetrix). Significance analysis of microarrays (129) was used to detect
differentially expressed ORFs. Then, a cutoff of 5% false discovery rate (FDR) was
chosen to analyze the data.
Activation of the TTSS Requires a Functional mucA Gene.
To identify P. aeruginosa genes that affect the expression of TTSS, a Tn insertion
mutant bank was constructed in PAK containing an exoT::lacZ (transcriptional fusion)
reporter plasmid (pHW0006) (see Materials and Methods). On plates containing X-Gal
and EGTA, the density of the blue color of each colony indicated the expression level of
the exoT gene in that particular Tn insertion mutant. To identify optimal screening
conditions, combinations of different concentrations of X-Gal and EGTA were tested. In
the presence of 20 [g of X-Gal/ml and 2.5 mM EGTA, wild-type PAK and the type III-
defective PAKexsA mutant harboring pHW0006 showed the greatest visual difference in
colony color (blue) and thus these concentrations were adopted for the screening
conditions. The mutant cells were grown on the screening plates, and we looked for
colonies with lighter blue color. About 40,000 Tn insertion mutants were screened.
Among four colonies with lighter blue color, two of them showed a mucoid phenotype
and the other two had Tn inserted in aprtR gene. The relationship between PrtR and
TTSS will be discussed in Chapter 3. The two mucoid mutants were picked to test their
TTSS activity by B-galactosidase assay. As shown in Fig. 2-1A, the exoTgene promoter
activity was three- to fourfold lower in the mutants than in the parent strain
PAK/pHW0006. To confirm this observation, the exoT: :lacZ reporter plasmid was cured
from the Tn insertion mutants by passage in the absence of antibiotic selection and a
pscN::lacZ reporter plasmid (pHW0024) was reintroduced. The resulting strain was
subjected to a B-galactosidase assay. The assay results shown in Fig. 2-1D indicated that
the expression of the pscN gene was also repressed in these mucoid mutants under both
TTSS-inducing and -noninducing conditions. Similar results were also obtained by
introducing exsA::lacZ (pHW0032) and exoS::lacZ (pHW0005) reporter plasmids and
testing B-galactosidase activities (Fig. 2-1B and C), confirming that the two Tn mutants
were indeed defective in TTSS expression.
The Tn and flanking DNA were rescued from the mutant strains and subjected to
sequencing analysis (see Materials and Methods). Sequencing results showed that the Tn
was inserted into two different positions in the mucA gene in these two mutants,
explaining the mucoid phenotype of the isolates.
PAK exsA A44 A61
PAK exsA A44 A61
PAK exsA A44 A61 PAK exsA A44 A61
Figure 2-1. Expression of type III secretion genes in Tn insertional mutants of mucA.
PAK, PAKexsA, and mucA mutants A44 and A61 harboring pHW0006
containing exoT::lacZ (A), pHW0032 containing exsA::lacZ (B), pHW0005
containing exoS::lacZ(C), or pHW0024 containing pscN::lacZ (D) were tested
for f-galactosidase activities. Bacteria were grown in LB (white bars) or LB
containing 5 mM EGTA (black bars) to an OD600 of 1 to 2 before B-
galactosidase assays. Each assay was done in triplicate, and the error bars
indicate standard deviations. *, P < 0.001, compared to the values in PAK.
Mutation in the mucA gene is commonly observed among P. aeruginosa isolates
from CF patients, such as mucA22, where a nucleotide G was deleted within five G
residues between positions 429 and 433 of the mucA coding region, causing protein
truncation (13, 111). The identical mucA22 mutant was constructed in the background of
PAK by allelic replacement with a mucA fragment amplified from P. aeruginosa FRD1
(78), which bears the mucA22 mutation (see Materials and Methods). Expression of the
effector genes exoS and exoTin the resulting mutant strain PAKmucA22 was compared to
that in PAK by Western blot analysis of the secreted and cell-associated proteins. A
polyclonal anti-ExoS antibody was used in the western blot experiment; however, it also
cross-recognizes ExoT due to a high sequence homology between the ExoS and ExoT
proteins. As shown in Fig. 2-2A, expression of ExoS and ExoT in the resulting
PAKmucA22 was greatly reduced in comparison to that in wild-type PAK when grown
under type III-inducing conditions. Reporter plasmids pHW0032 (exsA::lacZ) and
pHW0005 (exoS::lacZ) were further introduced into PAKmucA22 and tested for 3-
galactosidase activity. Similar to the original isolates of the mucA Tn insertional mutants,
expression of the exsA and exoS genes in PAKmucA22 was almost nonresponsive to low
Ca2+, compared to a three- to fourfold induction in the wild-type PAK background (Fig.
2-2B and C). Upon complementation of the PAKmucA22 mutant with the mucA gene in
pUCP 19, either driven by the algU promoter (pWW020) or lac promoter (pWW021),
expression of the exsA and exoS genes in the resulting strains was restored to the wild-
type level (Fig. 2-2C). These results clearly demonstrate that expression of the TTSS
genes requires a functional mucA gene.
mucA22 mucA22 mucA22 mucA22
/pWW020 /pWW021 algU::Gm algR::Gm
PAK mucA22 mucA22 mucA22 mucA22 mucA22
/pWW020 /pWW021 algU::Gm algR::Gm
Figure 2-2. Expression and secretion of ExoS protein. (A) Comparison of cellular and
secreted forms of ExoS in strains PAK and PAKmucA22 grown in LB or LB
plus 5 mM EGTA. Supernatants and pellets from equivalent bacterial cell
numbers were loaded onto sodium dodecyl sulfate-polyacrylamide gel
electrophoresis gels and immunoblotted with anti-ExoS antibody. Both ExoS
and ExoT are indicated by the arrow. Anti-ExoS polyclonal antibody also
recognizes ExoT due to high homology between these two proteins. (B)
Expression of exsA::lacZ (pHW0032) in the backgrounds of PAK,
PAKmucA22, with or without the mucA clone driven by an algU promoter
(pWW020) or lac promoter (pWW021), PAKmucA22algU::Gm and
PAKmucA22algR::Gm (C) Expression of exoS::lacZ (pHW0005) in the same
backgrounds as described above. Bacteria were grown to an OD600 of 1 to 2
in LB with (black bars) or without (white bars) EGTA before 3-galactosidase
assays. *, P < 0.05, compared to the values in mucA22.
Microarray Analysis of Gene Expression in the mucA Mutant
To further understand the mechanism of MucA-mediated regulation of TTSS
genes, global gene expression profiles were compared between PAKmucA22 and its wild-
type parent strain PAK grown under TTSS-inducing conditions. Previously, a microarray
analysis compared global gene expression patterns between mucoid (mucA mutant) and
wild-type P. aeruginosa under non-TTSS-inducing conditions (32). Under these
conditions, the TTSS activity in both strains was low; thus, no obvious effect of the mucA
gene on the TTSS was observed.
Results of our gene array analysis were consistent with the published data (32, 33);
genes under the control of AlgU are up regulated in the PAKmucA22 mutant background
compared to those in wild-type PAK, including genes for alginate biosynthesis (operon
PA3540-3551) and regulation (Table 2-2). Also up regulated was operon, PA4468-4471,
which includes the sodM gene (PA4468) encoding manganese superoxide dismutase,
whose production is known to be higher in mucoid than that in nonmucoid P. aeruginosa
(54), and thefumC gene (PA4470) encoding a tricarboxylic acid cycle enzyme fumarase
C, which is essential for alginate production (53). Their results validated our gene array
Table 2-2. Expression of AlgU regulon genes in PAKmucA22 (examed in microarray)
Group and ID no. Name Function Fold change
in mucA22 vs
PA3540 algD Alginate biosynthesis 64.2*
PA3541 alg8 Alginate biosynthesis 29.9*
PA3542 alg44 Alginate biosynthesis 28.9*
PA3543 algK Alginate biosynthesis 81.2*
PA3544 algE Alginate biosynthesis 47.9*
PA3545 algG Alginate biosynthesis 38.0*
Table 2-2. Continued
Group and ID no. Name Function
in mucA22 vs
Genes known to be up
regulated in mucA
PA0059 osmC Osmotically 3.8*
PA0376 rpoH Sigma factor 1.3
PA4876 osmE Osmotically 3.0*
PA5489 dsbA Thiol:disulfide 1.3
*, FDR<5%. **, Expression data is presented as fold change in mucA22 relative to wild-
Meanwhile, the expression levels of exoS, exoT, exoY, and other T3SS-related
genes were clearly down regulated in the mucA mutant background compared to those in
wild-type PAK under TTSS-inducing conditions (Table 2-3), which confirmed our 3-
galactosidase assay and the Western blotting results. However, no significant changes in
the expression of the exsA gene and a few other TTSS genes were observed. A previous
gene array study also showed that expression of the exsA gene and the exsD-pscL operon
is relatively nonresponsive to Ca2+ depletion (140), yet a clear difference in the 3-
galactosidase activities could be observed when PAK harboring exsA::lacZ (pHW0032)
was grown in LB with or without EGTA. Similarly, we have seen differences in the 3-
galactosidase activities between PAK(pHW0032) and PAKmucA22(pHW0032) under
type Ill-inducing conditions without observing such differences in gene array data,
suggesting possible involvement of posttranscriptional control of the exsA gene.
Table 2-3. Expression of TTSS-related genes in PAKmucA22 (examed in microarray)
ID no. Gene Function Fold change in mucA22 vs
PA0044 exoT Exoenzyme T -2.0*
PA2191 exoY Adenylate cyclase -1.3
PA3841 exoS Exoenzyme S -2.1*
PA1707 pcrH Regulatory protein -1.4
PA1708 popB Translocator protein -1.6
PA1709 popD Translocator outer membrane protein -1.5
PA1718 pscE Type III export protein -1.4
PA1719 pscF Type III export protein -1.5
*, FDR<5%. **, Expression data is presented as fold change in mucA22 relative to wild-
From the microarray analysis, genes that are differentially expressed more than
threefold between PAKmucA22 and PAK are listed in Tables 2-4 and 2-5. A number of
genes known to be inducible under iron deprivation was also elevated in the mucA22
mutant, including the sigma factor PvdS and genes regulated by PvdS for pyoverdine
synthesis (53), the operon PA4468-4471 (53), and the probable two-component
regulatory genes PA1300 and PA1301, encoding the extracytoplasmic function sigma-70
factor and a transmembrane sensor, respectively (97). Compared to the global gene
expression profile of PAK grown under TTSS inducing or noninducing conditions, none
of the above genes seem to be affected by Ca2+ depletion (140). The mechanism by
which these genes are activated is not clear.
Table 2-4. Genes up regulated in PAKmucA22* (examed in microarray)
ID no." Gene Function Fold change TSB" LBb
in mucA22 vs (fold) (fold)
PA0059 osmC Adaptation, protection 3.75 1.23 -1.29
PA2386 pvdA Adaptation, protection 3.89 -1.50 1.07
#PA2397 vvdE Adaptation. protection. 3.92 -3.00 -1.99
membrane proteins, transport
of small molecules
Antibiotic resistance and
proteins, transport of small
PA2019 Antibiotic resistance and
susceptibility, transport of
PA1985 pqqA Biosynthesis of cofactors,
prosthetic groups, and
PA1988 pqqD Biosynthesis of cofactors,
prosthetic groups, and
PA1989 pqqE Biosynthesis of cofactors,
prosthetic groups, and
#PA2414 Carbon compound
PA3158 wbpB Cell wall, LPS, and capsule;
PA0102 Central intermediary
PA2393 Central intermediary
PA2717 cpo Central intermediary
PA4470 fumCl Energy metabolism
PA5491 Energy metabolism
ID no.a Gene Function Fold change TSBb LBb
in mucA22 vs (fold) (fold)
PA5182 Hypothetical; membrane
PA5183 Hypothetical; membrane
PA2409 Membrane proteins, transport
of small molecules
PA4876 osmE Membrane proteins,
PA2407 Motility and attachment
ID no.a Gene Function Fold change TSBb LBb
in mucA22 vs (fold) (fold)
Table 2-5. Genes down regulated in PAKmucA22* (examed in microarray)
ID no." Gene Function Fold change TSBb LBb
in mucA22 vs (fold) (fold)
PA3450 Adaptation, protection -3.5 1.84 1.17
PA2138 DNA reDlication -3.2 -2.50 -3.19
transport of small molecules
transport of small molecules
PA2385 Putative enzymes 3.19 1.05 -2.36
PA2394 Putative enzymes 3.56 1.00 -1.75
PA2402 Putative enzymes 3.18 -1.70 -2.00
PA2413 Putative enzymes 5.97 1.46 -2.32
PA4785 Putative enzymes 4.51 1.70 -1.98
PA0724 Related to phage, transposon, 3.48 1.83 -2.39
PA1300 Transcriptional regulators 3.66 -1.70 -1.13
PA2426 pvdS Transcriptional regulators 4.39 -1.40 -1.38
PA2408 Transport of small molecules 4.09 2.08 -3.14
PA3049 rmf Translation, posttranslational 6.81 2.03 1.38
PA3188 Transport of small molecules 3.08 4.04 15.76
PA5470 Translation, posttranslational 3.31 -1.50 1.15
#PA2398 fpvA Transport of small molecules 6.65 -2.10 -1.51
*, genes with FDR<5% and changes greater than threefold. **, Expression data is
presented as fold change in mucA22 relative to wild-type PAK. a #, up regulated in mucA
mutant compared to PAK, but down regulated in PAK under type III-inducing conditions
versus noninducing conditions, and vice versa. Not included are those known to be
affected by the growth medium, such as those varied in TSB versus LB (140). b Change
in gene expression in PAK grown under TTSS inducing conditions versus PAK grown
under TTSS noninducing conditions (140). Bacteria were grown in TSB or LB.
Table 2-5. Continued
ID no.a Gene Function Fold change TSBb LBb
in mucA22 vs (fold) (fold)
PA1246 aprD Secreted factors (toxins, -3.4 2.05 -2.64
enzymes, alginate); protein
PA1312 Transcriptional regulators -3.1 -1.10 -1.55
PA3927 Transcriptional regulators -5.1 -1.70 -1.67
PA0198 exbB] Transport of small molecules -5.5 2.71 -3.68
PA0280 cysA Transport of small molecules -6.0 -1.00 -1.18
PA2204 Transport of small molecules -4.7 1.16 -1.16
*, genes with FDR<5% and changes greater than threefold. **, Expression data is
presented as fold change in mucA22 relative to wild-type PAK. a #, up regulated in mucA
mutant compared to PAK but down regulated in PAK under type III-inducing conditions
versus noninducing conditions and vice versa. Not included are those known to be
affected by the growth medium, such as those varied in TSB versus LB (140). b Change
in gene expression in PAK grown under TTSS inducing conditions versus PAK grown
under TTSS noninducing conditions (140). Bacteria were grown in TSB or LB.
TTSS Repression in the mucA Mutant is AlgU Dependent.
MucA is an anti-sigma factor which represses the activity of AlgU (022). In the
mucA mutant, AlgU is derepressed and activates the expression of genes for alginate
synthesis, resulting in a mucoid phenotype. AlgU can also activate the expression of
itself and downstream genes (mucA-B-C-D) in the same operon. To determine the role of
AlgU in the repression of TTSS in the mucA mutant, the algU gene was knocked out in
the background of PAKmucA22, resulting in a PAKmucA22algU::Gm double mutant.
Under TTSS inducing conditions, expression of the exsA and exoS genes in this double
mutant was similar to that in the wild-type (Fig. 2-2B and C), indicating that AlgU is
required for the TTSS repression in the mucA mutant. An algU::Gm mutant was further
generated in the background of PAK, and TTSS activity in the resulting mutant was
compared with that in PAK. As shown in Fig. 2-3, expression of the exsA and exoS
genes was the same in the PAKalgU::Gm mutant and wild-type PAK under both TTSS
inducing and noninducing conditions, suggesting that the basal level of AlgU in wild-
type P. aeruginosa does not play a significant role in the regulation of TTSS genes.
PAK mucA22 PAK PAK
Figure 2-3. Expression of exsA::lacZ (A) and exoS::lacZ (B) in strains PAK,
PAKmucA22, PAKalgU::Gm, and PAK harboring algU overexpression
plasmid pWW025. Bacteria were grown in LB (white bars) or LB plus 5 mM
EGTA (black bars) to an OD600 of 1 to 2 before 8-galactosidase assays. *,
P<0.05, compared to the values in PAK.
When the algU gene was overexpressed in wild-type PAK by introducing
pWW025, the TTSS activity was partially repressed under type Ill-inducing conditions
(Fig. 2-3B). Since AlgU mediates the activation of the algU-mucA operon, an extra copy
of algU also increased the expression of its repressor MucA; thus, overexpression of the
algU gene could not repress TTSS expression to the level seen in the mucA mutant.
AlgR has a Negative Regulatory Function on the TTSS
algR is a regulatory gene required for alginate synthesis and is under the control of
AlgU (78, 146). To investigate the role of AlgR in the regulation of TTSS, the algR gene
was knocked out in the background of PAKmucA22. In the PAKmucA22algR::Gm
double mutant, the expression of the exsA and exoS genes was restored to that of the wild
type (Fig. 2-2A and B), suggesting that the repression of TTSS in the mucA mutant is also
AlgR dependent. To test the function of AlgR on TTSS in wild-type P. aeruginosa, an
algR::Gm mutant was generated in the PAK background. The expression of the exoS
gene was consistently higher in the resulting PAKalgR::Gm mutant than in PAK under
both type III inducing and noninducing conditions (Fig. 2-4B). However, the expression
of the exsA gene was similar in the PAKalgR::Gm mutant and wild-type PAK.
Complementation of the algR mutant with an algR-expressing clone (pWW022)
decreased exsA and exoS expression under both type III inducing and noninducing
conditions (Fig. 2-4). However, higher expression of algR induced by increasing the
amount of isopropyl-1-D-thiogalactopyranoside (IPTG) could not further decrease exsA
and exoS expression (Fig. 2-4). These results indicate that AlgR has a negative
regulatory effect on the TTSS, but the up regulation of AlgR alone might not be sufficient
to repress TTSS activity to the level seen in the mucA mutant. It is likely that in the
mucA mutant, algR gene expression is activated by AlgU, which in turn represses TTSS
PAK mucA22 PAK PAK algR::Gm/pWW022
algR::Gm 0 gg/ml 250tg/ml 500tg/ml IPTG
PAK mucA22 PAK PAK alR::Gm/pWW022
algR::Gm 0 gg/ml 250tg/ml 500tg/ml IPTG
Figure 2-4. Expression of exsA::lacZ (A) and exoS::lacZ (B) in the backgrounds of PAK,
PAKmucA22, PAKalgR::Gm, and PAKalgR::Gm complemented with algR-
expressing plasmid pWW022. For algR gene complementation, various
concentrations of IPTG were added into the culture medium as indicated.
Bacteria were grown in LB (white bars) or LB plus 5 mM EGTA (black bars)
to an OD600 of 1 to 2 before 8-galactosidase assays. *, P < 0.05, compared to
the values in PAK; **, P < 0.01, compared to the values in mucA22.
Discussion and Future Directions
The Expression of exsA in the mucA Mutant
TTSS is an important virulence determinant for P. aeruginosa: it inhibits the host
defense system by inducing apoptosis in macrophages, polymorphonuclear phagocytes,
and epithelial cells. In our screen for mutants with lower TTSS activities, mucA mutants
were found defective in exoT expression under type III-inducing conditions.
Furthermore, the basal promoter activity of the type III master regulatory gene exsA was
decreased two- to threefold in the mucA mutant compared to that in wild-type PAK,
suggesting that the down regulation of TTSS genes occurs through repression of ExsA.
Since ExsA is an autoactivator (60), the repression could be on the transcriptional or
posttranscriptional level. Our microarray results showed that the transcript level of exsA
in the mucA mutant was similar to that in wild-type PAK under type III-inducing
conditions, which suggested that the activity of ExsA might be repressed at the
posttranscriptional level. However, the data from exsA::lacZ reporter plasmid indicates
that the promoter activity of exsA gene is much lower in the mucA mutant (Fig.2-1B).
Real-time PCR may be necessary to precisely determine the mRNA levels of exsA gene.
Further study is required to clarify the mechanism of exsA gene regulation.
The Regulatory Pathway of AlgU Regulon
MucA is a transmembrane protein, with its cytoplasmic domain binding to and
repressing the sigma factor AlgU. Mutation in the mucA gene leads to derepression of
AlgU, which in turn activates genes for alginate synthesis as well as others, such as dsbA,
oprF, osmE, and rpoH (32, 80). In the mucA mutant, not only the sigma factor AlgU but
also AlgQ, an anti-o70 factor, are activated (31), thus posing the possibility that sigma
factor competition by AlgU and AlgQ effectively decreases the availability of C70-
containing RNA polymerase for the expression of TTSS related genes (62). However,
the observation that AlgR, an AlgU-dependent transcriptional activator, is required for
the TTSS suppression makes it unlikely that sigma factor competition leads to the type III
gene suppression; instead, an AlgR-dependent repressor is likely involved. AlgR is a
global regulator, affecting expression of multiple genes. Proteomics analysis of an
algR::Gm mutant showed that more than 17 proteins were up regulated and 30 proteins
were down regulated (77). In the present study, AlgR was also found to mediate the
repression of type III secretion genes. In the PAKalgR::Gm mutant background,
expression of the exoS gene was higher than in wild-type PAK and, when complemented
by an algR gene clone, expression of exsA and exoS genes decreased to about 50% of that
seen in wild-type PAK (Fig. 2-4). The inability to suppress TTSS genes to the level seen
in the mucA mutant by pWW022 was possibly due to a lower level of expression of the
algR gene from pWW022 than that in the PAKmucA background, in which algR is
activated through the MucA-AlgU pathway. pMMIB67HE is a low-copy-number plasmid
(38), and the tac promoter is not as strong a promoter in P. aeruginosa as it is in E. coli.
AlgR is a DNA binding protein which binds to the promoter regions of algD (93) and
hcnA (hydrogen cyanide synthesis gene) (15). It is possible that AlgR represses exsA
expression by directly binding to the promoter region of the exsCEBA operon. The
protein-DNA binding can be tested by gel-shift assay and the algD promoter can be used
as a positive control. Alternatively, other regulatory genes might be involved in the
repression of TTSS. Further study is needed to understand this observation.
We propose a model for TTSS repression in the mucA mutant (Fig. 2-5). With the
activation of AlgU, the regulatory genes algP, algQ, algB, and algR are activated, which
up regulates the expression of the algD operon. AlgR is required for TTSS repression in
the mucA mutant, but whether the repression function is directly on ExsA or not is
unclear. The involvement of other regulatory genes (algP, algQ, and algB) in TTSS
regulation awaits further study.
The TTSS Activity in P. aeruginosa CF Isolates
During chronic infection of CF patient airways, P. aeruginosa overproduces
alginate and forms a biofilm (58). Alginate production is known to be activated by high
osmolarity, nitrogen limitation, and membrane perturbation induced by ethanol (10);
thus, the high salt concentration in the CF patient airway might be a signal for the
overproduction of alginate. The biofilm mode of growth can help the bacterium survive
in hostile environments and also render resistance against macrophages and
polymorphonuclear cells (58).
Figure 2-5. Proposed model of MucA-mediated coordination of alginate production and
TTSS expression. MucA is a transmembrane protein, with its cytoplasmic
portion binding and inhibiting the sigma factor AlgU. Upon sensing of certain
environmental stress signals by the periplasmic MucB, it signals MucA
through the periplasmic domain to release the bound AlgU. Free AlgU is
required for the expression of downstream transcriptional activators AlgP,
AlgQ, AlgB, and AlgR, all of which contribute to the optimal expression of
the algD operon, encoding enzymes for the synthesis of alginate. AlgR, on
the other hand, also activates downstream genes which are responsible for the
suppression of the type III secretion genes.
Our experimental data suggest that bacteria have evolved a mechanism to turn off
TTSS when they need to synthesize alginate to overcome environmental stress. Such
coordinated regulation of two energy-expensive processes is likely to render to the
bacterium a survival advantage under environmental stress conditions. In addition, when
the bacteria are surrounded by alginate, no intimate contact can be established between
the bacteria and host cells. Under this circumstance, the TTSS needle can not reach the
host cell membrane, which renders the TTSS unnecessary. This might be another reason
to turn off TTSS while over producing alginate. Indeed, a majority of P. aeruginosa
isolates from CF patients at a late stage in the disease displays the mucoid phenotype (34,
111) and are defective in type III gene expression (22). In a previous report, introduction
of the wild-type exsA gene into type III secretion-defective clinical isolates restored type
III secretion (22). However, our attempts to restore TTSS gene expression in 10 mucoid
CF isolates by introducing a mucA gene clone failed, although all of the transformants
were reverted back to the nonmucoid phenotype. It is possible that those mucoid clinical
isolates may harbor additional mutations in the TTSS genes.
Genes Differently Expressed in the mucA Mutant and Isogenic Wild-type PAK
Known TTSS regulators include ExsA, Vfr, CyaA/B, ExsD, ExsC and ExsE (27,
60, 89, 106, 130, 140). Recently, DsbA and AceAB were also found to be necessary for
the expression of TTSS. AceA and -B are subunits of pyruvate dehydrogenase,
suggesting that metabolic imbalance influences the expression of TTSS (23, 107). DsbA
is a periplasmic thiol-disulfide oxidoreductase and was shown to affect TTSS expression,
twitching motility, and intracellular survival of P. aeruginosa upon infection of HeLa
cells (48, 80). Interestingly, the dsbA gene is up regulated in the mucA mutant
background, and its expression was shown to be regulated by AlgU (80). However, the
role of DsbA on the TTSS is believed to be through its general effect on protein disulfide
bond formation in the periplasm, and up regulation of this gene may not be related to the
MucA-AlgU-AglR-mediated suppression of the TTSS.
From the microarray analysis of the mucA mutant and wild-type strain under TTSS
inducing conditions, alginate synthesis genes and genes known to be under the control of
AlgU were up regulated, while TTSS genes were down regulated in the mucA mutant
(Tables 2-2 and -3). In addition, pyoverdine synthesis genes as well as an operon,
PA4468-4471, which might be under the control of Fur (54), were up regulated in the
mucA mutant under TTSS-inducing conditions (Table 2-4). These findings are consistent
with published results, in which mucoid P. aeruginosa strains produced higher levels of
pyoverdine, pyochelin, manganese superoxide dismutase (PA4468), and fumarase
(PA4470) than wild-type strains (52) (53). However, pyochelin synthesis genes were not
seen up regulated in our microarray data. The mechanism by which these genes are up
regulated in the mucA mutant background is not known.
The mucA gene mutation-mediated suppression of the TTSS genes requires AlgR,
which is a transcriptional regulator; thus, it is likely that AlgR may repress TTSS genes
or an AlgR-regulated repressor mediates the suppression of TTSS genes. To identify
such candidate genes from the gene array data, I initially identified genes that were
differentially expressed in the mucA mutant compared to wild-type PAK under type III
inducing conditions. The selected genes include those that were up regulated in the mucA
mutant compared to PAK under type III inducing conditions but were down regulated in
PAK under type III inducing conditions versus noninducing conditions, and vice versa. I
further eliminated those known to be affected by the growth medium, such as those with
varied responses in tryptic soy broth (TSB) versus LB (140). Based on the above criteria,
13 genes were identified (Tables 2-4 and -5). For example, expression of the PA2172
gene in mucA22 was up regulated about fourfold compared to that in wild-type PAK
under TTSS inducing conditions. From published data, the expression of this gene was
down regulated twofold in wild-type PAK grown under type III inducing conditions
compared to that under noninducing conditions (140). Therefore, mutation in the mucA
gene reversed the expression of PA2172 in response to the type III-inducing signal.
Among the 13 genes, pvdE andfpvA are involved in pyoverdine synthesis and
absorption, respectively; PA2414 is involved in carbon compound catabolism. The
remaining 10 genes are all hypothetical genes. The expression ofPA0737, PA2167,
PA2176, and PA4785 seems to be ExsA dependent, since in the exsA mutant the
expression of these genes was lower than in wild-type PAK under type III inducing
conditions and overexpression of exsA could activate expression of these genes under
non-type III-inducing conditions (140). It is reasonable to hypothesize that one or more
of such differentially expressed genes mediate the repression of the TTSS in the mucA
mutant. It will be interesting to mutate each of these candidate genes in the background
of PAKmucA22 and test the TTSS activities.
Another approach to identify the TTSS repressor is to screen a random Tn library
generated in the background of PAKmucA22 for those mutants with restored wild-type
TTSS activity. In those mutants, the TTSS repressor should be knocked out by the
insertion of Tn. There are two potential pitfalls in this Tn mutagenesis strategy. One is
that the mucA mutant over produces alginate which might obstruct the intimate contact
between the E. coli donor strain and the P. aeruginosa recipient strain. To solve this
problem, I can knock out the alginate synthesis gene, algD, which would render the mucA
mutant non-mucoid. The other problem is that, when cultured statically, mucA mutants
tend to become non-mucoid, due to spontaneous mutations in the algU gene (143).
During the conjugation for Tn mutagenesis, algU mutants may accumulate in the
population. These mucAalgU double mutants display wild-type TTSS activity, which
may lead to wrong interpretation of Tn mutated genes. It was reported that cultures
containing the alternative electron acceptor nitrate may decrease the mutation rate of the
algU gene. So during the conjugation, nitrate can be added into the nutrient agar.
In conclusion, in mucA mutants, the TTSS is repressed and the repression is AlgU
and AlgR dependent. Most P. aeruginosa clinical isolates from CF patients display
mucoid phenotype and are defective in the TTSS. This study provides possible
explanation on the relationship between these two phenotypes and indicates that during
chronic infection, P. aeruginosa might over produce alginate, which might function as a
protection mechanism, and down regulate the TTSS, a virulence factor.
PtrB OF Pseudomonas aeruginosa SUPPRESSES THE TYPE III SECRETION
SYSTEM UNDER THE STRESS OF DNA DAMAGE
As described in Chapter 2, two mutants with Tn inserted into theprtR gene were
found to be defective in the TTSS activity. PrtR is a XCI homologue which binds to the
promoter region of the prtN gene and inhibits its expression. PrtN is an activator of
genes required for the production of a kind of bacteriocins, called pyocins. Three types
of pyocins, R-, F- and S-type, have been identified. R- and F-type pyocins resemble
phage tails. After they bind to their receptors, lipopolysaccharides (LPS), R-type pyocins
cause a depolarization of the cytoplasmic membrane, which leads to cell death. S-type
pyocins cause cell death by DNA breakdown due to their endonuclease activity (90). The
uptake of most S-type pyocins occurs through ferripyoverdine receptors so that their
killing activity is greatly increased when bacteria are grown under iron-limited conditions
(7). The production of pyocins is induced by DNA-damaging agents, such as UV light
and mitomycin C, when the bacterial SOS response is activated. Under these conditions,
the RecA protein is activated and cleaves PrtR. As a result, PrtN is up regulated and
actives the expression of pyocin synthesis genes (86, 90).
In this Chapter, I describe a coordinated repression of the TTSS under the stress of
DNA damage. The expression of TTSS genes was found to be repressed in the
background of aprtR mutant. Further analysis eliminated the possible involvement of the
prtNgene in the TTSS repression. A gene designatedptrB has been identified which is
specifically repressed by PrtR and mediates the suppression of the TTSS genes. PtrB has
a prokaryotic DskA/TraR C4-type zinc-finger motif but may not directly interact with the
master regulator, ExsA.
Material and Methords
Bacterial Strains and Growth Conditions
Plasmids and bacterial strains used in this study are listed in Table 3-1. Growth
conditions and antibiotic concentrations are the same as described in Chapter 2.
Table 3-1. Strains and plasmids used in this study
Strain or plasmid Description Source or
E. coli strains
P. aeruginosa strains
RP4-2-Tc::Mu-1 kan::Tn7 integrant leu-63::IS 10
recA1 zbf-5 creB510 hsdR 7 endA1 thi uidA (A
080dlacZAM15 A(lacZYA-argF)U169 recAl
hsdR 7 deoR thi-1 supE44 gyrA96 relA1/Apir
Wild-type P. aeruginosa strain
PAKprtR::Tn5 mutant isolate; Neor
PAK with prtN and prtR disrupted by replacement
of Gm cassette; Gmr
PAK with prtN disrupted by insertion of Gm
PAKAprtNprtR: :GmPA0612 :Tn5; Gmr Neor
PAKAprtNprtR::Gm with deletion of PA0612 and
PAKAprtNprtR::Gm with deletion of PA0612;
PAKAprtNprtR::Gm with deletion of PA0613;
PAK with deletion of PA0612 and PA0613
PAK with deletion of PA0612
PAK with deletion of PA0613
Cloning vector for the PCR products
exoS promoter of PAK fused to promoterless lacZ
on pDN191acZQd S)r Smr Tcr
Table 3-2. Continued
Strain or plasmid
exoT promoter of PAK fused to promoterless lacZ
on pDN191acZO; Spr Smr Tcr
Shuttle vector between E. coli and P. aeruginosa
Gene replacement vector; Gmr, ori7 sacB+
Gene replacement vector; Apr, oriT sacB+
Source of Gmr cassette; Apr Gmr
prtN gene of PAK on pUCP19 driven by lac
prtR gene of PAK on pUCP 19 driven by lac
prtN disrupted by insertion of Gm cassette on
pEX18Ap; Apr Gmr
prtN and prtR disrupted by replacement of Gm
cassette on pEX18Ap; Apr Gmr
PA0612 promoter of PAK fused to promoterless
lacZ on pDN191acZQ; Spr Smr Tcr
exsC promoter of PAK fused to promoterless lacZ
on pDN191acZQ; Spr Smr Tcr
Deletion of PA0612 and PA0613 on plasmid
Deletion of PA0612 and PA0613 on plasmid
Deletion of PA0612 on plasmid pEX18Gm; Gmr
Deletion of PA0613 on plasmid pEX18Gm; Gmr
PA0613 open reading frame cloned into pCR2.1-
PA0612 open reading frame cloned into pCR2.1-
Bait vector plasmid encoding full length bacterial
phage AcI protein; Chlr
Target vector plasmid encoding RNAP-alpha
subunit protein; Tcr
Interaction control plasmid containing dimerization
domain of Gal4 on bait vector; Chlr
Interaction control plasmid encoding mutant form
of Gal 11 on target vector; Tcr
PA0612 open reading frame cloned into pTRG; Tcr
PA0613 open reading frame cloned into pTRG; Tcr
PA0612 open reading frame cloned into pBT; Chlr
PA0613 open reading frame cloned into pBT; Chlr
exsA open reading frame cloned into pTRG; Tcr
exsA open reading frame cloned into pBT; Chlr
ForprtR gene complementation, aprtR containing fragment was amplified from
PAK genomic DNA by PCR (Table 3-2). The PCR product was cloned into pCR2.1-
TOPO (Invitrogen), resulting in pTopo-prtR. From pTopo-prtR, the prtR gene was
isolated as a HinclI-HindIIl fragment and cloned into Smal-HindII sites of pUCP19,
resulting in pWW037, where the prtR gene is driven by a lac promoter. ForprtN gene
overexpression, prtN coding sequence was amplified by PCR (Table 3-2), initially cloned
into pCR2.1-TOPO, and subcloned into Hindlll-Xbal sites of pUCP19, where the
expression of the prtN gene in the resulting plasmid, pWW031, was driven by the lac
promoter on the vector. The promoter region ofPA0612 was amplified from PAK
chromosomal DNA (Table 3-2), cloned into pCR2.1-TOPO, and subcloned into EcoRI-
BamHI sites of pDN191acZ, resulting in pWW048-1. For the construction of exsC::lacZ
reporter plasmid, a PCR product containing exsCEBA (Table 3-2) was cloned into
pCR2.1-TOPO. The exsC promoter was cut out with EcoRI and HincII, and subcloned
Chromosomal gene mutations were generated as described (55). A fragment
containing theprtN andprtR genes was amplified by PCR using the primers PrtR1 and
PrtN2. The PCR product was cloned into pCR2.1-TOPO and subcloned into HindlII-
Xbal sites of pEX18Ap, resulting in pEX18Ap-prtNR. For construction of aprtNprtR
double mutant, a SphI fragment containing 3'-terminal sequence ofprtR and 5'-terminal
sequence ofprtN was replaced with a gentamicin resistance cassette, resulting in
pWW035. For the construction ofPA0612-613, PA0612, and PA0613 mutants, a 2.4-kb
fragment was amplified from PAK chromosomal DNA with primers 612-3M1 and 612-
3M2 (Table 3-2), followed by cloning into pCR2.1-TOPO. A SacII fragment containing
both PA0612 and PA0613 was deleted to generate the PA0612-613 mutant. A 76-bp
SacII-PstI fragment within PA0612 was removed to generate the PA0612 mutant, while a
116-bp ClaI-SacII fragment was deleted to generate the PA0613 mutant. The resulting
plasmids were transformed into wild-type PAK or PAKprtNprtR::Gm and selected for
single and double crossover mutants as described previously (55). Construction of a
transposon (Tn5) insertion mutant library, plasmid rescue, and sequence analysis were
conducted as described in Chapter 2.
Table 3-2. PCR primers used in this study
Gene Amplicon Sequences of primers
exsAl: 5' -TGCAGCTCATCCAGCAGTACACCCAGAGCCATAAC-3'
RT-PCR and Quantitative Real-time PCR
Overnight cultures of bacterial cells were diluted 100-fold into fresh medium and
grown to an optical density at 600 nm (OD600) of 1.0. Total RNA was isolated with an
RNeasy Mini kit (QIAGEN). DNA was eliminated by column digestion as described by
the manufacturer (QIAGEN). cDNA was synthesized with an iScript cDNA synthesis kit
(Bio-Rad). Taq DNA polymerase from Eppendorf was used in PCRs. The cDNAs
synthesized by reverse transcription-PCR (RT-PCR) were used as templates in
quantitative real-time PCR. The cDNA was mixed with 5pmol of forward and reverse
primers (Table 3-2) and iQ SYBR Green Supermix (Bio-Rad). Quantitative real-time
PCR was conducted using the ABI Prism 7000 sequence detection system (Applied
Biosystems). The results were analyzed with ABI Prism 7000 SDS software. Transcript
for the 30S ribosomal protein (rpsL) was used as an internal standard to compensate for
differences in the amount of cDNA. The mRNA levels ofptrB in test strains were
expressed relative to that of PAK, which was set at 1.00.
HeLa cells (5 x 104) were seeded into each well of a 24-well plate. The cells were
cultured in Dulbecco's modified Eagle's medium with 5% fetal calf serum at 370C with
5% CO2 for 24 h. Overnight bacterial cultures were washed with LB and subcultured to
log phase before infection. Bacteria were washed once with phosphate-buffered saline
and resuspended in tissue culture medium. HeLa cells were infected with the bacteria at
a multiplicity of infection (MOI) of 20. A cell lifting assay was performed after 4 h of
infection. Culture medium in each well was aspirated. Cells were washed twice with
phosphate-buffered saline (PBS) and stained with 0.05% crystal violet for 5 min. The
stain solution was discarded, and the plates were washed twice with water. A 250-1tl
volume of 95% ethanol was then added into each well and incubated at room temperature
for 30 min with gentle shaking. The ethanol solution with dissolved crystal violet dye
was used to measure absorbance at a wavelength of 590 nm.
Application of BacterioMatch Two-hybrid System
PA0612 and PA0613 open reading frames were amplified from PAK chromosomal
DNA with primers 612BT1 plus 612BT2 and 613BT1 plus 613BT2, respectively (Table
3-2). The PCR products were cloned into pCR2.1-TOPO, and each was subcloned into
Notl-Xhol sites of pBT and pTRG, resulting in pWW079 (PA0612 in pBT), pWW077
(PA0612 in pTRG), pWW080 (PA0613 in pBT), and pWW078 (PA0613 in pTRG). The
exsA open reading frame was isolated from pHW0315 (exsA in pTRG) as a Notl-Spel
fragment. The Spel site was blunt ended and ligated into Notl-Smal sites of pBT,
resulting in pWW081 (exsA in pBT). Desired pairs of plasmids were cotransformed into
a reporter strain by electroporation, and the protein-protein interaction assays were
performed following the protocol supplied by the manufacturer (Stratagene). The
interaction between two proteins is indicated by the expression level of a lacZ reporter
gene. By testing the P-galactosidase activity of the reporter strains containing cloned
genes on pBT and pTRG, the interaction between the two proteins can be tested.
Western blotting, 1-Galactosidase activity assays and statistical assays were done
as described in Chapter 2. For twitching motility assays, bacteria were stabbed into a
thin-layer LB plate and incubated overnight at 370C. The LB plate was directly stained
with Coomassie blue at room temperature for 5 min and destined with destaining
TTSS Is Repressed in aprtR Mutant
As described in Chapter 2, by screening a Tn insertion library consisting of 40,000
independent mutants, two prtR mutants were found to be defective in TTSS activity (Fig.
3-1). Complementation of the original prtR::Tn mutants with aprtR gene partially
restored the TTSS activity (Fig. 3-1A). PrtR is a repressor of pyocin synthesis, which is a
set of bacteriocins synthesized by P. aeruginosa. PrtR binds to the promoter region of
the prtN gene and represses its expression. PrtN is also a DNA binding protein which
recognizes a highly conserved sequence (P box) present upstream of pyocin synthesis
genes and activates their expression (86). Based on this regulatory pathway, either the
up-regulated PrtN is responsible for the TTSS repression or another gene under the
control of PrtR mediates the TTSS repression. To test these possibilities, aprtRprtN
double mutant was generated in the background of wild-type PAK. The resulting mutant,
PAKAprtNprtR::Gm, had the same TTSS defect as theprtR::Tn5 mutant (Fig. 3-1A and
B), and complementation by aprtR gene (pWW037) but not by aprtNgene (pWW031)
restored the TTSS inducibility (Fig. 3-1A). Furthermore, introduction of aprtN-
expressing clone in a high-copy-number plasmid (pWW031) in wild-type PAK had no
effect on the TTSS activity (Fig. 3-1A and B). Thus, all of the above results indicated
that PrtN is not involved in the TTSS repression. Therefore, it is likely that another
gene(s) under the control of PrtR mediates the repression of the TTSS.
Identification of the PrtR-regulated Repressor of the TTSS
Since PrtR functions as a repressor, it might also repress the expression of a
hypothetical TTSS repressor. With the mutation inprtR, this hypothetical repressor
would be up regulated and therefore would repress the expression of the TTSS. Thus,
upon inactivation of this repressor gene in the prtR mutant background, the TTSS activity
should be restored to that of wild-type. To identify this hypothetical repressor, the
AprtNprtR::Gm double mutant containing exoT::lacZ (pHW0006) was subjected to
transposon mutagenesis. A plasmid containing a Tn5 transposon (pRL27) was
transferred from E. coli donor strain BW20767 into P. aeruginosa by conjugation. The
double mutant strain AprtNprtR::Gm was chosen as a recipient, since it has an identical
phenotype of a TTSS defect as the prtR::Tn mutant. More importantly, constitutive
production of pyocin by aprtR mutant seems to have a detrimental effect on the E. coli
donor strain which may lower the conjugation frequency.
A S 300
^ PAK prtR::Tn5 prtR::Tn5/ AprtNR::Gm AprtNR::Gm/ AprtNR::Gm/ PAK/
prtR-pUCP19 prtR-pUCP19 prtN-pUCP19 prtN-pUCP19
B PAK prtR::Tn JprtNR::Gm prtN-pUCP19
EGTA + + + +
Figure 3-1. Expression and secretion of ExoS. (A) Expression of exoS::lacZin the
backgrounds of PAK, prtR::Tn5, prtR::Tn5 containing prtR expression
plasmid pWW037 (prtR-pUCP19), AprtNR::Gm, AprtNR::Gm containing
pWW037 (prtR-pUCP19) orprtN expression plasmid pWW031 (prtN-
pUCP19), and PAK with pWW031 (prtN-pUCP19). Bacteria were grown to
an OD600 of 1 to 2 in LB with (black bars) or without (white bars) EGTA
before 1-galactosidase assays. (B) Cellular and secreted forms of ExoS in
strains PAK, prtR::Tn5, AprtNR::Gm, and PAK containing pWW031 (prtN-
pUCP19). Overnight bacterial cultures were diluted to 1% in LB or 3% in LB
plus 5 mM EGTA and grown at 370C for 3.5 h. Supernatants and pellets from
equivalent bacterial cell numbers were loaded onto SDS-PAGE gels and
immunoblotted with anti-ExoS antibody. Both ExoS and ExoT are indicated
by arrows. Anti-ExoS polyclonal antibody also recognizes ExoT due to high
homology between them. *, P < 0.01, compared to the values in PAK. **, P
< 0.05, compared to the values inprtR::Tn5.
The Tn insertion mutants were spread on LB agar plates containing 20 tg/ml X-
Gal, 2.5 mM EGTA, and proper antibiotics. Blue colonies were looked for in which the
TTSS repressor under the control of PrtR should have been knocked out. About 100,000
Tn insertion mutants were screened. Thirty blue colonies were picked and cultured in
liquid LB for 1-galactosidase assay. Sixteen mutants showed restored TTSS activity
compared to the parent strain. Sequence analysis of the Tn insertion sites showed that 14
mutants had Tn insertions at a single locus (PA0612) at nine different positions. PA0612
encodes a hypothetical protein with a consensus prokaryotic DksA/TraR C4-type zinc-
finger motif. The dksA gene product suppresses the temperature-sensitive growth and
filamentation of a dnaK deletion mutant of E. coli (66), while TraR is involved in
plasmid conjugation (30). These proteins contain a C-terminal region thought to fold into
a four-cysteine zinc finger (30). Its homologues also exist in other gram-negative
bacteria, such as Pseudomonas syringae, Pseudomonas putida, E. coli, Salmonella
enterica serovar Typhimurium, and .\/nge/ll flexneri. However, the functions of these
gene homologues have not been studied. The remaining two mutants contained a Tn
insertion in the genes PA2265 and PA5021, respectively. PA2265 encodes a putative
gluconate dehydrogenase. Promoter analysis
(http://www.fruitfly.org/seq tools/promoter.html) indicates it is in the same operon with
an upstream gene, PA2264, as well as a downstream gene, PA2266. PA2264 is an
unknown gene, while PA2266 encodes a putative cytochrome c precursor. PA5021
encodes a probable sodium:hydrogen antiporter. Promoter analysis indicated that two
downstream genes, PA5022 and PA5023, are in the same operon with PA5021, where
PA5022 and PA5023 encode two unknown proteins. We further pursued the regulation
and function of PA0612 in this study.
PA0612 and PA0613 Form an Operon Which Is Under the Control of PrtR
Promoter analysis predicted that PA0612 and PA0613 may form an operon, while
the pyocin synthesis gene PA0614 has its own promoter. The downstream gene
(PA0613) encodes an unknown protein. On the chromosome ofPAO1, PA0612 is
located next to the prtR gene in the opposite direction. In the promoter region of
PA0612, a 14-base sequence was observed that was also present as a direct repeat in the
predicted prtN promoter region, which might be the PrtR recognition site (Fig. 3-2) (86).
Therefore, it is highly likely that the expression of PA0612 is under the control of PrtR
and mediates the repression of TTSS.
Direct repeat Direct repeat
prtR promoter 1 PA0612 promoter
prtR promoter 2
Figure 3-2. Genetic organization and putative promoter regions ofprtN, prtR, PA0612-3.
Computer-predicted promoters of prtN, prtR, PA0612-613, and PA0614 are
indicated with arrows. Two promoters are predicted for the prtR gene and are
designated promoters 1 and 2. The potential PrtR binding sequences are
underlined. The arrow of each open reading frame represents the
To confirm the prediction that PA0612 and PA0613 are in the same operon, a pair
of primers annealing to the 5' end of PA0612 (612GS1) and 3' end of PA0613 (613BT2)
was designed for RT-PCR analysis (Table 3-2). A 649-bp PCR product was amplified
using total RNA isolated fromprtR::Tn or AprtNprtR::Gm (Fig. 3-3A), and the size was
the same as that when PAK genomic DNA was used as template (data not shown).
However, when total RNA from PAK or PAK/pWW031 (prtN overexpresser) was used
as template, a faint PCR product could be seen (Fig. 3-3A), indicating low abundance of
this transcript. These results suggested that PA0612 and PA0613 are in the same operon,
which is under the negative control of PrtR. Transcription of PA0612 was investigated
further by real-time PCR. Expression of PA0612 mRNA inprtR::Tn and AprtNR::Gm
was 30- and 38-fold greater than that in PAK, respectively, while overexpression of the
prtN gene had little effect on the transcript level of PA0612 (Fig. 3-3B). To further
confirm this, the promoter of PA0612 was fused with a promoterless lacZ gene on
plasmid pDN191acZ, and the resulting fusion construct (pWW048-1) was introduced into
various strain backgrounds for the 1-galactosidase assay. As shown in Fig. 3-4, the
expression of PA0612 was up regulated in bothprtR::Tn and AprtNprtR::Gm mutant
backgrounds compared to that in PAK or PAK overexpressingprtN (PAK/pWW031),
further proving that the expression of PA0612 and PA0613 is repressed by PrtR. The
above results also reaffirmed our earlier conclusion that prtN has no effect on the
expression of PA0612 and PA0613.
PA0612 Is Required for the Repression of the TTSS In the prtR Mutant
Since PA0612 and PA0613 are in the same operon, insertion of a Tn in PA0612
will have a polar effect on the expression of PA0613. To test which of the two genes is
required for the TTSS repression in the prtR mutant, deletion mutants of PA0612 and
PA0613 and the PA0612 PA0613 double mutant were generated in the background of the
AprtNprtR::Gm mutant. The production and secretion of ExoS, as judged by Western
blotting, were restored in the PA0612 and PA0612-013 mutants but not in the PA0613
mutant (Fig. 3-5A). The reporter plasmid of exoT: :lacZ (pHW0006) was further
transformed into these mutants and subjected to a B-galactosidase assay. As the results
show in Fig. 3-5B, transcription of the exoT gene was partially restored in the
backgrounds of AprtNprtR::GmAPA0612-013 and AprtNprtR::GmAPA0612 mutants,
while they remained repressed in the background of the AprtNprtR::GmAPA0613 mutant,
indicating that PA0612 is required for repression of the TTSS in the prtR mutant
Marker Genomic PAK prtR::Tn AprtNR::Gm PAK/
PAK prtR::Tn AprtNR::Gm PAK/
Figure 3-3. Expression of PA0612 is repressed by prtR. (A) RT-PCR of the PA0612-
0613 operon. Total RNA was isolated from PAK, prtR::Tn5, AprtNR::Gm,
and PAK/pWW031. One microgram of RNA from each sample was used to
synthesize cDNA, and the cDNA was diluted 100-fold for subsequent PCR
amplification. The primers used in the PCR anneal to the 5' end ofPA0612
and the 3' end of PA0613. (B) Quantitation of PA0612 gene expression by
real-time PCR. Data are expressed relative to the quantity of PA0612 mRNA
in PAK. *, P < 0.01, compared to the values in PAK.
PAK prtR::Tn J prtNR::Gm PAK/
Figure 3-4. Expression of PA0612::lacZ (pWW048-1) in PAK, prtR::Tn5, AprtNR::Gm,
and PAK/pWW031. Bacteria were grown in LB for 10 h before 8-
galactosidase assays. *, P < 0.01, compared to the values in PAK.
The TTSS of PAK can directly deliver ExoS, ExoT, and ExoY into the host cell,
resulting in cell rounding and lifting (46, 125, 131). HeLa cells were infected with wild-
type PAK and prtR mutants at a MOI of 20. Upon infection by PAK, almost all of the
HeLa cells were rounded after 2.5 h. Under the same conditions, the PAKexsA::Q
mutant, a TTSS-defective mutant, had no effect on HeLa cell rounding; similar to the
PAKexsA::Q mutant, low cytotoxicity was seen with mutant strains prtR::Tn,
AprtNprtR::Gm, and AprtNprtR::GmAPA0613. However, AprtNprtR::GmAPAO612-013
and AprtNprtR::GmAPA0612 caused comparable levels of HeLa cell lifting as that seen
with PAK. Quantitative assay of the cell lifting was further performed by crystal violet
staining of the adhered cells after 4 h of infection. As shown in Fig. 3-5C, mutant strains
AprtNprtR::GmAPAO612-013 and AprtNprtR::GmAPAO612 showed similar cytotoxicity
as wild-type PAK. However, PrtR::Tn, AprtNprtR::Gm, and AprtNprtR::GmAPA0613
showed much-reduced cytotoxicity. The above observations clearly indicated that
PA0612, but not PA0613, is required for the TTSS repression in theprtR mutant
background. We designate this newly identified repressor gene as pseudomonas type III
repressor gene B or, ptrB.
PAK AprtNR::Gm AprtNR::Gm AprtNR::Gm AprtNR::Gm
A612-3 A612 A613
EGTA + -- + +
PAK prtR::Tn5 AprtNR::Gm AprtNR::Gm AprtNR::Gm AprtNR::Gm AprtNR::Gm
612::Tn5 A612-3 A612 A613
Cell PAK exsA::fl prtR::Tn5 AprtNR::Gm AprtNR::Gm AprtNR::Gm AprtNR::Gm
A612-3 A612 A613
Figure 3-5. Characterization of ExoS expression and cytotoxicity. (A) Cellular and
secreted forms of the ExoS in strains PAK, AprtNR::Gm,
AprtNR::GmAPAO612-0613, AprtNR: :GmAPA0612, and
AprtNR::GmAPA0613. Overnight bacteria cultures were diluted to 1% in LB
or 3% in LB plus 5 mM EGTA and grown at 370C for 3.5 h. Supernatants
and pellets from equivalent bacterial cell numbers were loaded onto SDS-
PAGE gels and immunoblotted with anti-ExoS antibody. Both ExoS and
ExoT are indicated by arrows. (B) Expression of exoT::lacZ(pHW0005) in
the backgrounds of PAK, AprtNR::Gm, AprtNR::GmAPA0612-0613,
AprtNR::GmAPA0612, and AprtNR::GmAPA0613. Bacteria were grown to
an OD600 of 1 to 2 in LB with (black bars) or without (white bars) EGTA
before 1-galactosidase assays. (C) Cell lifting assay. HeLa cells were
infected with PAK, prtR: :Tn5, AprtNR: :Gm, AprtNR::GmAPA0612-0613,
AprtNR::GmAPA0612, and AprtNR::GmAPA0613 at an MOI of 20. After a
4-hour infection, cell lifting was measured with crystal violet staining (see
Materials and Methods for details). *, P < 0.01, compared to the values in
PAK; **, P < 0.01, compared to the values in AprtNR::Gm.
The Expression of exsA Is Repressed by PtrB inprtR mutants
The master activator of TTSS genes is ExsA. It is the last gene in the exsCEBA
operon (144). The great reduction of ExoS and ExoT inprtR mutants may occur through
the repression of exsA expression. To test the transcription of exsA, an exsC::lacZ
reporter plasmid was introduced into theprtR mutants. As shown in Fig. 3-6, the
expression of the exsCEBA operon was greatly reduced in prtR and AprtNR mutants.
Deletion ofPA0612-3 and ptrB, but not PA0613, partially restored the promoter activity
of exsC. Since ExsA is also the activator of its own operon (60), the repression may be
on the transcriptional, translational or protein level. So I further tested the interaction
between ExsA and PtrB.
ci PAK prtR::Tn5 AprtNR::Gm AprtNR::Gm AprtNR::Gm AprtNR::Gm
A612-3 AptrB A613
Figure 3-6. Expression of exsA operon inprtR mutants. Bacteria were grown to an OD600
of 1 to 2 in LB with (black bars) or without (white bars) EGTA before B-
galactosidase assays. *, P < 0.01, compared to the values in PAK; **, P <
0.01, compared to the values in AprtNR::Gm; ***, P < 0.001, compared to the
values in AprtNR::GmAptrB.
PtrB Might Not Directly Interact with ExsA
In earlier reports, it has been shown that ExsA activity can be repressed by
interaction with ExsD or PtrA (47, 89). We wanted to test if the TTSS repressor function
of PtrB is achieved through a direct interaction with the master regulator, ExsA. A
bacterial two-hybrid system (Stratagene) was used to test the interaction between the two
components. ptrB and exsA were each cloned into bait (pBT) or prey (pTRG) plasmids,
fused with XCI and RNA polymerase (RNAP) a-subunit at C terminus, respectively.
Interaction between the two tested proteins can stable XCI and RNAP in the promoter
region of a lacZ gene and activates its expression. Thus, the interaction of two proteins
was indicated by the expression of lacZ in the reporter strain. 1-Galactosidase assay
results, however, did not suggest a direct interaction between PtrB and ExsA, although
strong interaction was observed between the positive controls provided (Fig. 3-7).
Therefore, the mechanism of TTSS repression in the prtR mutant might not involve a
direct binding of PtrB to ExsA. Negative results were also obtained in similar tests
between PtrB and PA0613, indicating no direct interaction of the two small proteins
encoded in the same operon.
20 FF r
Figure 3-7. Monitoring of protein-protein interactions by the BacterioMatch two-hybrid
system. pBT, bait vector; pTRG, target vector; 2BT, ptrB cloned into bait
vector; 2TRG, ptrB cloned into target vector; 3BT, PA0613 cloned into bait
vector; 3TRG, PA0613 cloned into target vector; exsABT, exsA cloned into
bait vector; exsATRG, exsA cloned into target vector; positive, positive control
provided by the manufacturer. *, P < 0.01, compared to the values in the
Mitomycin C-mediated Suppression of the TTSS Genes Requires PtrB
Pyocin production can be triggered by mutagenic agents, such as mitomycin C. In
response to the DNA damage, RecA is activated and cleaves PrtR, similar to LexA
cleavage by RecA in E. coli during the SOS response (90). In the absence of PrtR, the
expression ofprtNis derepressed, resulting in up regulation of the pyocin synthesis
genes. Under this circumstance, theptrB gene should also be up regulated, resulting in
TTSS repression. To test this prediction, wild-type PAK was treated with mitomycin C
under TTSS inducing and noninducing conditions and the expression of ExoS was
monitored by Western blot analysis. In previous reports, 1 tg/ml of mitomycin C was
shown to be able to induce pyocin synthesis (90). After treatment with 1 tg/ml of
mitomycin C for 1.5 h, the OD600 of PAK began to decrease with or without EGTA due
to the toxic effect of the mitomycin C (Fig. 3-8A); therefore, we collected the samples 1.5
h after mitomycin C treatment. Two culture methods were used. One was to grow PAK
with mitomycin C for 30 min and then EGTA was added to induce TTSS for 1 hour. The
other was to add mitomycin C and EGTA at the same time and induce for 1 hour.
Experimental results showed that when wild-type PAK was treated with mitomycin C
and EGTA at the same time, normal TTSS activation was observed. However, when
cells were treated with mitomycin C 30 min before the addition of EGTA, a clear
repression of the TTSS was observed (Fig. 3-8B). To test whether theptrB gene
mediates the repression of the TTSS by mitomycin C, a deletion mutant ofptrB was
further generated in the background of wild-type PAK. Deletion ofptrB in PAK had no
effect on the expression of the TTSS (Fig. 3-8B and C). Interestingly, even with the 30-
min pretreatment of mitomycin C (1 ig/ml), production of ExoS in the PAKAptrB
mutant was activated by EGTA, even higher than that without mitomycin C treatment
(Fig. 3-7B). Clearly, mitomycin C-mediated suppression of the TTSS requires theptrB
A 1 0E+10
S,1 0,09 LB+Mitomycin C
o 1 2 3 4 5 hr
+ + + +
+ + + -
- + +
- + +
Figure 3-8. Effect of mitomycin C on bacteria growth and TTSS activity. (A) An
overnight culture of PAK was diluted to an OD600 of 0.8 in LB, LB plus 1
[g/ml mitomycin C, LB plus 5 mM EGTA, or LB plus 1 [g/ml mitomycin C
plus 5 mM EGTA. The OD600 of each sample was measured at 30-min
intervals. The cell densities were calculated based on the OD600. (B)
Overnight cultures of PAK, PA0612-0613, and AptrB were diluted to an
OD600 of 0.5 with LB or LB plus 1 [g/ml mitomycin C. After 30 min, EGTA
was added to the culture medium at a final concentration of 5 mM. One hour
later, each culture was mixed with protein loading buffer. Samples derived
from equivalent bacterial cell numbers were loaded onto SDS-PAGE gels and
immunoblotted with anti-ExoS antibody. *, PAK was grown in LB for 30
min, and then both mitomycin C and EGTA were added at the same time. (C)
Overnight cultures of PAK, PA0612-0613, and AptrB strains were diluted to
1% in LB or 3% in LB plus 5 mM EGTA and grown at 370C for 3.5 h.
Supernatants and pellets from equivalent bacterial cell numbers were loaded
onto SDS-PAGE gels and immunoblotted with anti-ExoS antibody.
Twitching Motility Was Not Affected by the prtR mutation
The TTSS genes have been shown to be affected by Vfr and CyaA/B, homologues
of CRP and cyclic AMP synthase (140). Vfr is well known for its involvement in the
regulation of twitching motility (8), flagellum synthesis (26), type II secretion (140), and
quorum sensing (3). Recently, FimL was found to regulate both the TTSS and twitching
motility through Vfr (137). To test whether mutation ofprtR affects twitching motility,
strains with prtR and ptrB mutations were subjected to a stab assay. Mutation in the prtR
orptrB gene had no effect on twitching motility (Fig. 3-9), indicating that the repression
of the TTSS in theprtR mutant does not go through the Vfr pathway.
Figure 3-9. Twitching motility ofprtR, ptrB and PA0613 mutants. The bacteria of each
strain were stabbed into a thin-layer LB agar. The plate was incubated at
370C over night. The whole plate was directly stained with Coomassie blue at
room temperature for 5 min and destined with destain solution.
During early infection of cystic fibrosis patients, P. aeruginosa produces S-type
pyocins (9); however, the exact physiological role played by pyocins is unclear. Pyocins
might ensure the predominance of a given strain in a bacterial niche against other bacteria
of the same species. The pyocin production starts when adverse conditions provoke
DNA damage. Under these conditions, the effect of pyocins is likely to preserve the
initial predominance of pyocinogenic bacteria against pyocin-sensitive cells (90). Upon
activation by DNA-damaging agents, RecA mediates the cleavage of PrtR, derepressing
the expression ofprtN, resulting in active synthesis of pyocins. Thus, the pyocin
synthesis is dependent on the SOS response, resembling those responses of temperate
bacteriophages in E. coli (16, 90). Indeed, DNA-damaging agents, such as UV
irradiation and mitomycin C, induce the synthesis of pyocins in a recA-dependent manner
(90). Apparently, in response to the DNA damage stress signal, P. aeruginosa not only
turns on the SOS response system for DNA repair and pyocin synthesis but also actively
represses the energy expensive type III secretion system, an example of coordinated gene
regulation for survival.
Along the regulatory pathway, mutation of the prtR gene results in the up
regulation ofprtN (86). We found that PrtN is not responsible for the repression of the
TTSS; rather, ptrB next to and under the control ofprtR is required for the TTSS
repression. We also found that the downstream gene PA0613 was in the same operon
with PA0612. Homologues of these genes are also found in Pseudomonas putida
(PP3039 and PP3037) and Pseudomonas syringae (PSPT03417 and PSPT03419), where
they seem to also form operon structures, although with one additional gene between
them (PP3038 or PSPR03418). The promoter ofptrB contains a 14-base sequence that
was also found in the prtN promoter (86), which may be a binding site for PrtR.
Considering that PrtR is the ortholog of XCI, which functions as a homodimer (16), PrtR
may also form a dimer. Whether PrtR recognizes these potential binding sites is not
known. Interestingly, the PtrB protein contains a prokaryotic DksA/TraR C4-type zinc-
finger motif (www.pseudomonas.com). The dksA gene product suppresses the
temperature-sensitive growth and filamentation of a dnaK deletion mutant ofE. coli (66),
while TraR is involved in plasmid conjugation (30). These proteins contain a C-terminal
region thought to fold into a four-cysteine zinc-finger (30). Yersinia sp. also encodes a
small-sized protein, YmoA (8 kDa), which negatively regulates the type III secretion
system (79). YmoA resembles the histone-like protein HU and E. coli integration host
factor; thus, it is likely to repress type III genes through its influence on DNA
conformation. Whether PtrB exerts its repressor function through interaction with
another regulator or through binding to specific DNA sequences present in the TTSS
operons or their upstream regulator genes is not known. It would also be interesting to
investigate on what other genes of the P. aeruginosa genome PtrB effects on.
It is not surprising that P. aeruginosa has multiple regulatory networks, since 8% of
its genome codes for regulatory genes, indicating that P. aeruginosa has dynamic and
complicated regulatory mechanisms responding to various environmental signals (108,
124). Also, due to the requirement of a large number of genes, construction of the type
III secretion apparatus is an energy-expensive process. Thus, P. aeruginosa might have
evolved multiple signaling pathways to fine-tune the regulation of the type III secretion
system in response to the environmental changes. Similarly, Yersinia has been reported
to have several regulators, such as an activator, VirF, and repressor molecules, LcrQ,
YscM1, YscM2, and YmoA, that are involved in the control ofyop gene transcription
(20, 139, 142). Current efforts are focused on the elucidation of the molecular
mechanism by which PtrB mediates suppression of the TTSS. Also, the relevance of the
two additional genes, PA2265 and PA5021, to the regulation of the TTSS needs more
Fige)- (a -ExsA'-1TTSS
Figure 3-10. Proposed model ofPtrB-mediated TTSS repression. In wild-type PAK,
PrtR represses the expression ofprtN andptrB. In response to DNA damage,
RecA is activated and cleaves PrtR, resulting in increased expression ofprtN
and ptrB. PrtN activates the expression of pyocin synthesis genes, while PtrB
represses the type III secretion genes directly or through additional
Based on our results, we propose a model for the repression of the TTSS induced
by DNA damage (treatment with mitomycin C) (Fig. 3-10). DNA damage induces the
SOS response, in which RecA is activated. RecA cleaves PrtR, resulting in the up
regulation ofprtN andptrB. PrtN activates the expression of pyocin synthesis genes,
while PtrB represses the TTSS genes. How PtrB represses the TTSS is not known. In the
bacteria two-hybrid system, I failed to detect the interaction between PtrB and ExsA.
However, PtrB is a small protein (-6.7 kDa), and when fused with either XCI or RNAP a-
subunit, its interaction with ExsA might be affected due to conformational change or
steric hindrance. Further experiments are needed to study the interaction between PtrB
DISCUSSION AND FUTURE DIRECTIONS
The TTSS and Environmental Stresses
Repression of the TTSS under Environmental Stresses
The TTSS ofP. aeruginosa is under the control of a complicated regulatory
network. ExsA, an AraC-type protein, is the master activator of the TTSS. Two proteins,
ExsD and PtrA, have been found to directly interact with ExsA. ExsD is an anti-
activator, inhibiting the activity of ExsA (89). PtrA is an in vivo inducible protein and
represses the activity of ExsA through direct binding. In vitro, the expression of PtrA is
inducible by high copper stress signal through a CopR/S two-component regulatory
system (47). Over expression of multi-drug efflux systems MexCD-OprJ and MexEF-
OprN leads to repression of the TTSS (75). The expression of multi-drug efflux systems
are usually triggered by antibiotics which is a detrimental stress. We also found that
mutation in the mucA gene not only results in overproduction of alginate but also causes
repression of the TTSS (Chapter 2). MucA-regulated alginate production is induced by
environmental stresses, such as high osmolarity, reactive oxygen intermediates, and
anaerobic environment (45, 84). Metabolic imbalance was also shown to cause
repression of the TTSS, which represents a nutritional stress (23, 107). In Chapter 3 we
reported that mutation in theprtR gene resulted in repression of the TTSS. PrtR is a
repressor whose activity is regulated by DNA damage (90), yet another stress signal.
Mitomycin C, a mutagenic agent, can indeed repress the activity of the TTSS. My
preliminary data showed that heat shock could also cause repression of the TTSS. These
discoveries indicate that the TTSS is effectively turned off under various environmental
stresses, which might be an important survival strategy for this microorganism. Since
mounting an effective resistance against stress requires a full devotion of energy, turning
off other energy-expensive processes, such as the TTSS, will be beneficial to the
Indication for the Control of P. aeruginosa Infection
Mutation in TTSS renders P. aeruginosa avirulence in a burned mouse model (59).
In a mouse model ofP. aeruginosa pneumonia and a rabbit model of septic shock,
antibodies against PcrV ( required for effectors translocation) are able to decrease lung
injury and ensure survival of the infected animals (37, 113, 120). These results indicated
that inactivation of the TTSS is a prospective therapeutic strategy. Since environmental
stresses can lead to the repression of the TTSS, drugs can be designed towards
components in the stress response signaling pathways, such as DNA damage, heat shock,
metabolism imbalance, copper stress, etc. The more we know about the regulatory
pathways, the more candidate targets we will have. This strategy might be extended to
the control of other virulence mechanisms, such as biofilm formation. During the chronic
infection in CF lungs, P. aeruginosa grows under a low oxygen environment in the form
of a biofilm. Quorum sensing mutants (lasR or rhlR) are unable to survive in the
anaerobic condition, due to the metabolic intoxication by nitric oxide (145). Therefore,
drugs targeting the quorum sensing system might facilitate the eradication of P.
aeruginosa biofilm (51). Indeed, some non-native AHLs (autoinducers of the las and rhl
quorum sensing systems) have been found to disrupt P. aeruginosa biofilm formation
Regulation of the TTSS under Environmental Stresses
Among all the environmental stresses that induce TTSS repression, only one
regulatory pathway (PtrA) is well understood (47). Based on the experimental data for
PtrA and PtrB, it is possible that each environmental stress involves a specific TTSS
repressor, such as PtrA for copper stress and PtrB for DNA damage stress (Chapter 3).
Some of these repressors may even have common regulators. For example, cAMP and
Vfr are required for the TTSS. Any environmental signals affecting cAMP level or Vfr
activity will affect the TTSS. It will be interesting to measure the expression level of Vfr
as well as cAMP level under various environmental stresses.
The ExsA activity is under the direct control of a regulatory cascade, consisting of
ExsE, ExsC and ExsD (Fig. 1-1) (27, 89, 106, 130). Each of the components can also be
the target of regulation under stress conditions. Activation of ExsA depends on the
secretion of ExsE through the TTSS machinery. Any environmental stresses that block
the ExsE secretion will result in inhibition of the ExsA function (106, 130). Furthermore,
expression of exsA may also be affected by stress responses.
Expression of ExsA
exsA is the last gene in the exsCEBA operon as shown in Fig. 4-1, which is
activated by ExsA itself (144). It is not known which sigma factor recognizes this operon
promoter. Interestingly, the predicted exsB open reading frame (ORF) seems not
translated in either P. aeruginosa or E.coli (43). Neither point mutation of the exsB start
cordon nor over expression of exsB had any effect on the TTSS activity (43, 106).
However, deletion of the exsE and exsB region (StuI sites, Fig 4-1) resulted in a drastic
reduction of the TTSS activity. Since ExsE is a TTSS repressor, mutation in exsE should
lead to derepression of the TTSS (106). These results indicate that the exsB region (DNA
or RNA) might affect the transcription or translation of ExsA. It will be interesting to
delete only the exsB region and test the effect on the expression of exsA.
I 'I I I I I
exsC exsE exsB exsA
Figure 4-1. Structure of the exsCEBA operon. The ORFs and transcription directions are
indicated as arrows.
The exsB DNA fragment might control the transcription of exsA gene through the
formation of a secondary structure. This type of regulation usually happens at the
promoter region, where RNA polymerase or regulators bind to (16). Since exsA does not
have its own promoter immediate upstream of its coding sequence (144), it is unlikely
that exsB DNA has this type of function.
Microarray analysis and lacZ transcriptional fusion experiments indicate that the
mRNA level of exsCEBA operon does not change much under TTSS inducing vs. non-
inducing conditions (72, 140). A real-time PCR experiment is needed to precisely
determine the mRNA levels of each ORF and the region between exsB and exsA. Despite
the minimal increase at the transcriptional level, the ExsA protein level increased
significantly under TTSS inducing conditions as judged by Western blot analysis (27),
suggesting that the expression of ExsA is under post-transcriptional control. Well known
mechanisms of the post-transcriptional controls include mRNA stability or formation of
secondary structures which affect translation efficiency (16, 74). In prokaryotes,
untranslated mRNA tends to be degraded quickly by endoribonucleases or exonucleases
(70). The translation of an mRNA can be affected by secondary structures formed by
endogenous sequences or with an antisense RNA. A likely hair-pin structure has indeed
been found in the exsB-exsA junction (Fig. 4-2) which may blocks the access of the
ribosome to its binding site for the translation for exsA. Experimental tests, deletion as
well as site-directed mutagenesis, are needed to confirm this possibility.
Start cordon of exsA
.* A I **IA
Figure 4-2. The secondary structure of exsA mRNA 5' terminus. The sequence was
analyzed by mfold (http://www.bioinfo.rpi.edu/applications/mfold/old/rna/).
The mRNA stability and secondary structure can also be controlled by small RNAs
(sRNAs). sRNAs, with length range from 50 to 200 nucleotides, are used by bacteria to
rapidly tune gene expression in responding to changing environments (83, 123). sRNAs
usually anneal to 5' untranslated region (5' UTR) of target mRNAs. The effects of sRNA
binding include increase or decrease of mRNA stability, exposure or blockage of
ribosome binding site. Most interactions between sRNA and target mRNA require a
small protein called Hfq. Mutation of Hfq in P. aeruginosa resulted in impaired
twitching motility and attenuation of virulence when injected intraperitoneally into mice
(122). It will be interesting to test the TTSS activity in the hfq mutant, which may give
us a clue whether sRNAs are involved in the TTSS regulation.
In summary, the transcriptional and translational control of exsA is not clear at
present time. Understanding the regulatory mechanism of exsA may help us to clarify the
relationship between TTSS and many other genes that affect its activity. Also, it will
help us to develop strategies to control P. aeruginosa infections.
My project started from the construction and screening of Tn insertional mutant
libraries. This strategy is a powerful tool in searching genes related to certain phenotype.
The success of this method relies on the high efficiency of transposition, special
characteristics of the Tn and sensitive screening methods.
Usually, the Tn is on a suicide plasmid and transferred into the recipient through
conjugation or sometimes by electroporation. In my experiments, the growth phase of
E.coli donor strain was important, with the highest efficiency achieved by using cells
grown to OD600=0.6-1.0. The growth phase of P. aeruginosa recipient strain seems less
important. The optimum donor to recipient ratio ranged between 3:1 and 8:1, with about
5X108 recipient cells in each conjugation mixture.
During the growth of the conjugation mixtures (121), P. aeruginosa seems to kill
E.coli, resulting in low conjugation efficiency. This killing can be repressed by
performing the conjugation on nutrient agar. Probably, P. aeruginosa produces fewer
bactericidal factors when grown on nutrient agar medium compared to the L-agar.
Another factor limiting the conjugation efficiency is the DNA modification and
restriction system of the recipient, which mediates the degradation of foreign DNA.
Growth of the recipient at 420C for at least 2 hours before conjugation can greatly
increase the mutagenesis efficiency, presumably due to the repression of the DNA
modification and restriction system.
In most of my experiments, 1-3x104 Tn insertion mutants can readily be obtained
from each conjugation. P. aeruginosa has about 5600 genes; thus theoretically, 3x104
mutants should provide about 5-fold coverage of these genes (63).
Characteristics of the Tn
Most Tn insertional mutagenesis do not ensure every target gene being hit by the
Tn, although statistically the number of the mutants should saturate the whole genome.
Tns seem preferentially to insert in certain regions while avoiding other regions, so called
hot and cold spots, respectively. The Tn used in my research is a derivative of Tn5 (71).
In my Tn5 mutagenesis experiments, no insertion was found in the TTSS region,
suggesting it is a cold spot for the Tn5. In agreement with my experience, a Tn5
insertion library constructed in strain PAO1 by Jacobs et al. (University of Washington
Genome Center, Seattle) has also concluded that the coding region of the TTSS apparatus
is a clod spot (63). Testing of different transposons might identify ones that can readily
transpose into the TTSS region.
The success of Tn mutagenesis experiments also depends on the screening strategy.
Two types of screening methods are widely used. One is to individually test for
phenotypes of interests, which provides a high accuracy. However, it takes a lot of
manpower and is cumbersome. The other one is to do large scale screening on the whole
library. By this method, a large number of mutants can be screened quickly, although the
accuracy is compromised. Usually, this method requires a reporter gene, either encoded
on the Tn or harbored by the recipient strain. In my experiments, an exoT::lacZ fusion on
plasmid was used as the reporter. On plates containing X-gal, the density of blue color of
each colony represents the exoTpromoter activity. With this method, 100,000 mutants
can be screened in less than one hour. The shortcoming of this method is that the color
density is judged by eyes; thus many mutants with interesting phenotypes might have
been missed. Indeed, although I successfully identified two genes, mucA andprtR, which
are required for the TTSS activity, no other genes known to regulate the TTSS were
identified. Possibly either I missed those colonies with minor changes in blue color or
the Tn insertion libraries were not saturated. Other Tn with more sensitive screening
methods might be needed to identify additional TTSS related genes.
In summary, I developed a screening system for the identification of the TTSS
related genes. From the Tn insertion libraries constructed in wild type PAK containing
exoT::lacZ reporter plasmid, two genes, mucA and prtR, were found to be related to the
TTSS. I further studied the regulatory relationship between MucA and the TTSS as well
as PrtR and the TTSS. In the mucA mutant, AlgU and AlgR are required for the
repression of the TTSS. In theprtR mutant, a newly identified gene, ptrB is up regulated
and responsible for the repression of the TTSS. Wild type P. aeruginosa strain will turn
into mucoid phenotype in response to some environmental stresses, such as anaerobic
environment, high osmolarity and reactive oxygen intermediates. PrtR is a regulator of
pyocin synthesis, it responses to DNA damage. All my results suggest that TTSS will be
repressed under environmental stresses (10), which may provide a potential strategy for
the control of the TTSS activity and improve the treatment of P. aeruginosa infection.
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