Downloaded from www.microbiologyresearch.org by

IP: 54.237.57.119

On: Mon, 23 May 2016 21:09:23

Microbiology (2000), 146, 1321–1332 Printed in Great Britain

Identification of a novel gene, fimV, involvedin twitching motility in Pseudomonasaeruginosa

Annalese B. T. Semmler, Cynthia B. Whitchurch, Andrew J. Leechand John S. Mattick

Author for correspondence: John S. Mattick. Tel : ­61 7 3365 4446. Fax: ­61 7 3365 4388.e-mail : j.mattick!cmcb.uq.edu.au

Centre for Molecular andCellular Biology, TheUniversity of Queensland,Brisbane, QLD 4072,Australia

Transposon mutagenesis was used to identify a new locus required fortwitching motility in Pseudomonas aeruginosa. Four Tn5-B21 mutants whichlacked twitching motility and a fifth which exhibited impaired motility werefound to map to the same KpnI restriction fragment at approximately 40 minon the P. aeruginosa genome. Cloning and sequencing studies showed that allfive transposon insertions occurred within the same 2<8 kb ORF, which wastermed fimV. The product of this gene has a putative peptidoglycan-bindingdomain, predicted transmembrane domains, a highly acidic C terminus andanomalous electrophoretic migration, indicating unusual primary or secondarystructure. The P. aeruginosa genome also possesses a paralogue of fimV.Homologues of fimV were also found in the sequenced genomes of the othertype-IV-fimbriated bacteria Neisseria gonorrhoeae, Neisseria meningitidis,Legionella pneumophila and Vibrio cholerae, but not in those of other bacteriawhich lack type IV fimbriae. A fimV homologue was also found in the genomesequence of Shewanella putrefaciens, along with many other homologues oftype IV fimbrial genes, indicating that this bacterium is also likely to producetype IV fimbriae. Wild-type twitching motility was restored to fimV mutants bycomplementation in a dosage-dependent manner. Overexpression of fimVresulted in an unusual phenotype where the cells were massively elongatedand migrated in large convoys at the periphery of the colony. It is suggestedthat FimV may be involved in remodelling of the peptidoglycan layer to enableassembly of the type IV fimbrial structure and machinery.

Keywords : type IV pili, fimbriae, twitching motility, surface translocation,Pseudomonas aeruginosa

INTRODUCTION

Pseudomonas aeruginosa is an opportunistic pathogenof animals and humans infecting immunocompromisedhosts (Sato et al., 1988). Pathogenesis by this bacteriuminvolves the production of a number of extracellularvirulence determinants including lipases and phospho-lipases, proteases, exopolysaccharides, alkaline phos-phatases, pyochelins and type IV fimbriae. Together,these factors contribute to the bacterium’s successfulattachment to and colonization of the host epithelial

.................................................................................................................................................

The GenBank accession number for the sequence determined in this workis U93274.

tissues and its resistance to host defences (Bodey et al.,1983).

Type IV fimbriae are flexible, filamentous surfaceappendages produced at the poles of the bacterial cellwhich mediate attachment to the host epithelial tissueand a form of surface translocation termed twitchingmotility. They also appear to act as receptors for certainbacteriophages. The mechanism of twitching motilityhas been proposed to be fimbrial retraction and ex-tension (Bradley, 1980). Bacteria that exhibit twitchingmotility can be seen as rough, spreading colonies on agarplates under humid conditions, and as very fine zones ofrapid colony expansion on smooth surfaces (Semmler etal., 1999). This phenotype has been used to distinguish

0002-3953 # 2000 SGM 1321

Downloaded from www.microbiologyresearch.org by

IP: 54.237.57.119

On: Mon, 23 May 2016 21:09:23

A. B. T. SEMMLER and OTHERS

Table 1. Plasmids used in this study

Plasmid Description* Reference

pBluescript II

KS}SK(­)

AmpR cloning vectors Stratagene

pUCP18 E. coli}P. aeruginosa shuttle vector Schweizer (1991)

pUCPKS}SK P. aeruginosa T7 expression vectors Watson et al. (1996a)

pUK21 KanR cloning vector Vieira & Messing (1991)

pRIC380 P. aeruginosa suicide vector Alm & Mattick (1996)

pMMB207 ChlorR cloning vector with inducible

tac promoter

Morales et al. (1991)

pSM-TET Source of TcR cassette Mongkolsuk et al. (1993)

pAB1 Source of pilZ probe Alm et al. (1996)

pUS1 Source of xcpY–Z probe, kindly

provided by M. Bally, CNRS,

Marseille, France

Filloux et al. (1990)

pUS13 Source of xcpQ probe, kindly provided

by M. Bally, CNRS, Marseille, France

Akrim et al. (1993)

pMO010323,

pMO011618,

pMO012140

pLA2917 containing partial Sau3A

PAO1 chromosomal DNA fragments,

kindly provided by B. Holloway,

Monash University, Melbourne,

Australia

Ratnaningsih et al. (1990)

pASP6 6 kb EcoRI fragment from pMO011618

in pUCP18

This study

pASB351 3±5 kb HindIII fragment from

pMO011618 in pBluescript II KS(­)

This study

pASB1 1 kb EcoRI fragment from pASB351 in

pBluescript II KS(­)

This study

pASE281 2±8 kb Ppu10I}BspHI fragment from

pMO011618 in pUCPKS (fimV in

direction of lac promoter)

This study

pASE280 2±8 kb Ppu10I}BspHI fragment from

pMO011618 in pUCPKS (fimV in

direction of T7 promoter)

This study

pASE230 2±4 kb EcoRI}KpnI fragment from

pASE281 in pUCPSK (truncated fimV

in direction of T7 promoter)

This study

pASE18a 2±2 kb NruI fragment from pASE280 in

EcoRV site of pUCPKS (truncated

fimV in direction of T7 promoter)

This study

pAS36 3±6 kb EcoRV}EcoRI fragment from

pMO011618 (containing usg-1) in

pUK21

This study

pAST36 pAS36 with TcR cartridge in PstI site of

usg-1

This study

pASS36 pRIC380 carrying usg-1 : :TcR on SpeI

fragment

This study

pAS14 1±4 kb XbaI}ClaI fragment from pASB1

(containing hisT) in pUK21

This study

pAST14 pAS14 with TcR cartridge in

NcoI}NdeI fragment of hisT

This study

pASS14 pRIC380 carrying hisT : :TcR on SpeI

fragment

This study

pASM281 2±8 kb XbaI}KpnI fragment from

pASE281 in pMMB207 (fimV in

direction of tac promoter)

This study

*AmpR, ampicillin resistance ; TcR, tetracycline resistance ; KanR, kanamycin resistance ; ChlorR,chloramphenicol resistance.

1322

Downloaded from www.microbiologyresearch.org by

IP: 54.237.57.119

On: Mon, 23 May 2016 21:09:23

P. aeruginosa FimV and twitching motility

between bacteria which have functional fimbriae andthose which possess a mutation in genes affectingfimbrial biogenesis or function (Hobbs et al., 1993; Alm& Mattick, 1997). Twitching motility also appears to bean important virulence factor as mutants which lackfunctional type IV fimbriae have reduced infectivity(Hazlett et al., 1991; Comolli et al., 1999). Twitchingmotility has also been shown to be involved in biofilmformation (O’Toole & Kolter, 1998), which may beimportant during infection (Potera, 1999; Costerton etal., 1999).

Twitching motility and type IV fimbriae have beendescribed in a wide range of bacteria, including P.aeruginosa, Neisseria gonorrhoeae, Neisseria menin-gitidis and other Neisseriaceae, various Moraxellaspecies, Dichelobacter nodosus, Branhamella catar-rhalis, Suttonella indologenes, Alteromonas putre-faciens, Pasteurella multocida, Xanthomonas malto-phila, Kingella denitrificans and many others (Matticket al., 1993). Related genes encoding the type IV fimbrialsubunit and other components have also been found ina number of bacteria not previously recognized topossess type IV fimbriae, including Aeromonas spp.(Pepe et al., 1996; Barnett et al., 1997), Legionellapneumophila (Liles et al., 1998; Stone & Abu Kwaik,1998), Pseudomonas syringae (Roine et al., 1998) andAzoarcus spp. (Dorr et al., 1998), the last two indicatingthat type IV fimbriae are important in bacterialcolonization not only of animals but also of plants, fungiand protozoa. Type IV fimbriae have also been found inMyxococcus xanthus, where they have been shown tobe required for social gliding motility, a process whichappears to be functionally equivalent to twitchingmotility (Wu & Kaiser, 1995; Semmler et al., 1999).

Type IV fimbriae are filaments of about 6 nm in diameterwhich range up to several µm in length. They areprimarily composed of a small (145–160 aa) structuralsubunit (pilin or PilA in P. aeruginosa) with a charac-teristic highly conserved and highly hydrophobic amino-terminal region. This forms the core of the helicalstructure, whose outer face is comprised of the morehydrophilic and more variable domains of the subunit(Folkhard et al., 1981; Paranchych & Frost, 1988;Dalrymple & Mattick, 1987; Parge et al., 1990; Forest& Tainer, 1997).

The biogenesis and function of type IV fimbriae in P.aeruginosa is dependent on at least 35 genes which arelocated in several clusters on the chromosome. Theseinclude genes encoding the fimbrial subunit (PilA), aleader peptidase (PilD), ancillary proteins with pre-pilin-like leader sequences (PilE, PilV, PilW, PilX, FimT,FimU), inner and outer-membrane proteins (PilC, PilQ),nucleotide-binding proteins (PilB, PilT, PilU), otherproteins whose functions are not clear (PilM-P, PilF,PilY1, PilY2, PilZ), the RpoN sigma factor, 2 two-component sensor–regulator pairs (PilS}PilR and FimS}AlgR) and a complex chemosensory signal transductionsystem (PilG-L, ChpA-C) (for a recent review see Alm &

Mattick, 1997). Here, we report the identification andcharacterization of a novel gene, fimV, which is alsorequired for twitching motility.

METHODS

Bacterial strains, plasmids and media. The Escherichia colistrain DH5α (recA endA1 gyrA96 hsdR17 thi-1 supE44 relA1φ80dlacZ∆M15) was used in all genetic manipulations and inthe preparation of DNA sequencing templates ; E. coli S17-1was used as the donor strain in the bacterial conjugation(Simon et al., 1983). The P. aeruginosa strains used werePAK (D. Bradley, Memorial University of Newfoundland,Canada), Tn5-B21 mutants of this strain (Hobbs et al., 1993),PAKpilA : :TcR (previously referred to as AWK; Watson et al.,1996b) and ADD1976 (Brunschwig & Darzins, 1992). A PAO1cosmid library (Ratnaningsih et al., 1990) was used in thesubcloning and sequence analysis of the fimV region. Detailsof plasmid construction are given in Table 1. P. aeruginosacompetent cells and transformations were prepared as de-scribed previously (Mattick et al., 1987). E. coli and P.aeruginosa liquid cultures were maintained in Luria–Bertani(LB) broth (Sambrook et al., 1989) and solid media wasprepared by adding 1±0–1±5% Select agar (Gibco-BRL). Lightmicrosocopy was performed using nutrient media (4 gtryptone l−", 2 g yeast extract l−" and 2 g NaCl l−") solidifiedwith 8 g GelGro (ICN) l−" for greater optical clarity. Thefollowing antibiotic concentrations were used for the selectionof E. coli : 12±5 µg tetracycline ml−" for plasmid selection and40 µg tetracycline ml−" for cosmid selection; 100 µg ampicillinml−" ; 25 µg chloramphenicol ml−" and 50 µg kanamycin ml−".The concentrations of antibiotics for the selection of P.aeruginosa were 500 µg carbenicillin ml−", 250 µg chloram-phenicol ml−", 20 µg rifampicin ml−" and 200 µg tetracyclineml−".

The ptac expression studies involved cloning the fimV geneinto pMMB207 (Morales et al., 1991), a chloramphenicol-resistant tac promoter expression shuttle vector that isinducible with IPTG. Following transformation of the con-struct into wild-type PAK and the Tn5-B21 mutants, thetransformants were exposed to varying concentrations (0±03–1±00 mM) IPTG with 250 µg chloramphenicol ml−" on 1%agar plates.

Construction of isogenic mutants. Allelic exchange mutantswere constructed of the genes usg-1 and hisT, which lieadjacent to fimV, using the sucrose selection system describedpreviously (Schweizer, 1992; Alm & Mattick, 1996). Briefly,these genes were subcloned into the vector pUK21 (formingpAS36 and pAS14, respectively). The tetracycline gene car-tridge from pSM-TET was cloned into the PstI site within usg-1 and into a blunted NcoI}NdeI site in hisT to disrupt thegenes. The resulting clones were then digested with SpeI,whose sites span the multiple cloning site of pUK21, and thedisrupted genes were inserted into the suicide vector pRIC380.This vector carries the genes sacBR, which promote sensitivityto sucrose, and oriT, enabling conjugal transfer. The con-structs were then transformed into the E. coli donor strainS17-1 in preparation for mating into P. aeruginosa. Followingconjugation, the transconjugates were selected on 5% sucrosemedia containing tetracycline. This forces the excision of theplasmid whilst leaving the homologously recombined mutatedgene in the chromosome. Mutants were confirmed usingSouthern analysis and examined using the subsurfacetwitching assay (see below).

1323

Downloaded from www.microbiologyresearch.org by

IP: 54.237.57.119

On: Mon, 23 May 2016 21:09:23

A. B. T. SEMMLER and OTHERS

Recombinant DNA techniques. The preparation of plasmidDNA, restriction endonuclease digestion (New EnglandBiolabs), ligation reactions, Southern blotting and radio-labelling of probe were carried out using standard protocols(Sambrook et al., 1989).

Sequence analysis. Sequence templates were generated by acombination of subcloning and shotgun cloning strategies.The dsDNA was prepared for sequencing using a modifiedalkaline lysis method involving PEG precipitation (AppliedBiosystems). Sequencing was performed using the AppliedBiosystems PRISM system on a 373A automated sequencer.Nucleotide and predicted protein sequences were analysedusing gapped (Altschul et al., 1997), (Schultz etal., 1998; Ponting et al., 1999) and (Nakai & Horton,1999) programs.

Protein expression and analysis. The FimV protein wassubcloned into a P. aeruginosa expression plasmid, pUCPKS(Watson et al., 1996a) and transformed into P. aeruginosaADD1976, which contains a chromosomal T7 RNA poly-merase gene under the control of an inducible lac promoter(Brunschwig & Darzins, 1992). Protein expression wasinduced in the presence of [$&S]methionine and analysed on7±5% SDS-polyacrylamide gels as described previously (Alm& Mattick, 1995).

Western blotting. Bacterial cells from plates were resuspendedto an OD

'!!of 1±0 in 50 mM sodium carbonate buffer pH 9±6.

Samples (1 ml) were centrifuged and the cell pellet wasresuspended in 100 µl sample buffer (60 mM Tris}HCl pH 6±8,2% SDS, 10% glycerol, 5% β-mercaptoethanol, 0±001%bromophenol blue). To remove DNA, the samples werecentrifuged at 45000 r.p.m. for 90 min and the supernatantwas heated to 100 °C for 5 min. Proteins in the samples werethen separated by SDS-PAGE using a 15% polyacrylamide geland a 5% stacking gel as described by Laemmli (1970) andtransferred electrophoretically to Hybond-C nitrocellulose(Amersham) in the Tris}glycine system described by Towbinet al. (1979). Proteins were detected with anti-PilA antiserum(1:5000) followed by goat anti-rabbit immunoglobulin Gconjugated to alkaline phosphatase (1:5000; BoehringerMannheim).

ELISA. This was based on a method described by Engvall &Perlmann (1972). The cells were resuspended in 50 mMsodium carbonate buffer pH 9±6 at an OD

'!!of 1±0 and 200 µl

of suspension was loaded into wells of a 96-well ELISA plate.After overnight incubation at 4 °C, the wells were washedwith PBS (137 mM NaCl, 2 mM KCl, 10 mM NaHPO

%,

pH 7±4) containing 0±1% Tween 20, blocked with 3% BSA for1 h and then exposed to an anti-PilA antibody at a startingdilution of 1:500 for 2 h at 37 °C. After removal of antisera,the wells were again washed with PBS containing 0±1% Tween20. Goat anti-rabbit immunoglobulin G conjugated withalkaline phosphatase was then added (1:5000) and the mixtureincubated for 2 h at 37 °C. Detection was carried out using20 mg p-nitrophenyl phosphate (Sigma) ml−" in 1 M Trisbuffer pH 8±0 and the plate was read at 405 nm using an ELISAreader (Bio-Rad).

Elastase assay. Aliquots (2 µl) of overnight broth cultureswere inoculated onto the surface of LB agar plates containing0±1% elastin (Sigma). After incubation at 37 °C for 2–3 d,plates were examined for zones of proteolytic clearingsurrounding the colonies.

Twitching motility assay. Twitching motility was assayed asdescribed previously (Alm & Mattick, 1995). Briefly, the P.

aeruginosa strain to be tested was stab-inoculated through a1% agar plate, and after overnight growth at 37 °C the zone oftwitching motility between the agar and Petri dish interfacewas visualized by staining with Coomassie brilliant blue R250.

Light still and video microscopy. Light microscopy wasperformed as described previously (Semmler et al., 1999).Sterile microscope slides were submerged in molten GelGromedia to obtain a thin layer of media coating the slide. Theslides were allowed to set in a horizontal position and air-dried briefly prior to use. The slides were then inoculated witha small loopful of bacteria taken from an overnight plateculture. A sterile glass coverslip was placed over the point ofinoculation and the slide transferred to a large Petri dishcontaining a moist tissue and sealed with Nescofilm (BandoChemical Industries) to maintain humid conditions. Incu-bation times ranged from 2–6 h at 37 °C.

Slide cultures were examined using a Zeiss Axioskop 50microscope with Nomarski facilities at ¬200 to ¬400magnification. Video microscopy was performed in a roomheated to 30 °C. Video images were recorded over a period of2–4 h at speeds of either 1 field per 3±22 s, 1 field per 0±66 s orreal time (1 field per 1}50 s) using a JVC TK-CI38IEG videocamera connected to a Sanyo TLS-S2500P time-lapse videorecorder. Video images were edited and converted to Quick-time movies using Avid Videoshop version 3.0 and can beviewed at http :}}www.cmcb.uq.edu.au}cmcb}PUBS}twitch.html.

RESULTS

Initial characterization of the transposon mutants

We have previously generated a library of twitchingmotility mutants through transposon mutagenesis of theP. aeruginosa strain PAK genome (Hobbs et al., 1993).The mutants were then classified according to theirsensitivity or resistance to infection by the fimbriae-specific bacteriophage PO

%and further subgrouped

according to the size of the KpnI restriction fragmentsinto which the transposon had inserted. Five of thesemutants (S4, S76, S125, S359 and S361) that retainedphage sensitivity were found to have transposoninsertions in the same large (" 25 kb) KpnI fragment.

DNA flanking the site of insertion of the transposon ineach of these mutants was cloned using a ‘markerrescue’ approach that involved digestion of mutantchromosomal DNA with restriction enzymes (eitherEcoRI or HindIII) which cut once within the transposonbeyond the tetracycline resistance marker, ligation intopBluescript II KS(­) and recovery of tetracycline-resistant E. coli colonies. The DNA adjacent to thetransposon insertion in each of the mutants was thensequenced using a primer which is specific for theinverted repeats of Tn5-B21 (Hobbs et al., 1993).Database searches with these sequences revealed thatthe transposon mutants were unique and not located inpreviously characterized genes.

Cloned chromosomal DNA flanking the site of trans-poson insertion in S76 was used for Southern analysis ofmutant genomic DNA. This analysis demonstrated thatthe transposon insertions in mutants S4, S76, S359 and

1324

Downloaded from www.microbiologyresearch.org by

IP: 54.237.57.119

On: Mon, 23 May 2016 21:09:23

P. aeruginosa FimV and twitching motility

.................................................................................................................................................................................................................................................................................................................

Fig. 1. Genetic organization of the fimV locus. The locations of transposon insertions are indicated by filled triangles. Thesites of insertion of the TcR cartridge are indicated by open triangles. The cloned inserts used for sequence analysis,complementation and expression studies are shown. Arrows indicate the orientation of fimV with respect to the externalpromoters. Relevant restriction sites are noted.

S361 were located in the same 10 kb HindIII and 6 kbEcoRI restriction fragments, whereas the S125 insertionsite was not located within these fragments. The clonedDNA from S76 was also used to screen a reference PAO1cosmid library (Ratnaningsih et al., 1990), from whichwe identified three cosmids (pMO010323, pMO011618and pMO012140) covering the region. Restriction map-ping of these cosmids and further Southern analysisshowed that the transposon insertion site of the S125mutant was located in 3±5 kb HindIII and 1 kb EcoRIfragments contained within these cosmids, adjacent tothe restriction fragments containing the other fourinsertions (Fig. 1).

Cosmids pMO011618 (leu-10 trpF pur-9013 met-9011)and pMO012140 (leu-10 trpF) have been previouslymapped to approximately 40 min on the P. aeruginosachromosome by the presence of metabolic markers(Ratnaningsih et al., 1990). This region of the P.aeruginosa genome is known to contain the fimbrialgene pilZ (Alm et al., 1996) and a cluster of genes(xcpQ–Z) which are required for protein secretion inthis organism (Filloux et al., 1990; Bally et al., 1992;Akrim et al., 1993). Southern analysis of the cosmidswith probes specific for pilZ, xcpQ and xcpY–Z (seeTable 1) demonstrated that the transposon insertionswere not in the vicinity of these genes (data not shown).The mutants were also found to have no defect in theirability to secrete elastase, an enzyme exported via theXcp pathway, indicating that the gene(s) into which thetransposons had inserted is not involved in this system(data not shown).

Cloning and sequence analysis of the fimV region

The 6 kb EcoRI (pASP6) and 3±5 kb HindIII (pASB351)fragments (Fig. 1) were subcloned from pMO011618and sequenced. The complete sequence of 8 kb of thisregion (GenBank accession no. U93274) identified sixORFs (Fig. 1), three of which were previously charac-terized genes from P. aeruginosa : leuB (3-isopropyl-malate dehydrogenase) ; asd (aspartate-β-semialdehydedehydrogenase) ; and orfA (Hoang et al., 1997; Hoang& Schweizer, 1997). Our sequence analysis of this regionrevealed a frameshift error in the previously reportedsequence of orfA. The revised sequence shows that thisputative gene encodes a protein which shows significanthomology to the product of the unknown genes termedusg-1 from Azotobacter vinelandii (50% identity and66% similarity over 333 aa) and E. coli (36% identityand 53% similarity over 339 aa). In light of this we haverenamed orfA as usg-1. Interestingly, the Usg-1 proteinsare also predicted to belong to the family of aspartate-β-semialdehyde dehydrogenases and in fact are hom-ologous to the asd gene products from Vibrio choleraeand Vibrio mimicus, as well as Shewanella sp. and L.pneumophila. These enzymes catalyse the second stepin the common biosynthetic pathway leading fromaspartate to the cell wall precursor meso-diamino-pimelate, lysine, methionine, isoleucine and threonine.Two of the remaining ORFs, which we have termedorfB and hisT, showed significant homology to genescharacterized in other bacteria. The product of orfB (187aa) shows close strong similarity to the hypotheticalproteins YafE from E. coli (58% identity and 68%

1325

Downloaded from www.microbiologyresearch.org by

IP: 54.237.57.119

On: Mon, 23 May 2016 21:09:23

A. B. T. SEMMLER and OTHERS

Pa FimV

Pa FimV homologue(32 % identical, 43% similar over 771 aa of Pa FimV )

Nm/Ng TspA(25% identical, 38% similar over 799 aa of Pa FimV )

Lp FimV homologue(28% identical, 48% similar over 404 aa of Pa FimV )

Vc FimV homologue(23% identical, 38% similar over 742 aa of Pa FimV )

Sp FimV homologue(24% identical, 38% similar over 733 aa of Pa FimV )

Putative peptidoglycan-binding domain

Homologous to FimV

Highly acidic

Putative transmembrane spanning domain

100 aa

927 aa

682 aa

875 aa

799 aa

1110 aa

1600 aa

.................................................................................................................................................................................................................................................................................................................

Fig. 2. Schematic diagrams of FimV and its homologues in P. aeruginosa (Pa), N. meningitidis (Nm), N. gonnorhoeae (Ng),L. pneumophila (Lp), V. cholerae (Vc) and Shewanella putrefaciens (Sp). Relevant features are indicated by filled andhatched boxes. Regions of shared homology to Pa FimV are indicated by light hatching and extend into the highly acidicregions.

similarity over 182 aa) and YcgJ from Bacillus subtilis(37% identity and 54% similarity over 166 aa), and toputative methyltransferases from a number of bacterialspecies including Lactococcus lactis, Bacillus stearo-thermophilus, Streptomyces hygroscopicus, Micro-cococcus luteus and E. coli. HisT shows strong hom-ology to pseudouridylate synthetases (involved in tRNAmodification) from a broad spectrum of bacterial species(Arps & Winkler, 1987). Sequence analysis of the regiondownstream of hisT indicated that the previouslycharacterized gene trpF (phosphoribosyl anthranilateisomerase) (Murata, 1996) is located immediately down-stream of hisT.

The remaining ORF in this region was found to containall five transposon insertions (Fig. 1). This ORF,designated fimV, is 2±8 kb in size and has an overallG­C content of 67±5 mol%, in agreement with theestimated 67 mol% for the P. aeruginosa genome as awhole (West & Iglewski, 1988). Further analysis of thefimV sequence showed a decrease in rare codon usagewithin the ORF and a high G­C bias (81±7%) in thethird codon position, suggestive of a likely coding region(West & Iglewski, 1988).

search analyses at NCBI revealed that FimVshows regions of homology with the recently describedprotein TspA of N. meningitidis (Kizil et al., 1999).Analysis of the Unfinished Microbial Genomes data-bases at NCBI also identified homologies betweenFimV and predicted proteins from N. gonorrhoeae(TspA equivalent), L. pneumophila, V. cholerae andShewanella putrefaciens (Fig. 2). FimV also showssignificant homology with the predicted product of asecond P. aeruginosa gene identified in the unfinishedgenome sequences (Fig. 2). Interestingly, except forShewanella putrefaciens, these bacterial species are all

known to possess type IV fimbriae. Further analysis ofthe Shewanella putrefaciens genome sequences indicatedthat this organism should also be capable of producingtype IV fimbriae as it possesses many close homologuesof P. aeruginosa proteins required for type IV fimbrialbiogenesis (including homologues of PilA-F, PilM-Q,PilT and PilU). It appears therefore that fimV is specificto type-IV-fimbriate bacteria.

FimV and its homologues show a number of features incommon. Each of these proteins is highly acidic with anestimated pI ranging from 3±24 to 4±64. Acidic residuesare located across the entire length of these proteins,with large numbers clustered in the carboxy termini(Fig. 2). All of these proteins are predicted to possess atleast one transmembrane spanning region (Fig. 2) and,except for TspA and the L. pneumophila FimV hom-ologue, have predicted signal sequences. It is expectedtherefore that these proteins are integral cytoplasmicmembrane proteins. analysis at EMBL alsopredicted that each of these proteins possesses a putativepeptidoglycan-binding domain in their N-terminus (Fig.2). This motif has been found in a variety of enzymesinvolved in bacterial cell wall degradation (Joris et al.,1992). Sequence homology between these proteins isstrongest in the region surrounding this domain.

In P. aeruginosa, fimV is situated between the genes usg-1 and hisT (Fig. 1). Interestingly, in E. coli usg-1 isknown to be located directly upstream of and within anoperon with hisT (Arps & Winkler, 1987). Thedifference therefore between the E. coli and P.aeruginosa genomic organization is the presence of fimVbetween usg-1 and hisT. A search of the E. coli genomeshows that it does not contain a homologue of FimV.We constructed allelic exchange mutants of the P.aeruginosa genes usg-1 and hisT to determine whether

1326

Downloaded from www.microbiologyresearch.org by

IP: 54.237.57.119

On: Mon, 23 May 2016 21:09:23

P. aeruginosa FimV and twitching motility

1 2 3 4 5kDa

220

160

120

9080

100

.................................................................................................................................................

Fig. 3. Expression of the fimV gene in P. aeruginosa ADD1976.Proteins encoded in plasmids under the direction of theexternal T7 promoter were labelled with [35S]methionineand separated on a 7±5% polyacrylamide gel. Rifampicin(200 µg ml−1) was added to inhibit host RNA polymerase. Theplasmids used were pUCPKS (lane 1), pASE280 (lane 2), pASE281(lane 3), pASE230 (lane 4) and pASE18a (lane 5). The mobility ofsize markers is indicated on the left.

(like fimV) either might be involved in type IV fimbrialbiogenesis or function (Fig. 1). Following confirmationby Southern blotting, the mutants PAKusg-1 : :TcR andPAKhisT : :TcR were examined for their ability toexhibit twitching motility using the subsurface twitchingassay and light microscopy. Both retained wild-typetwitching motility (data not shown). Further analysis ofthe sequences surrounding the fimV homologue genesin the other type IV fimbriate bacteria revealed thatShewanella putrefaciens has a similar genetic arrange-ment to that of P. aeruginosa, with the fimV homologuesituated between genes encoding homologues of Usg-1and HisT. The V. cholerae genetic arrangement also hasthe fimV homologue situated upstream of hisT. Theconserved genetic arrangement in these type-IV-fim-briate species perhaps further supports our hypothesisthat these genes encode proteins with analogousfunctions to FimV.

T7 expression of FimV

The protein encoded by fimV was examined using a T7expression system. fimV was cloned as a 3±161 kbPpu10I}BspHI fragment covering the predicted codingregion of fimV (from ®86 bp to the stop codon) into thebroad-host-range T7 expression vectors pUCPKS}SK(Fig. 1). The gene was cloned both in the forward andreverse direction (pASE280 and pASE281, respectively)relative to the T7 promoter. These constructs weretransformed into P. aeruginosa ADD1976, whichcontains a chromosomal T7 RNA polymerase geneunder lac promoter control. A unique band wasobserved in the pASE280 expressed lane that was notfound in the other samples (Fig. 3). The size of this bandwas estimated to be 145 kDa, which was 47 kDa greaterthan that predicted from the sequence for FimV(98 kDa). The fidelity of the insert including the stopcodon was confirmed by restriction enzyme profilingand end sequence analysis. To try to locate the source ofthe anomaly we then generated truncations of the acidicC terminus of FimV, by removing 455 bp (pASE230) and1005 bp (pASE18a) from the end of the fimV gene, within-frame stop codons close downstream in the vector

(a) (b) (c)

(d) (e) (f)

.................................................................................................................................................

Fig. 4. Subsurface twitching motility assay of P. aeruginosastrains PAK (a), S4 (b), S125 (c), PAK ­ pASE281 (d), S4 ­pASE281 (e) and S125 ­ pASE281 (f). Strains were incubated at37 °C for 24 h post-inoculation prior to staining. Panels (b)–(f)are at the same magnification. Bar, 1 cm.

(Fig. 1). However, both truncations still producedproteins with similar anomalies in electrophoretic mi-gration (Fig. 3). This suggests that the anomaly may notbe caused by the highly acidic C terminus but ratherresides in the N-terminal two-thirds of the protein,perhaps due to some other undetermined secondarystructure or possibly covalent modification of theprotein in this region.

Phenotypic characterization of the mutants

As previously described, the identification of transposoninsertions in fimbrial genes was initially based onalterations to colony morphology on the agar surface.We examined the twitching motility of these mutants inmore detail using the more sensitive subsurface stabassay where twitching motility results in a zone ofcolony expansion at the interstitial surface between theagar and Petri dish. Four of the fimV mutants (S4, S76,S359, S361) had the same phenotype, all showing a lossof twitching motility in comparison to wild-type. S4 wasused as the representative strain for further studies onthis group. The fifth mutant, S125, was found to have asmall irregular twitching zone (Fig. 4).

We examined whether the fimbrial subunit (PilA orpilin) is produced in the mutants and if so, if it isassembled on the cell surface into fimbriae. Whole-cellWestern analysis, using an anti-PilA antiserum, revealedthat all of the transposon mutants produced the fimbrialsubunit. In S4, S76, S359 and S361 there was nodetectable pilin on the surface of the cells shown byELISA, whereas S125 had a small amount of the proteinlocalized on the surface. This amount was greater thanin the PAKpilA : :TcR mutant but significantly less thanin the wild-type (data not shown).

We assessed quantitatively the degree of sensitivity ofeach of the fimV mutants for infection by the type-IV-

1327

Downloaded from www.microbiologyresearch.org by

IP: 54.237.57.119

On: Mon, 23 May 2016 21:09:23

A. B. T. SEMMLER and OTHERS

1·4

1·2

1·0

0·8

0·6

0·4

0·2

0·001 0·003 0·01 0·03 0·30·1 1 3 10

IPTG concn (mM)

Twit

chin

g z

on

e d

iam

eter

(cm

)

.................................................................................................................................................

Fig. 5. Twitching zone diameters obtained for PAK (E), S4 (+)and S125 (_) containing pASM281 (fimV under ptac control) atdifferent IPTG concentrations. Zone diameters were measuredafter 24 h incubation at 37 °C post-inoculation. Normaltwitching motility was seen in all strains at IPTG concentrationsbetween 0±01 mM and 0±03 mM. Higher concentrations of IPTGresulted in aberrant zones similar to those observed in Fig. 4(d),(e) and (f). No motility was observed with IPTG concentrationsabove 0±3 mM.

fimbriae-specific bacteriophage PO%. All mutants

demonstrated wild-type titres for this phage (data notshown). These data suggest that the S4 group of fimVmutants may fall into the same class as mutants of pilG,pilI and pilJ, which also appear incapable of producingassembled pilus filaments but remain sensitive tofimbriae-specific bacteriophage. It has been proposedthat these strains form a preliminary structure or pre-pilus complex consisting of an exposed pilus tip at thecell surface to which the phage can bind and subse-quently infect (Darzins, 1993, 1994).

Complementation of fimV

The plasmid pASE281 containing fimV cloned down-stream of the lac promoter (which is constitutivelyactive in P. aeruginosa) was used for complementationstudies of the fimV mutants. Subsurface twitching assaysshowed that the non-motile S4 mutant had twitchingmotility restored by pASE281 but not to wild-typelevels. Instead, the zones remained small and irregular(Fig. 4e). S125 was also not complemented to wild-typetwitching motility but appeared to exhibit exaggeratedmedusa-like structures erupting from the centre of thecolony (Fig. 4f). PAK containing this construct alsoresulted in a reduction and aberration of the wild-typetwitching zone (Fig. 4d). These results suggested that theoverexpression of fimV may be interfering with normaltwitching motility both in the mutants and in wild-typePAK.

To test this possibility, fimV was cloned into pMMB207,a vector that has an inducible tac promoter (carries therepressor lacI), to generate plasmid pASM281 (Fig. 1).By varying the concentrations of IPTG, the levels ofexpressed FimV could therefore be altered. Using aconcentration range of 0–10 mM IPTG, the twitchingzones from the S4 and S125 mutants and PAK wereobserved. The results showed that between 0±01 mMand 0±03 mM IPTG there was complete restoration ofnormal twitching motility in the mutants and the wild-type was unaffected (Fig. 5). (We presume that thetwitching motility observed in the mutants at 0 mMIPTG was due to leakage of the tac promoter.) Levels ofIPTG above 0±03 mM resulted in reduced and aberranttwitching zones in both wild-type and the mutants(Fig. 5). These data confirm that the loss of fimV isresponsible for the loss of twitching motility in themutants, that overexpression of fimV causes aberranttwitching motility and that a specific level of FimVexpression is required for normal motility.

Microscopic analysis of fimV mutants

We have recently examined twitching motility in wild-type P. aeruginosa using video microscopy and haveshown that this process involves leading edge rafts ofaggregated cells in tight cell–cell contact leading outfrom the colony edge. These rafts appear to moveprimarily in an outward manner with occasional limitedcell reversal. Behind these rafts the cells break up andform a network which resembles a lattice structure (Fig.6a), wherein the cells move bidirectionally with frequentcell reversal. They are also capable of moving quite longdistances within the lattice network but always withinclose contact of neighbouring cells. In comparison, thePAKpilA : :TcR mutant has a uniform leading edgewhere no cells are observed moving away from thecolony (Semmler et al., 1999).

We examined the micromorphological details of thetwitching zones produced by the fimV mutants. Themutant S4 (and the other non-motile mutants S76, S359,S361) resembled the PAKpilA : :TcR mutant (Fig. 6b). Incomparison, mutant S125 formed large rafts leading outfrom the centre of the colony, but lacked the latticenetwork of cells which is characteristic of wild-type (Fig.6c). These rafts also appeared to be larger than thoseexhibited by PAK. Individual cells were visible withinthese rafts but, unlike PAK, these cells appeared to besimply moving back and forth, resulting in a reductionin the net forward movement of the rafts. It appearstherefore that S125, with its reduced fimbriae pro-duction, is capable of exhibiting the first stage oftwitching motility, raft formation. It is clear that for thisstage to occur at least a small number of fimbriaeare required as mutants lacking surface fimbriae(PAKpilA : :TcR, S4, S76, S359, S361) are incapable ofexhibiting any raft formation or cellular movements.Time-lapse videos of these experiments may be viewedat our web site (http :}}www.cmcb.uq.edu.au}cmcb}PUBS}twitch.html).

1328

Downloaded from www.microbiologyresearch.org by

IP: 54.237.57.119

On: Mon, 23 May 2016 21:09:23

P. aeruginosa FimV and twitching motility

(a) (b)

(c) (d)

.................................................................................................................................................................................................................................................................................................................

Fig. 6. Light microscopy of zones of twitching motility showing typical colony expansion zones obtained at theinterstitial surface between the glass coverslip and Gelgro media for PAK (a), S4 (b), S125 (c) and S4 ­ pASE281 (d).Micrographs were taken after 4–6 h incubation at 37 °C post-inoculation. In all micrographs the colony is situated to theleft of the image. Bar, 50 µm.

In view of the complementation data, which suggestedthat fimV function is controlled by gene dosage, theeffect of overexpressing fimV in the wild-type, in thenon-motile fimV mutant S4 and in S125 was alsoexamined. Transformation of pASE281 into the mutantspartially restored the raft and lattice-type structurestypical of twitching motility, but also resulted indramatic cell elongation (Fig. 6d), which was alsoobserved when fimV was overexpressed in the wild-type(not shown). The most spectacular phenotype wasobserved in S125 with pASE281 which exhibited grosslyelongated cells, and a medusa-like phenotype at thecolony level (see Fig. 4f). These effects were not observedin controls with vector alone, indicating that theelongated phenotype was not an artefact of carbenicillinselection (data not shown).

Electron microscopy of cells taken from the colonyedges confirmed that overexpression of fimV in PAK, S4and S125 produces cells that are grossly elongated, oftenwith lengths up to 50–100 times normal (data notshown). Interestingly, only a small percentage of thepopulation demonstrated this abnormality, and cells atthe centre of the colony, away from the twitching edge,appeared largely normal. This suggests that only thosecells which are actively in twitching mode (see Semmler

et al., 1999) are affected by abnormally high levels ofFimV.

DISCUSSION

In this report we have identified a novel gene, fimV,which is required for twitching motility, bringing thetotal now to 36 genes required for the biogenesis andfunction of type IV fimbriae in P. aeruginosa. Searches ofthe Unfinished Microbial Genomes database at NCBIindicates that homologues of FimV may be found inN. meningitidis, N. gonorrhoeae, L. pneumophila, V.cholerae and Shewanella putrefaciens as well as a secondhomologue in P. aeruginosa. No homologues have yetbeen identified in any other genome. As far as we cantell, fimV only occurs in type-IV-fimbriate bacteria. Ittherefore appears that FimV is specific for the biogenesisand}or function of type IV fimbriae.

Although the precise role of FimV has not yet beendetermined, the examination of the mutant phenotypeshas revealed that this protein is essential for twitchingmotility. Four of the five fimV mutants (S4, S76, S359,S361) are incapable of twitching motility. Westernanalysis and ELISA data showed that these mutants

1329

Downloaded from www.microbiologyresearch.org by

IP: 54.237.57.119

On: Mon, 23 May 2016 21:09:23

A. B. T. SEMMLER and OTHERS

continue to produce the PilA subunit but do not producesurface-assembled fimbriae, suggesting a lesion in theprocess of fimbrial assembly. The fifth mutant, S125,produces a small, irregular twitching zone, demonstratesimpairment of cellular motility during twitching andexpresses a reduced number of fimbriae on the cellsurface. The differences observed between S125 and theothermutants is likely to be due to the sites of transposoninsertions, which in S125 is located C230 bp from thestop codon of fimV. We predict that this mutantproduces a truncated form of FimV, presumably withpartial function, whilst the others do not produce astable fimV product.

Our complementation data indicates that a precise levelof FimV is required for normal twitching motility.Microscopic examinations show that overexpression offimV results in the formation of dramatically elongatedcells, probably accounting for the observed defects intwitching motility when FimV is produced at high levels.The presence of a putative peptidoglycan-binding do-main in the N-terminus of FimV and its homologuesmay give some clue as to the function of these proteins.The elongated cell phenotype obtained when fimV isoverexpressed may be due to interference by high levelsof FimV during the remodelling of the peptidoglycanlayer in the process of cell division and}or fimbrialbiogenesis, perhaps indicating some direct associationof FimV with peptidoglycan components. It is also ofinterest to note that fimV is located downstream of thegenes asd and usg-1, the products of which are predictedto be involved in the production of the cell wall precursormeso-diaminopimelate. We propose that FimV may beinvolved in remodelling of the peptidoglycan layer toenable assembly of the type IV fimbrial structure andassociated machinery. Interestingly, upstream of thepilM–Q operon of P. aeruginosa is a gene, ponA,encoding a high-molecular-mass penicillin-binding pro-tein (PBP-1A), one of a class of proteins involved in theformation and maintenance of the peptidoglycan layer(Martin et al., 1993). A related gene (fimD) is alsoobserved in the same operon as the fimbrial subunitgenes (fimA and fimZ) in class II strains of D. nodosus(Hobbs et al., 1991). Taken together, these observationssuggest that cell wall structure and local remodelling isimportant for type IV fimbrial biogenesis and}or func-tion at the pole of the cells.

ACKNOWLEDGEMENTS

A. B. T. Semmler and C. B. Whitchurch contributed equallyto this work. This work was supported by grants to C.B.Wand J.S.M by the National Health and Medical ResearchCouncil of Australia. The Centre for Molecular and CellularBiology is a Special Research Centre of the AustralianResearch Council.

REFERENCES

Akrim, M., Bally, M., Ball, G., Tommassen, J., Teerink, H., Filloux,A. & Lazdunski, A. (1993). Xcp-mediated protein secretion in

Pseudomonas aeruginosa : identification of two additionalgenes and evidence for regulation of xcp gene expression. MolMicrobiol 10, 431–443.

Alm, R. A. & Mattick, J. S. (1995). Identification of a gene, pilV,required for type 4 fimbrial biogenesis in Pseudomonas aeruginosawhose product possesses a prepilin-like leader sequence. MolMicrobiol 16, 485–496.

Alm, R. A. & Mattick, J. S. (1996). Identification of two geneswith prepilin-like leader sequences required for type 4 fim-brial biogenesis in Pseudomonas aeruginosa. J Bacteriol 178,3809–3817.

Alm, R. A. & Mattick, J. S. (1997). Genes involved in the biogenesisand function of type-4 fimbriae in Pseudomonas aeruginosa. Gene192, 89–98.

Alm, R. A., Bodero, A. J., Free, P. D. & Mattick, J. S. (1996).Identification of a novel gene, pilZ, essential for type 4 fimbrialbiogenesis in Pseudomonas aeruginosa. J Bacteriol 178, 46–53.

Altschul, S. F., Madden, T. L., Schaffer, A. A., Zhang, J., Zhang, Z.,Miller, W. & Lipman, D. J. (1997). Gapped and - : anew generation of protein database search programs. NucleicAcids Res 25, 3389–3402.

Arps, P. J. & Winkler, M. E. (1987). Structural analysis of theEscherichia coliK-12 hisT operon by using a kanamycin resistancecassette. J Bacteriol 169, 1061–1070.

Bally, M., Filloux, A., Akrim, M., Ball, G., Lazdunski, A. &Tommassen, J. (1992). Protein secretion in Pseudomonasaeruginosa : characterization of seven xcp genes and processing ofsecretory apparatus components by prepilin peptidase. MolMicrobiol 6, 1121–1131.

Barnett, T. C., Kirov, S. M., Strom, M. S. & Sanderson, K. (1997).Aeromonas spp. possess at least two distinct type IV pilusfamilies. Microb Pathog 23, 241–247.

Bodey, G. P., Bolivar, R., Fainstein, V. & Jadeja, L. (1983).Infections caused by Pseudomonas aeruginosa. Rev Infect Dis 5,279–313.

Bradley, D. E. (1980). A function of Pseudomonas aeruginosaPAO pili : twitching motility. Can J Microbiol 26, 146–154.

Brunschwig, E. & Darzins, A. (1992). A two-component T7 systemfor the overexpression of genes in Pseudomonas aeruginosa. Gene111, 35–41.

Comolli, J. C., Hauser, A. R., Waite, L., Whitchurch, C. B., Mattick,J. S. & Engel, J. N. (1999). Pseudomonas aeruginosa gene productsPilT and PilU are required for cytotoxicity in vitro and virulencein a mouse model of acute pneumonia. Infect Immun 67,3625–3630.

Costerton, J. W., Stewart,P. S. & Greenberg, E. P. (1999). Bacterialbiofilms: a common cause of persistent infections. Science 284,1318–1322.

Dalrymple, B. & Mattick, J. S. (1987). An analysis of theorganization and evolution of type 4 fimbrial (MePhe) subunitproteins. J Mol Evol 25, 261–269.

Darzins, A. (1993). The pilG gene product, required for Pseudo-monas aeruginosa pilus production and twitching motility, ishomologous to the enteric, single-domain response regulatorCheY. J Bacteriol 175, 5934–5944.

Darzins, A. (1994). Characterization of a Pseudomonas aeruginosagene cluster involved in pilus biosythesis and twitching motility :sequence similarity to the chemotaxis proteins of enterics and thegliding bacterium Myxococcus xanthus. Mol Microbiol 11,137–153.

Dorr, J., Hurek, T. & Reinhold-Hurek, B. (1998). Type IV pili are

1330

Downloaded from www.microbiologyresearch.org by

IP: 54.237.57.119

On: Mon, 23 May 2016 21:09:23

P. aeruginosa FimV and twitching motility

involved in plant–microbe and fungus–microbe interactions. MolMicrobiol 30, 7–17.

Engvall, E. & Perlmann, P. (1972). Enzyme-linked immuno-absorbent assay, Elisa. III. Quantitation of specific antibodies byenzyme-labeled anti-immunoglobulin in antigen-coated tubes. JImmunol 109, 128–135.

Filloux, A., Bally, M., Ball, G., Akrim, M., Tommasson, J. &Lazdunski, A. (1990). Protein secretion in Gram-negative bac-teria : transport across the outer membrane involves commonmechanisms in different bacteria. EMBO J 9, 4323–4329.

Folkhard, W., Marvin, D. A., Watts, T. H. & Paranchych, W. (1981).Structure of polar pili from Pseudomonas aeruginosa strains Kand O. J Mol Biol 149, 79–93.

Forest, K. T. & Tainer, J. A. (1997). Type-4 pilus-structure : outsideto inside and top to bottom – a minireview. Gene 192, 165–169.

Hazlett, L. D., Moon, M. M., Singh, A., Berk, R. S. & Rudner, X. L.(1991). Analysis of adhesion, piliation, protease production andocular infectivity of several P. aeruginosa strains. Curr Eye Res10, 351–362.

Hoang, T. T. & Schweizer, H. P. (1997). Identification and geneticcharacterization of the Pseudomonas aeruginosa leuB geneencoding 3-isopropylmalate dehydrogenase. Mol Gen Genet 254,166–170.

Hoang, T. T., Williams, S., Schweizer, H. P. & Lam, J. S. (1997).Molecular genetic analysis of the region containing the essentialPseudomonas aeruginosa asd gene encoding aspartate-β-semi-aldehyde dehydrogenase. Microbiology 143, 899–907.

Hobbs, M., Dalrymple, B. P., Cox, P. T., Livingston, S. P., Delaney,S. F. & Mattick, J. S. (1991). Organization of the fimbrial generegion of Bacteroides nodosus : class I and class II strains. MolMicrobiol 5, 543–560.

Hobbs, M., Collie, E. S. R., Free, P. D., Livingston, S. P. & Mattick,J. S. (1993). PilS and PilR, a two-component transcriptionalregulatory system controlling transcription of type 4 fimbriae inPseudomonas aeruginosa. Mol Microbiol 7, 669–682.

Joris, B., Englebert, S., Chu, C. P., Kariyama, R., Daneo-Moore, L.,Shockman, G. D. & Ghuysen, J. M. (1992). Modular design of theEnterococcus hirae muramidase-2 and Streptococcus faecalisautolysin. FEMS Microbiol Lett 91, 257–264.

Kizil, G., Todd, I., Atta, M., Borriello, S. P., Ait-Tahar, K. &Ala’Aldeen, D. A. (1999). Identification and characterization ofTspA, a major CD4(­) T-cell- and B-cell-stimulating Neisseria-specific antigen. Infect Immun 67, 3533–3541.

Laemmli, U. K. (1970). Cleavage of structural proteins during theassembly of the head of bacteriophage T4. Nature 227, 680–685.

Liles, M. R., Viswanathan, V. K. & Cianciotto, N. P. (1998).Identification and temperature regulation of Legionella pneumo-phila genes involved in type IV pilus biogenesis and type II proteinsecretion. Infect Immun 66, 1776–1782.

Martin, P. R., Hobbs, M., Free, P. D., Jeske, Y. & Mattick, J. S.(1993). Characterization of pilQ, a new gene required for thebiogenesis of type 4 fimbriae in Pseudomonas aeruginosa. MolMicrobiol 9, 857–868.

Mattick, J. S., Bills, M. M., Anderson, B. J., Dalrymple, B., Mott,M. R. & Egerton, J. R. (1987). Morphogenetic expression ofBacteroides nodosus fimbriae in Pseudomonas aeruginosa. JBacteriol 169, 33–41.

Mattick, J. S., Hobbs, M., Cox, P. T. & Dalrymple, B. P. (1993).Molecular biology of the fimbriae of Dichelobacter (prev.Bacteroides) nodosus. In Genetics and Molecular Biology ofAnaerobic Bacteria, pp. 517–545. Edited by M. Sebald. NewYork: Springer.

Mongkolsuk, S.,Vattanaviboon, P., Rabibhadana, S.& Kiatpapan,P. (1993). Versatile gene cassette plasmids to facilitate theconstruction of generalized and specialized cloning vectors. Gene124, 131–132.

Morales, V. M., Backman, A. & Bagdasarian, M. (1991). A series ofwide-host-range low-copy-number vectors that allow directscreening for recombinants. Gene 97, 39–47.

Murata, T. (1996). The trpF nucleotide sequence and its promoteranalysis in Pseudomonas aeruginosa. Microbiol Immunol 40,107–114.

Nakai, K. & Horton, P. (1999). : a program for detectingsorting signals in proteins and predicting their subcellularlocalization. Trends Biochem Sci 24, 34–36.

O’Toole, G. A. & Kolter, R. (1998). Flagellar and twitching motilityare necessary for Pseudomonas aeruginosa biofilm development.Mol Microbiol 30, 295–304.

Paranchych, W. & Frost, L. S. (1988). The physiology andbiochemistry of pili. Adv Microb Physiol 29, 53–114.

Parge, H. E., Bernstein, S. L., Deal, C. D. & 9 other authors (1990).Biochemical purification and crystallographic characterization ofthe fiber-forming protein pilin from Neisseria gonorrhoeae. J BiolChem 265, 2278–2285.

Pepe, C. M., Eklund, M. W. & Strom, M. S. (1996). Cloning of anAeromonas hydrophila type IV pilus biogenesis gene cluster :complementation of pilus assembly functions and charac-terization of a type IV leader peptidase}N-methyltransferaserequired for extracellular protein secretion. Mol Microbiol 19,857–869.

Ponting, C. P., Schultz, J., Milpetz, F. & Bork, P. (1999). :identification and annotation of domains from signalling andextracellular protein sequences. Nucleic Acids Res 27, 229–232.

Potera, C. (1999). Forging a link between biofilms and disease.Science 283, 1837, 1839.

Ratnaningsih, E., Dharmsthiti, S., Krishnapillai, V., Morgan, A.,Sinclair, M. & Holloway, B. W. (1990). A combined physical andgenetic map of Pseudomonas aeruginosa PAO. J Gen Microbiol136, 2351–2357.

Roine, E., Raineri, D. M., Romantschuk, M., Wilson, M. & Nunn,D. N. (1998). Characterization of type IV pilus genes in Pseudo-monas syringae pv. tomato DC3000. Mol Plant–Microbe Interact11, 1048–1056.

Sambrook, J., Fritsch, E. F. & Maniatis, T. (1989). MolecularCloning: a Laboratory Manual, 2nd edn. Cold Spring Harbor,New York: Cold Spring Harbor Laboratory.

Sato, H., Okinaga, K. & Saito, H. (1988). Role of pili in thepathogenesis of Pseudomonas aeruginosa burn infection.Microbiol Immunol 32, 131–139.

Schultz, J., Milpetz, F., Bork, P. & Ponting, C. P. (1998). , asimple modular architecture research tool : identification ofsignaling domains. Proc Natl Acad Sci USA 95, 5857–5864.

Schweizer, H. P. (1991). Escherichia–Pseudomonas shuttle vectorsderived from pUC18}19. Gene 97, 109–112.

Schweizer, H. P. (1992). Allelic exchange in Pseudomonasaeruginosa using novel ColE1-type vectors and a family ofcassettes containing a portable oriT and the counter-selectableBacillus subtilis sacB marker. Mol Microbiol 6, 1195–1204.

Semmler, A. B. T., Whitchurch, C. B. & Mattick, J. S. (1999). Are-examination of twitching motility in Pseudomonas aeruginosa.Microbiology 145, 2863–2873.

Simon, R., Priefer, U. & Pu$ hler, A. (1983). A broad host rangemobilization system for in vivo genetic engineering: transposon

1331

Downloaded from www.microbiologyresearch.org by

IP: 54.237.57.119

On: Mon, 23 May 2016 21:09:23

A. B. T. SEMMLER and OTHERS

mutagenesis in gram negative bacteria. Bio}Technology 1,784–791.

Stone, B. J. & Abu Kwaik, Y. (1998). Expression of multiple pili byLegionella pneumophila : identification and characterization of atype IV pilin gene and its role in adherence to mammalian andprotozoan cells. Infect Immun 66, 1768–1775.

Towbin, H., Staehelin, T. & Gordon, J. (1979). Electrophoretictransfer of proteins from polyacrylamide gels to nitrocellulosesheets : procedure and some applications. Proc Natl Acad Sci USA76, 4350–4354.

Vieira, J. & Messing, J. (1991). New pUC-derived cloning vectorswith different selectable markers and DNA replication origins.Gene 100, 189–194.

Watson, A. A., Alm, R. A. & Mattick, J. S. (1996a). Construction of

improved vectors for protein production in Pseudomonasaeruginosa. Gene 172, 163–164.

Watson, A. A., Mattick, J. S. & Alm, R. A. (1996b). Functionalexpression of heterologous type 4 fimbriae in Pseudomonasaeruginosa. Gene 175, 143–150.

West, S. E. H. & Iglewski, B. H. (1988). Codon usage in Pseudo-monas aeruginosa. Nucleic Acids Res 16, 9323–9334.

Wu, S. S. & Kaiser, D. (1995). Genetic and functional evidence thatType IV pili are required for social gliding motility in Myxococcusxanthus. Mol Microbiol 18, 547–558.

.................................................................................................................................................

Received 23 December 1999; revised 29 February 2000; accepted 7 March2000.

1332