Center for Adaptation Genetics and Drug Resistance, and Department of Molecular Biology and Microbiology, Tufts University School of Medicine, 136 Harrison Avenue, Boston, MA 02111, USA1
Author for correspondence: Stuart B. Levy. Tel: +1 617 636 6764. Fax: +1 617 636 0458. e-mail: stuart.levy{at}tufts.edu
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ABSTRACT |
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Keywords: adhesion, flagella, transcription regulation
The GenBank accession number for the sequence reported in this paper is AF312695.
a Present address: Lawrence Livermore National Laboratories, 7000 East Avenue, Livermore, CA 94550, USA.
b Present address: Millennium Pharmaceuticals Inc., 640 Memorial Drive, Cambridge, MA 02139, USA.
c Present address: University of Massachusetts-Dartmouth, Department of Biology, Dartmouth, MA 02747, USA.
d Present address: Slippery Rock University, Department of Biology, Slippery Rock, PA 16057, USA.
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INTRODUCTION |
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Numerous traits contribute to the survival of a particular bacterial strain in the soil. Cells must be able to adapt to changing temperature, nutrient availability and osmolarity. Biotic factors, such as avoiding predation by other organisms and resistance to antibacterial compounds produced by other species, also play a role. One general survival strategy is attachment to a surface and growth of a biofilm (Costerton et al., 1995 , 1999
). By this mechanism, cells have access to the nutrients adsorbed by the surface and may be protected from exogenous antibiotics and competitive colonization by other species.
Motility is often recognized as a factor contributing to adhesion and colonization of both biologic (Piette & Idziak, 1992 ; Ramphal et al., 1991
; Scharfman et al., 1996
) and abiotic surfaces (Korber et al., 1994
; OToole & Kolter, 1998a
, b
; Williams & Fletcher, 1996
), but the exact relationship between the two is somewhat obscure. It may be that motility is required to overcome the repulsion between the negatively charged bacterial cell and a negatively charged surface. In the case of Pseudomonas aeruginosa however, the most recent data indicate that it is the FliD protein, located at the tip of the flagella, that mediates a specific interaction with mucins (Arora et al., 1998
).
In a previous study, 3500 Tn5 insertion mutants of Pseudomonas fluorescens strain Pf0-1 were screened for reduced binding to quartz sand columns (DeFlaun et al., 1990 ). Three defective mutants were identified. These mutants also displayed defects in adhesion to a variety of seeds and soil (DeFlaun et al., 1994
). Examination of these strains indicated that two mutants, Pf0-5 and Pf0-10, lacked flagella and were non-motile. The insertions Pf0-5 and Pf0-10 have now been localized and the disrupted genes identified as the same ORF, specifying a putative transcriptional regulator named AdnA belonging to the NtrC/NifA family of activators.
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METHODS |
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Isolation of the wild-type adnA gene.
P. fluorescens gene-specific DNA probes were isolated by digesting pPfA34 and pPfA32 with EcoRI and HpaI to produce 4·5 kb and 3·95 kb fragments, respectively. The fragments were labelled with [32P]CTP and random hexamer primers. Genomic DNA from strain Pf0-1 was digested to completion with BamHI. DNA fragments (913 kb) were electroeluted from agarose gel slices and ligated to pLAFR5 cosmid arms. Cosmid arms were produced by first linearizing pLAFR5 with ScaI and then digesting with BamHI to produce two DNA fragments of 19·75 and 1·75 kb (Keen et al., 1988 ). The ligation buffer contained 10 mM ATP to suppress blunt end ligation at the ScaI site. The ligation mix was packaged in vitro (Gigapack II gold packaging extract; Stratagene) and transduced into E. coli DH5
.
DNA sequence analysis.
To sequence the adnA gene, a 6·2 kb BamHIEcoRI fragment of pPBF2 was subcloned into pBluescript II to form plasmid pPf1B. Both strands of DNA were sequenced manually using a Sequenase kit (Amersham). Similarity searches were performed using the National Center for Biotechnology Information BLAST server (Altschul et al., 1990 ). The sequence was deposited in GenBank with the accession number AF312695.
Inducible adnA expression.
Plasmid pPC100 was constructed by inserting a PCR fragment consisting of the adnA ORF flanked by AflIII and EcoRI sites into the broad-host-range plasmid pJB866 (Blatny et al., 1997 ). This manipulation places adnA under control of the Pm promoter and XylS activator, both derived from the TOL plasmid pWWO of P. putida (Mermod et al., 1986
). pPC101 was constructed by deleting a 923 bp AflIII fragment from pPC100, thus removing residues encoding amino acids 1309 from adnA and shifting the translation reading frame of the residual adnA sequence. To verify that adnA expression from pPC100 was inducible, E. coli cells were grown with and without 3-methylbenzoate and examined for differences in whole-cell protein profiles. LB was inoculated with an overnight culture of DH5
carrying pPC100 or pPC101 and each was grown at 30 °C to OD550
0·4. The cultures were each divided into two aliquots and 3-methylbenzoate (2 mM in 95% ethanol) was added to one, and an equal volume of 95% ethanol was added to the control. Incubation was continued for 16 h at 30 °C. Cells were collected by centrifugation and lysed by resuspension in 100 µl 10 mM Tris/HCl, 0·1 mM EDTA, pH 7·5 and 100 µl 2xSDS loading buffer. 10 µl lysate was resolved by SDS-PAGE in 10% gels. Proteins were stained with 0·01% Coomassie blue for 2 h and photographed.
Complementation.
Cosmids pPBF2 and pPBF3 were mobilized from E. coli strains into P. fluorescens in triparental matings using pRK2013 to supply transfer functions in trans (Ditta et al., 1980 ). pPC100 and pPC101 (see above) were initially transformed into E. coli strain S17.1, then transferred to Pf0-1 and mutant strains in biparental matings. Relative to Pseudomonas strains carrying control plasmid pPC101, strains carrying pPC100 exhibited reduced growth rates in Luria broth. However pPC101 had little effect on the exponential growth rate in minimal media. When diluted into fresh media, overnight cultures carrying pPC100 also exhibited a longer lag phase than the parental strains before resuming exponential growth. Therefore care was taken to start complementation experiments with cultures at the same growth state and optical density.
Motility.
Motility was assayed in MMO, 0·2% glutamate, 0·3% agar at 30 °C. Strains were inoculated into the agar with a sterile needle and growth away from the inoculation point was recorded.
Adhesion.
The adhesion assay is based on the assay described by OToole & Kolter (1998b) . An exponential phase culture (20 µl) growing in MMO+0·2% glutamate was added to 1 ml MMO+glutamate in borosilicate glass tubes. The tubes were incubated for 6 h at 30 °C without agitation, then rinsed with ddH2O and stained with 1% crystal violet. Excess stain was removed by rinsing in ddH2O. Adhesion was detected as a ring of stain on the tube wall at the air/medium interface.
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RESULTS AND DISCUSSION |
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The Tn5 insertion junction sites were cloned by digesting chromosomal DNA from Pf0-5 and Pf0-10 with EcoRI and BamHI, ligating the fragments into pUC18 and selecting for kanamycin-resistant transformants. The insertion junctions in the resulting plasmid DNAs, pPfA34 and pPfA32, were sequenced using a primer complementary to the end of the transposon. Distinct DNA sequences were obtained for Pf0-5 and Pf0-10 (data not shown). Thus, although the Tn5 insertions in Pf0-5 and Pf0-10 are physically close, they are independent mutations.
Isolation of cosmid clones complementing the motility defect of Pf0-5 and Pf0-10
A library containing BamHI fragments from strain Pf0-1 was constructed in the cosmid pLAFR5, packaged in vitro and transduced into E. coli. The transductants were screened by colony hybridization using a probe containing DNA flanking the Tn5 insertion in Pf0-5. Two clones, containing cosmids pPBF2 and pPBF3, hybridized to the Pf0-5 DNA probe. Both clones also hybridized to a probe containing DNA flanking the Tn5 insertion in Pf0-10.
Digestion of these cosmids with several restriction enzymes showed that both clones contained an identical 11 kb BamHI fragment. Further subcloning demonstrated that adnA resided on a 6·2 kb BamHIEcoRI fragment. The ability of pPBF2 and the cosmid vector alone to complement motility defects was tested by conjugating these plasmids into mutant strains Pf0-5 and Pf0-10. Motility was complemented in the mutant strains by pPBF2 but not by pLAFR5 (data not shown).
DNA sequence of adnA
The 6·2 kb EcoRIBamHI subclone was sequenced on both strands. Eight ORFs with similarity to known genes were identified within this fragment. One gene, called adnA, contains the sites of Tn5 insertion in Pf0-5 and Pf0-10 (Fig. 2). The other genes encode homologues of flagella structural (fliD, fliE, fliF) and regulatory genes (fliS, fleS, fleR). The function of orf99 is unknown, but it shares similarity with its positional homologue in P. aeruginosa, orf97. Regions with a similar or identical genetic organization are found in P. aeruginosa, Vibrio cholerae and Vibrio parahaemolyticus (Arora et al., 1997
; Klose & Mekalanos, 1998a
; Kim & McCarter, 2000
).
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AdnA is a transcription regulator
The predicted adnA-encoded protein sequence was compared to known proteins using the BLAST alignment tool (Altschul et al., 1990 ). AdnA showed strong homology to the NtrC/NifA family of transcriptional activators of
54-dependent promoters (Fig. 3
) (Kustu et al., 1991
; Morett & Segovia, 1993
). These activators respond to environmental stimuli by activating transcription of adaptive genes, often as the response regulator component of a two-component signal transduction system. The closest matches to AdnA are FleQ of P. aeruginosa (83% identity), FlrA of V. cholerae (52% identity) and FlaK of V. parahaemolyticus (52% identity), all three of which are activators involved in flagellar synthesis (Arora et al., 1997
; Klose & Mekalanos, 1998a
, b
; Stewart & McCarter, 1996
). The preliminary genomic sequence of Pseudomonas putida also contains an adnA homologue. Of particular interest, FleQ regulates adhesion to respiratory mucins as well as motility (Arora et al., 1997
). The similarity with AdnA is greatest between residues 142375 (66% identical in all four proteins), which encompass amino acids involved in ATP binding and hydrolysis, and transcription activation. The amino-terminal 141 residues are expected to regulate AdnA activity and are less well conserved with the other homologues except for FleQ. The carboxyl terminal 116 residues are moderately conserved and contain the helixturnhelix DNA binding domain. The AdnA transcription regulator presumably affects adhesion by altering expression of structural genes required for adhesion. The demonstrated role of AdnA in flagella synthesis suggests that, as in P. aeruginosa, flagella are critical for adhesion.
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Complementation of Pf0-5 with a cloned adnA gene
A broad-host-range plasmid, pPC100, was constructed in which adnA expression is under control of the activator XylS and the inducible XylS-dependent promoter Pm, both from the TOL plasmid from P. putida (Blatny et al., 1997 ; Mermod et al., 1986
). When this plasmid was introduced into E. coli DH5
, a high level of a 55 kDa protein was synthesized in the presence of the inducer 3-methylbenzoate, indicating that the expected ORF is being transcribed and translated in E. coli (Fig. 4
).
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ACKNOWLEDGEMENTS |
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Received 12 July 2000;
revised 18 October 2000;
accepted 6 November 2000.