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1 Departamento de Microbiología, Facultad de Biología, Universidad de Barcelona, Diagonal 645, 08071 Barcelona, Spain
2 N. D. Zelinsky Institute of Organic Chemistry, Russian Academy of Sciences, Moscow 119991, Russia
Correspondence
Juan M. Tomás
jtomas{at}ub.edu
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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Present address: Institute for Biological Sciences, National Research Council, Ottawa, ON K1A 0R6, Canada.
The GenBank/EMBL/DDBJ accession no. for the nucleotide sequence of the A. hydrophila AH-3 galU region described in this paper is EF123040.
A table of 1H and 13C NMR data for oligosaccharide fractions 1 and 2 is available with the online version of this paper.
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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; 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), lipopolysaccharide (LPS) (Merino et al., 1996), flagella (Merino et al., 1997; Rabaan et al., 2001) and type III secretion system (Vilches et al., 2004). The O-antigen LPS plays an important role as a virulence factor in many Gram-negative bacteria (see Finlay & McFadden, 2006 for a review). We have fully characterized chemically the O-antigen and the core LPS of Aeromonas hydrophila strain AH-3 (serotype O34) (Fig. 1; Knirel et al., 2002, 2004), this serotype being frequently found among mesophilic Aeromonas strains from clinical sources (Merino et al., 1993). Mutants devoid of the O34-antigen LPS are drastically reduced in some pathogenic features (serum resistance or adhesion to HEp-2 cells) and less virulent for fish and mice in comparison to the wild-type strain (Canals et al., 2006a), thus the O34-antigen LPS is essential for the virulence of mesophilic Aeromonas O34 strains. Strain AH-3, like other mesophilic Aeromonas strains, possesses two kinds of flagella (Canals et al., 2006b, c); the lack of the O34-antigen molecules in this strain reduced the motility without any effect on the biogenesis of both polar and lateral flagella (Canals et al., 2006a).
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; Stimson et al., 1995). The galU gene was found to be important for pathogenesis of infections due to a number of Gram-negative pathogens, including Actinobacillus pleuropneumoniae (Rioux et al., 1999), Escherichia coli (Komeda et al., 1977), Klebsiella pneumoniae (Chang et al., 1996), Pseudomonas aeruginosa (Evans et al., 2002; Priebe et al., 2004), Shigella flexneri (Sandlin et al., 1995) and Vibrio cholerae (Nesper et al., 2001). The phenotypes of the K. pneumoniae and V. cholerae galU mutants were dominated by abnormal capsule synthesis rather than LPS O-antigen abnormalities, and several of the galU mutants also had defects in other surface proteins, such as IscA for S. flexneri (Sandlin et al. 1995) and flagella for E. coli (Komeda et al., 1977). Among all these studies, however, the common theme of impaired survival of galU mutants in the face of host factors such as complement does emerge, as for instance in the P. aeruginosa galU mutants (Priebe et al., 2004). We characterized the Aeromonas galU mutants by phenotypic characteristics, LPS structure, enzymic assays and some virulence traits. Furthermore, the mutants obtained in several mesophilic Aeromonas strains from different serotypes allowed us to conclude that the O-antigen LPS is an essential factor for the virulence of these strains.
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METHODS |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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. Aeromonas strains were routinely grown on tryptic soy broth (TSB) or tryptic soy agar (TSA) at 30 °C, while E. coli strains were grown on Luria–Bertani (LB) Miller broth and LB Miller agar at 37 °C (Atlas, 1993). When required, the strains were cultured in Davis minimal medium (Atlas, 1993) with glucose or galactose as the sole carbon source. Kanamycin (50 µg ml–1), ampicillin (100 µg ml–1), tetracycline (20 µg ml–1), rifampicin (100 µg ml–1), or chloramphenicol (20 µg ml–1) were added to the different media.
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pirKm-1 (De Lorenzo et al. 1990) to A. hydrophila AH-405 (AH-3 rifampicin-resistant) 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.
General DNA methods.
General DNA manipulations were done essentially as described by 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.
DNA sequencing and computer analysis of sequence data.
Double-stranded DNA sequencing was performed by using the dideoxy-chain-termination method (Sanger et al., 1977) with the ABI Prism dye terminator cycle sequencing kit (Perkin Elmer). Oligonucleotides used for genomic DNA amplification experiments and for DNA sequencing were purchased from Pharmacia LKB Biotechnology. The DNA sequence was translated in all six frames, and all ORFs were inspected. Deduced amino acid sequences were compared with those of DNA translated in all six frames from non-redundant GenBank and EMBL database by using the BLAST (Altschul et al., 1997; Bateman et al., 2002) network service at the National Center for Biotechnology Information and the European Biotechnology Information, respectively. CLUSTAL W was used for multiple sequence alignments.
Southern blot hybridizations.
Southern blotting was performed by capillary transfer (Sambrook et al., 1989). Probe labelling, hybridization and detection were carried out using the enhanced chemiluminescence labelling and detection system (Amersham) according to the manufacturer's instructions.
DNA amplification, plasmid and mutant construction.
Genomic DNAs from mesophilic Aeromonas strains with different O serotypes were isolated and used as template in PCR experiments using primers galUfor and galUrev (5'-TGCAAGGGCTAGGAACAAG-3' and 5'-AGCAAGGCAAGTTCACCAC-3') designed to amplify a 1274 bp band including the complete Aeromonas galU. These oligonucleotides were used also to amplify and to subclone galU in vector pGEMT (pGEMT-GALU). After NotI digestion of plasmid pGEMT-GALU, a 1724 bp band was obtained, Klenow-treated and ligated to plasmid vector pACYC184 digested with EcoRV, yielding plasmid pACYC-GALU where galU is orientated to the vector promoter. An inner 604 bp DNA fragment of galU was obtained from AH-3 genomic DNA by PCR using primers galU-A and galU-B (5'-ACGGCATATTCCACGTTGG-3' and 5'-CTGGAAAAACGGGTCAAGC-3') and subcloned in vector pGEMT, digested with EcoRI and subcloned in the pir replication-dependent plasmid pFS100 (Rubirés et al., 1997). This plasmid construction (pFS-GALU) was used to obtain galU-deficient mutants from several Aeromonas strains by a single recombination event as previously described (Rubirés et al., 1997). Plasmid pFS-GALU was isolated, transformed into E. coli MC1061 (pir) (Rubirés et al., 1997), and transferred by conjugation from E. coli MC1061 to the different rifampicin-resistant (Rifr) Aeromonas strains (from our laboratory collection) as previously described (Rubirés et al., 1997). Chromosomal DNA from 10 transconjugants obtained from each of the different mesophilic Aeromonas strains was analysed by Southern blot hybridization with appropriate galU DNA probe to obtain defined insertion galU mutants as previously described (Nogueras et al., 2000).
Complementation studies.
Complementation analysis of the different galU mutants was performed by conjugal transfer of wild-type galU (pACYC-GALU) cloned in pACYC184 vector. Recombinants were selected on LB agar containing chloramphenicol and rifampicin, and their LPS was isolated and analysed in gels (Darveau & Hancock, 1983).
Plasmid constructions for gene overexpression.
For galU overexpression, the pET-30 Xa/LIC vector (Novagen) and AccuPrime (Invitrogen, High-fidelity Taq) polymerase were used. The galU gene was amplified from plasmid DNA pACYC-GALU using primers AFw (5'-GGTATTGAGGGTCGCATGAAAAAGACACCGCTCGT-3') and ARv (5'-AGAGGAGAGTTAGAGCCCAGCTATTACAACTGCTTGACG-3'). The 918 nt PCR product was electrophoresed in agarose and the DNA band recovered and purified. Purified amplicons were treated with T4 DNA polymerase in the presence of 2.5 mM dGTP for 30 min at 22 °C to generate single-stranded ends (underlined letters above) complementary to Xa/LIC single-stranded ends in pET-30 Xa/LIC. After inactivation of the T4 DNA polymerase, the amplicon was mixed with pET-30 Xa/LIC vector and electroporated into NovaBlue GigaSingles competent cells, and plated on LB supplemented with kanamycin (30 µg ml–1). Transformants were analysed for proper construction of pET30-GalU by PCR amplification and sequencing. The plasmid expresses GalU with an in-frame His6 tag fused to the N terminus.
Preparation of cell-free extracts containing GalU.
The His6-GalU protein was overexpressed from E. coli BL21(DE3) (Novagen) containing pET30-GalU by growing the strain for 18 h at 37 °C in LB medium supplemented with kanamycin. The culture was then diluted 1 : 100 in fresh medium, and incubation was continued until the culture reached OD600 0.6. Expression of the fusion protein was induced by adding isopropyl-1-thio-β-D-galactopyranoside to the culture (1 mM final concentration) and an additional 3 h incubation. The cells were harvested, washed once with 50 mM HEPES (pH 7.5), and then frozen until needed. The cell pellet was resuspended in 50 mM HEPES (pH 7.5) and sonicated on ice (for a total of 2 min using 10 s bursts followed by 10 s cooling periods at 0.9 W). Unbroken cells, cell debris, and the membrane fraction were removed by ultracentrifugation at 100 000 g for 60 min. For comparison, a soluble extract was prepared using the same protocol from E. coli BL21(DE3) containing the pET30 vector. Protein expression was monitored by SDS-PAGE, and protein contents of the pET30 and pET30-GalU lysates were determined using the Bio-Rad Bradford assay as directed by the manufacturer.
Purification of His6-GalU.
Lysate containing the His6-GalU protein was prepared as described above, except that the pellet was washed and resuspended in 20 mM sodium phosphate buffer (pH 7.4) containing 500 mM NaCl (buffer A). The His6-GalU was purified on an ÄKTA Explorer 100 system (Amersham Biosciences) using a 1 ml HiTrap chelating HP column (Amersham Biosciences) previously loaded with nickel sulfate and equilibrated with buffer A as recommended by the manufacturer. The column was washed with 5 mM imidazole in buffer A for 10 column volumes, and a step gradient of 50–500 mM imidazole in buffer A was then applied. His6-GalU was eluted from the column at an imidazole concentration of 300 mM. The buffer was exchanged into 50 mM HEPES (pH 7.5) with 50 mM NaCl using a HiPrep 26/10 desalting column (Amersham Biosciences) according to the manufacturer's instructions. The protein was concentrated using a Centriplus 10 ml YM-30 centrifugal filter device (Amicon Bioseparations), and typical protein preparations contained yields of 0.07–0.08 g ml–1 as determined by the Bio-Rad Bradford assay.
Cell-surface isolation and analyses.
Cell envelopes were prepared by lysis of whole cells in a French press at 16 000 p.s.i (110.4 MPa). Unbroken cells were removed by centrifugation at 10 000 g for 10 min, and the envelope fraction was collected by centrifugation at 100 000 g for 2 h. Cytoplasmic membranes were solubilized twice with sodium N-laurylsarcosinate (Filip et al., 1973), and the outer-membrane fraction was collected as described above. Outer-membrane proteins were analysed by SDS-PAGE by the Laemmli procedure (Nogueras et al., 2000). Protein gels were fixed and stained with Coomassie blue. Cultures for chemical analysis of LPS were grown in TSB at 37 °C. LPS was purified by the method of Westphal & Jann (1965), resulting in a 2.3 % yield. 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 as previously described (Darveau & Hancock, 1983).
Chemicals.
All chemicals used in the LPS isolation and analyses were purchased from Sigma-Aldrich. Sephadex G-50 (S) was from Amersham Biosciences; phosphoglucomutase and glucose-6-phosphate dehydrogenase were from Sigma.
Isolation and mild acid degradation of the LPS.
Bacterial cells grown in 14 l TSB for 18 h at 30 °C (5.57 g dry weight) were digested with DNase, RNase (24 h, 1 mg g–1 each) and Proteinase K (36 h, 1 mg g–1) in water (60 ml) at 37 °C with stirring; the suspension was dialysed against distilled water and freeze-dried. Digested cells were extracted with aqueous 45 % phenol at 68 °C (Westphal & Jann, 1965); the extract was dialysed against tap water without separation of the layers and, after removal of residual cells by centrifugation, freeze-dried to give the LPS (300 mg).
Degradation of the LPS
N,O-Deacylation.
A LPS sample (100 mg) was deacylated with 4 M potassium hydroxide (4 ml) in the presence of sodium borohydride (20 mg) by heating at 100 °C for 16 h using a block heater. The product was fractionated by high-performance anion-exchange chromatography on a CarboPac PA1 column (Dionex; 250x9 mm) using a linear gradient of 0.1
0.8 M sodium acetate in 0.1 M sodium hydroxide at a flow rate of 3 ml min–1 for 1 h. Oligosaccharide fractions 1 and 2 were eluted at a concentration of sodium acetate 
0.5 M and desalted on a column (1.6x80 cm) of Sephadex G-15.
O-Deacylation.
A LPS sample (20 mg) was treated with anhydrous hydrazine (1 ml) for 1 h at 50 °C. The mixture was cooled down to 0 °C and poured into cooled acetone (200 ml) with stirring. The precipitated material was collected by centrifugation, dissolved in water and lyophilized to yield O-deacylated LPS (15 mg).
Mild acid degradation.
A LPS sample (40 mg) was treated with aqueous 2 % acetic acid (100 °C, 2 h), the precipitate was removed by centrifugation, and the supernatant was fractionated by gel-permeation chromatography on a column (80x2.5 cm) of Sephadex G-50 (S) using pyridinium acetate buffer (4 ml pyridine and 10 ml acetic acid in 1 l water) as the eluant and a Waters differential refractometer (USA) for monitoring. Two core oligosaccharide fractions, 3 and 4 (12 and 3 mg, elution volumes 2.84 and 2.98 related to the column void volume, respectively were obtained as well as a monosaccharide fraction, which was not studied further.
Monosaccharide analysis.
A sample of the O-deacylated LPS (2 mg) was N-acetylated with acetanhydride (0.1 ml) in aqueous saturated sodium hydrogen carbonate (1 ml) in the presence of an excess of solid sodium hydrogen carbonate with stirring for 30 min, desalted on Sephadex G-15, hydrolysed with 4 M trifluoroacetic acid (100 °C, 2 h), dried under a stream of nitrogen and conventionally reduced with sodium borohydride. After adding acetic acid and methanol (2x1 ml), the sample was dried, acetylated with acetanhydride (0.5 ml, 100 °C, 20 min), dried and analysed by GLC.
NMR spectroscopy and mass spectrometry.
1H and 13C NMR spectra were recorded using a Varian UNITY/Inova 500 spectrometer in deuterium oxide solutions at 25 °C with acetone standard (
2.225 for 1H and 31.5 for 13C) using software from the manufacturer and standard pulse sequences COSY (correlation spectroscopy), TOCSY (total correlation spectroscopy; mixing time 120 ms), NOESY (nuclear Overhauser effect spectroscopy; mixing time 300 ms), 1H,31P HMQC (heteronuclear multiple-quantum coherence), 1H,13C HSQC (heteronuclear single-quantum coherence), gHMBC (gradient-enhanced heteronuclear multiple-band correlation; optimized for 5 Hz coupling constant) and HSQC-TOCSY (mixing time 80 ms).
Electrospray ionization mass spectrometry (ESIMS) was performed in the negative ion mode using a Micromass Quattro spectrometer with direct injection in aqueous 50 % acetonitrile with 0.2 % formic acid at a flow rate of 15 µl min–1.
Cell-free extract production and enzymic activity measurements in cell-free extracts or purified recombinant protein (UDP-glucose pyrophosphorylase).
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 using the Bio-Rad Bradford assay as directed by the manufacturer with BSA as the standard.
The activity of UDP-glucose pyrophosphorylase was determined by the procedure of Albrecht et al. (1966) with some modification. The reaction mixture contained 100 mM Tris/HCl, pH 8.0, 2.0 mM MgCl2, 1.0 mM 2-mercaptoethanol, 1.0 mM UDP-glucose, 1.0 mM NADP, 1.0 mM sodium pyrophosphate, 8.0 µM glucose 1,6-bisphosphate, 5 units each of phosphoglucomutase and glucose-6-phosphate dehydrogenase, and the protein sample (either cell-free extract or purified recombinant protein). In a final volume of 1 ml, the reaction was started by the addition of the protein sample, and the change of corresponding absorbance at 340 nm was determined for 1 h. Specific activity was expressed as the amount of NADPH synthesized per min per mg protein at 25 °C.
Measurements of UDP-glucose in dried cells or cell-free extracts.
Dried bacterial cells were weighed and then suspended [50 mg dried cells (ml acid)–1] at 0 °C in 0.02 M HCl. The suspensions were centrifuged at 8900 g for 10 min at 4 °C, and supernatants collected for analyses. UDP-glucose was measured by a modification of the fluorometric assay described by Passonneau & Lowry (1992). Briefly, the HCl extracts were neutralized with NaOH, then after adding 0.1 mM EDTA [100 µl (ml extract)–1], the extracts were boiled for 5 min and centrifuged at 8900 g for 20 min at 4 °C. Aliquots of the these supernatants (500 µl) or cell-free extracts (500 µl) were added to 500 µl solutions containing 2 mM MgCl2, 2 mM NAD+, 0.016 unit (ml UDP-glucose dehydrogenase)–1, and 50 mM Tris/HCl, pH 8.1. The samples were incubated for 2 h at 24 °C, then the NADH generated was measured fluorometrically. After subtracting blank values from incubations conducted without UDP-glucose dehydrogenase, the UDP-glucose content of the samples was determined from a standard curve generated with known concentrations of UDP-glucose.
Study of some pathogenic features.
Bacterial survival in human serum and adherence assay to HEp-2 cells were performed as previously described (Canals et al., 2006a).
Virulence for fish and mice.
The virulence of the strains grown in TSB for 24 h at 20 °C was measured by monitoring their LD50 by the method of Reed and Muench, as previously described (Canals et al., 2006a).
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RESULTS |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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) in order to obtain mutants devoid of the O34-antigen LPS. Mutant AH-2886 was one of the 36 amongst 1500 colonies that were initially screened and showed a complete resistance to bacteriophage PM1 when compared to the wild-type (sensitive to this bacteriophage). Whole cells of mutant AH-2886 were unable to react in an enzyme immunoassay with specific antiserum against O34-antigen LPS and, as can be observed in Fig. 2 (lane 2), the LPS of strain AH-2886 was devoid of O34-antigen. The LPS-core showed two bands with a faster mobility in comparison with the wild-type strain. Neither whole AH-2886 cells nor its purified LPS were able to react with specific antiserum against O34-antigen LPS in enzyme immunoassays (Benedí et al., 1989). Strain AH-2886 was unable to grow in minimal medium with galactose as sole carbon source while the wild-type strain was fully able to do so. No differences between the wild-type and the mutant were observed using glucose as sole carbon source. No major differences were found in the growth rate (in Davis minimal medium) between mutant strain AH-2886 (generation time 45 min) and the wild-type strain AH-3 (generation time 40 min). Comparative SDS-PAGE analysis of the outer-membrane protein profile indicated that the mutant and the wild-type strain showed essentially the same protein bands (data not shown). Southern blot analysis using a specific probe for the transposon demonstrated that mutant AH-2886 had a single copy of the mini-transposon in its genome (data not shown). In order to identify the gene(s) responsible for the observed phenotype, genomic DNA was isolated from mutant AH-2886, partially digested with EcoRV, and DNA fragments of about 5.0 kb were ligated to vector pBluescript SK. The DNA ligation mixture was transformed into E. coli DH5
; colonies growing on kanamycin and ampicillin plates were recovered, and analysed again by Southern blotting with the mini-Tn5-specific probe to identify clones containing the transposon and surrounding chromosomal DNA from mutant AH-2886. The nucleotide sequence of the insert of one such recombinant plasmid was determined by using oligonucleotides 5'-AGATCTGATCAAGAGACAG-3' and 5'-ACTTGTGTATAAGAGTCAG-3' (from the mini-Tn5Km-1 flanking regions), and the oligonucleotides T3 and T7 from the plasmid vector. Analysis of the sequence allowed the identification of an ORF, interrupted by the mini-Tn5, which encoded a protein with identity/similarity (73/85 %, respectively) to several GalU proteins from different Gram-negative bacteria. For this reason we named this ORF galU.
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was constructed as previously described (Nogueras et al., 2000). This library was screened by colony blotting using a DNA probe to the galU ORF of A. hydrophila AH-3. Several positive recombinant clones were identified, of which clone COS-GALU was chosen for further analysis because it was able to completely complement the AH-2886 mutation by rescuing the wild-type strain pattern of bacteriophage sensitivity, growth in minimal medium with galactose as sole carbon source, and complete LPS as shown by SDS-PAGE (Fig. 2
). The DNA sequence of galU, where the mini-Tn5 was inserted, as well as the surrounding region in cosmid COS-GALU, indicates the presence of two flanking ORFs (ORF1 and ORF2) to galU (Fig. 3). The upstream incomplete ORF1 transcribed in the same direction as galU after a stop codon was found to be similar/identical (71/75 %) in the BLASTX to several dipeptide/tripeptide permeases from Photobacterium and Vibrio species. The downstream complete ORF2 transcribed in the same direction showed identity/similarity (50/67 %) (in the helix–hairpin–helix region) to competence proteins ComEA of several Gram-negative bacteria), such as Actinobacillus pleuropneumoniae and Shewanella sp. This genetic organization and the transcriptional direction for these genes (Fig. 3) strongly suggest that the mutant AH-2886 phenotype is attributable only to galU insertion mutation, since no polar effects on downstream genes should be expected. This was confirmed since reintroduction of the single galU (plasmid pACYC-GALU) rescues the complete wild-type phenotype in mutant AH-2886, i.e. phage sensitivity, growth in minimal medium with galactose as sole carbon source, and presence of O34-antigen LPS (Fig. 2).
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). The mesophilic Aeromonas strains used were environmental strains (n=15) isolated mainly from water samples (n=10) and shellfish (n=5) by using ampicillin dextrin agar or Tergitol agar, and clinical strains (n=35) isolated from blood agar supplemented or not with ampicillin depending upon their intestinal (n=25) or extraintestinal (n=10) origin. All the strains (not belonging to serotype O34) were identified to the species level by 16S rDNA restriction fragment length polymorphism analysis (Figueras et al., 2000). In all the strains tested a single 1274 bp band was amplified. To confirm that galU was indeed present, the nucleotide sequence of the DNA-amplified fragments from several of the Aeromonas strains was determined. Thus, galU was found in all the mesophilic Aeromonas strains tested.
To determine if galU is also involved in the production of non-O34-antigen LPSs we constructed galU-defined insertion mutants in several mesophilic Aeromonas reference strains belonging to different O serotypes (O1, O2, O11, O18, O21 and O44). LPS from the reference rifampicin-resistant strains O1, O2, O11, O18, O21 and O44 showed O-antigen LPS on SDS-PAGE (Fig. 4). All the galU mutants obtained with plasmid pFS-GALU from these strains are in agreement with the AH-2886 mutant phenotype: they lack the O-antigen LPS as shown by SDS-PAGE, have a LPS core with two bands with higher migration than the unique wild-type LPS-core band, and are unable to grow in minimal medium with galactose as sole carbon source. All the galU mutants can be complemented by the reintroduction of galU in plasmid pACYC-GALU as judged by recovery of the O-antigen LPS in gels (Fig. 4) or the ability to grow in minimal medium with galactose as sole carbon source.
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, lanes 1 and 2). This suggested that the mutant AH-2886 LPS has a truncated core moiety.
The LPS from mutant AH-2886 was fully deacylated by strong alkaline hydrolysis, and the product was fractionated by high-performance anion-exchange chromatography on CarboPac PA1 to give two oligosaccharides 1 and 2. They were studied by 1H, 13C and 31P NMR spectroscopy, including full assignment of the signals and fixation of chemical shifts correlations by two-dimensional NMR techniques, including 1H,1H COSY, TOCSY, NOESY, 1H,31P HMQC, 1H,13C HSQC, HMBC and HSQC-TOCSY experiments according to the published methodology (Duus et al., 2000) (for chemical shift assignments see supplementary Table S1, available with the online version of this paper). The results showed that the core region in the major compound 1 has almost the same structure as the corresponding oligosaccharide obtained in a similar manner from the R-type LPS of mutant AH-901 strain studied by us earlier (Knirel et al., 2004). The only difference between the two was the lack of the terminal galactose residue from 1, whereas both Gal-containing and Gal-lacking compounds were isolated from the LPS of mutant AH-901 (Knirel et al., 2004). The minor oligosaccharide 2 had a deeply truncated structure with the core restricted to one 3-deoxy-D-manno-oct-2-ulosonic acid (Kdo) and three L-glycero-D-manno-heptose (LD-Hep) residues. The structure of 2 was determined by NMR spectroscopy, and it was confirmed to be a part of the major oligosaccharide 1. The structures of oligosaccharides 1 and 2 are shown in Fig. 5.
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)] . The absence of Gal from this compound was finally confirmed by monosaccharide analysis, which revealed Glc, GlcN, DD-Hep and LD-Hep in approximate ratios 1 : 1 : 2 : 4.
The mass spectrum of the O-deacylated LPS contained no peak corresponding to the minor compound 2, probably because of its low content. However, ESIMS analysis of the products obtained by mild acid degradation of the mutant AH-2886 LPS showed the presence of both expected oligosaccharides 3 and 4 (molecular masses 1696.6 and 797.3 Da, respectively, Fig. 5) thus showing that 2 was no artefact induced by alkaline degradation of the LPS. The two types of the LPS core structures are evidently represented by two bands shown by the mutant AH-2886 LPS in SDS-PAGE (Fig. 2, lane 2).
UDP-glucose pyrophosphorylase activity
The genetic analysis as well as the LPS chemical structure of the mutants prompted us to study the enzymic activity that allows the production of UDP-glucose from glucose 1-phosphate (UDP-glucose pyrophosphorylase). A. hydrophila AH-3 (wild-type strain) grown on glucose or galactose showed a high enzymic activity (Table 2), when measured as described in Methods. However, mutant strain AH-2886 with or without the plasmid vector (pACYC184) showed a marked reduction in enzymic activity (Table 2), which could be rescued by the reintroduction of the single gene using plasmid pACYC-GALU. As described for other galU mutants (Nesper et al., 2001), AH-2886 (or with plasmid vector) was unable to grow on minimal medium with galactose, while the same strain plus plasmid pACYC-GALU was able to do so.
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Measurements of UDP-glucose in dried cells and cell-free extracts
The neutralized HCl extracts from dried cells obtained according to Methods from the wild-type strain AH-3 had UDP-glucose contents of 25±3.0 nmol (mg dry weight)–1. However, the values obtained for the mutant strain AH-2886 (either with or without the plasmid vector) were always about 10±0.4 nmol UDP-glucose (mg dry weight)–1. When strain AH-2886 was complemented with plasmid pACYC-GALU, it had a similar UDP-glucose content [24±2.0 nmol) (mg dry weight)–1] to that of the wild-type strain. Also, the cell-free extracts from strain AH-3 (wild-type) or the mutant complemented with plasmid pACYC-GALU had UDP-glucose contents at least three times higher than those obtained with mutant strain AH-2886 with or without the plasmid vector.
Serum killing
Wild-type mesophilic Aeromonas strains belonging to serotypes O1, O2, O11, O18, O21, O34 and O44 were resistant to the bactericidal activity of non-immune human serum (NHS) (98 % survival after 3 h incubation with NHS), while their respective galU insertion mutants or the same mutants with plasmid vector pACYC184 were sensitive (less than 1 % survival after 3 h incubation with NHS). The introduction of galU (pACYC-GALU) rendered galU insertion mutants resistant to the bactericidal activity of NHS (>90 % survival after 3 h incubation with NHS), as found for their respective wild-type strains.
Adherence to HEp-2 cells
Table 3 shows the adhesion of O34 wild-type strain, galU mutant and complemented mutant to HEp-2 cells. The wild-type strains showed a high percentage of adherence to HEp-2 cells, while the galU mutant (either with the plasmid vector alone) showed a large decrease (60 % reduction) in the percentage adhesion to HEp-2 cells. The introduction of galU (pACYC-GALU) rescued completely the adherence to Hep-2 cells in the mutant strain. As can be observed in Table 3, all the galU mutants obtained from several mesophilic Aeromonas strains (serotypes O1, O2, O11, O18, O21 and O44) showed a large reduction in the adhesion to HEp-2 cells.
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. The galU mutant AH-2886 showed a higher LD50 (an increase of 1.5–2 log units) in both fish and mice in comparison with the wild-type LD50. No differences were found in mortality between the wild-type strain and its rifampicin-resistant mutant. Complementation of the defined insertion mutant (AH-2886) with pACYC-GALU (carrying the single galU) completely restored its virulence for fish and mice (similar LD50 to the wild-type strain; Table 4), while no changes were observed with the plasmid vector alone.
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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). We proved that A. hydrophila GalU is a UDP-glucose pyrophosphorylase responsible for the chemical reaction mentioned above. Besides its function as a substrate for glucosyltransferases resulting in glucosylated surface structures, UDP-glucose plays a well-established biochemical role as a glycosyl donor in the enzymic biosynthesis of carbohydrates. Some examples in Gram-negative bacteria are synthesis of the osmoprotectants trehalose (under conditions of high osmolarity) and membrane-derived oligosaccharide (under conditions of low osmolarity) in E. coli (Csonka & Epstein, 1996); synthesis of UDP-4-amino-4-deoxy-L-arabinose, which is incorporated into lipid A of Salmonella enterica serovar Typhimurium to protect against antimicrobial cationic peptides, especially polymyxin B (Ernst et al., 1999); and synthesis of UDP-galactose, which serves as a donor for several surface structures, including glycosylated pili in Neisseria meningitidis (Stimson et al., 1995). It is less clear whether UDP-glucose additionally acts as an intracellular signal molecule. For one study, it was reported that UDP-glucose controls the expression of 
s in E. coli (Böhringer et al., 1995).
The LPS from several galU mutants in SDS-PAGE, growing in either TSB or minimal medium with glucose as sole carbon source, showed the absence of the O-antigen and a higher mobility of the LPS-core two bands as compared with the corresponding unique band of their respective wild-type strain's LPS-core. Taking advantage of the complete determined LPS structure of strain AH-3 (serotype O34), we could establish that the galU mutant from this strain showed two types (represented by two bands on LPS gels) of LPS structures by several chemical analyses: one (the major product representing the LPS band on the gels with slow migration), which is the complete LPS-core but devoid of galactose residue in the outer core where the O34-antigen LPS is linked; and a second one (the minor product representing the LPS band on the gels with fast migration), which is a deeply truncated structure with the core restricted to one Kdo and three LD-Hep residues (see Figs 1 and 5). The low amount of UDP-glucose in the mutant compared to the wild-type could explain its inability to complete the LPS-core structure. In approximately 10 % of the LPS molecules the mutant is unable to transfer the glucose residue to the inner core, and the LPS-core is not further extended (minor LPS product, oligosaccharide 2 in Fig. 5). In the rest of the LPS molecules (90 %), the mutant is able to incorporate the glucose residue to the inner core and the outer core is added (major LPS product, oligosaccharide 1 in Fig. 5). However, in this case also the mutant cannot complete the LPS-core because it is always unable to incorporate the final Gal residue. Of course the lack of galactose residue explains why the mutant is unable to express the O34-antigen LPS. Furthermore, not only the lack of the galactose residue from the outer core but also the lack of Ara4N residues from the lipid A backbone is due to the low level of UDP-glucose. In view of the results obtained in other galU mutants from different Aeromonas strains, these facts about LPS seem to be general, at least from what we observed in LPS gels (two bands in the core LPS region lacking the O-antigen LPS). Complementation with the single A. hydrophila galU gene completely restored all the LPS defects. To our knowledge this is the first time that well-characterized LPS structural defects have been correlated with a reduced level of a nucleotide diphosphate sugar (UDP-glucose) in the intracellular pool.
The Aeromonas galU mutation would prevent the growth on galactose as sole carbon source for similar reasons to those established in other bacteria. In E. coli, galactose is converted to galactose 1-phosphate, which in turn leads to the formation of glucose 6-phosphate, which enters the glycolytic pathway. To convert galactose 1-phosphate to glucose 1-phosphate, the GalT enzyme needs UDP-glucose. In the absence of GalU, not enough UDP-glucose would be present to form glucose 1-phosphate. In addition, the accumulation of galactose 1-phosphate could have a toxic effect and explain the inability to grow in minimal medium with galactose as sole carbon source (Priebe et al., 2004). Furthermore, the low amount of UDP-glucose found in Aeromonas galU mutants could not be explained by GalE (UDP-glucose epimerase) or by GalT, due to the inability of these mutants to grow in minimal medium with galactose as sole carbon source. Since there are several reactions where UDP-glucose is used in biosynthesis [e.g. trehalose production in E. coli (Purvis et al., 2005)], perhaps some of these reactions are reversible enough in Aeromonas galU mutants to generate the low amount of UDP-glucose needed to introduce a single glucose residue into the LPS-core.
The galU mutation also drastically affected the serum susceptibility of several Aeromonas strains from different serotypes, reduced the ability of these strains to adhere, and decreased the virulence in a septicaemic model either in fish or mice. The O-antigen LPS from several Gram-negative bacteria (smooth strains) impedes the attachment of C3b (complement opsonin) to the membrane, and thus the strain becomes resistant to the bactericidal activity of serum (Taylor, 1988). Furthermore, the Aeromonas O-antigen LPS has been described as an adhesin which facilitates the attachment to eukaryotic cells (Francki & Chang, 1994).
Results obtained with waaL mutants of P. aeruginosa (lacking the O-antigen LPS) indicated that LPS of P. aeruginosa plays a role in flagellar biogenesis in this species (Abeyrathne et al., 2005). However, this is not the case in A. hydrophila galU mutants because the flagellar biogenesis is unaffected. The alteration in motility observed for these mutants could be attributed to the lack of a hydrophilic surface component such as O-antigen LPS. It is well known that the O-antigen LPS contributes to the wettability of the bacterial surfaces (Toguchi et al., 2000).
All the changes observed in the galU mutants in these virulence experiments are rescued by the introduction of the corresponding single wild-type gene, but not by the introduction of the plasmid vector alone, i.e. by the recovery of O-antigen LPS. Thus, O-antigen LPS is essential for mesophilic Aeromonas virulence. To our knowledge this is the first report relating the intracellular level of UDP-glucose to virulence through the LPS structure.
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ACKNOWLEDGEMENTS |
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Edited by: P. van der Ley
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REFERENCES |
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Albrecht, G. J., Bass, S. T., Seifert, L. L. & Hansen, R. G. (1966). Crystallization and properties of uridine diphosphate glucose pyrophosphorylase from liver. J Biol Chem 241, 2968–2975.
Allen, L. N. & Hanson, R. S. (1985). Construction of broad host-range cosmid cloning vector: identification of genes necessary for growth of Methylobacterium organophilum on methanol. J Bacteriol 161, 955–962.
Altschul, S. F., Madden, T. L., Schäffer, A. A., Zhang, J., Zhang, Z., Miller, W. & Lipman, D. J. (1997). Gapped BLAST and PSI-BLAST: a new generation of protein database search programs. Nucleic Acids Res 25, 3389–3402.
Atlas, R. M. (1993). Handbook of Microbiological Media. Boca Raton, FL: CRC Press.
Austin, B. & Adams, C. (1996). Fish pathogens. In The Genus Aeromonas, pp. 197–243. Edited by B. Austin, M. Altwegg, P. J. Gosling & S. W. Joseph. New York: Wiley.
Bateman, A., Birney, E., Cerruti, L., Durbin, R., Etwiller, L., Eddy, S. R., Griffiths-Jones, S., Howe, K. L., Marshall, M. & Sonnhammer, E. L. (2002). The Pfam protein families database. Nucleic Acids Res 30, 276–280.
Benedí, V. J., Ciurana, B. & Tomás, J. M. (1989). Isolation and characterization of Klebsiella pneumoniae unencapsulated mutants. J Clin Microbiol 27, 82–87.
Böhringer, J., Fischer, D., Mosler, G. & Hengge-Aronis, R. (1995). UDPglucose is a potential intracellular signal molecule in the control of expression of s-dependent genes in Escherichia coli. J Bacteriol 177, 413–422.
Canals, R., Jiménez, N., Vilches, S., Regué, M., Merino, S. & Tomás, J. M. (2006a). A gene (uridine diphosphate N-acetylgalactosamine 4-epimerase) is essential for mesophilic Aeromonas serotype O34 virulence. Infect Immun 74, 537–548.
Canals, R., Ramirez, S., Vilches, S., Horsburgh, G., Shaw, J. G., Tomás, J. M. & Merino, S. (2006b). Polar flagella biogenesis in Aeromonas hydrophila. J Bacteriol 188, 542–555.
Canals, R., Altarriba, M., Vilches, S., Horsburgh, G., Shaw, J. G., Tomás, J. M. & Merino, S. (2006c). Analysis of the lateral flagella gene system of Aeromonas hydrophila AH-3. J Bacteriol 188, 852–862.
Chang, H. Y., Lee, J. H., Deng, W. L., Fu, T. F. & Peng, H. L. (1996). Virulence and outer membrane properties of a galU mutant of Klebsiella pneumoniae CG43. Microb Pathog 20, 255–261.[CrossRef][Medline]
Csonka, L. N. & Epstein, W. (1996). Osmoregulation. In Escherichia coli and Salmonella: Cellular and Molecular Biology, 2nd edn, pp. 1210–1223. Edited by F. C. Neidhardt and others. Washington, DC: American Society for Microbiology.
Darveau, R. P. & Hancock, R. E. (1983). Procedure for isolation of bacterial lipopolysaccharides from both smooth and rough Pseudomonas aeruginosa and Salmonella typhimurium strains. J Bacteriol 155, 831–838.
De Lorenzo, V., Herrero, M., Jakubzik, U. & Timmis, K. N. (1990). Mini-Tn5 transposon derivatives for insertion mutagenesis, promoter probing, and chromosomal insertion of cloned DNA in gram-negative eubacteria. J Bacteriol 172, 6568–6572.
Duus, J. Ø., Gotfredsen, C. H. & Bock, K. (2000). Carbohydrate structural determination by NMR spectroscopy: modern methods and limitations. Chem Rev 100, 4589–4614.[CrossRef][Medline]
Ernst, R. K., Guina, T. & Miller, S. I. (1999). How intracellular bacteria survive: surface modifications that promote resistance to host innate immune responses. J Infect Dis 179 (Suppl. 2), S326–S330.[CrossRef][Medline]
Evans, D., Kuo, T., Kwong, M., Van, R. & Fleiszig, S. (2002). Pseudomonas aeruginosa strains with lipopolysaccharide defects exhibit reduced intracellular viability after invasion of corneal epithelial cells. Exp Eye Res 75, 635–643.[CrossRef][Medline]
Figueras, M. J., Soler, L., Chacón, M. R., Guarro, J. & Martínez-Murcia, A. J. (2000). Extended method for discrimination of Aeromonas spp. by 16S rDNA-RFLP. Int J Syst Evol Microbiol 50, 2069–2073.[Abstract]
Filip, C., Fletcher, G., Wulff, J. L. & Earhart, C. F. (1973). Solubilization of the cytoplasmic membrane of Escherichia coli by the ionic detergent sodium lauryl sarcosinate. J Bacteriol 115, 717–722.
Finlay, B. B. & McFadden, M. (2006). Anti-immunology: evasion of host immune system by bacterial and viral pathogens. Cell 124, 767–782.[CrossRef][Medline]
Francki, K. T. & Chang, B. J. (1994). Variable expression of O-antigen and the role of lipopolysaccharide as an adhesin of Aeromonas sobria. FEMS Microbiol Lett 122, 97–102.[CrossRef][Medline]
Hanahan, D. (1983). Studies on transformation of Escherichia coli with plasmids. J Mol Biol 166, 557–580.[Medline]
Janda, J. M. & Abbott, S. L. (1998). Evolving concepts regarding the genus Aeromonas: an expanding panorama of species, disease presentations, and unanswered questions. Clin Infect Dis 27, 332–344.[Medline]
Knirel, Y. A., Shaskov, A. S., Senchenkova, S. N., Merino, S. & Tomás, J. M. (2002). Structure of the O-polysaccharide of Aeromonas hydrophila O34: a case of random O-acetylation of 6-deoxy-L-talose. Carbohydr Res 337, 1381–1386.[CrossRef][Medline]
Knirel, Y. A., Vinogradov, E., Jimenez, N., Merino, S. & Tomás, J. M. (2004). Structural studies on the R-type lipopolysaccharide of Aeromonas hydrophila. Carbohydr Res 339, 787–793.[CrossRef][Medline]
Komeda, Y., Icho, T. & Iino, T. (1977). Effects of galU mutation on flagellar formation in Escherichia coli. J Bacteriol 129, 908–915.
Merino, S., Camprubí, S. & Tomás, J. M. (1992). Characterization of an O-antigen bacteriophage from Aeromonas hydrophila. Can J Microbiol 38, 235–240.[Medline]
Merino, S., Camprubí, S. & Tomás, J. M. (1993). Incidence of Aeromonas spp serotypes O34 and O : 11 among clinical isolates. Med Microbiol Lett 2, 48–55.
Merino, S., Rubires, X., Aguilar, A., Guillot, J. F. & Tomás, J. M. (1996). The role of the O-antigen lipopolysaccharide on the colonization in vivo of the germfree chicken gut by Aeromonas hydrophila serogroup O34. Microb Pathog 20, 325–333.[CrossRef][Medline]
Merino, S., Rubires, X., Aguilar, A. & Tomás, J. M. (1997). The role of flagella and motility in the adherence and invasion to fish cell lines by Aeromonas hydrophila serogroup O34 strains. FEMS Microbiol Lett 151, 213–217.[CrossRef][Medline]
Nesper, J., Lauriano, C. M., Klose, K. E., Kapfhammer, D., Kraiss, A. & Reidl, J. (2001). Characterization of Vibrio cholerae O1 El Tor galU and galE mutants: influence on lipopolysaccharide structure, colonization, and biofilm formation. Infect Immun 69, 435–445.
Nogueras, M. M., Merino, S., Aguilar, A., Benedí, V. J. & Tomás, J. M. (2000). Cloning, sequencing and role in serum susceptibility of porin II from mesophilic Aeromonas sp. Infect Immun 68, 1849–1854.
Passonneau, J. V. & Lowry, O. H. (1992). Enzymatic Analysis: a Practical Guide. Clifton, NJ: Humana Press.
Priebe, G. P., Dean, C. R., Zaidi, T., Meluleni, G. J., Coleman, F. T., Coutinho, Y. S., Noto, M. J., Urban, T. A., Pier, G. B. & Goldberg, J. B. (2004). The galU gene of Pseudomonas aeruginosa is required for corneal infection and efficient systemic spread following pneumonia but not for infection confined to the lung. Infect Immun 72, 4224–4232.
Purvis, J. E., Yomano, L. P. & Ingram, L. O. (2005). Enhanced trehalose production improves growth of Escherichia coli under osmotic stress. Appl Environ Microbiol 71, 3761–3769.
Rabaan, A. A., Gryllos, I., Tomás, J. M. & Shaw, J. G. (2001). Motility and the polar flagellum are required for Aeromonas caviae adherence to Hep-2 cells. Infect Immun 69, 4257–4267.
Rioux, S., Galarneau, C., Harel, J., Frey, J., Nicolet, J., Kobisch, M., Dubreuil, J. D. & Jacques, M. (1999). Isolation and characterization of mini-Tn10 lipopolysaccharide mutants of Actinobacillus pleuropneumoniae serotype 1. Can J Microbiol 45, 1017–1026.[CrossRef][Medline]
Rubirés, X., Saigí, F., Piqué, N., Climent, N., Merino, S., Albertí, S., Tomás, J. M. & Regué, M. (1997). A gene (wbbL) from Serratia marcescens N28b (O4) complements the rfb-50 mutation of Escherichia coli K-12 derivatives. J Bacteriol 179, 7581–7586.
Sambrook, J., Fritsch, E. F. & Maniatis, T. (1989). Molecular Cloning: a Laboratory Manual, 2nd edn. Cold Spring Harbor, NY: Cold Spring Harbor Laboratory.
Sandlin, R. C., Lampel, K. A., Keasler, S. P., Goldberg, M. B., Stolzer, A. L. & Maurelli, A. T. (1995). Avirulence of rough mutants of Shigella flexneri: requirement of O antigen for correct unipolar localization of IcsA in the bacterial outer membrane. Infect Immun 63, 229–237.[Abstract]
Sanger, F., Nicklen, S. A. & Coulson, R. (1977). DNA sequencing with chain-terminating inhibitors. Proc Natl Acad Sci U S A 74, 5463–5467.
Stimson, E., Virji, M., Makepeace, K., Dell, A., Morris, H. R., Payne, G., Saunders, J. R., Jennings, M. P., Barker, S. & other authors (1995). Meningococcal pilin: a glycoprotein substituted with digalactosyl 2,4-diacetamid-2,4,6-trideoxyhexose. Mol Microbiol 17, 1201–1214.[CrossRef][Medline]
Taylor, P. W. (1988). Bacterial resistance to complement. In Virulence Mechanisms of Bacterial Pathogens, pp. 107–120. Edited by J. A. Roth. Washington, DC: American Society for Microbiology.
Toguchi, A., Siano, M., Burkart, M. & Harshey, R. M. (2000). Genetics of swarming motility in Salmonella enterica serovar typhimurium: critical role for lipopolysaccharide. J Bacteriol 182, 6308–6321.
Vilches, S., Urgell, C., Merino, S., Chacón, M. R., Soler, L., Castro-Escarpulli, G., Figueras, M. J. & Tomás, J. M. (2004). Complete type III secretion system of a mesophilic Aeromonas hydrophila strain. Appl Environ Microbiol 70, 6914–6919.
Westphal, O. & Jann, K. (1965). Bacterial lipopolysaccharides. Extraction with phenol-water and further applications of the procedure. In Methods in Carbohydrate Chemistry, vol. 5, General Polysaccharides, pp. 83–91. Edited by R. L. Whistler. New York: Academic Press.
Received 25 January 2007;
revised 8 March 2007;
accepted 10 April 2007.
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