Research Institute of Innovative Technology for the Earth, 9-2 Kizugawadai, Kizu, Soraku, Kyoto 619-0292, Japan
Correspondence
Hideaki Yukawa
mmg-lab{at}rite.or.jp
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ABSTRACT |
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The GenBank accession number for the sequence reported in this paper is AF508972.
Present address: Department of Biochemistry and Microbiology, Institute of Chemical Technology Prague, Technická 3, 166 28 Prague, Czech Republic.
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INTRODUCTION |
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The -glucoside utilization pathways that rely upon the PTS for carbohydrate uptake have been characterized in several bacteria. They include
-glucoside-specific Enzymes IIBgl and associated activities encoded within operons arbGFB from Erwinia chrysanthemi (el Hassouni et al., 1992
), abgGFA from Clostridium longisporum (Brown et al., 1998
), bglGPT from Lactobacillus plantarum (Marasco et al., 2000
), bglPH from Bacillus subtilis (Krüger & Hecker, 1995
), casRAB from Klebsiella oxytoca (Lai et al., 1997
), bvrABC from Listeria monocytogenes (Brehm et al., 1999
), and cryptic bglGFB from Escherichia coli (Schnetz et al., 1987
). Expression of these operons is controlled by multi-domain antiterminators of the BglG/SacY family (Stülke et al., 1998
), which, in active dimeric form, bind to nascent mRNA and prevent premature transcription termination. The activity of antiterminator proteins is controlled by the PTS in a substrate-dependent manner via antagonistic phosphorylations at two PTS regulatory domains (PRDs) (Stülke et al., 1998
; Görke & Rak, 1999
). Only a few of the permeases encoded by the above operons, namely the casA and bvrB products, have been reported to facilitate transport of cellobiose (Lai et al., 1997
; Brehm et al., 1999
).
It is believed that the IIC component of PTS permease is responsible for selective binding and transport of the carbohydrate. As yet unknown are the amino acid residues responsible for specific recognition of the substrate to be translocated and phosphorylated by the PTS permease. Mutations in the IIC domains of glucose- and mannitol-specific Enzymes II from E. coli that abolished sugar binding (Saraceni-Richards et al., 1997) or allowed facilitated diffusion of substrate without phosphorylation (Ruijter et al., 1992
) and/or its phosphorylation without transport (Buhr et al., 1992
) are known, however. Several point mutations that extend the substrate specificity of glucose-specific IICBGlc of E. coli were reported recently. These mutations are scattered along the IIC sequence and are believed to induce conformational changes in IICBGlc, which then promote facilitated diffusion of ribose or fructose (Oh et al., 1999
; Kornberg et al., 2000
; Notley-McRobb & Ferenci, 2000
). Other amino acid exchanges allowed IICBGlc-dependent uptake of mannitol (Begley et al., 1996
), glucosamine and mannose (Plumbridge, 2000
; Notley-McRobb & Ferenci, 2000
; Zeppenfeld et al., 2000
), which normally are not, or are poorly, transported by this PTS.
In the present study we describe a -glucoside phosphotransferase and utilization system from C. glutamicum R, which we believe represents the first example of such a system reported from Actinobacteria. Specifically, it comprises three genes, bglF, bglA and bglG, encoding PTS permease,
-glucosidase and the positive transcription regulator, respectively. We demonstrate the potential of this system in the utilization of
-glucosides and its repression exerted by glucose. We further show that a single amino acid substitution is required for activity of BglF on cellobiose in adaptive cellobiose-utilizing mutants of C. glutamicum R.
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METHODS |
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The DNA sequences were determined on both strands of the linear template by the dideoxy chain-termination method using the BigDye Terminator Cycle Sequencing kit in the presence of 5 % dimethyl sulfoxide and by use of the ABI Prism 377 DNA sequencer (Applied BiosystemsPerkin-Elmer). The DNA sequences were analysed by the Genetyx version 4.0 program (Software Development, Japan), and database searches were performed using the BLAST server of the National Center for Biotechnology Information (www.ncbi.nlm.nih.gov).
Isolation of bgl genes and construction of plasmids.
The C. glutamicum R FIX library (Kotrba et al., 2001
) was screened by plaque hybridization using a probe obtained in PCR with degenerate primers. To do this, the primers 5'-ACICAYTGYGCIACIMGIYTIMG-3' (PBG1, plus strand, [I, inosine; Y, T or C; M, A or C]) and 5'-GGIRTIACIGARCCIGC-3' (PBG8, minus strand [R, A or G]) were designed for amino acid sequences of highly conserved regions of the IIB (THCATRLR) and IIC (G[IV]TEPA) domains of
-glucoside-specific Enzymes II. The amplified DNA fragment was cloned in pGEM-T (Promega), sequenced and labelled with digoxigenin-dUTP (DIG), which was later detected by using the DIG Nucleic Acid Detection Kit (Roche Diagnostics) according to the protocol supplied. DNA from positive phages was isolated by using the Lambda Midi Kit (Qiagen), digested with SalI, and the resulting fragments of C. glutamicum DNA were subcloned in pUC118 (Takara). The sequences at the 5' and 3' ends of the cloned fragments were determined with the vector primers and fragments fr-FA and fr-AG (Fig. 1
A) in plasmids pUC-SB2 and pUC-SB3, respectively, by using a primer-walking strategy.
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Construction of bgl replacement mutants.
The bgl mutants were constructed by replacing the bgl promoter region and 1·6 kb of bglF with a Kmr cassette (npt) using homologous recombination. To achieve this, the 4·1 kb fr-FA (SalI) was first inserted into plasmid pHSG396 (Takara), which confers Cm resistance (Cmr) and does not replicate in C. glutamicum. The resulting plasmid, pHSG-SB2, was then digested with ApaLI to liberate a 1·9 kb DNA fragment from fr-FA and it was replaced with npt (Fig. 1A). The 1·2 kb npt gene bordered with ApaLI was obtained by PCR with primers NAF (5'-CATGTGCACGGAAAGCCACGTTGTGTC-3' [plus strand]) and NAB (5'-CATGTGCACTGAGGTCTGCCTCGTGAAG-3' [minus strand]) and pUC4K (Amersham Pharmacia Biotech) as a template (the ApaLI sites are underlined). The resulting plasmid, pDbglPF, with npt reading in the opposite direction to the former bglFAG was electroporated into C. glutamicum R and R-CEL and integrants were selected on AR plates with Km. The bgl : : npt strains resulting from two simultaneous crossover events were distinguished from Cmr Kmr integrants in which the pDbglPF integrated via a Campbell-like mechanism as those of Cms Kmr phenotype. Correct insertion of npt in selected strains was confirmed by Southern hybridization (not shown).
Analyses of mRNA by dot-blotting.
Total RNA was isolated from cells growing exponentially (OD590 about 3) in AA medium supplemented with 0·5 % carbon source by using the Qiagen RNA Mini Kit. Equal amounts of total RNA (1·5 µg each) in 10x SSC (1·5 M NaCl, 0·15 M sodium citrate, pH 7·0) were denatured at 90 °C for 10 min and spotted onto nylon membranes soaked in 10x SSC. After baking at 80 °C for 2 h, the membranes were prehybridized for 2 h, hybridized overnight at 42 °C and then washed at high stringency as described by Sambrook et al. (1989). The 1·9 kb DNA fragment covering bglF and the 1·5 kb fragment covering bglA were labelled with digoxigenin-dUTP (Roche Diagnostics) by PCR with primers ABF plus ABB and FKF plus FBB (see above), respectively, and used as a mixed hybridization probe. Detection was performed by using the Gene Images detection kit (Amersham Pharmacia Biotech) according to the recommended protocol. The signal was scanned in a luminescent image analyser model LAS-1000 CH (Fuji).
Analysis of sugar in culture media and in vitro assay of PTS activity.
For sugar uptake studies, the culture samples were removed at stated intervals and filtered through 0·2 µm filters (Millipore). Sugar concentrations were determined by HPLC using the model 8020 apparatus from TOSOH (Japan) with a 600 mm OA PAK-A TSK-GEL column (TOSOH). The chromatography was performed at 40 °C with 0·75 mM H2SO4 as a mobile phase (1·0 ml min-1) and sugars were detected with a refraction index detector.
The combined -glucoside phosphorylation and phospho-
-glucosidase activities were determined essentially by the method of Kricker & Hall (1987)
. The cells from a culture that had reached the desired OD590 were harvested by centrifugation (6000 g, 5 min, 4 °C), washed in ice-cold 50 mM NaKHPO4 buffer (pH 7·2) and resuspended in the same buffer to an OD590 of 200. Cells were disrupted for 2 min with 0·1 mm Al2O3 beads in a Mini-Beadbeater device (BioSpec) set to the maximum speed. Crude cell-free extracts were assayed at 33 °C in 50 mM NaKHPO4 buffer (pH 7·2) containing 5 mM MgCl2, 2 mM p-nitrophenyl
-D-1,4-glucopyranoside (PNPG) and 2 mM PEP. The unit of activity was defined as the amount (pmol) of p-nitrophenol released from PNPG per second per mg of total protein. Formation of p-nitrophenol was monitored by measuring the A410 at 30 s intervals in a period of 15 min. The protein content was determined by using the Bradford Reagent Kit (Bio-Rad).
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RESULTS |
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Identification of bgl genes by analysis of DNA sequence and homology search
Analysis of the nucleotide sequence revealed that the coding sequences of all three bgl genes, named bglF (orf2), bglA (orf3) and bglG (orf4), were in the same translational reading frame. The DNA sequence spanning approximately 200 bp upstream of bglF contains several characteristic elements (Fig. 1B). The hexamers TTGCTT and CATAAT separated by 18 nucleotides corresponded well with consensus -35 and -10 promoter sequences, respectively. Downstream from the putative promoter was found a region of dyad symmetry with the potential to form a stable RNA stemloop structure (-28·9 kcal mol-1, -120·9 kJ mol-1) which, followed by a T-rich stretch of nucleotides, resembled a factor-independent transcription terminator. Partially overlapping this region at the 5' end, a putative ribonucleic antiterminator (RAT) sequence was recognized. A comparison of this sequence with known RAT sequences (Fig. 1C
), and its position relative to the putative terminator, suggested a strong relationship of this element to regulatory features characteristic of operons controlled by antiterminators of the BglG/SacY family (Stülke et al., 1998
).
The choice of the translational start codons for putative bgl products was based on the position of a potential ribosome-binding site and on similarities to known proteins. The first bgl gene, bglF, encoded a 64·8 kDa protein, 618 amino acids long. It was highly similar to other Enzymes IIBgl containing IIA, IIB and IIC domains fused in one polypeptide. The highest scores found were with BglF from E. coli (36 % identity and 50 % similarity: Schnetz et al., 1987), BglP from L. plantarum (35 % identity and 52 % similarity: Marasco et al., 2000
) and CasA from K. oxytoca (33 % identity and 51 % similarity: Lai et al., 1997
). The observed homologies and pattern of hydrophobicity (not shown) suggested a central membrane-spanning IIC domain flanked by hydrophilic IIB and IIA domains located in the N terminus and C terminus of BglF, respectively (Fig. 2A
).
The translational start of the protein encoded by bglA is located 60 bp downstream from bglF (Fig. 1A). The deduced 52·7 kDa BglA of 469 amino acids has, throughout the full length of the protein, extensive homology to phospho-
-glucosidases, having 65 %, 64 % and 57 % identity to BglA from Streptococcus mutans (Cote et al., 2000
), AbgA from C. longisporum (Brown et al., 1998
) and BglB from E. coli (Schnetz et al., 1987
), respectively.
Assuming that the GTG codon positioned 39 bp downstream from bglA represents a translation initiation codon, a protein of 289 amino acids could be deduced from bglG. The predicted 31·7 kDa protein shares 53 % similarity with LicT from B. subtilis (Schnetz et al., 1996) and BglG from E. coli (Schnetz et al., 1987
), the transcriptional antiterminators belonging to the BglG/SacY family. The similarities were particularly significant for three distinct regions. The first 56 amino acid residues resembled the so-called co-antiterminator RNA binding domain, which was 75 % similar to that of LicT from B. subtilis (Schnetz et al., 1996
). Two other regions (amino acids 93169 and 208280) might, by similarity, represent duplicated PTS regulatory domains PRD-I and II (Stülke et al., 1998
).
The presence of a single promoter preceding the bgl genes which encode closely related activities, and the gene spacing and putative regulatory features, which are similar to those of other PTSs, suggested that bglFAG represents the -glucoside PTS operon. The inverted repeats with a putative stable RNA stemloop structure (-18·91 kcal mol-1, 79·12 kJ mol-1) located immediately (9 bp) downstream from the bglG translation termination codon may allow factor-independent termination of the putative tricistronic transcript.
Induction of bgl genes and utilization of different sugars in parental strains and bgl disruptant mutants
The induction of bglF and bglA was explored by using hybridization of total RNA from cells grown on various carbon sources with probes corresponding to these genes (Fig. 3A). The in vivo bgl transcripts were detected in C. glutamicum R grown on methyl
-glucoside, salicin and arbutin but not on acetate, glucose or cellobiose. Cellobiose induced transcription of bgl genes only in an adaptive Cel+ mutant of C. glutamicum R, named R-CEL (Table 1
), which is capable of cellobiose utilization.
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Functional analysis of bgl genes in C. glutamicum ATCC 13869
To further examine whether bgl genes confer the potential for utilization of -glucosides, we subcloned promoterless bglFA (plasmid pCbglFA) and individual bglF and bglA genes (plasmids pCbglF and pCbglA, respectively) to provide their constitutive transcription from the lac promoter of the vector pCRA1 in C. glutamicum ATCC 13869-C (Fig. 4
A). Unlike C. glutamicum R, C. glutamicum ATCC 13869 does not grow on methyl
-glucoside, salicin or arbutin. Southern hybridization further revealed the absence of the bglFA genes from the chromosome of this strain (data not shown). Phosphorylation and cleavage of
-glucoside analogue PNPG were also not detected under any conditions tested (Table 2
). The PTS and
-glucosidase (PTS/
-glucosidase) activities were evaluated as combined activities in vitro. The transformation of strain ATCC 13869-C with pCbglFA resulted in constitutive, carbohydrate-independent expression of PTS/
-glucosidase activity (Table 2
) and transformants grew on methyl
-glucoside and salicin (Fig. 4A
) or arbutin (data not shown). Regardless of the high constitutive PTS/
-glucosidase activity in ATCC 13869-C harbouring pCbglFA, this strain failed to grow on cellobiose (Figs 4A and 5B
), thereby suggesting that wild-type bglFA genes alone do not support cellobiose utilization.
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To examine whether bglG, encoding a putative transcriptional regulator, is required for induction of bgl genes, we compared growth and PTS/-glucosidase activities in C. glutamicum ATCC 13869-C transformed with plasmids containing the entire bglFAG sequence including the regulatory features (plasmid pRbglFAG) and its bglG deletion variant (plasmid pRbglFA). Whilst transformation with pRbglFAG resulted in utilization of methyl
-glucoside, salicin and arbutin, these
-glucosides were not utilized in cells harbouring pRbglFA (data not shown). As illustrated in Table 2
, methyl
-glucoside and salicin induced PTS/
-glucosidase activity in pRbglFAG-transformed ATCC 13869-C, but not in pRbglFA transformants, thereby suggesting that bglG product acts as a positive regulator.
Mutations that occur in Cel+ strains
A possible explanation for the occurrence of Cel+ strains was that a mutation within bglF and/or bglA was required to convert bglFAG into a cellobiose PTS. To test this hypothesis, we incubated C. glutamicum ATCC 13869-C transformed with pRbglFAG and pCbglFA on minimal BT medium supplemented with 0·5 % cellobiose. Cel+ colonies appeared with both transformants at a frequency of 10-7. Sequence analysis of plasmids isolated from 15 independent Cel+ mutants showed that all contained one of two different single-nucleotide mutations in codon 317 of bglF, which caused an amino acid exchange. The point mutation GTGGCG resulted in substitution V317A and GTG
ATG in V317M. bglF genes carrying V317A (bglF317A) and V317M (bglF317M) were the only mutated alleles identified and we did not find any mutations within bglA or bglG in the isolated plasmids. The respective mutant plasmids were named pRbglFAAG or pCbglFAA and pRbglFMAG or pCbglFMA. These plasmids were used to transform C. glutamicum R and ATCC 13869-C. All transformants harbouring the mutant plasmids grew on
-glucosides, including cellobiose (Fig. 4B
), indicating that further mutations are not required for the Cel+ phenotype. We also constitutively expressed bglF317AA and bglF317MA in the Enzyme I-deficient mutant of C. glutamicum R (strain R-PDI5; data not shown). The observation that pCbglFAA and pCbglFMA did not support growth of R-PDI5 on cellobiose was in good agreement with the idea that the transport of cellobiose is coupled to its phosphorylation.
Finally, to investigate whether a mutation in bglF is also responsible for the Cel+ phenotype in C. glutamicum R-CEL, we amplified the bgl locus from this strain in a PCR using VentR DNA polymerase and chromosomal DNA as a template. Sequencing of the products from three independent reactions showed that R-CEL carried the bglF317AAG genes. We were also able to isolate and, by sequencing of the bgl locus, identify a spontaneous Cel+ mutant of C. glutamicum R, which harboured mutation V317M. This newly identified mutant was designated R-CEL2. Both R-CEL and R-CEL2 strains grew equally well in minimal BT medium with glucose (as well as methyl -glucoside and salicin; data not shown) with a growth rate (µ) of about 0·65 h-1, but evaluation of growth on 0·5 % cellobiose revealed significant differences between strains (Fig. 5
A). The R-CEL strain both consumed cellobiose and grew (µ 0·23 h-1) faster than R-CEL2 (µ 0·10 h-1), indicating that mutations V317A and V317M do not provide an equally well-performing cellobiose PTS.
Effect of different carbon sources on PTS/-glucosidase activity
To investigate the -glucoside PTS and its regulation in more detail we measured the combined PTS and
-glucosidase activity in crude cell extracts from strains grown in AA medium supplemented with 0·4 % sodium acetate, 0·4 % glucose, 0·1 % methyl
-glucoside, 0·1 % salicin or 0·05 % cellobiose (Table 3
). Compared to the control (acetate), the presence of methyl
-glucoside and salicin resulted in a 20- to 30-fold increase of PTS/
-glucosidase activity in C. glutamicum R, R-CEL and R-CEL2. This activity was induced by the same spectrum of
-glucosides in bgl-deficient mutants also, suggesting the presence of another PTS responsible for the observed Bgl+ phenotype of bgl : : npt strains. The levels of the PTS/
-glucosidase activity were actually similar in wild-type and R(bgl : : npt) strains, suggesting that a second system may fully complement the bgl genes to provide sufficient activity for maximum (as compared to glucose) growth. Cellobiose induced the PTS/
-glucosidase activity only in R-CEL (28-fold) and R-CEL2 (9-fold).
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DISCUSSION |
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Functional analysis of bglF and bglA performed in Bgl- C. glutamicum ATCC 13869-C confirmed that BglF permease, along with the BglA hydrolase, supports transport and breakdown of methyl -glucoside, salicin and arbutin in vivo and that the system isolated in this study is specific for these
-glucosides. The bglFA genes are absent from both C. glutamicum ATCC 13869 and ATCC 13032, which cannot grow at the expense of
-glucosides (Kotrba et al., 2001
) and thus apparently also lack a functional homologue of the second, as yet unidentified,
-glucoside system of C. glutamicum R. Taking advantage of the available genome sequence of C. glutamicum ATCC 13032 (GenBank accession no. NC003450) and probing it with the bglFAG sequence in silico, we obtained the only positive hit in the DNA region which was identical with part of the bglG coding sequence corresponding to amino acids 96289 of BglG (data not shown). It is noteworthy that in strain ATCC 13032 this sequence is located 3 kb upstream of an insertion element designated ISCg2 (Quast et al., 1999
). It is, therefore, tempting to speculate that the bgl homologue in strain ATCC 13032 was knocked out due to DNA rearrangements related to the transposition event.
In addition to -glucosides transported by wild-type BglF, the permease that accumulated mutations V317A and V317M in strains R-CEL and R-CEL2, respectively, can also transport cellobiose, a disaccharide that is not normally utilized in C. glutamicum R. Considering that only phosphorylated
-glucosides can be hydrolysed by BglA, and given the fact that constitutive expression of mutant permeases along with phospho-
-glucosidase in the PTS-deficient ptsI : : npt strain R-PDI5 did not support growth on cellobiose, it appears that mutants BglF317A and BglF317M act on cellobiose as phosphorylating PTS Enzymes II. Our experiments did not fully exclude the possibility that phosphorylated cellobiose was hydrolysed by an as yet unidentified
-glucosidase in C. glutamicum strains. An examination of the amino acid sequence surrounding the V317 of BglF revealed that the site accumulating mutations is situated only three residues from H313. This histidine residue is highly conserved in all Enzymes IIBgl (Fig. 2B
) and may be considered a potential phosphotransferase-acting determinant. The corresponding H306 of the homologous BglF from E. coli was reported as being indispensable for phosphorylation of transported
-glucosides (Schnetz et al., 1990
). From the close proximity of H313 and V317, it is tempting to speculate that substitution of V317 with a residue having either smaller (Ala) or larger (Met) side chains causes a local conformational change affecting the side chain of H313, such that it may participate in phosphorylation of incoming cellobiose. Intriguingly, residues corresponding to position 317 of BglF are also different from valine in the other three permeases (Fig. 2B
) which recognize cellobiose as a substrate, namely BvrB from L. monocytogenes (Brehm et al., 1999
), CasA from K. oxytoca (Lai et al., 1997
) and AscF from E. coli (Hall & Xu, 1992
). Conversely, the valine residue is conserved in those Enzymes IIBgl, which do not transport cellobiose.
In view of the identification of the RAT/terminator overlap in the bgl sequence, and BglG, which may be regarded as a transcription antiterminator from the BglG/SacY family, and considering that bglG was confirmed as being indispensable for induction of the PTS/-glucosidase activity in C. glutamicum ATCC 13969-C (Table 2
), it appears that transcription of bgl genes is regulated via a transcription antitermination mechanism (Stülke et al., 1998
; Görke & Rak, 1999
). Accordingly, the RNA hybridization analysis demonstrated that the bgl genes are induced in C. glutamicum R and R-CEL by methyl
-glucoside, salicin and arbutin, and in the latter strain also by cellobiose. The availability of glucose further repressed PTS/
-glucosidase activity in C. glutamicum R and R-CEL (Table 3
), and the
-glucosides salicin and cellobiose, respectively, were appreciably taken up from the medium only after all the glucose was consumed (Fig. 3B, C
). It should be also noted that mutations V317A and V317M did not have equivalent phenotypes. Induction of bgl-borne PTS/
-glucosidase activity by cellobiose and the response to glucose availability was more pronounced in the R-CEL mutant. Accordingly, growth of R-CEL2 was slower on cellobiose than that of R-CEL, which was also reflected by the lower rate of cellobiose consumption. Our approach did not fully address the influence of V317A and V317M on transport of other
-glucosides, as these mutations are masked in C. glutamicum R-CEL and R-CEL2 by a complementary
-glucoside PTS. It is worth noting, however, that we did not observe any significant differences in growth and PTS/
-glucosidase activities in C. glutamicum ATCC 13869-C transformed with plasmids harbouring wild-type or mutant bglFAG when salicin or methyl
-glucoside was supplied as the only carbon and energy source (data not shown). This suggested that the reported substitutions at residue 317 only extended the substrate specificity towards cellobiose. The experimental data represented here encourage the concept that it is the IIC domain that is responsible for substrate specificity of Enzyme II and suggest that a specific PTS may adopt new substrates under unfavourable environmental conditions to support the growth and survival of bacteria.
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ACKNOWLEDGEMENTS |
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Received 14 October 2002;
revised 12 February 2003;
accepted 17 February 2003.
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