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PubMed2003

Virulence factors and in vitro adherence of Enterococcus strains to urinary catheters.

The ability to adhere in vitro to urinary catheters and the presence of enterococcal virulence factors was determined in 30 Enterococcus urinary isolates (12 E. faecalis, 12 E. faecium, 3 E. casseliflavus, 3 E. gallinarum). Silicone, siliconized latex and polyvinyl chloride (PVC) were examined by sonication quantitative culture technique and scanning electron microscope. As compared to E. faecalis and E. faecium, E. casseliflavus and E. gallinarum displayed lower adhesion to all synthetic materials. All the tests performed showed higher adherence of all tested strains to siliconized latex and silicone than to PVC. Biofilmforming ability was observed in 5 E. faecalis but in none of the remaining strains. The gene coding enterococcal surface protein (Esp) was detected in 7 E. faecalis and 6 E. faecium strains. Gelatinase was found in 1 E. faecalis, 2 E. faecium and hemolysins were found in 6 E. faecalis and 1 E. faecium strains. All E. casseliflavus and E. gallinarum strainswere negative for these traits. Hydrophobic type of cell surface (measured by its affinity for n-hexadecane) was shown in a few isolates. Bacterial adherence was not significantly associated with the above pathogenic factors.

Bacterial Adhesion↗
PubMed2002

The 2.4-A crystal structure of the penicillin-resistant penicillin-binding protein PBP5fm from Enterococcus faecium in complex with benzylpenicillin.

Penicillin-binding proteins (PBPs) are membrane proteins involved in the final stages of peptidoglycan synthesis and represent the targets of beta-lactam antibiotics. Enterococci are naturally resistant to these antibiotics because they produce a PBP, named PBP5fm in Enterococcus faecium, with low-level affinity for beta-lactams. We report here the crystal structure of the acyl-enzyme complex of PBP5fm with benzylpenicillin at a resolution of 2.4 A. A characteristic of the active site, which distinguishes PBP5fm from other PBPs of known structure, is the topology of the loop 451-465 defining the left edge of the cavity. The residue Arg464, involved in a salt bridge with the residue Asp481, confers a greater rigidity to the PBP5fm active site. In addition, the presence of the Val465 residue, which points into the active site, reducing its accessibility, could account for the low affinity of PBP5fm for beta-lactam. This loop is common to PBPs of low affinity, such as PBP2a from Staphylococcus aureus and PBP3 from Bacillus subtilis. Moreover, the insertion of a serine after residue 466 in the most resistant strains underlines even more the determining role of this loop in the recognition of the substrates.

Amino Acid Sequence↗
PubMed2002

Promoter analysis of the sodium-responsive V-ATPase (ntp) operon in Enterococcus hirae.

The Enterococcus hirae ntp operon encodes all subunits of the vacuolar-type ATPase (V-ATPase), which transports Na(+) or Li(+). This operon is expressed preferentially in response to Na(+), but not to Li(+). Deletion analysis of the ntp promoter region in plasmids indicated that the AT-tract between -198 and -132 is required for Na(+)-specific transcriptional regulation. In addition, lithium-tolerant (LTR) mutants were isolated in which functional V-ATPase levels were high even in Na(+)-depleted medium. Western blot and Northern blot experiments revealed an increase in basal Na(+)-independent transcription in one of the mutants (LTR1). The nucleotide sequences of the ntp promoter region of the LTR mutants showed mutational conversion of single base-pairs between positions -23 and +1. Na(+)-independent expression of a reporter gene linked to the ntp promoter in plasmids was elevated by base substitutions at -23 to +1, and promoter activity induced by these base substitutions was lost by deletion of the region between -198 and -132. These results suggest that the AT-tract between -198 and -132 is indispensable for transcription of the ntp operon.

Adenosine Triphosphatases↗
PubMed2001

Potassium uptake with low affinity and high rate in Enterococcus hirae at alkaline pH.

Two high-affinity K+ uptake systems, KtrI and KtrII, have been reported in Enterococcus hirae. A mutant, JEMK1, defective in these two systems did not grow at pH 10 in low-K+ medium (less than 1 mM K+), but grew well when supplemented with 10 mM KCl. In this mutant, we found an energy-dependent K+ uptake at pH 10 with a low affinity for K+ (Km of approximately 20 mM) and an extremely high rate [Vmax of 1.6 micromol x min(-1) (mg protein)(-1)]. Rb+ uptake [Km of approximately 40 mM and Vmax of 0.5 micromol x min(-1) (mg protein)(-1)], which was inhibited competitively by K+ and less prominently by Cs+, was also observed. The specificity of this transport is likely to be K+>Rb+>Cs+. This peculiar K+ transport plays a role as a salvage mechanism against defects in high-affinity systems in the K+ homeostasis of this bacterium.

Adenosine Triphosphatases↗
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↗
PubMed1998

Peptidoglycan synthesis by Enterococcus faecalis penicillin binding protein 5.

Low-affinity penicillin binding proteins (PBPs) are a particular class of proteins involved in beta-lactam antibiotic resistance of enterococci. The activity of these PBPs is just sufficient to allow the cells to survive in the presence of high concentrations of beta-lactams that cause saturation (and inhibition) of the other PBPs. For this reason, the low-affinity PBPs are thought to be multifunctional enzymes capable of catalyzing the entire peptidoglycan synthesis. To test the validity of this claim, we analyzed the muropeptide composition by reversed-phase high-performance liquid chromatography of the peptidoglycan synthesized by PBP5 (the low-affinity PBP) of Enterococcus faecalis, in comparison with the peptidoglycan produced normally by the concerted action of the usual PBPs (namely PBPs 1, 2, and 3). Cross-linked peptidoglycan was produced. The main difference consisted in the lack of oligomers higher than trimers, thus suggesting that this oligomer cannot be used as an acceptor/donor by the transpeptidase component of PBP5. The lack of higher oligomers had little impact on total cross-linking because of the increase observed in the dimer family. This increase was distributed among the various members of the dimer family with the result that minor dimer components figured among the prevalent ones in cells in which peptidoglycan was synthesized by PBP5. This also suggests that E. faecalis PBP5 is capable of catalyzing the synthesis of a peptidoglycan that is less precise and refined than usual, and for this reason PBP5 can be considered an enzyme endowed with poor specificity for substrates, as may be expected on the basis of its survival function.

Bacterial Proteins↗
PubMed1998

Isolation and characterization of enterocin EJ97, a bacteriocin produced by Enterococcus faecalis EJ97.

The bacteriocinogenic strain of Enterococcus faecalis EJ97 has been isolated from municipal waste water. It produces a cationic bacteriocin (enterocin EJ97) of low molecular mass (5,340 Da) that is very stable under mild heat conditions and is sensitive to proteolytic enzymes. The amino acid sequence of the first 18 N-terminal residues of enterocin EJ97 indicates that it is different from other known protein sequences. Enterocin EJ97 is active on several gram-positive bacteria including enterococci, several species of Bacillus, Listeria, and Staphylococcus aureus. The producer strain is immune to bacteriocin. Enterocin EJ97 has a concentration-dependent bactericidal and bacteriolytic effect on E. faecalis S-47.

Amino Acid Sequence↗
PubMed2005

Heterologous expression of enterocin A, a bacteriocin from Enterococcus faecium, fused to a cellulose-binding domain in Escherichia coli results in a functional protein with inhibitory activity against Listeria.

The genes for the bacteriocins enterocin A and B were isolated from Enterococcus faecium ATB 197a. Using the pET37b(+) vector, the enterocin genes were fused to an Escherichia coli specific export signal sequence, a cellulose-binding domain (CBD(cenA)) and a S-tag under the control of a T7lac promotor. The constructs were subsequently cloned into E. coli host cells. The expression of the recombinant enterocins had different effects on both the host cells and other Gram-positive bacteria. The expression of entA in Esc. coli led to the synthesis and secretion of functional active enterocin A fusion proteins, which were active against some Gram-positive indicator bacteria, but did not influence the viability of the host cells. In contrast, the expression of enterocin B fusion proteins led to a reduced viability of the host cells, indicating a misfolding of the protein or interference with the cellular metabolism of Esc. coli. Indicator strains of Gram-positive bacteria were not inhibited by purified enterocin B fusion proteins. However, recombinant enterocin B displayed inhibitory activity after the proteolytic cleavage of the fused peptides.

Bacteriocins↗
PubMed2006

High-level heterologous production and functional expression of the sec-dependent enterocin P from Enterococcus faecium P13 in Lactococcus lactis.

Enterocin P (EntP), a sec-dependent bacteriocin from Enterococcus faecium P13, was produced by Lactococcus lactis. The EntP structural gene (entP) with or without the EntP immunity gene (entiP) was cloned in (1), plasmid pMG36c under control of the lactococcal constitutive promoter P32, (2) in plasmid pNG8048e under control of the inducible PnisA promoter, and (3) in the integration vector pINT29. Introduction of the recombinant vectors in L. lactis resulted in production of biologically active EntP in the supernatants of L. lactis subsp. lactis IL1403 and L. lactis subsp. cremoris NZ9000, and the coproduction of nisin A and EntP in L. lactis subsp. lactis DPC5598. The level of production of EntP, detected and quantified by specific anti-EntP antibodies and a noncompetitive indirect enzyme-linked immunosorbent assay, by the recombinant L. lactis strains depended on the host strain, the expression vector, and the presence of the entiP gene in the constructs of the recombinant L. lactis strains. The highest amount of EntP was produced with derivatives containing entP and entiP, for both L. lactis IL1403 and L. lactis NZ9000. These derivatives produced up to five- to six-fold more EntP than E. faecium P13. Mass spectrometry analysis revealed that EntP purified from L. lactis IL1403 (pJP214) has a molecular mass identical to that purified from E. faecium P13, suggesting that the synthesis, processing, and secretion of EntP progresses efficiently in recombinant L. lactis hosts.

Bacterial Proteins↗
PubMed2006

Factors affecting tyramine production in Enterococcus durans IPLA 655.

The decarboxylation of tyrosine by certain lactic acid bacteria leads to the undesirable presence of tyramine in fermented foods. Tyramine is the most frequent biogenic amine found in cheese and is also commonly found in other fermented foods and beverages. The tyramine-producing strain Enterococcus durans IPLA 655 was grown in a bioreactor under different conditions to determine the influence of carbon source, tyrosine and tyramine concentrations, and pH on tyramine production. The carbon source appeared to have no significant effect on the production of tyramine. In contrast, tyrosine was necessary for tyramine production, while the presence of tyramine itself in the growth medium inhibited such production. pH showed by far the greatest influence on tyramine synthesis; tyramine was produced in the greatest quantities at pH 5.0, although this was accompanied by a reduced growth rate.

Biogenic Amines↗
PubMed2000

Cloning, characterization and expression of an Enterococcus faecalis gene responsive to heavy metals.

Gene encoding stress response proteins are induced by a variety of environmental stimuli including the presence of heavy metals. To address the utility of this response for pollutant detection, one cadmium-induced gene in Enterococcus faecalis was isolated, sequenced and studied at the transcriptional level. csrA contains an open reading frame encoding a protein of 168 amino acids with homology to the enzyme peptide methionine sulfoxide reductase. The csrA mRNA was barely present in unstressed E. faecalis cells grown in M17-glucose medium, but accumulated at higher levels in cadmium-treated cells. Mercury also had an effect on csrA expression, whereas lead, copper and manganese induced csrA expression only at the highest doses tested. Our results suggest that biosensors based on E. faecalis may have potential applications for environmental monitoring and should be constructed.

Bacterial Proteins↗
PubMed1996

Factors affecting the formation of 10-hydroxystearic acid from oleic acid by a ruminal strain of Enterococcus faecalis.

A ruminal strain of Enterococcus faecalis was characterised with respect to its ability to hydrate oleic acid to 10-hydroxystearic acid. Hydroxy fatty acid was produced after growth had ceased and the carbon source was almost exhausted. Hydroxy fatty acid production was equally rapid whether the inoculum had been grown in the presence of oleic acid or not, and almost complete conversion was achieved when oleic acid was present at a concentration of up to 0.5% (v/v). Incubation under a hydrogen headspace did not result in biohydrogenation of oleic acid. In pH-controlled batch culture the proportion of oleic acid hydrated varied with the pH of incubation, with more hydration at lower pH. Growth was retarded in the presence of 0.1% (v/v) linoleic acid, inhibited by the same concentration of linolenic acid and did not result in the formation of hydrated products from these substrates. If this organism is able to transform oleic acid in the rumen then the only product likely to be formed is 10-hydroxystearic acid.

Animals↗
PubMed2001

Optimization of enterocin P production by batch fermentation of Enterococcus faecium P13 at constant pH.

The influence of pH on growth, enterocin P production and glucose consumption by Enterococcus faecium P13 was studied during anaerobic batch fermentation in MRS broth at 32 degrees C in a fermentor. Growth and glucose consumption were maximal at pH 7.0. Enterocin P production displayed primary metabolite kinetics and was strongly dependent on pH. A maximum antimicrobial activity of 1,949 bacteriocin units (BU) ml(-1) was obtained at pH 6.0, which represented a four-fold increase compared with the antimicrobial activity obtained without pH regulation. The pH exerted a marked effect on the decrease in bacteriocin activity, with the decrease being maximal at pH 7.0. In this report, we propose models for the growth of E. faecium P13 as well as enterocin P production and inactivation. Enterocin P production decreased when potentially stress-inducing compounds (NaCl or ethanol) were included in the growth medium.

Adsorption↗
PubMed2002

Survival of Enterococcus faecalis in seawater microcosms is limited in the presence of bacterivorous zooflagellates.

The survival and persistence of growing and starved cells of Enterococcus faecalis in untreated and differentially filtered (20 microm, 5 microm, 3 microm, 1.2 microm, and 0.1 microm) seawater was analyzed in samples taken at different times over a 1-year period by plate counts and scanning electron microscopy. Whereas seawater filtered through a 0.1-microm mesh was not at all or only slightly bactericidal during incubation at 16 degrees C in the dark, culturability of E. faecalis in the other systems decreased as a function of increasing pore size of the filters. Recovery of culturable, glucose pre-starved cells was always higher than that of cells harvested from the exponential growth phase. Electron microscopic analysis showed that the disappearance of enterococci appeared related to the presence and multiplication of various zooflagellates.

Animals↗
PubMed2002

In vitro adhesion to human cells by viable but nonculturable Enterococcus faecalis.

The ability of viable but nonculturable (VBNC) Enterococcus faecalis to adhere to Caco-2 and Girardi heart cultured cells and to urinary tract epithelial cells (ECs) was studied. Enterococci were harvested during the vegetative growth phase (early exponential and stationary), in the VBNC state, and after recovery of the ability to divide. VBNC bacteria maintained their adherence capability but the efficiency of attachment was reduced by about 50 to 70%, depending on the target cell employed. The decrease was transient, since enterococci that regained their culturability showed adherence values similar to those observed for actively growing cells. Analysis of the invasive properties of E. faecalis revealed that the VBNC state caused a decrease in the number of bacteria that entered the cultured HEK cells as a result of the reduction in the number of adhering bacteria. These results highlight the importance of studies of the VBNC phenomenon, with respect to both microbial survival in the environment and the impact on human health.

Bacterial Adhesion↗
PubMed2002

Isolation and characterization of bile salts-sensitive mutants of Enterococcus faecalis.

A library of insertional mutants of Enterococcus faecalis was constructed; it allowed the isolation and the characterization of 10 mutants affected in resistance to bile salts. Insertion loci of two mutants corresponded to genes of unknown function, while the amino acid sequences deduced from the other loci were homologous to proteins related to DNA repair, oxidative response, transcriptional regulation, dGTP hydrolysis, membrane composition, or cell wall synthesis. Further characterization of one mutant revealed that the insertion within the E. faecalis sagA gene led to a decrease of the resistance towards numerous independent physicochemical stresses, to modifications of the cell wall integrity, and to perturbations of cell division with septation anomalies.

Bacterial Proteins↗
PubMed2003

Heat-resistance and heat-shock response in the nosocomial pathogen Enterococcus faecium.

We have characterized the heat-shock response of the nosocomial pathogen Enterococcus faecium. The growth of E. faecium cells was analyzed at different temperatures; little growth was observed at 50 degrees C, and no growth at 52 degrees C or 55 degrees C. In agreement, a marked decrease of general protein synthesis was observed at 52 degrees C, and very light synthesis was detected at 55 degrees C. The heat resistance of E. faecium cells was analyzed by measuring the survival at temperatures higher than 52 degrees C and, after 2 h of incubation, viable cells were still observed at 70 degrees C. By Western blot analysis, two heat-induced proteins were identified as GroEL (65 kDa) and DnaK (75 kDa). Only one isoform for either GroEL or DnaK was found. The gene expression of these heat-shock proteins was also analyzed by pulsed-labeled experiments. The heat-induced proteins showed an increased rate of synthesis during the first 5 min, reaching the highest level of induction after 10 min and returning to the steady-state level after 20 min of heat treatment.

Chaperonin 60↗