Biomedical subjects

Y Auffray

Publications and source records attributed to Y Auffray.

At least 19 recordsLinked to original sources

PubMed1998

A new theta-type thermosensitive replicon from Lactococcus lactis as an integration vector for Enterococcus faecalis.

We isolated a replication thermosensitive mutant of the theta-type lactococcal pUCL22 replicon. An improved version of this thermosensitive replicon was obtained by fusioning the replication repA gene with the downstream repB gene. The resulting plasmid was named pUCB3522Ts. It is highly instable at 42 degrees C in Enterococcus faecalis. Integration into the chromosome via homologous recombination was monitored using the npr gene of E. faecalis JH2-2 as a target. A 513 bp PCR amplification product from an internal region of this npr gene was cloned into pUCB3522Ts. Integration of this construction into the JH2-2 npr gene was selected by shift temperature, from 30 degrees C to 42 degrees C. 85% of the analysed clones showed integration into the npr gene, demonstrating the practicality of this thermosensitive replicon as a genetic integrative tool for E. faecalis.

Amino Acid Sequence↗

PubMed1998

The oxidative stress response in Enterococcus faecalis: relationship between H2O2 tolerance and H2O2 stress proteins.

The hydrogen peroxide (H2O2) stress response in Enterococcus faecalis ATCC19433 was investigated. A 2.4 mmol l-1 H2O2 pretreatment conferred protection against a lethal concentration (45 mmol l-1) of this agent. The relatively high concentrations of H2O2 used for adaptation and challenge treatments in Ent. faecalis emphasised the strong resistance towards oxidative stress in this species. Various stresses (NaCl, heat, ethanol, acidity and alkalinity) induced weak or strong H2O2 cross-protection. This paper describes the involvement of protein synthesis in the active response to lethal dose of H2O2, in addition to the impressive enhancement of synthesis of five H2O2 stress proteins. Combined results suggest that these proteins might play an important role in the H2O2 tolerance response.

Bacterial Proteins↗

PubMed1998

Survival of Enterococcus faecalis in an oligotrophic microcosm: changes in morphology, development of general stress resistance, and analysis of protein synthesis.

The ability of Enterococcus faecalis to metabolically adapt to an oligotrophic environment has been analyzed. E. faecalis is able to survive for prolonged periods under conditions of complete starvation established by incubation in tap water. During incubation in this microcosm, cells developed a rippled cell surface with irregular shapes. Exponentially growing cells survived to the same extent as cells starved for glucose prior to exposure to the multiple nutrient deficient stress. Chloramphenicol treatment during incubation in tap water led to a rapid decline in plate counts for exponentially growing cells but showed progressively reduced influence on stationary-phase cells harvested after different times of glucose starvation. During incubation in the oligotrophic environment, cells from the exponential-growth phase and early-stationary phase became progressively more resistant to other environmental stresses (heat [62 degreesC], acid [pH 3.3], UV254 nm light [180 J/m2], and sodium hypochlorite [0.05%]) until they reached a maximum of survival characteristic for each treatment. In contrast, cells starved of glucose for 24 h did not become more resistant to the different treatments during incubation in tap water. Our combined data suggest that energy starvation induces a response similar to that triggered by oligotrophy. Analysis of protein synthesis by two-dimensional gel electrophoresis revealed the enhanced synthesis of 51 proteins which were induced in the oligotrophic environment. A comparison of these oligotrophy-inducible proteins with the 42 glucose starvation-induced polypeptides (J. C. Giard, A. Hartke, S. Flahaut, P. Boutibonnes, and Y. Auffray, Res. Microbiol. 148:27-35, 1997) showed that 16 are common between the two different starvation conditions. These proteins and the corresponding genes seem to play a key role in the observed phenomena of long-term survival and development of general stress resistance of starved cultures of E. faecalis.

Autoradiography↗

PubMed1997

Relationship between the thermotolerance and the increase of DnaK and GroEL synthesis in Enterococcus faecalis ATCC19433.

Relationship between intrinsic thermal resistance, thermotolerance and heat shock proteins (hsp) synthesis is studied in Enterococcus faecalis. We showed that an impressive phenotypic heat resistance was induced by mild heat and a slight thermotolerance was developed by various sublethal pretreatments such as NaCl, SDS and bile salts. Hydrogen peroxide, acid and alkaline shifts or "thermomimetic" agent such as ethanol, did not enhance the survival of adapted cells against the lethal thermal shock (62 degrees C). The inhibition of protein synthesis by chloramphenicol or rifampin abolished thermotolerance. The immunological identification of the DnaK and GroEL proteins in E. faecalis allowed to study induction of these molecular chaperones under various conditions. Heat was the most efficient inductor of DnaK and GroEL synthesis. However, it was surprising that ethanol did not strongly induce these proteins. We also show that amplification of these hsp is not correlated to acquired thermotolerance with a linear relationship. A weak thermotolerance is not coupled from increased synthesis of DnaK and GroEL. So, we postulate that the high synthesis of the major hsp is not obligatory in the thermal cross-protection but that de novo protein synthesis is an absolute necessity in E. faecalis. Activation of preformed hsp or other factors depending or not on protein synthesis may be also necessary to enhance thermal resistance.

Chaperonin 60↗

PubMed1997

Differential induction of the chaperonin GroEL and the Co-chaperonin GroES by heat, acid, and UV-irradiation in Lactococcus lactis subsp. lactis.

Microsequencing of a polypeptide with MW of 14.5 and pI of 5.0 induced by heat treatment at 42 degrees C and 50 degrees C in Lactococcus lactis subsp. lactis revealed that it corresponds to the co-chaperonin GroES. Quantitative analysis of analytical 2-D gels showed a relative induction of 12- and 11-fold after 30 min of heat adaptation at 42 degrees C and 50 degrees C, respectively. GroES is also induced by an acid shift from pH 7 to pH 5.5 and by UV254 nm-irradiation, with relative induction factors of 3.8 and 2.3, respectively. To our knowledge this is the first report showing induction of GroES by mild acid treatment. Contrasting to the relative induction of the groEL gene product, the second protein encoded by the groESL operon, GroES shows significantly higher induction under all stress situations.

Amino Acid Sequence↗

PubMed1997

Sodium hypochlorite stress in Enterococcus faecalis: influence of antecedent growth conditions and induced proteins.

Compared with exponential growing bacteria, carbohydrate-starved cells of Enterococcus faecalis exhibit a high level of resistance to sodium hypochlorite with maximal resistance observed in cultures entering stationary phase. Chloramphenicol treatment, at various stages of growing phase, does not abolish the hypochlorite resistance of starved cells. However, Enterococcus faecalis conditioned by low sodium hypochlorite concentrations does not develop tolerance towards a lethal dose of the disinfectant. Two-dimensional gel analysis shows that protein synthesis is drastically turned off by hypochlorite treatment, whereas synthesis of a few proteins is enhanced by a low concentration of this chemical agent.

Bacterial Proteins↗

PubMed1997

Molecular analysis of the replication region of the theta-replicating plasmid pUCL287 from Tetragenococcus (Pediococcus) halophilus ATCC33315.

The complete nucleotide sequence of the 8.7-kb theta-replicating plasmid pUCL287 from Tetragenococcus halophilus (formerly Pediococcus halophilus) ATCC33315 has been determined. The replication region was identified and analyzed. Its nucleotide sequence contains an untranslated region, the replication origin, followed by two open reading frames (ORFs) encoding two proteins of 311 (RepA287) and 168 (RepB287) amino acids, respectively. Evidence is presented to show that RepA287 represents the plasmid replication protein. RepB287, which is non-essential for replication, is involved in the plasmid copy-number control and segregational stability. The roles of lactococcal proteins homologous to RepB287 have not been defined so far. Nevertheless, the structural organization of the pUCL287 replication region is remarkably similar to those of well known theta-replicating lactococcal plasmids despite the absence of homology of the replication origin and of the replication protein, and this suggests that pUCL287 uses the same mechanism of replication. Nucleotide sequence comparisons show that pSMB74, a pediococcal plasmid encoding bacteriocin production, is a member of the pUCL287 replicon family.

Amino Acid Sequence↗

PubMed1997

Glucose starvation response in Enterococcus faecalis JH2-2: survival and protein analysis.

We investigated the survival of Enterococcus faecalis following starvation provoked by energy source glucose exhaustion. Inhibition of protein synthesis by chloramphenicol before 3 h of starvation resulted in a dramatic decrease in viable bacteria. Antibiotic treatment of cells after 3 or 6 h of starvation had a progressively lesser influence on bacterial survival. During the first 24 h of deprivation, a total of 42 proteins were identified as glucose-starvation-inducible; 4 temporal classes of proteins (A, B, C and D) were defined in relation to their enhanced synthesis after glucose exhaustion. Our results show that proteins from the two early classes (A and B) seem to be the most important for long-term survival in E. faecalis. One protein of each of these classes was analysed at the molecular level. The N-terminal sequence of one of them, belonging to class A, showed strong homology with the N-terminal sequence of carbamate kinase from Streptococcus faecium. This enzyme could be implicated in the development of alternative metabolic pathways of energy production and could be compared to the Cst proteins of Escherichia coli.

Bacterial Proteins↗

PubMed1997

Alkaline stress response in Enterococcus faecalis: adaptation, cross-protection, and changes in protein synthesis.

The alkaline shock response in Enterococcus faecalis was studied in this work. Cells adapted to an optimum pH of 10.5 were tolerate to pH 11.9 conditions but acquired sensitivity to acid damage. An analysis of stress proteins revealed that 37 polypeptides were amplified. Two of these are DnaK and GroEL. The combined results show that bile salts and alkaline stress responses are closely related.

Acids↗

PubMed1997

[Enterococci in human environment].

Enterococci, formerly confounded with faecal streptococci, are recognized since the beginning of the century as being faecal in origin and are generally searched for in waste waters and food products; their detection may in fact indicate the presence of enteropathogenic organisms. Although nearly ubiquitous, their preferred ecological niche is the intestine sphere. Rejected in the environment by means of human faeces or animal dejecta, they are scattered afterwards in diverse niches. Once in the external environment, their survival is linked with their exceptional aptitude to resist or grow in hostile environments that are usually detrimental to the development of most mesophilic microorganisms. However, a certain ambiguity exists concerning their relationships with human beings. In fact, certain enterococcus strains or species are used in the elaboration of some milk products. Conversely, others are opportunists and may cause severe infections to people from infants to adults. Moreover, undergoing adaptation perpetually, they present a multiresistance pattern to antibiotics. Thus, the barrier that separates bacteria as nonoffensive contaminants from powerful pathogens appears most fragile, suggesting that people must systematically consider suspect the presence of enterococci in their near environment.

Animals↗

PubMed1996

Relationship between stress response toward bile salts, acid and heat treatment in Enterococcus faecalis.

Stress tolerance and cross-protection in Enterococcus faecalis ATCC19433 were examined after exposure to bile salts, acid or heat shock. Bile salts and heat adapted cells demonstrated induced homologous tolerance and cross-resistance. No cross-protection of heat adapted cells against acid stress is observed and pretreatment with bile salts even sensitized the cells to this challenge. Whole-cell protein extract analysis revealed that each treatment induced a battery of stress proteins. Some of these polypeptides are induced by more than one treatment. The greatest overlap is observed between bile salts and heat treatments. Eighteen stress proteins, including DnaK and GroEL, are common between these stresses.

Bile Acids and Salts↗

PubMed1996

Molecular analysis of the rpoD gene of Enterococcus faecalis.

The complete nucleotide sequence of the rpoD gene of Enterococcus faecalis ATCC19433 has been determined. This gene encodes a putative 368 amino acids (aa) polypeptide of a predicted Mr of 41842 named sigma 42. Upstream of the rpoD gene and beginning from the end of the cloned chromosomal fragment is an open reading frame encoding a putative 264-aa polypeptide. It is reminiscent of the C-terminal domain of known DNA primase from Gram-positive bacteria indicating that the E. faecalis rpoD gene is included in a macromolecular synthesis (MMS) operon, as it was described for Escherichia coli, Bacillus subtilis and Lactococcus lactis rpoD gene.

Amino Acid Sequence↗

PubMed1996

Unusual resistance and acquired tolerance to cadmium chloride in Enterococcus faecalis.

Enterococcus faecalis exhibits an extremely high natural resistance to cadmium which can be even raised by a conditioning treatment at a lower cadmium concentration as well as by a previous exposure to a sublethal temperature. By contrast, thermotolerance is not significantly induced by previous exposure to low cadmium concentration. The synthesis of several proteins is markedly enhanced by a low concentration of the chemical agent.

Adaptation, Biological↗

PubMed1996

Defense against lethal treatments and de novo protein synthesis induced by NaCl in Enterococcus faecalis ATCC 19433.

Enterococcus faecalis was strongly resistant to high osmotic pressure in complex medium; however, when it was subjected to a moderate osmotic stress [6.5% (w/v) NaCl or 52% (w/v) sucrose] for 2 h, it showed cross-protection against ethanol (22%), detergents stresses [bile sales (0.3%) and SDS (0.017%)], hydrogen peroxide challenge (45 mM), and to a minor extent against lethal temperature (62 degrees C). In response to salt stress [6.5% (w/v) NaCl], E. faecalis induced a large number of stress proteins. In addition, NaCl strongly induced the synthesis of many proteins more than tenfold. Although the acquired thermotolerance was inhibited markedly by chloramphenicol, the other NaCl-induced cross-tolerances seemed not to be correlated with de novo protein synthesis. The relationship between the stress protein synthesis and the induction of different types of cross-protection is discussed.

Adaptation, Physiological↗

PubMed1996

Starvation-induced multiresistance in Enterococcus faecalis JH2-2.

Compared with growing bacteria, carbohydrate-starved cells of Enterococcus faecalis show development of a multiresistance state against heat, H2O2, acid, and ethanol, but not against UV irradiation. The kinetics of acquisition of resistance is different according to the stress. Three hours of starvation provide maximal resistance against ethanol, while the tolerance to heat, H2O2, and acid increases progressively with the duration of starvation. Chloramphenicol treatment does not abolish the ethanol tolerance. Protein synthesis inhibition during the transitional growth phase and the first hours of starvation partially inhibit the acquisition of heat and oxidative resistances. Antibiotic treatment after 3 h of starvation does not affect the increase of these resistances. We suggest that synthesis of specific proteins revealed by 2-D gel analysis in the first 3 h of starvation, followed by a second mechanism related to protein degradation or alteration, is necessary for acquisition of maximal resistance towards heat and oxidative stresses.

Bacterial Proteins↗

PubMed1996

The Lactic Acid Stress Response of Lactococcus lactis subsp. lactis

The lactic acid tolerance response (LATR) of the lactic acid bacterium Lactococcus lactis subsp. lactis has been studied. A dramatic increase in survival to a severe acid stress (pH 3.9) was obtained by preexposing the cells for 30 min to a mildly acid shock at pH 5.5. Whole-cell protein extract analysis revealed that during the acid tolerance response 33 polypeptides are induced over the level of naive cells. Among these are the major heat shock proteins DnaK and GroEL. In conjunction with a previous report (Hartke et al. 1994), the results establish that L. lactis can adapt to lactic acid exposure in two different ways: a logarithmic phase LATR, which may be activated by protons, and a stationary-phase LATR, which needs no activation by protons. Both systems are independent of de novo protein synthesis.

Journal Article↗

PubMed1996

Physiological response of Enterococcus faecalis JH2-2 to cold shock: growth at low temperatures and freezing/thawing challenge.

Growth at low positive temperatures and induced phenotypic resistance to extreme cold temperature (freezing/thawing cycles) of Enterococcus faecalis were investigated. The effect of low temperatures on the specific growth rates was studied; use of Arrhenius profile and Ratkovsky 'square-root' model allowed determination of the 'temperature characteristic' (mu approximately equal to 13,800 cal mol-1), the critical temperature (Tcrit approximately equal to 17.9 degrees C) and the notional minimum growth temperature (T0 approximately equal to 3.6 degrees C). Preincubation of Ent. faecalis cells at low temperatures (8-16 degrees C) during periods corresponding to their generation time resulted in an increased ability of the bacterial cells to withstand short periods of freezing/thawing (-20 degrees C/+37 degrees C) challenge. Moreover, the increase of the incubation period at low positive temperature led to a higher degree of adaptation.

Cold Temperature↗

PubMed1996

Comparison of the bile salts and sodium dodecyl sulfate stress responses in Enterococcus faecalis.

The resistance to detergents and detergent-induced tolerance of a gastrointestinal organism, Enterococcus faecalis ATCC 19433, were examined. The most remarkable observation was the rapid response of cells in contact with bile salts and sodium dodecyl sulfate (SDS). The killing by high concentrations of detergents was nearly instantaneous. A 5-s adaptation with moderate sublethal concentrations of bile salts or SDS (0.08 or 0.01%, respectively) was sufficient to induce significant adaptation against homologous lethal conditions (0.3% bile salts or 0.017% SDS). However, resistance to a subsequent lethal challenge progressively increased further to a maximum reached after 30 min of adaptation. Furthermore, extremely strong cross-resistances were observed with bile salts- and SDS-adapted cells. However, no relationship seems to exist between levels of tolerance and de novo-synthesized proteins, since blockage of protein synthesis during adaptation had no effect on induction of resistance to bile salts and SDS. We conclude that this induced tolerance to detergent stress is independent of protein synthesis. Nevertheless, the stress-induced protein patterns of E. faecalis ATCC 19433 showed significant modifications. The rates of synthesis of 45 and 34 proteins were enhanced after treatments with bile salts and SDS, respectively. In spite of the overlap of 12 polypeptides, the protein profiles induced by the two detergents were different, suggesting that these detergents trigger different responses in E. faecalis. Therefore, bile salts cannot be substituted for SDS in biochemical detergent shock experiments with bacteria.

Adaptation, Physiological↗