The Pseudomonas fluorescens transcription activator AdnA is required for adhesion and motility

Paul Casaz1, Anne Happela,1, Joann Keithanb,1, Dorothy L. Readc,1, Steven R. Straind,1 and Stuart B. Levy1

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


   ABSTRACT
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ABSTRACT
INTRODUCTION
METHODS
RESULTS AND DISCUSSION
REFERENCES
 
The locations of two mutations that prevent adhesion of Pseudomonas fluorescens Pf0-1 to sand columns and seeds (adn, adhesion) were identified. Both lie in a single gene showing homology to the NtrC/NifA family of transcription activators. The predicted 55 kDa protein encoded by adnA is most closely related to activators involved in expression of flagellar proteins, consistent with the lack of flagella in adnA strains. Constitutive adnA expression restored motility and adhesion to an adnA strain, demonstrating that the observed phenotypes are due to lack of AdnA and not a consequence of other mutations or polar effects of mutations in adnA on other genes.

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.


   INTRODUCTION
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ABSTRACT
INTRODUCTION
METHODS
RESULTS AND DISCUSSION
REFERENCES
 
Fluorescent pseudomonads are common soil bacteria. They have been investigated for their potential use in bioremediation and for biocontrol of agricultural pathogens (de Lorenzo et al., 1993 ; Lajoie et al., 1993 ; Simons et al., 1996 ). However, performance of strains introduced for these purposes into soil in field trials has been inconsistent (van Veen et al., 1997 ). One likely source of this inconsistency is the failure of the introduced cells to become established and colonize the soil to a sufficient degree when faced with an established community of micro-organisms in the soil.

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 ; O’Toole & 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.


   METHODS
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ABSTRACT
INTRODUCTION
METHODS
RESULTS AND DISCUSSION
REFERENCES
 
Bacterial strains and media.
The bacterial strains and plasmids used in this study are listed in Table 1. Pf0-5 and Pf0-10 are Tn5 insertion mutants (DeFlaun et al., 1990 ) derived from the wild-type P. fluorescens strain Pf0-1, which was isolated from agricultural soils in Sherborn, MA, USA (Compeau et al., 1988 ). Pseudomonas strains were grown on nutrient agar, Luria–Bertani agar (LB) or minimal medium MMO (Stanier et al., 1966 ) at 30 °C. Escherichia coli strain DH5{alpha} was used for cosmid and plasmid constructions and grown at 37 °C. The following antibiotics were used for selection: tetracycline, 15 µg ml-1; kanamycin, 50 µg ml-1; ampicillin, 100 µg ml-1.


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Table 1. Strains and plasmids used

 
Cloning of P. fluorescens DNA flanking Tn5 insertion mutations.
A Tn5-specific probe was prepared by labelling a 1·55 kb BamHI–BglII fragment of pKan1 (Hächler et al., 1991 ) with [32P]CTP by extension of random hexamer primers using the Klenow fragment of E. coli DNA polymerase. Chromosomal DNA from P. fluorescens Pf0-5 and Pf0-10 was isolated by the method of Beji et al. (1987) . DNA from each mutant was cleaved with restriction endonucleases and Southern blot analysis was performed using the Tn5-specific probe (Sambrook et al., 1989 ). Tn5 contains a single BamHI site 3' to the kanamycin resistance gene and does not contain an EcoRI site. BamHI–EcoRI fragments were isolated from Pf0-5 and Pf0-10 and ligated into pUC18. Selection for kanamycin-resistant transformants yielded plasmids pPfA34 and pPfA32.

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 (9–13 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{alpha}.

DNA sequence analysis.
To sequence the adnA gene, a 6·2 kb BamHI–EcoRI 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 1–309 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{alpha} 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 O’Toole & 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.


   RESULTS AND DISCUSSION
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ABSTRACT
INTRODUCTION
METHODS
RESULTS AND DISCUSSION
REFERENCES
 
Co-localization of Tn5 insertions in adhesion mutants
The Tn5 insertion sites of the adhesion-defective mutants Pf0-5 and Pf0-10 were compared by Southern blot analysis of chromosomal DNA using a Tn5-specific probe (Fig. 1a). Digestion of Pf0-5 and Pf0-10 DNA with KpnI and ClaI yielded identically sized Tn5-containing fragments, while digestion of these DNAs with BamHI and EcoRI yielded fragments of distinct sizes. This hybridization pattern suggested that the Tn5 insertion sites in these two mutants were physically close.



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Fig. 1. Southern analyses of adnA. (a) Genomic DNA from Pf0-5 (odd-numbered lanes) and Pf0-10 (even-numbered lanes) was digested with BamHI (lanes 1, 2), Kpn I (lanes 3, 4), EcoRI (lanes 5, 6) and ClaI (lanes 7, 8) and probed with a Tn5-specific sequence. (b) Genomic DNA from Pf0-1 was digested and probed with DNA sequences adjacent to the Tn5 insertions from Pf0-5 (lanes 1–5) and Pf0-10 (lanes 6–10). Lanes 1–5 show digestions with KpnI, EcoRI, BamHI, ClaI and ClaI/KpnI double digest, respectively. The same enzymes were used in lanes 6–10.

 
Chromosomal DNA from the wild-type strain Pf0-1 was digested with a number of restriction enzymes and analysed by Southern analysis using probes generated from DNA flanking the Tn5 insertion sites in Pf0-10 and Pf0-5 (Fig. 1b). Identical bands were observed in all cases, clearly showing that the Tn5 insertions in Pf0-5 and Pf0-10 were located in the same region of the DNA sequence. The difference in size between Pf0-5 and Pf0-10 BamHI fragments (Fig. 1a) are due to the different insertion sites of Tn5 in the chromosome. The difference in size of EcoRI fragments hybridizing with the Tn5 probe may be due to sequence rearrangements that occurred following the Tn5 insertion into one of these strains.

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 BamHI–EcoRI 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 EcoRI–BamHI 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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Fig. 2. Organization of the adnA locus. ORFs were identified by BLAST searches. Asterisks mark the positions of Tn5 insertions in Pf0-5 and Pf0-10. A potential promoter () and transcription terminator () for adnA are marked. The predicted genes are labelled based on homology to flagella structural genes (‘fliD’, ‘fliE’ and ‘fliF’) or regulatory genes (‘fliS’, ‘fleS’, ‘fleR’). A protein homologous to the predicted orf99 is found in the same position in P. aeruginosa. The sequences of fliD and fliF homologues are partial.

 
The predicted adnA ORF encodes a 491 residue protein with a predicted molecular mass of 55·5 kDa. A potential ribosome-binding site is located 11 nt upstream of the initiation codon. There is a 172 nt intergenic region upstream of the adnA initiation codon, but no {sigma}54, {sigma}28 or {sigma}38 promoters were identified. A potential {sigma}70-dependent promoter was identified beginning 135 nt upstream of the adnA coding region (TTGACTgtgcacggttttttgacTTAACT: upper case letters, conserved nucleotides; lower case letters, less well conserved ‘spacer’ region). A potential stem–loop structure followed by a T-rich sequence overlaps the translation termination site, suggesting that this is also a transcription termination site. The Tn5 insertion sites in Pf0-5 and Pf0-10 map to amino acid residues 284 and 462, respectively.

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 {sigma}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 142–375 (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 helix–turn–helix 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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Fig. 3. Alignment of AdnA with homologues. Alignments were created with BLAST. Amino acid residues identical to AdnA are boxed. FleQ, P. aeruginosa; FlaK, V. parahaemolyticus; FlrA, V. cholerae; NtrC, Salmonella typhimurium. Conserved residues involved in Mg2+ binding (aa 11–12) and potential phosphorylation sites (aa 59) are underlined. The double underline indicates the predicted position of a helix–turn–helix motif based on alignment with S. typhimurium NtrC.

 
It is striking that in P. fluorescens, P. aeruginosa, V. cholerae and V. parahaemolyticus there appears to be a conserved regulatory system controlling polar flagella synthesis. The regulation of flagella synthesis in these species is distinct from the synthesis of peritrichous flagella in enteric bacteria in that {sigma}54 plays an important role in early stages of flagella synthesis, before the flagella-specific {sigma}28 is required (Helmann, 1991 ). In all four cases, there is an activator homologous to AdnA that does not have an adjacent sensor protein (Klose & Mekalanos, 1998a ; Stewart & McCarter, 1996 ; Arora et al., 1997 ). Signal transduction through {sigma}54-dependent activators occurs by one of three mechanisms (Morett & Segovia, 1993 ). In the case of NtrC, the sensor kinase NtrB phosphorylates the amino-terminal regulatory domain of NtrC in response to the nitrogen status of the cell. A second mechanism, illustrated by the regulation of the activator NifA by NifL in response to oxygen and fixed-nitrogen levels, requires an interaction between the proteins, but not phosphorylation of the activator. A third mechanism is illustrated by P. putida XylR. Here, binding of a small aromatic ligand (toluene) directly to the amino terminus of the activator regulates XylR activity. AdnA, FleQ, FlrA and FlaK all contain two conserved acidic residues common to phosphorylation-regulated activators (Fig. 3), but a third highly conserved aspartate phosphorylation site is changed to threonine, serine or asparagine (Stock et al., 1990 ). Although the conserved aspartate residue is missing, serine and threonine can be phosphorylated, leaving open the possibility that an unrecoginzed kinase exists for AdnA. The regions immediately downstream of fleQ, flaK and flrA contain {sigma}54-dependent, two-component systems named fleSR, flaLM and flrBC (Ritchings et al., 1995 ; Klose & Mekalanos, 1998a ; Kim & McCarter, 2000 ). Transcription of fleSR and flrBC is activated by FleQ and FlrA, respectively, so it seems likely that the P. fluorescens fleSR homologues will be regulated by AdnA.

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{alpha}, 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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Fig. 4. Overexpression of AdnA in E. coli. (a) Diagram of a portion of the vector pPC100. The xylS promoter is constitutive, Pm is induced by XylS and 3-methylbenzoate (3-MB). (b) AdnA expression from plasmid pPC100 was induced by the addition of 2 mM 3-MB for 16 h. Lanes 1, 2: whole cell lysate of DH5{alpha} carrying pPC100 (Pm–adnA). Lanes 3, 4: whole cell lysate of DH5{alpha} carrying pPC101 (Pm–{Delta}adnA). Odd-numbered lanes contained no 3-MB. Even-numbered lanes contained 3-MB. Lane 5, molecular mass markers. The arrow indicates the overexpressed 55 kDa protein.

 
Plasmids pPC100 or pPC101 (a control plasmid with a deletion in adnA), were transferred to Pf0-1, Pf0-5 or Pf0-10, and motility and adhesion assays were performed. Strains carrying pPC100 re-establish exponential growth more slowly than strains carrying pPC101 when an overnight culture is used as an inoculum. Therefore, all complementation assays were initiated with actively growing cultures adjusted to equal cell density just prior to inoculation. The defective motility and adhesion of Pf0-5 and Pf0-10 were complemented by pPC100 but not by pPC101 (Fig. 5). The constitutive low-level transcription from Pm was sufficient for complementation in these experiments. These results demonstrate that the phenotypic effects of the Tn5 insertion in Pf0-5 and Pf0-10 are due to a lack of AdnA and not mutations elsewhere in the chromosome or polar effects of the insertion on expression of other genes.



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Fig. 5. Complementation of adhesion and motility defects by cloned adnA. (a) Adhesion of Pf0-1, Pf0-5 and Pf0-10 to borosilicate tubes, without plasmid, or carrying pPC100 (‘adnA’) or pPC101 (‘{Delta}adnA’). (b) Motility assay of Pf0-1, Pf0-5 or Pf0-10 alone or carrying plasmids pPC100 or pPC101. The presence of adnA restored adhesion and motility to Pf0-5 and Pf0-10. Pf0-1 is the wild-type control.

 
As is the case with other bacteria, it is likely that synthesis of flagella proteins in P. fluorescens will proceed through a cascade mechanism in which one activator regulates expression or activity of more activators (Arora et al., 1997 ; Helmann, 1991 ; Klose & Mekalanos, 1998a ) causing the flagella to be assembled in a defined order. Future experiments directed at identifying genes regulated by AdnA and the proteins that control adnA expression will provide clues to the position of AdnA in this hierarchy.


   ACKNOWLEDGEMENTS
 
This work was supported by grants from the Department of Energy (DE-FG02-97ER62493) and the National Science Foundation (DEB 9120897) and an NRSA F32 fellowship GM171G5 (S.S)


   REFERENCES
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ABSTRACT
INTRODUCTION
METHODS
RESULTS AND DISCUSSION
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Received 12 July 2000; revised 18 October 2000; accepted 6 November 2000.