Department of Microbiology, Faculty of Pharmacy, University of Granada, Campus Universitario de Cartuja, 18071, Granada, Spain
Correspondence
Ana del Moral
admoral{at}ugr.es
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ABSTRACT |
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The GenBank/EMBL/DDBJ accession number for the sequence reported in this paper is AY918062.
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INTRODUCTION |
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Moderate halophiles, the group to which Halomonas species belong, already have numerous applications in various fields of industry (Jones, 2004; Margesin & Schinner, 2001
; Quesada et al., 2004
; Ventosa et al., 1998
). The genus Halomonas contains about 30 species of halophilic Gram-negative bacteria, most of which have been isolated from saline environments and grow best in media containing 0·5 to 2·5 M NaCl. Four of these species, Halomonas eurihalina (Quesada et al., 1990
), Halomonas maura (Bouchotroch et al., 2001
), Halomonas ventosae (Martínez-Cánovas et al., 2004b
) and Halomonas anticariensis (Martínez-Cánovas et al., 2004a
), all isolated from the rhizosphere of xerophytic plants, have the ability to produce large quantities of EPSs with novel physical and chemical characteristics. Among these, mauran, the EPS produced by strain S-30 of H. maura, is notable for its high viscosifying capacity, similar to that of xanthan, and for the pseudoplastic and thixotropic behaviour of its solutions. In addition, the stability of its functional properties under a wide range of pH, saline and freezingthawing conditions makes this polymer a good candidate for use in foodstuffs, pharmaceutical products and other fields of biotechnology (Arias et al., 2003
). Although extensive studies have been made into the biotechnological applications of these halophilic EPSs (Arias et al., 2003
; Béjar et al., 1998
; Martínez-Checa et al., 2002
), little is known about the molecular mechanism of their biosynthesis or how they are regulated (Llamas et al., 2003
).
The mechanisms involved in assembly, polymerization and translocation across the outer membrane in Gram-negative bacteria follow a similar pathway to that of some capsule EPSs in Escherichia coli groups 1 and 4 (Rahn et al., 1999) and Klebsiella pneumoniae (Arakawa et al., 1995
) and of extracellular polysaccharides, including those produced by Erwinia spp. (Bugert & Geider, 1995
), Methylobacillus sp. strain 12S (Yoshida et al., 2003
), Rhizobium spp. (Reuber & Walker, 1993
) and Xanthomonas campestris (Katzen et al., 1998
). Undecaprenol-pyrophosphate-linked oligosaccharide repeating units are formed at the cytoplasmic face of the inner membrane, transported through this membrane by a Wzx-protein-dependent process, and then polymerized by a mechanism involving a Wzy protein. Some of the polysaccharide-biosynthesis gene clusters have been cloned and sequenced, and found to form long operons with similarities in their genetic organization. In general, the first three genes, which are necessary for high-level polymerization and surface assembly, are conserved in the above-mentioned micro-organisms; the genes are wza (encoding an outer-membrane protein), wzb (encoding an acid phosphatase) and wzc (encoding an inner-membrane tyrosine autokinase) (Drummelsmith & Whitfield, 2000
). It has been suggested that this high level of conservation is the result of lateral transfer events occurring between some of these species (Rahn et al., 1999
). The rest of the genes included in the operon encode glycosyltransferases and components of a Wzy-dependent polymerization system (Rahn et al., 1999
; Stevenson et al., 1996
).
We describe here the strategy used to identify the constituents required for the synthesis of the EPS mauran by H. maura strain S-30. We initially isolated an EPS-defective mutant that carried a single insertion of mini-Tn5 in its genome. An analysis of the regions located both upstream and downstream of the insertion revealed a typical polysaccharide-biosynthesis gene cluster. We identified three conserved genes, epsA, epsB and epsC, and demonstrated their role in the assembly and translocation of mauran. We also found a wzx homologue, epsJ, which indicates that this EPS is formed by a Wzy-dependent polymerization system. Preliminary transcriptional expression assays using a derivate of H. maura S-30, which carries an epsA : : lacZ transcriptional fusion, proved that the eps gene cluster reaches maximum activity during stationary phase, in the presence of high salt concentrations (5 % w/v).
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METHODS |
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Cloning of the epsABCD genes.
To isolate the DNA fragment carrying the mini-Tn5 insertion, and including the Km resistance gene, genomic DNA of H. maura TK71 was completely digested with BamHI, BglII, EcoRI, NcoI and SalI. DNA fragments were separated on an agarose gel and transferred onto a nylon filter by standard techniques (Sambrook & Russell, 2001). Fragments containing the mini-Tn5 Km2 insertion were analysed by Southern blot hybridization using a digoxigenin DNA labelling and detection kit (Boehringer Mannheim) as described previously (Llamas et al., 1997
, 2003
). Chromosomal DNA fragments digested with NcoI and SalI were selected, and ligated into pGEM-T and pUC19, creating pN71K and pS71K.
The genomic sequences located upstream (promoter region) and downstream of the known NcoISalI nucleotide sequence were obtained by inverse PCR (Ochman et al., 1988; Llamas et al., 2003
). Briefly, the chromosomal DNA of H. maura S-30 was completely digested by restriction endonucleases with no restriction sites within the 500 bp region at the 5' end (upstream) or the 500 bp region at the 3' end (downstream) of the sequenced fragment. The appropriate restriction enzymes were selected empirically on the basis of Southern blot hydridization using a digoxigenin DNA labelling and detection kit (Boehringer Mannheim). A 200 bp fragment of known sequence located at the end of the 500 bp sequence was used as a probe and labelled with digoxigenin-11dUTP by PCR. The following oligonucleotides were used as primers for amplifying both probes: 5'-CACCAATGTGCCGATGACCGTGCTGGAT-3' (forward) and 5'-GCTCTCGCTCTGGTAATCCACATGGCTG-3' (reverse) (upstream probe), and 5'-CGAATCGCGCTTTCGTTCGCGTTTCCAG-3' (forward) and 5'-ACAGGAAAGTGCCGCCGATCAACAACAG-3' (reverse) (downstream probe). The selected DNA fragments, of about 1 to 4 kb, were diluted and religated with T4 DNA ligase (Promega) for 16 h at 16 °C under conditions favouring the formation of monomeric circles (Collins & Weissman, 1984
). The resulting intramolecular ligation products were then used as substrates for DNA amplification by PCR using oligonucleotide primers homologous to the ends of the known DNA sequence, but facing in opposite directions. PCR reactions were performed by using 35 cycles of 30 s at 95 °C, 30 s at 72 °C and 1 min at 72 °C. The PCR products were purified and ligated into pBluescript SK (+), thus creating two plasmids, pXmn S-30 and pHind S-30.
DNA manipulations.
Extraction of plasmids from E. coli strains, agarose gel electrophoresis with TBE buffer, and other routine cloning procedures, were carried out according to standard protocols (Sambrook & Russell, 2001).
Nucleotide sequence analysis.
DNA sequences were determined by the dideoxynucleotide-chain-termination method of Sanger et al. (1977) with both universal and specific oligonucleotide primers (Table 2
), and double-stranded plasmid templates. Nucleotide and amino acid sequences were analysed using the DNASTAR program. Sequence comparison and multiple alignments were carried out using the BLAST (Altschul et al., 1990
) and CLUSTAL W (Thompson et al., 1994
) programs. Promoter regions and the putative transcription start site (+1) were predicted with the BDGP Neural Network Promoter Prediction Input program (Waibel et al., 1989
; Reese & Eeckman, 1995
). Transmembrane regions and signal-peptide sequences in the predicted amino acid sequences were found by the TopPred II and SignalP v. 3.0 programs (Claros & Von Heijne, 1994
; Nielsen et al., 1997
).
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Reverse transcription was carried out with 5 µg of RNA in a final volume of 20 µl using an Enhanced Avian HS RT-PCR kit following the protocol provided by the manufacturer (Sigma). The annealing temperature was 42 °C, and PCR reactions were performed using 35 cycles of 15 s at 94 °C, 30 s at 5560 °C and 30 s at 68 °C. Positive and negative controls were included in all assays. The primers used are listed in Table 2.
The primer extension experiment was performed using a specific oligonucleotide, 5'-GCCGTGCAGCAACAGTAGCG-3', complementary to the sense strand. This oligonucleotide was radioactively labelled at its 5' end with [-32P]ATP and T4 polynucleotide kinase, and hybridized to 20 µg total RNA isolated from H. maura S-30. The extension was carried out using avian myeloblastosis virus reverse transcriptase in accordance with the manufacturer's protocol (Roche). The cDNA product and sequencing reactions were analysed on a 6·5 % (w/v) urea-polyacrylamide sequencing gel.
Construction of epsA : : lacZ transcriptional fusion.
A 300 bp internal piece from the epsA gene of H. maura S-30 chromosomal DNA was amplified using the following primers, both containing EcoRI and XbaI restriction sites (underlined) at their respective 5' ends: 5'-GAATTCATGCGTCATTCCACG-3' and 5'-TCTAGATCAGATGTCATCCCG-3'. PCR was performed by using 30 cycles of 30 s at 95 °C, 30 s at 65 °C and 30 s at 72 °C. The PCR fragment was purified, digested with EcoRI and XbaI, and cloned into the suicide plasmid pVIK112, containing a promoterless lacZ gene (Kalogeraki & Winans, 1997). The resulting plasmid, pVIKepsA, containing an epsA : : lacZ transcriptional fusion, was then transformed into S17-1
pir and transferred into a rifampicin-resistant H. maura derivative (S30R3) by biparental mating, where the epsA gene was replaced via a single recombination event. The resulting fusion strain (S-30 epsA : : lacZ) contains the lacZ gene inserted after base pair 342 (codon 114) of the epsA gene. H. maura clones carrying the chromosomal lacZ transcriptional fusions were selected by plating on SW medium (Nieto et al., 1989
) containing 2 % (w/v) salts (Rodríguez-Valera et al., 1981
), rifampicin (50 µg ml1) and kanamycin (50 µg ml1).
-Galactosidase assays.
H. maura S-30 epsA : : lacZ was grown to exponential (OD6000·5), mid-exponential (OD600
1) and stationary (OD600
2) phases in 2 ml MM and MY media (composition described above) at 32 °C. This medium was supplemented with a mixture of sea salts to a final concentration of 1, 2·5, 5, 7·5 and 10 % (w/v) (Rodríguez-Valera et al., 1981
). Cells were resuspended in Z-buffer to eliminate salts, which might interfere in the assay. They were then diluted to 1 : 10 (MM cultures) or 1 : 100 (MY cultures) in Z buffer (0·06 M Na2HPO4, 0·04 M KCl, 0·001 M MgSO4, 0·05 M
-mercaptoethanol) and assayed in triplicate (Miller, 1972
); each experiment was repeated at least three times.
Production, chemical composition and molecular mass of the EPS synthesized by H. maura strains.
To isolate the EPS, the S-30 and TK71 strains of H. maura were grown for 5 days at 32 °C in MY medium (Moraine & Rogovin, 1966) containing a 5 % (w/v) salt solution (Rodríguez-Valera et al., 1981
). The EPSs were extracted following the protocol described by Quesada et al. (1993)
. The purified polymers were analysed for total carbohydrates (Dubois et al., 1956
), proteins (Bradford, 1976
), uronic acids (Blumenkrantz & Asboe-Hansen, 1973
) and acetyl residues (McComb & McCready, 1957
) using colorimetric assays.
The EPSs from H. maura strains S-30 and TK71 were purified by chromatography in a column packed with 16/10 Q Sepharose Fast Flow (Pharmacia), and their apparent molecular masses were determined by high-performance size-exclusion chromatography (HPSEC) done with a 9012 chromatograph (Varian) as described by Arias et al. (2003).
Transmission electron microscopy of H. maura strains.
Mid-exponential-phase cells were fixed in 2·5 % (v/v) glutaraldehyde, 0·05 % (w/v) ruthenium red buffered with 0·05 M sodium cacodylate, 2 mM MgCl2.6H2O (pH 7·4) at 4 °C for 4 h. After washing in 0·1 M sodium cacodylate, the cells were post-fixed with 1 % (w/v) OsO4 in 0·1 M sodium cacodylate at room temperature for 1 h. The cells were dehydrated in an ethanol series (30, 50, 70, 90 and 100 % v/v) at room temperature, each dehydration step taking 20 min. Infiltration with an embedding resin : ethanol mixture (1 : 1, v/v) was done overnight at room temperature, followed by overnight pure-resin infiltration. Cells were transferred into a gelatin capsule and filled with resin monomer. Polymerization was done at 60 °C for 8 h. Ultrathin sections were cut and collected on grids (300 mesh, Cu). The sections were stained for 10 min with 1 % (w/v) aqueous uranyl acetate solution and lead citrate. The samples were examined under a transmission electron microscope (Zeiss TEM EM 10C, 30 µm objective aperture, 80 kV acceleration voltage) and photographed on Agfa Scientia EM film.
Nucleotide sequence accession number.
The nucleotide sequence reported in this paper has been submitted to the EMBL database and assigned accession no. AY918062.
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RESULTS AND DISCUSSION |
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Mutations in several such homologues (Bik et al., 1996; Bugert & Geider, 1995
; Drummelsmith & Whitfield, 1999
; Huang & Schell, 1995
; Yoshida et al., 2003
) inhibit polysaccharide production, just as occurs in the mutant H. maura S-30 epsA : : lacZ strain. This finding, together with the characteristics shown in EpsA common to other OMA proteins, suggests that EpsA may be located in either the periplasm or the outer membrane, and play a role in the process of exporting mauran to the cell surface in H. maura S-30.
Encoded by ORF2 (EpsB) and ORF3 (EpsC) are a pair of 15·85 kDa tyrosine phosphatase and 79·51 kDa tyrosine autokinase proteins, which display 38 to 46 % homology with the Wzb and Wzc proteins of E. coli (Drummelsmith & Whitfield, 1999), ORF5/ORF6 from K. peumoniae (Arakawa et al., 1995
; Fang et al., 2004
), AmsI/AmsA from E. amylovora (Bugert & Geider, 1995
) and EpsP/EpsB from R. solacearum (Salanoubat et al., 2002
) (Table 3
). The order of transcription of these two proteins within the eps operon is similar to those of their analogues in a number of large gene clusters involved in the production and export of various EPSs (Arakawa et al., 1995
; Bugert & Geider, 1995
; Rahn et al., 1999
; Stevenson et al., 1996
). Wzb is a cytoplasmic protein often produced by systems involving Wza and Wzc, in which it seems that Wzb dephosphorylates the spent undecaprenyl pyrophosphate carrier to allow its re-entry into the polymerization cycle, and also removes a phosphate group from the Wzc protein (Vicent et al., 1999
). Protein Wzc is a representative of the PCP (polysaccharide copolymerase protein) family (Morona et al., 2000
), previously known as the MPA1 (cytoplasmic-membrane periplasmic auxiliary protein) family. These proteins seem to play a part in the translocation-surface assembly process by regulating the chain length of the EPS, CPS and LPS O antigen (Whitfield et al., 1997
), as they do with ExoP from Sinorhizobium meliloti (González et al., 1998
) and Wzc from E. coli (Vicent et al., 1999
).
A hydropathy analysis of ORF3 by the TopPred II program predicted that the EpsC protein is located in the plasma membrane, and possesses a short hydrophilic N-terminus followed by a transmembrane segment (TMS), a large periplasmic loop of about 386 residues, a second TMS, and finally a C-terminal tail of about 273 residues showing an ATP-binding motif (Fig. 3a). The alignment of EpsC and several related proteins contains conserved Walker A and B ATP-binding motifs, and a tyrosine-rich domain close to the carboxy terminus in all of these PCP proteins (Fig. 3b
). Some of these proteins (Glucksmann et al., 1993
; Grangeasse et al., 1997
; Morona et al., 2000
; Vicent et al., 1999
) have been shown to have autophosphorylating protein-tyrosine-kinase activity.
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A 47·95 kDa protein encoded by ORF5 (EpsJ) showed low homology with some members of the PST family (Table 3), within which the PST(1) protein subfamily, or Wzx homologues, act as flippases to transfer assembled lipid-linked repeating units across the cytoplasmic membrane. Despite the low homology shown, the protein's hydropathy profile was very similar to those of other PST-family members. ORF5 has the characteristic 12 putative transmembrane regions, and a cytoplasmic loop of 30 aa between TMS6 and TMS7 was predicted, suggesting that EpsJ is probably located at the cytoplasmic membrane and acts as a flippase. Since PST(1) exporter proteins seem to function together with each OMAPCP pair in polysaccharide transport in Gram-negative bacteria (Paulsen et al., 1997
), we believe that mauran may well be synthesized via a Wzy-like system. In this type of system the lipid-linked repeating units are normally polymerized by a Wzy polymerase enzyme, a process that takes place at the periplasmic face of the plasma membrane. Nevertheless, although we have sequenced about 8 kb downstream of the epsJ gene, in which more genes involved in the biosynthesis process are located, we have not as yet been able to identify a Wzy-like protein in H. maura.
Transcriptional organization and identification of a conserved regulatory region upstream of the epsABCDJ genes
DNA sequencing upstream of the epsABCDJ genes showed some regulatory elements characteristically appearing in gene clusters that are involved in bacterial polysaccharides. A conserved 39 bp DNA sequence involved in transcript elongation, known as the JUMPstart motif (just upstream of many polysaccharide starts) (Hobbs & Reeves, 1994), which includes shorter sequences, termed ops (operon polarity suppressor) sequences, was found upstream of epsA in H. maura S-30 (Fig. 2b
). The DNA sequence of a 5 kb chromosomal region upstream of the regulatory region did not reveal the presence of any ORF related to the biosynthesis of mauran.
Both regulatory elements are believed to play a role in the transcriptional antitermination of gene clusters (Artsimovitch & Landick, 2002; Bailey et al., 1996
, 1997
; Marolda & Valvano, 1998
; Nieto et al., 1996
). The ops motif is believed to ensure the transcription of distal genes in long operons, such as the ones located immediately downstream of stemloop structures in E. coli (Rahn et al., 1999
), by recruiting the antitermination protein RfaH into the transcription complex (Artsimovitch & Landick, 2002
; Bailey et al., 1996
, 1997
; Marolda & Valvano, 1998
; Nieto et al., 1996
). In fact, a homologous rfaH partial gene was amplified from H. maura S-30. What is more, a predicted stemloop structure was identified in the intergenic region between epsC and epsD (Fig. 2a
) in H. maura. These findings suggest that mauran may be transcriptionally controlled via antitermination, as happens in polysaccharides such as amylovoran (Bugert & Geider, 1995
), colanic acid (Stevenson et al., 1996
) and capsule (Rahn & Whitfield, 2003
).
To test whether epsABCDJ genes formed part of the same operon we carried out RT-PCR assays with primers based on their intergenic-region sequences (see Methods). The results shown in Fig. 2(c) confirmed that all of these genes are transcribed as a single transcriptional unit (Fig. 2c
, lanes 25) and that there seems to be a promoter region upstream of epsA (Fig. 2c
, lane 1).
To locate the transcription initiation point of this operon we did a primer extension experiment, as described in Methods, and the result suggested that the gene cluster was transcribed from a single promoter, the mRNA 5' end of which starts at a C base (Fig. 2a, b; data not shown).
Transcriptional regulation of the eps gene cluster
Since a conserved regulatory region for polysaccharide gene clusters, containing a JUMPstart sequence, precedes the eps gene cluster in H. maura, we sought to determine whether this gene cluster was constitutively expressed and the conditions that might affect its expression. To this end we cloned an internal epsA fragment of 300 bp upstream of a promoterless lacZ gene in pVIK112 (Kalogeraki & Winans, 1997; Llamas et al., 2004
), referred to as pVIKepsA. This construction was transferred by biparental mating into H. maura S30R3 and integrated into the chromosome by single recombination. The resulting strain (S-30 epsA : : lacZ) forms non-mucoid colonies that do not produce EPS, suggesting that the epsA gene was disrupted. The S-30 epsA : : lacZ transcriptional fusion candidates were checked by PCR using primers from the beginning of the gene and the end of the lacZ gene.
An analysis of the activation of the epsA : : lacZ fusion under different culture conditions (see Methods) revealed that expression of the eps cluster seemed to start at low cell density (OD600 0·5) and reached maximum induction after 5 days' incubation, during the stationary phase in the presence of a medium salt concentration (5 % w/v). These results agree with those reported elsewhere in studies of mauran production (Arias et al., 2003). We also confirmed that expression was higher in complex medium (2000 Miller units) than in MM medium (225 Miller units). Further work is needed, however, to confirm whether the eps gene cluster is regulated by a JUMPstart-antitermination RfaH mechanism, including an analysis of the expression of a lacZ transcriptional fusion to genes located downstream of the stemloop structure.
EpsA and EpsC are involved in the cell-surface assembly of EPS mauran
To find out more about the involvement of EpsC (Wzc homologue) in mauran synthesis we first confirmed by RT-PCR assays that the transposon insertion had no polar effect on the downstream genes in strain TK71 (S-30 : : epsC : : mini-Tn5 Km2). To study the effect of the epsC mutation on the synthesis of EPS, cultures of H. maura S-30 wild-type strain and the mutant TK71 strain were prepared for cell-morphology analysis, and extracellular materials were also quantified and analysed. Transmission electron microscopy showed that although both micro-organisms were similar in size their cell surfaces seemed quite different; in contrast to the wild-type strain (Fig. 4a), the TK71 cells did not have any extracellular material either attached to their surface or nearby in the surrounding medium (Fig. 4b
). In fact, epsC mutation resulted in an almost complete lack of mauran production: strain TK71 produced 98 % less than the wild-type strain. In addition, the molecular mass of extracellular material synthesized by strain TK71 was 2·5x104 Da whilst that produced by the wild-type strain S-30 was 4·7x106 Da (Arias et al., 2003
).
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ACKNOWLEDGEMENTS |
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Received 22 February 2005;
revised 6 June 2005;
accepted 22 June 2005.
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