Bacterial strains, plasmids, and growth conditions.

The bacterial strains and plasmids used in this study are listed in Table1.E. coli strains were grown on Luria-Bertani (LB) Miller broth and LB Miller agar at 37°C,Aeromonas strains were grown in either tryptic soy broth (TSB) or tryptic soy agar (TSA) at 30°C, andVibrio cholerae strains were grown in either LB or LB agar with 2 mM glutamine at 37°C. When required, ampicillin (50 μg/ml), kanamycin (50 μg/ml), chloramphenicol (25 μg/ml), rifampin (100 μg/ml), spectinomycin (50 μg/ml), and tetracycline (20 μg/ml) were added to the different media.

Motility assays (swarming and swimming).

Freshly grown bacterial colonies were transferred with a sterile toothpick into the center of swarm agar (1% tryptone, 0.5% NaCl, 0.6% agar) or swim agar (1% tryptone, 0.5% NaCl, 0.25% agar). Agar plates consisting of LB with 0.3% agar and 2 mM glutamine were used to measureVibrio cholerae motility. The plates were incubated face up for 16 to 24 h at 30°C, and motility was assessed by examining the migration of bacteria through the agar from the center towards the periphery of the plate. Moreover, swimming motility was assessed by light microscopy observations with liquid media.

Transmission electron microscopy (TEM).

Bacterial suspensions were placed on Formvar-coated grids and negative stained with a 2% solution of uranyl acetate, pH 4.1. Preparations were observed with a Hitachi 600 transmission electron microscope.

MiniTn5Km-1 mutagenesis.

Conjugal transfer of transposition element miniTn5Km-1 fromE. coli S17-1λpirKm-1 (16) toA. hydrophila AH-405 (AH-3, rifampin resistant) was carried out in a conjugal drop incubated for 6 h at 30°C with the ratio 1:5:1, corresponding to S17-1λpirKm-1, AH-405, and HB101 pRK2073 (helper plasmid), respectively. Serial dilutions of the mating mix were plated on TSA supplemented with rifampin and kanamycin in order to select mutants.

DNA techniques.

DNA manipulations were carried out essentially as previously described (56). DNA restriction endonucleases, T4 DNA ligase,E. coli DNA polymerase Klenow fragment, and alkaline phosphatase were used as recommended by the suppliers. PCR was performed usingTaq DNA polymerase (Invitrogen) in a gene amplifier PCR system 2400 thermal cycler (Perkin-Elmer). Colony hybridizations were carried out by colony transfer onto positive nylon membranes (Roche) and then lysed according to the manufacturer's instructions. Probe labeling with digoxigenin, hybridization, and detection (Amersham) were carried out as recommended by the suppliers.

Cloning of DNA flanking miniTn5Km-1 insertions.

Chromosomal DNA of miniTn5Km-1 mutants was digested with EcoRI, PstI, and EcoRV, purified, ligated into the vector pBCSK (Stratagene), and introduced intoE. coli XL1-Blue. Recombinant plasmids containing the transposon with flanking insertions were selected in LB plates supplemented with kanamycin and chloramphenicol. The miniTn5Km-1 flanking sequences were obtained by using primers specific to the I and O ends of miniTn5Km-1 (5′-AGATCTGATCAAGAGACAG-3′ and 5′-ACTTGTGTATAAGAGTCAG-3′, respectively), as well as M13for and T3 primers from the plasmid vector used.

Nucleotide sequencing and computer sequence analysis.

Plasmid DNA for sequencing was isolated with a QIAGEN plasmid purification kit (QIAGEN, Inc., Ltd.) as recommended by the suppliers. Double-stranded DNA sequencing was performed by using the Sanger dideoxy-chain termination method (57) with an ABI Prism dye terminator cycle sequencing kit (Perkin-Elmer). Custom-designed primers used for DNA sequencing were purchased from Amersham Biosciences.

The DNA sequence was translated in all six frames, and all open reading frames (ORFs) greater than 100 bp were inspected. Deduced amino acid sequences were inspected in the GenBank, EMBL, and SwissProt databases by using the BLASTX, BLASTP, or PSI-BLAST network service at the National Center for Biotechnology Information (NCBI) (3). A protein family profile was performed using the Protein Family Database Pfam at the Sanger Center (6). Determination of possible terminator sequences was done by using the Terminator program from the Genetics Computer Group package (Madison, Wisconsin). Other online sequence analysis services were also used.

RT-PCR.

Total RNA was isolated fromA. hydrophila AH-3 grown in liquid medium (TSB) and plates by Trizol reagent (Invitrogen). To ensure that RNA was devoid of contaminating DNA, the preparation was treated with RNase-free DNase I, amplification grade (Invitrogen). The isolated RNA was used as a template in reverse transcriptase (RT) PCR, utilizing a Thermoscript RT-PCR system (Invitrogen) according to the manufacturer's instructions. PCR without reverse transcriptase was also performed to confirm the absence of contaminating DNA in the RNA samples. RT-PCR amplifications were performed at least twice with total RNA preparations obtained from minimum of two independent extractions. The RT-PCR and PCR products were analyzed by gel electrophoresis.

Mutant construction.

To obtain single defined insertion mutants with mutations in genesflaA,maf-1,pomB,motX, andflrC, we used a method based on suicide plasmid pFS100 (55). Briefly, an internal fragment of the selected gene was amplified by PCR, ligated into pGEM-Teasy (Promega), and transformed intoE. coli XL1-Blue. The DNA insert was recovered by EcoRI restriction digestion and was ligated into EcoRI-digested and phosphatase-treated pFS100. The ligation product was transformed intoE. coli MC1061 (λpir) and selected for kanamycin resistance (Km^r). Triparental mating with the mobilizing strain HB101/pRK2073 was used to transfer the recombinant plasmid intoA. hydrophila AH-405 rifampin-resistant (Rif^r) strains to obtain defined insertion mutants, selecting for Rif^r and Km^r.

To obtain strains with mutations inflaH,fliM,flhA, andfliA, the genes were amplified by PCR, ligated into vector pGEMTeasy (Promega), and transformed intoE. coli XL1-Blue. The Tn5-derived kanamycin resistance cartridge (nptll) from pUC4-KIXX was inserted into each of these genes. The cartridge contains an outward-reading promoter that ensures the expression of downstream genes when inserted in the correct orientation (9); however, such an insertion will alter the regulation of such genes. The SmaI-digested cassette was inserted into a restriction site internal to each gene, and the presence of a single HindIII site in the SmaI-digested cassette allowed its orientation to be determined. Constructs containing the mutated genes were ligated into suicide vector pDM4 (44), electroporated intoE. coli MC1061 (λpir), and plated on chloramphenicol plates at 30°C. Plasmids with mutated genes were transferred intoA. hydrophila AH-405 rifampin-resistant (Rif^r) strains by triparental mating usingE. coli MC1061 (λpir) containing the insertion constructs and the mobilizing strain HB101/pRK2073. Transconjugants were selected on plates containing chloramphenicol, kanamycin, and rifampin. PCR analysis confirmed that the vector had integrated correctly into the chromosomal DNA. To complete the allelic exchange, the integrated suicide plasmid was forced to recombine out of the chromosome by addition of 5% sucrose to the agar plates. The pDM4 vector containssacB, which produces an enzyme that converts sucrose into a product that is toxic to gram-negative bacteria. Transconjugants surviving on plates with 5% sucrose (Rif^r, Km^r, and chloramphenicol sensitive [Cm^s]) were chosen and confirmed by PCR.

The chromosomal in-frameflaB deletion mutantA. hydrophila AH-4425 was constructed by allelic exchange as described by Milton et al. (44). Briefly, DNA regions flanking theflaB gene were amplified using the primer pairs A (5′-CGCGGATCCCCTGGTCAAGAGATGGAA-3′) and B (5′-CCCATCCACTAAACT TAAACAGGCGTTCAGTGATGAAGT-3′) and C (5′-TGTTTAAGTTTAGTGGATGGGTCCCTGCTGGGTTAACAG-3′) and D (CGCGGATCCCTGGGCATTGGTCTT TTT-3′) in two sets of asymmetric PCRs to amplify DNA fragments of 592 (AB) and 646 (CD) bp, respectively. DNA fragment AB contains nucleotide 1194, outsideflaB, to nucleotide 1786, corresponding to the 14th codon offlaB. DNA fragment CD contains nucleotide 2636, corresponding to the first base in the 328 codon offlaB, to nucleotide 3282, inside theflaH gene. DNA fragments AB and CD were annealed at their overlapping regions (underlined letters in primers B and C) and amplified as a single fragment by using primers A and D. The fusion product was purified, BamHI digested (the BamHI site is double underlined in primers A and D), ligated into BglII-digested and phosphatase-treated pDM4 vector (44), electroporated intoE. coli MC1061 (λpir), and plated on chloramphenicol plates at 30°C to obtain plasmid pDM-FLAB. Introduction of plasmid pDM-FLAB into theA. hydrophila AH-405 Rif^r strain was performed as previously described. Transconjugants were selected on plates containing chloramphenicol and rifampin. PCR analysis confirmed that the vector had integrated correctly into the chromosomal DNA. After sucrose treatment, transformants (Rif^r and Cm^s) were chosen and confirmed by PCR.

Plasmid construction.

Plasmid pACYC-MAF, containing the completemaf-1 gene, and plasmid pACYC-FLR1, containingflrB andflrC genes fromA. hydrophila AH-3, were obtained by PCR amplification of genomic DNA using oligonucleotides 5′-GGGGTGGGATTAGGATACC-3′ and 5′-GGGATACATGAGCCTACGG-3′ to generate a band of 1,829 bp and 5′-TGGAGCACAGTGCCAGTTA-3′ and 5′-TCCCCCTGCTCTACCAATA-3′ to generate a band of 2,857 bp, respectively. The amplified bands were ligated into pGEM-Teasy (Promega) and transformed intoE. coli XL1-Blue. The DNA insert was recovered by EcoRI restriction digestion and ligated into EcoRI phosphatase-treated pACYC184 vector (56), introduced intoE. coli DH5α to generate plasmid pACYC-MAF.

Whole-cell protein preparation and immunoblotting.

Whole-cell proteins were obtained fromAeromonas strains grown at 30°C. Equivalent numbers of cells were harvested by centrifugation, and the cell pellet was suspended in 50 to 200 μl of sodium dodecyl sulfate-polyacrylamide gel electrophoresis loading buffer and boiled for 5 min. Following sodium dodecyl sulfate-polyacrylamide gel electrophoresis and transfer to nitrocellulose membranes, the membranes were blocked with bovine serum albumin (3 mg/ml) and probed with either polyclonal rabbit anti-polar or anti-lateral flagellin antibody (1:1,000), previously obtained (23). The unbound antibody was removed by three washes in phosphate-buffered saline (PBS), and a goat anti-rabbit horseradish peroxidase-conjugated secondary antibody (1:1,000) was added. The unbound secondary antibody was removed by three washes in PBS. The bound conjugate was then detected by the addition of 2 ml 0.5% 4-chloro-1-naphthol (Sigma) prepared in methanol and diluted in 8 ml PBS containing 50 μl of 30% H₂O₂.

Adherence assay to HEp-2 cells.

Tissue culture was maintained as described by Thornley et al. (63). The adherence assay was conducted as a slight modification of that described by Carrello et al. (12). Bacteria were grown statically in brain heart infusion broth at 37°C, harvested by gentle centrifugation (1,600 ×g for 5 min), and resuspended in PBS (pH 7.2) at approximately 10⁷ CFU/ml (A₆₀₀ of ∼0.07). The monolayer was infected with 1 ml of the bacterial suspension for 90 min at 37°C in 5% CO₂. Following infection, the nonadherent bacteria were removed from the monolayer by three washes with PBS. The remaining adherent bacteria and the monolayers were then fixed in 100% methanol for 5 min. Methanol was removed by washing monolayers and bacteria with PBS, and the HEp-2 cells with the adherent bacteria were stained for 45 min in 10% (vol/vol) Giemsa stain (BDH) prepared in Giemsa buffer. The coverslips were air dried, mounted, and viewed by oil immersion under a light microscope. Twenty HEp-2 cells/coverslips were randomly chosen, and the number of bacteria adhering to HEp-2 cells was recorded. Assays were carried out in duplicate or triplicate.

Biofilm formation.

Quantitative biofilm formation was performed in a microtiter plate as described previously (51), with minor modifications. Briefly, bacteria were grown on TSA and several colonies were gently resuspended in TSB (with or without the appropriate antibiotic); 100-μl aliquots were place in a microtiter plate (polystyrene) and incubated for 48 h at 30°C without shaking. After the bacterial cultures were poured out, the plate was washed extensively with water, fixed with 2.5% glutaraldehyde, washed once with water, and stained with 0.4% crystal violet solution. After solubilization of the crystal violet with ethanol-acetone (80/20, vol/vol), the absorbance was determined at 570 nm.

Statistical analysis.

The differences in adherence to HEp-2 cells or biofilm formation in vitro between the wild-type and mutant strains were analyzed by thet test using Microsoft Excel software.

Nucleotide sequence accession numbers.

The nucleotide sequences reported in this paper have been submitted to the GenBank/EMBL database under accession numbersDQ119104,DQ124696,DQ124697, andDQ124698.

Isolation and characterization ofA. hydrophila AH-3 mutants with reduced swimming phenotype in plates.

In order to find the chromosomal regions ofA. hydrophila AH-3 involved in polar flagellum biogenesis, we performed miniTn5Km-1 mutagenesis usingA. hydrophila AH-405 (AH-3, rifampin resistant) as the recipient strain, and transconjugants were screened for reduced swimming motility in swim agar. From 7,000 transconjugants analyzed by light microscopy, 23 transposon insertion mutants exhibited a reproducible reduction in swim agar and inability to move in liquid media. Transposon insertion mutants unable to swim were analyzed by transmission electron microscopy after growth in liquid media and on plates; these were subsequently divided in two groups on the basis of their ability to produce polar flagella. The first group was unable to produce polar flagella but was able to produce lateral flagella, and the second group was unable to swim but showed both flagellum types.

As no EcoRV restriction sites were present in the transposon, all altered-motility mutants were analyzed for the presence of the transposon by Southern hybridization of EcoRV chromosomal DNA digestions. A single band was detected in every mutant, indicating that each mutant had a single copy of the mini-transposon in its genome.

Sequence analysis of miniTn5Km-1 interrupted genes.

The DNA flanking the transposon was isolated and cloned into pBCSK (see Materials and Methods). From 22 mutants of the first transposon insertion mutant group, 18 ORFs that shared homology with differentA. hydrophila AH-3 polar flagellum structural genes in theflg locus (2) were revealed: two mutants showed the transposon inserted inflgN, two inflgM, four inflgB, three inflgE, three inflgK, and four inflgL (2). However, the encoded product of sequences flanking the insertion of four more mutations had not previously been described forAeromonas. The predicted amino acid sequences from mutant AH4422 encoded a product similar to an uncharacterized protein ofShewanella oneidensis (GenBank no.SO3259),Clostridium acetobutylicum (CAC2196), andCampylobacter jejuni (Cj1337), a group of proteins thought to be involved in phase-variable flagellum-mediated motility and flagellin modification (33). Of the other ORF products, AH-4440 shared homology with FlhA ofShewanella oneidensis andV. parahaemolyticus, a component of the polar flagellar export machinery (60). The only representative mutant of the second mutant group, AH-4460, revealed an ORF whose predicted amino acid sequence shared homology with the sodium-type flagellar motor component MotX ofVibrio alginolyticus.

Organization ofA. hydrophila AH-3 polar flagellum region 2.

A cosmid genomic library ofA. hydrophila AH-3 (41) was screened by colony blotting using a DNA probe to themaf-like transposon flanking sequences from mutant AH-4422. Several positive recombinant clones were identified, of which clone pLA-FLA was chosen because it was able to complement mutant AH-4422.

Nucleotide sequencing of pLA-FLA revealed six complete ORFs (Fig.1) transcribed in the same direction, five of which are related toAeromonas salmonicida andA. caviae polar flagella (53,65). Flanking the 3′ end offla polar flagella loci, 70 bp downstream of ORF6, and transcribed in the opposite direction was the stop codon of a incomplete ORF whose amino acid sequence exhibited homology to the OrfB transposase ofPseudomonas putida, as well as transposases of other bacteria. ORF1 (flaA) was separated from ORF2 (flaB) by 585 bp, and ORF5 (flaJ) was separated from ORF6 (maf-1) by 1,060 bp. The other ORFs were located one behind the other, with intergenic regions less than 80 bp. Sequences defining putative ribosome binding sites were found upstream of each of the ORF's start codons. Data summarizing the locations of the six complete ORFs are shown in Table2. Sequence analysis in silico showed three possible transcriptional terminator rho-independent sequences downstream of ORF1 (flaA), ORF5 (flaJ), and ORF6 (maf-1) and putative σ²⁸ promoter sequences upstream of ORF1 (flaA), ORF2 (flaB), and ORF6 (maf-1), as well as putative σ⁵⁴ promoter sequences upstream of ORF2 (flaB) (Fig.1). RT-PCR using specific primers and total RNA fromA. hydrophila AH-3 grown in liquid (TSB) and solid (TSA) media demonstrated amplifications between ORFs 2 and 5 (flaB toflaJ) under both conditions. However, no amplifications were obtained with oligonucleotide pairs from ORFs 1 (flaA) to 2 (flaB) and ORFs 5 (flaJ) to 6 (maf-1), confirming thatflaA andmaf-1 are transcribed independently (Fig.1).

Table2 summarizes the characteristics of the individual proteins and predicted functions analyzed using the BLASTP program (3) of the NCBI database. ORFs 1 to 5 (flaA toflaJ) had the same organization and were similar to those previously reported for the polar flagellins ofA. salmonicida andA. caviae (53,65). However, downstream of ORF5 (flaJ) was ORF6, whose deduced amino acid sequence contained the DUF115 protein family domain and exhibited homology to the motility accessory factor Maf-1 ofCampylobacter jejuni (26% identity), thought to be involved in a slipped-strand mispairing mechanism of phase variation (33).

Organization ofA. hydrophila AH-3 polar flagellum region 3.

The cosmid library ofA. hydrophila AH-3 (48) was screened by colony blotting using DNA probes to theA. hydrophila AH-3flhA-like gene. Several positive recombinant clones were identified, of which pLA-FLIP1 and pLA-FLIP2 were selected, as they were able to complement the AH-4440 mutant.

Nucleotide sequencing of the overlapping cosmids pLA-FLIP1 (ORFs 1 to 20) and pLA-FLIP2 (ORFs 13 to 29) revealed 29,799 bp which contained 29 ORFs transcribed in the same direction (Fig.1), most of which encoded homologues ofproteins involved polar flagellum biosynthesis and chemotaxis in different bacteria, such asPhotobacterium profundum,Shewanella oneidensis, andV. parahaemolyticus (41,60). ORFs 1 to 23 (fliE tocheB) and ORFs 26 to 29 had the same organization as region 2 of theV. parahaemolyticus polar flagellum system (Fig.2). However, unlikeV. parahaemolyticus,A. hydrophila lacked theflaF toflaM genes in this chromosomal region but contained thepomAB genes (ORFs 24 to 25) which are located in region 3 of theV. parahaemolyticus polar flagellum system (Fig.2). Upstream ofA. hydrophila ORF1 (fliE) was a truncated ORF transcribed in the opposite direction, with homology to different bacterial regulatory proteins related to the AraC family, and downstream of ORF29 was another truncated ORF transcribed in the same direction, which shared homology with the CcmA ATP-binding subunit of a putative ABC-type heme exporter. Sequences defining putative ribosome binding sites were found upstream of each ORF's start codons. Most ORFs were overlapping or with intergenic regions less than 80 bp. Data summarizing the locations of the 29 complete ORFs are shown in Table3. Sequence analysis in silico showed possible transcriptional terminator rho-independent sequences downstream of ORF15 (flhB) and ORF29. Putative σ⁵⁴ promoter sequences were found upstream of ORF1 (fliE), ORF16 (flhA), and ORF24 (pomA), and a putative promoter sequence was found upstream of ORF26 (Fig. 1). RT-PCRs using specific primers showed amplifications between ORFs 1 and 15 (fliE toflhB), ORFs 16 and 27 (flhA tocheB), ORFs 24 and 25 (pomAB), and ORFs 26 to 29. However, no amplifications were obtained with oligonucleotide pairs from ORFs 15 (flhB) to 16 (flhA), ORFs 23 (cheB) to 24 (pomA), and ORFs 25 (pomB) to 26. The RT-PCR results showed thatfli andflh A. hydrophila polar genes are distributed in four clusters, although no rho-independent terminator sequences were found downstream of ORF23 (cheB) and ORF25 (pomB).

Table3 shows the characteristics of the individual proteins and their protein homologues analyzed using the BLASTP program (3) of the NCBI database. ORF1, ORF2, ORF7, and ORF8 encoded proteins that exhibited more than 40% identity to different polar flagellum basal body and hook proteins FliE (57% identity) ofVibrio vulnificus, FliF (50% identity) ofS. oneidensis, FliK (43% identity) ofV. parahaemolyticus, and FliL (55% identity) ofP. profundum, respectively. ORF3, ORF9, and ORF10 predicted amino acid sequences exhibited high homology (>70% identity) to the polar flagellum motor switch proteins FliG, FliM, and FliN of different bacteria, respectively. ORFs 4 to 6 and ORFs 11 to 16 exhibited homologies to different export and assembly polar flagellum proteins, with ORF4 and ORF6 having 38% identity to the ATP synthase-negative regulator FliH ofPhotobacterium profundum and 39% identity to a potential nonspecific chaperone FliJ ofPseudomonas syringae, respectively. ORF5, which is homologous to FliI, a polar flagellum ATP synthase, and ORFs 11 to 16 (FliOPQR and FlhBA homologues) displayed homologies greater than 43% identity toVibrio spp.,S. oneidensis, andP. profundum export and assembly proteins. ORFs 17 and 18 matched with a pair of proteins that could be involved in the control of site selection of flagellum insertion as well as flagellum number. ORF17 (FlhF homologue) showed homology to FlhF ofV. vulnificus andV. parahaemolyticus (50% identity), a GTP-binding signal recognition particle pathway protein required for flagellar synthesis inBacillus subtilis (38),Campylobacter jejuni (28), andHelicobacter pylori (47) and required for polar flagellar placement inP. putida (50). Furthermore, it has recently been demonstrated withV. cholerae that FlhF can regulate class III flagellar transcription (46). ORF18 (FlhG homologue) encoded a protein with a CbiA domain and is a MinD-related protein involved in septum site determination inBacillus subtilis (11,38) as well as in regulation of flagellum number inP. aeruginosa (14). The predicted amino acid sequence of ORF19 (FliA homologue) exhibited 66% identity to the RNA polymerase σ²⁷ factor ofS. oneidensis and 62% identity to the RNA polymerase σ²⁸ factor ofV. vulnificus. ORFs 20 to 23 and ORF28 shared 58 to 91% identity to different chemotaxis proteins (7,10,37). ORFs 20 to 23 and ORF28 matched with CheY, CheZ, CheA, CheB, and CheW, respectively (Table 3). Proteins encoded by ORF24 and ORF25 showed homology (50% and 30% identity, respectively) to the cytoplasmic membrane proteins MotA and MotB (ofPseudomonas spp.), respectively, which constitute the stator of the flagellum motor. Together, these proteins are involved in the formation of a proton or sodium-conducting channel to generate rotational motion in the proton-type or sodium-type flagellum motor (31,61). ORF25 (MotB) contained an OmpA domain at the C-terminal sequence, which is probably involved in peptidoglycan interaction (17). Although ORF26, ORF27, and ORF29 appear not to be involved in polar flagellum formation, their locations, downstream of chemotaxis genes, are conserved in different bacteria that possess polar flagella. ORF26 encodes a putative protein with a CbiA domain that exhibits high homology to Soj-like proteins as well as other ATPase proteins involved in chromosome partitioning (36). The predicted protein for ORF27 had a CheW-like domain which is found in proteins involved in the two-component signaling systems regulating bacterial chemotaxis (7) and matched to VP2226 ofV. parahaemolyticus (Table3). ORF29 showed sequence homology (41 to 50% identity) (Table3) to hypothetical proteins located at the end of polar flagellum regions of different bacteria (41).

Sequence analysis ofA. hydrophila AH-3 polar flagellum region 4.

TheA. hydrophila AH-3 genomic library (48) was screened by colony blotting using a DNA probe to themotX-like gene ofA. hydrophila AH-3, leading to the identification of clone pLA-MOTX, which was able to complement the AH-4460 mutant. By sequence analysis of pLA-MOTX, we found a complete ORF of 933 bp, encoding a protein of 311 amino acids and a predicted molecular weight of 32.8 kDa, that was homologous to MotX ofV. alginolyticus (54% identity), involved in the sodium-type motor formation inV. parahaemolyticus (35) (Table2). A putative Shine-Dalgarno sequence is positioned 7 bp upstream of the ATG start codon, and there isalso a putative σ²⁸ promoter sequence (CTAAG-N15-GCCGATAA) 26 bp upstream of the start codon. The nucleotide sequence GACCCACAGATCTGCATCTGTGGGTC downstream of themotX-like TGA stop codon could provide a termination stem-loop structure (Fig.1). Moreover, 86 nucleotides upstream of themotX-like start codon is found a putative terminator sequence, AAGGGGAGCTTCGGCTCCCCTT, and the stop codon (125 bp upstream) of an incomplete ORF whose deduced amino acid sequence is similar to that of the malate dehydrogenase ofV. cholerae. Downstream ofmotX, transcribed in the opposite direction, is an incomplete ORF which showed 55% identity to aYersinia pestis flavohemoprotein which plays a central role in the inducible response to nitrosative stress.

Cloning and sequence analysis ofA. hydrophila AH-3 polar flagellum regulatory region 5.

TheA. hydrophila AH-3 genomic library (48) was transferred by mating into the rifampin-resistantVibrio cholerae flrC in-frame deletion mutant strain KKV98 (35). Transconjugants were selected for rifampin and tetracycline resistance and inoculated onto 0.3% agar LB plates supplemented with 2 mM glutamine and tetracycline. The complemented colonies which spread on the plates were isolated, and the plasmid pLA-FLR was recovered. Sequence analysis of pLA-FLR revealed three complete ORFs transcribed in the same direction, which encoded proteins related to the polar flagellum master regulatory proteins of differentVibrio species (35,41,52,60). ORF2 (flrB) began 106 bp downstream of the ORF1 (flrA) stop codon, and ORF2 and ORF3 (flrC) are overlapping sequences. Moreover, 220 nucleotides upstream of the ORF1 (flrA) start codon is found a putative rho-independent terminator sequence and the stop codon (260 bp upstream) of a incomplete ORF whose encoded protein has a topoisomerase DNA binding C4 zinc finger domain.

Nucleotide analysis in silico shows putative Shine-Dalgarno sequences upstream of each ORF, two putative σ⁵⁴ promoter sequences upstream of ORF1 and ORF2 (70 and 35 bp, respectively), and two rho-independent terminator sequences downstream of ORF1 and ORF3 (3 and 31 bp, respectively). RT-PCR using specific primers showed amplifications between ORFs 2 and 3 (flrB toflrC), and no amplifications were obtained with oligonucleotide pairs from ORFs 1 (flrA) and 2 (flrB) (Fig.1). A search of the protein databases revealed that ORF1 (flrA) encoded a cytoplasmic protein of 475 amino acids which displayed more than 50% identity to the polar flagellum proteins FlrA ofV. cholerae and FlaK ofV. parahaemolyticus. ORF2 (flrB) encoded a transmembrane protein of 344 amino acids and ORF3 (flrC) a cytoplasmic protein of 447 amino acids, which are homologous to two-component flagellar regulatory proteins. ORF2 (flrB) encoded a protein which exhibited 38 to 40% identity to different two-component sensor kinases, such as FlrB ofV. cholerae and FlaK ofV. parahaemolyticus, and ORF3 (flrC) encoded a protein which shared high homology with the two-component response regulator proteins FlaM ofV. parahaemolyticus (60% identity) and FlrC ofV. fischeri (58% identity) (Table2).

Construction of polar flagellum mutants and complementation studies.

We constructed several mutants ofA. hydrophila AH-3 to study theAeromonas polar flagellum regions and to determine if they had any role in lateral flagellum formation. We obtained an in-frameflaB deletion mutant (AH-4425) and the following defined insertion mutants:flaA,flaH, andmaf-1, with mutations in polar flagellum region 2;fliM,flhA,fliA, andpomB, with mutations in polar flagellum region 3;motX, with a mutation in polar flagellum region 4; andflrC, with a mutation in polar flagellum region 5. Amplified internal fragments offlaA,maf-1,pomB,motX, andflrC were ligated into pFS100 to construct plasmids pFS-FLAA (flaA), pFS-MAFP (maf-1), pFS-POMB (pomB), pFS-MOTX (motX), and pFS-FLRC (flrC), respectively. The Km cassette was inserted intoflaH,fliM,flhA, andfliA and constructs ligated into pDM4, resulting in pDM-FLAH (flaH), pDM-FLIM (fliM), pDM-FLHA (flhA), and pDM-FLIA (fliA), respectively. Plasmids were independently introduced intoA. hydrophila AH-405 by mating. Using this method, we obtained the followingA. hydrophila mutant strains, with mutations in the genes in parentheses: AH-4426 (flaA), AH-4423 (flaH), AH-4424 (maf-1), AH-4441 (fliM), AH-4442 (flhA), AH-4443 (fliA), AH-4444 (pomB), AH-4461 (motX), AH-4462 (flrC), and AH-4427 (flaA flaB double mutant). The correct construction of all mutants was verified by Southern blot hybridization (data not shown). Swarming motility ofA. hydrophila mutant strains was then assessed on swarm agar, swimming motility was assessed by light microscopy after growth in liquid media, and the presence/absence of both flagellum types (lateral and polar) was analyzed by TEM after growth in solid and liquid media. The motility phenotypes of the mutant strains are shown in Table4. Mutations of eitherflaA (AH-4426) orflaB (AH-4425), located inA. hydrophila polar flagellum region 2, did not abolish motility and produced only a minor reduction in the swarm size on semisolid plates compared to the wild type, such as withflaA orflaB A. caviae mutant strains (53). Because single insertions inflaA orflaB donot abolish motility, we introduced the suicide plasmid pFS-FLAA into theA. hydrophila mutant AH-4425 in order to construct a double mutant strain (AH-4427), in which both genes were mutated in tandem. Motility assays showed that the double mutation abolished swimming motility and also caused a large decrease in swarming motility. TEM analysis and specific immunoblotting of the single mutant and double mutant whole cells, after growth in solid or liquid media, using lateral flagellin- or polar flagellin-specific antibodies, showed thatflaA andflaB single mutant strains possess both flagellum types; however, the double mutant strain is unable to form polar flagella but is able to form lateral flagella (Fig.3). This suggests that either of the flagellin proteins is able to substitute for the other to a certain extent, causing only a slight loss of motility, and that these flagellins are not involved in lateral flagellum formation. TheflaH (AH-4423),maf-1 (AH-4424),fliM (AH-4441),flhA (AH-4442),fliA (AH-4443), andflrC (AH-4462) defined insertion mutants were unable to swim (examined by microscopy) and exhibited a 60% decreased swarming motility, and under TEM, all of these mutant strains showed lateral flagella but no polar flagella, as seen previously for the miniTn5 mutants AH-4422 and AH-4440 (Fig.4). In contrast, thepomB (AH-4444) defined insertion mutant did not show any differences in swimming or swarming motility from the wild-type strain. Finally, themotX defined insertion mutant (AH-4461) was unable to swim and had a 60% decrease in swarming motility, and TEM assays showed both flagellum types.

Complementation studies were undertaken to determine if wild-type motility could be restored to the mutants by providing polar flagellum genes intrans. The plasmid pLA-FLA, containingA. hydrophila polar flagellum region 2, was introduced into the mutant strains AH-4423, AH-4424, AH-4425, AH-4426, and AH-4427. Swimming motility and polar flagellum production were restored in AH-4423, AH-4424, and AH-4427 (Fig.3), whereas in AH-4425 and AH-4426, full swarming motility was recovered to the level of the wild type (data not shown). Furthermore, plasmid pACYC-MAF (see Materials and Methods) was able to fully complement mutant AH-4424 (Fig.4) and the mini-Tn5 mutant AH-4422. Plasmids pLA-FLIP1 and pLA-FLIP2, containing theA. hydrophila fliE tocheY andfliQ toorf29 genes, respectively, of polar flagellum region 3, were introduced into AH-4441, AH-4442, and AH-4443 mutant strains. TheflhA andfliA mutants (AH-4442 and AH-4443, respectively) were able to swim and to produce polar flagella when pLA-FLIP1 or pLA-FLIP2 was introduced independently. In contrast, AH-4441 (fliM) recovered the wild-type swimming phenotype and the ability to produce polar flagella only when plasmid pLA-FLIP1 was introduced (data not shown). When plasmid pLA-MOTX, containing theA. hydrophila motX gene of polar flagellum region 4, was introduced into AH-4461, the mutant strain was fully able to swim (data not shown). Introduction of plasmid pLA-FLR (containingA. hydrophila polar flagellum region 5) and plasmid pACYC-FLR1 (containing theflrBC genes [see Materials and Methods]) into the AH-4462 mutant strain rescued the swimming phenotype and the ability to synthesize polar flagella, as determined by TEM (Fig.4). Moreover,V. cholerae KKV59 (ΔflrA) and KKV98 (ΔflrC) (35) mutant strains complemented with plasmid pLA-FLR spread onVibrio motility plates as fast as the wild type, and the formation of polar flagella was confirmed by TEM (data not shown). No complementation was observed when only the vector pLA2917 or pACYC184 was introduced into the mutants.

Distribution of polar flagellum genes in mesophilicAeromonas strains.

The distribution of polar flagellum genes in mesophilicAeromonas strains (n = 50) was analyzed by dot blot hybridization experiments against total genomic DNA, using independent PCR probes. The distribution of polar flagellum region 2 was analyzed using three PCR probes (flaB toflaH,flaA, andmaf-1); region 3 was analyzed using three PCR probes (fliK tofliM,flhA tofliA, andpomAB); region 4 was determined by an intragenicmotX probe; and master regulatory genes on region 5 were analyzed using two PCR probes (flrA andflrBC).

Polar flagellum probes hybridized to the chromosomal DNA of all mesophilicAeromonas strains tested, whether or not the strains were able to produce lateral flagella.

Adhesion to HEp-2 cells and biofilm formation.

In order to correlate polar flagellum presence and motility with adherence to mammalian cells, we examined the interaction of polar flagellum mutants with cultured monolayers of HEp-2 cells. Differences in adherence were calculated by determining the average numbers of bacteria adhering to HEp-2 cells (Fig.5A). Also, we compared the abilities of the wild-type and polar mutant strains to form biofilms in microtiter plates (Fig.5B). TheA. hydrophila wild-type strain, AH-3, exhibited an adhesion value of 18.3 (±1.7) bacteria adhered per HEp-2 cell and a biofilm formation ability with an optical density at 570 nm of 1.3 (±0.1). Polar flagellum mutant strains AH-4423 (flaH), AH-4424 (maf-1), AH-4427 (flaA flaB), AH-4441 (fliM), AH-4442 (flhA), AH-4443 (fliA), and AH-4462 (flrC), which produce lateral flagella but are unable to produce polar flagella, showed approximately an 86% reduction in adhesion to HEp-2 cells and a 55% reduction in biofilm formation. These results were similar to those forA. hydrophila flg polar flagellum mutants described previously (2). Mutant strains AH-4425 (flaB) and AH-4426 (flaA), which possess both flagellum types but show a small reduction in motility phenotype, exhibit a slight reduction in adhesion to HEp-2 cells, as well as in ability to form biofilms (approximately 35% and 10%, respectively). Finally, AH-4461 (motX), which has both flagellum types but is unable to swim, exhibited approximately half of the adhesion capacity to HEp-2 cells compared to the wild-type strain and had a 37% reduction in its ability to form biofilms. No changes in adhesion or biofilm formation were observed for the AH-4444 (pomB) mutant in comparison to the wild-type strain. Complementation studies using polar flagellum mutants AH-4423, AH-4425, AH-4426, and AH-4427 with plasmid pLA-FLA; AH-4424 with plasmid pLA-FLA or pACYC-MAF; AH-4441 with plasmid pLA-FLIP1; AH-4442 and AH-4443 with plasmid pLA-FLIP1 or pLA-FLIP2; AH-4461 with plasmid pLA-MOTX; and AH-4462 with plasmid pLA-FLR or pACYC-FLR1 showed values similar to those for the wild-type strain regarding adhesion to HEp-2 cells and biofilm formation. These adhesion and biofilm formation assays showed that polar flagella have an important role in cellular adhesion and biofilm formation, as was previously reported (2).

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