Microbiology (2002), 148, 703–711 Printed in Great Britain The Enterococcus faecalis gene encoding the novel general stress protein Gsp62 Alain Rince  , 1 Marilyne Uguen, 2 Yoann Le Breton, 1 Jean-Christophe Giard, 1 Sigrid Flahaut, 1 † Alain Dufour 2 and Yanick Auffray 1 Author for correspondence : Alain Rince  . Tel: 33 2 31 56 55 23. Fax: 33 2 31 56 53 11. e-mail : rinceibba.unicaen.fr 1 Laboratoire de Microbiologie de l’Environnement, IRBA, Universite  de Caen, 14032 Caen Cedex, France 2 Laboratoire de Biologie et Chimie Mole  culaires, EA 2594, Universite  de Bretagne Sud, 56000 Vannes, France The Enterococcus faecalis general stress protein Gsp62 was purified using two- dimensional gel electrophoresis and its 25 N-terminal amino acid sequence determined. Analysis of the corresponding gene revealed that the gsp62 product is a 172 aa protein. Transcriptional analysis of gsp62 gave evidence for a monocistronic mRNA, the synthesis of which was induced at the onset of stationary phase and in response to heat shock, acid pH, detergents (i.e. SDS or bile salts), ethanol, tert-butyl hydroperoxide, sodium chloride and, to a lesser extent, hydrogen peroxide. 5 rapid amplification of cDNA ends by PCR experiments showed that gsp62 transcription initiates 30 nt upstream of the ATG start codon. Although gsp62 expression was induced in response to various stresses, its disruption had no significant effect on the cell survival after each individual stress. Two-dimensional protein gels from wild-type and mutant cells revealed no pleiotropic effect of the mutation on protein synthesis. Transcriptional fusions with the lacL lacM β-galactosidase genes showed that an inverted repeat located upstream of the promoter is required for transcriptional induction by environmental stresses but not by entrance into stationary phase. Two distinct mechanisms responding to different signals are thus involved in gsp62 induction. Keywords : Enterococcus faecalis, general stress protein, Gsp INTRODUCTION When grown under moderate stress conditions, bacteria may develop an adaptive response, allowing them to cope with subsequent more severe stresses. In general, this adaptation phenomenon appears to involve multiple genes encoding stress proteins, which can be specifically induced by a particular stress factor (specific stress proteins) or induced by several conditions (general stress proteins). 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 ................................................................................................................................................. † Present address : Laboratoire de Biologie Cellulaire et Mole  culaire, Universite  du Littoral – Co  te d’Opale, Bassin Napole  on, BP120, 62327 Boulogne sur mer, Cedex, France. Abbreviations : tBOOH, tert-butyl hydroperoxide ; 2-D, two-dimen- sional; IR, inverted repeat; RACE, rapid amplification of cDNA ends. 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 suble- thal 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 im- portant physiological role in the non-specific stress response. These general stress proteins were named Gsp62 to Gsp67 (Rince  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 0002-5113  2002 SGM 703