Molecular Microbiology (1995) 15(4). 703-717

The Pseudomonas aeruginosa pilK gene encodes achemotactic methyltransferase (CheR) homologue that istranslationally regulated

Aldis Darzins^Department of Microbiology, The Ohio State University,484 West 12th Avenue. Coiumbus, Ohio 43210, USA.

Summary

A new locus, designated pilK, located immediatelyadjacent to the previously described Pseudomonasaeruginosa pilG-J gene ciuster, has been identified.Sequence analysis of a 1.3 kb region revealed thepresence of a singie open reading frame of 291amino acid residues (Afr, 33 338) that contained signifi-cant homoiogy to the chemotactic methyitransferaseproteins of Escherichia coii. Bacillus subtilis and thegliding bacterium Myxococcus xanthus. The 60 bppilJ-piiK intergenic region was devoid of promoterconsensus sequences, suggesting that piU and pilKare contained within the same transcriptional unit.The intergenic region did contain, however, a large,highly GC-rich, inverted repeat that prevented PilKproduction in expression studies. To investigate theregulatory roie of these sequences, pilK-iacZ genefusions, as well as derivatives containing sequencealterations in the potentiai stem-loop region, wereconstructed and analysed in E. coli and P. aerugi-nosa. Modification of the inverted repeat region inpilK-lacZ protein fusion constructs resulted in asmuch as a 24'foid increase in p-galactosidaseactivity, whereas similar modifications in pilK-lacZtranscriptional fusions had only a marginai effect on(J-galactosidase ieveis. These results indicated thatPilK production may be iargely regulated at the levelof translation. In stark contrast to pilG-J mutants,which are dramatically impaired in piius productionand/or function, a PA01 pilK deietion mutant wasindistinguishabie from the wiid type. In addition,complementation studies suggested that the PitKand E. coli CheR proteins are not functionally inter-changeable.

Received 29 July. 1994; revised 18 October, 1994; accepted 28October, 1994. tPresent address: M6 Pharmaceuticals, Inc., 200Corporate Boulevard South. Yonkers, New York 10701, USA. Tel.(914) 476 6799: Fax (914) 476 6798.

Introduction

Twitching motiiity is a novei. yet pooriy understood, modeof flagelia-independent bacterial surface translocation.This unusual form of locomotion, initialiy described inAcinetobacter caicoaceticus (Lautrop, 1961; 1965), hasbeen reported in a diverse group of Gram-negative bacteriawhich includes Pseudomonas aenjginosa, Neisseria gonor-rhoeae, Neisseria meningitidis. Moraxeiia bovis. Dichelo-bacter nodosus. Eii<eneila corrodens and Pastuereilamultocida (Henrichsen, 1972; 1975; 1983; Henrichsenand Blom, 1975; Henrichsen and Froholm, 1975). Strepto-coccus sanguis. a normal inhabitant of the oropharyngealcavity, is the only Gram-positive organism that has beenlinked to this form of surface transiocation (Henricksenand Henrichsen, 1975).

To the unaided eye, twitching motility typically appearsas a slowly spreading, irregularly shaped zone that sur-rounds bacterial colonies. The term 'twitching motility',which was originally used by Lautrop (1965). describesthe characteristic intermittent and often jerky movementsof individual cells on solid surfaces. Using agar-platemicroscopy, it has been shown that twitching proficientbacteria form 'raft-like' cell aggregates at the extremeperiphery of the spreading zone that move outward ata rate of approximately 2-5 nm min ^ (Bradley, 1980;Darzins, 1993; Henrichsen, 1972). Even though individualcells within these motile rafts are constantly moving in reia-tion to each other (i.e., twitching) there appears to be aco-ordinated effort on part of the cell aggregate to moveas a single entity. This motility pattern is reminiscent of thesociai type of movement described for the gliding bacteriasuch as Myxococcus xanthus (for review, see Kim et ai.,1992).

The exact mechanism responsible for twitching motilityhas not been satisfactorily elucidated. Early eiectron-microscopic studies revealed a strong correlation betweensurface iocomotion (Twf) and the presence of polar pili(Henrichsen, 1972). This finding was consistent with theresults of subsequent studies which showed that twitchingmotility does not occur in either PU strain variants thatlack polar pili or in microorganisms that produce peri-trichous pili (Henrichsen, 1975; 1983).

Bradley (1972; 1974) proposed a 'retraction' model inorder to explain changes in the length of P. aeruginosa

704 A. Darzins

0.7B

pilG pilH pill

2.5 3.8N RV

pilf

pADD693EEB BB E BE

5 kb

5.0 kbB

H

Fig. 1. Physical and genetic map of the P.aeruginosa pilG-J gene region.Top line. Shown above are the pilG-J genes,their organization and relevant restriction sites(Darzins. 1993; 1994),Bottom line. Partial restriction map of cosmidclone pADD693 which harbours the pilG-Jgene cluster and the pyrB gene (A, Darzins,unpublished). Abbreviations for restrictionendonucteases: B, BamHI; E, EcoRI; H,H/ndlll; N, NotV. RV, EcoRV; S. Stu\; X, Xho\.

pili during infection with pili-spedfic phages. According tothis model, phage particles, upon binding to the sides orthe tip of the pilus, would be drawn to the ceil surfacethrough the action of a hypothetical retraction mechan-ism. Once at the cell surface the virions would then beable to inject their nucleic acid. In support of this model,Bradley (1972; 1974) identified hyperpiliated, non-retractile(pil^^) mutants of P. aeruginosa that were resistant toinfection by pilus-specific phages and incapable of twitchingmotiiity (Bradley, 1980). Taken together, the accumulatedexperimental evidence to date suggests that surfacemovement via twitching motiiity is dependent upon thepresence of polar, functional (i,e.. retractable) pili.

Using P. aeruginosa as a mode! system to dissectthe process of type-4 pilus biogenesis, genetic studiescarried out in several different laboratories have identifieda number of genes whose products are absolutely requiredfor normal pilus production and/or function (Darzins, 1993;1994; Hobbs et al.. 1993; Ishimoto and Lory, 1989; Ishi-moto and Lory, 1992; Johnson and Lory, 1987; Martinet aL. 1993; Nunn et al., 1990; Nunn and Lory, 1991;Russell and Darzins, 1994; Whitchurch et aL, 1991).Many of these pil genes reside in two physically unlinkedregions of the PAO chromosome (Russell and Darzins,1994). Recently, however, a third set of unlinked pilgenes with very novel mutant phenotypes has been phys-ically localized to approximately 20 min on the PAO geneticmap (Darzins, 1993; O'Hoy and Krishnapillai, 1987; Rat-naningsih et aL, 1990; Romling et aL. 1992). The pilG,-H, -I, and -J gene cluster (Fig, 1) encodes proteins thatdemonstrate remarkable similarity to the chemotaxisproteins of enterics and the gliding bacterium M. xanthus(Darzins, 1993; 1994). For example, the pilG and pilHgene products are both homologous to the enteric CheYprotein, a single-domain response-regulator (Albright efaL. 1989; Stock efa/., 1989), The p/7/gene product demon-strates similarity to the M. xanthus FrzA protein, a CheW

homologue (McBride et at., 1989) and the pllJ geneproduct shows homology to a large group of integralmembrane sensory proteins known as methyl-acceptingchemotaxis proteins (MCPs) (Bollinger et al., 1984; BoydetaL, 1983; Krikos etal., 1983), Based on their similarityto the che and frz gene products that control flagellar andgliding motiiity in enterics and M. xanthus, respectively(Macnab, 1987; McBride et aL, 1989; McCleary andZusman, 1990), it has been postulated that the P. aerugi-nosa PilG-J proteins are part of a signal-transductionsystem that controls pilus biosynthesis and twitchingmotiiity (Darzins, 1993; 1994).

In this study, I describe the moiecuiar characterizationof a new P. aeruginosa gene located downstream of thepreviously identified piU locus. This gene, designatedpilK, encodes a chemotactic methyltransferase (CheR)homoiogue that is translationally regulated by sequencespresent within the p/7J-p/7/C intergenic region. In order toinvestigate the biological properties of the piiK geneproduct, a defined chromosomal mutation was constructedand analysed for its effect on twitching motiiity and pilusproduction.

Results

Nucleotide analysis of sequences downstream o/pil j

The nucleotide sequence downstream of pilJ (Fig. 1) wasdetermined in an attempt to locate additional genes thatmay be invoived in P. aeruginosa pilus biosynthesisand/or twitching motiiity. As a cluster of genes encodingCheY, CheW and MCP homologues (i.e., PilG-J) hasbeen previously identified (Darzins, 1993; 1994), it wasconceivable that genes encoding P. aeruginosa homo-logues of other chemotaxis proteins such as CheR,CheB, CheA, or CheZ could be situated nearby. Sequenceanalysis of this region was further justified en the basisof two additional findings. First, a preliminary examination

Pseudomonas aeruginosa pilus biogenesis 705

Sail RBSI CCTGAGCRTfcGGCGCQGCGGCCGCCTGGCGGGCGCCGCGTCGACACCTTGGA.C£fi£2GGC

61

181

241

301

361

421

481

541

601

661

721

781

841

901

961

1021

1081

1141

1201

1261

ACGGCATGCAGGCGAACGGCQTCTGGTCACTGCAGCCGCTGGCCGATATGTCGGCAGCGGH Q A N G V H S L Q P L A D M S A A E

AGTTCCGCGACTGGCAGGTCCTGTTGGAGAACCGCACCGGCGTGGTGCTCAACGAGCAACF R D W Q V L L E N B T G V V L H E Q B

GCCGGACGTTCCTGCAGGCCAGCCTGACCGCGCGCATGCGTGAACTGGGCATCGGCGACTR T F L Q A S L T A R H R B X . G I G D Y

ACCACAGCTACTACCGCCAGGTGACCGACGGTCCGCGTGGCGCAGTGGAGTGGGCGACCCH S Y Y R Q V T D G P R G A V E H A T L

120

180

300

Fig. 2. Nucleolide sequence of the pilK (orfi)gene and the deduced amino acid sequence.The sequence starts with lhe partial Stu\ site(CCT) present at the end of the pilJ gene(Darzins, 1994) and extends to the rightmostSamHi site shown in the top line of Fig, 1.Relevant restriction sites are labelled. Theputative p//^Shine-Dalgarno sequence isunderlined and labelled (RBS), Translation ofthe sequence is shown by use of the single-letter abbreviations of the amlno acids.Inverted repeats are underlined with arrows.Possible orf3' ATG start codons locateddownstream of pitK{ort1) are underlined.These sequence data appear in the EMBL/

TGCTGGACCGCCTGACCGTCCAGGAAACCCGTTTCTTCCGTCATCCCCCGTCTTTCGAGC 360 GenBank/DDBJ Nuclsotide Sequence DataL D R L T V Q E T R V r R H P P S F E L Libraries under the accession numberJ £ M i _ . . . . . . U11382,

TGCTCGAGCGCTACCTGGGCGAGCGCCTGCGCCGCGAAGGCATGCCGCGGCCCTGGGCCC 4 2 0

L E R Y L G E R L R R E G M F R P W A L

TGTGGAGCGTCGGCTGCTCCAGCGGCGAGGAACCCTACTCGCTGGCGATGTGCGCCGCGC 4 80

W S V G C S S G E E P y S L A M C A A Q. E c o R V

AGGTGTTGCGCGGGCAGGAACGGGAAGATTTTTTCGGCGTCACCGGAACGGATATCAGCC 5 4 0V L R G Q E R E D F F G V T G T D I S L

TTCACGCCCTGCAGCGGGCGCGGCAGGCGAACTATCCGGCCCGCAAGCTGGAGCAACTGG 600B A Z . Q R A R Q A H X P A R I C L E Q L E

AGGCTGGGCTGGTCGAACGCTATTGCGAACGCCAGGCCGACGGCAGCTTCAGCGTGAAAA 660A G L V E R Y C E R Q A D G S F S V K T

CGATACTGACCGAGCGCGTCTGCTGCGCCCGGCTGAATGTGCTGGACCTGGCGAAGGCGC "720I L T E R V C C A R L H V I . D L A K A P

CCTGGTCCGGCATGGRCGTGATTTTTTGTCAGAACCTGCTGRTCTACTTCCGTCGCTGGC 780W S G M D V I F C Q N L L I Y F R R W R

GACGGCGCGAGATCCTCAACCGGTTGGCCGAACGGCTGGCGCCGGGAGGCTTGCTGGTGA 840R R E I L N R L A E R L A P G G L L V Z

Sail . Sacl XholTCGGGGTCGGCGAAGTGGTCGACTGGAGCCATCCGGAGCTCGAGCCGGTCGCCGACGAGC 900G V G E V V D V r S H P E L E P V A D E R

GGGTCCTGGCCTTTACCCGTAAGGGGTACTCAGGCACiXGAATGGAGTGGCXaXGGGTGA 960V L A F T R K G Y S G T *

CCGGCACGACTACGTCGCTCTGGAGTGGGTGAAAGGCGAGATCGCCGAAACCCTGAAACA 1020

GGCGCOCCAGGCGCTGGAAGCGTTCGTCGAGAACCCGCAGGACCCGACCCGCilfiCGGTT 1080

CTGCCTGACCTACGTGCACCAGGTGCAGGGCACCCTGCAGaifiGTCGAGTTCTACGGCGC 1140

GGCCCTGCTCGCCGAGGAAAISGAGCAGTTGGTCCAGGCCTTGCTGGACGGTCGCGTGCC 1200

GAACCAGGGCGAGGCCCTGGAAGTGCTGATGCAGGCGATCCTGCAACTGCCGGTCTACCT 12 60

BamHICGftCCGGATCC 1271

Of the 90 bp immediately downstream of plU (Dar2ins,

1994) revealed the start of another possible open reading

frame (ORF). The translationai start codon of this hypothe-

tical ORF was preceded by an appropriately spaced puta-

tive ribosome-binding site. Second, the absence of typical

promoter consensus sequences within this downstream

region suggested that pilJ and the hypothetical ORF may

be linked transcriptionally.

The nucleotide sequence of the c. 1.3 kb region immedi-

ately downstream of piU is shown in Fig. 2. Computer

analysis reveaied the presence of two complete ORFs,

designated orfi and orf2. and one incomplete ORF. desig-

nated orf3\ The sequences of orfi, which were arranged

in the same transcriptional direction as the pilG-J genes,

predicted a protein of 291 amino acids {M,, 33338). orf2,

which was slightly smaller (261 amino acid residues; Mf,

28105), was arranged in the opposite orientation with

respect to this gene cluster. orf3' was located immedi-

ately downstream of orfi and contained no less than six

ATGs in the same reading frame (Fig. 2). In fact, the first

706 A. Darzins

of these ATGs overlapped the orfi stop codon. Of thethree ORFs, only orf1 and orf3' demonstrated the charac-teristic preference for either G or C in codon positions 1and 3 {70 and 85% and 74 and 88%, respectively). In addi-tion, orf1 and orf3' contained characteristic P. aeruginosacodon-bias patterns (West and Iglewski, 1988).

An appropriately spaced, potential Shine-Dalgarnosequence (GAGGG) was located approximately sevennucleotides upstream of the putative orfi start codon(Fig. 2). Reasonable ribosome-binding sites were alsoidentified upstream of four out of the six potential ATGcodons in orf3\ However, no such sequences werefound upstream of orf2. An examination of the 60 bppilj-orfi intergenic region revealed the absence oftypical promoter consensus sequences. However, alarge, highly GC-rich inverted repeat that spanned aregion of 31 nucleotides was found immediately down-stream of the p;yj termination codon (Fig. 2).

The 291-amino-acid Orf1 is homologous tochemotactic methyltransferase (CheR) proteins

A TFASTA search of the GenBank/EMBL database with thepredicted sequences of the putative orf1 gene productrevealed considerable homology to several chemotacticmethyltransferase proteins. Because of this homology,orf1 was redesignated pilK. A search with orf2- andorG'-derived sequences, however, revealed no signifi-cant similarities to known proteins.

The deduced amino acid sequences of pilK and theEscherichia coli cheR gene (Mutoh and Simon, 1986)shared 32% identity (55% overall similarity; Fig. 3). The

PilK and CheR proteins are very similar in size (c.33 kDa) and are generally homologous over their entirelengths (Fig. 3). However, the highest levels of sequenceidentity were found in the central and C-terminal regions ofthese proteins (Fig. 3, boxes A and B). A similar findinghas been previously reported from a study comparingthe CheR proteins from E coli and B. subtilis (Kirsch etaL, 1993b). Not surprisingly, PilK also shows significantsimilarity to the CheR proteins of Salmonella typhimurium(30% identity, 52% similarity) and R subtilis (27% identity.52% similarity) (Kirsch etaL, 1993b; Simms etaL. 1987),and to the N-terminal domain of FrzF (30% identity, 537osimilarity), a CheR homologue of the gliding bacteriumM. xanthus (McCleary et aL, 1990).

Overexpression of the pilK gene

A 1.3 kb H/ndlll-EcoRI fragment of pADD3092, harbouringthe pilK gene, was cloned into the broad-host-range T7expression vector pADD3261, The resulting construct,PADD3275, was introduced into strains BL21(DE3) andADD1976, and examined for PilK production. Followingfluorography, however, no expression of the PilK proteincould be detected (data not shown). To investigatewhether a larger DNA fragment harbouring upstream pilgenes was required for PilK production, a 4.3 kb SamHIfragment containing the pilH. -I, -J, and K genes (Fig. 1;Darzins, 1994) was cloned into pT7-6 in such a way asto direct transcription toward the pil cluster. Expressionstudies with BL21(DE3) harbouring the resulting plasmid,pADD3280, clearly revealed the induction of a 73 000 Dapolypeptide previously identified as the PilJ protein

PilK 1 MQANGVWSLQPLAD. . .MSAAEFRDHQVLLEHRTGWLNEQRRTFL 43: f .: . ; : I . I . I I I : .1.1:11.:::!.::

CheR 1 MTSSLPCGQTSLLLQMTSRLALSDAHFRRISQLIYQRAGIVLADHKRDMV 50

PiiK

CheR

AA QASLTARKRELGrGDYHSYYRQVTDGPRGAVEWATLLDRLTVQETRFFRH 93. . I . I : 1 . I 1 : . I : ;. I .: :: . . . I | - - : : = - I I • - I I I I •

51 YNRLVRRLRSLGLTDF.GHYLNLLESNQHSGEWQAFINSLTTNLTAFFRE 99

PPSFELLERYLGERl^REGMPRPWALWSVGCSSGEEPYSLAMCAAQVll^GPilK 94: . I.I

CheR 100 AHHFPL LADHA^RGS. : : I I . : . I . I I I I 1 1 : I I . I : . I

.GEYRVKSAAASTGEEPYSIAMTLADTLfeT

143

113

PiiK 144 QEREDFFGVTGTDISLHALQRARQANYPARKLEQLEAGLVERYCERQA.. 191. .: ; I :.| I . . . 1 : : i I : I . . I.. I-:- :: I 1 I..

C h e R H 4 A . . P G R W K V F A S D I D T E V L E K A R S G I Y R H E E L K N L T P Q Q L Q R Y F M R G T G P 1 9 1

PilK 192 .DGSFSVKTILTERVCCARLNVLDLAKAPWSG^3VIFCQNLLIYFRRWRR

CheR i92 HEGLVRVRQELANYVDFAPLNLLAKQYTVPGPfDAIFCRNVMIYFDQTTQ 241

240

PiiK 241

CheR 24 2

SEILNRLAERLAPGGLLVIGVGEWDWSHPEJLE. i I 1 - I :- . I 1:111. 1 : I :: I I i3EILRRFVPLLKPDGLLFAGHSE..

TPUADERVLAFTRKGYSGT 29i1.1:.:.

287NFSHLE ̂ RFTLRGQTVYALSKD'

Fig. 3. Comparison of ihe deduced aminoacid sequence of P. aeruginosa PilK with thatot the E. CO//CheR, Vertical lines indicateidentical residues conserved in the twoproteins. Conservative amino acid substitu-tions are represented by dots and colons.Gaps are introduced into the sequence tomaximize the homotogy. Regions ot extensivehomology (A and B) are boxed. The aminoacids located between the vertical arrowsindicate those residues deleted in Hie pilKmutant FA9.

Pseudomonas aeruginosa pilus biogenesis 707

Not I

C G

C- G

G- C

C-G

C-G

[G G

G- C

C-G

G'C

G-C

C-G

kc I

PstI SailT701O

G - C

C - GG "TG - C

A GT AAC A

-G-c'̂A-T

BamHI * G • TT* G m^mmm

^TCCpCACGTTGCATGCCTGCAGGTCCC - GACGAGGGCACGGCATGCAGGCGAACM Q A N

PilK

PstI Sail BamHI Sal I RBSr ' 0^0 |CTGCA^TCGACJCTAGfl|GGATCCt:CACGTTGGC^TGCAG)3TCGAcKcCTTGGACGAGGGCACGGCATGCAGGCGAAC

M Q A N

PilK

43-

29-

18.4-

PilK

• P i l K '

(Darzins, 1994). However, no PilK-specific band wasdetected, even after prolonged exposure to X-ray film(data not shown).

Each of the constructs used in the previous attempts to

Fig. 4. T7 expression of PilK, Sequences immediately upstream of theputative prfK" ribosome-binding site (RBS) in plasmids pADD338i (A) andPADD3382 (B), Watson-Crick base pairing is denoted by - , while G"Tindicates a non-Watson-Crick base pair The asterisk (•) near the bottomot the stem-loop in (A) denotes the pilJ stop codon. An autoradiograph of^H-amino-acid-label!ed polypeptides in BL21{DE3) is shown (C), Lanes: 1,molecular mass markers wilh lhe indicated vaiues on the left in kiiodaltons;2, pTG79; 3, pADD3381; 4, pADD3382; 5, pADD3434- The locations of thePilK protein and a truncated derivative (PilK') are indicated by arrows onthe right.

Visualize the p(/Kgene product harboured the entire piU~pilK intergenic region. This region contained a iarge,highly GC-rich palindromic sequence (Fig. 2) that couldconceivably prevent the production of PilK at the ievel of

708 A. Darzins

Not I

B

c-G -

C -

C -

G -

C -

G -

G -

C

G

cGG

G

C

G

G

(

C

G

- C

- C

- G

- C

MJotl-Sall

G G

C C

G GC - GG - C

r G - C

G = -5.1 kcal/mol

RBSCCUGAGCAUA - OCGACACCOUGGRCGAGGGGCACGGCAUGCAGGCGAAC

AOAC

C I

G = -23.3 kcal/mol

Sal I

Dcc

PilK

AC

c•u

G £ ^ M Q AGACGAGGGCACGGCAUGCAGGCGAAC

RBS Wot IN

D

C G

C-G

G-C

C-G

C-G

tG G

G-CC-GG-CG-CC-G

G-C

C-GG -O

G = -23 .3 kcal/mol

PilK

ASalX RBSCCOGAGCADAG - CGAUCGACACCUUGGACGAGGGCACGGCAUGCAGGCGAAC

Fig. 5. Proposed mRNA secondary structure ot the pf/K" initiation region. prfK-/acZ transcriptional (operon} and translational (protein) fusionswere constructed and transferred to the broad-host-range plasmid pMMB24 (see the Experimental procedures tor construction details).Predicted secondary structure of the pitJ-pilK intergenic region in unmodified p;/K-/acZfusion constructs (A), AWod-Sa/l-modrfied constnjcts(17bp deletion) (B), and ASa/l-modified constnjcfs (4bp insartron) (C) are shown. An asterisk denotes the TGA (erminafion codon on the piUgene. The putative starl of PilK is shown as a line with an an-ow. The putative p/V/C ribosome-binding site (RBS) is overtined. Relevant restric-tion sites are labelled, Watson-Crick base pairing is denoted by - , while GBU indicates a non-Watson-Crick base pair.

transcription by acting as a terminator or, alternatively, atthe level of translation through the formation of inhibitorysecondary structures in the mRNA. With this possibility inmind, a pair of ptasmids containing the pilK gene with(pADD3381) and without (pADD3382) the intergenicregion were constnjcted (Fig. 4, A and B). Figure 4Cshows that pADD3382 (lane 4) directed the synthesis ofa polypeptide whose size (33 kDa) was consistent withthe size of the putative PilK protein. No such polypeptidewas seen in extracts of BL21 (DE3) harbouring the vectorpTG79 or pADD3381 (Fig. 4C, ianes 2 and 3, respec-tively). To determine if the presence of the 33 kDaprotein was due to expression of the pilK gene, an in-frame deletion of pilK was constructed by removing theinternal Xho\ fragment (Fig. 2) from plasmid pADD3382.As predicted, this new construct, designated pADD3434,directed the synthesis of a 15 kDa band which is thoughtto represent a truncated form of the PilK protein (Rg. 4C,lane 5).

Regulation of PilK production

The presence of the p/7J-p//K intergenic region was shownto have a dramatically negative effect on the production ofthe pilK gene product (Fig. 4). In order to distinguishwhether PilK production was being regulated at the levelof transcription or translation, a pilK-lacZ operon andprotein fusions were constructed and placed onto thebroad-host-range, fac promoter vector pMMB24. In addi-tion, plasmid derivatives containing sequence modifi-cations in the putative stem-loop structure were alsoconstructed. These included a deletion plasmid, desig-nated ANot\~$al\, which lacked the 17bp between theNot\ and Sail sites in the putative stem-loop structure(Fig. 5, A and B) and a construct, designated ASa/l(Fig. 5C), which contained an additional 4bp at theunique Sal\ site.

Table 1 shows that following (PTG induction the activityof p-galactosidase in E coli MC1061 harbouring the

Pseudomonas aeruginosa pilus biogenesis 709

Table 1. Analysis of trar>scriptional (operon) and translational(protein) p//K-/acZ fusions in E. coli.

IacZ fusion

Transcriplional

Translational

Ptasmid^

plD1plD1ASa/-/Vof

plD3plD3a Sal-No)plD3ASa/

Beta-galactosidase activity^(Miller Units)

8556 ±1699957±17

9 ±0.3125±4228 ±16

a. See Mettiods for details on plasmid construction.b. Bacterial cultures were grown in LB plus Amp (SO^gmr') untilvery early log phase (approx, ODaoo = 0-2)- 'PTG (2 mM, final concen-tration) was added and the cultures were harvested at an ODBOO of0,5-1,2, Beta-galactosidase activity was determined according lothe method of Miller (1972). Values are the averages of two orthree independent experiments.

transcriptional fusion constnjcts pIDI and piDIAA/oM-Sa/I varied only slightly (c. 1.2-fold). However, cells con-taining the modified translational fusion derivativesp\D3ANot\~Sat\ and plD3ASa/l produced significantlyhigher levels of p-gatactosidase than cells harbouringplD3 (13- and 24-fold, respectively). Similar results havebeen obtained In a P. aeruginosa host (data not shown).These results, therefore, strongly suggest that the putativestem-loop structure in the pilJ-pilK intergenic region playsa major role in regulating PilK production primarily at thelevel of translation.

Analysis ofthe pilK mutant phenotype

In order to investigate the biological properties of the P.aeruginosa CheR homoiogue, a defined mutation in pilKwas constructed and introduced into the chromosome ofstrain PAOl. Briefly, the internal 520bp Xho\ fragmentof pilK (Fig. 6A) was replaced with the tetracycline-resistance (Tc") gene from pBR322. The allelic exchangesystem developed by Schweizer (1992) was then used toreplace the wild-type copy of pilK on the PAOl chromo-some with the disrupted copy constructed in vitro.Several Tc'^. sue rose-resistant colonies obtained by genereplacement were verified by Southern blot hybridization(data not shown) using a 3.8 kb EcoRV probe (Fig. 6,top). One such verified pilK insertion mutant was desig-nated FA7 (Fig, 6B) and chosen for further study.

Strain FA7 produced dramatically reduced zones(approximately 50%) when compared to PAOl in twitch-ing assays (data not shown). This mutant also appearedto be slightly more resistant to the pilus-specific phageD3112 as determined by plaque assays, but remained assensitive as PA01 to the pilus-specific phages B3 andF116. Previous studies have shown that P. aeruginosapilG, 'H, -/, and -J mutants can be complemented intrans with plasmids harbouring the respective wild-typegene (Darzins, 1993; 1994). In order to determine if FA7(p//K;:Tc") could be complemented in trans, a pilK* frag-ment lacking the pilJ-pilK intergenic region was clonedinto the SamHI site of pRO1614, thereby placing thegene under the transcriptional control of the tet pro-moter. Following mobilization of the resulting plasmid.

probe = 3.8 kb E«I

probe = 1.5 kb Xhoi

Not: Xhol tcoRV Xhol B.uninI J L . 1 1

ficoRI

200 bp

Fig. 6. Strategy used in the construction otpilK chromosomal mutants.A, Structure of the wild-type (PAOl) pilKregion and flanking sequences,B, Structure of the pilK region in which aninternal Xhol fragment of pilK has beenreplaced by a tef (Tc") cassette.C, Structure ot the pilK region in which aninternal Xho\ fragment of pilK has beendeleted. Relevant restriction sites are shown.Thin lines with arrows at the top represent theprobes used to verify the structure of eachmutant-

Xhol/Aval . , EcoRI/XholBamHI EcoRI

.Nfoll Xhol BamHI EcoRI

pill ' pilK'

710 A. Darzins

designated pADD3450, into FA7 it was discovered thatthe phenotype of individual FA7/pADD3450 carbenicillin-resistant (Cb"^) transconjugants was identical to thephenotype of FA7. This result indicated that FA7 was notcomplemented in trans with the pilK gene. To rule out thepossibility that multiple copies of pilK have a negativeeffect on twitching motiiity, pADD3450 was also intro-duced into PAOl, The presence of pADD3450 did nothave a detrimental effect on twitching motiiity sincePAOl Cb^ transconjugants produced zones that werecomparable in size and appearance to the zones pro-duced by PAOl.

The inability of the cloned pilK gene to complementFA7 suggested the possibility that the tet insertion withinthe disruption mutant might be negatively affecting theneighbouring pil genes. To test this hypothesis, plasmidpADD699, which contains the entire pilG-K gene cluster(Darzins, 1993), was introduced into the pilK mutant.Stab and D3112 plaque assays revealed that pADD699was capable of complementing FA7, In order to determinewhich of the genes present on pADD699 was responsiblefor complementation, plasmids containing the individuallycloned pil genes (i,e., pilG-J) were tested. Of theseplasmids, it was found that only the subclone harbouringan intact pilJ gene, pADD2953 (Darzins, 1994), couldcomplement FA7 (data not shown). As the pilJ and tetgenes in FA7 are convergently transcribed (Fig. 6B) it ispossible that readthrough transcription past the end ofthe tet gene may reduce the production of PilJ by eitherinterfering with pilJ transcription originating from theopposite direction (Darzins, 1994}, or by generating inhibi-tory pilJ antisense RNA. Consistent with this hypothesiswas our ability to isolate second-site suppressor muta-tions in the tet gene (presumably promoter mutations)that relieved the interference (A. Darzins, unpublished).

In order to mutate py/K without perturbing expression ofthe piU gene, an in-frame pilK deletion mutant of PAOlwas constructed, Briefly, a 520 bp Xho\ fragment, whichcontains approximately 60% of the pilK coding region,was removed and the resulting deletion was used toreplace wild-type PAOl p//K sequences by allelic exchange(Fig. 6C). The resulting pilK deletion mutant, designatedFA9, was indistinguishable, both macroscopically in twitch-ing assays, and microscopically in slide culture assays,from PAOL The in-frame deletion mutant was also justas motile (Fla*) as the parent in semi-solid swarm assays.Furthermore, FA9 was as susceptible as PAOl to ail ofthe pilus-specific phages used in this study (data notshown).

Expression of pilK in an E. coli cheR mutant

Previous studies have shown that not onty can the B.subtilis cheR gene complement an E coli cheR mutant

but that the B. subtilis CheR protein is capable of methy-lating E, coli MCPs in vivo (Kirsch et aL, 1993b). In orderto investigate whether the P. aeruginosa pilK gene cancomplement an E. coli cheR mutant, strain RP1254(cheR) was transformed with pADD3450 ipilK*) andpRO1614 (vector control) and individual ampicillin-resistant (Ap") transformants were tested in swarmassays. Unlike previous complementation studies withthe S, subtilis cheR gene, the presence of the pilK genedid not restore chemotaxis to RP1254. To rule out thepossibility that multiple copies of the pilK gene were insome way inhibiting chemotaxis, a wild-type Che* strain(RP437) was also transformed with pADD3450 andpRO1614, The presence of the pilK gene did not diminishthe chemotactic abiiity of wild-type strain RP437, indicatingthat the inability of the pilK gene to complement the E. coliCheR mutant was not due to an inhibitory effect of PilK onthe enteric chemotactic signal-transduction process.

Discussion

Previous studies have shown that the enteric CheRprotein (Simms etaL, 1987; Springer and Koshland, 1977)acts in concert with several other che gene products (i.e.,CheA. CheB, CheW, CheY, CheZ, and MCPs) to regulatethe frequency with which flagella reverse their directionof rotation in response to certain chemoeffectors (for areview, see Macnab, 1987), After exposure to stimuli,cells adapt to the new levels of chemoeffector and even-tually return to their pre-stimulus motiiity pattern. Twoproteins, the methyltransferase CheR. along with itsmethylesterase counterpart, CheB, are involved in thissensory adaptation process. Adaptation to attractants ismediated by CheR. which transfers a methyl group froman S-adenosylmethionine donor to specific glutamateresidues located in the cytoplasmic domain of transmem-brane receptors known as MCPs (Kehry and Dahlquist,1982). In the presence of negative stimuli (ie., repel-lants) the activated methylesterase, CheB, removesmethyl groups from modified glutamyl residues. Receptorsignalling is, therefore, controlled by the reversible methy-tation of specific glutamate residues,

Chemotactic methyltransferase (cheR) mutants areseverely impaired in their ability to respond to chemoeffec-tors. For example, E coli and S. typhimurium mutantslacking CheR are smooth swimming (i.e., counter-clockwisebiased) whereas cheB mutants are tumbly (i.e,, clockwisebiased), 6, subtilis cheR and cheB mutants, like theirenteric counterparts, also lack the ability to adapt andare non-chemotactic. However, recent studies have shownthat the signal-transduction process in B. subtilis is the'opposite" of that in the enteric system; CheR is responsiblefor the adaptation to repeliants while CheB is required foradaptation to attractants (Kirsch et al., 1993a,b). In

Pseudomonas aeruginosa pilus biogenesis 711

addition, M. xanthus 'frizzy' mutants lacking FrzF, a CheRhomologue required for gliding motiiity, cannot respond toattractants. As a result, these mutants, which are glidingdefective, reverse their direction very infrequently whencompared to the wild type (Blackhart and Zusman, 1985;McCleary e/a/,, 1990).

The phenotypes of cheR and frzF mutants are in starkcontrast to the phenotype of the P. aeruginosa pilKmutant (FA9), which was indistinguishable from the wildtype (PirTwt*). This result was particularly surprising inview of the fact that pUK is apparently transcriptionatlylinked to a che-iike pii gene cluster in which mutationshave a dramatic effect on twitching motiiity (Darzins,1993; 1994). pilG, -I, and -J mutants are defective inpilus biosynthesis (Pil") and twitching motiiity (Twt~),pilH mutants, on the other hand, retain piliation andtwitching motiiity but display an altered motiiity pattern(Darzins. 1993; 1994), In some respects the phenotypeof pilH mutants closely resembles that of enteric and Mxanfhus chemotaxis mutants (Armstrong et al, 1967;Zusman, 1982),

There are several possible explanations that couldaccount for the phenotype of the pilK deletion mutantobserved in this study. First, the 15 kDa PilK' proteinproduced by the truncated gene in strain FA9 (Fig. 4C)might retain wild-type activity, thereby preserving thefunctional integrity of the pil signal-transduction pathway.This explanation is unlikely since as much as 60% of thep//K coding sequences has been removed. Moreover, thedeleted sequences span a region that appears to behighly conserved in CheR homologues (Fig. 3), Thisregion, which comprises the middle and C-terminalportions of PilK. not only probably contains residuescritical for methyltransferase activity but also may containthe residues necessary for specific recognition of the PiUmethylation domains.

Another possible explanation is based on the findingthat CheB (methylesterase) activity in enteric and S.subtitis CheR null mutants is dramatically reduced (KirschetaL, 1993b; Stock and Koshland, 1978). The phenotypeof such a strain wouid, therefore, closely mimic that of acheR CheB double mutant. While the biochemical basisfor this finding has not been elucidated, Kirsch et aL(1993a) hypothesized that CheB and CheR may form astabilizing complex which protects CheB from degra-dation. If a similar situation exists in the putative signal-transduction cascade which reguiates pilus productionand twitching motiiity, pilK mutants may also have dimin-ished levels of the corresponding CheB homologue.

Lastly, it is also conceivable that the P. aeruginosaCheR protein involved in flagella-mediated chemotaxismay be able to substitute for the defective PiiK protein.Previous studies have shown that P. aenjginosa possesseselements of a chemotactic system that regulates the

direction of flagellar rotation in response to certain stimuli(Craven and Montie, 1983; Moulton and Montie, 1979;Starnbach and Lory, 1992; Tsuda and lino. 1983a,b).However, since the genes encoding chemotaxis proteinsinvolved in P aeruginosa flageilar motiiity have not beenextensively studied, questions concerning possible inter-actions between the two motiiity systems must awaitfurther analysis.

Previous studies have demonstrated that the E co//andB. subtilis CheR proteins are not only similar at the aminoacid level (31% identity) but that they are aiso functionallyhomologous as well (Burgess-Cassler and Ordal, 1982;Kirsch et aL, 1993b). This conclusion is based on thefollowing results: (I) the B. subtilis cheR gene can comple-ment an E. coli cheR null mutant to Che*, (ii) expression ofthe B. subtilis cheR gene in an E. coli cheR mutant canrestore MCP methylation. and (iii) CheR proteins obtainedfrom both E. coli and B. subtilis will methylate MCPs fromboth organisms. These findings are in contrast to theresults of this study, which have shown that despite thesimilarity in their sizes (c. 33000) and their considerablebomology (32% identity), the PilK and E coli CheRproteins are probably not functionally interchangeable.The inability of the pilK gene to complement an E. colicheR mutant may be explained by the possible failure ofthe PilK protein to recognize the E. coli MCP methylationdomains, Rl andKI (Krikos e/a/,, 1983). Adirectcompari-son between the known methylation domains of the E, coliTsr protein and the corresponding regions in PilJ hasrevealed very little homology (Darzins, 1994), This, there-fore, raises the possibility that PilK may recognize methy-lation domain(s) in PilJ that are significantly different fromthe Rl and Kl regions of the E. coli MCPs.

The transcriptional signals that govern pilK expressionhave not been determined, but an inspection of the regiondirectly upstream of the gene revealed the absence of con-sensus promoter sequences. As previous genetic studieshave indicated the possible existence of a promoter withinthe pill-pilJ intergenic region (Darzins, 1994). it is likelythat pilJ and pilK are cotranscribed. Yet despite this tran-scriptional organization it appears that PilJ and PilK arenot produced in equivalent amounts. The PilJ protein canbe quite easily detected by in vivo labelling experimentscarried out in either E coli or P aeruginosa (Darzins,1994; A. Darzins, unpublished). The findings of thisstudy show that unless the pilJ-pilK intergenic region isremoved, PilK production remains essentially undeteet-able. Subsequent p/y/<-/acZprotein fusion studies demon-strated that specific sequences within the pilJ-pilK inter-genic region presumably play a major role in regulatingproduction of PilK at the level of translation.

A variety of translational regulatory mechanisms havebeen identified that involve the formation of either RNA-RNA or RNA-protein interactions. For example, the

712 A. Darzins

secondary structure of mRNA can dramatically affectthe accessibility of the ribosome-binding site which, inturn, has a negative effect on translational initiation (DeSmit and van Dujn, 1990). In addition, several typesof translational repressor/activator interactions withsecondary mRNA structures have also been proposed toexplain certain experimental observations (McCarthy andGualerzi, 1990). The finding that sequences presentwithin the pilJ-pilK intergenic region regulate PilK pro-duction in both £. coli and P. aeruginosa appears to ruleout the action of a host-specific protein (i.e,, repressor).Instead, to account for our observations, we favour amechanism that involves translational coupling of pilJand pilK.

Two models have been proposed to explain trans-lationa! coupling, The first involves a single ribosomethat reinitiates translation soon after terminating at anupstream stop codon (Adhin and van Duin, 1990). Thesecond, also known as the 'tv '̂o-ribosome model', involvesthe activation of downstream genes by ribosomes termi-nating at the upstream cistron (De Smit and van Duin,1990). This activation Involves the destabilization of aninhibitory secondary structure in the mRNA (Mayford andWeisblum, 1985} which allows a second ribosome toinitiate at the downstream Shine-Dalgarno sequence. Insome cases the ribosome-binding site {RBS) of the trans-lationally regulated gene is actually sequestered withinthe inhibitory secondary structure (Dallman and Dunn,1994). However, in other situations (as appears to be thecase for pilK) the RBS is not sequestered in a RNAiRNAduplex but is located close enough to the stem of thesecondary structure to inhibit ribosomal access (Blumeret aL, 1987). Based on the p/7K-/acZ fusion data pre-sented in Table 1 and the rather large distance {c. 60 bp)between the piiJ stop and pilK start codons (Fig, 5A), wefavour the "two-ribosome-activation' model to explain theregulation of pilK expression, The scanning reinitiationmodel does not appear to be a viable choice since thisprocess is apparently sensitive to even slight increasesin distances between the stop and start codons of theupstream and downstream genes, respectively.

According to the activation model, a ribosome nearingthe translation termination site of pUJ wouid disrupt thesecondary mRNA structure within the intergenic region.As a result of this action, the pilK Shine-Dalgarnosequence would be displaced away from the base of theinhibiting hairpin (Fig. 5A), thereby making it much morelikely to interact with a second ribosome. The efficiencyof coupling or the degree to which the downstream gene(i.e., pilK) is expressed, therefore, depends upon thelength of time for which the 'activated' sequences remainavailable for translation initiation (Blumer et al.. 1987;Dallman and Dunn, 1994). In theory, it should be possibleto estimate the duration of p/7K translation initiation region

availability and the approximate frequency with whichpilK is translated. A terminating ribosome at the end ofpiU would normally protect approximately 12 bases ofmRNA downstream of the UGA stop codon (Steitz,1979). Using a translation rate of 20 codons per second(Dalimann and Dunn, 1994), it can be estimated that thehairpin structure upstream of pilK should be destabilizedfor approximately 0.2 s. Since it has been determinedthat approximately 3.2 s is required to initiate translationof the well-expressed IacZ gene (Kennell and Reizman,1977), it is possible to estimate that less than 6% of theribosomes terminating at the end of p/U would lead to anactivation of pilK. This translational coupling value sug-gests that PilK would be produced at an extremely lowlevel in P. aeruginosa. Our interpretation of the calculatedtranslationai coupling frequency is consistent with T7expression-labelling (Fig. 4) and pZ/K-ZacZ protein fusion(Table 1) results presented in this study.

The opportunistic pathogen P. aeruginosa is uniqueamong motile bacteria in the sense that it apparentlycontains a duplicate set of cfte-like signal-transductiongenes: one set presumably controls flagellar locomotion,while another set controls twitching motiiity and/or pilusproduction. As mutants defective in either pilG, -H, -I, -Jor -K appear to be unaffected in flagellar motiiity andchemotaxis (Darzins, 1993; 1994). it is unlikely that thetwo motiiity systems share signal-transduction compo-nents. Future studies aimed at elucidating the preciserole of each of the proteins encoded by the pilG~K genecluster will contribute greatly to our general knov r̂ledgeof flagella-independent surface translocation and pilusbiogenesis. Nevertheless, these results do not njleout the potential for 'cross-talk'. In fact, communicationbetween the two signal-transduction systems mayexplain why a pilK deletion mutant is able to retain thetwitching and phage-sensitivity properties of a wild-typestrain. For this reason, future research should also targetthe possible interactions between individual componentsof the flageltar and pilus-mediated (i.e., twitching) loco-motion processes.

Experimental procedures

Bacterial strains, phages and ptasmids

The bacterial strains, phages and plasmids used in this studyare described in Table 2.

Selective media

LB medium was routinely used to propagate P. aeruginosaand Escherichia coli. LB broth was 1% tryptone (Difco).0,5% yeast extract {Difco), and 0.5% NaCl. M9 minimalmedium has been described previously (Sambrook et ai,1989). For most solid media, agar (Difco) was added at a

Pseudomonas aeruginosa piius biogenesis 713

Table 2. Strains, phages and piasmids used in this study.

Strain/Phage/Plasmid Relevant characteristics^ Source/Reference

Strain

£. coli

BL21(DE3)

RP1254

RP437

MCI 061

P. aeruginosaADDI 976PAOl

BRLStudier and Moffatt (1986)

recA endAI gyrA96 thi-1 hsdR17supE44 relAI ^60 6iacZAM15f~ ampT rgniB lambda D69 tysogen carrying phage T7 gene / under control of

PiacUVS(cheR) Dem58-13 zec::TniO-2 thr (Am)-1 leuB6 his-4 metF {Am) 159 rpsL136 [thi-i J. S. Parkinson

ara-14 IacY mtl-1 xyl-5 tonA31 tsx-78]thrlAm)-1 leuB6 his-4 metFlAm) 159 eda-50 rpsL 136 [thi-1 ara-14 lacY1 metl-1 xyl-5 J. S. Parkinson

tonA31 tsX'78]F" araD139 A{ara-leu)7696 gaiE15 galK16 A(/flc)X74 rpsL (Str") hsdR2 mcrA M. Casadaban

mcrBI

PAO1::miniD180 (T7 RNA polymerase) (Tc")Prototroph FP ^ ^ ^

Brunschwig and Darzins (1992}Holloway (1955)

Bacteriophage

D3112 ctsB3 cts-3F1l6Lcts53

cts (temperature-sensitive repressor)cts (temperature-sensitive repressor)cts (temperature-sensitive repressor)

Krylov era/. (1980)A. DarzinsKrishnapiiiai (1971)

Plasmid

pADD693 pCP13 H/ndltl cosmid clone pilG" (Tc")pADD698 6.2kb EcoRI (ragment from pADD693 cloned into the EcoRI site of pROi614 (Tc")pADD699 6.2kb EcoRI fragment from pADD693 in pROi614 (Tc") — opposite orientation

from pADD698pADD2953 2.2 kb Aat\~Notl fragment containing piU blunted and cloned into the EcoRV site of

pRO1614(Ap")pADD3092 1.3kb Slu\-BamH\ fragment (pilK) in H/noll-SamHi sites of pN0T19 (Ap")pADD3261 Broad-host-range T? expression vector oriVpRoieoo onTRKs onVpwiBi 4.3kb (Ap'')PADD3275 pADD3261 with 1.3 kb H/ndili-BamHI p/W fragment from pADD3092 (Ap")PADD3280 4.3kb SamHI fragment {pilH, -I. -J. and -K) in SamHl site of pT7-6 (Ap")pADD3381 1.3kb pi/Kfragmeni plus pilJ-pilK inlergenic region cloned into pTG79 (Ap")pADD3382 1.3kb p///^fragment minutes p/yj-p/'/K"intergenic region cioned into pTG79 (Ap")PADD3434 pADD3382 AXho\ (Ap")pADD3450 1.2kb BamH\ pilK* fragment lacking p(W-p//K'intergenic region oloned into the

eamHI site of pRO1614 (Ap")pBR322 reft,Msi 4.4 kb (Ap*^Tc")piDl py/K-ZacZtranscriptional fusion (Ap")plD1ASa/-Wof py/K-ZacZtranscriptional fusion pIDI ASa/l-WoM (Ap")piD3 p/yK-ZacZtransiational fusion (Ap")plD3 ASal-Not p/VK'-fecZ translational fusion plD3 ASa/l-Wo(l (Ap")piD3 ASal pf/K'-ZacZtranslational fusion plD3 ASa/l (Ap")pMMB24 repR330B (ac promoter, lacl'^ 12.7kb (Ap")pM0B3 pHSS21 sacB sacR oriT, (Km"Cm")pN0T19 pUC19 with lObp Nde\-Noi\ adaptor in A/del site, (Ap")pRK2013 re/^MBi Tra* (RK2) (Km")pRO16l4 Broad-host-range vector, 6.2kb (Ap" [Cb"] Tc")pRS551 lac operon lusion vector, 12.5 kb (Ap", Km*̂ )pRS552 lac protein fusion vector, 12.4kb (Ap", Km")pT7-6 T7 4iiO promoter, 2.208kb (Ap")pTG79 T7 (bio promoter, 2.4kb (Ap")

Darzins (1993)Darzins (1993)Darzins (1993)

This work

This workThis workThis workThis workThis workThis workThis workThis work

Sutclitfe(1979)This workThis workThis workThis workThis workBagdasarian et al. (19S3)Schweizer (1992)Schweizer (1992)Figurski and Heiinski (1979)Oisen etal. (1982)Simons etai. (1987)Simons etal. (1987)S, TaborGiordano etal. (1989)

Ap", ampiciiiin resistance: Cb", carbeniciilin resistance; Cm", chloramphenicol resistance; Km", kanamycin resistance; Sm", streptomycin resis-tance; Tc", tetracycline resistance: Tp", trimethoprim resistance; rep, repiicon; Tra*. seif transmissible; oriT, RK2 origin of transfer; orfV. origin ofvegetative replication.

concentration of 1.5%. LB top agar for titering phage lysatescontained agar at a concentration of 0.7% and 1 mM MgS04.The antibiotic concentrations for E. coli were as follows: ampi-cillin, 100j.igml" ^ kanamycin. 30ngml" ' ' ; tetracycline, 25 jtgm l " ' , and chloramphenicol, 3 0 n g m r ' . For selection of P.aeruginosa drug-resistant transconjugants, Pseudomonas

Isolation Agar (PIA; Difco) was supplemented with carbeni-cillin (300 (.ig m l " ' ) or tetracycline (300 ).ig m l " ' ) ,

DNA manipulations and sequence anaiysis

DNA fragmente were cloned into vectors by using techniques

714 A. Darzins

described by Sambrook et al. (1989), DNA fragments werepurified from agarose gels after electrophoretic separationby the method of Vogetstein and Gillespie (1979). Blunt-ended ligations were performed after filling in of 5' cohesivetermini with Klenow fragment (Boehringer Mannheim). Pro-cedures for Southern hybridization analysis were performedas previously described (Darzins and Chakrabarty, 1984).

A1.3 kb Siful-BamHI fragment (Fig. 1) harbouring pilKwascloned into the Hinc\\-BamH\ sites of pN0T19. The resultingplasmid, designated pADD3092, was used as the DNA sourcefor sequence analysis. Nucleotide sequences for both strandswere determined on denatured double-stranded plasmid DNAcontaining fragments cloned into pUCi8 or pUC19. DNAsequencing was accomplished by the dideoxy chain termi-nation method (Sanger ef al.. 1977) with Sequenase 2.0(United States Biochemical) using deaza-dGTP, [̂ ^S]-dCTPand commercial oligonucleotide primers. Labelled sampleswere run in 8M urea/6% polyacrylamide gels. DNA andprotein sequence analysis was performed with the IBIMACVECTOH softwars program. Nucleotide and amino acidsequences were also analysed with the Wisconsin GeneticsComputer Group (GCG) software (Devereux et ai, 1984).The TFASTA algorithm for protein homology was used tocompare the deduced protein product of pilG to sequencesin the GenBank database (Release 82.0). PUBLISH was usedto generate the DNA sequence in Fig. 2. GAP and PUBLISHwere used to generate the comparisons between PilK andCheR (Fig. 3).

Genetic procedures

Transformation of E. coti and the Introduction of recombinantpiasmids into P. aeruginosa from E coli by triparental mat-ings were performed as previously described (Darzins, 1993).P. aeruginosa cells were transformed by the method of Olsenet al. (1982). Preparation of phage and phage-sensitivityassays were performed as described previously (Darzins,1994).

Expression studies with the T7 RNApolymerase-promoter system

The E. coli and P. aeruginosa T7 expression systems wereused as described by Tabor and Richardson (1985) andBrunschwig and Darzins (1992). respectively. BL21(DE3) orADDI 976 celis containing the piasmids to be analysed weregrown in M9 medium, induced with IPTG (1 mM final cone),and labelled with ^H-labelled amino acids (Amersham37 MBq, imCimr ' ) . Proteins were separated by SDS-PAGE (12.5% acrylamide (w/v)) and the resulting gels wereprepared for fluorography as described previously (Darzins.1993).

Construction of pilK-lacZ fusions

The 5.2 kb and 5.1 kb EcoR\-Stu\ fragments {IacZ*. -Y*. and•/^*) from pRS551 and pRS652. respectively, were cloned intopUC18 which had been digested with EcoRI and H/nc!l. Theresulting constructs, pADD3551 and pADD3550. were theneach digested with Smal and ligated with a 540 bp Stu\~EcoRV fragment which contains the pitJ-pilK intergenic

region and approximately half of the pilK coding region(Fig. 2). The largest EcoRI-Scal fragments of the resultingpiasmids were cloned into pRO1614. which had beendigested with the same restriction enzymes. The intact pilK-IacZ fusions were removed from each of the previousplasmid constructs by digestion with H/ndlll and placed intothe unique H/ndlll site of the broad-host-range (ac promoterpiasmid pMMB24 (Bagdasarian et ai, 1983). Screening ofseveral potential clones yielded the piiK-lacZ transcriptionalfusion plasmlds pIDI and the corresponding translationalfusion plasmid plD3.

One set of p///<-/acZ fusion piasmids containing sequencemodifications to the inverted repeat was constructed bydeleting the 17bp between the Not\ and Sa/I sites (Fig. 2)in the pilJ-pilK intergenic region after filling-in and blunt-endligation. The resulting constructs, designated plD1A/VoM-Sa/I and plD3AWo/l-Sa/l, contained the modified pilK-lacZIranscriptional and translational fusion, respectively (Fig. 5B).Plasmid plD3ASa/l, which contained a 4bp insertion inthe descending stem of the putative secondary structure(Fig. 5C). was constructed by filling-in the unique Sa/I sitedirectly upstream of the putative pilK ribosome-binding sitein the pZ/ZC-ZaoZ translational plasmid plD3.

Beta-galactosidase assays

Cells were grown at 37'C in LB broth containing ampicillinor carbenicillin overnight, diluted 1/200 into 15ml of thesame medium and grown to late-log phase (-4600= 1.0). Beta-galactosidase activity in SDS-CHCIs-permeabilized cells wasdetermined as previously described by Miller (1972). Enzymeactivity was expressed in Miller units (defined as nanomoles ofnitrophenol produced per minute per cell culture turbidity(•̂ eoo)) The background p-galactosidase activity in MC1061cells lacking the fusions was undetectable (<1).

Construction of pWK gene-replacement mutants

Gene-replacement techniques developed for P. aeruginosa(Schweizer. 1992) were carried out as previously described(Darzins, 1994). The initial chromosomal mutant constructedcontained a tet cassette within the piiK gene (Rg. 5).Construction of FA7 began with liberation of a 2,6 kb Not\-H/r7dlll fragment harbouring the pilK gene from pADD699and ligation into the Not\-Hin6\\\ sites of pN0T19. Theresulting plasmid, pADD2686, was digested with Xho\.thereby removing the internal 520 bp fragment. The ends ofpADD2686 were made flush with Klenow and ligated to a1.4 kb blunted EcoR\-Ava\ tet cassette from pBR322 tomake pADD2687. The oriT sacB Cm" (chloramphenicol-resistance) cassette from pM0B3 was cloned into the uniqueNot\ site of pADD2687 and the resulting plasmid was intro-duced into PAOl by triparental matings. Several sucrose-resistant. Tc'̂ transconjugants were isolated and verified bySouthern hybridization. One mutant, designated FA7, waschosen for further analysis.

The second pilK chromosomal mutant constructed was anin-frame deletion which lacked an antibiotic-resistance deter-minant. This mutant was constructed by first cloning the1.3kb BamH\-Not\ fragment harbouring the pilK gene(Fig. 2) into pN0T19. The resulting plasmid was then

Pseudomonas aeruginosa pHus biogenesis 715

digested with Xho\ in order to facilitate removal of the internal520 bp Xtiol fragment. The resulting plasmid, designatedpADD3526. contained an in-frame piiK deletion. PlasmidpADD3526 was further modified, first by the addition of thepBR322 tet cassette (see above) in the Seal site of the Ap"determinant, and second by the addition of the orlT sacBCm" cassette from pM0B3. The resuiting construct was mobi-lized into PA01, and several sucrose-sensitive Tc" transcon-jugants were isolated. Several of these transconjugantswere then streaked onto LB 5% sucrose plates which yieldedmany sucrose-resistant, Tc-sensitive (Tc^) colonies. Twelveindividual sucrose-resistant, Tc^ colonies were subjected toSouthern hybridization in order to verify the absence of the520 bp internal XAJOI fragment. Of the colonies tested, eighthad the expected wild type pattern and four had the expectedin-frame deletion pattern. One confirmed deletion mutant,designated FA9, was chosen for further analysis.

Motility assays

Twitching-motility assays were performed essentially asdescribed previously (Darzins, 1993). Swarm assays usedto assess f . coli and P. aeruginosa flage I la-mediated motilitywere carried out by inoculating the strains to be tested into thecentres of LB agar (0.3%) plates. After 8-12 h of incubation at37 C, the plates were inspected for radial zones of bacteriaigrowth indicating a motile, chemotactic response.

Acknowledgements

I would like to thank Dr J. S. Parkinson for the E. co//strainsRP437 and RP1254, and for his helpful suggestions. I wouldaiso like to thank Lynn O'Donnell, Patricia Truax and MaryRussell for helpful discussions.

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