Laboratoire de Microbiologie de lEnvironnement, IRBA, Université de Caen, 14032 Caen Cedex, France1
Laboratoire de Biologie et Chimie Moléculaires, EA 2594, Université de Bretagne Sud, 56000 Vannes, France2
Author for correspondence: Alain Rincé. Tel: +33 2 31 56 55 23. Fax: +33 2 31 56 53 11. e-mail: rince{at}ibba.unicaen.fr
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ABSTRACT |
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Keywords: Enterococcus faecalis, general stress protein, Gsp
Abbreviations: tBOOH, tert-butyl hydroperoxide; 2-D, two-dimensional; IR, inverted repeat; RACE, rapid amplification of cDNA ends
a Present address: Laboratoire de Biologie Cellulaire et Moléculaire, Université du Littoral Côte dOpale, Bassin Napoléon, BP120, 62327 Boulogne sur mer, Cedex, France.
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INTRODUCTION |
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The Gram-positive bacterium Enterococcus faecalis is an ubiquitous micro-organism. Resident of the human and animal gut, it is introduced to the environment by means of faeces and subsequently disseminated to diverse niches. Ent. faecalis also has importance as a pathogen, ranking the second most important agent in total nosocomial infections. Its survival in the external environment is linked to its exceptional aptitude for coping with harsh conditions (Jett et al., 1994 ; Mundt, 1986
). Physiological studies showed that Ent. faecalis is able to develop adaptive responses towards diverse stresses (Boutibonnes et al., 1993
; Flahaut et al., 1996a
, b
, c
; 1997a
, b
, c
; 1998
; Laplace et al., 1996
). Moreover, analysis of protein synthesis during incubation of exponentially growing cells of Ent. faecalis with sublethal stresses led to the detection of the overexpression of 167 proteins. Six of these are induced by at least six different stress conditions and probably play an important physiological role in the non-specific stress response. These general stress proteins were named Gsp62 to Gsp67 (Rincé et al., 2000
). Studies of proteins overexpressed during glucose exhaustion led to the identification of several glucose starvation proteins (Glsp) and to the characterization of a seventh general stress protein, Gls24, which is involved in bile salt resistance (Giard et al., 2000
). Western blot analyses identified Gsp66 and Gsp67 as DnaK and GroEL chaperonins, respectively (Flahaut et al., 1997b
), and their induction characteristics implied that their genes belong to the same regulon (Rincé et al., 2000
). Recently, Gsp65 was identified as an organic hydroperoxide resistance protein (Ohr) involved in resistance to oxidative stress (Rincé et al., 2001
). mRNA studies revealed a transcriptional induction of gsp65 in response to tert-butyl hydroperoxide (tBOOH), heat shock, acid pH, detergents (bile salts, SDS), ethanol, sodium chloride and H2O2.
In this paper, we describe Gsp62, a novel Ent. faecalis general stress protein. This was purified from two-dimensional (2-D) protein gels and its N-terminal sequence determined. The identification of the corresponding gene allowed sequence analyses, gene inactivation and expression studies.
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METHODS |
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Adapted and non-adapted cells (treated as described above) were harvested by centrifugation. Cells were resuspended in 10 ml BHI and incubated at 37 °C (control) or at 62 °C to determine resistance to a thermal challenge. Other challenges were carried out at 37 °C in medium supplied with (i) 22% (v/v) ethanol, (ii) 0·3% (w/v) bile salts, (iii) 0·017% (w/v) SDS, (iv) 28·5% (w/v) NaCl, (v) 45 mM H2O2, (vi) lactic acid to adjust pH to 3·2, (vii) NaOH to adjust pH to 11·9, (viii) 20 mM tBOOH. Challenges were performed for 30 min except for the bile salts and SDS detergents (30 s). Samples (0·5 ml) were removed, diluted in 0·9% NaCl and poured in GM17 agar for the determination of c.f.u. numbers. Plates were incubated at 37 °C for 48 h. Each point of an experiment is the mean of duplicate platings and all experiments were repeated at least twice.
Analysis of mRNA by Northern and dot blot experiments.
Total RNA of Ent. faecalis JH2-2 was isolated from exponentially growing cells, from cells entering stationary phase or from stressed cells by using the RNeasy Midi Kit (Qiagen). For Northern blots, 10 µg RNA was electrophoretically resolved per lane and transferred onto Hybond-N+ membranes (Amersham International) using standard procedures (Sambrook et al., 1989 ). Sizes of transcripts were estimated by comparison with an RNA ladder (0·569·4 kb) (Amersham International). For dot blots, 1 µg total RNA from cells incubated 10 min under the different individual stress conditions was spotted onto Hybond-N+ membranes. Membrane-bound nucleic acids were hybridized at a temperature of 55 °C in 1 M sodium phosphate buffer (pH 7·0) containing 5% SDS with a single-strand labelled probe complementary to gsp62mRNA. After hybridization, membranes were washed twice in 2xSSC, 0·1% SDS (10 min), twice in 0·5xSSC, 0·1% SDS (10 min) at 55 °C, and exposed to a storage phosphor screen (Packard Instrument Company) for 5 h.
Preparation of the single-strand labelled probe was as follows: first, a DNA fragment was amplified by PCR from chromosomal DNA of Ent. faecalis JH2-2 with the primers P4 and P5 (Fig. 1a; Table 2
). The probe was then synthesized by elongating the oligonucleotide P4 with Taq DNA polymerase, 2 µM dCTP, dGTP and dTTP, 2 µCi (74 kBq) [
-32P]dATP and 10 ng of the previously obtained PCR DNA fragment as template. Thirty cycles of 1 min at 94 °C, 1 min at 55 °C and 1 min at 72 °C were performed.
Mapping of the transcriptional start site.
The 5' end of gsp62 mRNA was mapped from a 5' RACE (rapid amplification of cDNA ends) PCR product obtained with the 3'/5' RACE kit (Roche Molecular Biochemicals) using primer P6 (Fig. 1a; Table 2
) for the reverse transcriptase reaction and polyA tailing and primer Ppe for PCR amplification (Fig. 1a
; Table 2
). The cDNA was then purified, analysed on a 6% polyacrylamide gel and sequenced by the dideoxy chain termination method with the ABI prism sequencing system (PE Biosystems) and primer Pext (Fig. 1a
; Table 2
).
Construction of promoter fusions.
A 353 bp fragment containing the wild-type gsp62 promoter (nucleotide region -246 to +107, relative to the transcriptional start site) was amplified by PCR using the primers P62S and P62NS (Fig. 1a; Table 2
), digested with BamHI and PstI, and cloned into the BglII and PstI sites of the promoter probe vector pAK80, creating plasmid pFWT62. The insert was sequenced to ensure that the PCR reaction did not induce any mutations. A variant of pFWT62, bearing an EcoRI deletion which removes a part of the inverted repeat located upstream of the promoter (Fig. 1a
) was constructed as follows. First, the 353 bp fragment containing the wild-type gsp62 promoter was cloned into the BamHI and PstI sites of the pBluescript vector and its sequence was determined to make sure that the PCR did not create any mutations. Then, the EcoRI site of the vector was eliminated by inserting the kanamycin resistance cassette from p17635 (Bardonnet & Blanco, 1992
) between the PstI and ApaI sites. The resulting plasmid was linearized by EcoRI and self-ligated to obtain the 15 bp deletion within the gsp62 promoter region. The resulting 338 bp fragment was excised by digestion with BamHI and PstI, purified and subcloned into the BglII and PstI sites of pAK80, yielding plasmid pFDE62. For both constructions, conservation of the three stop codons immediately upstream of the ribosome-binding site of lacL in pAK80 ensured that the coding region of gsp62 was not translationally fused to the reporter gene. Plasmids pFWT62 and pFDE62 were introduced by electroporation as described below.
ß-Galactosidase assays.
Cells grown in BHI and exposed to 4% ethanol or 0·3 M sodium chloride were harvested by centrifugation and concentrated fivefold in Z buffer (Miller, 1972 ). Two hundred microlitres of bacterial suspension were treated as described by Israelsen et al. (1995)
and ß-galactosidase activity was expressed in Miller units calculated according to the equation: activity=(OD420x1000)/(OD600x0·2xt(min)) (0·2 corresponding to the volume of cell suspension in ml). Each point is the mean of at least three experiments.
Two-dimensional protein gel electrophoresis.
Sample preparation and 2-D protein gel electrophoresis were carried out as described by Giard et al. (1997) .
General molecular methods.
Restriction endonucleases, T4 polynucleotide kinase, alkaline phosphatase and T4 DNA ligase were obtained from Roche Molecular Biochemicals, Amersham International and Eurogentec, and used according to the manufacturers instructions. PCRs were carried out with 5 µg chromosomal DNA from Ent. faecalis JH2-2 and 20 pmol primers, using Taq DNA polymerase (Sigma) or Ready To Go PCR Beads (Pharmacia Biotech). When necessary, PCR products were purified using the QIAquick Kit (Qiagen). Esc. coli and Ent. faecalis were transformed by electroporation with the Gene Pulser Apparatus (Bio-Rad), as described by Dower et al. (1988) and Holo & Nes (1995)
, respectively. Plasmids were purified by using QIAprep Miniprep Kit (Qiagen). DNA and amino acid sequences were analysed using the Mac Vector program (Kodak, Scientific Imaging Systems) and database searches were performed with the BLAST program (Altschul et al., 1990
). Other standard techniques were carried out as described by Sambrook et al. (1989)
.
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RESULTS |
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Phenotypic study of the gsp62 mutant
Because of the induction of Gsp62 synthesis under stress conditions, we examined whether a knockout of the gene affected stress resistance. A gsp62 mutant was constructed by homologous recombination as described in Methods. Growth studies of the gsp62 mutant did not reveal any significant difference with respect to the wild-type JH2-2 strain when cultured at 37 °C in BHI broth. Gsp62 is thus dispensable for optimal growth. When assessing the survival of bacteria after different individual lethal treatments, we observed no significant differences between the two strains after heat, NaCl, pH, detergents, tBOOH and H2O2 challenges applied either with or without previous adaptation.
We examined whether the knockout confers modifications to the protein pattern observable on 2-D PAGE. 2-D PAGE of proteins extracted from exponentially growing JH2-2 and gsp62 mutant cells exposed to 0·08% bile salts (Fig. 3) or 2 mM H2O2 (data not shown) for 30 min confirmed the absence of Gsp62 in the mutant, but revealed no other significant difference in protein synthesis between the gsp62 mutant and the wild-type strains.
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Mapping of the transcriptional start site of gsp62
To identify the promoter of gsp62, 5' RACE PCR experiments were carried out with RNA extracted from cells treated for 10 min with 2 mM tBOOH. A unique band was observed after electrophoresis of the 5' RACE PCR product and the sequence of the corresponding cDNA showed that the transcriptional start site of gsp62 lies 21 nt upstream of the ribosome-binding site (Fig. 1b). Seven base pairs upstream of this transcriptional start site, we found the putative -10 box TATACT separated by 17 bp from the putative -35 box ATGATT. This promoter matches the consensus sequence for promoters depending on the primary sigma factor (TATAAT-1619 bp-TTGACA, respectively). Immediately upstream of the -35 box of the gsp62 promoter, an inverted repeat (IR) (AGgCACGAATTCA-7 bp-TGAATTCGTGtCT) was identified (Fig. 1a
).
Construction of promoter fusions and ß-galactosidase expression analyses
Two promoter fusions with the lacL and lacM ß-galactosidase genes were constructed as described in Methods. The first fusion corresponded to the wild-type gsp62 promoter (region -246 to +107) cloned upstream of the ß-galactosidase genes of the pAK80 vector (plasmid pFWT62), while the second differed by a 15 bp deletion which removed a part of the IR located immediately upstream of the -35 box (Fig. 1a) (plasmid pFDE62).
ß-Galactosidase activity was monitored during the growth of Ent. faecalis JH2-2 harbouring pFWT62 or pFDE62. A strong increase (6·8-fold) of the ß-galactosidase activity was observed at the end of the exponential growth phase with the wild-type promoter (Fig. 5a). This confirms the gsp62 transcriptional induction at the entry into stationary phase. The transcriptional induction at the entry into stationary phase (7·2-fold) was also observable with the deleted version of the promoter fragment (Fig. 5b
), showing that this phenomenon does not rely on the IR located immediately upstream of the -35 box.
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DISCUSSION |
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We recently identified and characterized gsp65, another general stress protein encoding gene in Ent. faecalis (Rincé et al., 2001 ). The transcription of gsp62 and of gsp65 is induced to comparable levels by the same treatments, suggesting that those two genes belong to the same regulon.
5' RACE PCR product sequencing allowed us to locate the gsp62 promoter, which matches the consensus sequence for the primary sigma factor. Immediately upstream of this promoter, an IR was identified. Upstream of the previously described gsp65 promoter, an IR of distinct sequence was also identified (Rincé et al., 2001 ). As gsp65 is preceded by a gene located on the other DNA strand, we suggested that the gsp65 IR was unlikely to be a rho-independent terminator, but rather a structure potentially involved in transcriptional regulation (Rincé et al., 2001
). Here, results from gsp62 promoter fusions with ß-galactosidase genes revealed that the disruption of the IR led to a reduction of the basal level of transcription and prevented environmental stress induction of gsp62. This IR is thus very likely a target of a regulator (activator) of general stress response in Ent. faecalis. The induction of gsp62 expression in response to environmental stresses is thus not due to a secondary sigma factor. Whereas a gene encoding a
B homologue has been identified in some non-sporulating Gram-positive bacteria (Becker et al., 1998
; Wiedmann et al., 1998
; Wu et al., 1996
), no alternative sigma factor has been identified in the Ent. faecalis V583 chromosome sequence. Moreover, in Lactococcus lactis, which is closely related to Ent. faecalis and the genome of which has been entirely sequenced (Bolotin et al., 1999
), no
S or
B-like transcription factor has been found. Surprisingly, the deletion of a part of the IR had no effect on the activation of gsp62 promoter in response to entry into stationary phase. This implies that in contrast to B. subtilis class II gsp genes, gsp62 relies on two completely distinct pathways of induction responding to different signals.
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ACKNOWLEDGEMENTS |
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Received 18 July 2001;
revised 8 October 2001;
accepted 11 October 2001.
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