regulator algr andan sensorfims for - pnas · homologyto algr(fig. 2b) andthat share the sametandem...

5
Proc. Natl. Acad. Sci. USA Vol. 93, pp. 9839-9843, September 1996 Microbiology The alginate regulator AlgR and an associated sensor FimS are required for twitching motility in Pseudomonas aeruginosa (twitching motility/fimbriae/pili/cystic fibrosis/pathogenesis) CYNTHIA B. WHITCHURCH, RICHARD A. ALM, AND JOHN S. MATriCK* Centre for Molecular and Cellular Biology, University of Queensland, Brisbane, QLD 4072, Australia Communicated by Salih J. Wakil, Baylor College of Medicine, Houston, TX, June 3, 1996 (received for review January 2Z 1996) ABSTRACT Mucoid strains of Pseudomonas aeruginosa isolated from the lungs of cystic fibrosis patients produce large amounts of the exopolysaccharide alginate. AlgR has long been considered a key regulator of alginate production, but its cognate sensor has not been identified. Here we show that AlgR is required for twitching motility, which is a form of bacterial surface translocation mediated by type 4 fimbriae. Adjacent to algR we have identified a sensor gene (fimS), which is also required for twitching motility. However, FimS does not appear to be required for alginate production in mucoid strains. FimS and AlgR are representative of a new subclass of two-component transmitter-receiver regulatory systems. The alternative sigma factor AlgU also affects both alginate production and twitching motility. Therefore, these two vir- ulence determinants appear to be closely associated and coordinately regulated. Pseudomonas aeruginosa is an opportunistic pathogen of hu- mans and other species (1). It causes severe chronic infections in patients who are immunocompromised as a result of cancer chemotherapy or AIDS, or who are suffering from burns or cystic fibrosis. It produces an extensive suite of virulence factors including extracellular proteases and toxins, lipases, pyochelins, exopolysaccharides, and type 4 fimbriae. In cystic fibrosis, P. aeruginosa produces recurrent and chronic lung infections, which impose substantial morbidity and often mor- tality as a consequence of accumulated damage to the lung. Isolates from such chronic infections exhibit a "mucoid" colony phenotype due to the production of large amounts of the exopolysaccharide alginate, apparently as a means of evading immune responses (for reviews see refs. 2 and 3). Transcriptional regulation of the alginate biosynthetic genes involves a variety of components whose interrelationships are not entirely clear. They include the response regulators AlgR and AlgB; the alternative sigma factor AlgU (or AlgT), which is required for the transcription of alginate biosynthetic genes; the anti-sigma factors MucA and AlgN (or MucB), which counteract the function of AlgU; the histone-like protein AlgP; and AlgQ, the exact function of which is unclear (2, 3). AlgR and AlgB have been the subject of considerable study and appear to represent the receiver components of classical sensor-regulator pairs, but their cognate sensors have not been identified. We have been examining the molecular genetics of the biogenesis and function of type 4 fimbriae in P. aeruginosa (4, 5). Type 4 fimbriae are polar filaments produced by a wide range of pathogenic bacteria, including Neisseria gonorrhoeae, Neisseria menigitidis, Dichelobacter nodosus, various Moraxella species, and Vibrio cholerae (for review, see ref. 6). These filaments mediate adhesion to epithelial cells (7) and surface translocation via a phenomenon termed twitching motility, which is probably their primary function. The mechanical basis of this process is unknown, but is thought to involve fimbrial extension and retraction (8). Loss of fimbriae and/or twitching motility renders the cells avirulent (6, 9). Type 4 fimbriae are assembled by a system closely related to those involved in the formation of cell surface complexes for protein secretion and DNA uptake in a wide variety of bacteria (10, 11), and are composed of a major structural subunit (PilA) whose tran- scription in P. aeruginosa is tightly controlled by a classical RpoN(or54)-dependent two-component sensor-regulator pair, PilS and PilR (4). Here we show that another sensor-regulator pair, AlgR and FimS, is also required for fimbrial function. MATERIALS AND METHODS Bacterial Strains and Plasmids. The Escherichia coli strain DH5a (hsdR recA lacZYA #80 lacZAM15) was used for standard DNA manipulations and preparation of sequencing templates. The P. aeruginosa strains used were wild-type PAK (David Bradley, Memorial University of Newfoundland) and Tn5-B21 (12) mutants of this strain. Construction of these strains has been described (4). Transformation of P. aeruginosa strains was performed as described (13). Antibiotics were used at the following concentrations: E. coli, ampicillin (50 ,g/ml)/ tetracycline (40 ,g/ml); P. aeruginosa, carbenicillin (500 ,tg/ ml)/tetracycline (200 ,ug/ml). DNA Manipulations. Preparation of plasmid DNA, restric- tion endonuclease digestions, Southern blotting, ligation re- actions, and transformation of E. coli were carried out using standard procedures (14). algR was cloned as an 895-bp ClaI-SmaI fragment into the broad-host range shuttle vector pUCPKS (15). fimS was cloned as a 2.3-kb EcoRI fragment into pUCPSK (15). algU was cloned by PCR amplification from P. aeruginosa PAO1 with amplimers designed against the sequence of algU (GenBank accession no. L04794) with flank- ing 5' BamHI and 3' XbaI restriction sites and cloned into the corresponding sites of the P. aeruginosa-E. coli shuttle vector, pUCPKS. The integrity of the cloned gene was checked by sequencing. DNA sequence analysis was performed using the dideoxy chain termination Applied Biosystems PRISM kit on a 373A automated sequencer. The sequences were completed on both strands. Assays. ELISA and Western blot analysis were performed as described (16). Twitching motility assays were performed as described using stab inoculation followed by overnight growth and Coomassie stain of the zone of movement at the agar- plastic interface (17). Alginate and total cell protein were assayed as described by May and Chakrabarty (18). Alginate was isolated as described by May and Chakrabarty (18) from a lawn of cells generated by plating 100 ,ul of an overnight broth culture onto standard 85-mm plates containing 15 ml of agar and grown for 24 hr at 37°C. The media used for alginate assays was Pseudomonas isolation agar (PIA, Difco) containing 1% Data deposition: The sequence reported in this paper has been deposited in the GenBank data base [accession no. L48729 (fimS)]. *To whom reprint requests should be addressed. 9839 The publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. §1734 solely to indicate this fact. Downloaded by guest on October 4, 2020

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Page 1: regulator AlgR andan sensorFimS for - PNAS · homologyto AlgR(Fig. 2b) andthat share the sametandem genetic arrangement asfimS andalgR. AlgRand FimSAre Members of a NewFamily ofTrans-mitter-ReceiverResponseRegulators.Allresponseregulators

Proc. Natl. Acad. Sci. USAVol. 93, pp. 9839-9843, September 1996Microbiology

The alginate regulator AlgR and an associated sensor FimS arerequired for twitching motility in Pseudomonas aeruginosa

(twitching motility/fimbriae/pili/cystic fibrosis/pathogenesis)

CYNTHIA B. WHITCHURCH, RICHARD A. ALM, AND JOHN S. MATriCK*Centre for Molecular and Cellular Biology, University of Queensland, Brisbane, QLD 4072, Australia

Communicated by Salih J. Wakil, Baylor College of Medicine, Houston, TX, June 3, 1996 (received for review January 2Z 1996)

ABSTRACT Mucoid strains of Pseudomonas aeruginosaisolated from the lungs of cystic fibrosis patients producelarge amounts of the exopolysaccharide alginate. AlgR haslong been considered a key regulator of alginate production,but its cognate sensor has not been identified. Here we showthat AlgR is required for twitching motility, which is a formofbacterial surface translocation mediated by type 4 fimbriae.Adjacent to algR we have identified a sensor gene (fimS), whichis also required for twitching motility. However, FimS does notappear to be required for alginate production in mucoidstrains. FimS and AlgR are representative of a new subclassof two-component transmitter-receiver regulatory systems.The alternative sigma factor AlgU also affects both alginateproduction and twitching motility. Therefore, these two vir-ulence determinants appear to be closely associated andcoordinately regulated.

Pseudomonas aeruginosa is an opportunistic pathogen of hu-mans and other species (1). It causes severe chronic infectionsin patients who are immunocompromised as a result of cancerchemotherapy or AIDS, or who are suffering from burns orcystic fibrosis. It produces an extensive suite of virulencefactors including extracellular proteases and toxins, lipases,pyochelins, exopolysaccharides, and type 4 fimbriae. In cysticfibrosis, P. aeruginosa produces recurrent and chronic lunginfections, which impose substantial morbidity and often mor-tality as a consequence of accumulated damage to the lung.Isolates from such chronic infections exhibit a "mucoid"colony phenotype due to the production of large amounts ofthe exopolysaccharide alginate, apparently as a means ofevading immune responses (for reviews see refs. 2 and 3).

Transcriptional regulation of the alginate biosynthetic genesinvolves a variety of components whose interrelationships arenot entirely clear. They include the response regulators AlgRand AlgB; the alternative sigma factor AlgU (or AlgT), whichis required for the transcription of alginate biosynthetic genes;the anti-sigma factors MucA and AlgN (or MucB), whichcounteract the function of AlgU; the histone-like proteinAlgP; and AlgQ, the exact function of which is unclear (2, 3).AlgR and AlgB have been the subject of considerable studyand appear to represent the receiver components of classicalsensor-regulator pairs, but their cognate sensors have not beenidentified.We have been examining the molecular genetics of the

biogenesis and function of type 4 fimbriae in P. aeruginosa (4,5). Type 4 fimbriae are polar filaments produced by a widerange of pathogenic bacteria, including Neisseria gonorrhoeae,Neisseria menigitidis, Dichelobacter nodosus, various Moraxellaspecies, and Vibrio cholerae (for review, see ref. 6). Thesefilaments mediate adhesion to epithelial cells (7) and surfacetranslocation via a phenomenon termed twitching motility,which is probably their primary function. The mechanical basis

of this process is unknown, but is thought to involve fimbrialextension and retraction (8). Loss of fimbriae and/or twitchingmotility renders the cells avirulent (6, 9). Type 4 fimbriae areassembled by a system closely related to those involved in theformation of cell surface complexes for protein secretion andDNA uptake in a wide variety of bacteria (10, 11), and arecomposed of a major structural subunit (PilA) whose tran-scription in P. aeruginosa is tightly controlled by a classicalRpoN(or54)-dependent two-component sensor-regulator pair,PilS and PilR (4). Here we show that another sensor-regulatorpair, AlgR and FimS, is also required for fimbrial function.

MATERIALS AND METHODSBacterial Strains and Plasmids. The Escherichia coli strain

DH5a (hsdR recA lacZYA #80 lacZAM15) was used forstandard DNA manipulations and preparation of sequencingtemplates. The P. aeruginosa strains used were wild-type PAK(David Bradley, Memorial University of Newfoundland) andTn5-B21 (12) mutants of this strain. Construction of thesestrains has been described (4). Transformation ofP. aeruginosastrains was performed as described (13). Antibiotics were usedat the following concentrations: E. coli, ampicillin (50 ,g/ml)/tetracycline (40 ,g/ml); P. aeruginosa, carbenicillin (500 ,tg/ml)/tetracycline (200 ,ug/ml).DNA Manipulations. Preparation of plasmid DNA, restric-

tion endonuclease digestions, Southern blotting, ligation re-actions, and transformation of E. coli were carried out usingstandard procedures (14). algR was cloned as an 895-bpClaI-SmaI fragment into the broad-host range shuttle vectorpUCPKS (15). fimS was cloned as a 2.3-kb EcoRI fragmentinto pUCPSK (15). algU was cloned by PCR amplificationfrom P. aeruginosa PAO1 with amplimers designed against thesequence of algU (GenBank accession no. L04794) with flank-ing 5' BamHI and 3' XbaI restriction sites and cloned into thecorresponding sites of the P. aeruginosa-E. coli shuttle vector,pUCPKS. The integrity of the cloned gene was checked bysequencing. DNA sequence analysis was performed using thedideoxy chain termination Applied Biosystems PRISM kit ona 373A automated sequencer. The sequences were completedon both strands.

Assays. ELISA and Western blot analysis were performed asdescribed (16). Twitching motility assays were performed asdescribed using stab inoculation followed by overnight growthand Coomassie stain of the zone of movement at the agar-plastic interface (17). Alginate and total cell protein wereassayed as described by May and Chakrabarty (18). Alginatewas isolated as described by May and Chakrabarty (18) froma lawn of cells generated by plating 100 ,ul of an overnight brothculture onto standard 85-mm plates containing 15 ml of agarand grown for 24 hr at 37°C. The media used for alginate assayswas Pseudomonas isolation agar (PIA, Difco) containing 1%

Data deposition: The sequence reported in this paper has beendeposited in the GenBank data base [accession no. L48729 (fimS)].*To whom reprint requests should be addressed.

9839

The publication costs of this article were defrayed in part by page chargepayment. This article must therefore be hereby marked "advertisement" inaccordance with 18 U.S.C. §1734 solely to indicate this fact.

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Page 2: regulator AlgR andan sensorFimS for - PNAS · homologyto AlgR(Fig. 2b) andthat share the sametandem genetic arrangement asfimS andalgR. AlgRand FimSAre Members of a NewFamily ofTrans-mitter-ReceiverResponseRegulators.Allresponseregulators

9840 Microbiology: Whitchurch et al.

glycerol (vol/vol), which is normally used for assaying alginateproduction by mucoid P. aeruginosa strains (18).

RESULTS AND DISCUSSIONThe Alginate Regulator AlgR and Its Related Sensor FimS

Affect Twitching Motility. We have previously constructed alibrary of transposon mutants that affect twitching motility inP. aeruginosa as determined by altered surface colony mor-phology (4). These mutants were grouped according towhether they had also acquired resistance to fimbrial-specificbacteriophage and according to the size of the chromosomalrestriction fragment into which the transposon had inserted(4). The corresponding loci have since been progressivelycloned and sequenced to identify the affected genes. In thisway, a large number of genes affecting fimbrial biogenesis orfunction have been identified and at least 25 genes at severalloci are now known to be involved (4, 5).

This study describes the characterization of four phage-sensitive mutants in the library that mapped to a new locus.The mutants S31, S39, S47, and S62 containing the transposonTn5-B21 (12) were grouped to the same KpnI P. aeruginosaPAK genomic DNA fragment by Southern blotting using thetransposon as a probe. DNA flanking the site of transposoninsertion in each mutant was cloned by digestion with EcoRI(which cuts the transposon once beyond the tetracyclineresistance marker), ligation into pBluescript II (Stratagene),and recovery of tetracycline-resistant E. coli colonies. TheDNA adjacent to the transposon was then sequenced using theprimer "Ollie" (4) and in two cases (S31 and S39) corre-sponded to the published algR sequence (GenBank accessionno. M23230) (Fig. 1). Screening a reference PAO1 cosmidlibrary (kindly supplied by B. Holloway, Monash University,Melbourne) with DNA flanking the transposon from S62enabled isolation of the wild-type locus. Southern blots usingthe cloned flanking DNA from S39 and S62 showed that thesetransposons had inserted into the same 2.3-kb EcoRI fragment(Fig. 1). This fragment was cloned on the broad host-rangeplasmid pUCPSK (15) and shown to restore twitching motilityto S47 and S62, but not to S31 or S39. This fragment was thensequenced and shown to represent the previously uncharac-terized region between algR and argH (19) (Fig. 1). Sequenceanalysis of this region identified a gene, which we havedesignatedfimS (GenBank accession no. L48729), the productof which shares homology to LytS, a sensor or transmitter thatregulates autolysis (murein hydrolase production) in Staphy-lococcus aureus (21) and YehU, a hypothetical protein from E.coli (GenBank accession no. U00007) (Fig. 2a). Both the lytSand yehU genes are directly followed by genes (lytR and yehT,respectively) encoding putative receivers that have overallhomology to AlgR (Fig. 2b) and that share the same tandemgenetic arrangement as fimS and algR.AlgR and FimS Are Members of a New Family of Trans-

mitter-Receiver Response Regulators. All response regulators

EcoRIA

or receivers share a common N-terminal domain showinghomology to CheY and include a conserved lysine and threeconserved aspartate residues, the third of which is phosphor-ylated by a cognate sensor or transmitter. (For reviews of thestructures of proteins involved in signal transduction by mod-ular phosphorylation in sensor-regulator or receiver-transmitter pairs, see refs. 24-27.) These receivers differsignificantly in their C termini and can be subgrouped accord-ing to similarities of this region. AlgR clearly has a receiverdomain, but shows significant differences from other well-characterized receivers in the remainder of the protein (24,27). It is however homologous to the recently-described LytRand YehT across their entire sequence, suggesting that theseproteins may constitute a new class of response regulators (Fig.2b). Similarly, FimS, LytS, and YehU are also closely related,but differ significantly from other histidine kinases and appearto form a new family of sensors or transmitters (Fig. 2a).Together, these protein pairs form a novel subclass of thesensor-regulator or transmitter-receiver superfamily.There are several differences between FimS, LytS, and

YehU and other known types of transmitters such as NtrB andCheA (24-26). First, FimS is similar to LytS and YehU, butdiffers from known transmitters in the region around theputative histidine phosphorylation site (Fig. 2a). It has beenproposed that the local structure around the critical histidinein such transmitters can influence the ratio of phosphoryla-tion-dephosphorylation exchange between the histidine andthe aspartate in the receiver (24-26). Second, FimS lacks theDXGXG and GXG motifs in region III (Fig. 2a), which arethought to comprise nucleotide binding domains (24-26),suggesting that FimS may be incapable of autophosphoryla-tion. YehU lacks the first site, whereas LytS retains both. It ispossible that proteins such as LytS may be capable of bothautophosphorylation and of blocking or reversing the phos-phorylation of its partner(s). The conserved histidine on theseproteins may either be able to accept phosphate groups fromanother transmitter (in which case these proteins may functionas an intermediate receiver-transmitter) or act as an acceptorof phosphates from the conserved aspartate in their receiver-partners (24-26). In any case the lack of nucleotide bindingdomains in FimS suggests that it may operate by an unusualmechanism.The Role of FimS, AlgR, and AlgU in Alginate Production

and Twitching Motility. AlgR and FimS appear to be requiredfor twitching motility in P. aeruginosa (Fig. 3). Mutations inalgR and fimS result in either complete loss or severe retar-dation of twitching motility (Fig. 3 e and i). Both types ofmutants essentially lack surface fimbriae as shown by ELISA(Fig. 4), which is a sensitive quantitative assay, but producenormal amounts of the fimbrial subunit PilA as determined bywhole cell Western blots with -anti-PilA antiserum (data notshown). Thus AlgR and FimS are not required for pilAtranscription. These mutants can be restored to essentiallywild-type levels of twitching motility by complementation with

Clal EcoRIIL K

Smal

sX ~ C -;.. n.....................................r_......................................................................Me....................................................................................................

N

pUCPfimS LS47 S62

ArAA rS39 S31

V

0.5 KbIpUCPalgR I

FIG. 1. Map of the fimS-algR locus and site of transposon insertions. The sites of transposon Tn5-B21 (12) insertion in the mutants S31, S39,S47, and S62 were mapped as described in the text. The 2.3-kb EcoRI fragment containing the sites of transposon insertion of S47 and S62 wassequenced and shown to represent the previously uncharacterized region between algR and argH (19), which encodes arginosuccinate lyase. It hasbeen shown that downstream of algR is hemC, which encodes porphobilinogen deaminase (20). Triangles indicate the sites of transposon insertionin the different mutants.

Proc. Natl. Acad. Sci. USA 93 (1996)

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Page 3: regulator AlgR andan sensorFimS for - PNAS · homologyto AlgR(Fig. 2b) andthat share the sametandem genetic arrangement asfimS andalgR. AlgRand FimSAre Members of a NewFamily ofTrans-mitter-ReceiverResponseRegulators.Allresponseregulators

Microbiology: Whitchurch et al. Proc. Natl. Acad. Sci. USA 93 (1996) 9841

aYehULytS

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AigR A VI F C T A H D ELF A|L E'A F 61V SIA V|G Y L,V[KP V R S E DAL!A L!K[K A S R P N R vONRLAAAL T K PFPA S G G S G P R S HIIS A R T R K GI E LI PFL1E ENVYehT Y V F L T A - F D E Y Al iKIA FIE E HI AF D Y L L K P D E AIRLEIKTLARLIRQ E R S K Q D V SL L PE NIQ Q - - A L K F PICTGHSRIIYLLIOM K D VIALytR A F A T A - H D OIY A V IAF' E LNIAIT!D YI L K PFFIG Q KiR. EQ AVNK1V,RATKAKD N N AHS A,IA NiD MS F D Q L FV El DID K HM LIK OQN|NtrC V!iI|M AH S D L D A G A F D YLFKPFDI DEAVALVDIRAI SHYOEIOPRNAF NS TAjD GE RPAMOD V R G L . 312aaCheY VL M V -TAEAKKEIN K P FIT A A T L E E K L N K F E K L G M

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YehT F S S R M S G'V Y V T- S HIE G K E G F T E L T'LIR T,L E S R T - - - P L L R CGH RDQ Y L V N L A H;L Q E R L! E DNGOAE L HR -N - - - G L T V P V S RR Y L K

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YehT S L K E A: I G L

LytH D F. K A S G L L

FIG. 2. Comparison of the predicted FimS and AlgR sequences with other known transmitters and receivers. Sequence homologies wereidentified using the BLAST network service at the National Center for Biotechnology Information (22). Proteins were aligned using the CLUSTALVprogram (23). (a) Multiple alignment of the new class of sensor molecules FimS, YehU, and LytS. Identical residues between at least two membersare boxed and the conserved His and Asn residues are shaded. The region III motifs of DXGXG and GXG are also shaded. (b) Multiple alignmentof regulator proteins AlgR, YehT, LytR, NtrC, and CheY [GenBank accession nos. M19277 (NtrC) and K02175 (CheY)]. The identical residuesbetween at least two members are boxed and the critical Lys and Asp residues are shaded. A second putative phosphate-accepting Asp residueis indicated in boldface type. Only the conserved N-terminal domain of NtrC is included, as the remainder of the protein possesses little similarityto this class of regulators.

their respective genes in trans (Fig. 3 f and k). The level oftwitching motility in algR-complemented cells is somewhatreduced, but this is also observed in the wild type (Fig. 3b),suggesting an inhibitory effect of AlgR overproduction fromthe multicopy plasmid. This effect has been observed previ-ously in relation to alginate production (28, 29). We have alsoobserved that overproduction of the response regulator PilR,which is required for transcription of the fimbrial subunit genepilA, can substantially overcome the effects of mutations in itscognate sensor PilS (4). Similarly, overexpression of algR canovercome fimS mutations (Fig. 3j), thereby strengthening theconclusion from their related phenotypes and chromosomaljuxtaposition that FimS is a sensor or transmitter that interactswith AlgR.The mucoid colony phenotype associated with high levels of

alginate production is observed in fresh clinical isolates of P.

aeruginosa from cystic fibrosis patients, but this characteristicis unstable in culture (2, 3). As a consequence, studies on theregulation of alginate production have been carried out largelyin variants selected for the stable expression of mucoidy (3, 19,30,32). Such variants have been subsequently shown to containmutations in the anti-sigma factors algN or mucA (whichsuppress the sigma factor AlgU) and thus result in up-regulation of AlgU activity (2, 3, 33). Mucoidy can also beinduced by increasing algU gene dosage by transformation witha multicopy plasmid containing this gene (33), presumably dueto increased levels of AlgU. We therefore reproduced themucoid phenotype in our P. aeruginosa strains by transforma-tion with the multicopy vector pUCPKS containing algU(pUCPalgU). Under these conditions we confirmed that thealgR mutant S39 is severely reduced in alginate production(Table 1), in agreement with previous studies in mucoid P.

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9842 Microbiology: Whitchurch et al.

Control + algRb c

+ fimSd

+ aIgU

PAK

S39

h

i k

S62

FIG. 3. Subsurface twitching motility assays were performed on P. aeruginosa PAK (a-d), P. aeruginosa S39 (algR) (e-h), and P. aeruginosa S62(fimS) (i-1) carrying plasmids pUCPKS (a, e, and i), pUCPalgR (b, f, and j), pUCPfimS (c, g, and k), or pUCPalgU (d, h, and 1).

aeruginosa strains (19, 32, 34). However, under these condi-tions, the fimS mutant S62 still produces large amounts ofalginate similar to the control (Table 1). This does not discountthe possibility that FimS may have a role in regulating alginateproduction under other circumstances.

Overproduction of AlgU from pUCPalgU restores twitchingmotility to thefimS mutant (Fig. 31) suggesting that the lack ofFimS can be overcome by increasing the level of AlgU. Thismay be due to a direct effect of upregulating transcription fromthe relevant promoters thereby bypassing the normal environ-mental signals. Because of this, it is unlikely that the effect ofFimS on twitching motility would have been discovered instable mucoid strains, which have been selected or constructedin vitro to overproduce AlgU. On the other hand algR muta-tions cannot be overcome by AlgU overproduction (Fig. 3h),indicating that AlgR and AlgU are both required for tran-scription of the genes involved.FimS and AlgR appear to have different effects on twitching

motility and alginate production, although in other respectsthey appear to constitute a receiver-transmitter pair. Oneexplanation for this dichotomy is that there are two forms of

1.2

1.0

0.8

A405 0.6

0.4

0.2

0.0 '-

2.4 2.7 3.0 3.3 3.6 3.9 4.2- log dilution

FIG. 4. ELISA against whole cells of the following Pseudomonasstrains: PAK (-); R94 (-); S39 (A); S62 (0) using anti-fimbrialantiserum. Bovine serum albumin was used as the antigen control.

AlgR, which (in combination with AlgU) recognize differentpromoters. It is possible that AlgR contains more than onephosphorylation site, one of which is recognized by FimS andthe other of which receives independent signals. It is worthnoting here that AlgR shares a second aspartate in a conserveddomain with LytR and YehT (Fig. 2b). AlgR has been reportedto be capable of being phosphorylated by both histidinekinases, such as CheA, and by small molecular weight phos-phate donors, such as carbamoyl phosphate and acetyl phos-phate (35). Alternatively, it is possible that FimS blocks orreverses AlgR phosphorylation. The latter is consistent withthe apparent lack of crucial residues required for autophos-phorylation activity in FimS.The mechanism by which AlgR and FimS might regulate

twitching motility is unclear at this point. AlgR is thought tobe a transcription factor (2, 3), although it has an unusualstructure (27). It does not affect the expression of the fimbrialsubunit gene, piLA (data not shown), but there are more than30 gene products involved in fimbrial assembly and function(16, 17), one or more of which may require AlgR for expres-sion. Alternatively, AlgR may participate in phosphotransferinteractions with components of the fimbrial system.An alternative explanation for our observations is that FimS

may be a bona fide part of the signal transduction pathway thatregulates alginate production in wild-type cells, but that itsfunction is masked by the upregulation of algU in mucoidstrains, wherein alginate production is normally studied andmeasured. This occurs with twitching motility (Fig. 31). Be-cause it is difficult to measure alginate production in wild-type

Table 1. Alginate production by P. aeruginosa PAK, algR, andfimS mutants containing pUCPalgU confluently grown for 24 hr ona standard 85-mm Pseudomonas isolation agar (Difco) platecontaining 1% glycerol (vol/vol)

Alginate conc.,Strain mg/mg cell

(+pUCPalgU) Mutation protein % PAK

PAK 0.93 100S39 algR NDS62 fimS 0.99 103

Assays were performed in duplicate. ND, not detectable; conc.,concentration.

Proc. Natl. Acad. Sci. USA 93 (1996)

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Page 5: regulator AlgR andan sensorFimS for - PNAS · homologyto AlgR(Fig. 2b) andthat share the sametandem genetic arrangement asfimS andalgR. AlgRand FimSAre Members of a NewFamily ofTrans-mitter-ReceiverResponseRegulators.Allresponseregulators

Proc. Natl. Acad. Sci. USA 93 (1996) 9843

strains under standard media and assay conditions, this issueremains to be resolved. However, if FimS is in fact required foralginate production in the wild type, it is then also possible thatthe effect of FimS (and AlgR) on twitching motility may be aconsequence of a direct link between alginate production andtwitching motility. In this respect, it is worth noting thattwitching motility appears to be closely related to glidingmotility in Myxococcus xanthus, which is known to be depen-dent on the production of extracellular "slime" comprised ofcomplex polysaccharides and proteins (36, 37).

Conclusion. This report shows that the alginate regulatorAlgR and an associated sensor FimS are required for twitchingmotility in P. aeruginosa. Although algR and fimS mutationsappear to produce different phenotypes, it seems that they are

a genuine transmitter-receiver pair, which are likely to interactwith each other, based on the following evidence: (i) the algRand fimS genes are juxtaposed on the chromosome, as iscommon with other sensor-regulator pairs; (ii) AlgR and FimSeach show particular sequence characteristics that have onlybeen found in other related sensor-regulator pairs, LytR/LytSand YehU/YehT, the former of which at least is known tofunction as a cognate pair in vivo (21); (iii) both AlgR and FimSaffect twitching motility; and (iv) algR overexpression over-comes mutations infimS, indicating that they are on the samepathway, as has been observed for other sensor-regulator pairs(4).The discovery of a linkage between alginate production and

twitching motility is unexpected and has considerable impli-cations. Understanding the details of this linkage will providefurther insight into the process of infection by P. aeruginosa ofpatients suffering cystic fibrosis and other diseases.

We thank Kym Brown and Alison Watson for technical assistance.We also thank the University of Queensland DNA Sequencing Facil-ity. This work was supported by grants from the Australian ResearchCouncil and the National Health and Medical Research Council.

1. Fick, R. (1993) Pseudomonas aeruginosa the Opportunist: Patho-genesis and Disease (CRC, Boca Raton, FL).

2. Zielinski, N. A., Roychoudhury, S. & Chakrabarty, A. M. (1994)Methods Enzymol. 235, 493-502.

3. Deretic, V., Schurr, M. J., Boucher, J. C. & Martin, D. W. (1994)J. Bacteriol. 176, 2773-2780.

4. Hobbs, M., Collie, E. S. R., Free, P. D., Livingston, S. P. &Mattick, J. S. (1993) Moi. Microbiol. 7, 669-682.

5. Alm, R. A., Bodero, A. J., Free, P. & Mattick, J. S. (1996) J.Bacteriol. 178, 46-53.

6. Tennent, J. M. & Mattick, J. S. (1994) in Fimbriae: Aspects ofAdhesion, Genetics, Biogenesis and Vaccines, ed. Klemm, P.(CRC, Boca Raton, FL), pp. 127-146.

7. Doig, P., Todd, T., Sastry, P. A., Lee, K. K., Hodges, R. S.,Paranchych, W. & Irvin, R. T. (1988) Infect. Immun. 56, 1641-1646.

8. Bradley, D. E. (1980) Can. J. Microbiol. 26, 146-154.9. Hazlett, L. D., Moon, M. M., Singh, A., Berk, R. S. & Rudner,

X. L. (1991) Curr. Eye Res. 10, 351-362.10. Hobbs, M. & Mattick, J. S. (1993) Mo. Microbiol. 10, 233-243.11. Mattick, J. S. & Alm, R. A. (1995) Trends Microbiol. 3, 411-414.12. Simon, R., Quandt, J. & Klipp, W. (1989) Gene 80, 161-169.13. Mattick, J. S., Bills, M. M., Anderson, B. J., Dalrymple, B., Mott,

M. R. & Egerton, J. R. (1987) J. Bacteriol. 169, 33-41.14. Sambrook, J., Fritsch, E. F. & Maniatis, T. (1989) Molecular

Cloning: A Laboratory Manual (Cold Spring Harbor Lab. Press,Plainview, NY).

15. Watson, A. A., Alm, R. A. & Mattick, J. S. (1996) Gene 172,163-164.

16. Watson, A. A., Mattick, J. S. & Alm, R. A. (1996) Gene, in press.17. Alm, R. A. & Mattick, J. S. (1995) Moi. Microbiol. 16, 485-496.18. May, T. B. & Chakrabarty, A. M. (1994) Methods Enzymol. 235,

295-304.19. Mohr, C. D. & Deretic, V. (1990) J. Bacteriol. 172, 6252-6260.20. Mohr, C. D., Sonsteby, S. K. & Deretic, V. (1994) Mol. Gen.

Genet. 242, 177-184.21. Brunskill, E. W. & Bayles, K. W. (1996) J. Bacteriol. 178, 611-

618.22. Altschul, S. F., Gish, W., Miller, W., Myers, E. W. & Lipman,

D. J. (1990) J. Mo. Bio. 215, 403-410.23. Higgins, D. G. & Sharp, P. M. (1989) Comput. Appl. Bio. Sci. 5,

151-153.24. Parkinson, J. S. & Kofoid, E. C. (1992) Annu. Rev. Genet. 26,

71-112.25. Parkinson, J. S. (1993) Cell 73, 857-871.26. Swanson, R. V., Alex, L. A. & Simon, M. I. (1994) Trends Bio-

chem. Sci. 19, 485-490.27. Pao, G. M. & Saier, M. H., Jr. (1995) J. Mol. Evol. 40, 136-154.28. Kimbara, K & Chakrabarty, A. M. (1989) Biochem. Biophys. Res.

Commun. 164, 601-608.29. Deretic, V. & Konyecsni, W. M. (1989) J. Bacteriol. 171, 3680-

3688.30. Fyfe, J. A. M. & Govan, J. R. W. (1980) J. Gen. Microbiol. 119,

443-450.31. Darzins, A. & Chakrabarty, A. M. (1984) J. Bacteriol. 159, 9-18.32. Deretic, V., Dikshit, R., Konyecsni, W. M., Chakrabarty, A. M. &

Misra, T. K. (1989) J. Bacteriol. 171, 1278-1283.33. Goldberg, J. B., Gorman, W. L., Flynn, J. L. & Ohman, D. E.

(1993) J. Bacteriol. 175, 1303-1308.34. Mohr, C. D., Martin, D. W., Konyecsni, W. M., Govan, J. R. W.,

Lory, S. & Deretic, V. (1990) J. Bacteriol. 172, 6576-6580.35. Deretic, V., Leveau, J. H. J., Mohr, C. D. & Hibler, N. S. (1992)

Mol. Microbiol. 6, 2761-2767.36. Wu, S. S. & Kaiser, D. (1995) Mol. Microbiol. 18, 547-558.37. Shimkets, L. J. (1990) Microbiol. Rev. 54, 473-501.

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