IDENTIFICATION OF pilE AND ANALYSIS OF THE GENE PRODUCT, A PROTEIN INVOLVED IN PILUS BIOGENESIS IN Pseudomonas aeruginosa
DISSERTATION
Presented in Partial Fulfillment of the Requirements for the Degree
Doctor of Philosophy in the Graduate School of The Ohio State
University
By
Mary Alice Russell, M. S.
*****
The Ohio State University 1996
Dissertation Committee: Approved by N. R. Baker
K. M. Coggeshall
C. J. Daniels 1 Advisor Microbiology A. Darzins
W. R. Strohl UMI Number: 9639340
UMI Microform 9639340 Copyright 1996, by UMI Company. All rights reserved. This microform edition is protected against unauthorized copying under Title 17, United States Code.
UMI 300 North Zeeb Road Ann Arbor, MI 48103 ABSTRACT
A new locus required for type 4 pilus biogenesis by Pseudomonas aeruginosa has been identified. A pilE mutant, designated MJ- 6, was broadly resistant to pili-specific phages and unable to translocate across solid surfaces by the pilus-dependent mechanism of twitching motility. Immunoblot analysis demonstrated that MJ -6 was devoid of pili but was unaffected in the production of unassembled pilin pools. Genetic studies were used to localize the pilE mutation to the 71 min. region of the P. aeruginosa PAO chromosome. A PAOl library was mobilized into MJ -6 and a clone was identified that restored sensitivity to pili-specific phage, twitching motility and pilus production. The nucleotide sequence of a 1 kb EcoRV-Clal fragment revealed a single complete open reading frame with characteristic P. aeruginosa codon bias ( pilE). PilE (15,278 kDa), showed significant identity to the N-terminal 40 amino acids of the pilin precursors of P. aeruginosa and to other type 4 prepilin proteins. PilE contains a seven-residue basic leader sequence followed by a consensus cleavage site for prepilin peptidase and a largely hydrophobic region which contains tyrosine residues (Tyr-24 and Tyr-
28) previously implicated in maintaining pilin subunit-subunit interactions. A chromosomal pilE insertion mutant constructed in our lab lacks pili and is twitching-motility deficient, thus confirming the requirement of PilE in pilus biogenesis.
Immunological experiments indicated that PilE may be capable of homomultimeric interaction but does not seem capable of interaction with
PilA. We also found no evidence that PilE is a minor pilin protein. Based on these findings and drawing parallels from other bacterial systems, we believe that PilE may form a multimeric structure with itself and possibly other prepilin-like proteins (such as PilV) that acts as a scaffold or conveyor for the pilus filament. Dedicated to the memory of my father. He always believed I would make it. ACKNOWLEDGMENTS
I wish to thank my advisors, Drs. A1 Darzins and Neil Baker, for their support, encouragement, and patience throughout the entire project. I also wish to thank my committee members, Drs. Bill Strohl and Chuck Daniels for their time and involvement in this project. I want to especially thank Dr.
Mark Coggeshall for all the help, advise and encouragement in the last year of this endeavor.
I am indebted to Patty Truax, Lynn O'Donnell and Ellen Ostrofsky for their helpful scientific discussions and friendship.
I must also thank my family, Brant and my mom, for all their patience, love and understanding over the years.
v VITA
May 16,1966 Born-Columbus, Ohio
1988 Microbiologist, Vector Born Disease Unit, Ohio Department of Health Labs
1994 M.S. Microbiology, The Ohio State U niversity
1989-present Graduate Teaching Associate, The Ohio State University
PUBLICATIONS
RESEARCH PUBLICATIONS
1. Russell, M. A. and A. Darzins. 1994. The pilE gene product of Pseudomonas aeruginosa, required for pilus biogenesis, shares amino acid sequence identity with the N-termini of prepilin proteins. Mol. Microbiol. 13:973-985.
FIELDS OF STUDY
Major Field: Microbiology TABLE OF CONTENTS
Page Dedication ...... i v
Acknowledgments ...... v
Vita...... vi
List of Tables ...... x
List of Figures ...... xi
List of Abbreviations ...... xiii
Chapters:
1. Introduction ...... 1 Ecology of Pseudomonas aeruginosa...... 2 P. aeruginosa as a pathogen ...... 2 P. aeruginosa infections in the CF patient ...... 4 P. aeruginosa virulence factors ...... 5 Adhesins...... 6 Adhesin receptors ...... 8 Exoproducts ...... 9 T ype4pili...... II The type 4 pili family...... 11 Structure, function, and occurrence ...... 12 The P. aeruginosa pilin gene and gene product 15 Accessory pil genes ...... 18 pilB, pilC, and pilD...... 18 pilM, pilN, pilO, pilP, and pilQ...... 22 pilV and pilE...... 24 pilR and pilS...... 25 pilG, pilH, pill, pil], and pilK...... 26 pilT and pilU...... 27
vii pilZ...... 28 Pil homologs in other bacterial systems ...... 28 Protein secretion in P. aeruginosa...... 29 Pullulanase secretion in Klebsiella oxytoca...... 30 Other protein secretion systems ...... 31 Bacillus subtilis...... 32 Goals of this study ...... 32
2. Materials and methods ...... 33 Bacterial strains, phages, and plasmids ...... 33 Media ...... 33 DNA methods ...... 40 Genetic procedures ...... 40 Phage sensitivity testing and motility assays ...... 41 DNA sequence analysis ...... 41 Construction of pilE and pil A gene replacement mutants... 42 Expression studies with the T7 RNA polymerase-promoter system...... 46 Pilus purification ...... 46 Nucleotide sequence accession number ...... 47 Construction and purification of GST fusion proteins 47 PCR...... 50 Antibody production ...... 51 Cell fractionation ...... 52 Immunoblotting ...... 53 Coimmunoprecipitation techniques ...... 54 Capture experiment ...... 54
3. The identification and characterization of pilE and preliminary characterization of the gene product ...... 55 Introduction ...... 55 Results...... 59 Characterization of the P. aeruginosa pit m utant MJ-6...... 59 Mapping of the MJ -6 pilE m utation ...... 63 Complementation of MJ -6...... 65 Sequence analysis of the MJ -6 complementing region ...... 66 Homology searches ...... 71 Expression of p ilE...... 78 Construction and analysis of a P. aeruginosa pilE gene replacement mutant ...... 80 Discussion ...... 81 Characterization of the pilE gene ...... 81
viii Characterization of the pilE gene product ...... 82 Possible role(s) of the PilE N-terminus ...... 83 Homology between PilE and type 4 prepilin proteins 84 Homology between PilE and Xcp proteins ...... 85 Possible role(s) of PilE ...... 86
4. Analysis of PilE ...... 89 Introduction...... 89 Results...... 97 Sequencing of the MJ -6 pilE m utation ...... 97 Production of antibody against PilE ...... 99 Level of PilE during growth in broth culture ...... 103 Localization of PilE and processing by PilD ...... 103 Immunoprecipitation using antiPAO pilin and antiPilE antisera ...... 112 Capture experiment with GSTmPilE fusion protein.. 115 Site-directed mutagenesis of Tyr 24 and 27 to Phe in pilE...... 118 Discussion ...... 121 Production of PilE in PAOl ...... 121 Production of PilE in DA11 ...... 122 Role of Tyr 24 and 27 in PilE function ...... 122 Possible role(s) of PilE ...... 123 Summary...... 133
Appendices ...... 134 A. Sequence alignment of P. aeruginosa ORFB' with E. coli ORF316 ...... 134 B. Cartoon depiction of Pil A and PilE proteins ...... 136
References ...... 138
ix LIST OF TABLES
Table Page
1 Summary of the known pil genes ...... 19
2 Strains, phages and plasmids used in this study ...... 34
3 Strains and plasmids generated in this study ...... 37
4 Conjugational and transductional mapping of the MJ -6 mutation 64
5 Homologs of the P. aeruginosa Pil proteins and proteins with prepilin leader sequence ...... 92
6 Percent identity of prepilin-like proteins to the consensus sequence for their respective groups ...... 122 LIST OF FIGURES Figure Page
1 Summary of method used to generate chromosomal gene replacement mutants ...... 43
2 P. aeruginosa PAO genetic map and location of pil loci ...... 56
3 Twitching motility assay of PAOl, MJ- 6, Rl, and R 8 ...... 61
4 Western blot analysis of whole cell lysates of P. aeruginosa...... 62
5 Cloning and localization of the MJ -6 complementing activity 67
6 Nucleotide sequence of the P. aeruginosa pilE gene ...... 69
7 Sequence alignment of P. aeruginosa PilE with PilA...... 72
8 Comparison of N-terminal sequences of prepilin peptidase dependent proteins ...... 75
9 Expression of the P. aeruginosa pilE gene in P. aeruginosa...... 79
10 Comparison of prePilE and GST fusion proteins ...... 100
11 Inhibition studies of antiPilE' antisera ...... 102
12 Western analysis of purified pili and GSTPilE' ...... 104
13 Localization of PilD to the inner membrane and visualization of outer membrane proteins ...... 106
14 Western blot analysis determining the subcellular location of PilE under physiological conditions ...... 109
15 Western blot analysis determining the size and subcellular location of PilE in DA 11 ...... I l l 16 Western blot analysis of immunoprecipitation experiments 113
17 Western blot analysis of fishing experiment using PAOl, Rl, and PAO-A- inner membrane fractions ...... 117
18 Western blot analysis of fishing experiment using PAOl, Rl and Phe2427 inner membrane fractions...... 118
19 Comparison of PilE to PilA and other prepilin-like proteins 124
20 Sequence alignment of P. aeruginosa ORFB' with E. coli ORF316.. 133
21 Cartoon depiction of PilA and PilE proteins ...... 135
xii LIST OF ABBREVIATIONS
ADP adenosine diphosphate ATP adenosine triphosphate ATPase adenosine triphosphatase bp base pairs Cm chloramphenicol dCTP deoxycytidine triphosphate dGTP deoxyguanosine triphosphate DNA deoxyribonucleic acid 8 gravity h hours kb kilobases kDa kilodaltons LD lethal dose LPS Hpopolysaccharide |ig microgram pi microliter min minutes mg milligram ml milliliter m M millimolar mmol millimoles mRNA messenger RNA PAGE polyacrylamide gel electrophoresis PCR polymerase chain reaction Phe phenylalanine RNA ribonucleic acid s seconds SDS sodium dodecyl sulfate Tyr tyrosine
xiii CHAPTER 1
INTRODUCTION
The genus Pseudomonas is large and varied, and, one of the most striking features of this group is the nutritional versatility of its members.
Genus Pseudomonas includes species known to be opportunistic human pathogens ( Pseudomonas aeruginosa, P. cepacia), phytopathogens (P. aeruginosa, P. syringae), plant growth-promoting organisms (P. aeruginosa,
P.putida), and scavengers of environmentally toxic compounds (P. putida, P. oleovorans). This diversity alone makes Pseudomonas an exciting area of research. In addition, pseudomonads are amendable to many of the techniques used in molecular biology and thus have been studied extensively.
The result is a large volume of data that not only furthers our knowledge of this genus, but also can be applied to other, closely related bacteria. This large group of soil and water borne gram-negative bacillary organisms has greatly increased our knowledge of the microbial world.
1 Ecology of Pseudomonas aeruginosa. P. aeruginosa is a ubiquitous organism. It has been isolated from a large variety of soil and water environments. P. aeruginosa is a common isolate from water sources polluted by humans or animals. It has been found in sink drains, toilets and
showers in hospitals and private residences. Even medical devices that work with water and disinfectants have been contaminated. P. aeruginosa is rarely
found in nonpolluted water but it is able to subsist in distilled water suggesting that purifying water so that it does not support growth of P. aeruginosa is difficult (Botzenhart and Doring, 1993).
P. aeruginosa is found in the soil, primarily in the rhizosphere, but does require moisture to survive. It is a common contaminant of fruits and vegetables (Botzenhart and Doring, 1993). The plant-bacteria interaction is often harmful for the plant (Cother el al., 1976; Elrod and Braun, 1942; Lebeda et al, 1984), but a strain of P. aeruginosa has been identified that increases the growth of plants by 10-25% (Hofte et al, 1990).
P. aeruginosa as a pathogen. Normally nonpathogenic P. aeruginosa is also a formidable opportunistic human pathogen. This organism is found at low numbers in the healthy population but is responsible for approximately
10% of all hospital acquired infections and was the third most common nosocomial pathogen in the 1980's (Botzenhart and Doring, 1993; Tonner et a l, 1993). In addition, the mortality rate associated with P. aeruginosa
2 infections is higher than infections caused by other gram-negative, bacillary organisms (Artenstein and Cross, 1993). For instance, the mortality rate among patients with nosocomial pneumonia can be as high as 70% and complications in cystic fibrosis (CF) patients due to Pseudomonas infections are responsible for 90% of CF deaths (Fick, 1993).
A common factor among patients that acquire a P. aeruginosa infection is a disruption of their defense barriers against bacterial infections. Common susceptible groups include burned patients (Sato et al., 1988), patients with a corneal ulcer, cancer or acute leukemia (Bodey et at., 1983), and, more recently, patients with AIDS (Dropulic et al, 1995). Patient intubation and antibiotic treatment also increase the risk of P. aeruginosa infection
(Artenstein and Cross, 1993; Bodey et al., 1983; Tonner et a l, 1993).
Some of the more common types of P. aeruginosa infections include respiratory infections, both acute and chronic, urinary tract infections usually due to intubation and catheterization, and bacteremia prevalent in immunocompromised patients with an underlying disease (Aksamit, 1993;
Artenstein and Cross, 1993; Dropulic et al, 1995; Tonner et a l, 1993). P. aeruginosa corneal infections are particularly destructive. The infection progresses rapidly and is thus difficult to treat. Further, Fleiszig et a l (1994) suggest that the bacteria are able to invade the corneal epithelial cells resulting in evasion of both the host defense system and antibiotic treatment.
3 The high incidence of mortality associated with P. aeruginosa infections is due primarily to the innate resistance of this organism to many commonly used antibiotics (Bellido and Hancock, 1993). This resistance can be attributed to a combination of low outer membrane permeability; a chromosomally encoded, inducible gene coding for a product that cleaves p- lactams; plasmid borne p-lactamases and aminoglycoside-modifying enzymes; either phenotypic or genotypic changes that lead to quinolone resistance by altering the A subunit of DNA gyrase; and, the exopolysaccharide surrounding mucoid strains found primarily in CF patients (Bellido and
Hancock, 1993; Gimeno et al., 1996).
P. aeruginosa infections in the CF patient. CF, the most common lethal genetic disease in Caucasians, is caused by a mutation in the gene coding for the cystic fibrosis transmembrane conductance regulator (CFTR; Doring, 1993;
Gilligan, 1991; McCubbin and Fick, 1993). CFTR is a member of the ABC transporter superfamily and specifically functions as a chloride channel
(Deretic et a l, 1994). The mutation affects ion transport causing production of thick, viscous secretions in the airways, liver, pancreas, intestines and gall bladder (Doring, 1993). In the lungs, the thick secretions are difficult to clear and lead to chronic bacterial colonization (Gilligan, 1991). Progressive loss of lung function due to recurrent infections is responsible for 90% of CF deaths
(Gilligan, 1991). Initially, the lungs of CF patients are colonized by Staphylococcus aureus. As these patients age, P. aeruginosa establishes a chronic infection and becomes the dominant pathogen (McCubbin and Fick,
1993).
The initial adherence step of infection is probably mediated by nonmucoid strains of P. aeruginosa, but there is a switch to a mucoid phenotype soon after (Irvin, 1993). This switch signals the onset of chronic infection. The mucoid exopolysaccharide (alginate) is proposed to be important in the ability of P. aeruginosa to colonize the lungs of a non immunocompromised host and to allow the organism to establish a chronic infection (Baltimore, 1993). Alginate seems to be involved in increased resistance to antibiotics and surfactants, and may inhibit phagocytosis (Ames et al., 1985; Baltimore and Mitchell, 1980; Govan and Fyfe, 1978; Lam et a l,
1980; Schwarzman and Boring, 1971).
P. aeruginosa virulence factors
P. aeruginosa has a multitude of virulence factors at its disposal, including several adhesins and numerous exoproducts. The exact role and extent of the importance of many exoproducts is still unclear. Therefore, only the better characterized virulence factors will be discussed. Because this study involved a gene associated with pili, an adhesin, the adhesins will be examined in more detail than the other virulence factors.
5 Adhesins. Studies aimed at understanding the initial step of P. aeruginosa colonization date back to 1980 when Woods et al. first recognized the role of pili in adherence to buccal epithelial cells (BECs). Since then, alginate and possibly flagella have also been identified as mediators of adherence by P. aeruginosa.
Piliated strains of P. aeruginosa are more virulent than isogeneic, nonpiliated strains (Sato et al., 1988; Tang et al., 1995), which indicates that pili are a virulence factor. The fact that purified pili, antipilus antibodies and pilus peptides each inhibit binding of homologous P. aeruginosa whole cells to respiratory epithelial cells (Doig et al., 1988; 1990; Woods et al., 1980) points to the role of pili in adherence. The adhesion moiety of P. aeruginosa pili is located exclusively at the tip of the pilus filament (Lee et al., 1994) and has been mapped to the C-terminal domain of the pilin subunit (Irvin et al., 1989;
Farinha et al. 1994). The binding of P. aeruginosa cells to BECs could not be completely inhibited with monoclonal antibodies directed against the binding domain of pili (Doig et al., 1990) which suggests that other adhesins are involved.
Comparison of the adherence of mucoid and nonmucoid strains of P. aeruginosa to an inert substrate (i.e. dacron fiber; Mai et al., 1993) also points to a role for alginate in adherence. Alginase and monoclonal antibodies
6 against alginate were able to inhibit the adherence of P. aeruginosa to dacron fibers (Mai et al., 1993). Earlier studies with purified alginate also show that it is able to mediate binding to BECs and TECs (Doig et al., 1987).
Recent results indicate that flagella may also be an important adhesin of P. aeruginosa (Wahl and Baker, 1996, manuscript submitted). An earlier report that exoenzyme S acts as an adhesin is not correct as the preparation used in that study contained a significant amount of flagellin (Baker et al.,
1991). Flagella has been shown to bind to asialoganglioside 1 (ASGMi; one of the possible receptor for P. aeruginosa identified on host epithelial cell discussed below) by independent investigations, but the results of one study concluded that this binding is non-specific (Gupta et al., 1994). Flagella were shown to be a virulence factor in the burned mouse model, but their role seemed to be primarily related to motility and not adhesion (Drake and
Montie, 1988). Motility deficient (mot-) and flagella deficient (fla-) strains were compared to a wild type strain. The results demonstrated that all three types of cells colonized the site of infection, but the mot- and fla- strains did not cause a systemic infection as the wild type strain did, which is often the most serious type of infection for a burn patient. Consequently the two mutant strains were less virulent ( 102 to 105 fold less) than the wild type organism. McEachran and Irvin (1985) also found that purified flagella did not affect binding of whole bacterial cells to BECs. The role of flagella in colonization by P. aeruginosa needs to be further clarified.
7 Adhesin receptors. Studies have shown that P. aeruginosa is capable of binding BECs, tracheal epithelial cells (TECs; Doig et al., 1988; Irvin et al., 1989;
W oods et al., 1980), and mucin (Carnoy et al., 1994; Simpson et a l, 1992).
There appears to be two classes of adhesins, one which recognizes both epithelial cells and mucins and one which only recognizes mucins (Simpson et a l, 1992). Pili play a minimal role in mucin adherence (Simpson et a l,
1992). They, instead, appear to be a primary mediator of initial attachment to respiratory epithelial cells with alginate contributing to colonization of the lower airway (Baker and Svanborg-Eden, 1989 ). The mucin adhesin is still undefined. A nonpiliated, flagellar deficient mutant was still able to bind mucin (Simpson et a l, 1992). However, Carnoy et a l (1994) have isolated outer membrane proteins from nonpiliated strains and identified multiple adhesins capable of binding mucin.
The receptors for P. aeruginosa binding have been identified as glycolipids and glycoproteins found on the surface of respiratory epithelial cells (Baker et a l, 1990; Krivan et a l, 1988) and in respiratory mucin (Carnoy et a l, 1994). One study showed that canine kidney cells which were defective in glycosylation were less susceptible to P. aeruginosa probably due to a lack of appropriate receptors (glycolipids and/or glycoproteins) for the bacterial cells
(Apodaca et a l, 1995). P. aeruginosa pili are capable of binding the glycolipids
ASGMi and ASGM 2, the carbohydrate unit necessary for recognition is
8 PGalNAc(l-4)pGal (Lee et al., 1994; Shethet al., 1994). Whether or not sialylated glycolipids are also a receptor for P. aeruginosa is still not clear. One study shows that P. aeruginosa cannot bind GMi or GM 2 (Krivan et al., 1988), others show thatP. aeruginosa can bind sialylated glycolipids (Baker et a l,
1990; Komiyama et a l, 1987; Vishwanath and Ramphal, 1985),while still others indicate that it might, but to a lesser extent than to ASGMi and ASGM 2
(Lee et a l, 1994; Siaman et a l, 1992). If GMi is a receptor, then the adhesin is unlikely to be pili because purified pili do not recognize the sialylated glycolipid (Siaman et a l, 1992). Studies employing cell culture techniques also indicate that P. aeruginosa adhesins recognize unsialyated carbohydrates.
Siaman et al. (1992) found an increased number of receptors for P. aeruginosa on CF respiratory epithelial cells. Their results indicated that exoproducts secreted by P. aeruginosa enhanced adherence to both CF and normal primary cell cultures by exposing asiaologanglioside binding sites. A more recent study reported that CF epithelial cells undersialylate apically secreted proteins and that a tetrasaccharide moiety of ASGMi is the receptor on CF cells
(Imundo et al, 1995). Antibodies against ASGMi inhibited adherence of whole bacterial cells and dislodged bacteria already bound.
Exoproducts. Exotoxin A (ETA) is an ADP-ribosyltransferase enzyme that transfers ADP-ribose to elongation factor 2 (EF2; Iglewski and Kabat,
1975). This results in the inhibition of protein synthesis and leads to cell
9 death. ETA has an LD50 of 2.5 |ig m l 1 in mice (Taylor and Pollack, 1978) and
is produced in human infections (Cross et al., 1980). It has been demonstrated
that ETA is required for maximum virulence of P. aeruginosa (Woods et al.,
1982), and passive immunization against ETA is protective against strains
making ETA (Pavlovskis et al., 1977).
Exoenzyme S (Exo S) is also an ADP-ribosyltransferase enzyme but the
target of its actions does not appear to be EF2 (Iglewski et al., 1978). Instead,
Exo S was found to be able to modify several small GTP-binding proteins
from the H-ras and K-ras family in vitro (Coburn and Gill, 1991; Coburn et al.,
1989). Exo S activity is dependent on a eucaryotic protein that is a member of
the 14-3-3 family of proteins (Coburn et al., 1991; Fu et al., 1993). Exo S
producing strains of P. aeruginosa were found to be significantly more
virulent than isogenic exo S- strains in the burned mouse model and in rat
lungs (simulating a chronic lung infection) and antibodies against Exo S
provided protection against Exo S producing strains (Nicas and Iglewski, 1984;
1985).
The elastolytic activity of two different proteins produced by P. aeruginosa, elastase and alkaline protease, require LasA which has been
shown to act on elastin (Galloway, 1993). Woods et al. (1982) showed that
elastolytic activity was required for maximum virulence of P. aeruginosa.
Purified elastase is able to degrade complement components (Schultz and
Miller, 1974), IgG (Fick et al..,1985), and IgA (Heck et al. ,1990). Elastase and
10 alkaline protease were shown to be able to degrade components of connective tissue, contributing to the virulence of P. aeruginosa (Heck et al., 1986a;
1986b).
P. aeruginosa produces two phospholipase enzymes, hemolytic phospholipase C (PLC; Kurioka and Liu, 1967) and nonhemolytic phospholipase C-N (PLN; Ostroff and Vasil, 1987). These two enzymes together degrade phospholipids common in the eucaryotic membrane but not the procaryotic membrane (Ostroff et al., 1990). As evidence that PLC plays a role in virulence, the purified enzyme caused an inflammatory response and death when injected into mice (Berk et al., 1987; Myers and Berk, 1990).
Furthermore, when the operon encoding PLC is mutated, the resulting strain was less virulent than the parental strain (Ostroff et al., 1989).
Type 4 pili
The type 4 pilus family. The production of type 4 pili is not restricted to P. aeruginosa. They are found on a number of diverse gram-negative organisms including Neisseria gonorrhoeae, N. meningitidis (Hermodson, et al., 1978), Dichelobacter nodosus (McKern et al., 1983), Moraxella bovis ,M . nonliquefaciens (Marrs et al., 1985), Eikenella corrodens (Rao and Progulske-
Fox, 1993) and the more distantly related type 4 producer Vibrio cholerae
(Ottow, 1975; Paranchych and Frost, 1988; Rao and Progulske-Fox, 1993; Shaw
11 and Taylor, 1990). In addition, the N-terminal amino acid sequences of pilin proteins from two Aeromonas spp. have recently been shown to contain significant homology to type 4 pilins (Hokama and Iwanaga, 1991; Iwanaga and Hokama, 1992). The members of this family share extensive amino acid homology at the N-termini (first 32 amino acids) of their pilin proteins and members of the same species within the type 4 family also share partial homology at the C-termini of their pilin proteins (Dalrymple and Mattick,
1987; Paranchychet a l, 1979; Pugsley, 1993b). These systems also share a common assembly machinery evidenced by the cloning and expression of the type 4 pilin gene from N. gonorrhoeae, D. nodosus, and M. bovis in P. aeruginosa, and a dependence on RpoN for transcription of the pilin gene
(Beard et al., 1990; Dalrymple and Mattick, 1987; Hoyne et al., 1992; Johnson et al., 1986; Mattick et a l, 1987). Comparison of the type 4 pilin proteins in
Pseudomonas, Neisseria, Dichelobacier, and Moraxella indicated that each also has a similar predicted secondary structure and overall structure of the pilin subunit (Dalrymple and Mattick, 1987).
Since most of the research on type 4 pili has centered on P. aeruginosa the remainder of the discussion on will focus primarily on P. aeruginosa pili.
Structure, function, and occurrence. P. aeruginosa type 4 pili are receptors for pilus specific phage (Bradley,1972a; 1972b; Bradley, 1973; Bradley,
1974a, 1974b; Pemberton, 1973; Roncero et a l, 1990 1974a), m ediate twitching
12 motility (Bradley, 1980; Henrichsen, 1972; 1983; Paranchych et a l, 1979), and act as adhesins as discussed above (Doig et at., 1988; Johanson et al., 1980;
Paranchych and Frost, 1988; Ramphalet al, 1984; Sato et al, 1988; Woods et a l, 1980). In addition to the type 4 family members mentioned above whose pili are involved in these three functions, Myxococcus xanthus are also thought to posses type 4 pili which are involved in gliding motility. Recently three genes have been identified whose predicted amino acid sequence have homology to PilR, PilS and PilA (these proteins are discussed below; Wu and
Kaiser, 1995). A mutation in these three genes causes a loss of piliation and a loss of social motility, cellular swarming at high cell densities. The putative
Pil proteins do not appear to be involved in adventurous motility, cellular swarming at both high and low cell densities. Because the M. xanthus Pil- strains are not deficient in adventurous motility, the type 4 pili might not act to directly mediate the movement but might instead participate in the sensory apparatus for motility (Arnold and Shimkets, 1988).
Type 4 pili of P. aeruginosa are polar, flexible rod shaped filaments approximately 6 nm in diameter and range from 1000 to 2500 nm in length
(Bradley, 1972a; Weiss, 1971). Each filament is made up of 500-1000 subunits of a 15 kDa protein (Sastry et al, 1983; Strom and Lory, 1993). X-ray diffraction studies indicated that the subunits form a hollow cylinder with approximately 5 subunits per turn; the external hydrophobic surface is
13 comprised of alpha-helices approximately parallel to the axis arranged around a core of beta-sheets (Dalrymple and Mattick, 1987; Folkhard et al., 1981;
Paranchych and Frost, 1988).
Piliation has been found to vary with the growth phase. The maximal number of pili per cell occur during logarithmic growth phase. The mono or bipolar distribution does not vary as both types of distribution are evident within the same culture (Weiss, 1971). One early study found that in log phase growth, the number of pili per cell was evenly distributed between 1 to
10 among the 64% of the cells with pili. In stationary phase most of the cells with pili had only 1 or 2 pilus filaments (Weiss, 1971).
Pilus retraction is thought to be involved in phage adsorption and twitching motility. The location of the pilin pool needed for pilus extension and retraction was originally determined to be in both the inner and outer membrane fractions, with a slightly larger pool in the inner membrane fraction as determined in both PAK and a hyperpiliated PAK mutant strain
(W atts et al., 1982b). The membrane fractions in the above study were separated via sucrose density gradients. However, when differential solubilization in Sarkosyl was used to separate membrane fractions, pilin was completely soluble (inner membrane fraction) at 0.5 and 2.0% Sarkosyl (Nunn and Lory, 1993; this study). Therefore, the actual location of the pilin pool(s) is currently unclear.
14 Dissociation of pilin fibers in octyl-glucoside to primarily dimers
(Watts et al., 1983) led to speculation that the dimer is the building block of the pilus filament. However, recent data on the structure of N. gonorrhoeae type 4 pilin protein indicates that a dimer building block would not fit the criteria for pilus fibers and a monomer building block is more likely (Parge et al., 1995). The model of a pilus fiber built from dimers did match the appearance of the detergent-resolubilized dimers in the earlier studies (Watts et al., 1982a; 1982b; Watts et al., 1983). The dimers were about twice the diameter of native pili and were unable to bind the pilus-specific phage P04 indicating that the dimers were incapable of normal pilus function.
The P. aeruginosa pilin gene and gene product. The gene encoding the pilus subunit ( pilA) is located on a 1.2kb H indlll chromosomal fragment in P. aeruginosa strains PAO and PAK (Paranchych et al., 1979). These two strains produce serologically distinct type 4 pili (Sastry et al., 1985). Not surprisingly, the amino acid sequences of the two pilin proteins are highly homologous at the N-terminus and semi-conserved at the C-terminus (Sastry et al., 1985).
The middle, variable region is also the immunodominant region, which explains the serological differences (Sastry et al., 1984). The C-termini contain two cysteine residues that have been shown to form a disulfide loop in PAK which is responsible for the adhesin function of pili (Lee et al,, 1989).
15 The N-terminal homology of the type 4 pilin proteins includes an N- methylated phenylalanine as the first residue of the mature protein (giving rise to the alternative name for this family, NMePhe) and a short basic leader sequence that is cleaved by the novel bifunctional enzyme PilD that also N- methylates the protein (Nunn and Lory, 1991). The basic leader region is followed by a stretch of hydrophobic residues. Because the N-terminus is conserved among the type 4 pili, it is thought to be involved in subunit- subunit interaction and/or interaction with the secretion machinery. Early biochemical work on P. aeruginosa pili also suggested that the JV-terminus of the P. aeruginosa pilin protein is involved in subunit-subunit interaction
(Watts et at., 1983). Alkaline pH titration and tryptophan fluorescence quenching with acrylamide indicated that the only two tyrosine residues in the protein (positions 24 and 27 in the mature protein) were exposed at a dimer-dimer interface in native and detergent-resolubilized dimers which were normally buried in the native protein.
The N-terminus of the pilin subunit is also involved in the correct localization of the protein. The 6-7 residue leader itself is not sufficient to direct localization (Strom and Lory, 1987). This is in contrast to the classical signal sequence that is cleaved by signal peptidase I found on most secreted proteins including the Escherichia coli prepilin proteins of Pap and type 1 pili
(Pugsley, 1993b). Alkaline phosphatase fusions with the P. aeruginosa pilin gene required 45 residues of the hydrophobic portion of the mature protein to
16 direct translocation of alkaline phosphatase across the inner membrane in both P. aeruginosa and E. coli (Strom and Lory, 1987). Further, the lack of signal sequence cleavage (or methylation) did not affect the final location of pilin (Strom and Lory, 1991). The amino-terminal region of type 4 pilin proteins resemble the classical leader sequences found on proteins secreted from E. coli (such as Pap and type 1 pilin proteins) and in P. aeruginosa (such as ETA and PLC) and it seems as though the entire region acts as the export signal for the pilin subunit (Strom and Lory, 1987). The role then of the cleaved leader sequence is unknown. Nunn and Lory (1993) have suggested that it coordinates polymerization into a multimeric structure and Paloske et al. (1989) suggest that it is required for recognition of the assembly machinery.
A recent study found that even though there is a high degree of homology in the amino-terminus of the type 4 pilin proteins, many residues could be changed without affecting pilus biogenesis (Strom and Lory, 1991).
The pili assembled from these mutant proteins were shown to be functional receptors for the pilus-specific phage P04 and were indistinguishable from wild type pili as determined by electron microscopy. This was true if the mutation was either a conserved change in the mature, hydrophobic portion of the protein or a change of the charge of the leader from +2 to either to 0, -1 or -2. However, if the leader was deleted or if more than one hydrophobic residue was changed to a polar residue, the cells were nonpiliated and P04 resistant. The glycine residue at -1 is necessary for PilD cleavage and the
17 glutamic acid at +5 is necessary for methylation. If the leader sequence is not cleaved, the proteins are not assembled into pili. Nevertheless, like the wild
type proteins, the proteins localize to both membrane fractions (determined by sucrose density gradients). The purpose of the N-methylation is still not
clear. Substitutions at the phenylalanine residue resulted in one nonmethylated mutant which was still piliated and another mutant which was nonpiliated but partially methylated (50% of the proteins; Strom and
Lory, 1991). When nonmethylated pilins proteins were expressed in a wild type background (PAO), the mutant pilin could be assembled into heteropolymers with the wild type protein but were unable to assemble into homopolymers in a Pil- background (Pasloske et al., 1989).
Accessory p il genes
pilB, pilC, and pilD . Three genes located upstream of and transcribed in opposite orientation to the pilA gene are also required for pilus production
(Nunn et al., 1990). The PilB protein (65 kDa) is believed to reside in the cytoplasm (Table 1). The pilC open reading frame contains 5 potential start sites. T7 expression-labelling experiments indicated that the size of the PilC
18 pil gene Putative and known characteristics of the gene product 3
p il A 15 kDa; pilin subunit pilB 65 kDa; membrane associated, cytoplasmic protein; nucleotide binding motif; involved in pili biogenesis pilC 37 kDa; inner membrane protein; involved in pili biogenesis pilD 27 kDa; inner membrane protein; prepilin leader peptidase; involved in pili biogenesis and protein secretion pilE 15 kDa; inner membrane protein; high homology to PilA at amino-terminus; involved in pili biogenesis pilG 15 kDa; cytoplasmic protein; homologous to CheY; involved in twitching motility pilH 13 kDa; cytoplasmic protein; homologous to CheY; involved in twitching motility p ill 20 kDa; cytoplasmic protein; homologous to CheW; involved in pili biogenesis pil} 73 kDa; periplasmic protein; homologous to MCPs; involved in pili biogenesis pi IK 33 kDa; cytoplasmic protein; homologous to CheR p ilM 38 kDa; cytoplasmic protein; actin-like ATPase pocket; involved in pili biogenesis p ilN 22 kDa; inner membrane protein; involved in pili biogenesis pilO 23 kDa; inner membrane protein; involved in pili biogenesis pilP 19 kDa; outer membrane lipoprotein; involved in pili biogenesis pilQ 77 kDa; outer membrane protein, forms multimers of 10-12; involved in pili biogenesis p ilR 50 kDa; inner membrane protein; response regulator, activates pilA transcription; involved in pili biogenesis pilS 59 kDa; inner membrane protein, sensor that relays signal to PilR; involved in pili biogenesis
Table 1. Summary of the known pil genes. 19 Table 1 (continued)
pil gene Putative and known characteristics of the gene product
38 kDa; cytoplasmic; nucleotide binding motif; involved in p ilT twitching motility 42.5 kDa; cytoplasmic; nucleotide binding motif; involved in pilU twitching motility pilZ 13 kDa; cytoplasmic protein; involved in pili biogenesis a Involvement in pili biogenesis or twitching motility as determined by phenotype of mutant strain. For references, see text.
20 protein was 37 kDa which agrees with the size of the smaller possible open reading frames (Table 1). PilC is predicted to reside in the inner membrane due to the prediction of several membrane-spanning helices (Nunn et al.,
1990). PilB and PilC mutants were deficient in assembly of extracellular pilus filaments but they did make wild type levels of the PilA protein which seemed to localize correctly (Nunn et a l, 1990). PilB contains a consensus sequence found in ATP-binding domains (Walker box A) and changing a single nucleotide of this sequence resulted in loss of piliation (Turner et at.,
1993). The presence of a nucleotide-binding domain indicates that PilB may act to supply energy for membrane translocation during pilus biogenesis or it may participate as a protein kinase (Turner et al., 1993).
While the role of PilB and PilC seem to be restricted to pilus biogenesis, the pilD gene product is also involved in extracellular secretion (Table 1;
Strom et al., 1991). Initially, PilD mutants were found to make wild type levels of PilA, but the PilA protein was about 1 kDa larger than in wild type or
PilB and PilC m utant cells (Nunn et al., 1991). This led to speculation that
PilD was the prepilin peptidase which cleaved the 6-7 amino acid leader sequence found on type 4 pilin proteins (Nunn and Lory, 1991). PilD mutants also accumulated extracellular enzymes (ETA, PLC, alkaline phosphatase, and elastase) in the periplasmic space rather than secreting them into the extracellular milieu (Nunn and Lory, 1991). This second phenotype is explained by the fact that PilD acts on components of the terminal branch of
21 the general secretion pathway (GSP) in P. aeruginosa (XcpT,U,V,W; discussed below) by cleaving their prepilin-like leader sequence and methylating the N- terminus of the mature protein (Bally et al., 1992; Nunn and Lory, 1992; 1993).
PilD is thus a bifunctional enzyme. It cleaves and N-methylates its target proteins (Strom et al., 1993b). PilD targets contain a short leader sequence with the consensus sequence -Gly-Phe-Thr-Leu/Ile-Glu. Cleavage occurs between the glycine (-1) and phenylalanine (+1) residues (Strom et al.,
1993a). The N-methyltransferase activity of PilD requires a glutamic acid at position +5 in the target protein (Strom and Lory, 1991). PilD, which spans the inner membrane, is believed to have two distinct active sites which are on the cytoplasmic side of the inner membrane; its activity may be Sec independent (Kaufman et al., 1991; Strom et al., 1993b).
pilM, pilN, pilO, pilP, and pilQ . ThepilM through pilP genes comprise an operon that likely also contains pilQ (Martin et al., 1995). A mutation in any of these 5 pil accessory genes results in nonpiliated stains resistant to phage P04 and deficient in twitching motility (Table 1; Martin et al., 1993,
1995). Whole cell extracts of these mutants contained wild type levels of PilA which were the same size as wild type PilA processed by PilD (Martin et al.,
1995). Due to the presence of a potential membrane spanning region in the predicted amino acid sequence, PilN and PilO are believed to be anchored in the inner membrane; PilP is predicted to be a lipoprotein and may localize to
22 the outer membrane (Martin et al., 1995). PilM is likely to be localized in the cytosol and PilQ, which has a classical signal sequence, is believed to be an outer membrane protein due to both the hydropathy profile and homology to several proteins that have been localize to the outer membrane (Martin et al.,
1993; 1995). Mutants of the last two proteins resulted in a dominant-negative phenotype when the genes were mobilized into a wild type background indicating that the proteins are likely to be involved in a multimeric structure (Martin et a l, 1995). In further support of multimerization of PilQ, the PilQ homologs pIV, a filamentous phage protein, and N. gonorrhoeae
PilQ have been shown to form multimers of 10-12 monomers (Kazmierczak et al. 1994; Newhall et al., 1980).
Until recently, most researchers in this field believed that phage sensitivity and twitching motility were both tied directly to the presence of external functional pili. However, Martin et al., (1995) found that phage sensitivity was restored to mutants without restoring twitching motility. In addition, Darzins (1993; 1994) found that PilG, Pill, and PilJ mutants
(discussed below) were differentially sensitive to different pilus specific phage.
In addition, a PilV mutant (discussed below) was complemented to P04 sensitivity by low level expression of the wild type gene but was still twitching deficient (Aim and Mattick, 1995). A hyperpiliated PilU mutant
(discussed below) was differentially susceptible to different phage but also unable to twitch (Whitchurch and Mattick, 1994). One explanation for all of
23 these results is that the mutants are not expressing wild type levels of PilA and thus not forming functional pili needed for twitching motility, but a minimal amount of pili, or a preliminary pilus structure is expressed and allows at least some phage to recognize their pilus specific receptor (Aim and
Mattick, 1995; Darzins, 1993; Martin et al. 1995). An alternative theory suggested by Whitchurch and Mattick (1994) is that phage sensitivity is dependent on some factor(s) involved in both systems but that phage sensitivity is not directly dependent on pili assembly and a loss of one results in the loss of the other if a mutation occurs early in the pathway. However, if there are two pathways that diverge at some point then mutants that are phage resistant but able to twitch would also be expected. Presently the first theory is more probable of the two because no mutants capable of twitching motility but still resistant to "pilus-specific" phage have been found which would be expected if the two pathways diverged and were independent at some point.
p ilV and pilE. Two recently identified genes ,pilE and pilV, encode proteins involved in pili biogenesis and also have a prepilin-like leader sequence cleaved by PilD (Table 1; Aim and Mattick, 1995; Darzins and
Russell, 1996; Russell and Darzins, 1994). A PilV mutant had wild type levels of PilA in the membrane fraction but did not express extracellular pili. It was also capable of secreting alkaline phosphatase and elastase at wild type levels,
24 indicative of its exclusive role in pilus biogenesis (Aim and Mattick, 1995).
When thepilV gene was introduced into a mutant background and expressed at low levels, the mutant became sensitive to P04 but was still unable to exhibit twitching motility (Aim and Mattick, 1995). Electron microscopy and immunoblot analysis indicated that there were no pili associated with these cells. ThepilE gene and gene product are the subject of this study. The identification and characterization of the gene is presented in Chapter 3 and characterization of the protein is presented in Chapter 4. Discussion of possible roles for PilE in pilus biogenesis in P. aeruginosa is presented at the end of both chapters.
p ilR and pilS. The PilR and PilS proteins are members of the two component sensor/regulator system involved in pilA transcription (Table 1).
The necessity of a transcriptional activator for pilA is not surprising since all genes identified to date that are transcribed by RpoN are also dependent on transcriptional activators. PilR is the cytoplasmic response regulator that activates transcription of pilA by binding upstream of the promoter. PilS is the inner membrane sensor that phosphorylates and dephosphorylates PilR
(Boyd et al., 1994; Boyd and Lory, 1996; Hobbs et al., 1993; Ishimoto and Lory,
1992; Jin et al., 1994). The signal that activates PilS is currently unknown
(Boyd and Lory, 1996).
25 Even though the production of all of the type 4 pilin proteins is dependent on the alternative sigma factor RpoN, none of the others have
PilR binding sites upstream of the pilin gene, suggesting that each system has its own unique signal(s) activating pilin transcription (Jin et a l, 1994).
pilG, pilH, pill, pil] and pilK. The pilG-K genes encode gene products that are homologous to proteins involved in chemotaxis (Table 1; Darzins
1993; 1994; 1995). PilG and PilH are homologous to CheY, a single domain response regulator protein involved in E. coli flagellar rotation (Darzins, 1993;
1994). A PilG mutant was resistant to pilus specific phage D3112 but susceptible to F116L and B3 and it did not produce extracellular pili or exhibit twitching motility (Darzins, 1993). In comparison, a PilH mutant was piliated, phage sensitive, and able to exhibit twitching motility (Darzins, 1994).
However, closer inspection of a PilH mutant revealed that it was altered in its twitching motility pattern (Darzins, 1994). Pill is homologous to CheW and
PilJ is homologous to a methyl-accepting chemotaxis protein (MCPs; Darzins,
1994). The phenotype of Pill and PilJ mutants were the same as the PilG mutant (Darzins, 1994). The PilK protein is homologous to the chemotactic methyl transferase CheR. A mutation in the pilK gene, however, did not effect any observable phenotype (Darzins, 1995).
The proteins encoded by pilG-K are thought to be a part of a signal transduction network involved in regulating pili biogenesis and twitching
26 motility. PilJ probably acts as the sensor which stimulates a complex, proposed to be made up of PilJ, Pill and a newly identified CheA homolog,
PilL (Darzins and Russell, 1996). The CheA homolog is then believed to transfer a phosphate group to PilG and/or PilH. The role of PilK may be to work with an as yet unidentified CheB homolog to modulate the level of PilJ methylation. As in the PilR-S system, the environmental cues being sensed by the bacterium are still unknown.
p ilT and pilU . Pilus-deficient mutants have been identified that are phage resistant and do not exhibit twitching motility but that do not lack external pili, they are instead hyperpiliated but have nonretractile pili
(Bradley, 1974a; 1980; Bradley and Pitt, 1974). Two genes, pilT and pilU , have been identified that, when mutated, resulted in a hyperpiliated phenotype
(Table 1; Whitchurchet al., 1990; W hitchurch and Mattick, 1994). N orthern analysis showed that the pilA mRNA level in these hyperpiliated mutants was not elevated and thus was not the cause of hyperpiliation (Whitchurch and Mattick, 1994). The putative amino acid sequence of PilT indicates that it is a nucleotide bind protein (containing a Walker box A consensus sequence), as is PilB (Whitchurchet al., 1990). PilU, homologous to PilT, also contains a
Walker box A sequence (Whitchurch and Mattick, 1994). A PilT mutant was phage P04 resistant and unable to twitch while a PilU mutant was differentially susceptible to pilus specific phage (sensitive to P04) and unable
27 to twitch, the implications of which are discussed above (Whitchurch and
Mattick, 1994). The relationship between PilT and PilB is unclear, perhaps
they have an antagonistic relationship where PilB is involved in the incorporation of pilin subunits and PilT is involved in the disassembly of the subunits thought to be necessary for retraction during twitching and phage adsorption (Whitchurch et al., 1990).
p ilZ . ThepilZ gene product has a predicted weight of 12.9 kDa and is
thought to reside in the cytosol (Table 1; Aim et a l, 1996). A PilZ m utant was deficient in extracellular pili, unable to twitch and phage resistant (Aim et a l,
1996). The pools of PilA were unaffected by a pilZ mutation (Aim et a l, 1996).
No proteins homologous to PilZ have been identified in the protein database and its function is currently unknown (Aim et a l, 1996).
Pil homologs in other bacterial systems
It is not surprising that homologs of pil genes have been found in other type 4 family members; proteins homologous to PilB, PilC, PilD, PilT and PilQ have been identified in N. gonorrhoeae, PilF, PilG, PilD, PilT and
PilQ respectively (Drake an Koomey, 1995; Freitag et a l, 1995; Lauer et a l,
1993; Tonjum et a l, 1995) and PilB, PilC and PilD homologs have been identified in D. nodosus, FimN, FimO, and FimP respectively (Johnston et a l,
28 1995). Even the m ore distantly related V. cholerae system in which the cleaved leader sequence is 25 amino acids long and the first residue in the mature protein is methionine has a prepilin peptidase analogous to PilD and a methyl-accepting chemotaxis protein homolog (Harkey et al ., 1994;
Kaufman et al.r 1991; Strom and Lory, 1993). What was unexpected however, was that other bacterial systems such as protein export and DNA uptake systems have Pil-like proteins. The emerging picture indicates that these systems are involved in macromolecular transport either into (DNA uptake) or out of (protein secretion and pilus biogenesis) the cell. These other bacterial systems have homologs to PilB ( a nucleotide-binding protein), PilC
(an inner membrane protein), PilD (prepilin peptidase), and PilQ (outer membrane protein) along with at least 3 proteins that have a prepilin-like signal sequence acted upon by the PilD homolog. The current theory is that the PilB-like and PilC-like proteins aid in assembly of the proteins with the prepilin-like leader sequence and that the PilQ-like protein acts as a channel or pore in the outer membrane. The multimeric structure, often referred to a pseudopilus or pilus-like structure, is believed to be the conduit for the macromolecular transport. These systems are introduced below and discussed more in depth in Chapter 4.
Protein secretion in P. aeruginosa. P. aeruginosa contains two systems that fall into the first category, the Pil system for pilus biogenesis and the Xcp
29 system for the second step of protein secretion also called the main terminal branch of the GSP. Several exoproducts made by P. aeruginosa have a classical signal sequence (ETA, PLC, elastase, and alkaline phosphatase); that is they cross the inner membrane in a signal sequence dependent manner
(Akrim et al., 1993). If there is a block in the second step of secretion, these proteins are found in the periplasmic space folded into a fully active form
(Strom and Lory, 1991). Currently eleven xcp genes have been sequenced which include XcpA (later determined to be PilD), Xcp Q (homologous to
PilQ), XcpP and XcpR, S, T, U, V, W, X, Y, and Z (Akrim et a l, 1993; Bally et a l,
1991; 1992; Filloux et al., 1990; N unn and Lory, 1992).
Pullulanase secretion in Klebsiella oxytoca. This system is the best characterized of those discussed here and is currently the best model system for the generalized bacterial protein secretion pathway. The pullulanase secretion pathway has been completely reconstituted in E. coli (d'Enfert et a l,
1989). It is responsible for the transport of the lipoprotein pullulanase to the outer surface of the outer membrane of K. oxytoca. The release of the lipoprotein is believed to be a spontaneous event (Pugsley, 1993b). All but one of the genes required for pullulanase secretion is in an operon upstream of the pulA structural gene, pulD, E, F, G, H, I, /, K, L, M , N, and O; pilS is located downstream of the structural gene (Pugsley, 1993b). There is much more overlap of the main terminal branch of the GSP in P. aeruginosa and
30 the K. oxytoca pullulanase secretion pathway than just the Pil-like proteins.
For example, each of the first eleven pul genes ( pulC -M) has a counterpart in the xcp system (Akrim et al., 1993).
Other protein secretion systems. Erwinia carotovora and E. chrysanthemi are plant pathogens that secretes pectinases and cellulases via a
two step process (Pugsley, 1993b). Thirteen out genes have been identified
(OutD; Hobbs and Mattick, 1993; Lindeberg and Collmer, 1992). While four gene products have been shown to contain the type 4 peptidase consensus cleavage site, their leader sequences are longer than the 6-8 amino acids usually seen (OutG-22, OutH-lO, OutI- 6, and OutJ-25; Reeves et al., 1994).
Xanthonionas campestris is another plant pathogen that secretes extracellular degradative enzymes and also uses a GSP containing Pil-like proteins (Hu et a l, 1992; 1995).
The system in Aeromonas hydrophila with Pil-like proteins is not only involved in protein secretion but is also involved in outer membrane assembly (Howard et a l, 1993). ThisexeC-hJ cluster has also been identified in
A. salmonicida , a fish pathogen (Karlyshev and MacIntyre, 1995).
31 Bacillus subtilis competence. B. subtilis , a gram positive organism, is able to bind and take up extracellular DNA. This competence system also contains
Pil-like proteins, except for a homolog to the Pil protein found in the outer membrane (Albano et al., 1989; Chung and Dubnau, 1995).
Goals of this study
Strains acquired by this lab were routinely screened for phage (pilus specific) sensitivity. One such strain, MJ-6 kindly supplied by J. Rowe, was found to be broadly resistant to D3112, F116L and B3. MJ -6 was initially described as a P. aeruginosa mutant unable to assimilate or dissimilate nitrate
(Goldfam and Rowe, 1983). This strain also grew as domed colonies on agar media, an indication that the cells are unable to twitch. The phage resistance and colony morphology were preliminary indications that the strain was deficient in functional pili. We wanted to:
1) confirm that MJ-6 was deficient in functional pili,
2) determine the gene responsible for the pil- phenotypes,
3) characterize the gene product,
4) determine the function of the gene product; and,
5) try to determine the role it plays in pilus biogenesis.
32 CHAPTER 2
MATERIALS AND METHODS
Bacterial strains, phages and plasmids. The bacterial strains, phages and plasmids used in this study are described in Table 2. The strains and plasmids constructed in this study can be found in Table 3.
M edia. P. aeruginosa and E. coli were routinely propagated in LB medium {Difco Laboratories, Detroit, MI) which was 1% tryptone, 0.5% yeast extract, and 0.5% NaCl. Minimal medium for the selection of P. aeruginosa recombinants was described by Brammer and Clarke (1964), except that the trace element solution was omitted. When necessary, amino acids were added to a final concentration of 1 mM and glucose was added to a final concentration of 50 mM. In conjugation studies, naladixic acid (300 pg ml-i) was used to counterselect against the donor. For most solid media, select agar
(Gibco BRL, Gaithersburg, MD) was added at a concentration of 1.5%. LB top agar for titering phage lysates contained agar at a concentration of 0.7% and 1 mM MgSC> 4. The antibiotic concentrations for E. coli were as follows:
33 Strain, phage or Relevant characteristics 21 Source or reference plasmid
E. coli DH5a recA endAl gyr A96 thi-1 hsdR\7 sup44, relAl lacZAMIS N. Pagratis BL21 (DE3) F- ompT (re-mg- )lambda D69 lysogen carrying Studier and Moffatt, 1986 phage T7 gene 1 under control of P kcUV5
P. aeruginosa ADD1976 PAOl::miniD180 (Tcr) Brunschwig and Darzins, 1992 CD10 PA04141:: D3112cts Darzins and Casadaban, 1989a DAI pilR mutant of PAOl This study DA11 pilD mutant of PAOl This study HOD5 pilB mutant of PAOl This study MJ-6 nar D3112' F1161 B3 1 Goldflam and Rowe, 1983 PA103 Fla- Pavlovskis et aL, 1977 PAOl prototroph FP- Holloway, 1955 PA0222 ilv-226 his-4 lys-12 proA82 met-28 trp-6 FP- Haas and Holloway, 1976 PA0381 leu -10 strA FP2+ R. V. Miller PAOl 042 pur-67 cys-59 proB65 thr-9001 FP- Darzins and Casadaban, 1989b
Table 2. Strains, phage and plasmids used in this study Table 2 (continued)
Strain, phage or Relevant characteristics Source or reference plasmid
Phages D3112cts cts (temperature-sensitive repressor) Krylov et al., 1980 B3 cts-3 cts (temperature-sensitive repressor) A. Darzins F116Lcts53 cts (temperature-sensitive repressor) R.V. Miller; Krishnapillai, 1971 G101 generalized transducing phage Holloway and van de Putte, 1968
Plasmids pBR322 reppMBb Apr, Ter (4.361 kb) Sutcliffe, 1979 pCP13 IncP, Tcr, Kmr (23 kb) cos Darzins and Chakrabarty, 1984 pEB12 T7 olO promoter, repKsnoio, Apr (10.2 kb) Brunschwig and Darzins, 1992 pGEX2T GST fusion vector, tac promoter, lac Iq, Apr (4.9 kb) Pharmacia Biotech pK19 ColEl ori, general cloning vector, Kmr Pridmore, 1987 pMOB3 pHSS21, sacB sacR oriT, Kmr, Cmr Schweizer, 1992 pNOT19 pUC19 with 10 bp Ndel-Notl adaptor in Schweizer, 1992 NdeI site, Apr pRK2013 reppAiBi,Tra+ (RK2) Kmr Figurski and Helinski, 1979 pR01614 broad host range vector, Apr (Cbr), Tcr ( 6.2 kb) Olsen et a l, 1982 pTJS140 repRK2 > oriT, reppMBi, lac ' IPOZ Cbr (8.0 kb) Darzins and Casadaban, 1989a pT7-5 T7 0IO promoter, Apr (2.777 kb) United States Biochem. Corp. Table 2 (continued)
Strain, phage or Relevant characteristics Source or reference plasmid
Plasmids (continued) pUC18 lac' IP O Z , repPMBb Apr (2.7 kb) Vieira and Messing, 1982 FP2 Hgr, Tra+, Cma+ Holloway et al., 1979 a Apr, ampicillin resistance; Cbr, carbenicillin resistance; Cmr, chloramphenicol resistance; Cma+, chromosome mobilization ability; Hgr, mercury resistance; Kmr, kanamycin resistance; Nalr, naladixic acid resistance; Tcr, tetracycline resistance; Tra+, self transmissible; oriT, RK2 origin of transfer; reppMBi, replicon from pMBl; repm , replicon from RK2; repRSF 10io, replicon from RSF1010; Smr, streptomycin resistance. P. aeruginosa gene designations are as described by Royle et al. (1981). Strain or plasmid designation Relevant characteristics
P. aeruginosa R1 pilE chromosomal insertional mutant of PAOl, Ter in N o tl site PAOl-A- pilA chromosomal insertional mutant of PAOl, Tcr in Kpn site Plasmids pADD1544 pCP13 + 30 kb H in d lll fragment, complements MJ -6 pADD1795 pTJS140 + 5.5 kb BamHl fragment, complements MJ -6 pMR2385 pR01614 + 3.5 kb EcoRV-EcoRI fragment, complements MJ -6 pMR2422 pRO!614 + 3.0 kb EcoRV-Scal fragment, complements MJ -6 pMR2714 pR01614 + 1 kb EcoRV-Clal fragment, complements MJ -6 pMR2715 pUC18 + 1 kb EcoRV-Clal fragment, used for sequencing pMR2716 same construct as pMR2715 with fragment in opposite orientation, used for sequencing pMR2717 pT7-5 + 1 kb EcoRV-C/fll fragment, used for protein overexpression in E. coli
Table 3. Strains and plasmids generated in this study.
37 Table 3. (continued)
Strain or plasmid designation Relevant characteristics
pMR2718 same construct as pMR2717 with fragment in opposite orientation pMR2762 pR01614 + 0.7 kb Smal-Clal fragment, does not complement MJ-6 pMR2822 pMR2717 + Tcr gene (EcoRI-Ai?aI) from pBR322 cloned into the Notl site within pilE, Tcr is in opposite orientation to pilE pMR2823 pEB12 + 1 kb EcoRV-CM fragment, pilE in negative orientation with respect to T7 promoter, used for protein overexpression in P. aeruginosa pMR2824 same construct as pMR2823 with fragment in opposite orientation pMR2967 pNOT19 + 2.4 kb from pMR2822 containing pi7E::Tc pMR2968 pMR2967 + MOB cassette from pMOB3, containing sacB oriT Cmr pMR3764 pGEX2T + Smal-EcoRV fragment from pMR2715, contains final 2/3 of pilE, used for overproduction of GSTPilE' for Ab production pMR3819 pK19 + 5.5 kb BamHl fragment from MJ-6 containing mutated form of pilE pMR3820 pRO!614 + 5.5 kb BamHI fragment from MJ -6 containing mutated form of pilE
38 Table 3. (continued)
Strain or plasmid designation Relevant characteristics
pMR3839 pUC18 + 1 kb EcoRV-C/fll fragment from pMR3820, used for sequencing the MJ-6 copy of pilE pMR3890 pUC18 + Kpnl-Xbal PCR fragment, codes for mature PilE protein pMR3940 pNOT19 + 1.2 kb Hwdlll fragment containing pilA pMR3961 pMR3940 + Tcr gene from pBR322 (EcoRI-AwjI) cloned into Kpnl site within pilA gene pMR3962 pMR3961 + MOB cassette from pMOB3 pMR3964 pMR2715 with the X m nl-N otl fragment within pilE replaced with Phe2427 pMR4034 pRO!614 + Eco RI-Baw HI from pMR3964 a Ab, antibody; Cim, chloramphenicol resistance gene; oriT, RP4 origin of transfer; sacB, sucrose resistance gene from B. subtilis; Tc^, tetracycline resistance gene.
39 ampicillin (Ap), 100 pg ml-1; chloramphenicol (Cm), 30 pg ml-1; kanamycin
(Km), 30 pg ml-1; tetracycline (Tc), 25 pg ml-L For selection of P. aeruginosa drug-resistant transconjugants, Pseudomonas Isolation Agar (PIA; Difco
Laboratories, Detroit, MI) was supplemented with carbenicillin (Cb; 300 pg ml-1) and tetracycline (300 pg ml*1).
DNA methods. Restriction enzymes and T4 DNA ligase, were purchased from International Biotechnologies Inc., (IBI, New Haven, CT) and used as recommended by the supplier. The isolation of chromosomal or plasmid DNA from P. aeruginosa and E. coli was carried out as previously described (Darzins, 1993). Procedures for Southern hybridization analysis were performed as previously described (Darzins and Chakrabarty, 1984). Colony hybridizations were performed according to the Schleicher & Schuell (Keene,
NH) protocol. DNA fragments were purified from agarose gels after electrophoretic separation by the method of Vogelstein and Gillespie (1979).
Genetic procedures. Procedures for the transformation of E. coli and P. aeruginosa, the introduction of recombinant plasmids into P. aeruginosa by triparental matings and G101 transductions in P. aeruginosa have been previously described (Darzins, 1993).
40 Phage sensitivity testing and motility assays. The preparation of phage lysates was as previously described (Darzins and Casadaban, 1989a; Miller and
Ku, 1978). An overnight culture of PAOl cells was diluted, and 3 x 10 7 cells were mixed with 3 x 1010 D3112 cts phage. Following addition of soft agar, the mixture was poured onto LB agar plates and incubated for 24 h at 42°C.
Surviving colonies were streaked onto individual LB agar plates. Phage sensitivity was measure by two methods. For screening purposes, strains were streaked onto LB agar media and spotted with 10 jxl of phage lysate (10 9 to 10*0 p fu ml-*). Zones of clearing were indicative of phage sensitivity.
Phage sensitivity was also measured by the plaque assay. Ten ( 4.1 spots of diluted phage lysates were spotted onto LB top agar seeded with 0.2 ml of a culture of indicator bacteria grown to stationary phase in LB broth.
In order to assay twitching motility, LB agar (1%) plates were poured to an average depth of 3 mm and dried briefly. The strains to be tested were stab inoculated with a needle to the bottom of the polystyrene dish and the plates incubated at 37°C for 24 h. The zone between the agar and polystyrene was m easured (McMichael, 1992).
DNA sequence analysis. A series of deletion plasmids used for sequence analysis were constructed as described previously by Henikoff (1984). pMR2715 was the basis for nested deletions and sequencing the wild type pilE gene from PAOl and pMR3839 for the mutated copy of pilE in MJ- 6. Double
41 stranded DNA sequencing was accomplished by the dideoxy chain
termination method (Sanger et ai, 1977). Sequenase 2.0 (United States
Biochemical, Cleveland, OH) was employed for chain elongation and 7-
Deaza-dGTP was used in the place of dGTP to reduce the number of sequencing artifacts. 35S-dCTP labeled samples were run in 8 M urea-6% polyacrylamide gels. Sequence analysis was performed with the IBI
MacVector software program. Nucleotide and derived amino acid sequences were also analyzed with the Wisconsin Genetics Computer Group (GCG) software (Devereux et ai, 1984). The TFASTA algorithm for protein homology was used to compare the deduced protein products of pilE and orfB' to sequences in the GenBank data base (Release 73.0). PUBLISH was used to generate the DNA sequence in Fig. 6. GAP and PUBLISH was used to generate the comparison between PilE and PilA (Fig. 7) and between ORFB' and ORF316 (Fig. 20).
Construction of pilE and pilA gene replacement mutants. Gene replacement techniques were carried out essentially as described by Schweizer
(1992). Briefly, thepilE gene within the 1 kb EcoRV-Cifll fragment cloned in pUC18 was disrupted by inserting a filled-in EcoRI-Aual Tcr cassette from pBR322 into the unique, filled-in Notl site (Fig. 1, step 2). The entire 1 kb
EcoRV-C/al fragment including the Tcr insert was removed from plasmid pMR2822 with digestion by EcoRI and H/ttdlll and cloned between the EcoRI
42 Fig. 1 Summary of method used to generate chromosomal gene replacement mutants. The dark grey box represents pilE. The lighter grey box represents the Tcr gene from pBR322. The arrows in step 1 (pilE) and 2 (Tcr) denote direction of transcription. The striped boxes in step 3 represent the pNOT19 vector including the Apr gene (large box with large stripes) and the MOB cassette (large box with small stripes). See text for description of the steps.
43 lkb pMR2715
2. pMR2822
3.
£ pMR2968
4. PAOl chromosome
5. R1 chromosome
figure 1 and Hmdlll sites of plasmid pNOT19 (Schweizer, 1992). This plasmid, designated pMR2967, was digested with Notl and ligated with the sacB oriT
CmT cassette from plasmid pMOB3 (Fig. 1, step 3; Schweizer, 1992). The resulting construct, known as pMR2968, was mobilized into P. aeruginosa strain PAOl by filter matings and Tcr transconjugants were isolated by plating the mating mixture onto PIA containing Tc (Fig. 1, step 4). Approximately
70% of the Tcr colonies that arose were flat, spready with ragged edges which is indicative of a p ib phenotype, while the remaining 30% grew as compact, domed colonies with well-defined edges which is indicative of a pib phenotype. One representative spready (R7) and compact (Rl) Tcr transconjugant was streaked onto a LB agar plate containing Tc (50 pg mH) and sucrose (5% [w/v]). The spready Tcr strain R7, which was found to be sucrose-sensitive, yielded many sucrose-resistant, compact colonies. On the other hand, the compact Tcr strain Rl was sucrose-resistant and grew to confluence on the selection plate. The two independently isolated Tcr Suer colonies, designated Rl and R8, were chosen for further analysis.
The construction of a pilA gene replacement mutation was carried out in much the same way except that the Tcr gene from pBR322 was inserted into the unique Kpnl site within the PAOlpilA gene (pMR3961) and the orientation is unknown. Once the MOB cassette was introduced (pMR3962), this plasmid was mobilized into PAOl as above. In this case, approximately
45 6% of the colonies were compact when plated on PIA containing Tc. One of the compact colonies was chosen, verified using Southern analysis and designated the PAO-A- strain.
Expression studies with the T7 RNA polymerase-promoter system.
The T7 expression system was used as described by Tabor and Richardson
(1985). BL21(DE3) cells containing the plasmids to be analyzed were induced with IPTG (ImM, final concentration), treated with rifampicin (50 mg ml-1 in
N, N'-dimethylformamide) at a final concentration of 200 pg mb1, and the polypeptides were labeled with 35S-labeled methionine and cysteine (ICN
Biomedicals, Inc. 43 MBq mmol-1, 1.2 mCi ml-1). Following separation by SDS-
PAGE (12.5% acrylamide [w/v]) the gel was prepared for fluorography as described previously (Chamberlain, 1979). Expression studies with the T7 promoter constructs in P. aeruginosa were carried out as described by
Brunschwig and Darzins (1992).
Pilus purification. Pili, isolated from cells that were grown on the surface of a LB agar plate, were purified as previously described by Roncero et a i (1990). Briefly, an overnight culture was diluted (1/100) and 10 plates were inoculated with 100 pi each. The plates were incubated for 7 h at 37°C. The cells were then gently scraped from the plates in 2 ml of sterile water and pooled. The final volume from the 10 plates was adjusted so that the optical density at 600 nm was approximately 5.0. The suspension was vortexed three times 30 s each to release more than 95% of the pili, and cells were removed by centrifugation twice at 2 000 x g . The suspension was supplemented with
10X phosphate-buffered saline to a final concentration of IX and centrifuged at 12 000 x g for 20 min, and the supernatant was equilibrated to pH 4.5 with
0.1 M sodium acetate (pH 3.9). After overnight incubation at 4°C, the pili precipitated by isoelectric equilibration were recovered by centrifugation at
12 000 x g for 20 min and the pellet obtained from 20 ml of a suspension with an optical density at 600 nm of 5.0 was suspended in 1 ml of IX phosphate- buffered saline.
Nucleotide sequence accession number. The nucleotide sequence of the pilE gene region (Fig. 6) has been deposited in the GenBank data library under accession number U02552.
Construction and purification of GST fusion proteins. Two glutathione-S-transferase (GST) fusions were generated, GSTPilE' and
GSTmPilE. In each case, thepilE portion of the chimera was inserted into the pGEX2T vector (Pharmacia Biotech, Sweden) so as to create an in frame fusion. The GSTPilE' fusion was created by blunting and inserting the final
47 two-thirds of the pilE gene (0.7 kb Smal-Clal from pMR2715) into the blunted
BamHl site of pGEX2T producing pMR3764. Restriction analysis was used to ensure correct orientation of the insert.
A DNA fragment used to construct the GSTmPilE fusion was generated using PCR so that the PilE portion of the protein would correspond to the mature PilE protein. The primers were constructed so that a Kpnl and BamHl site would be introduced just 5' of the +1 phenylalanine codon (5'-
ACAGGTACCGGATCCTTCACGTTGCTGGA-3'; Integrated DNA
Technologies, INC., Coralville, IA) and HimcII and Xbal sites would be just 3' of the stop codon (5'-AGGTCTAGAGTCGACTCAGCGCCAGCAG-3'). The
PCR product was first cloned into pUC18 using the Kpnl and Xbal sites
(pMR3890) and sequenced to verify the 5' end and to ensure no mutations were introduced during PCR. Using the BamHl and HincII sites to clone into pGEX2T (pMR3839), an in frame fusion was constructed.
Overproduction and purification conditions were the same for both proteins. An overnight culture of the appropriate strain, grown in LB + Ap, was reinoculated into a large volume of LB + Ap (a 1/500 dilution into either
250 ml to 1 L). The culture was grown until an ODsoo of approximately 0.5 was reached. Isopropyl P-D-thiogalactoside (IPTG; BioWorld, Dublin, OH) was then added to a final concentration of ImM and the incubation continued for
4 h. The cells were then harvested, washed in PBS and pelleted. The pellets were kept at -20° until use.
48 To purify the fusion protein, the cell pellets were thawed on ice and 5 ml of Tris-buffered saline (TBS; lOOmM Tris[hydroxymethyl]aminomethane,
0.5M N aC l) was added along with lysozyme (Sigma Chemical Co., St. Louis,
MO) to a final concentration of 1 pg ml-L Following incubation on ice for 20 min, 5 ml of RIPA buffer (150 mM NaCl; 10 mM Na 4P207; 10 mM NaF; 0.5%
Deoxycholate; 10 mM EDTA; 50 mM Tris, pH 8; 0.1% SDS; 10% glycerol; 1%
NP-40 ) containing protease inhibitors (aprotinin and leupeptin to 20 pg ml-i and phenylmethylsulfonyl fluoride [PMSF] to ImM; Sigma Chemical Co., St.
Louis, MO) was added and the cells were sonicated 3 times at 50% output
(Sonifier Cell Disrupter; Branson Sonic Power, Melville, NY). The samples were spun at 10 000 rpm for 10 min to clear the cell debris. The supernatant was removed to a disposable 50 ml tube and 2 ml of glutathione-sepharose
(Pharmacia Biotech, Sweden) was added. Following an overnight incubation, the sepharose was wash twice with RIPA, 3 times with TBS + 1% NP-40 and twice with TBS alone. The sepharose was then transferred to a column and the eluate was monitored at A 280 * The column was washed with TBS and the fusion protein was eluted using 5 ml of 20 mM glutathione (pH8; Sigma
Chemical Co., St. Louis, MO), dialyzed against TBS (sterile PBS for GSTPilE' for use in raising antisera), and concentrated using a Centriprep-10 microseparation device (Amicon, Beverly, MA). Protein concentration was determined using the Bradford protein assay from Bio-Rad (Hercules, CA).
The diluent used was 20mM Tris (pH 7.0). PCR. Conditions for PCR reactions were as according to manufactures recommendations (Gibco BRL, Gaithersburg, MD) including 0.5 pg template
DNA and 1 pi of a 50 pM solution of each primer. Because a PTC-100
Programmable Thermal Controller (MJ Research, Watertown, MA) thermal cycler with a heated bonnet was used, no mineral oil overlay was needed.
The tem perature cycle was as follows: 95°, 2 min; 45°, 2 min; and 72°, 1 min for 30 cycles followed by one cycle of 95°, 2 min; 45°, 2 min; and 72°, 10 min.
The PCR reaction was then held at 4° overnight. The PCR product was precipitated using 95% ethanol, resuspended in Tris-EDTA (TE) and stored at
- 20°.
In order to generate the coding sequence for mPilE of the GSTmPilE fusion, the primers were as indicated above. Overlapping PCR site-directed mutagenesis involved 4 primers; two external primers (FI [5'-CCCGATGAG
GACAAGACAGAAG-3'] and B1 [5'-TACAGGTTGTTCGGCGAGTTGG-3'];
Ransom Hill Bioscience, Inc., Ramona, CA) that flanked the entire region involved in the mutagenesis and two that hybridized to the same sequence on opposite strands containing the Tyr 24 and 27 to Phe 24 and 27 changes
(BPhe2 [5'-GGATCACGAAGTTCTGGAAACTGGGA-3'l and FPhe2 [5'-
TCCCAGTTTCCAGAACTTCGTGATCC-3']). Two PCR reactions were carried out simultaneously, one with FI and BPhe2 and another with B1 and FPhe2.
These products contained the mutation at either the extreme 3' or 5' end. These two products were visualized by agarose-gel electrophoresis and purified using Gene Clean (BIO 101, La Jolla, CA). The two products were the template DNA for the last round of PCR with FI and B1 as the primers. The final product spanned two sites within the pilE gene allowing for replacement of the region within the wild type gene with the phe2427 mutation. The final
PCR product (210 bp Kpnl-Xbal) was first cloned into pUC18 (pMR3890) and sequenced. Following verification of the sequence the DNA fragment was then placed into the pi!E gene in pMR2715 creating a gene that codes for Phe instead of Tyr at positions 24 and 27 (pMR3964).
Antibody production. The purified GSTPilE' protein, at concentrations ranging from 0.3 to 0.5 mg ml-i, was used to immunize and boost a rabbit at
Lampire Biological Laboratories (Pipersville, PA). The initial injection and subsequent boosts at week 1 and 2 contained Freund's Complete adjuvant while the boost at week 4 and 8 contained Freund's Incomplete adjuvant.
The final bleed was 10 weeks after the initial injection.
The GSTPilE' antisera was purified by immunoaffinify chromatography. First, the antisera was loaded on a GST-Affi-GellO/15 column (prepared according to manufactures specification; Bio-Rad, Hercules,
CA) and the eluate, containing antisera that did not recognize GST, was then loaded on a GSTPilE'-Affi-GellO/15 column. The antibodies that recognized
PilE' were eluted with 20 mM glycine at pH 2.5 which was quickly neutralized
51 with 1M Tris at pH 8. The affinity purified antisera was concentrated using a
Centriprep-10 microseparation device. Sodium azide was added to a final concentration of 1 % to small aliquots of the antisera that were kept at 4° and the rest was kept at -20°.
Cell fractionation. Cell fractionation was based on the method of Bally et a i (1992). Overnight cultures were reinoculated into 250 ml of LB (and appropriate antibiotics added) and grown to an ODsoo of 0.7. The cells were harvested and washed in 20 mM Tris (pH 7.5). The cells were then resuspended in 5 ml of 20 mM Tris and protease inhibitors added (aprotinin and leupeptin to 20pg mb1 and PMSF to ImM). The cells were lysed using sonication (except for when the fractions were to be used in capture studies in which case the cells were broken by two passages through a French pressure cell at 15 000 lb/in2) and the soluble and insoluble fractions separated via ultracentrifugation at 100 000 x g for 1 h. The soluble fraction (supernatant) was concentrated in a Centriprep-10. The membrane fraction was then resuspended in 2% Sarkosyl (IBI, New Haven, CT) and rocked at room temperature for 20 min. The membranes were then separated by ultracentrifugation at 100 000 x g for 1 h. The inner membrane fraction
(soluble in 2% Sarkosyl) was collected and concentrated. The soluble fractions
(cytoplasmic and periplasmic fraction), inner membrane fractions and the outer membrane pellets were stored at -70°. Immunoblotting. Whole cell lysates (WCLs), soluble and inner membrane fractions were mixed with 2X sample buffer (125 mM Tris, pH6.8;
20% glycerol; 10% 2-mercaptoethanol; 4% SDS; 20 pg bromphenol blue) and the outer membrane pellets were resuspended in IX sample buffer. All samples were boiled for 5 min prior to analysis by 15% SDS-PAGE according to the method of Laemmli (1970). The proteins were transferred electrophoretically to Optitran with a 0.2 pm pore size (Schleicher & Schuell,
Keene, NH) using the Towbin buffer method (Towbin et a i, 1979) and a semi dry electrophoretic transfer cell (Bio Rad, Richmond, CA ). Blots were blocked with 5% skim milk (Difco Laboratories, Detroit, MI) in TBS at room temperature for 1-2 h (if the samples under analysis were from a capture experiment, TBS with 0.1M NaCl was used throughout). Proteins were detected using the appropriate antibody (anti-PilE antisera at 1:3000; anti-PAO pilin antisera at 1:5000; anti-PilD at 1:500) in TBS + 1% skim milk during an overnight incubation at 4° followed by a 30 min incubation at room temperature with goat antirabbit IgG conjugated to horseradish peroxidase
(1:10 000; Gibco BRL, Gaithersburg, MD), both incubations were followed by 3-
5 washes in TBS+ 0.1% Tween 20 (Sigma Chemical Co., St. Louis, MO). The interactions were detected using the ECL detection kit (Amersham Life
Science, England) and the resulting bands were visualized within 1 min to 4 h using XR 100 high speed blue sensitive X-ray film (BioWorld, Dublin, OH). Coimmunoprecipitation techniques. Inner membrane fractions at a
concentration of 1.4 mg ml-i of total protein were used in the
coimmunoprecipitation reactions. The immunoprecipitating antibody (30 pi)
was added and the volume was brought up to 1 ml using TNI (1% NP-40; 20
mM Tris, pH8; 150 mM NaCl; 10 mM EDTA; 10 mM NaF; 10 mM Na3V0 4; 10
mM NaP~P). This mixture was allowed to incubate overnight at 4° with
rocking. Protein A-sepharose (Sigma Chemical Co., St. Louis, MO) was added
and incubation continued for at least 1 h. The samples were washed 4 times
with TNI and resuspended in 25-50 pi of IX sample buffer for SDS-PAGE
analysis.
Capture experiment. These experiments were carried out in a manner
similar to the coimmunoprecipitation experiments. Inner membrane
fractions were used to yield 1.4 mg mH of total protein per reaction and the
mild detergent TNI was used to bring the sample volume up to 1 ml.
GSTmPilE (30 pg) was added to the inner membrane-TNl solution and the
samples were incubated overnight at 4° with rocking. Glutathione-sepharose
(30 pi) was added after 12-18 h and the samples rocked for at least 1 h further.
The glutathione-sepharose was then washed 4 times with TNI and resuspended in 25 pi of IX sample buffer and analyzed via SDS-PAGE.
54 CHAPTER 3
THE IDENTIFICATION AND CHARACTERIZATION OF pilE AND PRELIMINARY CHARACTERIZATION OF THE GENE PRODUCT
Introduction. Hobbs et al. (1993) have recently estimated that as many as 20-40 chromosomal genes may be required for type 4 pilus production by P. aeruginosa. Genetic studies from a number of laboratories have shown that the genes required for pilus biogenesis are not tightly clustered. The 15 pil loci examined to date have been localized to three distinct regions of the chromosome (Fig. 2). A large region containing several pil genes has been localized to the SpeI fragment 5 of the P. aeruginosa PAO physical map, which corresponds to approximately 71-75 min on the PAO genetic map (Hobbs et al., 1993; Ratnaningsih et ai, 1990; Romling et a i, 1992; Shortridge et a i, 1991;
Whitchurchet a i, 1990). This region includes pilA, the pilus subunit gene,
(Pasloske et a i, 1985; Sastry et a i, 1985), several accessory pilus biosynthetic genes, designated pilB, C, and D (Nunn et ai, 1990; Nunn and Lory, 1991), the two component regulator/sensor pair pilR and S (Ishimoto and Lory, 1992;
Hobbs et a i, 1993) and the alternative sigma factor encoded by the rpoN gene
55 Fig. 2 P. aeruginosa PAO genetic map and location of pil loci. The genetic map
(in minutes) shown is the previously recalibrated map of PAOl (O'Hoy and
Krishnapillai, 1987).
56 rpoN pilA, B, C, D pilE pilR, S
r h isl proB ur-8001 rys-59 pur-67
cys-54 aeruginosa pilT, U CJ1 met-28 pyrB pilG, H, I, J, K
Z a ,%F met-900KamiE leu-10J
figure 2 (Ishimoto and Lory, 1989). pilB, C, and D, which are located adjacent to the whose products are likely involved in export and assembly of various surface- pilA gene but transcribed in the opposite direction, are part of a gene family associated protein complexes (Nunn et al., 1990; Nunn and Lory, 1991; Strom et al. 1993a; 1993b; Whitchurchet al., 1991). pilR ,pilS, and rpoN encode proteins which are involved in activating pilA transcription. pilR and S have been shown to reside approximately 25 kb from the pilA,B, C, D cluster (Hobbs et al., 1993).
An unlinked set of pil genes has been localized to the Spel fragment 8 which corresponds to approximately 20 min on the PAO genetic map
(Darzins, 1993), the pilG, H, I, /, and K gene cluster. A nearby gene, designated pilT, encodes a protein that appears to be involved in pilus retraction and is required for the twitching motility phenotype (Bradley, 1974a; Whitchurch et al. 1991). ThepilT locus also contains an additional recently characterized gene designated pilU (Martin et al., 1993).
Recent studies by Martin et al. (1993) have identified a gene required for
P. aeruginosa pilus biosynthesis that maps to yet a third unlinked region of the chromosome. This gene, designated pilQ, has been localized to the Spel fragment 2 and corresponds to approximately 0-5 min on the PAO genetic map.
In this report, we describe the identification and characterization of a new gene involved in P. aeruginosa pilus biogenesis. This gene, designated
58 pilE, resides at approximately 71 min on the PAO genetic map and is located in a region of the chromosome previously shown to harbor a number of genes required for pilus assembly (i.e., pil A, B, C, D, R, S and rpoN). The protein product predicted from the pilE nucleotide sequence contains significant amino acid identity to the N-terminal domain of PilA as well as to the pilin precursors from a variety of type 4 pilus producers. Included within this region is a short, positively charged leader sequence which is followed by a prepilin peptidase cleavage site. In addition, the JV-terminal domain of PilE contains a relatively hydrophobic region that has been shown to be important for PilA membrane translocation and in maintaining the structural integrity of pilin subunit-subunit interactions. Possible functions of PilE in P. aeruginosa pilus assembly are discussed.
Results
Characterization of the P. aeruginosa pil mutant MJ-6. This laboratory has previously described the identification of P. aeruginosa PAO pil m utants by screening strains with pili-specific phages (Darzins, 1993; 1994). One strain in our collection, designated MJ-6, which was originally isolated as a spontaneous mutant unable to assimilate or dissimilate nitrate (Goldfam and
Rowe, 1983), was discovered to be broadly resistant to the pili-specific phage
D3112, B3 and F116L. This initial result indicated that MJ-6 possibly contained
59 a defect in pilus biogenesis. Upon further testing, we found that on agar plates
MJ-6 grew, like most other pil mutants, as domed colonies with well defined edges, while the wild-type PAOl produced flat, spready colonies with irregular borders (Bradley, 1980). In addition, MJ-6 was not capable of translocating across solid surfaces via twitching motility when compared to
PAOl (Pil+ Twt+) in a twitching assay developed previously (Fig.3, quadrant B;
Darzins, 1993). Therefore, these results suggested that MJ-6 was impaired in its ability to produce functional pili.
The ability of MJ-6 to physically elaborate pili was determined by immunoblot analysis. Cultures of agar grown PAOl and MJ-6 were suspended in PBS, vortexed to shear off pili and centrifuged to remove intact cells.
Following isoelectric precipitation of the pili, samples from each culture were then analyzed by SDS-PAGE and Western blot analysis with anti-pilin antiserum. While the pilin-specific antiserum recognized pilin protein (15 kDa) in PAOl samples, no detectable pilin protein was found in the MJ-6 samples (Fig. 4A, lane 3). This result suggested that the MJ-6 pil defect affected pilus assembly. In order to determine whether the membrane pool of unassembled pilin was affected in the MJ-6 mutant, WCLs of P. aeruginosa were also subjected to immunoblot analysis. Figure 4B shows that the pool of
60 Fig. 3 Twitching motility assay of PAOl, MJ-6, Rl and R8. The cells were stabbed to the bottom of 1% agar plates and the twitching zone was visible after 24-48 h of incubation. Quadrants: A, PAOl; B, MJ-6; C, Rl; D, R8.
61 A 1 2 3 4 5
6 1 2 3 4 5
Fig. 4 Western blot analysis of the sheared fraction and whole cell lysates of P. aeruginosa. The blots were incubated with antiserum to P. aeruginosa PA O pilin. (A)The sheared fraction of strains of P. aeruginosa; (B) W hole cell lysates of strains of P. aeruginosa. Lanes: 1, PAOl; 2, DAI; 3, MJ-6; 4, R l; 5, R8.
62 unassembled pilin in MJ-6 (lane 3) was unaffected when compared to the
PAOl control (lane 1). Pools of unassembled pilin, on the other hand, were severely diminished in the pilR mutant, DAI (lane 2). PilR is a transcriptional activator of the P. aeruginosa pilin gene, pilA (Hobbs et al.,
1993; Ishimoto and Lory, 1992).
Mapping of the MJ-6 pil mutation. Genetic experiments were performed in order to elucidate the location of the MJ-6 pil mutation on the
PAO chromosome. FP-2 mediated conjugation experiments demonstrated that genetic markers located within the first 23 min of the PAO chromosome showed only a moderate level of co-linkage to the MJ-6 phage- resistance phenotype. The phage resistance mutation was more closely associated with m arkers located late on the PAO genetic m ap (i.e., 66-71 min) (Table 4).
Transduction studies with phage G101 showed that the pilus-specific phage resistance mutation of MJ-6 was tightly linked (78%) to the proB m arker located at approximately 71 min on the PAO genetic map (Table 4).
Additional genetic studies were performed to rule out to the possibility that the MJ-6 mutation resided in a known pil gene described previously.
Transduction analysis utilizing a pilB mutant revealed that phage resistance was not linked to proB (Table 4). This result indicated that the MJ-6 pH mutation was not likely to be located in the pil A, B, C, and D gene cluster
(N unn et al., 1990; Pasloske et al., 1985; Sastry et al., 1985). A pilR m utation,
63 Donor Recipient Markers Selected Marker Coinheritance Location3 Frequency1*
MJ-6/FP2 PA0222 Nal-1 ilv+ his+ lys+ met+ [0-20] 22% (31/143)
MJ-6/FP2 PAO1042 Nal-1 pro+ cys+ pur+ [66-71] 100% (87/87)
MJ-6/FP2 PAO1042 Nal-1 pur+ cys+ [66-69] 13% (8/63)
MJ-6/FP2 PAO1042 Nal-1 cys+ pro+ [69-71] 94% (83/88)
MJ-6/FP2 PAO1042 Nal-1 pro+ [71] 95% (96/101)
MJ-6/FP2 PAO1042 Nal-1 cys+ [69] 100% (93/93)
MJ-6 (G101) PAO1042 Nal-1 pro+ [71] 78% (88/113)
HODS(GIOI) PAO1042 Nal-1 pro+ [71] 0% (0/111)
DAl(GlOl) PAO1042 Nal-1 pro^ [71] 57% (57/100) a The numbers in the brackets indicate marker locations in minutes on the revised chromosomal map (O'Hoy and Krishnapillai, 1987). b The numbers in the parentheses indicate the number of transconjugants or transductants containing the unselected marker (phage D3112 resistance) per number of transconjugants or transductants scored.
Table 4. Conjugational and Transductional Mapping of the MJ-6 mutation.
64 however, was linked to proB by transduction, but at a consistently lower level when compared to the colinkage frequency of the MJ-6 pil mutation to proB (Table 4). Lastly, we were able to rule out a mutation in the gene encoding the alternative sigma factor RpoN simply because MJ-6 grew in a minimal glucose medium without amino acid supplements. Previous work by Ishimoto and Lory (1989) has shown that an RpoN- m utant of P. aeruginosa was unable to grow in minimal medium without the addition of glutamate (i.e., auxotrophic for glutamate). Therefore, it was evident from these studies that the MJ-6 defect must represent a new pil locus which resides in the late region of the PAO chromosome.
Complementation of MJ-6. Since the phage resistance mutation of MJ-
6 was shown to be tightly linked to the proB marker, it was reasonable to assume that a cosmid clone harboring the proB gene might also contain the pil gene(s) capable of complementing the MJ-6 mutant. Therefore, a Hindlll- generated cosmid library of PAOl in vector pCP13 (Tcr) was mobilized into
PAO1042 (proB) and proB+ Tcr transconjugants were selected on minimal agar plates. Restriction endonuclease analysis of plasmid DNA of four individual proB+ Tcr transconjugants revealed the presence of an identical 27-30 kb
Hmdlll cloned fragment. One proB+ plasmid, designated pADD1544, was introduced into PAO1042 and every Tcr transconjugant tested was able to grow on minimal medium without the addition of proline. Following
65 mobilization of pADD1544 into MJ-6 the resulting Tcr transconjugants grew as flat colonies with spready or irregular borders (i.e., wild-type colony morphology). More importantly, MJ-6/pADD1544 transconjugants also regained the ability to plaque the pilus-specific phage D3112, B3 and F116L, translocate across solid surfaces by twitching motility and produce pili (data not shown). Plasmid pADD1544 was not, however, able to complement the nitrate defect of MJ-6.
Subcloning of the large genomic fragment in plasmid pADD1544 revealed that the ability to complement MJ-6 and the proB auxotrophic mutant PAO1042 resided in different DNA fragments. The MJ-6 complementing activity was found to reside on a 5.5 kb Bam HI fragment, while theproB complementing ability resided on a nearby 12 kb Sacl fragment
(Fig. 5). Further subcloning analysis of the 5.5 kb BamHl fragment
(pADD1795) revealed that the ability to restore phage sensitivity, twitching motility and pilus production to MJ-6 resided within an approximately 1 kb
EcoRV-Clal fragment (Fig. 5; pMR2714).
Sequence analysis of the MJ-6 complementing region. The complete nucleotide sequence of the approximately 1 kb EcoRV-C/al fragment of pMR2715 was determined on both strands (Fig. 6). An open reading frame
66 Fig. 5 Cloning and localization of the MJ-6 complementing activity. (A)
Partial restriction endonuclease map of the proB+ cosmid pADD1544. The proB gene has been mapped to 71 min on the PAO chromosome (Holloway et aL, 1987). (B) Subcloning of the MJ-6 complementing ability. Open regions under the restriction map of pADD1795 represent DNA fragments unable to complement MJ-6. Filled-in regions represent DNA fragments able to complement MJ-6. Abbreviations: B, BamHl; C, Clal; H, Hiwdlll; N, Notl; R V,
EcoRV ; S, SacI; Sm, Sm al.
67 5kb 1 B C BC S c h A H ------1— U-rz------1------2----- ? pADD1544 / proB / I i lk b i 1 3 RV Sm C S B I------L-£------1------1 1 pADD1795 .03 B
pMR2422
pMR2714 : pMR2762
figure 5 Fig. 6 Nucleotide sequence of the P. aeruginosa pilE gene. A potential pilE promoter (-10 and -35) and ribosome binding site (RBS) are indicated. The asterisk denotes the pilE stop codon. Arrows indicate a palindromic sequence that is capable of forming a stem-loop structure. The nucleotide sequence of the pilE gene region has been deposited in the GenBank data library under accession number U02552.
69 RBS 1 ATCTACATTCACAAACAGATGAGCGAACAGGAGCTGGCCGAGATGATCGAGAAAGAACAGCCCCGCCAGGACGGGGAGGAGCAGCCCCGATGAGGACAAG 100 -35 -10 M R T R pilE
101 ACAGAAGGGCTTCACGTTGCTGGAAATGGTGGTGGTAGTGGCGGTGATCGGCATCCTCCTCGGCATCGCCATTCCCAGTTACCAGAACTACGTGAiCCGC 200 QKGFTLLEMVVVVAVIGILLGIAIPSYQNYVIR Notl
201 TCCAACCGCACCGAGGGCCAGGCCCTGCTCTCGGACGCGGCCGCGCGCCAGGAACGCTACTACTCGCAGAACCCCGGGGTCGGCTACACCAAGGACGTGG 3 0 0 snrtegqallsdaaarqeryysqupgvgytkdva
301 ccaagctgggcatgagttcggccaactcgccgaacaacctgtacaacctcaccatagcgacgcccaccagcaccacctataccctgaccgccacgccgat 4 0 0 KLGKSSANSPNNLYNLTIATPTSTTYTLTATPI
4 01 CAACTCGCAGACCCGCGACAAGACCTGCGGCAAGCTGACCCTCAATCAGCTCGGCGAACGCGGCGCAGCCGGCAAGACCGGCAACAACAGCACCGTCAAC 5 0 0 NSQTRDKTCGKLTLNQLGERGAAGKTGNNSTVN
501 GACTGCTGGCGCTGAAACGAAAGAGCCCCTCTACGAGAGGGGCTCTTTCATTGCGCACGGTTCACAAAGCCTTGACCCGCAGTTCCTTGGGCATCGAGAA 6 0 0 D C W R * ^ ^
601 GGTAATGTTCTCCTCCCGCCCCTCCAGTTCCTGCTCCTCCGACGCCCCCCACTCGCGTAGCTGGGCGATCACGCCGCGCACCAGCACTTCCGGCGCGGAA 7 0 0
101 GCGGCTGCGGTGATTCCGATGCGACGCACACCGTCGAACCAGCCGCGTTGCATGTCCTCGGCGCCGTCGATCAGGTAGGCCGGCGTGCCCATCCGCTCGG 8 0 0
8 0 1 cgagttcgcgcaggcggttggagttggaactgttggggctgcccaccaccaggaccatgtcgcactggtcggccagttccttcacggcatcctggcggtt 900
901 CTGGGTGGCATAGCAGATGTCGTTCTTGCGCGGTCCCTGGATCTGCGGGAACTTGGCGCGCAGGGCATCGAT 972
figure 6 search of the sequence revealed the presence of multiple partial and complete
ORFs on each strand. Of these, only one complete ORF, designated pilE, with characteristic P. aeruginosa codon bias and a high preference (91%) for C or G in the third codon position was identified. The G+C content of pilE is 63%, a value which closely approximates the 67% overall G+C content reported for
P. aeruginosa (West and Iglewski, 1988). The pilE open reading frame, which extends from nucleotide position 90 (ATG) to 512 (TGA), predicted a protein product of 141 amino acids with a molecular weight of 15,278. A putative ribosome binding site (RBS) with homology to the 3' end of the P. aeruginosa
16S RNA (AGGAG; Shine and Dalgarno, 1975) and sequences resembling a sigma-70 promoter with -10 and -35 regions were found upstream of the putative PilE start codon (Fig. 6). A partial P. aeruginosa open reading frame, designated orfB', was found on the opposite strand and extends from nucleotide 972 to 565. A palindromic sequence that could possibly function as a transcriptional terminator (<4G, -25.1 kcal; Jaeger et al., 1989) was found between nucleotides 516 and 552 (Fig. 6).
Homology searches. The deduced amino acid residues of PilE and
ORFB' were used to conduct a search of the GenBank database using the
TFASTA program (Devereux et a l, 1984). The PilE sequence was homologous to the prepilin proteins of P. aeruginosa. Figure 7 shows that over their entire lengths PilE and PilA of P. aeruginosa strain PAOl are 32% identical.
71 Fig. 7 Sequence alignment of P. aeruginosa PilE with PilA. The GAP and
PUBLISH programs from the University of Wisconsin Genetics Computer
Group (GCG) were used to generate the alignments. The vertical bars indicate identical amino acids and colons and periods indicate two degrees of similarity of amino acids.
72 PilE 1 MRTRQKGFTLLEMWWAVIGILLGIAIPSYQNYVIRSNRTEGQALLSDA 50 • - I I I I I I : I : : : I I I : I I I I : I I I I I I 1 I I I I : . . : I : . . PilA 1 .MKAQKGFTLIELMIVVAIIGILAAIAIPQYQNYVARSEGASALATINPL 49
PilE 51 AARQERYYSQNPG VGYTKDVAK...LGMSSANSPNNLYNLTIATP 92 [ • I - • : : I I - • I • : I : : . . : . | . PilA 50 KTTVEESLSRGIAGSKIKIGTTASTATETYVGVEPDANKLGVIAVAIEDS 99
PilE 93 TSTTYTLTATPINSQTRDKTCGKLTLNQLGERGAAGKTGNNS..TVNDCW 140
* • • * • | l"l| I * * I \ *111 ■III* * • "• • II| • | + + • “• • I | • « *1» | S3 PilA 100 GAGDITFTFQTGTSSPKNAT.KVITLNRTADGVWACKSTQDPMFTPKGCD 148
PilE 141 R 141
PilA 149 N 149
figure 7 However, it is evident from the comparison that this homology is limited to the N-terminal approximately 37 amino acid residues. Over the first 37 amino acids PilE and PilA were found to be 68% identical and 89% similar, whereas the remaining regions were only 18% identical. The amino terminus of PilE was also considerably homologous to the pilin precursors from a number of type 4 pilus producers, such as D. nodosus, M. bovis, and N. gonorrhoeae.
This result was not unexpected since it has been shown that the type 4 pilins are remarkably similar, particularly within the amino terminus (Elleman,
1988; Strom and Lory, 1993).
A closer examination of the N-terminus of PilE revealed the presence of a consensus cleavage site for the P. aeruginosa prepilin peptidase (Fig. 8A).
The prepilin peptidase, encoded by the pilD gene, removes the six residue leader of PilA by cleaving between the glycine and phenylalanine residues
(N unn and Lory, 1991; N unn and Lory, 1992). After cleavage, PilD also catalyzes the methylation of the N-terminal phenylalanine (Strom et al.,
1993a; 1993b), The putative PilE prepilin peptidase cleavage site was preceded by a seven residue basic leader sequence which included the invariant glycine residue at the cleavage site (Fig. 8A). In a region encompassing the 31 amino acids following the putative PilD cleavage site, PilE and PilA were found to be
71% identical and 90% similar (Fig. 8A). In addition, the first 31 amino acids of PilE and the corresponding regions of pilins from the type 4 pilus
74 Fig. 8 Comparison of N-terminal sequences of prepilin peptidase dependent proteins. (A) Sequence alignment of the N-terminal regions of PilE and type 4 prepilin proteins. The line above the PilE sequence represents the PilD consensus cleavage site. Asterisks above the PilE sequence represent the conserved Tyr-24 and Tyr-27 residues (Watts et al., 1983). (B) Sequence alignment of the N-terminal regions of PilE and the P. aeruginosa XcpT, U, V, and W proteins. Arrows indicate PilD cleavage sites. The phenylalanine (+1 residue) has been shown to be methylated by PilD. Residues homologous to
PilE are boxed.
75 A t +1 10 P . a e ru g in o sa P i l E QKG FTLyE[W\WA IGILLG IAIPSYQNYVIRS P ilA MKft QKG FTLIEL vn W A IG IL Aft IAIPQYQNYVftRS D. n o d o su s FimA E fcsi <£3g FTLIEL XI] WA IGILAA AIF : i m s M. b o v is TfpQ MNft QKG FTLIEL Al VIA 'ism N. gonorrhoeae P i l E |m^ i QKG FTLIEL Al VIA iJTAgA B 30 P . a e ru g in o sa P i l E yivvw PSjYQNYVIRS XcpT FTI GILAALV^_ 'VMSRPDQA XcpU FTL I@^lJsjrGFASTSR XcpV FTLLEIVL Ift IFAMVAASVLSASARSLQNA XcpW FTLLEiLLIAIft IFAgLALATYRMFDSVMQTD
figure 8 producers D. nodosus, M. bovis, and N. gonorrhoeae were from 52 to 71% identical (Fig. 8A).
Four components of the P. aeruginosa secretory apparatus, which have been designated XcpT, U, V, and W, are also processed by PilD (Bally et al.,
1992; Nunn and Lory, 1992; 1993). These proteins are similar to type 4 prepilin proteins in that they also contain a 6 or 8 residue leader sequence and a PilD consensus cleavage site which is followed by a relatively hydrophobic region.
A comparison between PilE and the P. aeruginosa XcpT, U, V, and W proteins revealed that they were only 23 - 39% identical over the first 31 amino acids in the mature proteins. This identity, however, was primarily limited to the proximal region of the N-terminus (Fig. 8B).
A search of the Genbank database with ORFB' amino acid sequences revealed that it is highly homologous to a hypothetical 34.8 kDa E. coli protein encoded by orf316 (Appendix A; Bouvier and Stragier, 1990; Vura et al., 1992), located in a region between the Isp and dapB genes. Isp encodes the lipoprotein signal peptidase (Innis et al., 1984) and dapB encodes dihydrodipicolinate reductase (Bouvier et al., 1984), which is used in the biosynthesis of diaminopimelate. or/316, which is a part of the same operon as Isp (Miller et al., 1987) encodes a hypothetical protein of unknown function. The C-terminal 137 amino acids of ORF316 were found to be 61% identical to the partial amino acid sequence of ORFB' (Appendix A). The function of the putative P. aeruginosa ORFB is not known.
77 Expression of pilE, In order to verify that pilE encodes a protein of the size predicted from the nucleotide sequence, the 1 kb EcoRV-C/al fragment was cloned into the T7 promoter plasmid pT7-5. Constructs representing the fragment cloned in both orientations were identified and expressed using the
T7 RNA polymerase system as described previously by Studier and Moffatt
(1986). The expression studies in E. coli BL21(DE3), however, were inconclusive since an orientation dependent production of an approximately
15 kDa polypeptide could not be demonstrated.
The inability to unambiguously identify PilE in E. coli prompted an attempt to express pilE in a recently developed P. aeruginosa T7 expression system. The 1 kb EcoRV-C/al fragment containing pilE was cloned into the broad-host-range T7 expression vector pEB12 (Brunschwig and Darzins, 1992) and constructs, designated pMR2823 and pMR2824, which contained the insert in both orientations were mobilized into the P. aeruginosa strain
ADD1976 (Brunschwig and Darzins, 1992). Plasmid pMR2824, which contained the pilE fragment in the correct orientation with respect to the T7 promoter, was capable of directing the synthesis of what appeared to be two proteins (see below) with approximate molecular weights of 15,000 and 14,300
(Fig. 9, lane 1 ). No such protein bands were detected in cells harboring the
78 prePilE PilE
Fig. 9 Expression of the P. aeruginosa pilE gene in P. aeruginosa.
Autoradiograph of [3^S] methionine and cysteine-labeled polypeptides expressed with the T7 system in P. aeruginosa ADD1976 (Brunschweig and
Darzins, 1992). Lanes: 1, pMR2823 + IPTG; 2, pMR2824 + IPTG; 3, pEB12.
Positions of molecular mass markers, in kilodaltons, are indicated on the right. The location of the pilE gene product is indicated by the arrows on the left.
79 expression vector pEB12 (Fig. 9, lane 3) or plasmid pMR2823, which contained the pilE fragment in the opposite orientation with respect to the T7 promoter
(Fig. 9, lane 2).
Construction and analysis of a P. aeruginosa pilE gene replacement mutant. The gene replacement techniques described previously by Schweizer
(1992) were used to generate a chromosomal pilE insertion mutant of P. aeruginosa strain PAOl. Southern blot analysis, which was used to verify the gene replacements in PAOl, revealed that the hybridization patterns of the two putative pilE insertion mutants, designated R1 and R8, were consistent with the expected sizes of the chromosomal region containing the gene disruption (data not shown). The two pilE insertion mutants, R1 and R8, produced colonies with a distinctive morphology. These strains grew as compact, domed colonies with well-defined edges as compared to the parent
PAOl which grew as larger, flat and spready colonies. R1 and R8 were able to assimilate nitrate, were broadly resistant to the pilus-specific phage D3112, B3 and F116L and were unable to translocate across solid surfaces in a twitching motility assay (Fig. 3, quadrant C and D). Furthermore, immunoblot analysis revealed that the marker exchange strains R1 and R8 produced unassembled pilin pools (Fig. 4B, lanes 4 and 5), but like MJ-6 failed to produce assembled pili (Fig. 4A, lanes 4 and 5). In addition, the pil defect of R1 and R8 was complemented in trans by the 1 kb EcoRV-Clal fragment of
80 pMR2714 (Fig. 5). R1 and R8 cells containing pMR2714 regained phage sensitivity, twitching motility, and pilus production (data not shown). Taken together, therefore, these results suggested that R1 and R8 were not capable of forming functional pili due to the disruption of the pilE gene.
Discussion
Characterization of the pilE gene. A new gene, designated pilE, has been identified and characterized and its gene product was shown to be required for the production of P. aeruginosa pili. One of the most significant findings of this study was the homology between the N-terminus of the protein product predicted from the nucleotide sequence of pilE and the N- terminal domains of type 4 prepilins (Fig. 7 and 8A). The N-terminal region of the P. aeruginosa PilA precursor has been previously shown to contain important signals for both pilin processing and membrane translocation prior to pilus assembly (Strom and Lory, 1993). During pilus biogenesis the pilin
(PilA) subunit is translocated across the cytoplasmic membrane with a concomitant removal of the short, basic leader sequence and methylation of the resulting N-terminal phenylalanine (Frost et al, 1978). Both the removal of the short leader sequence and modification of the N-terminal amino acid residue are catalyzed by the pilD gene product (Nunn and Lory, 1991; Strom et a l, 1993b). The prepilin peptidase PilD cleaves between the glycine and
81 phenylalanine residues in the sequence -Gly-Phe(Met)-Thr-Leu-Ile(Leu)-Glu- which is conserved among all members of the type 4 pilin family (Nunn and
Lory, 1991). In addition, PilD is responsible for processing several components of the P. aeruginosa protein-secretion machinery (XcpT, U, V, and W) (Bally et al., 1992; Nunn and Lory, 1992; 1993).
Characterization of the pilE gene product. A close inspection of the N- terminus of PilE revealed the presence of a short, positively charged putative type 4 leader sequence (Met-Arg-Thr-Arg-Gln-Lys-Gly-). This was followed by a relatively hydrophobic region which contained a consensus cleavage site for
PilD, the P. aeruginosa prepilin peptidase (Fig. 8A; Nunn and Lory, 1991;
Strom et al., 1993a). The presence of a prepilin peptidase consensus cleavage site at the N-terminus of the putative PilE protein (Fig. 8A) suggests that this polypeptide is processed in a PilD-dependent manner along with PilA and other prepilin like proteins (Bally et al., 1992; Nunn and Lory, 1991; 1992) and, furthermore, that this processing may be critical for its function. The presence of two PilE specific bands on the autoradiogram following expression of pilE in P. aeruginosa ADD1976 (PilD+) may be interpreted as preliminary evidence for the existence of PilE processing (Fig. 9). The presence of these bands can be explained by an incomplete processing of the overproduced PilE protein by
82 PilD (Nunn and Lory, 1991; Strom et al., 1993b), The upper band most likely represents the unprocessed form of PilE, while the lower band likely represents the mature, processed form of PilE.
Possible role(s) of the PilE N-terminus. Previous studies have shown that residues located in both the amino and carboxyl termini of PilA are involved in subunit-subunit interactions. Antigenic analysis employing PilA peptides revealed that the protein can be divided into four distinct epitopes
(Watts et al., 1983; Sastry et al., 1985). A weak, cross-reactive epitope, has been localized to the N-terminus (amino acid residues 1-30). The fact that this epitope was not recognized in intact pili but, could be detected in denatured pili, suggested that residues located in the N-terminus are likely to be found at the interfacial regions between subunits. This hypothesis was supported by spectral studies which have shown that specific tyrosine residues present in the N-terminal hydrophobic domain (i.e., Tyr-24 and Tyr-27) are involved in subunit-subunit interactions (Watts et al., 1983). Therefore, the N-terminal, hydrophobic segment of a pilin precursor plays a dual role. First, it acts as a signal that allows nascent molecules to be recognized for transfer to a specific location in the cell (PilD processing and membrane translocation) and, second, it serves as a structural element that preserves the integrity of the pilus filament.
83 Evidence to support the role of amino acid residues located in the C- terminus in pilin subunit interactions has also been obtained. Monoclonal antibodies, PK3B and PK99H, which are specific for the C-terminus of pilin, have the ability to recognize an epitope which is found only at the tip of intact pili (Doig et a l, 1990). In addition, these antibodies have been found to inhibit pilus-mediated adherence to buccal epithelial cells. This epitope, which comprises a 12 residue disulfide loop, is not accessible at the sides of an intact pilus filament due to its potential role in subunit interactions.
Reduction of this disulfide loop has been shown to partially disassemble pili into both pilin monomers and dimers (Irvin, 1990). These studies, therefore, suggest the sequences present at the C-terminus also likely constitute a portion of the subunit interfacial region.
Homology between PilE and type 4 prepilin proteins. Previous comparisons of the amino terminal domain of the P. aeruginosa PilA protein with other type 4 pilins have revealed that the first 31 amino acids of the mature proteins are well conserved; these sequences are between 71 and 97% identical (Fig. 8A). The amino acid similarity among the type 4 pilins indicates a possible compatibility in pilin processing and in the pilus biosynthetic machinery. Evidence to support this hypothesis has been collected from several studies investigating the expression of heterologous type 4 pilins in P. aeruginosa. These studies show that the pilus assembly
84 machinery of P. aeruginosa is capable of correctly processing, transporting and polymerizing pilin subunits from a number of bacterial species such as D. nodosus, M. bovis, and N. gonorrhoeae (Beard et al., 1990; Elleman et al., 1986;
Hoyne et al., 1992; Mattick et al., 1987). These results, combined with more recent findings by Lauer et al. (1993), argue in favor of the notion that the pilus biosynthetic machinery among type 4 pilus producers is most likely conserved. The evolutionary pressure to retain the residues within the amino terminus of type 4 pilins may be due, in part, to the function of this region in maintaining pilus integrity through subunit-subunit interactions. It is conceivable, however, that this region of the protein may also be responsible for making specific contacts with the conserved components of the type 4 pilus assembly machinery.
Homology between PilE and Xcp proteins. The amino terminus of the putative PilE protein, described in this study, also contained significant homology to type 4 pilins over the first 31 amino acids. More importantly, the
PilA tyrosine residues (Tyr-24 and Tyr-27), which are thought to be involved in pilin subunit-subunit interactions (Watts et al., 1983), are conserved in PilE
(Fig. 8A). This result suggests that these residues may also play some important role in the function of PilE. The N-termini of the four PilD
85 dependent, secretion proteins XcpT, U, V, and W (Bally et al., 1992; Nunn and
Lory, 1992, 1993) were, as a group, less homologous to amino terminus of PilE and lacked the conserved tyrosine residues (Tyr-24 and Tyr-27; Fig. 8B).
Possible role(s) of PilE. Since the N-terminus of pilin has a multi functional role (i.e., contains signals for processing, membrane translocation and subunit-subunit interactions), it is tempting to speculate, based on what is known about pilin biochemistry, as to the function of PilE in pilus assembly.
The simplest possibility is that PilE might be able to form protein complexes with itself and the sequences present at the N-terminus might stabilize the
PilE protein interactions. However, the extensive homology between the N- termini of PilA and PilE suggests an alternate hypothesis; PilE might be capable of interacting with PilA at some point during pilus biogenesis. If PilE has the capability of interacting with PilA, then it could do so in a number of ways. One hypothesis is that PilE could be a minor pilin protein. Analogies could be drawn to the P or Pap pilus system of uropathogenic E. coli which contains, in addition to the major pilin protein (PapA) several minor pilins
Pap E, F, G and K (Hultgren et al, 1991; Kuehn et al, 1992). Minor Pap subunits are not necessary for the assembly of the rigid PapA-composed filament but are essential for proper assembly of the tip fibrillum (for review
86 see Pugsley, 1993b). The possibility that PilE is a minor pilin subunit seems
remote since, to date, no pilin subunit other than PilA has been detected in P. aeruginosa (Pasloske et al ., 1989).
Another hypothesis is that PilE functions in a manner analogous to the
PapH protein of the Pap pilus. PapH, also known as the anchor protein, is
located at the base of the pilus and serves as a signal that controls pilus length
(Hultgren, 1991). Mutants lacking PapH release unusually long pilus
filaments into the medium while the overproduction of PapH leads to the
production of shorter, stubby pili (Pugsley, 1993b). This explanation also
seems unlikely in light of the fact that strain MJ-6 and the pilE gene
replacement mutants R1 and R8 lacked pili.
Elleman (1988) has suggested that the role of the short leader peptide
sequence of prepilin proteins may be to prevent aggregation of the subunits
prior to assembly. However, once the pilin has been translocated across the
cytoplasmic membrane, what mechanism prevents processed pilin subunits
from aggregating prior to polymerization? Therefore, a third hypothesis is
that PilE functions as a component of the pilus assembly apparatus by capping
and uncapping the interactive surfaces of pilin subunits that have been
translocated across the cytoplasmic membrane. PilE might also serve as a pilot
that guides PilA subunits to and from the assembly platform during the
alternating mechanisms of pilus polymerization (extension) and retraction.
Its function would be directly comparable to the PapD chaperone protein of
87 the P pilus biogenesis system which guides pilin molecules to the outer
membrane protein PapC for assembly (Kuehn et al., 1991; Lindberg et at.,
1989).
The contents of Chapter 3 have been published elsewhere (Mol
Microbiol 13:973-985) with minor changes in the text along with the
appropriate sections of Materials and Methods in Chapter 2. Analysis of the
PilE protein is presented in the next chapter and discussed with respect to possible roles of PilE including that of a periplasmic chaperone.
88 CHAPTER 4
ANALYSIS OF PilE
Introduction. Further analysis of PilE and the recent finding of another prepilin-like protein involved in P. aeruginosa pili biogenesis prompted us to expand our theories of possible roles of PilE to include participation in a pseudopilus structure as proposed for prepilin-like proteins in the systems involved in macromolecular transport introduced in Chapter 1. The systems are further described below so as to illustrate the similarities between them and pilus biogenesis of P. aeruginosa.
Pullulanase secretion by K. oxytoca is the best characterized system containing Pil homologs and is considered the model for the main terminal branch of the GSP. Secretion of pullulanase is directed by the products of 14 genes, pulC-O which are in a single transcriptional unit and pulS which is just upstream of the operon (d'Enfert and Pugsley, 1989; Possot et al., 1992;
Pugsley and Reyss, 1990; Reyss and Pugsley, 1990). Between these two regions on the chromosome is an operon containing the gene encoding pullulanase
(pulA) followed by pulB, a gene that is not required for secretion in E. coli but
89 maybe required in K. oxytoca (Pugsley, 1993b). The pulO gene encodes the prepilin peptidase that is 52% identical to PilD (Table 5; Pugsley and Dupuy,
1992), PulG, H, I, and J contain the consensus sequence for the prepilin peptidase and are cleaved by PulO (Pugsley and Dupuy, 1992; Reyss and
Pugsley, 1990). PulE, 43% identical to PilB, is associated with the inner membrane and contains a Walker box A and Walker box B motif (Possot et a i, 1992; Possot and Pugsley, 1994). PulD was localized to the outer membrane and is homologous to the PilQ protein of P. aeruginosa (d'Enfert et a i, 1989;
Martin et a i, 1993). Currently little is known about the other Pul proteins.
There are Pul homologs and consequently Pil homologs in many bacteria, including Erwinia spp. (Out), P. aeruginosa (Xcp), X. campestris (Xps),
A. hydrophila (Exe), and B. subtilis (Com; Table 5). Some E. coii isolates also contain genes predicted to encode proteins homologous to PilB, C, D, and Q
(hopB, C, D, and Q) and five predicted to contain a prepilin-like leader sequence ( ppdA, B, C, hopG, and H; Stojiljkovic et a i, 1995; Whitchurch and
Mattick, 1994). Only one of the open reading frames has been shown to encode a protein using T7 expression experiments, hopG, and insertional mutations of hopB and hopD yielded no identifiable change in phenotype
(Stojiljkovic et a i, 1995; Whitchurch and Mattick, 1994). Furthermore, when the pilin genes from P. aeruginosa and N. gonorrhoeae were expressed in E.
90 proteins w ith PilB PilC PilD PilQ prepilin- hom olog hom olog hom olog hom olog like signal sequence
Protein secretion P. aeruginosa XcpR XcpS PiU> XcpQ XcpT-W Klebsiella oxytoca PulE PulF PulO PulD PulG-J Aeromonas hydrophila ExeE ExeF ExeD ExeG-J Xanthomonas campestrisXpsE XpsF XpsO XpsD XpsG-J Erwinia spp. OutE OutF OutO OutD OutG-J
DNA uptake Bacillus subtilis ComG-1 ComG-2 ComC ComG3-5 a PilD in P. aeruginosa processes proteins in both the Pil and Xcp systems (Nunn and Lory, 1993; Strom et at., 1993b). t> References: Aim and Mattick, 1995; Howard et a i, 1993; Hu et ai, 1992; Pugsley, 1993b.
Table 5. Homologs of the P. aeruginosa Pil proteins and proteins with prepilin leader sequence13. coli, the proteins were not processed or polymerized into a pilus filament
(Dupuy et al, 1991; Strom and Lory, 1986) so the role, if any, of the E. coli Pil- like system is unclear.
PilB and the PilB homologs each contain a Walker box A and some contain the less well conserved Walker box B sequence. The significance of the nucleotide binding sites is unknown but they have been shown to be indispensable for function in the case of XcpR, PilB, and PulE (Pugsley, 1993b;
Turner et ai, 1993). These proteins may supply energy via ATP hydrolysis for the assembly of a multicomponent structure or they might phosphorylate and activate another component of their system. The VirB-11 protein of
Agrobacterium tumefaciens is also homologous to PilB and has been shown to be an autophosphorylating ATPase involved in T-DNA transport process
(Christie et ai, 1989) suggesting that this might be the mechanism by which these proteins are involved in macromolecular transport. In this case the autophosphorylation of PilB-like proteins may alter their conformation which might allow them to bind other components of their systems. This category of Pil-like proteins is large and includes several not included in Table
5 such as VirB-11 mentioned above; KilB, encoded by the plasmid pRK2, which is involved in conjugation (Motallebi-Veshareh et a i, 1992); and the
PilT and PilU proteins of P. aeruginosa. A second nucleotide binding protein
92 has also been identified in A. hydrophila that is involved in protein secretion along with the ExeC-N proteins but it is not homologous to PilB (Jahagirdar and H ow ard, 1994).
The function of PilC and the PilC-like proteins is probably the least clear of the proteins discussed here. These proteins have been localized to the inner membrane and phenotypic analysis of mutant strains indicate that they are clearly involved in their respective systems. It seems as though little work has been done to date to elucidate the nature of their role. In his review, Pugsley (1993) speculated that because the pseudopilus structures are not external to the cell, PilB and PilC do not function to transport pilin across the outer membrane because that would not be the role of their homologs. A similar argument can be made that PilB and PilC do not function in pilin transport across the inner membrane. Pullulanase transport in E. coli across the inner membrane is dependent on Sec proteins and does not require any
Pul proteins (Pugsley et al.r 1991) so the Pul equivalents of PilB and C (PulE and F) are not involved in inner membrane translocation. However, it should be noted that unlike the proteins exported by the type II secretion pathway (such as those discussed here containing Pil homologs), type 4 prepilin proteins do not have a classical signal sequence and may not interact with the proteins that are involved in the first step of the GSP. Pugsley (1993) speculates that the role of PilC and similar proteins may be to catalyze the assembly of a pilin or pseudopilin filament on the periplasmic side of the
93 inner membrane and hydrolysis of ATP by the PilB and PilB-like proteins could be used to aid in the expulsion of the proteins from the inner membrane during their assembly.
The prepilin peptidase, PilD in P. aeruginosa, is the best characterized component of this system. PilD was shown to cleave and methylate PilA and
XcpT-W (Nunn and Lory, 1991; 1993; Strom et a i, 1993b). PilD also cleaves the leader sequence from PilV and PilE and presumably methylates both because they contain the conserved +5 glutamic acid residue (this study; Aim and
Mattick, 1995; Strom et a i, 1993a). Recently, a PilD homolog that is plasmid encoded has been identified in E. coli (the hop genes described above are chromosomally encoded) and is found in association with the gene encoding the structural protein of bundle-forming pili, a variety of type 4 pili more closely related to V. cholerae toxin-coregulated type 4 pili (Zhang et a\., 1994).
Some of these enzymes are interchangeable and can process heterologous substrates; for example, the prepilin from N. gonorrhoeae was processed by
PulO, ComC, and PilD (Dupuy et a i, 1991) and PulO was able to compensate for a PilD m utation in P. aeruginosa (Bally et a i, 1992). However, neither
PilD or ComC could correct for a PulO defect in the pullulanase secretion system reconstituted in E. coli (Pugsley and Dupuy, 1992) and the V. cholerae prepilin peptidase was not able to process P. aeruginosa prepilin when both
94 were expressed in E. coli (Zhang et a i, 1994). These data indicate that while the prepilin peptidases have a somewhat broad specificity, there must be some constraint dependent on sequences in the mature protein.
As in the PilB family, there are proteins homologous to PilQ that are not included in Table 5 because there are no other Pil homologs in that system. PilQ homologs are involved in the type III secretion pathway responsible for Yops secretion in pathogenic Yersiniae and Ipa protein secretion in Shigella flexneri. PilQ homologs are also found in the system responsible for the invasive property of Salmonella typhimurium and the transformation ability of Haemophilus influenzea (Russel, 1994b). All of the
PilQ-like outer membrane proteins are highly homologous over 60 amino acids at or near the C-terminus of the protein (Martin et a i, 1993) which is the region thought to be involved in subunit-subunit interaction in the multimeric complex (Russel, 1994a). The N-termini are poorly conserved and are thought to confer the functional specificity of these proteins (Martin et a i, 1993, Russel and Kazmierczak, 1993). Only the protein involved in type
4 pili biogenesis in N. gonorrhoeae (PilQ) and the homolog involved in filamentous phage morphogenesis (pIV) have been shown to form multimers (Kazmierczak et ai, 1994; Tsai et ai, 1989), but pIV was able to interact with the OutD protein from E. chrysanthemi so it is expected that the other members of this group can also form large multimeric complexes.
95 pIV, PulD, and OutD induce the expression of the psp operon in E. coli which is also induced by osmotic shock and other stimuli that affect the ion flux across the cell membrane (Russel and Kazmierczak, 1993). This finding supports the theory that PilQ and PilQ-like proteins act as a gated channel in the outer membrane. The induction of the psp operon might be due to an occasional opening that would connect the cytoplasm and the extracellular environment (Kazmierczak et al, 1994; Russel and Kazmierczak, 1993).
The proteins listed in Table 5 that have a prepilin-like leader sequence also share amino acid homology at their N-termini (Aim and Mattick, 1995;
Hobbs and Mattick, 1993; Whitchurch et al, 1990). These relatively small proteins contain a leader sequence that is cleaved by a specific leader peptidase at a site following an invariant glycine residue. A glutamic acid at the +5 position in the mature protein is also completely conserved and many contain a glutamine residue at the -3 position. All have a hydrophobic core of approximately 20 residues at the N-terminus of the mature protein (Aim and Mattick, 1995) which probably anchors the protein in the inner membrane after cleavage of the leader sequence (Nunn and Lory, 1993;
Reeves et al., 1994). Alkaline phosphatase fusion studies with the Pul proteins indicated that the C-termini of the proteins are exposed in the periplasm (Pugsley and Dupuy, 1992). The ComG3-5 proteins were also shown to be imbedded in the cell membrane via their hydrophobic regions with the C-termini of the proteins located on the surface of the cell (Chung
96 and Dubnau, 1995). Even though practically every report on these proteins proposes that they form a pilin-like structure, there is little evidence to support this theory. Recently, however, ComG3 and PulG have been shown to be capable of forming homodimers as evidenced by cross-linking studies
(Chung and Dubnau, 1995; Pugsley and Possot, 1993).
In this study we wanted to further characterize the PilE protein which along with PilV are the only proteins identified and published to date in the pilus biogenesis system with a PilD-dependent leader sequence other than the prepilin protein (two more PilD-dependent proteins have also been identified but work on them are not yet published; R. Aim, personal communication).
In order to determine the subcellular location of PilE under physiologically conditions, antiPilE antisera was generated using a GST fusion protein. We established that prePilE is cleaved by PilD and that PilE is capable of forming homomultimers. We found no evidence that PilE is capable of protein- protein interaction with PilA or that PilE is a minor pilin protein. There is also further discussion as to the possible roles of PilE in P. aeruginosa pili biogenesis.
Results
Sequencing of the MJ-6 pilE mutation. In order to determine if the mutation in the pilE gene of MJ-6 would yield any clues as to the function of
97 the protein, the gene was cloned and sequenced. MJ-6 chromosomal DNA was digested with BamHl and a 1 kb window centered around 5.5 kb was cloned into pK19. Following transformation in E. coli, a single colony containing the pilE gene was identified using colony hybridization. The resulting 1 kb EcoRV-Clal pilE fragment was not able to complement R1 confirming that a mutation in pilE was the cause of the pil phenotype of MJ-6.
The entire nucleotide sequence of one strand of the 1 kb EcoRV-Clal in pMR3839 was determined. A single C to T transition was found. This change forms a nonsense mutation that would result in a truncated protein 107 amino acids long (compared with 141 residues in the wild type preprotein). A
200 bp subclone containing the change was used to replace the wild type sequence in pMR2715 and the wild type sequence from pMR2715 was placed into the MJ-6 copy of pilE. The newly constructed MJ-6 gene was then able to complement MJ-6 and R1 while the newly constructed PAOl gene was not.
This confirmed that the only change in the MJ-6 pilE gene was within the small subcloned region. This 200 bp region was subsequently sequenced on both strands to confirm the single C to T mutation. The predicted molecular weight of the truncated protein is 11.5 kDa, however the protein could not be detected by Western analysis of MJ-6 WCLs possibly due to increased sensitivity of the truncated protein to proteolytic degradation (data not shown).
98 Production of antibody against PilE. Initial attempts to use a polyhistidine-PilE' fusion protein for antibody production were unsuccessful.
The fusion protein did not bind well to the nickel-agarose column. The inability of polyhistidine fusion proteins to bind nickel-agarose column has been observed previously (Pugsley, 1993a). A GSTPilE' fusion protein (Fig.
10), however, was successfully overproduced and purified. This protein lacks the N-terminal portion of PilE that strongly resembles PilA so as to reduce the chance that the antisera raised with the fusion protein would cross react with
PilA.
Figure 11 shows that most of the binding of the antiPilE' antisera to
PilE overproduced in P. aeruginosa strain PAOl could be blocked with purified GSTPilE' fusion protein (lane 2) and that GSTRas could inhibit binding to a lesser extent (lane 3). This indicates that the antisera is specific for GSTPilE' and at least a portion of the antibodies are directed against the
PilE' moiety of the fusion. The inhibition of antiPilE' binding to PilE with
GSTRas could be due to titrating out any antibodies that recognize a conformational epitope which includes both GST and PilE'. The smaller bands in lane 1 are probably breakdown products of the overproduced PilE protein as they are also inhibited in the inhibitions studies (lanes 2 and 3).
We also demonstrated that antiPilE' antisera does not cross react with
PilA and antiPAO pilin antisera does not cross react with PilE. AntiPilE'
99 Fig. 10 Comparison of prePilE and GST fusion proteins. The striped region represents the region of high homology within the type 4 pilin family. The grey region represents the glutathione-S-transferase portion of the fusion protein. The vertical arrow indicates the site of PilD cleavage. The proteins are drawn to scale.
100 * ] prePilE j GSTPilE'
■ GSTmPilE figure Fig. 11 Inhibition studies of antiPilE' antisera. Whole cell extracts from
PAOl /pMR2824 were run on a SDS-polyacrylamide gel and transferred to nitrocellulose membrane. Western analysis was then carried out using either antiPilE' antisera alone (lane 1) or antiPilE' antisera and purified GSTPilE' together (lane 2) or antiPilE' antisera and GSTRas together (lane 3). The purified GST fusion proteins were added to a concentration of 30 gg ml*1.
102 antisera does not recognize purified pilin protein {Fig. 12, lanes 5 and 6) and
antiPAO pilin antisera does not recognize purified GSTPilE' (Fig. 12, lanes 3
and 4). The smaller bands in lanes 7 and 8 (Fig. 12; approximately 26 kDa) are
probably GST protein as discussed below, which would be expected to be
detected by the antisera prior to affinity purification.
In order to eliminate the antibodies that recognized GST and other
antibodies which were cross reactive with proteins other than PilE, the
antisera was affinity purified in two steps. First, the antibodies directed
against the GST moiety of the fusion were removed and then the antibodies
that recognized PilE' portion were affinity purified and concentrated.
Levels of PilE during growth in LB broth culture. WCL samples of
PAOl and R1 were taken at OD6oo=0-5 and at 2, 4, and 6 h later along with an overnight sample. The samples were normalized with respect to optical density and subjected to Western blot analysis in order to determine if PilE is
differentially produced over time. The results demonstrated that PilE was continuously produced at nearly the same level during early, mid, late log phase and stationary phase in LB broth cultures (data not shown).
Localization of PilE and processing by PilD. The similarity between the
PilE and PilA proteins raised the possibility that PilE may be incorporated as a minor subunit into the pilin filament. Samples of purified pili and GSTPilE'
103 Fig. 12 Western analysis of purified pili and GSTPilE' protein. Samples of purified pili and purified GSTPilE' fusion protein were run on a SDS- polyacrylamide gel and transferred to nitrocellulose. Either antiPAO pilin antisera (lanes 1-4) or antiPilE' antisera (lanes 5-8) was used to detect. Lanes: 1 and 5,1.0 |ig mH purified pilin; 2 and 6, 5.0 |ig mH purified pilin; 3 and 7,1.0
|Xg mH purified GSTPilE' protein; 4 and 8, 5.0 gg mH purified GSTPilE' protein. The top arrow indicates PilA and the bottom indicates GSTPilE'.
104 were loaded on a SDS-polyacrylamide gel using the same concentrations for each and antiPilE' antisera was unable to recognize the purified pili (Fig. 12, lanes 5 and 6) while it did recognize GSTPilE' fusion protein (Fig. 12, lanes 7 and 8). This suggests that PilE may not be a minor pilin protein. This does not rule out the possibility that PilE is such a minor component of pili that it would be undetectable under the conditions used here. In the past, immunogold electron microscopy has been used to localize the minor pilin protein located at the tip of N. gonorrhoeae pili (Rudel et al., 1995) or to determine that the disulfide loop region of PilA is exclusively exposed at the tip of P. aeruginosa pili (Lee et a i, 1994). This sensitive technique could be used to unambiguously determine if PilE is a minor pilin protein; however, the antibody used in that type of experiment must be non-cross reactive unlike the antisera prep used in these studies.
We also wanted to determine the subcellular location of PilE. Cells were fractionated as described in the Materials and Methods section in order to examine localization under nonoverproducing, physiological conditions.
Separating membrane fractions using differential extraction with 2% Sarkosyl exploits the difference between the inner membrane and the LPS containing outer membrane (Nakaido, 1994). In order to ensure that the method used accurately reflected protein localization, the location of PilD was determined using antipeptide antisera (gratefully supplied by W. Paranchych) and the outer membrane proteins were stained with Coomassie blue. Figure 13A
105 Fig. 13 Localization of PilD to the inner membrane and visualization of outer membrane proteins. (A) Western blot analysis of inner membrane fractions.
The blot was incubated with antiserum to PilD at 1:500. Lanes: 1, PAOl; 2, Rl.
(B) Coomassie stained polyacrylamide gel. Proteins commonly found in the outer membrane of P. aeruginosa (Bally et al., 1992; Hancock and Carey, 1979) are marked by arrows on the right and the molecular weight markers are as indicated in kilodaltons on the left.
106 6
1 2
43
30
21.5
14.3
6.5
figure 13
107 shows that PilD was correctly localized to the inner membrane and several
typical outer membrane proteins are visible in Figure 13B. Furthermore, PilA was shown to localize exclusively to the inner membrane as shown previously (Fig. 14B; Nunn and Lory, 1993). PilE was also found exclusively in the inner membrane fraction in PAOl (Fig. 14A). Occasionally, small
amounts of PilE were detected in the soluble fraction (cytoplasm and periplasm) but could have been due to contamination of the soluble fraction with the insoluble membrane fraction.
To examine the PilD-dependent cleavage of PilE in P. aeruginosa under physiological conditions, the size and location of PilE in DA11, a PAO mutant strain lacking PilD, was determined. PilE in strain DA11 was found to localize to the inner membrane but the size of the protein was slightly larger than that found in strain PAOl (Fig, 15). Processing of prePilE does not change its subcellular location. Only one report found that processing by a specific leader peptidase led to a change in subcellular localization of the target protein (XcpU; Bally et al ., 1992). However the preprotein in that study was overproduced which may have led to aberrant localization. Many more studies have reported that processing does not alter target protein localization including one study that also reported on XcpU (Nunn and Lory, 1993;
Pugsley, 1993a; Strom and Lory, 1991). N unn and Lory (1993) determ ined the location of XcpU under physiological conditions and found no change in its localization compared to preXcpU.
108 Fig. 14 Western blot analysis determining the subcellular location of PilE under physiological conditions. Lanes: 1 and 2, inner membrane fractions; 3 and 4, outer membrane fractions; 1 and 3, PAOl; 2 and 4, Rl. (A) Membrane fractions are compared using antiPilE antisera at 1:3000. PilE is indicated by the arrow. (B) Membrane fractions are compared using antiPAO pilin antisera at 1:5000. PilA is indicated by the arrow.
109 A 1 2 3 4
« ► ~ m \ < ~
B
PilA
figure 14
110 1 2
prePilE PilE
Fig. 15 Western blot analysis determining the size and subcellular location of
PilE in DA11. Inner membrane fractions are compared using antiPilE antisera. Lanes: 1, PAOl; 2, DA11. PrePilE and PilE are as indicated by the arrows.
I l l Immunoprecipitation using antiPAO pilin and antiPilE antisera. The
N-terminal region of type 4 pilin protein is thought to be involved in subunit-subunit interaction and/or contain important signals for pilus morphogenesis. This region is highly conserved in the pilin proteins from different species. The N-termini of PilE and type 4 pilins are also highly homologous (Chapter 3; Dalrymple and Mattick, 1987). When the pilin gene from M . bovis was expressed in P. aeruginosa, the pilus filaments produced were composed of both types of pilin monomers (Beard et al.f 1990), indicating that the different pilin proteins are capable of subunit-subunit interaction.
We do not currently believe that PilE is a minor pilus protein (see above and
Chapter 3). However, we did want to determine if a PilE-PilA interaction occurs in vivo, possibly to inhibit nonproductive PilA-PilA interaction or to chaperone the pilin subunit to the site of pilin assembly.
Immunoprecipitation experiments using antiPilE and antiPAO pilin antisera were carried out using inner membrane fractions from PAOl, DA11 (PilD-),
R1 (PilE ), and PAOl-A-. Once the immunoprecipitating antibody and the inner membrane fraction had incubated together overnight, any complexes that had formed were detected by immunobloting after complex precipitation and SDS-PAGE. Western analysis (with antiPAO pilin antisera) indicated that antiPAO pilin antisera immunoprecipitated PilA from PAOl, DA11, and R1
(Fig. 16, lanes 3, 6, and 9 respectively) but not from the strain lacking PilA (Fig.
112 Fig. 16 Western blot analysis of immunoprecipitation experiments. Antisera was added to inner membrane fractions; immunocomplexes were precipitated and separated by SDS-PAGE. The blots were detected using antiPAO pilin antisera at 1:5000. PilA is indicated by the arrows. Lanes: 1, 4,
7, and 10, antiPilE prebleed control sera used as immunoprecipitating antibody; 2, 5, 8, and 11, antiPilE antisera used as immunoprecipitating antibody; 3, 6, 9, and 12, antiPAO pilin antisera used as immunoprecipitating antibody; 1-3, PAOl; 4-6, DA11; 7-9, Rl; 10-12, PAO-A-.
113 1 2 3 4 5 6
figure 16
114 16, lane 12). Additional Western analysis of these same samples with antiPilE antisera yielded no visible unique bands, indicating that PilE is not coimmunoprecipitated with PilA and that PilE is not immunoprecipitated by antiPilE (data not shown). Because antiPilE did not immunoprecipitate PilE, the corresponding coimmunoprecipitation experiment to determine if PilA coimmunoprecipitates with PilE could not be done. However, it would be expected that if PilE was capable of interacting with PilA, PilE would coimmunoprecipitate with PilA. The lack of detection of PilE in the above experiment where antiPAO pilin antisera precipitated PilA indicates that PilE may not be capable of interaction with PilA.
It should be noted that PilE might be involved in protein-protein interaction with PilA but that the interaction is not detectable under the conditions used here such as inner membrane fractions solubilized in 2%
Sarkosyl and the entire experimental sample suspended in the mild detergent
TN-1. However, as the next series of experiments indicates, a GSTmPilE-PilE interaction was detected under the same conditions suggesting that the lack of
PilE-PilA interaction is a true representation of their actions in vivo.
Capture experiment with GSTmPilE fusion protein. The fact that PilE was not shown to interact with PilA does not mean that the N-terminus does not contribute to protein-protein interactions. PilE might form a multimer with itself in a manner analogous to PilA interaction in the pilus filament.
115 A second GST gene fusion was constructed so as to generate a protein that contained a moiety identical to the mature PilE protein (lacking the N- terminal methylation). GSTmPilE (Fig. 10) was overexpressed and purified.
For unknown reasons, this fusion protein was much more difficult than
GSTPilE' to purify in large amounts. A one liter cultures yielded less than 0.5 mg of fusion protein compared to an average of 200 mg of protein from a 300 ml culture for GSTPilE'. In both cases the full length fusion protein was always the minor product and two breakdown products, one with the same relative mobility as GST itself, were more abundant as determined by
Coomassie blue staining. No steps were taken to remove these two truncated products. GSTmPilE was used to determine which proteins, if any, from the inner membranes of PAOl, R1 and PAOl-A- with which the fusion protein was capable of interacting. The fusion protein was incubated along with inner membrane fractions from strains PAOl, R1 and PAO-A- and
Glutathione-sepharose was used to precipitate any complexes that formed.
The complexes were then separated by SDS-PAGE and transferred to nitrocellulose for Western analysis. When antiPilE was the probing antibody, a single 15 kDa band was visible when in PAOl and PAOl-A- extracts (Fig. 17, lanes 4 and 6) which was lacking in the R1 extract (Fig. 17, lane 5). However, no bands in the 15 kDa range were visible when antiPAO pilin was used (data not shown). Control experiments indicated that 20mM
116 Fig. 17 Western blot analysis of capture experiment using PAOl, Rl, and
PAO-A- inner membrane fractions. GSTmPilE was used to capture out proteins participating in protein-protein interaction with PilE in inner membrane fractions (lanes 4-6). As controls either Sepharose alone (lanes 1-3) or glutathione-Sepharose + 20mM glutathione was also use in capture experiments. Precipitated complexes were separated by SDS-PAGE and transferred to nitrocellulose. The blot was detected using antiPilE antisera at
1:3000. PilE is indicated by the arrow. Lanes: 1, 4 and 8, PAOl; 2,5 and 8, Rl;
3,7 and 10, PAO-A-.
117 reduced glutathione was able to compete with PilE for GST (lanes 8-10) and that PilE could not bind to Sepharose alone (lanes 1-3). The abundant 26 kDa protein in lanes 4-10 is most likely GST which would be uncoupled from the
Sepharose during sample preparation for SDS-PAGE. This experiment indicated that PilE is capable of protein-protein interaction with itself but that
PilE is incapable of interacting with PilA (as would have been detected in the immunoblot probed with antiPAO pilin antisera). The results of this experiment are consistent with the results from the immunoprecipitation experiment that PilE most likely does not interact with PilA. It is highly unlikely that PilA interacts with PilE and is just not detectable here. In every experiment using antiPAO pilin antisera in which PilA was found, results were quickly visible (within 10-30 sec) while results with antiPilE could take up to 3 h to develop. In these experiments PilA was not detected even after an overnight exposure. Consequently, if PilA was actually captured and precipitated in the capture experiments, we believe that it would have been detected.
Site-directed mutagenesis of Tyr 24 and Tyr 27 to Phe in pilE. The two
Tyr residues that are conserved within all type 4 pilin proteins are also found in PilE at the same relative locations (residues 24 and 27 in the mature protein). These residues in P. aeruginosa PAK pilin have been implicated in subunit-subunit interaction via tryptophan fluorescence quenching with
118 acrylamide and alkaline pH titrations (Watts et al., 1983). These two residues are predicted to reside at a location within the pilus filament that is not exposed to the environment unless the filament is disassociated in octyl glycoside. By analogy, these residues may also play a role in PilE-PilE interaction and thus in PilE function. Both of the Tyr codons were changed to
Phe codons and this mutated copy of pilE designated phe2427 was introduced into PAOl and Rl on a low copy number plasmid (pBR322 based pR01614). A wild type copy of pilE and the vector alone were also mobilized into PAOl and Rl as controls. Following fractionation, PilE and Phe2427 were found to localize to the inner membrane.
Rl harboring the mutated copy of pilE along with all of the control constructs were tested for phage sensitivity and twitching ability. Each of the strains was found to be sensitive to D3112, B3, and F116L and each produced an equivalent zone on twitching assay plates. These data indicate that the mutant PilE protein is capable of correct localization and that it functions properly. The ability of GSTmPilE to interact with this protein was also tested. Figure 18 (lane 3) shows that the fusion protein was able to interact with Phe2427 indicating that the mutant protein was also still capable of protein-protein interaction. Either these two residues are not involved in this interaction or a conserved substitution to Phe from Tyr does not impede the ability of the residues to participate in the interaction. If the reactive hydroxyl groups of the Tyr residues are responsible for the PilE-PilE
119 12 3 b ig ] ^~
Fig. 18 Western blot analysis of capture experiment using PAOl, Rl, and
Phe2427 inner membrane fractions. GSTmPilE was used to fish out proteins participating in protein-protein interaction with PilE in inner membrane fractions. Precipitated complexes were separated by SDS-PAGE and transferred to nitrocellulose. The blot was detected using antiPilE antisera at
1:3000. PilE is indicated by the arrow. Lanes: 1, PAOl; 2, Rl; 3, Phe2427.
120 interaction (and presumably PilA-PilA interaction) then the substitutions
used here should have disrupted the function of the protein. If, however, the
aromatic rings interact with other aromatic rings to stabilize the interaction
then the Phe residues would be able to supply the necessary rings for correct
interaction and function.
DISCUSSION
Production of PilE in PAOl. According to the results presented here,
the level of PilE protein does not vary over time in a LB broth grown culture.
Because PilE is involved in pilus biogenesis, one would expect that PilE
would only be needed when functional pili are being produced. Whether P. aeruginosa produces extracellular pili in broth grown cultures is unclear.
Roncero el al. (1990) concluded that functional pili are not produced in broth grown cultures. They showed that adsorption of bacteriophage D3112 and B3
was enhanced when cells were grown on solid media compared to cells grown in broth and that cells lacking pili were unable to adsorb either phage.
This seems to indicate that pili are differentially expressed on solid grown
cells compared to broth grown cells. However, it may have simply been that
the PAOl cells did make pili but they were sheared off due to shaking during incubation and thus the bacterial cells were unable to absorb phage. Electron
micrograph studies by Weiss (1971) of broth grown cells that were not shaken
121 also supports the theory that pili are made in broth grown cultures. It was shown that 64% of cells in logarithmic phase and 52% of cells in stationary phase from broth cultures were piliated. So while at least two levels of PilA regulation have been identified, control of subunit synthesis via PilR-S
(Hobbs et ai, 1993; Jin et ai, 1994; Ishimoto and Lory, 1992) and chemotactic control of pili function via PilG-K (Darzins, 1993; 1994), the environmental signal PilS responds to and the stimuli involved in chemotactic control is currently unclear. Since pili seem to be constitutively produced under laboratory growth conditions, it is not surprising that PilE is also produced under these conditions.
Production of PilE in DA11. PilE is cleaved by PilD and is also presumably methylated because it contains the conserved glutamic acid residue at the +5 position in the mature protein (Nunn and Lory, 1991). In each case that it has been determined, a wild type protein that is cleaved by
PilD is also methylated (Nunn and Lory, 1993; Strom et ai, 1993b).
Role of Tyr 24 and Tyr 27 in PilE function. The supposition the Tyr 24 and Tyr 27 residues are important in PilA-PilA interaction led us to ask the question of whether they might be important in PilE function. Only one study has attempted to analyze the role of the Tyr residues in P. aeruginosa
PilA subunit-subunit interaction (Watts et ai, 1983). We decided to use
122 genetic methods to determine if the Tyr residues at positions 24 and 27 in the mature PilE protein are necessary for pilus biogenesis in PAOl. Changing both residues to Phe did not affect the pil phenotype of PAOl; it was still phage sensitive and able to translocate over a solid surface via twitching motility. The mutant protein was also still capable of interacting with the
GSTmPilE fusion. Thus, either the conservative change of Tyr to Phe was not drastic enough to disrupt protein-protein interaction as discussed in the
Results section or the residues do not participate in the interaction. Further analysis is obviously warranted to determine which might be the case. Such experiments might include more drastic point mutations that would interrupt any type of interaction that the Tyr residues might be involved in analyzed in a manner similar to the Phe2427 mutation.
Possible role(s) of PilE. The export and assembly of type 4 pili found on many gram-negative pathogens has been compared to several extracellular secretion and DNA uptake systems (Hobbs and Mattick, 1993; Pugsley, 1993b).
The comparison of these diverse systems stems from the fact that a number of the components have significant amino acid homology. The findings in this study further support this comparison. The protein identified in this study brings to two the number of proteins that have been identified in the P. aeruginosa type 4 pilus biogenesis system that have AMerminal homology to
PilA. This is analogous to the finding of either three or four proteins in the
123 extracellular secretions systems of P. aeruginosa, K. oxytoca, A, hydrophila, X. campestris, E. carotovora, and E. chrysanlhemi and the DNA uptake system of
B. subtilis (Table 5) which have a prepilin-like leader sequence followed by a highly hydrophobic region. The prepilin-like proteins might form a multicomponent structure, possibly spanning the inner and outer membrane in gram-negative organisms. This structure may act as a conveyor system for macromolecules such as extracellular enzymes or DNA. The formation of a pseudopilus is the simplest explanation for the homology of these systems to the seemingly unrelated pilus biogenesis system of P. aeruginosa.
Aim and Mattick (1995) categorized the prepilin-like proteins into four groups based on amino acid homology within the first 20 residues of the mature proteins: XcpT, ExeG, OutG, FulG; XcpU, ExeH, OutH, PulH; XcpV,
Exel, OutI, Pull; and XcpW, ExeJ, OutJ, PulJ. The groupings as defined in
Table 6 have been expanded to include the Xps proteins and the percent homologies are determined over the first 30 residues of the mature proteins.
While the Xps proteins have a lower degree of homology to their groups than the other members, they clearly fall within the groupings (without the Xps proteins the homologies within the groups range from 47% to 97% identical while the Xps proteins are 33% to 43% identical to their respective groups).
124 proteins w ith PilB PilC PilD PilQ prepilin- hom olog hom olog hom olog hom olog like signal sequence
Protein secretion P. aeruginosa XcpR XcpS PilDa XcpQ XcpT-W Klebsiella oxytoca PulE PulF PulO PulD PulG-J Aeromonas hydrophila ExeE ExeF ExeD ExeG-J Xanthomonas campestris XpsE XpsF XpsO XpsD XpsG-J Erwinia spp. OutE OutF OutO OutD OutG-J
DNA uptake Bacillus subtilis ComG-1 ComG-2 ComC ComG3-5 a PilD in P. aeruginosa processes proteins in both the Pil and Xcp systems (Nunn and Lory, 1993; Strom et a i, 1993b). b References: Aim and Mattick, 1995; Howard et ai, 1993; Hu et ai, 1992; Pugsley, 1993b.
Table 5. Homologs of the P. aeruginosa Pil proteins and proteins with prepilin leader sequenceb. On the other hand, the ComG3-5 proteins of B. subtilis do not fall into this group. They are instead more homologous to the type 4 pilin proteins (Fig.
19; Albano et al ., 1989).
PilV is most closely aligned with the XcpV group but its homology to the consensus sequence is lower than the other members of that group
(27%;Alm and Mattick, 1995). PilV is 33% identical to the N-terminus of PilA which is much lower than PilE homology to PilA, but PilV does have two Cys residues capable of forming a 12 amino acids disulfide loop at the same relative location as P. aeruginosa pilin proteins. The function of this region in PilA is to mediate adherence to cellular receptors (Lee et ai, 1989; 1994).
Early work in E. coli seemed to indicate that the C-terminus was also involved in subunit-subunit interaction (Pasloske et a i, 1988), but recently a mutant was generated in which 9 amino acids of the C-terminus in PilA were replace with 11 new amino acids. This mutant was still phage sensitive and intact pili were seen via electron microscopy (Farinha et at., 1994). This mutant was, however, reduced in its ability to bind target cells in vitro indicating that the disulfide loop domain is only involved in adherence. The function of this region in PilV is unclear but the addition of a 10 amino acid tag to the C-terminus rendered cells resistant to pilus-specific phage and unable to translocate across solid surfaces (Farinha et at., 1994). Unlike PilV,
PilE is most homologous to the type 4 pilin proteins but it is still more homologous to the XcpT and XcpU groups (37% and 33% respectively)
126 Fig. 19 Comparison of PilE to PilA and other prepilin-like proteins. Upper case letters represent residues identical to PilE. T/G, XcpT/PulG group; U/H,
XcpU/PulH group; V/I, XcpV/PulI group; W/J, XcpW/PulJ group. Sequence alignment generated by a BLAST search and consensus sequences were generated by DNA Star. The sequences are followed by the percent identity of the first 30 residues of the mature protein or group to PilE.
127 figure 17 FTLLEMWWAVIGILLGIAIPSYQNYVIR + 1 1 + 10 20 30 T/G T/G FTLLEimWivilGILaslvvPnlmgnkek 37% PILE PILA FTLiElmiWAilGILaalAIPqYQNYVaR 73% ComG3 ComG3 ComG4 ilsILLUtIPnvtkhnqt FTLvEMliVlf FTLLEsllVlslasILLvavfttlppatdn 43% 30% U/H FTLLEMmlVxllIGvaaGlvvlafggardd 33% ComG5 ComG5 FstiEtmsalslwlfvLltvvplwdklmad 10% W/J FTLLEMllaiAifa alslaayQvlqgv 27% V/I V/I mTLiEvlValAVfalaglavlqatarqasq 20%
128 than PilV is to the XcpV group (Fig. 19). PilE may act in a manner analogous to that proposed for the other prepilin-like proteins and participate in the formation of a pilus-like structure. The function of such a structure containing PilE might be to act as a scaffolding for pilus biogenesis or an anchor for the filament in the cell envelope. While no evidence was found that PilE interacts with PilA, PilE was able to interact with the PilE fusion protein indicating that PilE might be capable of homomultimer formation.
The hypothetical structure may be composed of PilE multimers along with other prepilin-like proteins such as PilV or other, as yet unidentified proteins.
In support of a complex containing PilE and PilV, both have been localized to the inner membrane and contain homology the N-terminal region of pilin protein implicated in type 4 pilin subunit interaction (Aim and Mattick, 1995;
Watts et ai, 1983). Further studies such as a capture experiment or cross- linking analysis via silver stained polyacrylamide gels are needed to determine if PilE is capable of protein-protein interactions with proteins other than PilA or itself.
To date very little data has been collected to support pseudopilus formation. The lack of supportive evidence may simply be because the structure is transient and difficult to detect. It is also possible that the conditions used may destablized the hypothetical structure (Pugsley and
Possot, 1993), Despite searching for evidence of a multiprotein complex,
Pugsley and Possot (1993) were unable to identify mixed multimers
129 containing the prepilin-like protein PulG involved in pullulanase secretion in K. oxytoca. PulG was shown to exist mainly as homodimers and did not seem to interact with other Pul proteins. One encouraging piece of evidence is that PulG initially extracted from membranes at pH 9.5 was induced to form a sedimentable complex when the pH was lowered to 7.5 as do type 4 pilin proteins. This is the first indication of protein complex formation in a manner similar to type 4 pili by prepilin-like proteins.
Why PilE is highly similar to the pilin proteins like the ComG3-5 proteins instead of exclusively falling into one of the Xcp groups as PilV does is unclear. For reasons discussed in Chapter 3 and the lack of evidence found in this study, it is unlikely that PilE is a minor pilin protein, so the homology to PilA is perplexing and lends credence to the possibility that PilE may act as a PilA chaperone as initially discussed in Chapter 3. Perhaps PilE is a molecular chaperone which shuttles pilus subunits between the inner membrane and the site of pilus biogenesis as do the periplasmic chaperones found in all other well characterized gram-negative pilus systems (Holmgren et ai, 1992). The periplasmic pilus chaperones have a three-dimensional structure similar to that of immunoglobulin variable domains and the proteins are highly homologous to each other (Holmgren et a i, 1992; Kuehn et a i, 1993). Examination of the P pilus system in E. coli illustrates why a periplasmic chaperones is necessary. The P pilus is made up of several different proteins, the major structural protein, two different initiator
130 proteins, an adaptor protein, a terminator protein and the tip-associated adhesin (Hultgren et al, 1991) and each must travel from the cytoplasm to the site of pilus assembly, the outer membrane protein PapC. While traversing the periplasm, each type of protein is capped by PapD, a periplasmic chaperone, to prevent nonproductive interactions and proteolytic degradation (Kuehn et al., 1991).
Type 4 pili differ from P pili in that, to date, only one protein has been identified in the pilus structure and the adhesin moiety is associated with that protein. It is currently unclear as to how the type 4 pilus filament is attached to the bacterial cell; whether it is embedded in the outer membrane or at a site of junction between the membranes is not known. In either case, PilA is found in pools in the inner membrane prior to pilus biogenesis (Nunn and
Lory, 1993) and probably during retraction (Pasloske et al, 1989). PilE may function to cap interactive surfaces and prevent nonproductive interaction while the pilin subunits are in a monomeric state. If this is the role of PilE, it must carry out this role in a novel manner unlike the members of the periplasmic chaperone family. The predicted secondary structure of PilE does not indicate the level of beta sheets found in the immunoglobulin superfamily and there is no homology between PilE and the periplasmic chaperones. Furthermore, PilE resides in the inner membrane, not the periplasm, and we were unable to demonstrate a PilE-PilA interaction.
However, it should be noted that in order to demonstrate chaperone-pilin
131 protein interactions in E. coli, the genes encoding the proteins of interest were mobilized into host cells on plasmids lacking the gene coding the major pilin subunit and the gene coding the outer membrane protein essentially eliminating pilus formation which caused a build up of the desired complex and allowed for its detection (Hultgren et a i, 1989; Jones et a i, 1993; Kuehn et a i, 1991; Lindberg et a i, 1989). The pilus biogenesis system in P. aeruginosa is not plasmid borne complicating the process of generating a strain lacking type
4 pili by creating the need for a chromosomally located gene to be inactivated.
And, since the only pilin protein identified so far is PilA, knocking out the pilA gene would not aid in identifying a chaperone-pilin protein complex in
P. aeruginosa. Assuming PilQ is the site of pilus assembly, a pilQ strain would presumably generate a situation similar to that used in £. coli chaperone-pilin protein investigations and might allow for isolation of a presumably transient PilE-PilA interaction if one occurs in vivo.
Due to the absence of supportive data for the latter theory, I believe that the former theory more likely approximates the role of PilE; however, there is still much work to be done to actually determine the role of PilE in pilus biogenesis by P. aeruginosa.
132 Sum m ary
The goal of this project was to identify the mutation responsible for the pil phenotype in P. aeruginosa strain MJ-6 and to characterize the gene and the gene product. The MJ-6 complementing locus was narrowed to a 1 kb
EcoKV-Clal fragment. The entire fragment was sequenced and only one complete open reading frame characteristic of P. aeruginosa ORFs was found, pilE. It appears to be a single transcriptional unit preceded by a typical -10 and
-35 region and a possible rbs and followed by a potential r/io-independent terminator.
A protein of approximately 15 kDa was predicted from the deduced amino acid sequence and was also seen in overexpression experiments in P. aeruginosa. The N-terminus of PilE was found to be highly homologous to
PilA, the P. aeruginosa pilin protein, including a consensus for PilD cleavage and methylation. Cleavage by PilD was demonstrated in this study. The phenotype of strains lacking functional PilE indicated that it is involved in pilus biogenesis. PilE may be capable of homomultimerization but does not appear to interact with PilA. Due to this finding and parallels with other homologous systems, it is proposed that PilE is involved in a multicomponent structure that plays a role in pilus biogenesis possibly by acting as a scaffold or conveyor system for the filament.
133 APPENDIX A
Fig. 20 Sequence alignment of P. aeruginosa ORFB' with E. coli ORF316. The
GAP and PUBLISH programs from the University of Wisconsin Genetics
Computer Group (GCG) were used to generate the alignments. The vertical bars indicate identical amino acids and colons and periods indicate two degrees of similarity of amino acids.
134 ORFB' 1 ...... ALRAKFPQIQGPRKNDICYAT 21 IIIr i i *11-1-if i iiii*ijiiii1111*111111 ORF316 151 WKLTVKNEEKLSFMTQTTLSVDDTSDVIDALRKRFPKIVGPRKDDICYAT 200
■ « • • • ORFB' 22 QNRQDAVKELADQCDMVLWGSPNSSNSNRLRE LAERMGTPAYLIDGAED 71 1111*11* 1 1 * 1 1 * ■ 11*11 1 * 1 - **111111* ■ 1 1 I 11 1 • 1 1II 1 1 1 1 1[ 1 111*111lil-lll**l*!ll*l*l | . | | | . I | ORF 316 201 T NRQE AVRAL AE QAEWLWGSKNSS NS NRL AE L AQRMGKRAF LID DAKD 250
ORFB' 72 MQRGWFDGVRRIGITAAASAPEVLVRGVIAQLREWGASEEQELEGREENI 121 • !1 *1• 1 • * • 1 *• • 1 *• Ii 1 • 1 I1 I1 1 • • 1 1 • • 1 1*1 * 1 * t 1 • •* *• 1 *• *• 1 * * llllllll1 II I I 1 1 1 ORF316 251 IQEEWVKEVKCVGVTAGASAPDILVQNWARLQQLGGGEAIPLEGREENI 300
ORFB' 122 TFSMPKELRVKAL... 134 * 1 • ■ 1 1 1 1 1 I * ORF316 301 VFEVPKELRVDIREVD 316
figure 19 APPENDIX B
Fig. 21 Cartoon depiction of PilA and PilE proteins. The dark grey regions are the regions of highest homology among type 4 pilin proteins (and PilE). The lighter grey regions are semi-conserved among type 4 pilin proteins. The vertical arrows mark the position of PilD cleavage in each protein. The horizontal arrows mark the positions of the two Cys residues in each protein.
These residues form a disulfide loop in P. aeruginosa PAK pili which is only exposed at the tip of the filament and act as the adhesin moiety of P. aeruginosa pili. It is unknown if a disulfide loop is formed in PilE. The proteins are drawn to scale.
136 PilE
figure 20 REFERENCES
Akrim, M., Bally, M., Ball, G., Tommassen, J., Teerink, H., Filloux, A., and Lazdunski, A. (1993) Xcp-mediated protein secretion in Pseudomonas aeruginosa: identification of two additional genes and evidence for regulation of xcp gene expression. Mol Microbiol 10:431-443.
Aksamit, T.R. (1993) Pseudomonas aeruginosa pneumonia and bacteremia in the immunocompromised patient. In Pseudomonas aeruginosa the opportunist: pathogenesis and disease. Fick, Jr.R.B. (eds.).Florida: CRC Press, pp. 177-188.
Albano, M., Breitling, R., and Dubnau, D.A. (1989) Nucleotide sequence and genetic organization of the Bacillus subtilis comG operon. J Bacteriol 171:5386-5404.
Aim, R.A., Bodero, A.J., Free, P.D. and Mattick, J.S. (1996) Identification of a novel gene, pilZ , essential for type 4 fimbrial biogenesis in Pseudomonas aeruginosa. J Bacteriol 178:40-53.
Aim, R.A., and Mattick, J.S. (1995) Identification of a gene, pilV, required for type 4 fimbrial biogenesis in Pseudomonas aeruginosa, whose product possesses a pre-pilin-like leader sequence. Mol Microbiol 16;485-496.
Ames, P., DesJardins, D., and Pier, G.B. (1985) Opsonophagocytic killing activity of rabbit antibody to Pseudomonas aeruginosa m ucoid exopolysaccharide. Infect Immun 49:281-285.
Apodaca, G., Bomsel, M., Lindstedt, R., Engel, J., Frank, D., Mostov, K.E., and Weiner-Kronish, J. (1995) Characterization of Pseudomonas aeruginosa- induced MDCK cell injury: glycosylation-defective host cells are resistant to bacterial killing. Infect Immun 63:1541-1551.
Arnold, J.W., and Shimkets, L.J. (1988) Cell surface properties correlated with cohesion in Myxococcus xanthus J Bacteriol 170:5771-5777.
138 Artenstein, A.W., and Cross, A.S. (1993) Local and disseminated diseases caused by Pseudomonas aeruginosa. In Pseudomonas aeruginosa as an opportunistic pathogen. Campa, M.„ Bendinelli, M., and Friedman, H. (eds.). New York: Plenum Press, pp. 223-244.
Baker, N.R. (1993) Mucosal adherence of Pseudomonas aeruginosa. In Pseudomonas aeruginosa the opportunist: pathogenesis and disease. Fick Jr. R.B. (ed.). Florida: CRC Press, pp. 7-23.
Baker, N., Hansson, G.C., Leffler, H., Riise, G., and Svanborg-Eden, C. (1990) Glycosphingolipid receptors for Pseudomonas aeruginosa. Infect Immun 58:2361-2366.
Baker, N.R., Minor, V., Deal, C., Shahrabadi, M.S., Simpson, D.A., and Woods, D.E. (1991) Pseudomonas aeruginosa exoenzyme S is an adhesin. Infect Immun 59:2859-2863.
Baker, N.R., and Svanborg-Eden, C. (1989) Role of alginate in the adherence of Pseudomonas aeruginosa. Antibiot Chemother 42:72-79.
Bally, M., Ball, G., Badere, A., and Lazdunski, A. (1991) Protein secretion in Pseudomonas aeruginosa: the xcpA gene encodes an integral inner membrane protein homologous to Klebsiella pneumoniae secretion function protein PulO. J Bacteriol 173:479-486
Bally, M., Filloux, A., Akrim, M., Ball, G., Lazdunski, A., and Tommassen, J. (1992) Protein secretion in Pseudomonas aeruginosa: characterization of seven xcp genes and processing of secretory apparatus components by prepilin peptidase. Mol Microbiol 6:1121-1132.
Baltimore, R.S. (1993) Mucoid colony variants: the exopolysaccharide of Pseudomonas aeruginosa and microcolony formation. In Pseudomonas aeruginosa the opportunist: pathogenesis and disease. Fick, Jr., R.B. (ed.). Florida: CRC Press, pp. 25-40.
Baltimore, R.S., and Mitchell, M. (1980) Immunological investigations of mucoid strains of Pseudomonas aeruginosa: Comparison of susceptibility to opsonic antibody in mucoid and nonmucoid stains. J Infect Dis 141:238- 247.
Beard, M.K.M., Mattick, J.S., Moore, L.J., Mott, M.R., Marrs, C.F., and Egerton, J.R. (1990) M orphogenetic expression of Moraxella bovis fimbriae (pili) in Pseudomonas aeruginosa. J Bacteriol 172:2601-2607.
139 Bellido, F., and Hancock, R.E.W. (1993) Susceptibility and resistance of Pseudomonas aeruginosa to antimicrobial agents. In Pseudomonas aeruginosa as an opportunistic pathogen. Campa, M., Bendinelli, M., and Friedman, H. (eds.). New York: Plenum, pp. 297-319.
Berk, R.S., Brown, D., Coutinho, I., and Meyers, D. (1987) In vivo studies with two phospholipase C fractions from Pseudomonas aeruginosa. Infect Im m un 55:1728-1730.
Bodey, G.R., Bolivar, R., Fainstein, V., and Jadeja, L. (1983) Infections caused by Pseudomonas aeruginosa. Rev Infect Dis 5:279-313.
Botzenhart, K., and Doming, G. (1993) Ecology and epidemiology of Pseudomonas aeruginosa. In Pseudomonas aeruginosa as an opportunistic pathogen. Campa, M., Bendinelli, M., and Friedman, H. (eds.). New York: Plenum Press, pp. 1-18.
Bouvier, J., Richaud, C., Richaud, F., Patte, J.-C., and Stragier, P. (1984) Nucleotide sequence and expression of the Escherichia coli dapB gene. J Biol Chem 259:14829-14834.
Bouvier, J. and Stragier, P. (1990) Nucleotide sequence of the Isp-dapB interval in Escherichia coli. Nuc Acids Res 19:180.
Boyd, J.M., Koga, T., and Lory, S. (1994) Identification and characterization of PilS, an essential regulator of pilin expression in Pseudomonas aeruginosa. Mol Gen Genet 243:565-574.
Boyd, J.M., and Lory, S. (1996) Dual function of PilS during transcriptional activation Pseudomonas aeruginosa pilin subunit gene. J Bacteriol 178:831-839.
Bradley, D.E. (1972a) A study of pili on Pseudomonas aeruginosa. Genet Res Camb 19:39-51.
Bradley, D.E. (1972b) Shortening of Pseudomonas aeruginosa pili after RNA- phage adsorption. J Gen Microbiol 72:303-319.
Bradley, D.E. (1973) Basic characterization of a Pseudomonas aeruginosa pilus- dependent bacteriophage with a long noncontractile tail. J Virol 12:1139- 1148.
140 Bradley, D.E. (1974a) The adsorption of Pseudomonas aeruginosa pilus- dependent bacteriophages to a host mutant with nonretractile pili. Virology 58:149-163.
Bradley, D.E. (1974b) Pilus-dependence of four Pseudomonas aeruginosa bacteriophages with non-contractile tails. J Gen Virol 23:1-15. Bradley, D.E. 1980. A function of Pseudomonas aeruginosa PAO polar pili: twitching motility. Can J Microbiol 26:146-154.
Bradley, D.E., and Pitt, T.L. (1974) Pilus-dependence of four Pseudomonas aeruginosa bacteriophages with non-contractile tails. J Gen Virol 23:1-15.
Brammer, W.J., and Clarke, P.H. (1964) Induction and repression of Pseudomonas aeruginosa amidase. J Gen Microbiol 37:307-319.
Brunschwig, E. and Darzins, A. (1992) A two-component T7 system for the overexpression of genes in Pseudomonas aeruginosa. Gene 111:35-41.
Carnoy, C., Scharfman, A., Van Brussel, E., Lamblin, G., Ramphal, R., and Roussel, P. (1994) Pseudomonas aeruginosa outer membrane adhesins for human respiratory mucus glycoproteins. Infect Immun 62:1896-1900.
Chamberlain, J.P. (1979) Fluorographic detection of radioactivity in polyacrylamide gels with the water soluble fluor, sodium salicylate. Anal Biochem 98:132-135.
Christie, P.J., Ward, J.E., Gordon, M.P., and Nester, E.W. (1989) A gene required for transfer of T-DNA to plants encodes an ATPase with autophosphorylating activity. Proc Natl Acad Sci USA 86:9677-9681.
Chung, Y.S., and Dubnau, D. (1995) ComC is required for the processing and translocation of ComGC, a pilin-like competence protein of Bacillus subtilis. Mol Microbiol 15:543-551.
Coburn, J., and Gill, D.M. (1991) ADP-ribosylation of p21ras and related proteins by Pseudomonas aeruginosa exoenzyme S. Infect Immuno 59:4259-4262.
Coburn, J., Kane, A.V., Feig, L., and Gill, D.M. (1991) Pseudomonas aeruginosa exoenzyme S requires a eucaryotic protein for ADP- ribosyltransferase activity. J Biol Chem 266:6438-6446.
141 Coburn, J., Wyatt, R.T., Iglewski, B.H., and Gill, D.M. (1989) Several GTP- binding proteins, including p21e-Hras,are preferred substrates of Pseudomonas aeruginosa exoenzyme S. J Biol Chem 264:9004-9008.
Cother E.J., Darbyshire, B., and Brewer, J. (1976) Pseudomonas aeruginosa: cause of internal brown rot of onion. Phytopathology 66:828-834.
Cross, A.S., Sadoff, J.C., Iglewski, B.H., and Sokol, P.A. (1980) Evidence for the role of toxin A in the pathogenesis of infection with Pseudomonas aeruginosa in humans. J Infect Dis 142:538-546.
Dalrymple, B., and Mattick, J.S. (1987) An analysis of the organization and evolution of type 4 fimbrial (MePhe) subunit proteins. J Mol Evol 25:261- 269.
Darzins, A. (1993) The pilG gene product, required for Pseudomonas aeruginosa pilus production and twitching motility, is homologous to the enteric, single domain response regulator CheY. J Bacteriol 175: 5934-5944.
Darzins, A. (1994) Characterization of a Pseudomonas aeruginosa gene cluster involved in pilus biosynthesis and twitching motility: Sequence similarity to the chemotaxis proteins of enterics and the gliding bacterium Myxococcus xanthus. Mol Microbiol 11:137-153.
Darzins, A. (1995) The Pseudomonas aeruginosa pilK gene encodes a chemotactic methyl transferase (CheR) homologue that is translationally regulated. Mol Microbiol 15:703-717.
Darzins, A. and Casadaban, M.J. (1989a) Mini-D3112 bacteriophage transposable elements for genetic analysis of Pseudomonas aeruginosa. J Bacteriol 171:3909-3916.
Darzins, A. and Casadaban, M.J. (1989b) In vivo of Pseudomonas aeruginosa genes with mini-D3112 transposable bacteriophage. J Bacteriol 171:3917- 3925.
Darzins, A., and Chakrabarty, A.M. (1984) Cloning of genes controlling alginate biosynthesis from a mucoid cystic fibrosis isolate of Pseudomonas aeruginosa. J Bacteriol 159:9-18.
Darzins, A., and Russell, M.A. (1996) Molecular genetic analysis of type-4 pilus biogenesis and twitching motility using Pseudomonas aeruginosa as a model system. Gene, in review.
142 d'Enfert, C., and Pugsley, A.P. (1989) Klebsiella pneumoniae pulS gene encodes an outer membrane lipoprotein required for pullulanase secretion. J Bacteriol 171:3673-3679. d'Enfert, C., Reyss, I. Wandersman, C., and Pugsley, A.P. (1989) Protein secretion by Gram-negative bacteria. Characterization of two membrane proteins required for pullulanase secretion by Escherichia coli K-12. J Biol Chem 264:17462-17468.
Deretic, V., Gill, J.F., and Chakrabarty, A.M. (1987) Gene algD coding for GDP mannose dehydrogenase is transcriptionally activated in mucoid Pseudomonas aeruginosa. J Bacteriol 169:351-358.
Deretic, V., Schurr, M.J., Boucher, J.C., and Martin, D.W. (1994) Conversion of Pseudomonas aeruginosa to mucoidy in cystic fibrosis: environmental stress and regulation of bacterial virulence by alternative sigma factors. J Bacteriol 176:2773-2780.
Devereux, J., Haeberli, P., and Smithies, O. (1984) A comprehensive set of sequence analysis programs for the VAX. Nucl Acids Res 12:387-395.
Doig, P., Sastry, P.A., Hodges, R.S., Lee, K.K., Paranchych, W. and Irvin, R.T. (1990) Inhibition of pilus-mediated adhesion of Pseudomonas aeruginosa to human buccal epithelial cells by monoclonal antibodies directed against pili. Infect Immun 58:124-130.
Doig, P., Smith, N.R., Todd, T., and Irvin, R.T. (1987) Characterization of the binding of Pseudomonas aeruginosa alginate to human epithelial cells. Infect Immun 55:1517-1522.
Doig, P., Todd, T., Sastry, P. A., Lee, K.K., Hodges, R.S., Paranchych, W. and Irvin, R.T. (1988) Role of pili in adhesion of Pseudomonas aeruginosa to human respiratory epithelial cells. Infect Immun 56:1641-1646.
Doring, G. (1993) Chronic Pseudomonas aeruginosa lung infection in cystic fibrosis patients. In Pseudomonas aeruginosa as an opportunistic pathogen. Campa, M., Bendinelli, M., and Friedman, H. (eds.). New York: Plenum Press, pp. 245-273.
Drake, D., and Montie, T.C. (1988) Flagella, motility and invasive virulence of Pseudomonas aeruginosa. J Gen Microbiol 134:43-52.
143 Drake, S.L., and Koomey, M. (1995) The product of the pilQ gene is essential for the biogenesis of type IV pili in Neisseria gonorrhoeae. Mol Microbiol 18:975-986.
Dropulic, L.K., Leslie, J.M., Eldred, L.J., Zenilman, J., and Sears, C.L. (1995) Clinical manifestations and risk factors of Pseudomonas aeruginosa infection in patients with AIDS. J Infect Dis 171:930-937.
Elleman, T.C. (1988) Pilins of Bacteroides nodosus: Molecular basis of serotypic variation and relationships to other bacterial pilins. Microbiol Rev 52:233-247.
Elleman, T.C., Hoyne, P.A., Stewart, D.J., McKern, N.M., and Peterson, J.E. (1986) Expression of pili from Bacteroides nodosus in Pseudomonas aeruginosa. J Bacteriol 168:574-580.
Elrod, R.P., and Braun, A.C. (1942) Pseudomonas aeruginosa; its role as a plant pathogen. J Bacteriol 44:633-644.
Farinha, M.A., Conway, B.D., Glasier, L.M.G., Ellert, N.W., Irvin, R.T., Sherburne, R., and Paranchych, W. (1994) Alteration of the pilin adhesin of Pseudomonas aeruginosa PAO results in normal pilus biogenesis but a loss of adherence to human pneumocyte cells and decreased virulence in mice. Infect Immun 62:4118-4123.
Fick, Jr., R. B. (1993) Pseudomonas aeruginosa-the microbial hyena and its role in disease: an introduction. In Pseudomonas aeruginosa the opportunist: pathogenesis and disease. Fick, Jr., R.B. (ed.). Florida: CRC Press, pp. 1-5.
Fick, Jr.,R.B., Baltimore, R.S., Squire, S.U., and Reynolds, H.V. (1985) IgG proteolytic activity of Pseudomonas aeruginosa in cystic fibrosis. J Infect Dis 151:589-597.
Figurski, D.H., and Helinski, D.R. (1979) Replication of an origin-containing derivative of plasmid RK2 dependent on a plasmid function in trans. Proc Natl Acad Sci USA 76:1648-1652.
Filloux, A., Bally, M., Ball, G., Akrim, A., Tommassen, J., and Lazdunski, A. (1990) Protein secretion in Gram-negative bacteria: transport across the outer membrane involves common mechanisms in different bacteria. EMBOJ 9:4323-4329.
144 Fleiszig, S.M.J., Zaidi, T.S., Fletcher, E.L., Preston, M.J., and Pier, G.B. (1994) Pseudomonas aeruginosa invades corneal epithelial cells during experimental infection. Infect Immun 62:3485-3493.
Folkhard, D., Marvin, D.A., Watts, T.H., and Paranchych, W. (1981) Structure of polar pili from Pseudomonas aeruginosa strains K and O. J Mol Biol 149:79-93.
Freitag, N.E., Steven, H., Seifert, S., and Koomey, M. (1995) Characterization of the pilF-pilD pilus-assembly locus of Neisseria gonorrhoeae. Mol Microbiol 16:575-586.
Frost, L.S., Carpenter, M. and Paranchych, W. (1978) N-methyl-phenylalanine at the N-terminus of Pseudomonas aeruginosa K pili. Nature 271:87-89.
Fu, H., Coburn, H., and Collier, R.J. (1993) The eukaryotic host factor that activates exoenzyme S is a member of the 14-3-3 protein family. Proc Natl Acad Sci USA 90:2320-2324.
Galloway, D.R. (1993) Role of exotoxins in the pathogenesis of P. aeruginosa infections. In Pseudomonas aeruginosa as an opportunistic pathogen. Campa, M., Bendinelli, M., and Friedman, H. (eds.). New York: Plenum Press, ppl07-127.
Gilligan, P.H. (1991) Microbiology of airway disease in patients with cystic fibrosis. Clin Microbiol Rev 4:35-51.
Gimeno, C., Navarro, D., Savall, F., Millas, E., Farga, M.A., Garau, J., Cisterna, R., and Garcia-de-Lomas, J. (1996) Relationship between outer membrane protein profiles and resistance to ceftazidime, imipemen, and ciprofloxacin in Pseudomonas aeruginosa isolates from bacteremic patients. Eur J Clin Micrbiol Dis 15:82-85.
Goldflam, M. and Rowe, J.J. (1983) Evidence for gene sharing in the nitrate reduction systems of Pseudomonas aeruginosa. J Bacteriol 155:1446-1449.
Govan, J.R.W., and Fyfe, J.A.M. (1978) Mucoid Pseudomonas aeruginosa and cystic fibrosis: Resistance of the mucoid form to carbencillin, flucloxacillin and tobramycin and the isolation of mucoid variants in vitro. J Antimicrob Chemother 4:233-240.
Gupta, S.K., Berk, R.S., Masinick, S., and Hazlett, L.D. (1994) Pili and lipopolysaccharide of Pseudomonas aeruginosa bind to the glycolipid asialo GM1. Infect Immun 62:4572-4579.
145 Haas, D. and Holloway, B.W. (1976) R factor variants with enhanced sex factor activity in Pseudomonas aeruginosa. Mol Gen Genet 144:243-251.
Hancock, R.E.W., and Carey, A.M. (1979) Outer membrane of Pseudomonas aeruginosa: Heat- and 2-Mercaptoethanol-modifiable proteins. J Bacteriol 140:902-910.
Harkey, C.W., Everiss, K.D., and Peterson, K.M. (1994) The Vibrio cholerae toxin-coregulated-pilus gene tcpl encodes a homolog of methyl-accepting chemotaxis proteins. Infect Immun 62:2669-2678.
Heck, L.W., Alarcon, P.G., Lulhavy, R.M., Morihara, K., Russell, M.W., and Mestecky, J.F. (1990) Degradation of IgA proteins by Pseudomonas aeruginosa elastase. J Immun 144:2253-2257.
Heck, L.W., Morihara, K., and Abrahamson. D.R. (1986a) Degradation of soluble laminin and depletion of tissue-associated basement membrane laminin by Pseudomonas aeruginosa elastase and alkaline protease. Infect Immun 54:149-153.
Heck, L.W., Morihara, K., McRae, W.B., and Miller, E.J. (1986b) Specific cleavage of human type III and IV collagens by Pseudomonas aeruginosa elastase. Infect Immun 51:115-118.
Henikoff, S. (1984) Unidirectional digestion with exonuclease III creates targeted breakpoints for DNA sequencing. Gene 28:351-359.
Henrichsen, J. (1972) Bacterial surface translocation: a survey and a classification. Bacteriol Rev 36:478-503.
Henrichsen, J. (1983) Twitching motility. Ann Rev Microbiol 37:81-93.
Hermodson, M.A., Chen, K.C.S., and Buchanan, T.M. (1978) Neisseria pili proteins: amino-terminal amino acid sequences and identification of an unusual amino acid. Biochem 17:442-445.
Hobbs, M., Collie, E.S.R., Free, P.D., Livingston, S.P., and Mattick, J.S. (1993) PilS and PilR, a two component transcriptional regulatory system controlling expression of type 4 fimbriae in Pseudomonas aeruginosa. Mol Microbiol 7:669-682.
Hobbs, M., and Mattick, J.S, (1993) Common components in the assembly of type 4 fimbriae, DNA transfer systems, filamentous phage and protein
146 secretion apparatus: a general system for the formation of surface- associated protein complexes. Mol Microbiol 10:233-242.
Hofte, M., Mergeay, M., and Verstraete, W. (1990) Marking the rhizopseudomonas strain TNSKj with a Mu d (lac) element for ecological studies. Appl Environ Microbiol 56:1046-1052.
Hokama, A. and Iwanaga, M. (1991) Purification and characterization of Aeromonas sobria pili, a possible colonization factor. Infect Immun 59:3478-3483.
Holloway, B.W, (1955) Genetic recombination in Pseudomonas aeruginosa. J Gen Microbiol 13:572-581.
Holloway, B.W., Krishnapillai, V., and Morgan, A.F. (1979) Chromosomal genetics of Pseudomonas. Microbiol Rev 43:73-102.
Holloway, B.W., O'Hoy, and M atsumoto, H. (1987) Pseudomonas aeruginosa PAO. In Genetic Maps. O'Brien, S.J. (ed). Cold Spring Harbor Laboratory, Cold Spring Harbor, NY., pp.213-221.
Holloway, B.W. and van de Putte, P. (1968) Lysogeny and bacterial recombination. In Replication and recombination of genetic material. Peacock, W.J. and Brock, R.D. (eds). Australian Academy of Sciences, Canberra, pp. 175-183.
Holmgren, A., Kuehn, M.J., Branden, C-I, and Hultgren, S.J. (1992) Conserved immunoglobulin-like features in a family of periplasmic pilus chaperones in bacteria. EMBO 11:1617-1622.
Howard, S.P., Critch, J., and Bedi, A. (1993) Isolation and analysis of eight exe genes and their involvement in extracellular protein secretion and outer membrane assembly in Aeromonas hydrophila. J Bacteriol 175:6695-6703.
Hoyne, P.A., Haas, R., Meyer, T.F., Davies, J.K. and Elleman, T.C. (1992) Production of Neisseria gonorrhoeae pili (fimbriae) in Pseudomonas aeruginosa. J Bacteriol 174:7321-7327.
Hu, N.-T., Hung, M.-N.,Chiou, S.-J., Tang, F., Chiang, D.-C, Huang, H.-Y., and Wu, C.-Y. (1992) Cloning and characterization of a gene required for the secretion of extracellular enzymes across the outer membrane by Xanthomonas campestris pv. campestris. J Bacteriol 174:2679-2687.
147 Hu, N.-T., Lee, P.-F., and Chen, C. (1995) The type IV pre-pilin leader peptidase of Xanthomonas campestris pv. campestris is functional without conserved cysteine residues. Mol Microbiol 18:769-777.
Hultgren, S.J., Lindberg, F., Magnusson, G., Kihlberg, J., Tennent, J.M., and Normark, S. (1989) The PapG adhesion of uropathogenic Escherichia coli contains separate regions for receptor binding and for the incorporation into the pilus. Proc Natl Acad Sci 86:4357-4361.
Hultgren, S.J., Normark, S. and Abraham, S.N. (1991) Chaperone-assisted assembly and molecular architecture of adhesive pili. Ann Rev Microbiol 45:383-415.
Iglewski, B.H., and Kabat, D. (1975) NAD-dependent inhibition of protein synthesis by Pseudomonas aeruginosa toxin. Proc Natl Acad Sci USA 72:2284-2288.
Iglewski, B.I„ Sadoff, J., Bjorn, M.J., and Maxwell, E.S. (1978) Pseudomonas aeruginosa exoenzyme S: an adenosine diphosphate ribosyltransferase distinct from toxin A. Proc Natl Acad Sci USA 75:3211-3215.
Iglewski, B.H. Sadoff, J.C., Bjorn, M.J., and Maxwell, E.S. (1976) Pseudomonas aeruginosa exoenzyme S: an adenosine diphosphate ribosyltransferase distinct from toxin A. Proc Natl Acad Sci USA 75:3211-3215
Imundo, L., Barasch, J., Prince, A., and Al-Awqati, Q. (1995) Cystic fibrosis epithelial cells have a receptor for pathogenic bacteria on their apical surface. Proc Natl Acad Sci USA 92:3019-3023.
Innis, M.A., Tokunaga, M., Williams, M.E., Loranger, J.M., Chang, S.-Y., Chang, S., and Wu, H.C. (1984) Nucleotide sequence of the Escherichia coli prolipoprotein signal peptidase (Isp) gene. Proc Natl Acad Sci USA 81:3708- 3712.
Irvin, R.T. (1990) Hydrophobicity of proteins and bacterial fimbriae. In Microbial Cell Surface Hydrophobicity. Doyle, R.J. and Rosenberg, M. (eds.). Washington, D.C.: American Society for Microbiology, pp.137-177.
Irvin, R.T. (1993) Attachment and colonization of Pseudomonas aeruginosa: role of the surface structures. In Pseudomonas aeruginosa as an opportunistic pathogen. Campa, M., Bendinelli, M., and Friedman, H. (eds.). New York: Plenum, pp.19-42.
148 Irvin, R.T., Doig, P., Lee, K.K., Sastry, P.A., Paranchych, W., Todd, T., and Hodges, R.S. (1989) Characterization of the Pseudomonas aeruginosa pilus adhesin: confirmation that the pilin structural protein subunit contains a human epithelial cell-binding domain. Infect Immun 57:3720-3726.
Ishimoto, K.S. and Lory, S. (1989) Formation of pilin in Pseudomonas aeruginosa requires the alternative sigma factor (RpoN) of RNA polymerase. Proc Natl Acad Sci USA 86:1954-1957.
Ishimoto, K.S. and Lory, S. (1992) Identification of pilR, which encodes a transcriptional activator of the Pseudomonas aeruginosa pilin gene. J Bacteriol 174:3514-3521.
Iwanaga, M. and Hokama, A. (1992) Characterization of Aeromonas sobria TAP13 pili: a possible new colonization factor. J Gen Microbiol 138:1913- 1919.
Jaeger, J.A., Turner, D. H. and Zuker, M. (1989) Predicting optimal and suboptimal secondary structure for RNA. Methods in Enzymology 183: 281-306.
Jahagirdar, R., and Howard, P. (1994) Isolation and characterization of a second exe operon required for extracellular protein secretion in Aeromonas hydrophila. J Bacteriol 176:6819-6826.
Jin, S., Ishimoto, K.S., Lory, S. (1994) PilR, a transcriptional regulator of piliation in Pseudomonas aeruginosa, binds to a ds-acting sequence upstream of the pilin gene promoter. Mon Microbiol 14:1049-1057.
Johanson, W. G., Higuchi, J.H., Chaudhuri, T.R., and Woods, D.E. (1980) Bacterial adherence to epithelial cells in bacillary colonization of the respiratory tract. Am Rev Resp Dis 121:55-63.
Johnson, K., Parker, M.L., an Lory, S. (1986) Nucleotide sequence and transcriptional initiation site of two Pseudomonas aeruginosa pilin genes. J Biol Chem 261:15703-15708.
Johnston, J.L., Billington, S.J., Haring V., and Rood, J.I. (1995) Identification of fimbrial assembly genes from Dichelobacter nodosus: evidence that fim P encodes the type-IV prepilin peptidase. Gene 161:21-26.
Jones, H.C., Pinkner, J.S., Nicholes, A.V., Slonim, L.N., Abraham, S.N., and Hultgren, S.J. (1993) FimC is a periplasmic PapD-like chaperone that directs assembly of type 1 pili in bacteria. Proc Natl Acad Sci 90:8397-8401.
149 Karlyshev, A.V., and MacIntyre, S. (1995) Cloning and study of the genetic organization of the exe gene cluster of Aeromonas salmonicida. Gene 158:77-82.
Kaufman, M.R., Seyer, J.M., and Taylor, R.K. (1991) Processing of TCP pilin by TcpJ typifies a common step intrinsic to a newly recognized pathway of extracellular protein secretion by gram-negative bacteria. Genes Dev 5:1834-1846.
Kazmierczak, B.I.l, Mielke, D.L., Russel, M., and Model, P. (1994) pIV, a filamentous phage protein that mediates phage export across the bacterial cell envelope, forms a multimer. J Mol Biol 238:187-198.
Komiyama, K., Habbick, B.F., and Tumber, K. (1987) Role of sialic acid in saliva-mediated aggregation of Pseudomonas aeruginosa isolated from cystic fibrosis patients. Infect Immun 55:2364-2369.
Krishnapillai, V. (1971) A novel transducing phage. Its role in recognition of a possible new host controlled modification system in Pseudomonas aeruginosa. Mol Gen Genet 114:134-143.
Krivan, H.C., Roberts, D.D., and Ginsberg, V. (1988) Many pulmonary pathogenic bacteria bind specifically to the carbohydrate sequence GalNAc(3l-4Gal found in some glycolipids. Proc Natl Acad Sci USA 85:6157-6161.
Krylov, V.N., Bogush, V.G and Shapiro, }. (1980) Bacteriophage of Pseudomonas aeruginosa with DNA similar in structure to that of phage Mul. I. General description, localization of sites sensitive to endonucleases in DNA, and structure of homoduplexes of phage D3112. Genetika 16:824-832.
Kuehn, M.J., Derek, J.O., Kihlberg, J., Slonim, L.N., Flemmer, K., Bergfors, T., and Hultgren, S.J. (1993) Structural basis of pilus subunit recognition by the PapD chaperone. Science 262:1234-1241.
Kuehn, M.J., Heuser, J., Normark, S., and Hultgren, S.J. (1992) P pili in uropathogenic E. coli are composite fibers with distinct fibrillar adhesive tips. Nature (London) 356:252-255.
150 Kuehn, M.J., Normark, S., and Hultgren, S.J. (1991) Immunoglobulin-like PapD chaperone caps and uncaps interactive surfaces of nascently translocated pilus subunits. Proc Natl Acad Sci USA 88:10586-10590.
Kulich, S.M., Fran, D.W., and Barbieri, J.T. (1993) Purification and characterization of exoenzyme S from Pseudomonas aeruginosa 388. Infect Immun 61:307-313.
Kurioka, S., and Liu, P. (1967) Effect of the hemolysin on Pseudomonas aeruginosa on phosphatides and on phospholipase c activity. J Bacteriol 93:670-674.
Laemmli, U.K. (1970) Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature 227:680-685.
Lam, J., Chan, R., Lam, K., and Costerton, J.W. (1980) Production of mucoid microcolonies by Pseudomonas aeruginosa with infected lungs in cystic fibrosis. Infect Immun 28:546-556.
Lauer, P., Albertson, N.H., and Koomey, M. (1993) Conservation of genes encoding components of a type IV pilus assembly/two-step protein export pathway in Neisseria gonorrftoeae. Mol Microbiol 8:357-368.
Lebeda, A., Kudela, V., and Jedlickova, Z. (1984) Pathogenicity of Pseudomonas aeruginosa for plants and animals. ACTA Phytopathol Acad Aci H ung 19:271-284.
Lee, K.K., Doig, P., Irvin, R.T., Paranchych, W., and Hodges, R.S. (1989) Mapping the surface regions of Pseudomonas aeruginosa PAK pilin: the importance of the C-terminal region for adherence to human buccal epithelial cells. Mol Microbiol 3:1493-1499.
Lee, K.K., Sheth, H.B., Wong, W.Y., Sherburne, R., Paranchych, W., Hodges, R.S., Lingwood, C.A., Krivan, H., and Irvin, R.T. (1994) The binding of Pseudomonas aeruginosa pili to glycosphingolipids is a tip-associated event involving the C-terminal region of the structural pilin subunit. Mol Microbiol 11:705-713.
Lindberg, F., Tennent, J.M., Hultgrem, S.J., Lund, B., and Normark, S. (1989) PapD, a periplasmic transport protein in P-pilus biogenesis. J Bacteriol 171:6052-6058.
Lindeberg, M., an Collmer, A. (1992) Analysis of eight out genes in a cluster required for pectic enzyme secretion by Erwinia chrysanthemi: sequence
151 comparison with secretion genes from other gram-negative bacteria. J Bacteriol 174:7385-7397.
Luiten, R.G.M., Putterman, D.G., Schoennmakers, J.G.G., Konings, R.N.H., and Day, L.A. (1985) Nucleotide sequence of the genome of Pf3, and IncP-1 plasmid specific filamentous bacteriophage of Pseudomonas aeruginosa. J Virol 56:268-276.
Mai, G.T., McCormack, J.G., Seow, W.K., Pier, G.B., Jackson, L.A., and Thong, Y.H. (1993) Inhibition of adherence of mucoid Pseudomonas aeruginosa by alginase, specific monoclonal antibodies, and antibiotics. Infect Immun 61:4338-4343.
Marrs, C.F., Schoolnik, G., Koomey, M., Hardy, J., Rothbard, J., and Falkow, S. (1985) Cloning and sequencing of a Moraxella bovis pilin gene. J Bacteriol 163:132-139.
Martin, P.R., Hobbs, M., Free, P.D., Jeske, Y, and Mattick, J.S. (1993) Characterization of pilQ, a new gene required for the biogenesis of type 4 fimbriae in Pseudomonas aeruginosa. Mol Microbiol 9:857-868.
Martin, P.R., Watson, A.A., McCaul, T.F., an Mattick, J.S. (1995) Characterization of a five-gene cluster required for the biogenesis of type 4 fimbriae in Pseudomonas aeruginosa. Mol Microbiol 16:497-508.
Mattick, J.S., Bills, M.M., Anderson, B.J., Dalrymple, B., Mott, M.R., and Egerton, J.R. (1987) M orphogenetic expression of Bacteroides nodosus fimbriae in Pseudomonas aeruginosa. J Bacteriol 169:33-41.
McCubbin, M., and Fick, Jr., R.B. (1993) Pathogenesis of Pseudomonas lung disease in cystic fibrosis. In Pseudomonas aeruginosa the opportunist: pathogenesis and disease. Fick, Jr., R.B. (ed.). Florida: CRC Press, pp. 189- 211.
McEachran, D.W., and Irvin, R.T. (1985) Adhesion of Pseudomonas aeruginosa to human buccal epithelial cells: evidence for two classes of receptors. Can J Microbiol 31:563-569.
McKern, N.M., O'Donnell, I.J., Inglis, A.S., Stewart, D.J., and Clark, B.L. (1983) Amino acid sequence of pilin from Bacteroides nodosus (strain 198), the causative organism of ovine footrot. FEBS Letters 164:149-153.
152 McMichael, J.C. (1992) Bacterial differentiation within Moraxella bovis colonies growing at the interface of the agar medium with the Petri dish. J Gen Microbiol 138:2687-2695.
Meyers, D.J., and Berk R.S. (1990) Characterization of phospholipase C from Pseudomonas aeruginosa as a potent inflammatory agent. Infect Immun 58:659-666.
Miller, K.W., Bouvier, J., Stragier, P., and Wu, H.C. (1987) Identification of the genes in the Escherichia coli ileS-lsp operon: Analysis of multiple polycistronic mRNAs made in vivo. J Biol Chem262:7391-7397.
Miller, R.V., and Ku, C-M. (1978) Characterization of Pseudomonas aeruginosa mutants deficient in the establishment of lysogeny. J Bacteriol 134:875-883.
Motallebi-Veshareh, M., Balzer, D., Lanka, E., Jagura-Birdzy, G., and Thomas, C.M. (1992) Conjugative transfer functions of the broad-host-range plasmid RK2 are coregulated with vegetative replication. Mol Microbiol 6:907-920.
Newhall, W.J., Wilde, C.E., Sawyer, W.D., and Haak, R.A. (1980) High- molecular-weight antigenic protein complex in the outer membrane of Neisseria gonorrhoeae. Infect Immun 27:475-482.
Nicas, T.I., and Iglewski, B.H. (1984) Isolation and characterization of transposon-induced mutants of Pseudomonas aeruginosa deficient in production of exoenzyme S. Infect Immun 45:470-474.
Nicas, T.I., and Iglewski, B.H. (1985) Contribution of exoenzyme S to the virulence of Pseudomonas aeruginosa. Antibiot Chemother 36:40-48.
Nikaido, H. (1994) Isolation of outer membranes. Methods Enzymol 235:225- 234.
Nunn, D. N., and Lory, S. (1991) Product of the Pseudomonas aeruginosa gene pilD is a prepilin leader peptidase. Proc Natl Acad Sci USA 88:3281-3285.
Nunn, D. N., and Lory, S. (1992) Components of the protein-excretion apparatus of Pseudomonas aeruginosa are processed by the type IV prepilin peptidase. Proc Natl Acad Sci USA 89:47-51.
153 Nunn, D. N. and Lory, S. (1993) Cleavage, methylation, and localization of the Pseudomonas aeruginosa export proteins XcpT,-U,-V, and -W. J Bacteriol 175:4375-4382.
Nunn, D., Bergman, S., and Lory, S. (1990) Products of three accessory gene pilB, pilC, and pitD are required for biogenesis of Pseudomonas aeruginosa pili. J Bacteriol 172:2911-2919.
O'Hoy, K., and Krishnapillai, V. (1987) Calibration of the Pseudomonas aeruginosa strain PAO chromosome map in time units using high- frequency-of-recombination donors. Genetics 115:611-618.
Olsen, R.H., DeBusscher, G. and McCombie, W.R. (1982) Development of broad-host-range vectors and gene banks: self-cloning of Pseudomonas aeruginosa PAO chromosome. J Bacteriol 150:60-69.
Ostroff, R.M., Vasil, A.I., and Vasil, M.L. (1990) Molecular comparison of a nonhemolytic and hemolytic phospholipase C from Pseudomonas aeruginosa. J Bacteriol 172:5915-5923.
Ostroff, R.M., and Vasil, M.L. (1987) Identification of a new phospholipase C activity by analysis of an insertional mutation in the hemolytic phospholipase C structural gene of Pseudomonas aeruginosa. J Bacteriol 169:4597-4601.
Ostroff, R.M., Wretlind, B., and Vasil, M.L. (1989) Mutations in the hemolytic- phospholipase C operon results in decreased virulence of Pseudomonas aeruginosa PAOl grown under phosphate-limiting conditions. Infect Immun 57:1369-1373.
Ottow, J.C.G. (1975) Ecology, physiology, and genetics of fimbriae and pili. Ann Review of Microbiol 29:79-108.
Paranchych, W. and Frost, L.S. (1988) The physiology and biochem istry of pili. Adv in Microbial Physiol 29:53-113.
Paranchych, W., Sastry, P.A., Frost, L.S., Cropenter, M., Armstrong, D., and Watts, T.H. (1979) Biochemical studies on pili isolated from Pseudomonas aeruginosa strain PAO. Can J Microbiol 25:1175-1181.
Parge, H.E., Forest, K.T., Hickey, M.J., Christensen, D.A., Getzoff, E.D., and Tainer, J.A. (1995) Structure of the fibre-forming protein pilin at 2.6 A resolution. Nature 378:32-38.
154 Pasloske, B.L., Carpenter, M.R., Frost, L.S., Finlay, B.B., and Paranchych, W. (1988) The expression of Pseudomonas aeruginosa PAK pilin gene mutants in Escherichia coli. Mol Microbiol 2:185-195.
Pasloske, B.L., Finlay, B.B., and Paranchych, W. (1985) Cloning and sequencing of the Pseudomonas aeruginosa PAK pilin gene. FEBS Lett 183:408-412.
Pasloske, B.L., Scraba, D.G., and Paranchych, W. (1989) Assembly of mutant pilins in Pseudomonas aeruginosa: formation of pili composed of heterologous subunits J Bacteriol 171:2142-2147.
Pavlovskis, O.R., Pollack, M., Callahan, L.T., Iglewski, B.H. (1977) Passive protection by antitoxin in experimental Pseudomonas aeruginosa burn infections. Infect Immun 18:596-602.
Pemberton, J.M. (1973) FI 16: A DNA bacteriophage specific for the pili of Pseudomonas aeruginosa strain PAO. Virology 55:558-560.
Possot, O., d'Enfert, C , Reyss, I., and Pugsley, A.P. (1992) Pullulanase secretion in Escherichia coli K-12 requires a cytoplasmic protein and a putative poly topic cytoplasmic membrane protein. Mol Microbiol 6:95-105.
Possot, O., and Pugsley, A.P. (1994) Molecular characterization of PulE, a protein required for pullulanase secretion. Mol Microbiol 12:287-299.
Pridmore, R.D. (1987) New and versatile cloning vectors with kanamycin- resistance markers. Gene 56:309-312.
Pugsley, A.P. (1993a) Processing and methylation of PulG, a pilin-like component of the general secretory pathway of Klebsiella oxytoca. Mol Microbiol 9:295-308.
Pugsley, A.P. (1993b) The complete general secretory pathway in Gram negative bacteria. Microbiol Rev 57:50-108.
Pugsley, A.P., and Dupuy, B. (1992) An enzyme with type IV prepilin peptidase activity is required to process components of the general extracellular protein secretion pathway of Klebsiella oxytoca.. Mol Microbiol 6:751-760.
Pugsley, A.P., Poquet, I., and Kornacker, M.G. (1991) Two distinct steps in pullulanase secretion by Escherichia coli K12. Mol Microbiol 5:865-873.
155 Pugsley, A.P., and Possot, O. (1993) The general secretory pathway of Klebsiella oxytoca: no evidence for relocalization or assembly of pilin-like PulG protein into a multiprotein complex. Mol Microbiol 10:665-674.
Pugsley, A.P., and Reyss, I. (1990) Five genes at the 3' end of the Klebsiella pneumoniae pulC operon are required for pullulanase secretion. Mol Microbiol 4:365-379.
Ramphal, R., Saddoff, J.C., Pyle, M., and Silipigni, J.D. (1984) Role of pili in the adherence of Pseudomonas aeruginosa to injured tracheal epithelium. Infect Immun 44:38-40.
Rao, V.K. and Progulske-Fox, A. (1993) Cloning and sequencing of two type 4 (N-methylphenylalanine) pilin genes from Eikenella corrodens. J Gen Microbiol 139:651-660.
Ratnaningsih, E., Dharmsthiti, S., Krishnapillai, V., Morgan, A., Sinclair, M., and Holloway, B.W. (1990) A combined physical and genetic map of Pseudomonas aeruginosa PAO. J Gen Microbiol 136:2351-2357.
Reeves, R.J., Douglas, P., and Saimond, G.P.C. (1994) Beta-lactamase topology probe analysis of the OutO NMePhe peptidase, and six other Out protein components of the Erwinia carotovora general secretion pathway apparatus. Mol Microbiol 12:445-457.
Reyss, I., and Pugsley, A.P. (1990) Five additional genes in the pilC-0 operon of the gram-negative bacterium Klebsiella oxytoca UNF5023 which are required for pullulanase secretion. Mol Gen Genet 222:176-184.
Romling, U., Duchene, M., Essar, D.W., Galloway, D., Guidi-Rontani, C., Hill, D., Lazdunski, A., Miller, R.V., Schleifer, K.H., Smith, D.W., Toschka, H.Y., and Tummler, B. (1992) Localization of alg, opr, phn, 4.5S RNA, 6S RNA, tox, trp, and xcp genes, rrn operons, and the chromosomal origin on the physical genome map of Pseudomonas aeruginosa PAO. J Bacteriol 174:327-330.
Roncero, C., Darzins, A. and Casadaban, M.J. (1990) Pseudomonas aeruginosa transposable bacteriophages D3112 and B3 require pili and surface growth for adsorption. J Bacteriol 172:1899-1904.
Royle, P.L., Matsumoto, H. and Holloway, B.W. (1981) Genetic circularity of the Pseudomonas aeruginosa PAO chromosome. J Bacteriol 145:145-155.
156 Rudel, T., Scheuerpflug, I., and Meyer, T.F. (1995) Neisseria PilC protein identified as type-4 pilus tip-located adhesin. Nature 373:357-359.
Russel, M. (1994a) Mutants at conserved positions in gene IV, a gene required for assembly and secretion of filamentous phages. Mol Microbiol 14:357- 369.
Russel, M. (1994b) Phage assembly: a paradigm for bacterial virulence factor export? Science 265:612-614.
Russel, M., and Kazmierczak, B. (1993) Analysis of the structure and subcellular location of filamentous phage pIV. J Bacteriol 175:3998-4007.
Russell, M.A., and Darzins, A. (1994) The pilE gene product of Pseudomonas aeruginosa, required for pilus biogenesis, shares amino acid sequence identity with the N-termini of type 4 prepilin proteins. Mol Microbiol 13:973-985.
Saiman, L., Cacalano, G., Gruenert, D., and Prince, A. (1992) Comparison of adherence of Pseudomonas aeruginosa to respiratory epithelial cells from cystic fibrosis patients and healthy subjects. Infect Immun 60:2808-2814.
Sanger, F. A., Nicklen, S. and Coulson, A.R. (1977) DNA sequencing with chain-terminating inhibitors. Proc Natl Acad Sci USA 74:5463-5467.
Sastry, P.A., Finlay, B.B., Pasloske, B.L., Paranchych, W., Pearlstone, J.R., and Smillie, L.B. (1985) Comparative studies of the amino acid and nucleotide sequences of pilin derived from Pseudomonas aeruginosa PAK and PAO. J Bacteriol 164:571-577.
Sastry, P.A., Pearlstone, J.R., Smillie, L.B., and Paranchych, W. (1983) Amino acid sequence of pilin isolated from Pseudomonas aeruginosa PAK. FEBS letters 151:253-256.
Sastry, P.A., Pearlstone, J.R., Smillie, L.B., and Paranchych, W. (1984) Studies on the primary structure and antigenic determinants of pilin isolated from Pseudomonas aeruginosa K Can J Biochem Cell Biol 63:284-291.
Sato, H., Okinaga, K., and Saito, H. (1988) Role of pili in the pathogenesis of Pseudomonas aeruginosa burn infection. Microbiol Immunol 32:131-139.
Schultz, D.R., and Miller, K.D. (1974) Elastase of Pseudomonas aeruginosa:inactivation of complement components and complement- derivied chemotactic and phagocytic factors. Infect Immun 10:128-135.
157 Schwarzmann, S., and Boring, J.R.III. (1971) Antiphagocytic effect of slime from a mucoid strain of Pseudomonas aeruginosa. Infect Im mun 3:762- 767.
Schweizer, H.P. (1992) Allelic exchange in Pseudomonas aeruginosa using novel ColEl-type vectors and a family of cassettes containing a portable oriT and the counter-selectable Bacillus subtilis sacB marker. Mol Microbiol 6:1195-1204.
Shaw, C.E. and Taylor, R.K. (1990) Vibrio cholerae 0395 tcpA pilin gene sequence and comparison of the predicted protein structural features to those of type IV pilins. Infect Immun 58:3042-3049.
Sheth, H.B., Lee, K.K., Wong, W.Y., Srivastava, G., Hindsgaul, O., Hodges R.S., Paranchych, W., and Irvin, R.T. (1994) The pili of Pseudomonas aeruginosa strains PAK and PAO bind specifically to the carbohydrate sequence pGalNAc(l-4)PGal found in glycosphingolipids asialo-GMi and asialo-GM 2- Mol Microbiol 11:715-723.
Shine, J. and Dalgarno, L. (1975) Determinant of cistron specificity in bacterial ribosomes. N ature 254:34-38.
Shortridge, V.D., Pato, M.L., Vasil, A.I., and Vasil, M.L. (1991) Physical mapping of virulence-associated genes in Pseudomonas aeruginosa by transverse alternating field electrophoresis. Infect Immun 59:3596-3603.
Simpson, D.A., Ramphal, R., and Lory, S. (1992) Genetic analysis of Pseudomonas aeruginosa adherence: distinct genetic loci control attachment to epithelial cells and mucins. Infect Immun 60:3771-3779.
Stojiljkovic, I., Schonherr, R., and Kusters, J.G. (1995) Identification of the hopG gene, a component of Escherichia coli K-12 type II export system, and its conservation among different pathogenic Escherichia coli and Shigella isolates. J Bacteriol 177:1892-1895.
Strom, M.S., Bergman, P., and Lory, S. (1993a) Identification of active-site cysteines in the conserved domain of PilD, the bifunctional type IV pilin leader peptidase//V-methyltransferase of Pseudomonas aeruginosa. J Biol Chem 268:15788-15794.
Strom, M.S., and Lory, S. (1986) Cloning and expression of the pilin gene of Pseudomonas aeruginosa PAK in Escherichia coli. J Bacteriol 165:367-372.
158 Strom, M.S., and Lory, S. (1987) Mapping of export signals of Pseudomonas aeruginosa pilin with alkaline phosphatase fusions. J Bacteriol 169:3181- 3188. Strom, M.S., and Lory, S. (1991) Amino acid substitutions in pilin of Pseudomonas aeruginosa. Biol J Chem 266:1656-1664.
Strom, M.S. and Lory, S. (1993) Structure-function and biogenesis of the type IV pili. Ann Rev Microbiol 47:565-596.
Strom M.S., Nunn, D., and Lory, S. (1991) Multiple roles of the pilus biogenesis protein PilD: involvement of PilD in excretion of enzymes from Pseudomonas aeruginosa. J Bacteriol 173:1175-1180.
Strom, M.S., Nunn, D.N., and Lory, S. (1993b) A single bifunctional enzyme, PilD, catalyzes cleavage and N-methylation of proteins belonging to the type IV pilin family. Proc Natl Acad Sci USA 90:2404-2408.
Studier, F. W. and Moffatt, B.A. (1986) Use of bacteriophage T7 RNA polymerase to direct selective high-level expression of cloned genes. J Mol Biol 189:113-130.
Sutcliffe, J.G. (1979) Complete nucleotide sequence of the Escherichia coli plasmid pBR322. Cold Spring Harbor Symp Quant Biol 43:77-90.
Tabor, S. and Richardson, C.C. (1985) A bacteriophage T7 RNA polymerase/promoter system for controlled expression of specific genes. Proc Natl Acad Sci USA 82:1074-1078.
Tang, H., Kays, M., and Prince, A. (1995) Role of Pseudomonas aeruginosa in acute pulmonary infection. Infect Immun 63:1278-1285.
Taylor, N.S., an Polack, M. (1978) Purification of Pseudomonas aeruginosa exotoxin by affinity chromatography. Infect Immun 19:66-70.
Tomb, J.-F., El-Hajj, H., and Smith, H.O. (1991) Nucleotide sequence of a cluster of genes involved in the transformation of Haemophilus influenzae Rd. Gene 104:1-10.
Tommassen, J., Filloux, A., Bally, M., Murgier, M., and Lazdunski, A. (1992) Protein secretion in Pseudomonas aeruginosa. FEMS Microbiol Rev 103:73-90.
159 Tonjum T., Freitag, N.E., Narmork, E., and Koomey, M. (1995) Identification and characterization of pilG, a highly conserved pilus-assembly gene in pathogenic Neisseria . Mol Microbiol 16:451-464.
Tonner, M.P., Zabner, J., and Doebbeling B.N. (1993) Nosocomial pneumonia due to Pseudomonas aeruginosa. In Pseudomonas aeruginosa the opportunist: pathogenesis and disease. Fick, Jr., R.B (ed.). Florida: CRC Press, pp. 214-233.
Towbin, H., Staehelin, T., and Gordon, J. (1979) Electrophoretic transfer of proteins from polyacrylamide gels to nitrocellulose sheets: procedure and some applications. Proc Natl Acad Sci USA 76:4350-4354.
Tsai, W.-M., Larsen, S.H., and Wilde III, C.E. (1989) Cloning and DNA sequence of the omc gene encoding the outer membrane protein- macromolecular complex from Neisseria gonorrhoeae. Infect Immun 57:2653-2659.
Turner, L.R., Lara, C., Nunn, D.N., and Lory, S. (1993) Mutations in the consensus ATP-binding sites of XcpR and PilB eliminate extracellular protein secretion and pilus biogenesis in Pseudomonas aeruginosa. J Bacteriol 175:4962-4969.
Vieira, J. and Messing, J. (1982) The pUC plasmids, an M13mp7-derived system for insertion mutagenesis and sequencing with synthetic universal primers. G enel9:259-268.
Vishwanath, S., and Ramphal, R. (1985) Tracheobronchial mucin receptor for Pseudomonas aeruginosa: predominance of amino sugar binding sites. Infect Immun 48:331
Vogelstein, B., and Gillespie, D. (1979) Preparative and analytical purification of DNA from agarose. Proc Natl Acad Sci USA 76:615-619.
Watts, T.H., Kay, C.M., and Paranchych,W. (1983) Spectral properties of three quaternary arrangements of Pseudomonas pilin. Biochemistry 22:3640- 3646.
Watts, T.W., Scraba, D.G., and Paranchych, W. (1982a) Formation of 9-nm filaments form pilin monomers obtained by octyl-glucoside dissociation of Pseudomonas aeruginosa pili. J Bacteriol 151:1508-1513.
160 Watts, T.H., Worobec, E.A., and Paranchych, W. (1982b) Identification of pilin pools in the membranes of Pseudomonas aeruginosa. J Bacteriol 152:687- 691.
Weiss, R.L. (1971) The structure and occurrence of pili (fimbriae) on Pseudomonas aeruginosa. J Gen Microbiol 67:135-143.
West. S.E.H. and Iglewski, B.H. (1988) Codon usage in Pseudomonas aeruginosa. Nucl Acids Res 16:9323-9334.
Whitchurch, C.B., Hobbs, M., Livingston, S.P., Krishnapillai, V., and Mattick, J.S. (1990) Characterization of a Pseudomonas aeruginosa twitching motility gene and evidence for a specialized protein export system widespread in eubacteria. Gene 101:33-44.
Whitchurch, C.B., and Mattick, J.S. (1994) Characterization of a gene, pilU, required for twitching motility but not phage sensitivity in Pseudomonas aeruginosa. Mol Microbiol 13:1079-1091.
Woods, D.E., Cryz, S.J., Friedman, R.L., and Iglewski, B.H. (1982) Contribution of toxin A and elastase to virulence of Pseudomonas aeruginosa in chronic lung infections of rats. Infect Immun 36:1223-1228.
Woods, D.E., Strauss, D.C., JohansonJr., W.G., Berry, V.K., and Bass, J.A. (1980) Role of pili in adherence of Pseudomonas aeruginosa to mammalian buccal epithelial cells. Infect Immun 29:1146-1151.
W retlind, B, and Pavlovsksis, O.R. (1984) Genetic m apping and characterization of Pseudomonas aeruginosa mutants defective in the formation of extracellular proteins. J Bacteriol 158:801-808.
Wu, S.S., and Kaiser, D. (1995) Genetic and functional evidence that Type IV pili are required for social gliding motility in Myxococcus xanthus. Mol Microbiol 18:547-558.
Yura, T., Mori, H., Nagai, H., Nagata, T., Ishihama, A., Fujita, N., Isono, K., Mizobuchi, and Nakata, A. (1992) Systemic sequencing of the Escherichia coli genome: analysis of the 0-2.4 min region. Nuc Acids Res 20:3305-3308.
Zhang, H.Z., Lory, S., and Donnenberg, M.S. (1994) A plasmid-encoded prepilin peptidase gene from enteropathogenic Escherichia coli. J Bacteriol 176:6885-6891.
161