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1 Departamento de Microbiología, Facultad de Biología, Universidad de Barcelona, Diagonal 645, 08071 Barcelona, Spain
2 Division of Molecular and Genetic Medicine, University of Sheffield Medical School, Sheffield S10 2RX, UK
Correspondence
Juan M. Tomás
jtomas{at}ub.edu
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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The Genbank/EMBL/DDBJ accession number for the nucleotide sequence determined in this work is DQ119104.
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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). They can be isolated as a part of the faecal flora of a wide variety of other animals, including some used for human consumption, such as pigs, cows, sheep and poultry. In humans, Aeromonas hydrophila belonging to hybridization groups 1 and 3 (HG1 and HG3), Aeromonas veronii biovar sobria (HG8/HG10) and Aeromonas caviae (HG4) have been associated with gastrointestinal and extraintestinal diseases, such as wound infections in healthy humans, and less commonly, with septicaemia of immunocompromised patients (Janda & Abbott, 1998). The pathogenicity of mesophilic aeromonads has been linked to a number of different determinants, such as toxins, proteases, outer-membrane proteins (Nogueras et al., 2000), LPS (Merino et al., 1996) and flagella (Merino et al., 1997; Rabaan et al., 2001).
Motility and flagella have been reported as virulence factors from several pathogenic bacteria, such as Salmonella enterica (McDermott et al., 2000), Escherichia coli (Pratt & Kolter, 1998), Helicobacter pylori (Eaton et al., 1996) and Vibrio cholerae (Gardel & Mekalanos, 1996). Mesophilic Aeromonas express constitutively a single polar unsheathed flagellum which allows the bacteria to swim in liquid environments, and it is required for adherence and invasion of human and fish cell lines (Gryllos et al., 2001; Canals et al., 2006a). Certain strains are able to produce many unsheathed peritrichous lateral flagella when grown in viscous environments or on surfaces; these increase bacterial adherence, and are required for swarming motility and biofilm formation (Gavín et al., 2002; Canals et al., 2006b). This second flagella system is only present in 50–60 % of mesophilic aeromonad species, most commonly associated with diarrhoea (Kirov et al., 2002). The expression of two distinct flagella systems is relatively uncommon, although it has been observed in Vibrio parahaemolyticus (McCarter, 1999), Azospirillum brasilense (Moens et al., 1996), Rhodospirillum centenum (McClain et al., 2002), Helicobacter mustelae (O'Rourke et al., 1992) and Plesiomonas shigelloides (Inoue et al., 1991). Recently, an E. coli 042 lateral flagella gene cluster (Flag-2) has been reported (Ren et al., 2005), and the presence of Flag-2-like gene clusters in Yersinia pestis, Yersinia pseudotuberculosis and Chromobacterium violaceum suggests that the coexistence of two flagella systems within the same species is more common than previously suspected (Ren et al., 2005).
Mesophilic Aeromonas strains, such as A. hydrophila AH-3 and A. caviae Sch3N, are able to express two entirely distinct flagella whose flagellins are glycosylated (Gavín et al., 2002; Rabaan et al., 2001). In this study, we have identified several new genes involved in glycosylation of polar and lateral flagella in mesophilic Aeromonas. These genes are not present in the complete sequenced genome of V. parahaemolyticus, a bacterium also able to express two flagella systems (polar and lateral).
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METHODS |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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. E. coli strains were grown on Luria–Bertani (LB) Miller broth and LB Miller agar at 37 °C, while Aeromonas strains were grown either in tryptic soy broth (TSB) or on tryptic soy agar (TSA) at 30 °C. When required, ampicillin (50 µg ml–1), kanamycin (50 µg ml–1), rifampicin (100 µg ml–1), spectinomycin (50 µg ml–1), chloramphenicol (25 µg ml–1) or tetracycline (20 µg ml–1) were added to the media. All the Aeromonas strains were identified to the species level by 16S rDNA RFLP analysis.
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pirKm-1 (De Lorenzo et al., 1990) to A. hydrophila AH-405 (AH-3 rifampicin-resistant, RifR) was carried out in a conjugal drop incubated for 6 h at 30 °C, with the ratio 1 : 5 : 1, corresponding to S17-1pirKm-1, AH-405 and HB101 pRK2073 (helper plasmid), respectively. Serial dilutions of the mating mix were plated on TSA supplemented with rifampicin and kanamycin, in order to select mutants.
Motility assays (swarming and swimming).
Freshly grown bacterial colonies were transferred with a sterile toothpick into the centre of swarm agar (1 % tryptone, 0.5 % NaCl, 0.6 % agar) or swim agar (1 % tryptone, 0.5 % NaCl, 0.25 % agar). The plates were incubated face up for 16–24 h at 30 °C, and motility was assessed by examining the migration of bacteria through the agar from the centre towards the periphery of the plate. Moreover, swimming motility was assessed by light microscopy.
Transmission electron microscopy (TEM).
Bacterial suspensions were placed on Formvar-coated grids and negatively stained with a 2 % solution of uranyl acetate, pH 4.1. Preparations were observed on a Hitachi 600 transmission electron microscope.
DNA techniques.
DNA manipulations were carried out essentially as described elsewhere (Sambrook et al., 1989). 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 using Taq DNA polymerase (Invitrogen) in a PerkinElmer Gene Amplifier PCR System 2400 thermal cycler. Amplifications over 4000 bp were performed using Platinum Taq DNA Polymerase High Fidelity (Invitrogen), as recommended by the supplier.
Nucleotide sequencing and computer sequence analysis.
Plasmid DNA for sequencing was isolated by the Qiagen plasmid purification kit, as recommended by the suppliers. In some cases, inverse PCR was used to amplify for sequencing a chromosomal DNA fragment that was not present in the A. hydrophila library. dsDNA sequencing was performed using the Sanger dideoxy-chain-termination method (Sanger et al., 1977), with an ABI Prism dye terminator cycle sequencing kit (PerkinElmer). Custom-designed primers used for DNA sequencing were purchased from Amersham Biosciences.
The DNA sequence was translated in all six frames, and all ORFs >100 bp were inspected. Deduced amino acid sequences were inspected in the GenBank, EMBL and SWISS-PROT databases by using the BLASTX, BLASTP or PSI-BLAST network service at the National Center for Biotechnology Information (NCBI) (Altschul et al., 1997). Protein family profiling was performed using the Protein Family Database Pfam at the Sanger Institute (Bateman et al., 2002). Determination of possible terminator sequences was done by using the Terminator program from the Genetics Computer Group package (Madison, WI, USA). Other online sequence analysis services were also used. The Genbank/EMBL/DDBJ accession number for the nucleotide sequence determined in this work is DQ119104.
RT-PCR.
Total RNA was isolated by Trizol reagent (Invitrogen) from A. hydrophila AH-3 grown in TSB or on TSA. The isolated RNA was used as a template in RT-PCRs, utilizing the Thermoscript RT-PCR system (Invitrogen), according to the manufacturer's instructions. RT-PCR amplifications were performed at least twice with total RNA preparations obtained from a minimum of two independent extractions. The RT-PCR and PCR products were analysed by agarose gel electrophoresis.
Southern, dot and colony blot hybridizations.
Dot and Southern hybridizations were performed by capillary transfer (Sambrook et al., 1989). Colonies were transferred onto nylon membranes for colony hybridization (Roche), and then lysed according to the manufacturer's instructions. Probe labelling with DIG (Amersham), and hybridization and detection were carried out according to the supplier's instructions.
Mutant construction.
To obtain non-polar mutants in maf-2, neuB-like, flmD and neuA-like, the genes were amplified by PCR using the specific oligonucleotides from the DNA sequence of COS-FLA, ligated into vector pGEM-T Easy (Promega), and transformed into E. coli XL-1 Blue. The Tn5-derived kanamycin resistance cartridge (nptII) 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 (Bott et al., 1995); however, such insertion alters 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 to suicide vector pDM4 (Milton et al., 1996), transformed into E. coli MC1061 (pir), and selected on chloramphenicol plates. Plasmids with mutated genes were transferred into A. hydrophila AH-405 RifR by triparental matings using chloramphenicol, kanamycin and rifampicin to select transconjugants. PCR analysis confirmed the correct integration of the vector into the chromosomal DNA. To complete the allelic exchange, the integrated suicide plasmid was forced to recombine out of the chromosome by adding 5 % sucrose to the agar plates. The pDM4 vector contains sacB, which produces an enzyme that converts sucrose into a product that is toxic to Gram-negative bacteria. Transconjugants surviving on plates with 5 % sucrose, which were rifampicin resistant (RifR) kanamycin resistant (KmR) and chloramphenicol sensitive (CmS), were selected, and allelic exchange was confirmed by PCR. The same methodology was used to generate a non-polar flmA mutant (AH-4515).
Plasmid construction for mutant complementation studies.
Plasmid pACYC-FLA2 containing the complete A. hydrophila AH-3 maf-2, neuA-like, flmD and neuB-like genes was obtained by genomic DNA amplification using paired oligonucleotides: 5'-ACACCCTTCGACATCAAAA-3' and 5'-GCCGTACCTCATCCTGATT-3' (4860 bp DNA band amplification for maf-2, neuA-like, flmD and neuB-like genes). The amplified band was ligated into ScaI-digested and phosphatase-treated pACYC184 vector to generate pACYC-FLA2.
A DNA fragment of 
5 kb containing the A. caviae Sch3N genes (flmB, neuA, flmD, neuB) was obtained after HindIII restriction of pDI54 (Gryllos et al., 2001). The HindIII fragment was treated to generate blunt ends, and cloned into the ScaI restriction site of pACYC184 to generate pACYC-FLA-3 (tetracycline resistant, TcR). This plasmid, containing the same genes as pDI54, was generated due to the incompatibility of antibiotic markers among pDI54 and A. hydrophila AH-3 mutants (KmR).
Plasmids pACYC-CJ1293 containing the complete Cj1293 gene, pACYC-CJ1294 containing the complete Cj1294 gene, and pACYC-CJ1317 containing the complete Cj1317 gene from C. jejuni NCTC 11168 were obtained by PCR amplification of genomic DNA, using oligonucleotides CJ1293-FOR (5'-CGCGGATCCAAGGCGATGAAAAATGTG-3') and CJ1293-REV (5'-CGCGGATCCGGCTGAAGTGGCTGAGTTT-3') to generate a band of 1268 bp; CJ1294-FOR (5'-CGCGGATCCGAAAAAGGGCAAAAGGTA-3') and CJ1294-REV (5'-CGCGGATCCATTCCATTCATCAGGGACT-3') to generate a band of 1465 bp; and CJ1317-FOR (5'-CGCGGATCCTGGCACAAAAATACGATGG-3') and CJ1317-REV (5'-CGCGGATCCAAATTCCATTTCCAAAACCA-3') to generate a band of 1532 bp, respectively. The amplified bands were purified, BamHI-digested (the BamHI site is underlined in the above primers), ligated to BamHI-digested and phosphatase-treated pACYC184 vector, and introduced into E. coli DH5
to generate the recombinant plasmids pACYC-CJ1293, pACYC-CJ1294 and pACYC-CJ1317.
Whole-cell protein preparation and immunoblotting.
Whole-cell proteins were obtained from Aeromonas strains grown at 30 °C. Equivalent numbers of cells were harvested by centrifugation, and the cell pellet was resuspended in 50–200 µl SDS-PAGE loading buffer and boiled for 5 min. Following SDS-PAGE and transfer to nitrocellulose membranes, the membranes were blocked with BSA (3 mg ml–1), and probed with either polyclonal rabbit anti-polar or anti-lateral flagellin antibodies (1 : 1000), previously obtained (Gavín et al., 2002; Rabaan et al., 2001). The unbound antibody was removed by three washes in PBS, and a goat anti-rabbit horseradish peroxidase-conjugated secondary antibody (1 : 1000) 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), which was prepared in methanol and diluted before use in 8 ml PBS containing 50 µl 30 % H2O2.
Glycosyl group detection in cell-free extracts.
Suspensions of bacteria (25 % w/v), washed in 25 mM Tris/HCl buffer (pH 7.5) containing 1 mM MgCl2, were disrupted in a Branson model 350 sonifier at 0 °C. Disrupted bacteria were subjected to high-speed centrifugation (180 000 g for 2 h) at 5 °C to obtain cell-free extracts. Protein concentrations of extracts were determined by the Bio-Rad Bradford assay with BSA as the standard. To determine the presence of glycosylated proteins, cell-free extracts were transferred onto nitrocellulose membranes and treated with periodate to oxidize any glycosyl groups present to produce reactive aldehydes, and then allowed to react with biotin hydrazide. This assay was performed with the Carbohydrate Detection kit from Oxford Glyco Systems, as directed by the manufacturer.
LPS isolation and electrophoresis.
Cultures for analysis of LPS were grown in TSB at 37 °C. For screening purposes, LPS was obtained after proteinase K digestion of whole cells (Darveau & Hancock, 1983). LPS samples were separated by SDS-PAGE or SDS-Tricine-PAGE and visualized by silver staining.
Adherence to HEp-2 cells.
Cultured cells were maintained as described by Thornley et al. (1997). The adherence assay was conducted as a slight modification of that described by Carrello et al. (1988). Bacteria were grown statically in brain heart infusion broth (BHIB) at 37 °C, harvested by gentle centrifugation (1600 g, 5 min), and resuspended in PBS (pH 7.2) at 107 c.f.u. ml–1 (OD600 0.07). The monolayer was infected with 1 ml bacterial suspension for 90 min at 37 °C in 5 % CO2. Following infection, the non-adherent 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 with PBS, and the HEp-2 cells with the adherent bacteria were stained for 45 min in 10 % (v/v) 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 per coverslip were randomly chosen, and the number of bacteria adhering per HEp-2 cell was recorded. Assays were carried out in duplicate or triplicate.
Biofilm formation.
Quantitative biofilm formation was performed in a microtitre assay, as described elsewhere (Pratt & Kolter, 1998), 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 then placed in a microtitre 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. After solubilization of the crystal violet with ethanol/acetone (80 : 20, v/v) the A570 was determined.
Statistical analysis.
The differences in adherence to HEp-2 cells or biofilm formation in vitro between the wild-type and mutant strains were analysed by the t test using Microsoft Excel software.
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RESULTS |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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), indicating that the transposon affected a gene implicated in the expression of the polar flagellum. Furthermore, lateral flagella were shown to be absent by TEM (Fig. 1), and the cells were unable to swarm on plates. Western blot analysis using whole cells showed the presence of polar and lateral flagellins in the mutant (Fig. 2A). It should be noted that the polar flagellin of the mutant appeared to be smaller in size and was reduced in amount when compared to that of the wild-type. No changes were observed for the lateral flagellin or LPS (Fig. 2B, C). Cell-free-extract proteins from mutant AH-4429 grown in liquid medium showed no glycosylation, while the corresponding extract from the wild-type strain showed a unique glycosylated band at the molecular mass of the polar flagellin (Fig. 2D). Southern blot analysis using a specific probe for the transposon demonstrated that mutant AH-4429 had a single copy of the mini-transposon in its genome (data not shown). The mini-transposon-containing fragment and flanking DNA from the genome of the mutant (EcoRV digestion) were cloned into pBCSK (Stratagene) and sequenced. The fragment flanking the insertion in AH-4429 encoded a product similar (43–62 %) to a series of conserved hypothetical proteins of Pseudomonas fluorescens, Shewanella and Clostridium, and to the motility accessory factor encoded by Cj1337 of C. jejuni (41 %). This group of proteins is thought to be involved in phase-variable flagellum-mediated motility and flagellin modifications (Karlyshev et al., 2002). We used this sequence data to synthesize an internal probe for the Cj1337-like locus of A. hydrophila AH-3.
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has previously been constructed (Nogueras et al., 2000). This library was screened by colony blotting using a DNA probe to the Cj1337-like locus (maf-2) of A. hydrophila AH-3. Several positive recombinant clones were identified, of which clone pLA-FLA2 was chosen because it was able to completely complement the AH-4429 mutation.
pLA-FLA2 DNA sequence
Analysis of the 15 419 bp nucleotide sequence revealed 14 complete ORFs; the initial six complete ORFs (Fig. 3) transcribed in the same direction have previously been described as region 2 genes of the polar flagellum (Canals et al., 2006a). Fig. 3 depicts the ORFs (named as their homologues) and the main characteristics of the DNA sequence. Sequences defining putative ribosome-binding sites were found upstream of each of the ORF start codons. Proteins homologous to the putative products of the 14 complete ORFs were identified using the BLASTX program of the NCBI. Data summarizing the location of the complete ORFs and characteristics of the putative encoded proteins, excluding the six initial ORFs, are shown in Table 2.
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, ORFs 7 and 8 were similar to different transposase proteins. ORF9 contained the miniTn5 insertion in mutant AH-4429. Its predicted amino acid sequence (Cj1337) contained a DUF115 conserved domain. In this gene (named maf-2), as can be observed in Fig. 3, this insertion did not cause a polar effect. The deduced amino acid sequences of ORFs 10, 11 and 12 showed similarity to those of three A. caviae proteins previously described by our laboratory (Table 2). Accordingly, they were named neuB-like, flmD and neuA-like, respectively.
Transcribed in the opposite direction to ORF12 was ORF13, whose encoded protein shares a high degree of homology with the putative flagella-modification protein FlmH of Caulobacter crescentus (Weissborn et al., 1982). Finally, 229 bp upstream of ORF13 was ORF14, which encodes a highly conserved protein implicated in the first step of the fatty acid biosynthesis pathway (Table 2). No terminator sequences or ORFs were found in the next 300 bp sequenced. Four putative promoter regions were identified upstream of genes orf8, neuB-like, neuA-like and flmH, and two rho-independent terminator sequences have also been identified downstream of the genes maf-1 and orf7. Using DNA-sequencing-derived oligonucleotides and RT-PCR, we detected the transcription of orf8–9, neuB-like–maf-2, neuA-like–flmD and flmH. However, no transcription was obtained using oligonucleotide pairs from maf-1 to orf7, maf-2 to orf8, flmD to neuB-like and neuA-like to flmH, thus confirming the putative operons of this entire pLA-FLA2 sequenced region (data not shown, Fig. 3).
Mutant analysis
Due to the characteristics of protein similarity observed from ORF6 to 13, and their location close to some structural genes for polar flagella production, we decided to construct defined insertion mutants in these genes and study their phenotypes. Non-polar insertion mutants in maf-2, neuB-like, flmD and neuA-like (mutants AH-4509, AH-4510, AH-4511 and AH-4512, respectively) were unable to swim or swarm on plates, and lacked both polar and lateral flagella under TEM (Fig. 4, data shown for neuB-like mutant). These data are in agreement with the phenotype found for insertion mutant AH-4429. These mutants seem to be able to produce polar or lateral flagellin when tested with specific immunoblots using whole cells, but the amounts of both flagellins were slightly reduced compared to that of the wild-type (Fig. 5, data shown for neuB-like mutant). Cell-free extracts from the above-mentioned mutants grown on solid medium showed no glycosylation, while the wild-type extract showed two glycosylated bands of the same molecular masses as the polar and lateral flagellins (Fig. 5C). It is important to remember that A. hydrophila AH-3 polar flagellum has shown constitutive expression in all the growth media tested (Canals et al., 2006a). No differences in LPS profiles in gels were observed between the mutants and the wild-type strain (data not shown).
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, data shown for IAG1419 mutant). In addition, the A. caviae mutants regained both the O antigen and the highest-migrating band of the outer-core LPS (Fig. 6B, C). pACYC-FLA2 was also able to complement the corresponding mutants in A. hydrophila AH-3 (Figs 1, 2, 4 and 5), while no complementation was observed with the plasmid vector alone (pACYC184). pACYC-FLA2 and pACYC-FLA3 containing neuA-B like genes from A. hydrophila AH-3 and A. caviae Sch3N, respectively, were unable to complement E. coli K1 neuA or neuB mutants (Zapata et al., 1989).
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). This result prompted us to create maf-2, neuA-like, flmD and neuB-like mutations in a strain that is only able to produce a polar flagellum (A. hydrophila ATCC 7966), using the same plasmids constructed to mutate A. hydrophila AH-3. The mutants were completely unable to move, as shown by light microscopy, and lacked the polar flagellum, as demonstrated by TEM. No differences in LPS profile could be observed between the wild-type and the corresponding mutants. pACYC-FLA2, but not the plasmid vector alone (data not shown), was able to fully complement ATCC 7966 maf-2, neuA-like, flmD and neuB-like mutants, as judged by recovery of motility and of a polar flagellum, observed by TEM. The only tested strain that did not give a clear positive reaction to any of the DNA probes was A. caviae Sch3N.
Adhesion to HEp-2 cells and biofilm formation
Polar and lateral flagella in Aeromonas are important factors in two pathogenic features, adhesion to HEp-2 cells and biofilm formation, as previously described (Canals et al., 2006a, b; Kirov et al., 2004). For this reason, we decided to study the mutants obtained for these two assays, and to prove the functionality of the complemented mutants.
Table 3 shows that A. hydrophila mutants in maf-2, neuA-like, flmD and neuB-like lacking the polar and lateral flagella were drastically reduced (80 %) in their adherence to HEp-2 cells and ability to form biofilms, in comparison with the values obtained for the wild-type strain. Reintroduction of the corresponding A. hydrophila genes (pACYC-FLA2) resulted in the recovery of both flagella, as well as similar values to those of the wild-type for adherence to HEp-2 cells and biofilm formation. A similar situation was observed when the genes from A. caviae Sch3N (pACYC-FLA3) were introduced into the A. hydrophila mutants, i.e. full complementation was achieved, as judged by adherence to HEp-2 cells and biofilm formation. A. caviae Sch3N mutants in flmD and neuA showed a more drastic reduction in adherence (>90 %) because they also lack O-antigen LPS (Gryllos et al., 2001). Complementation with the corresponding A. hydrophila genes rendered adherence to HEp-2 cells and biofilm formation similar to those of the wild-type strain (Sch3N). Complementation also restored the presence of O-antigen LPS, as indicated above.
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Complementation of A. hydrophila mutants by genes from C. jejuni
The similarity between A. hydrophila proteins FlmA, FlmB and NeuB-like and those encoded by the C. jejuni genes Cj1293, Cj1294 and Cj1317, respectively, and the fact that a derivative of pseudaminic acid (Pse5Ac7Ac), a nine-carbon sugar that is similar to sialic acid, has been identified to be linked to the polar flagella of one A. caviae strain (Schirm et al., 2005), prompted us to study the complementation of the corresponding A. hydrophila mutants with the equivalent C. jejuni genes. An A. hydrophila AH-3 defined insertion mutant in flmB (AH-1726) has previously been created (Gryllos et al., 2001). Using the methodology previously indicated, we obtained a non-polar insertion flmA mutant (AH-4515) in A. hydrophila AH-3. Mutants flmA and flmB showed the same phenotype as maf-2, neuA-like, flmD and neuB-like mutants, and were unable to produce polar and lateral flagella, as shown by TEM, but did show the presence of unglycosylated polar and lateral flagellins.
pACYC-CJ1293, pACYC-CJ1294 and pACYC-CJ1317 carrying the C. jejuni genes Cj1293, -1294 and -1317, respectively, were able to fully complement A. hydrophila AH-3 flmA, flmB and neuB-like mutants, respectively, as judged by the recovery of either the polar or the lateral flagella, observed by TEM. However, the plasmid vector alone was unable to rescue flagella formation in the corresponding A. hydrophila mutants. Complemented mutants showed similar values for adhesion to HEp-2 cells and biofilm formation as the corresponding wild-type strain (Table 3).
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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; Gavín et al., 2002; Canals et al., 2006a, b). The polar flagellum is constitutive, whereas lateral flagella are induced when the bacteria are grown on solid or semisolid media. We have previously described two genes (flmA and flmB) involved in flagella expression (now confirmed for both polar and lateral flagella) that are present in both strains. However, in A. caviae Sch3N, but not in other Aeromonas strains tested (Gryllos et al., 2001), three other genes were found together with flmA and flmB, and were named neuA-like, flmD and neuB-like. These genes were found in a putative cluster involved in flagella and O- antigen LPS production. In the original report (Gryllos et al., 2001), the A. caviae Sch3N cluster did not contain a maf-2 gene, but one has recently been found (data not shown). The flmA and flmB genes in other Aeromonas strains are not involved in LPS changes. We tried unsuccessfully to isolate the neuA-like, flmD and neuB-like genes from AH-3 by using DNA probes generated from A. caviae Sch3N (Gryllos et al., 2001). The very low DNA homology observed between the genes from both strains explains this previous lack of success, even though the proteins encoded by these genes are able to complement the corresponding mutants. The location of these genes (maf-2, neuA-like, flmD and neuB-like) in A. hydrophila AH-3 and other strains (data not shown) is near to those encoding proteins for the polar flagellum (region 2; Canals et al., 2006a) (see Fig. 3), as we found by sequencing the AH-3 cosmid (pLA-FLA2). Furthermore, we also found these genes in an Aeromonas strain that is naturally lateral-flagella negative. Non-polar mutations in the A. hydrophila AH-3 maf-2, neuA-like, flmD and neuB-like genes resulted in the complete loss of both flagella types (polar and lateral), without any effect on LPS profiles. In A. caviae Sch3N, mutations in the neuA or flmD genes resulted not only in the loss of both flagella types but also in the alteration of the LPS profile (lack of O-antigen LPS). We decided to designate these genes neuA-like and neuB-like because they are unable to complement E. coli neuA or neuB mutants. Cross-complementation studies using the genes from either AH-3 or Sch3N were able to fully complement the corresponding AH-3 or Sch3N mutants, as was demonstrated by the recovery of both flagella types, and in addition, strain Sch3N showed the restoration of the LPS profile.
It is well established that C. jejuni polar flagellins are glycosylated (Goon et al., 2003), and here we demonstrated that Aeromonas mutants of these genes (maf-2, neuA-like, flmD and neuB-like) produce unglycosylated polar and lateral flagellins. The following protein functions have recently been assigned: C. jejuni Cj1293 (PseB) as a UDP-N-acetylglucosamine (GlcNAc) C6 dehydratase/C4 reductase (Goon et al., 2003), Cj1294 as a UDP-4-keto-6-deoxy-GlcNAc aminotransferase (Obhi & Creuzenet, 2005), and Cj1317 (PseI) as a pseudaminic acid synthase (Chou et al., 2005); all of these are required for the formation of pseudaminic acid (Pse5Ac7Ac), a nine-carbon sugar that is similar to sialic acid. The protein similarity between A. hydrophila FlmA, FlmB and NeuB-like and Cj1293, Cj1294 and Cj1317, respectively, and the fact that a derivative of Pse5Ac7Ac has been identified linked to the polar flagella of one A. caviae strain (Schirm et al., 2005), prompted us to study the complementation of the corresponding Aeromonas mutants with the equivalent C. jejuni genes. Plasmids carrying Cj1293, Cj1294, and Cj1317 were fully able to complement A. hydrophila AH-3 flmA, flmB and neuB-like mutants, respectively, as judged by TEM showing the recovery of the polar and lateral flagella. Due to the fact that both Aeromonas flagellins (polar and lateral) are glycosylated, we suggest that these genes are involved in flagella glycosylation by producing Pse5Ac7Ac or a Pse5Ac7Ac derivative. Furthermore, the genes described here, believed to be for the first time, in A. hydrophila AH-3 (maf-2, neuA-like, flmD and neuB-like), together with flmA and flmB, are involved in the glycosylation of polar and lateral flagellins. We could not find any similar genes in the complete sequenced genome of V. parahaemolyticus, a bacterium that also expresses two different kinds of flagella (polar and lateral), probably because its flagella are not glycosylated.
The HEp-2 cell adhesion and biofilm formation studies performed with mutants and complemented strains allowed us to conclude that the recovery observed by TEM of both flagella types was fully functional. These experiments clearly demonstrated that when the polar and lateral flagella are recovered, so is the ability of the bacterial cells to adhere to eukaryotic cells and form biofilms.
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ACKNOWLEDGEMENTS |
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Edited by: S. MacIntyre
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REFERENCES |
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Received 31 July 2006;
revised 13 November 2006;
accepted 1 December 2006.
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