Gene, 101 (1990) 33-44

0 1991 Elsevier Science Publishers B.V. 0378-1119/91/$03.50 33

GENE 03995

Characterisation of a Pseudomonas aeruginosa twitching motility gene and evidence for a specialised protein export system widespread in eubacteria

(Fimbriae; pili; nucleotide sequence; nucleotide-binding protein; recombinant DNA; DNA uptake)

Cynthia B. Whitchurch”, Matthew Hobbs”, Susan P. Livingstona, Viji Krishnapillaib and John S. Mattick”

a Centrefor Molecular Biology and Biotechnology, The University of Queensland, Brisbane, Qld. 4072 (Australia); and b Department of Genetics and Developmental Biology, Monash University, Clayton, Vie. 3168 (Australia) Tel. (61-3)5653853

Received by P.A. Manning: 9 October 1990 Revised: 19 November 1990 Accepted: 20 November 1990

SUMMARY

Type-4 timbriae (pili) are associated with a phenomenon known as twitching motility, which appears to be involved with

bacterial translocation across solid surfaces. Pseudomonas aeruginosu mutants which produce fimbriae, but which have lost

the twitching motility function, display altered colony morphology and resistance to limbrial-specific bacteriophage. We have

used phenotypic complementation of such mutants to isolate a region of DNA involved in twitching motility. This region

was physically mapped to a SpeI fragment around 20 min on the P. aeruginosu PA0 chromosome, remote from the major

fimbrial locus (around 75 min) where the structural subunit-encoding gene (JmA/pU) and ancillary genes required for

limbrial assembly (pilB, C and D) are found. A gene, pilT, within the twitching motility region is predicted to encode a

344-amino acid protein which has strong homology to a variety of other bacterial proteins. These include the P. aeruginosu

PilB protein, the ComG ORF-1 protein from the Bacillus subtilis comG operon (necessary for competence), the PulE protein

from the Klebsiellu oxytocu (formerly K. pneumoniue) pulC-0 operon (involved in pullulanase export), and the VirB- 11 protein

from the virB operon (involved in virulence) which is located on the Agrobucterium tumefuciens Ti plasmid. We have also

identified other sets of homologies between P. ueruginosu limbrial assembly (Pil) proteins and B. subtilis Corn and K. oxytocu

Pul proteins, which suggest that these are all related members of a specialised protein export pathway which is widespread

in the eubacteria.

INTRODUCTION

The fimbriae (or common pili) of P. ueruginosu are long

thin filaments approx. 6 nm in diameter and extending from

Correspondence to: Dr. J.S. Mattick, Centre for Molecular Biology and

Biotechnology, University ofQueensland, Brisbane, Qld. 4072 (Australia)

Tel. (61-7)3654447; Fax (61-7)3654388; Internet e-mail address:

cmbmattick@uqvax.cc.uq.oz.au.

Abbreviations: aa, amino acid(s); bp, base pair(s); jhA/piL4, limbrial

subunit-encoding gene; kb, kilobase or 1000 bp; nt, nucleotide(s);

oligo, oligodeoxyribonucleotide; ORF, open reading frame; I’., Pseudo-

monas; pilB-D, ancillary genes involved in timbrial biosynthesis; pi1 NR,

pil allele encoding multiple ‘non-retractile’ timbriae; proC, gene encoding

A’-pyrroline 5-carboxylate reductase; RBS, ribosome-binding site; SDS,

sodium dodecyl sulfate; wt, wild type.

the cell surface for up to several pm in length (Bradley,

1972a; Folkhard et al., 1981). They are comprised of a

small structural subunit of about 16 kDa (Sastry et al.,

1985), which is apparently arranged in a helical manner

(approx. five subunits/turn) around the long axis of the

filament (Folkhard et al., 1981; Watts et al., 1983).

P. ueruginosu is an opportunistic pathogen of a variety of

mammals, including humans (Bodey et al., 1983), and the

limbriae are thought to play an important role during

colonization by mediating attachment of the bacterium to

host epithelial surfaces (Doig et al., 1988; Irvin et al., 1989;

Sato et al., 1988; Woods et al., 1980).

P. ueruginosu limbriae are closely related to those found

in a number of other bacterial pathogens, including Neisseriu

gonorrhoeue, N. meningitidis, Moruxellu bovis, and Diche-

34

lobacter [prev. Bacteroides (Dewhirst et al., 1990)] nodosus and are collectively termed type-4 (Ottow, 1975; Dalrymple

and Mattick, 1987). Type-4 limbrial subunits range from

about 145-160 aa in length, and have several distinctive

features, including a short (6-7 aa) positively charged

leader sequence, an unusual modified aa (N-methylphenyl-

alanine) as the first residue in the mature protein, and a

highly hydrophobic and highly conserved N-terminal

domain (see Dalrymple and Mattick, 1987).

Type-4 fimbriae are also characterized by a polar loca-

tion on the cell, and their association with a phenomenon

termed twitching motility. On this basis type-4 limbriae

appear to occur widely in the /I and y subdivisions of the

Proteobacteria, as well as in other species of less certain

phylogenetic position (Dalrymple and Mattick, 1987 ;

Henrichsen, 1975). However, little is known about

twitching motility. It is manifested as a spreading of

colonies with ‘ground-glass’ edges under appropriate

(humid) conditions on agar plates, as well as a jerky motion

in suspension (Henrichsen, 1983). This has led to the sug-

gestion that the limbriae mediate bacterial translocation

across cell surfaces and that this constitutes an important

virulence factor in vivo (Bradley, 1980a; Pedersen et al.,

1972; Depiazzi and Richards, 1985).

Some years ago, Bradley conducted a series of studies on

P. aeruginosa timbriae and twitching motility using electron

microscopy and fimbrial-specific phage (Bradley 1972a,b,c;

1974; 1980a,b; Bradley and Pitt, 1974). Phage-resistant

mutants were found to fall into two categories, those lacking

fimbriae (pi/-) and those that appeared to have multiple

but non-retractile fimbriae (pilNR) (Bradley, 1972a; 1974;

1980a). Moreover, both types of mutant had lost the ability

to spread on agar plates, and grew as small domed, rather

than large flat, colonies (Bradley, 1974; 1980a,b; see also

Pedersen et al., 1972). In wt cells (discharged), phage par-

ticles bound to limbriae were located at the cell surface,

whereas in the multilimbriate mutants phage were attached

at various points along the fimbriae, whose mean length was

twice that of the wt (Bradley 1972~; Bradley and Pitt, 1974).

Bradley proposed that wt limbriae are retractile and that

this is the basis of twitching motility, as well as being

required for phage susceptibility (Bradley 1980a; Bradley

and Pitt, 1974). The multilimbriate phenotype was inter-

preted to be a consequence of the lack of ability to retract

the limbriae. Preliminary genetic mapping showed that

pilNR was chromosomally remote from pil- (Bradley,

1980b) which presumably affected either the structural sub-

unit gene (jrnA/pifA) (Sastry et al., 1985; Strom and Lory,

1986; Hobbs et al., 1988) or the recently identified ancillary

genes (pilB, piIC and pilD) located adjacent to it (Nunn

et al., 1990).

To understand more about the function of type-4

fimbriae, we isolated a region of P. aeruginosa DNA that

could complement pilNR mutations, using colony mor-

phology and the restoration of phage sensitivity as pheno-

typic markers. Here we report the similarity of a twitching

motility gene (pilT) identified within this region, and of

previously reported limbrial assembly genes, to genes in

other bacterial systems involved in DNA transport and

protein export.

RESULTS AND DISCUSSION

(a) Cloning of the twitching motility gene of Pseudomonas

aeruginosa P. aeruginosa sequences necessary for twitching motility

were isolated by phenotypic complementation, as follows.

Genomic DNA from the P. aeruginosa wt prototype strain

PA01 was cleaved with BarnHI, inserted into the vector

pME290 (Itoh and Haas, 1985) and transformed into the

pilNR (non-twitching) P. aeruginosa mutant K2.2 (originally

designated K/2pfS; Bradley, 1974; 1980a). These transfor-

mants were then screened for complementation of the mu-

tation as judged by restoration of the wt colony mor-

phology. Three distinct colony types were identified in

which the characteristic small domed appearance of the

host was altered. Examination of a number of clones con-

firmed that each of these colony types was consistently

associated with a particular fragment of cloned genomic

DNA. Representative recombinant plasmids conferring

these phenotypes were designated pOT301 (25-kb insert),

pOG302 (4-kb insert) and pSB2 (5.9-kb insert). To deter-

mine which colony type represented true restoration of the

wt phenotype, clones containing these plasmids were tested

for their sensitivity to the fimbrial-specific bacteriophage

P04. K2.2 cells harbouring pOT301 and pOG302 retained

their resistance to the phage, whereas in the case of pSB2

phage sensitivity was completely restored, with the plaque

titre being identical to the wt control. Thus the 5.9-kb

BamHI insert of pSB2 is able to complement the

P. aeruginosa K2.2 pilNR mutation, as judged by both

colony morphology and phage sensitivity. The pSB2 was

also able to restore the wt phenotype to another pilNR mutant, P. aeruginosa PA02001.2 (Bradley, 1980a), indi-

cating that the mutations in both of these strains are at the

same locus.

(b) Mapping of the twitching motility gene region

The locations on the PA0 chromosome of the twitching

motility region cloned in pSB2 and of the limbrial subunit

gene region were physically mapped by hybridisation of

cloned DNA to genomic ,SpeI fragments (Fig. 1). These two

regions hybridised respectively to spe1 fragments 8 and 5

(data not shown) which are remote from one another

(Ratnaningsih et al., 1990). The twitching motility region

35

EXEN n / , Ikb , X N \r\ s ; \

I I I \ H

M A A,’ AAS 4

A’,AAA / \

/ \ / \

/ \ / \

/ 111. I ’

OKFI

Fig. 1. P. aeruginosa fimbrial genes. At the top of the figure the positions

of the pi/,4 and twitching motility loci on the P. aeruginosa PA0 chromo-

some are depicted on the 75min map of Holloway and Zhang (1990). The

two loci were mapped by hybridising the inserts of the recombinant

plasmids pJSM249 and pJRSB to ,SpeI fragments of the PA0 chromo-

some separated by pulsed-field gel electrophoresis. Plasmid pJSM249

(Hobbs et al., 1988) contains a 6.2-kb EcoRI insert which carries the

P. aerugimsu PAK timbrial subunit gene piL4, as well as the ancillary

genes pilB, pilC and a portion of pilD (Nunn et al., 1990). These frag-

ments hybridised respectively to SpeI fragments 5 and 8, which have

recently been mapped on the P. aeruginosa genome (Ratnaningsih et al.,

1990). In the centre of the figure the 5.9-kb BamHI fragment cloned from

the twitching motility region is shown in greater detail. B, BamHI; E,

EcoRI; N, NruI; S, SalI; X, XhoI. Transposon insertion at sites marked

by blackened arrowheads interrupted the ability of pKTSB5.9 to restore

phage sensitivity, whereas insertion at sites marked by open arrowheads

did not affect this property. Arrows in the lower part of the figure

represent ORFs deduced from nt sequencing. The entire nt sequence of

the proC gene has been determined by Savioz et al. (1990). The pilT gene

is heavily shaded, and other ORFs with a high probability of encoding

proteins as predicted by RNY score are lightly shaded. Methods. Cloning

and transformation procedures were as previously described (Mattick

et al., 1984; 1987). Restriction mapping and hybridisation studies were

performed with the plasmid pJRSB. The plasmid pKTSB5.9 was

randomly mutagenised with the transposon y6 (Davidson et al., 1975;

Guyer, 1978). 76, which is located on the F plasmid of E. coli, was

introduced into pKTSB5.9 by first transforming the recombinant plasmid

into the F + E. coli K-12 strain LC707[F ‘1 and selecting transformants

on LB medium containing 100 pg Ap/ml. Mating with the streptomycin-

resistant F- recipient JM83 (araD-139, d(lac-proAB), rpsL, $80,

lacZAM15) then allowed selection for transconjugants on selective

medium containing both Ap (100 pg/ml) and streptomycin (100 pg/ml).

Screening for phage sensitivity was done by streaking a 20-~1 suspension

of the timbrial-specific bacteriophage PO4 (Bradley, 1973) on selective

agar plates. The colony to be tested was then picked with a sterile

toothpick and cross-streaked across the phage, and the plate then

incubated at 37°C overnight. Phage resistance was detected as uninhibit-

ed growth across the phage streak. Quantitative phage sensitivity was

performed by growing a P. aeruginosa culture to an-t,,, = 0.3-0.5; mixing

100 ~1 of this culture with 10 ~1 of serial dilutions of high titre phage stock

at room temperature for 30 min; adding 3 ml of soft agar; and then

pouring this mixture onto LB agar plates which were incubated overnight

at 37°C. Plaque numbers quantitatively indicated phage sensitivity.

thus maps to a region at about 20 min on the P. aeruginosa

chromosome whilst pihI, B, C and D are located at about

75 min (Fig. 1).

The 5.9-kb BamHI insert of pSB2 was subcloned into

the E. coli multiple-restriction-site plasmid pJRD 184

(Heusterspreute et al., 1985) to give pJRSB, which was

used for restriction mapping (Fig. 1). Subcloning proved

difficult and despite numerous attempts this fragment could

only be obtained in one orientation in this vector. Attempts

to clone this fragment into the high-copy-number vector

pBluescript were also unsuccessful, suggesting that some

product of the 5.9-kb BamHI fragment is toxic to E. coli. Preliminary deletion experiments gave some indication of

the approximate site within pSB2 of the sequences

necessary for complementation of the K2.2 pilNR mutation.

Two overlapping fragments from within the 5.9-kb BamHI

fragment, a lefthand 3.6-kb BamHI-Sal1 fragment and a

righthand 2.5-kbXhoI-BamHI fragment (Fig. l), were sub-

cloned into pKT240 and transformed into K2.2. Although

there was no immediate complementation of the mutation

by either of these plasmids, it was noted that after two days

growth colonies containing the 2.5-kb XhoI-BamHI plas-

mid developed a morphology characteristic of twitching

motility. An explanation for this observation is that

although neither of these fragments contains the complete

region sufficient for immediate complementation by pro-

duction of a functional protein, the 2.5-kb fragment

contains an incomplete coding region which includes the wt

version of the region which is defective in K2.2. Reconsti-

tution of a functional gene in the chromosome by

homologous recombination could then give rise to the de-

layed complementation which was observed. The pilNR mutation in K2.2 therefore probably lies to the right of the

Sal1 site in Fig. 1.

The 5.9-kb BamHI fragment was next subcloned into the

shuttle vector pKT240 (Bagdasarian et al., 1983) to give

pKTSB5.9 which, as expected, was still able to restore the

twitching motility phenotype to K2.2 as indicated by sensi-

tivity to phage P04. pKTSB5.9 was then randomly muta-

genised in E. coli with the transposon y6 (Davidson et al.,

1975; Guyer, 1978) to more closely define the region re-

quired for twitching motility. Fifteen pKTSB5.9 derivatives

containing y6 insertions within the 5.9-kb cloned segment

were chosen for transformation into K2.2 determine if the

insertion had disrupted the ability of the plasmid to restore

phage sensitivity. As shown in Fig. 1, the sites of y6insertion

which prevent complementation are clustered within a

900-bp region. Since transposon insertions 300 bp up-

stream and 500 bp downstream from this region do not

disrupt the ability of pKTSB5.9 to complement the K2.2

pilNR mutation, it appears that the area of interest lies

within a 1.7-kb segment (Fig. 1).

36

CGCTA’~GCGGGGTGTGCTCATA GGGGATTCCTTCAAGAAGCCGGCGCGCCGTAGTCGCGGGCGCCGAACAGGGCGGTACCGATGCGGACCCAGGTCGCACCTTCG~CGATGGCTGCCTCG 10 20 RBS 40 50 60 70 80 90 100 110 *hoI

GCGATACGCCCCACACGAGTATCCCCTAAGGAAGTTCTTCGGCCGCGCGGCATCAGCGCCCGCGGCTTGTCCCGCCATGGCTACGCCTGGGTCCAGCGTGGAAGCGGCTACCGACGGAGC A I R P T S M <--- ProC * S A P A G Y D R A G F I, ATGIRVWTAGEG I A A E

AGGTCGTCGCTCATGCCCATGGACAGGGTGTCCAGGCCAAGGTTCAGGTCCAGCAGCAGTTCGCGCA~GCGGGCGAACGCGGCGTGTTGCGCGGCGCGTTCGGCGGTGGGTTCGGGGATG 130 140 150 160 170 180 190 200 210 220 230 240

TCCAGCAGCGAGTAJGGGTACCTGTCCCACAGGTCCGGTTCCAAGTCCAGGTCGTCGTCAAGCGCGTCCGCCCGCTTGCGCCGCACAACGCGCCGCGCAAGCCGCCACCCAAGCCCCTAC LDDSMGMSLTDLG LNLDLLLERLRAFAAHQAARERTPEPi

GCCATCAGGCCACGCAATCGGAGGTTGGGCAGTTGCTTCACGGCCTCGGCCAGGGCCGGCAGGTCCTCGGGGGCGCAGCCGGACTTGCTGGCTTCGCCGCTGACGTTGACCTGCAGGCAG 250 260 270 280 290 300 310 320 330 340 350 360

CGGTAGTCCGGTGCGTTAGCCTCCAACCCGTCAACGAAGTGCCGGAGCCGGTCCCGGCCGTCCAGGAGCCCCCGCGTCGGCCTGAACGACCGAAGCGGCGACTGCAACTGGACGTCCGTC AMLGRLRLNPLQKVAEA LAP LDEPACGSXSAEGSVNVQLC

ACATTCAGGGGCGGCAGCCCGGCCGGGCGTTGCTCCGACAGGCGCTGCGCGATCTTCAACCGGTCCACCGAGTGCACCCACTGGAAATGCTCGGCGATGGGCCGCGTCTTGTTCGACTGG 370 380 390 400 410 420 430 440 450 460 470 480

TGTRAGTCCCCGCCGTCGGGCCGGCCCGCAACGRGGCTGTCCGCGACGCGCTAGAAGTTGGCCAGGTGGCTCACGTGGGTGACCTTTACGAGCCGCTACCCGGCGCAGAACAAGCTGACC V N L P P LGAPRQES L R Q A I KLRDVS HVWQFHEAIPRTXNSQ

ATGGGGCCGATGAAGTGCCAGTTCAAGGGCAGGTCGGCCAGTTCGGCCTGCTTGCCGAGGGCCTCCTGCAGGTAGTTTTCGCCGAAGTCGCGAAGGCCGGC~CGTGCGCCTC~GCACC 490 500 510 520 530 540 550 560 NnlI 580 Promoter 600

TACCCCGGCTACTTCACGGTCAAGTTCCCGTCCAGCCGGTCAAGCCGGACGAACGGCTCCCGGAGGACGTCCATCAAAAGCGGCTTCAGCGCTTCCGGCCGCCGCACGCGGAGCGCGTGG IPGIFHWNLPLDALEAQXGLAEQLYNEGFDRLGAAHAERV

0RF2 ---> MRAATFAI F S A I v GCGGCGGCGGGCTTGGTCTTGCTCACGGCGAGCAGGCCGACCGTGGCCGGATCGCGCCCCGCAGCTTGCGCTGCCTCACGGATGCGC~CGGCAACCTTTGCAATAT~CTCTGCTATCGTG

610 620 630 640 650 660 670 680 690 700 710 720 CGCCGCCGCCCGAACCAGAACGAGTGCCGCTCGTCCGGCTGGCACCGGCCTAGCGCGGGGCGTCGAACGCGA~GGAGTGCCTACGCGCGCCGTTGGAAACGTTATAAGAGACGATAGCAC AAAPXTKSVALLGVTAPDRGAAQAAER IRAAVKAINEAiT

DITCALWKCGRRXLDALE TAGLDGHLPVPVSRFRVWRRTP GACATTACCTGCGCCCTATGGAAGTGCGGTCGGCGTCGGCTCGACGCCCTCGAAACCGCCGGCC?CGATGGCCATCTTCCTGTTCCAGTGTCGAGGTTCCGCGTCTGGCCCAGGACACCA

730 RBS 740 750 760 770 780 790 800 810 820 830 840 CTGTAATGGACGCGGGATACCTTCACGCCAGCCGCAGCCGAGCTGCGGGAGCTTTGGCGGCCGGAGCTACCGGTAGAAGGACAAGGTCACAGCTCCAAGGCGCAGAC~GCGTCCTGTGGT S M <--- 0RF5

RVSWKHPGSSGVF LVRDGGFGGILPACL* PilT ---> M D I T E L L CGCGTGTCCTGGAAGCACCCTGGCTCATCCGCTGTGTTTTCCTTGTCCGAGACGGCGGCTTTGGCGGCATTCTACCTGCTTGCTTGTAATTGGGGAGTCCCATGGATATTACCGAGCTGCTC

850 860 870 860 890 900 920 920 930 RBS 940 950 960

AFSAKQGASDLHLSAGLPPMI RVDGDVRR I N L P P LEHKQV GCCTTCAGTGCCAAACAGGGCGCTTCGGACCTGCACCTCTCCGCCGGCCTGCCACCCATGATCCGGGTGGATGGCGATGTACGCCGGATCAACCTGCCACCGC'rGGAACACAAGCAGGT~

970 980 990 1000 1010 1020 1030 1040 1050 1060 1070 1080

H A L I Y ff IMNDKQRKDFEE FLETDFSFEVPGVARFRVNAFN CATGCGCTGATCTACGACATCATGAACGACAAGCAGCG~AAGGAC?TCGAGGAATTCCTCGAGACCGACTTCTCCTTCGAGGTGCCGGGCGTCGCGCGTTTCCGGG'~CAACGCCTTCAAC

1090 1100 1110 1120 1130 EcoRI XhOI 1150 1160 1170 1180 1190 1200

QNRGAGAVFRT IPSKVLTMEELGMGEVFKRVSDVPRGLVL CAGAACCGTGGCGCCGGCGCGGTATTCCGGACCATTCCCTCCAAGGTACTGACCATGGAGGAGCTT~GCATGGGAGAAGTGTTCAAACGTGTTTCAGACGTCCCGCGCGGGTTGGTACTG

1210 1220 1230 1240 1250 1260 1270 1280 1290 1300 1310 1320

VT G P T G S G K S T T I. A AM L D Y L N N T K Y H H I L T I E D P IEFVHE GTCACCGGGCCGACCGGTTCGGGCAAGTCCACCACCACCCTGGCGGCGATGCTCGATTACCTGAACAACACCAAGTACCACCACATCCTCACCATCGAGGACCCGATCGAATTCGTCCACGAA

1330 1340 1350 1360 1370 1380 1390 1400 1410 1420 ECORI 1440

SKKCLVNQREVHRDTLGFSEA LRSALREDPDII LVGEMKD TCGAAGAAGTGCCTGGTCAACCAGCGCGAGGTGCATCGCGACACCCTCGGCTTCAGCGAAGCGCTGCGCTCGGCGCTGCGGGAGGACCCGGACATCATCCTGGTCGGCGAGATGCGCGAC

1450 1460 1470 NrUI 1490 1500 1510 1520 1530 1540 1550 1560

L E.T I R L A L T A A E T G H L V F G T L il T T 5 A A KT IDRVVDVFPAE CTGGAAACCATCCGCCTGGCCCTGACCGCGGCGGAGACCGGCCACCTGGTATTCGGCACCCTGCACACCACCTCGGCGGCGAAGACCATCGACCGGGTGGTCGACGTGTTCCC~GCCGAG

1570 1580 1590 1600 1610 1626 1630 1640 1650 1660 Sal1 1670 ,680

EXAMVRSMLSESLQSVISQTLIKKIGGGRVAAHEIM I G T P GAAAAGGCCATGGTTCGCTCGATGCTCTCCGAGTCGCTGCAATCGGTGATCTCGCAGACCCTGATCAAGAAGATCGGCGGCGGCCGGGTGGCGGCCCACGAGATCATG~rCGGCACCCCG

1690 1700 1710 1720 1730 1740 1750 1760 1170 1780 1790 1800

AIRNLIREDKVAQMYSAIQTGGSLG M Q T LDMCLKGLVAK'G GCGATCCGCAACCTGATCCGCGAGGACAAGGTCGCGCAGATGTATTCGGCGATCCAGACCGGCGGCTCGCTGGGCATGCAGACCCTCGACATGTGCCTCAAGGGCCTGGTCGCCAAGGGC

1810 1820 1830 1840 1850 1860 1870 1880 1890 1900 1910 1920

LISRENAREKAKIPENF* CTGATCAGCCGCGAGAACGCCCGCGAGAAGGCGAAGATCCCGGAAAACTTCTGATCCTGGCGCCGAT~CGCCGCGC~TCGCCCGAATCCCGGAACTGCGTCTAGGATTCAAGAGCGGGAG

1930 1940 1950 1960 1970 1980 1990 2000 2010 2020 2030 2040

AGCGGTACCGTAGGCGCATTCCTACCTATCCTTGCCGGCGAGCGCGTCCAGCGCCCGCTCGCCCGCATCCGGCTCGCCGTCGCGGCGCGCCCCTGAGTGAGCGAGAAC~rCATGGAATTC 2050 2060 2070 2080 2090 2100 2110 2120 2130 2140 2150 EcoRi

(c) Sequence analysis of the pilT gene

A 2.1-kb region which spans the relevant sites of transpo-

son insertion was sequenced (Fig. 2). Comparison of this

sequence with the GenBank database showed that this

sequence overlaps the sequence of the proC gene region

recently reported by Savioz et al. (1990) (Fig. 1). Gene

proC encodes the proline biosynthetic enzyme d’-pyrroline

5-carboxylate reductase. These sequence data were then

examined for the presence of potential coding regions to

identify candidate genes responsible for twitching motility.

Since the G + C content of this segment of DNA is 65%

there is a low incidence of stop codons encountered in

noncoding reading frames. Thus there are six ORFs which

are greater than 80 codons in length (Figs. 1 and 2), some

of which are presumably not bona fide coding regions.

Based on the RNY gene search technique described by

Sheperd (1981) the most likely coding regions are ORF2

and 3 (reading frame 1) and ORF5 (reading frame 6).

Consistent with this, ORF3 and 5 also possess upstream

sequences which might act as RBSes (Fig. 2).

ORF5 is not involved in twitching motility, since y6

insertions within this gene do not disrupt the ability of

pKTSB5.9 to restore phage sensitivity to the K2.2 mutant.

Furthermore, the 3’ end of ORF5 is just upstream from

proC (Fig. 1; Savioz et al., 1990), suggesting that these two

genes may be cotranscribed and are perhaps both involved

in proline biosynthesis. In contrast, disrupting y6 mutations

do map within ORFs 2 and 3 (Fig. l), which are in the

opposite transcriptional orientation to proC (Fig. 1) and

which may form the first two genes of an operon involved

in twitching motility. The deletion studies (see section b)

suggested that the complementing sequences are located in

the 3’ region of ORF3. This indicates that the relevant gene

is ORF3, and that transposon mutations in 0RF2 have a

polar effect on its expression and ability to complement the

37

pilNR mutation. Moreover, homologues of ORF3 exist in

other systems (see next paragraph), and so we have

designated ORF3 pilT because of its probable involvement

in the twitching motility phenotype, although ORF2 may

also play a role. Interestingly, there is a potential RpoN-

dependent promoter located about 100 bp upstream from

ORF2 (Fig. 2). This class of promoter, which also governs

transcription ofthe subunit gene piZA (Johnson et al., 1986),

is characteristically controlled by a protein activator.

Further evidence that piZT is a gene was obtained from

sequence database searches, which showed that of ORFs

l-6 only the translation product of piZT (ORF3) had a

significant degree of homology with previously reported

sequences (Fig. 3). The matching proteins identified were

the predicted products of ORFl from the Bacillus subtilis comG operon necessary for competence in DNA uptake

(Albano et al., 1989); the recently identified P. aeruginosa fimbrial assembly gene, piZB (Nunn et al., 1990); the puZE

gene of K. oxytoca (formerly K. pneumoniae) involved in

secretion of the extracellular enzyme pullulanase (d’Enfert

et al., 1989); and to a lesser extent ORFll from the virB operon of the A. tumefaciens Ti plasmid, involved in the

transport of A. tumefuciens bacterial DNA to the recipient

host plant tissue (Ward et al., 1988). The similarity of

ComG-1 and VirB-11 has been previously reported

(Albano et al., 1989).

Examination of the alignment of PilT, ComG-1, PulE,

PilB and VirB-11 (Fig. 3) shows that these proteins exhibit

homology across their entire sequences, although there are

two highly conserved regions. The first contains an

nt-binding consensus sequence GXXXXGK(T) which is

common to a wide variety of nt-binding proteins from both

prokaryotes and eukaryotes (Walker et al., 1982; see also

Thompson et al., 1988). The second corresponds to the less

strongly conserved secondary domain (R/KXXXGXXXL-

Fig. 2. The nt sequence of the twitching motility region beginning 116 bp upstream from the XhoI site and extending to an EcoRI site (see Fig. 1). The

first 120 bp overlap with the sequence of the proC gene region reported by Savioz et al. (1990). The complementary strand is shown as far as position

840. Restriction enzyme sites are marked. Predicted aa sequences of the products of ORFs 2, 3 and 5, which are predicted by RNY analysis to have

a high probability of encoding proteins, are given next to the appropriate DNA strand. Putative RBSes are indicated. The positions ofthe other ORFs

shown in Fig. 1 are ORFl: 82-552; ORF4: 447-2021; 0RF6: 1892-1377 (complementary strand). The characteristic GG and GC dinucleotides of a

potential RpoN-dependent promoter are underlined. Nucleotide sequence data were obtained by the dideoxynucleotide chain-termination method (Sanger

et al., 1977). The bacteriophage vectors M13mp18, M13mp19 and M13tg130 (Amersham Corp.) were used for sequence analysis. The sequencing strategy

utilised relevant internal restriction sites within the cloned segment for Ml3 phage-directional ‘forced’ cloning (Ml3mp18, M13mp19 and M13tg130;

Amersham Corp.) or blunt-end cloning (HincII-cut M13mp18; Amersham Corp.). The E. coliK-12 strain JM109 (recA 1, endA l,gvrA96, thi, hsdR17 (r[ ,

ml ), supE44, reL4 1,1-, d(lac-proAB), [F’traD36 proA ‘B + luql QZdM15]) was used for the preparation of single-stranded Ml3 template DNA. The

M 13 universal primer was purchased from Pharmacia Inc. In addition, synthetic oligo primers were synthesised with a Milligen 7500 DNA synthesizer

and were used to prime sequencing reactions in areas where no convenient restriction sites were present, to confirm sequence across cloning sites and

in some cases to clarify problems with sequence compressions. It should be noted that due to the high G + C content of the P. aeruginosa genome, many

problems were encountered with electrophoretic compressions, thereby necessitating the routine use of deazaguanine in the sequencing reactions

(Mizusawa et al., 1986) and the addition of 20% (v/v) formamide to the polyacrylamide gels. Most of the sequence data were obtained from both strands,

although single-strand data were accepted when this was clear and unambiguous. The nt sequences were compiled and analysed with the Macintosh

applications DNA Inspector II (Gross, 1986) and MacVector (International Biotechnologies Inc.). RNY analysis was performed with ANALYSEQ

(Staden, 1984). This nt sequence has been submitted to the GenBank/EMBL sequence databases under accession No. M55524. Last digits of numerals

are aligned with the corresponding nt.

38

I 0 2 0 3 0 4 0 5 0 6 0 PilB MNDSIQLSCLSRQLVQANLLDEKTALQAQTQAQRNKLSLVTHLVQNKLVSGLALAELSAE

7 0 8 0 120

PilB PUIE

PilB PUIE VirB

PilT

2 5 0 260 2 7 0 2 8 0 2 9 0 3 0 0

3 1 0 3 2 0 3 3 0 3 4 0 350 3 6 0

3 7 0 3 8 0 390 4 0 0 4 1 0 4 2 0 PilT TIEDPIEFVHESKKCLVNQREVHR-DTLGFSEA--LRSALREDPDIlLVGEMRDLETIRL ComC-ITLEDPVE--- -TRDEDVLQVQVNEKAGVTYsAG-- PilB

p$,j. .,,.I, 1 G[ TQVNAKVDMTFARG--LRAILRQDPD~VVLVGEIRDGETAQI 0 $j mfjHemAQE[ TAEDPVE----INLEGINQVNVNPRQGMDFSQA--LRAFLRQDPD~~MVGEIRDLETAEI

PUIE

VirB TIEDTLELVIPHDNHVRLLYSKNGAGLGAVSAEHLLQASLRMRPDRILLGEMRD-DAAWA

4 3 0 440 4 5 0 4 6 0 4 7 0 4 8 0

VirB RAYEDVYEVGEIWLAADARRRGETIGDLLNQ

5 5 0 5 6 0 5 7 0 5 8 0 590 PilT NARnKAKIPENF

Fig. 3. Sequences homologous to PilT were identified by searching the translated GenBank database (release 65) with TFASTA (Pearson and Lipman,

1988). The predicted translation products of the following genes have been aligned with PilT: ORFl from the B. subtilis comG operon (Albano et al., 1989);

the P. aeruginosa timbrial assembly gene pilB (Nunn et al., 1990); pulE from the K. oxytocapulc-0 pullulanase export operon (d’Enfert et al., 1989; the

3’ sequence of pulE has not been published but is available from the GenBank entry with locus name KPNPULL); and ORFI 1 from the A. tumefaciens

virB operon located on the Ti plasmid (Ward et al., 1988). Gaps (dashes) have been introduced to improve the alignment. Matches to a two-part m-binding

consensus sequence (see section c) are shaded. Positions at which at least two identical residues are found where there are four or fewer sequences aligned,

or three identical residues where five sequences are aligned, are boxed. Last digits of numerals are aligned with the corresponding aa.

hydrophobic-hydrophobic-hydrophobic-hydrophobic-D),

present in only some nt-binding proteins (Walker et al.,

1982; Fry et al., 1986; see also Albano et al., 1989). The

presence of these sequences indicates that PilT is likely to

be an nt-binding protein. A second characteristic shared by

all of these proteins is that they are largely hydrophilic and

do not appear to possess any stretches of hydrophobic aa

which could act as membrane-spanning domains, nor

leader sequences which could direct their export from the

cell. It appears likely, therefore, that PilT is located within

the cytoplasm.

Further comparison of these homologues highlights the

fact that they can be subgrouped according to their degree

of homology and size similarities. PulE, PilB and ComG-1

are most alike in aa sequence across the entire length of the

proteins. ComG-1, however, is significantly smaller than

either PilB and PulE but is closer in size to PilT. PilT is not

as strongly homologous to PilB, PulE and ComG-1 but is

closer to these three in aa sequence than the VirB-11 protein

which is the most dissimilar of the group. These size

similarities and sequence homology may reflect the degree

of functional similarity between the proteins.

(d) Similarities between bacterial fimbrial assembly, pro- tein export and competence systems

Because of these unexpected relationships, we carried

out further comparisons of P. aeruginosa fimbrial assembly

(Pil) proteins with B. subtilis Corn proteins involved in de-

40

ComC PiD

Pdo

ComC PilD Pdo

come PilD Pdo URF

come PilD PulO URF

Cod PilD

10 MLSILFIF~ILGSFYYTAGC~I~HLSIIA-.---

u?O o i ‘I’ a0 50 o 60

MPLLDYLASHPLAFVLCAILLGLLVGSFLNVVVHRLPKMMERNWKAEAREALGLEPEPKQ MVENIALLPEFAAQYPFLWGSFLFLSIGAFGSFFNVVIH~LIPMMEQAE-----

130 140 150 160 170 180

190 200 2 10 220 230 240

250 260 270 280 290

Fig. 5. Alignment ofpredicted products ofthe P. aeruginosa fimbrial assembly gene pilD (Nunn et al., 1990), the B. subtilis competence gene comC (Mohan

et al., 1989), pu/O from the K. oxytoca pulC-0 pullulanase export operon (Pugsley and Reyss, 1990), and a partially sequenced unassigned ORF (URF)

which lies downstream from the bfr gene in E. cofi (Andrews et al., 1989). Gaps (dashes) have been introduced to improve the alignment. Positions at

which at least two identical residues are found are boxed. Pairs of Cys residues at positions 72/75 and 97/100 (see section d) are printed in bold face

and shaded. Numeral alignment as in Fig. 3.

A Type 4 Fimbrial Subunit Consensus MK&bLQKG F T L I EUM I VWA I I,VG I L A Al&A

B. subfilis ComG-3 MN-EKG ComG-4 MNIKLNEEKG ComG-5 MWRENKG

K. oqoca PulG MQRQRG pull MKKQSG Puu MIRRSSG

V. cholerae TcpA MQLLKQLFKKKFVKEEHDKKTGQEG MTLLEVmlVLm%jGVVSAGV

COIlSellSllS M (x)4.9 G FTLiEvmivLaligiLIlav ms I ml lamYlIS”YaYia

P ” ” i s a s a &?

Y

E. coli %:

MPVKEQG MSASLKNQQG

Fig. 6. Putative signal sequences. (A) Alignment of the N-terminal aa sequences of proteins with type-4 timbrial subunit-like leader sequences. The

consensus sequence for type-4 subunits derived by Dalrymple and Mattick (1987) is given at the top of the figure. The symbol ‘b’ represents any small

aa. An arrow indicates the point at which the short ‘leader’ sequence is removed during export of subunits and assembly of timbriae (see Dalrymple and

Mattick, 1987). It is not yet known whether the other proteins shown here are cleaved in a similar manner. The other protein sequences are the predicted

products of ORF3,ORF4 and 0RF5 from the B. subtilis comG competence operon (Albano et al., 1989); and of pulG, pull and puLJ from the K. oxytoca

pulC-0 pullulanase export operon (Reyss and Pugsley, 1990). The predicted sequence of a major subunit of the toxin coregulated pilus of Vibrio cholerue

(Faast et al., 1989), which seems to be related to the type-4 timbrial system, is also included. A two-part consensus sequence (shown in bold) was derived

from this alignment and is presented at the bottom of part A. The first part represents an N-terminal sequence of 5-10 aa including two or more charged

residues. In the second part of the consensus upper-case letters represent the residue found in at least six of the eight sequences, and lower-case letters

residues found in at least two sequences. Within the first part of the alignment, charged residues have been highlighted (shaded), and in the second part

matches to the consensus sequence have been boxed. (B) The deduced products oftwo unassigned ORFs (URFl and URF3) found in the E. coli thyA-recC

intergenic region (Finch et al., 1986) have been aligned with the consensus sequence, and matches indicated as in part A.

(e) A specialised eubacterial protein export system that in other type-4 fimbriate organisms (Mouuxellu and

The homologies between the Pi1 proteins of P. aeruginosa Neisseria species) the presence of fimbriae is correlated with

and those involved in DNA transport in B. subtilis and competence for transformation by DNA (Bsvre and

A. tumefaciens are intriguing, especially in view of the fact Froholm, 1972; Biswas et al., 1977; Seifert et al., 1988). In

N. g~~~~~~oe~e this competence involves the reco~ition of specific nt sequences in DNA released from lysed cells (Graves et ai., 1982; Goodman and Scocca, 1988). Type-4 fimbriae are thought to be retractile (see INTRODUCTION),

which perhaps provides a mechanism by which DNA may be brought into contact with the cell, as appears to be the case with fimbrial-specific bacteriophage (Bradley, 1980a; Bradley and Pitt, 1974). It has been suggested that the sex pilus, a plasmid-encoded organelle responsible for DNA uptake by bacterial conjugation, brings cells into contact by retracting (Marvin and Hohn, 1969; Novotny and Fives- Taylor, 1974). However, there is no evidence that type-4 limbriae bind DNA (Mathis and Scocca, 1984), and the correlation of the presence of fimbriae with competence in some species may simply be due to the co-ordinate expres- sion of the ~mb~a1 subunit with a number of other outer- membrane proteins, including those responsible for the up- take of DNA (see Klimpel and Clark, 1988).

Irrespective of the relationship between type-4 fimbriae and competence per se, the existence of Pi1 protein homologues in different species, including those involved in

41

pulluIanase secretion, suggests that they are all components of a specialised protein export system which is widespread in the eubacteria. Although fimbriae are attached to the cell, the fimbrial subunit can be considered a secreted protein since it is transported through the outer membrane. In both Gram + and Gram- organisms, proteins exported from the cytoplasm are usually synthesised with a transitory N-ter- minal leader sequence which directs the protein into the membrane, probably either by interacting with membrane- associated component of the secretion apparatus, or by stabilising a conformation which is energetically consistent with spontaneous insertion. The classical signal sequence is 20-30 ad in length and consists of three parts: one or more positively charged aa near the N terminus, a hydrophobic core, and a leader peptidase cleavage reco~ition site. Sub- sequent to or during translocation of a protein to the membrane, the leader peptide is proteolytically removed. Type-4 fimbrial subunits are synthesised with an N-termi- nal sequence which resembles this canonical description (see Strom and Lory, 1987) and indeed a fimbrial subunit- alkaline phosphatase fusion protein containing the N-termi-

---” ‘.‘- 4 P. aerugiwzzz fimbrial genes

pilA pilC

B. subtilis competence genes

oRF2 oFtF3 oRF4 ow5

comG operon

K. ~xy~~c~ pu~~u~anase secretion genes

pulE pulF pulG pulH puM pulJ pul0

puZC-0 operon

Fig. 7. Comparison of the organisation of P. aerzrginosa fimbrial genes found at the fimA-D (Nunn et al., 1990) and pilT loci, B. sub&s late competence genes found at the comG (Albano et al., 1989) and comC (Mohan et al., 1989) loci, and the K. o.xytocapulC-0 operon (d’Enfert et al., 1989; Pugsley and Reyss, 1990; Reyss and Pugsley, 1990). Arrows represent the size and transcriptional orientation of genes. Similarly shaded areas indicate regions of homology as discussed in section d.

42

na15 1 aa of the subunit is translocated across the cytoplas-

mic membrane (Strom and Lory, 1987). However, although

it appears that the early stages of fimbrial export rely upon

a general mechanism, the subunit subsequently follows an

alternative pathway to the majority of exported proteins.

The preprotein is not processed by leader peptidase I, but

instead is cleaved at an atypical site: only a 6- or 7-aa long

peptide (region Ia; see Dalrymple and Mattick, 1987) is

removed during export and assembly of the limbrial

subunits.

The P. aeruginosa fimbrial accessory proteins PilB, PilC,

PilD and PilT have homologues in B. subtilis and K. oxytoca (Fig. 7), as well as in E. coli and A. tumefaciens. Since it is

thought that none of these other species produces type-4

fimbriae (see Dalrymple and Mattick, 1987) it appears that,

rather than having evolved specifically for fimbrial assem-

bly, the Pi1 proteins are representatives of a class of proteins

with a conserved protein export function which has been

adapted to a variety of bacterial systems. For example, we

envisage that in B. subtilis this specialised export system

(represented by the ComG- 1, ComG-2 and ComC proteins)

has been adapted to the processing of some components of

the membrane-associated protein complex that has been

proposed to be responsible for DNA uptake (Albano et al.,

1989). The substrates of this export system, which possibly

all share functional similarity in macromolecular transport,

are predicted to be proteins which possess type-4 subunit-

like leader sequences matching the consensus sequence

given in Fig. 6. In the case of the type-4 fimbrial subunits

processing by this system involves proteolytic cleavage of

the short positively charged leader sequence and the

methylation of the phenylalanine residue which forms the N

terminus of the mature protein. We predict that the other

proteins aligned in Fig. 6 will be also cleaved at the homol-

ogous glycyl-phenylalanyl or glycyl-methionyl site. Nunn

et al. (1990) observed that in P. aeruginosapilD mutants the

size of the subunit protein produced was about 1 kDa larger

than in the wt, and suggested that this might be due to the

absence of cleavage of the 6-aa leader sequence. Thus the

PilD/ComC/PulO proteins may share a related proteolytic

function, although the recognition of the preprotein sub-

strate could depend upon other factors specific to be bac-

terial system involved.

The relationship between the P. aeruginosa homologues

PilT and PilB remains unclear. The presence of conserved

sequences in the two proteins suggests that they function

similarly, perhaps by interacting with and transporting

limbrial subunits, although PilT is not necessary for fimbrial

assembly. If as has been suggested fimbrial retraction is the

molecular basis for twitching motility and phage sensitivity,

then it is possible that PilB and PilT act antagonistically, the

former involved in the incorporation of additional subunits

into the limbrial strand, and the latter in the disassembly

process which has been postulated to underly fimbrial re-

traction. Alternatively, PilT might modulate the action of

PilB through the formation of heteropolymeric complexes.

It is also possible that PilB and PilT are positive and nega-

tive regulators of the system. Curiously, none of the

P. aeruginosa ancillary pi1 genes exhibit any obvious

homology to those implicated in fimbrial biosynthesis in

other type-4 fimbriate bacteria (Taha et al., 1988; Fulks

et al., 1989; Hobbs et al., 1991) although their limbrial

subunits are interchangeable (Mattick et al., 1987; Beard

et al., 1990). This suggests that there may be a large number

of genes involved in the type-4 fimbrial assembly system.

This system may provide a useful model for studying

mechanisms involved in the translocation of proteins

across bacterial cell membranes.

(f) Conclusions

(1) A gene pilT necessary for twitching motility in

P. ueruginosa is located on SpeI fragment 8 (about 20 min

on the P. aeruginosa chromosome), remote from the fimbrial

subunit gene pilA and assembly genes pilB, C and D which

map to Spel fragment 5 (about 75 min).

(2) The pilT gene at the twitching motility locus poten-

tially encodes a 344 aa product which is probably a

nt-binding cytoplasmic protein.

(3) Sequence homologies indicate that PilT and other

proteins involved in type-4 limbrial assembly are members

of a specialised protein export pathway which is widespread

in the eubacteria.

ACKNOWLEDGEMENTS

This work was supported in part by grants from

the Australian Meat and Livestock Research and Develop-

ment Corporation and the National Health and Medical

Research Council. Cynthia Whitchurch is supported by an

Australian Postgraduate Research Award. The technical

assistance of Karen Theobold is gratefully acknowledged.

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(1) Since this manuscript was accepted for publication, Bally et al. [J. Bacterial. 173 (1991) 479-4861 have shown that

pilD is equivalent to xcpA, a gene necessary for the secretion of a number of proteins from P. aeruginosa, which reinforces

the basic conclusions of this paper. In addition, Filloux et al. [ EMBO J. 9 (1990) 4323-43291 have also described other xcp protein secretion genes which encode homologues of PulL and PulM. (2) An alternative pulH translational initiation site,

upstream from that suggested by Reyss and Pugsley (1990) would give rise to a predicted protein product with a putative

signal sequence of the type shown in Fig. 6.