Institute of Food Research, Norwich Research Park, Colney Lane, NR4 7UA Norwich, UK1
Author for correspondence: Annette Griffin. Tel: +44 1603 255 354. Fax: +44 1603 507 723. e-mail: annette.griffin{at}bbsrc.ac.uk
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ABSTRACT |
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Keywords: capsule polysaccharide, exopolysaccharide, Streptococcus thermophilus, glycosyltransferase, IS1193
Abbreviations: CPS, capsule polysaccharide; EPS, exopolysaccharide
The GenBank accession number for the sequence reported in this paper is Y17900.
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INTRODUCTION |
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METHODS |
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General DNA manipulations.
DNA digestion and ligation was performed as recommended by the enzyme manufacturer. Electroporation into E. coli TG1 cells was carried out as described previously (Dower et al., 1988 ). Plasmid DNA from E. coli was extracted using SV-Plus miniprep columns from Promega or by centrifugation through caesium chloride/ethidium bromide gradients (Sambrook et al., 1989
). Total DNA was extracted from Str. thermophilus as described by Lewington et al. (1987
). PCRs were carried out on Hybaid Omnigene thermocyclers. Template concentration was 600 ng per 50 µl reaction for chromosomal DNA; each primer was added to 0·1 µM. A mpliTaq polymerase enzyme (Perkin Elmer; 5 U µl-1) was used at 0·2 µl per 50 µl reaction. Annealing and extension conditions were dependent on the nature of primers and the expected product; these were as predicted by Oligo 4.0 for Macintosh.
DNA sequencing and sequence analysis.
A primer-walking strategy was used to obtain the DNA sequence from the lambda inserts containing the cps genes. For the sequencing reactions, the Dye Terminator DNA Sequencing Kit (Applied Biosystems) was used and primer was added at 3·2 pmol per 20 µl reaction. Lambda DNA template for sequencing was used at 23 µg per 20 µl reaction. Sequencing reactions were electrophoresed and analysed using a Perkin Elmer 373A Fluorescence DNA sequencer. Primer design was checked in Oligo 4.0 for Macintosh. Sequence data were analysed using the Wisconsin GCG package Version 10, Laser Gene Package for the Macintosh version 1.58 (DNAStar), TFASTA (Pearson et al., 1997 ), BLASTP (Altshchul et al., 1990
), CLUSTAL W (Higgins et al., 1994
) and PHDsec (Columbia University; http://www2.ebi.ac.uk). Hydrophobicity domains were determined using DNA STRIDER (version 1.1). Prediction of transmembrane domains and cytoplasmic/extracytoplasmic loops was also carried out using six different programs available from the internet at the ExPASY site (PHDhtm, DAS, HMMTOP, TopPred2, TMpred and TMHMM).
Cloning of the Str. thermophilus cps genes.
A Lambda DASH II (Stratagene) library was constructed according to the manufacturers instructions. This was screened for the presence of the cpsE gene, using PCR primers designed from the 3' end of a 4 kb insert in pAG14 as described previously (Griffin et al., 1993 ). Lambda DNA from positive clones was extracted as described by Sambrook et al. (1989
) or using the
-DNA extraction kit (Qiagen), and their inserts sequenced and analysed.
Cloning and expression of cpsE in E. coli.
Primers for the amplification of the cpsE gene designed from positions 47074726 and 61886206, were as follows: E60 5'-GGGATGATGCGGTTCCTTAT-3' and E61 5'-CCCTTAGAACCAACGATAT-3', respectively. These were used to amplify a 1·48 kb fragment from lambda clone 9L3, using a proof-reading polymerase mixture (Boehringer). This fragment, which contained the entire cpsE gene and the upstream RBS (but no promoter), was cloned into the vector pCR2.1 (Stratagene), in both the sense and antisense orientations in respect of the promoter for the ß-galactosidase gene (Plac) (confirmed by nucleotide sequencing). The resulting plasmids were called p13 and p34 respectively. E. coli TG1 cells were transformed with plasmids p13 and p34 by electroporation. Diluted (1/10) cultures of the clones containing the cpsE gene constructs were grown in 100 ml Terrific Broth until mid-exponential phase (OD600= 0·475). At this stage induction was carried out with 1 mM IPTG. Cultures of TG1 containing the pCR2.1 vector or no vector at all were treated in the same way and used as negative controls. Membrane extracts of all the E. coli clones were prepared as described by Kolkman et al. (1996 ). The efficiency of the membrane extraction was checked by loading 12 µl aliquots on a denaturing 10% polyacrylamide gel as described by Laemmli (1970
) and gels stained with Coomassie brilliant blue (Gibco-BRL). The protein concentration was determined using a simplification of the Lowry method (Peterson, 1977
).
Glucosyltransferase assay and TLC.
Membrane extracts (40 µl of 5 mg ml-1) from clones containing the cpsE gene construct as well as negative controls were incubated with UDP-[14C]glucose as described by Kolkman et al. (1996 ). One-third of the lipid fraction was used for scintillation counting and the remainder was vacuum dried and analysed by TLC. For TLC, the dried reacted samples were resuspended in 100 µl N-butanol and mild hydrolysis was carried out by adding 100 µl 50 mM trifluoroacetic acid and incubating at 95 °C for 20 min. The hydrolysed samples were vacuum dried and resuspended in 10 µl 40% 2-propanol, 8 µl of which was run on a silica gel plate (Merck HPTLC Alufolien Kieselgel 60). The developer used was a mixture of butanol/ethanol/water (5:3:2 by vol.). Standards of monosaccharides (40 µg each) were run simultaneously. Detection of radioactivity was done by exposing an X-ray sensitive film directly to the TLC plate, previously sprayed with a ß-enhancer spray (En3Hance, New England Nuclear), for 5 d at -80 °C. The standards were visualized by spraying with 5% sulfuric acid in ethanol and heating at 110 °C for 10 min. The assays described here were repeated and identical results were obtained.
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RESULTS AND DISCUSSION |
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Nucleotide sequence analysis of the cps cluster
In a previous study, we identified, cloned and nucleotide sequenced a 4074 bp chromosomal fragment containing part of the cps gene cluster (Griffin et al., 1996 ). To identify the remaining genes, primers designed from the 3' end of the NCFB 2393 cpsE gene were used to screen a lambda library of Str. thermophilus genomic DNA. This was used to identify three positive lambda clones;
9L3,
3K1 and
8FJ1 (see Fig. 1
). Nucleotide sequencing of the inserts from the lambda clones was performed to obtain the complete sequence data for a 14·62 kb region. Analysis of these sequence data revealed the presence of 16 ORFs including the 3' end of cpsA and the cpsBCDE genes previously reported by Griffin et al. (1996
) with strong conformity with the streptococcal codon-usage table (not presented) (Fig. 1
). Potential ribosome-binding sites were identified upstream of all genes (see accession no. Y17900) and putative promoters were identified upstream of six genes (see Fig. 1
and accession no. Y17900). An AT rich region, 5'-AAAACGTTTTTTTGTTTTTTTTTGAAAAAAA-3', similar to the consensus sequence of an upstream promoter (UP) element, 5'-NNAAA(AT)(AT)T(AT)TTTTNNAAAANNN-3' (Estrem et al., 1998
) was identified upstream of the -35 box of the cpsA gene between positions -72 and -42. UP elements have been reported to stimulate promoter activity up to 30-fold, and are found both in Gram-negative and Gram-positive bacteria (Estrem et al., 1998
). Thus strong transcription may occur from the cpsA promoter, resulting in the generation of a single long transcript. The short intergenic spacing between cps genes suggestive of translational coupling, supports this view. In Staphylococcus aureus, Streptococcus pneumoniae type 1 and Str. thermophilus Sfi6, it has been shown that expression of the eps or cps gene loci results in a single transcriptional unit despite the presence of putative promoter sequences within the operons (Muñoz et al., 1997
; Sau et al., 1997
; Stingele et al., 1999
). No hairpin loops resembling putative rho-independent transcription terminators were found within the cps genes; however one was identified downstream of orf14.9.
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The topological model of CpsE (Fig. 3), depicts CpsE spanning the cytoplasmic membrane five times (segments TM I, II, III, IV and V) and having two large loops, the first one extracytoplasmic, the second one intracytoplasmic (C-terminal region). The transmembrane domains are composed mainly of hydrophobic residues, which are predicted to adopt an
-helix conformation. The C-terminal domain, containing the active site, is in the cytoplasm. This contains three domains, A, B and C, reported to be common to glycosyltransferases by Wang et al. (1996
). Domains A and B are reported to be associated with the interaction of the glycosyltransferase with the membrane lipid, while block C seems to be specific for the transferred sugar. The model presented here is in agreement with reports that EPS biosynthesis occurs at the internal side of the cytoplasmic membrane, with subsequent translocation of the repeat unit across the membrane (Kolkman et al., 1997
; Stingele et al., 1996
).
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Analysis of cpsK and cpsL.
The cpsK and cpsL genes encode proteins with predicted molecular masses of 14·0 and 35·0 kDa respectively. When the amino acid sequence of cpsK was compared to the databases (see Table 2), similarity to several proteins involved in transport was detected. CpsL displayed significant homology to only one protein: WaaS from the LPS biosynthetic pathway of E. coli. A specific function has not been assigned to WaaS, however it has been suggested that it may act as an accessory protein required for the assembly of outer-core oligosaccharide, interacting either with glycosyltransferases or with the inner core LPS moiety (Pradel et al., 1992
). Alternatively, Heinrichs et al. (1998
) speculate that WaaS may be a rhamnosyltransferase, thus CpsL may be a rhamnosyltransferase. The position of a glycosyltransferase downstream of a putative export gene has been reported before for the cps/eps clusters of L. lactis and Str. pneumoniae (Van Kranenburg et al., 1999
; Morona et al., 1999
). More experiments are required to resolve the function of cpsL.
Expression of cpsE in E. coli
The NCFB 2393 cpsE gene was cloned into a high copy number vector and expressed in E. coli TG1. Expression from plasmid p13 (containing cpsE under the control of Plac) resulted in a protein of approximately 42 kDa, which was smaller than the expected molecular mass of 51·8 kDa (Fig. 4a). The reason for this is unclear. A glycosyltransferase assay using membrane extracts from E. coli cells containing either p13, p34 (cpsE cloned in the opposite direction to Plac) or pCR2.1 (vector alone) revealed enzymic activity, detected as the incorporation of UDP-[14C]Glc in extracts from TG1/p13 and TG1/p34 (Table 3
). No protein band was detected in extracts from p34, therefore we were surprised to detect glycosyltransferase activity in these. This activity may have resulted from expression from a non-specific promoter within the vector, yielding a level of protein too low to be detected by SDS-PAGE. Use of extracts from E. coli containing the vector alone confirmed that the glycosyltransferase activity detected was genuinely due to expression of the NCFB 2393 cpsE gene. No significant difference in glucoslytransferase activity was detected between the induced and uninduced clones, suggesting that the chromosomal lacIq gene in the background E. coli TG1 is insufficient to completely repress expression from Plac in pCR2.1. TLC analysis demonstrated that CpsE transferred only 14C-labelled glucose to the lipid fraction (Fig. 4b
), demonstrating that the conversion of glucose to galactose by the activity of an epimerase that was reported to occur in some strains of E. coli by Kolkman et al. (1997
), did not occur here. In addition, only labelled monosaccharide was detected in this study, demonstrating that CpsE transfers only a single glucose residue and is not a processive glucosyltransferase. Results from these assays combined with the data for the homology of NCFB 2393 CpsE to Xanthomonas campestris GumD (Ielpi et al., 1993
), imply that NCFB 2393 CpsE transfers a glucose 1-phosphate from UDP-glucose to undecaprenyl phosphate.
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A partial copy of the ISS1 transposase was identified next to orf14.9 in Str. thermophilus NCFB 2393. This contains only one long terminal repeat (at the 3' end) identical to that found in the CNRZ 368 ISS1. The NCFB 2393 ISS1 has 64% identity (78% similarity) to its homologue from Str. thermophilus CNRZ 368 (Bourgoin et al., 1999 ); 32% identity (58% similarity) to the IS257 transposase from Sta. aureus Tn4003 (accession no. P14506) and 28% identity (54% similarity) to the ISS1 transposase from L. lactis IL946 (accession no. L35176). Copies of ISS1 members are widespread among industrial strains of Str. thermophilus and L. lactis, where they have been found in different positions on the chromosome including the EPS gene clusters (Bourgoin et al., 1999
; Duwat et al., 1997
; Mercenier & Lemoin, 1989
; Stingele et al., 1996
).
A second IS element, IS1193, was also identified; this contained two 19 bp imperfect inverted repeats and two 8 bp perfect direct repeats, flanking a transposase gene (1246 bp). The NCFB 2393 IS1193 displayed 88% identity (91% similarity) at the amino acid level to IS1193 from Str. thermophilus CNRZ 368 (Bourgoin et al., 1999 ), 62% identity (75% similarity) to TnpA from Str. pneumoniae WU2 (accession no. U66845), and 57% identity (72% similarity) to IS1167 from Str. pneumoniae RX1 (accession no. M31680). A short ORF (orfM, 234 bp) has been identified within IS1193 in the opposite orientation to the transposase gene. orfM, including its putative RBS, is also present in Str. thermophilus CNRZ 368, although this was not reported by the authors. Recently, a new IS element from strain CNRZ 368, IS1194, was identified in the eps cluster (Bourgoin et al., 1998
). IS1194 and other members of the IS5 subfamily all contain two ORFs, the shorter one (of unknown function) in the opposite direction to and overlapping the larger ORF which encodes the transposase gene. orfM might be a remaining part of some element needed for the transposition of all IS5 members, or might be the vestige of a gene picked up during transposition between organisms.
Copies of ISS1 and IS1193 are widely present in strains of Str. thermophilus and L. lactis and it has been suggested that these could allow horizontal transfer to occur between these two organisms during co-culture in dairy manufacture (Bourgoin et al., 1996 , 1999
). Horizontal transfer and generation of new capsular polysaccharides or LPS as a consequence of homologous recombination between insertion elements has also been reported to occur in the CPS and LPS loci of strains of Str. pneumoniae and Sal. enterica (Morona et al., 1997
; Muñoz et al., 1997
; Xiang et al., 1994
). The G+C content of the NCFB 2393 cps cluster is 35·6 mol%; this is close to the average G+C content reported for Str. thermophilus of 3740 mol% (Farrow & Collins, 1984
). Interestingly, the G+C content of four genes at the 3' end of the cluster, cpsIJKL, is only of 30 mol%. These genes are located next to IS1193, suggesting that foreign genes could have been introduced into the NCFB 2393 cps locus by horizontal transfer. Recombination could have been facilitated by the presence of mobile elements such as IS1193 and ISS1, or by the presence of conserved genes within these clusters. Additionally, given the similarity of some of our genes to the cps genes from Str. pneumoniae, horizontal transfer between different strains of streptococci might also have occurred at some time during the evolution of these two species.
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ACKNOWLEDGEMENTS |
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Received 20 March 2000;
revised 30 June 2000;
accepted 26 July 2000.