Groupe de Recherche en Ecologie Buccale, Département de Biochimie et de Microbiologie, Faculté des Sciences et de Génie, and Faculté de Médecine Dentaire, Université Laval, Québec, Canada G1K 7P41
Author for correspondence: Michel Frenette. Tel: +1 418 656 2131 ext. 5502. Fax: +1 418 656 2861. e-mail: Michel.Frenette{at}greb.ulaval.ca
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ABSTRACT |
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Keywords: enzyme II, streptococci, man operon, phosphotransferase system
Abbreviations: PEP, phosphoenolpyruvate; PTS, phosphotransferase system
The GenBank accession number for the sequence reported in this paper is AF130465.
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INTRODUCTION |
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An analysis of several nucleotide sequences of EII genes allowed the PTS permeases to be classified into six families according to sequence similarity and domain organization (Postma et al., 1993 ; Lengeler et al., 1994
). These families are: (a) glucose- and sucrose-PTS, (b) mannitol- and fructose-PTS, (c) lactose- and cellobiose-PTS, (d) mannose-PTS, (e) glucitol-PTS, and (f) galactitol-PTS. EIIs of the mannose-PTS family are considered to be evolutionarily distinct from the other EIIs (Reizer et al., 1996
). The members of the mannose-PTS family that have been identified are EIIMan, EIIAga and EIIAga' of Escherichia coli (Erni et al., 1987
; Reizer et al., 1996
), EIILev of Bacillus subtilis (Martin-Verstraete et al., 1990
), EIISor of Klebsiella pneumoniae (Wehmeier & Lengeler, 1994
), EIIMan/Glc of Vibrio furnissii (Bouma & Roseman, 1996
) and EIIMan of Lactobacillus curvatus (Veyrat et al., 1996
).
In Streptococcus salivarius, the mannose-PTS is constitutively produced (Vadeboncoeur, 1984 ), and is responsible for transporting mannose, glucose, fructose and 2-deoxyglucose (Vadeboncoeur, 1984
). In addition to the permease moiety, this mannose-PTS was shown to contain two biochemically and antigenically related proteins,
and
, with molecular masses of 35·2 kDa and 38·9 kDa respectively (Bourassa et al., 1990
). Both forms of IIABMan are present only in S. salivarius and Streptococcus vestibularis; 15 other species of streptococci, Lactococcus lactis and Lactobacillus casei possess only one protein that reacts with
or
polyclonal antibodies. It has been shown that mutations affecting the expression of S. salivarius mannose-PTS components, more specifically
, have pleiotropic consequences on the expression of a wide variety of membrane and cytoplasmic proteins (Gauthier et al., 1990
; Bourassa & Vadeboncoeur, 1992
; Lapointe et al., 1993
; Brochu et al., 1993
), as well as on urease activity (Chen et al., 1998
).
The biochemical characterization of S. salivarius revealed that the IIA and IIB domains are phosphorylated on histidine residues, confirming that this enzyme belongs to the EII-mannose family (Pelletier et al., 1998
). Further characterization also showed that
is required for the phosphorylation of mannose and 2-deoxyglucose by the mannose-PTS, while the
is unable to fulfil this function (Pelletier et al., 1998
). The current work presents the isolation and characterization of the genes encoding the components of the mannose-PTS of S. salivarius.
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METHODS |
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DNA manipulations.
The genomic DNA of S. salivarius was isolated as previously described (Lortie et al., 1994 ). Unless otherwise mentioned, all DNA manipulations were performed using standard procedures (Ausubel et al., 1990
). DNA sequencing was completed on both strands by the DNA sequencing service of Université Laval using appropriate synthesized oligonucleotides. Computer-assisted DNA and protein analyses were performed using the Genetics Computer Group Sequence Analysis software package, Version 9.1 (Devereux et al., 1984
). At the time of writing, the relevant data from the genome sequencing project of Streptococcus mutans, Streptococcus pyogenes, Streptococcus pneumoniae and Enterococcus faecalis are available at http://www.ncbi.nlm.nih.gov/BLAST/unfinishedgenome.html.
RNA manipulations.
Total RNA was extracted from S. salivarius as previously described (Gagnon et al., 1995 ). Briefly, S. salivarius cells were harvested at mid-exponential phase by centrifugation and resuspended in 4 ml ice-cold 10 mM Tris (pH 8·0) containing 1 mM EDTA. Resuspended cells were mixed with a solution containing 3·5 g glass beads (150212 µm diameter, Sigma), 3 ml phenol/chloroform/isoamyl alcohol (100:24:1, by vol.) and 0·25 ml 10% (w/v) SDS, and vortexed for 3 min at 4 °C. Lysed cells were centrifuged and the supernatant was extracted five times with phenol/chloroform/isoamyl alcohol (100:24:1, by vol.). Ribonucleic acids were selectively precipitated by adding 0·1 vol. 10 M LiCl and 2 vols 95% ethanol. The RNA pellet was washed with 70% ethanol, air-dried, resuspended in 500 µl diethylpyrocarbonate-treated water, and stored in aliquots at -80 °C.
Denaturing formaldehyde agarose gels (1%, w/v), buffers and samples were prepared according to Ausubel et al. (1990) . RNA from S. salivarius ATCC 25975 (10 µg) was transferred to positively charged nylon membranes (Roche) using a PosiBlot pressure blotter according to the manufacturers instructions (Stratagene). Total RNA was fixed by UV cross-linking. RNA molecular mass markers (Gibco-BRL) were used to determine the size of the transcripts. Primer extension analysis was performed as described by Ausubel et al. (1990)
using primer 424R (5'-AATAATACCGATACCCATTCGTGTT-3') labelled with T4 polynucleotide kinase and [
-32P]ATP. The radiolabelled oligonucleotide (10 ng) was hybridized with 20 µg S. salivarius total RNA, and the extension was performed using 200 U murine leukaemia virus (MLV) reverse transcriptase (Gibco-BRL) for 1 h at 42 °C. The extended product was denatured and analysed by electrophoresis on a 9% polyacrylamide gel containing 7 M urea.
Western blot analysis.
Crude extracts of E. coli XL-1 Blue bearing pML17D or pML18D were prepared by sonication as previously described (Gagnon et al., 1995 ). Membrane-free cell extracts of S. salivarius were submitted to SDS-PAGE, electrotransferred to nitrocellulose membranes, and probed using rabbit anti-IIABMan polyclonal antibodies as previously described (Pelletier et al., 1995
).
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RESULTS AND DISCUSSION |
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To circumvent this toxicity problem, PCR using the E3N29 primer and RevlacZ universal primer was performed on a ligation mixture of a 47 kb Tsp509I partial genomic library of S. salivarius cloned into pUC18. The amplicons were ligated to pCR-II (Invitrogen) and transformed into E. coli XL-1 Blue. Screening using the 364 bp amplicon as a probe allowed the identification of one clone (pML17D) carrying a 1·2 kb amplicon. Analysis of the nucleotide sequence revealed the presence of 990 bp ORF encoding a 330 aa protein that shared a high degree of identity with IIAB proteins of the mannose-PTS family. The predicted molecular mass of this putative protein was 35·5 kDa, which was almost identical to the molecular mass of (35·2 kDa) determined by SDS-PAGE (Bourassa et al., 1990
). Moreover, E. coli cells bearing pML17D produced a 35 kDa protein, which was recognized by anti-IIABMan antibodies. This protein was not found in extracts from E. coli cells bearing pML18D, which carried the same DNA fragment inserted in the opposite orientation (Fig. 1
). This result proved that
is not toxic for E. coli under these growth conditions. The 35 kDa protein from pML17D was phosphorylated in the presence of PEP, EI and HPr proteins of S. salivarius, and allowed the phosphorylation of 2-deoxyglucose in the presence of membrane fractions from a S. salivarius mutant (G77) lacking
and
(Pelletier et al., 1998
). These results confirmed that the product of the gene (manL) is the
of S. salivarius.
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Characterization of Pman
Localization of the man operon promoter was confirmed by primer extension analysis (Fig. 4). The Pman promoter (TTGACA-N17-TAGAAT) is only one nucleotide different from the E. coli
70 and Bacillus subtilis
A-type consensus promoter sequences (TTGACA-N17-TATAAT) (Lisser & Margalit, 1993
; Moran et al., 1982
), which is consistent with the constitutive expression of
in S. salivarius (Vadeboncoeur, 1984
). Moreover, Szoke et al. (1987)
have demonstrated that a promoter with this T/G substitution in the -10 region is the second strongest next to the perfect consensus promoter. The potential strength of the Pman promoter could explain the toxicity of this chromosomal portion for E. coli cells. Every attempt to clone the chromosome region containing Pman and the upstream terminator-like structure into E. coli has failed. The fact that pML17D, which contains the entire manL gene, is stable in E. coli led to the conclusion that the toxicity was caused by transcriptional determinants located upstream from manL. Other authors have previously reported that DNA fragments from streptococci are toxic for E. coli due to the presence of strong promoters or promoter-like sequences (Stassi & Lacks, 1982
; Chen & Morrison, 1987
; Martin et al., 1989
; Dillard & Yother, 1991
).
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Characterization of the genes composing the man operon
manL.
The percentages of identity of the inferred amino acid sequence of with the corresponding domains of homologous proteins are presented in Table 1
. It is interesting to note that the IIB domains share a slightly higher degree of conservation than the IIA domains, possibly reflecting structural constraints imposed for recognition of the IIC and IID mannose permeases. Analyses of sequences available in the databases revealed that in addition to S. salivarius and E. coli, genes encoding IIABMan polypeptides are also present in the genomes of S. mutans, S. pyogenes, S. pneumoniae and Ent. faecalis. The IIA and the IIB domains of other mannose-PTSs are on separate polypeptides. A comparison of the amino acid sequences surrounding the IIA phosphorylation sites revealed a high level of identity (Fig. 5a
). These levels of identity are particularly high among IIAs from streptococci and enterococci. The amino acid sequences of the IIA domains from E. coli and S. salivarius only shared 35% identity (Table 1
, Fig. 5a
). Nevertheless, their structures have numerous points in common. Among the residues determined to be in the vicinity of the IIA phosphorylated histidine (H10) of the E. coli IIABMan by crystallographic studies (M23, L24, D67, G71, S72 and P73) (Nunn et al., 1996
), several were conserved in the S. salivarius
(M23, I24, D67, G71, T72 and P73). The hydroxyl group of S72, which was shown to be essential for PTS activity (Nunn et al., 1996
), is replaced in the S. salivarius homologue by another OH-bearing residue, T72. In addition, in the S. salivarius
, the hydrophobic L24 residue is conservatively replaced by I24. Consequently, the molecular environment of the IIA phosphorylation site of the S. salivarius
should be highly similar to that of the E. coli of IIABMan.
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manM.
The manM gene was located 67 bp downstream from manL and coded for a hydrophobic 271 aa polypeptide (27·2 kDa). An analysis of database sequences revealed that ManM shared identity with the mannose-PTS IIC domains of S. pyogenes (79%), S. pneumoniae (77%), Ent. faecalis (71%), S. mutans (68%), E. coli (46%), Lb. curvatus (44%) and K. pneumoniae (41%), and with B. subtilis levF (43%), E. coli agaC (27%) and E. coli agaW (23%). A putative RBS (AAAGGA) is present 9 bp upstream from the manM start codon. Sequence alignments of homologous IICMan proteins from different bacteria revealed that: (i) IICMan of streptococci and enterococci share high levels of identity (7179%) and (ii) the residues that were previously identified by Reizer et al. (1996) as being strictly conserved among the IICMan enzymes of the man, aga and aga' systems of E. coli and the lev system of B. subtilis (G20, P33, G41, G45, G50, G54, G55, P73, D126, P176, G182, G187, V193, G194, M202, P209, G214, F215, L226, G233, A237) were also conserved in the IICMan of S. salivarius, S. mutans, S. pyogenes, S. pneumoniae, Ent. faecalis and Lb. curvatus (data not shown). This strengthens the hypothesis that these residues are important for the structure and/or function of the enzyme. However, other residues reported by the same authors to be conserved in IICMan (L7, Q8, R32, L44, D46, T49, E58, G63, Q108, T180, G188 and Y240) were not maintained among streptococcal, enterococcal and lactobacillus homologues, suggesting that these residues are subject to more important evolutionary divergence than previously believed. None of the 12 variations occurred in the central cytoplasmic loop of IICMan (Huber & Erni, 1996
; Reizer et al., 1996
). Among the 13 nonconserved residues, four (L44, D46, T49, E58) were part of the IICMan family signature proposed by Reizer et al. (1996)
(Fig. 5b
). Taking into account the sequences of Gram-positive bacteria genomes currently available, we propose a modified IICMan signature corresponding to GX3G[DNH]X3G[LIVM]2XG2[STL][LT][EQ]. Reizer et al. (1996)
reported that E58 is a candidate for a crucial role in enzyme activity, and could correspond to the glutamyl residue involved in sugar binding and the catalytic function of IIC proteins (Jacobson & Saraceni-Richards, 1993
). This glutamate is substituted by a glutamine in the IICMan of the six Gram-positive species, suggesting that a negative charge at this position does not appear to be essential for IIC activity. Interestingly, the IICMan of all the Gram-positive bacteria possess strictly conserved glutamate (E142) and aspartate (D130) residues that might compensate for the absence of E58 and maintain PTS activity. The Cys37 residue proposed to be involved in the interactions of IICMan and IIDMan (Rhiel et al., 1994
) is conserved in IICMan proteins. However, despite its ubiquitous presence in IICMan proteins, mutagenesis of this residue in the E. coli IICMan demonstrated that it is not essential for catalytic activity (Rhiel et al., 1994
).
manN.
The manN gene is located 14 bp downstream from the manM stop codon. It encodes a putative 303 aa protein (33·4 kDa), which shared strong identity with the mannose-PTS IID domains of S. pyogenes (80%), S. pneumoniae (74%), S. mutans (74%), Ent. faecalis (68%), E. coli (55%), K. pneumoniae (49%), and with B. subtilis levG (48%) and E. coli agaD (37%). The gene is preceded by a putative RBS (GGGGTG) 5 bp upstream from the ATG start codon. The KLTEG motif reported to be involved in permeasesugar interactions (Jacobson & Saraceni-Richards, 1993 ; Lengeler et al., 1994
; Wehmeier & Lengeler, 1994
) was not conserved in streptococcal IID enzymes (Fig. 5c
), as in IIDAga of E. coli (Reizer et al., 1996
). The motif was replaced by a [DN]ITKG motif. A careful comparison of the motifs showed that the L residue was replaced by an I residue, a modification that could be regarded as conservative. Furthermore, with the exception of the S. mutans protein, the positively and negatively charged residues were inverted (K+LTE-G for D-ITK+G), leaving open the possibility that these residues are equally involved in the permeasesugar interaction. A highly conserved region (residues 2448) on the cytoplasmic side of the permease (Huber & Erni, 1996
) included a strictly conserved glutamate residue (E29), which might be involved in the enzymesubstrate interaction. Interestingly, the IIDMan of several Gram-positive bacteria contains an additional segment of about 30 aa at the cytoplasmic-membrane junction. The previously reported IIDMan family signature (K[LIVM][GA][LIVM]2GP[LIVM]AG[LIVM]GD[PA][LIVMF]2W) (Reizer et al., 1996
) was strictly conserved among the IIDMan domains presented in Fig. 5(c)
. Thus, manM and manN encoded the permease portions of the mannose-PTS system of S. salivarius.
manO.
The fourth and last gene of the man operon, manO is located 78 bp downstream from manN, and encodes a small basic (estimated pI 10·7) 124 aa protein (13·7 kDa). The basic pI suggested that it might interact with nucleic acids. However, in silico structure analyses of the ManO protein failed to assign the classical helixturnhelix motif to a portion of the polypeptide. A BLAST search of databases revealed high levels of identity with putative proteins from S. pneumoniae (79%), S. pyogenes (65%), S. mutans (64%), Ent. faecalis (32%) and Lc. lactis (33%). No function has been assigned to these proteins, but the genes are also located downstream from the IIDMan genes in S. pyogenes and Ent. faecalis.
Conclusions
We have reported the first isolation and characterization of a complete man operon for a Gram-positive eubacterium. This operon was comprised of four genes encoding , a IICMan, a IIDMan and a protein of unknown function. Identification of the precise functions of ManO and the putative regulatory roles of
is currently under investigation using insertional mutagenesis.
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ACKNOWLEDGEMENTS |
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Received 6 July 1999;
revised 22 October 1999;
accepted 6 December 1999.