Faecalis Genes Research Articles

Oral inoculation of Enterococcus faecalis, DNA quantification and histopathological evaluation of gingival, heart and kidney tissue samples in rats.

Enterococcus faecalis is known as common pathogen for endodontic infections and cause secondary and refractory pulp periapical periodontitis. The bacteria can opportunistically colonize periodontal pockets and presents a possibility of infection developing in other organs. This research will investigate the dissemination of E. faecalis from the gingival tissue to the heart and kidney. Three groups were formed, consisting of twelve male Sprague Dawley rats: a control group designated as 0-day, and experimental groups labeled as 7-days and 14-days. Periodontitis induced by concurrent infection with sterile wire 0.2 mm insertion and E. faecalis inoculation is performed into the gingival sulcus located between the maxillary right 1st and 2nd molar teeth area. After euthanasia, tissue samples around the maxillary gingiva, maxillary jaw samples, kidney and heart tissues were obtained for quantitative Real-Time PCR assay and histopathological analysis. Results showed at 7-days, there was an upregulation of E. faecalis gene expression in the gingiva, heart, and kidney samples as well as infiltration of the inflammatory cells at 7-days post induction, which consequently decreased at 14-days. Thus, the study suggests dissemination of E. faecalis from gingival tissue to the heart, kidney which could be probable link between periodontal disease, heart, and kidney disease.

Genome-wide analysis of acid tolerance genes of Enterococcus faecalis with RNA-seq and Tn-seq

Enterococcus faecalis, a formidable nosocomial and community-acquired opportunistic pathogen, can persist a wide range of extreme environments, including low pH and nutrient deficiency. Clarifying the survival mechanism of E. faecalis in low-pH conditions is the key to combating the infectious diseases caused by E. faecalis. In this study, we combined transcriptome profiling (RNA-seq) and transposon insertion sequencing (TIS) to comprehensively understand the genes that confer these features on E. faecalis. The metadata showed that genes whose products are involved in cation transportation and amino acid biosynthesis were predominantly differentially expressed under acid conditions. The products of genes such as opp1C and copY reduced the hydrion concentration in the cell, whereas those of gldA2, gnd2, ubiD, and ubiD2 mainly participated in amino metabolism, increasing matters to neutralize excess acid. These, together with the folE and hexB genes, which are involved in mismatch repair, form a network of E. faecalis genes necessary for its survival under acid conditions.

New Multilocus Sequence Typing Scheme for Enterococcus faecium Based on Whole Genome Sequencing Data.

The MLST scheme currently used for Enterococcus faecium typing was designed in 2002 and is based on putative gene functions and Enterococcus faecalis gene sequences available at that time. As a result, the original MLST scheme does not correspond to the real genetic relatedness of E. faecium strains and often clusters genetically distant strains to the same sequence types (ST). Nevertheless, typing has a significant impact on the subsequent epidemiological conclusions and introduction of appropriate epidemiological measures, thus it is crucial to use a more accurate MLST scheme. Based on the genome analysis of 1,843 E. faecium isolates, a new scheme, consisting of 8 highly discriminative loci, was created in this study. These strains were divided into 421 STs using the new MLST scheme, as opposed to 223 STs assigned by the original MLST scheme. The proposed MLST has a discriminatory power of D = 0.983 (CI95% 0.981 to 0.984), compared to the original scheme's D = 0.919 (CI95% 0.911 to 0.927). Moreover, we identified new clonal complexes with our newly designed MLST scheme. The scheme proposed here is available within the PubMLST database. Although whole genome sequencing availability has increased rapidly, MLST remains an integral part of clinical epidemiology, mainly due to its high standardization and excellent robustness. In this study, we proposed and validated a new MLST scheme for E. faecium, which is based on genome-wide data and thus reflects the tested isolates' more accurate genetic similarity. IMPORTANCE Enterococcus faecium is one of the most important pathogens causing health care associated infections. One of the main reasons for its clinical importance is a rapidly spreading resistance to vancomycin and linezolid, which significantly complicates antibiotic treatment of infections caused by such resistant strains. Monitoring the spread and relationships between resistant strains causing severe conditions represents an important tool for implementing appropriate preventive measures. Therefore, there is an urgent need to establish a robust method enabling strain monitoring and comparison at the local, national, and global level. Unfortunately, the current, extensively used MLST scheme does not reflect the real genetic relatedness between individual strains and thus does not provide sufficient discriminatory power. This can lead directly to incorrect epidemiological measures due to insufficient accuracy and biased results.

Interactions between Candida albicans and Enterococcus faecalis in an Organotypic Oral Epithelial Model.

Candida albicans as an opportunistic pathogen exploits the host immune system and causes a variety of life-threatening infections. The polymorphic nature of this fungus gives it tremendous advantage to breach mucosal barriers and cause oral and disseminated infections. Similar to C. albicans, Enterococcus faecalis is a major opportunistic pathogen, which is of critical concern in immunocompromised patients. There is increasing evidence that E. faecalis co-exists with C. albicans in the human body in disease samples. While the interactive profiles between these two organisms have been studied on abiotic substrates and mouse models, studies on their interactions on human oral mucosal surfaces are non-existent. Here, for the first time, we comprehensively characterized the interactive profiles between laboratory and clinical isolates of C. albicans (SC5314 and BF1) and E. faecalis (OG1RF and P52S) on an organotypic oral mucosal model. Our results demonstrated that the dual species biofilms resulted in profound surface erosion and significantly increased microbial invasion into mucosal compartments, compared to either species alone. Notably, several genes of C. albicans involved in tissue adhesion, hyphal formation, fungal invasion, and biofilm formation were significantly upregulated in the presence of E. faecalis. By contrast, E. faecalis genes involved in quorum sensing, biofilm formation, virulence, and mammalian cell invasion were downregulated. This study highlights the synergistic cross-kingdom interactions between E. faecalis and C. albicans in mucosal tissue invasion.

Identification of the general stress stimulon related to colonization in Enterococcus faecalis.

Enterococcus faecalis has to cope with major stress conditions during colonization. To understand the effects of stress encountered during infection, the present study assessed the transcriptomic response of the bacteria facing exposure to serum, urine, bile salts, acid pH, or oxidative stress. Compared to non-stressed culture, 30% of the E. faecalis genes were differentially expressed. The transcriptome analysis reveals common but also specific responses, depending on stresses encountered: thus, urine exposure has the most important impact, and the highest number of genes with modified expression is involved in transport and metabolism. The results also pinpoint many stress-related sRNA or intergenic regions not yet characterized. This study identified the general stress stimulon related to infection: when the commensal bacterium initiates its response to stress related to infection, it increases its ability to survive to rough conditions for colonization, rather than promoting expression of virulence factors, and becomes this opportunistic pathogen that thrives in hospital settings.

Role of epaQ, a Previously Uncharacterized Enterococcus faecalis Gene, in Biofilm Development and Antimicrobial Resistance.

Enterococcus faecalis is a commensal of the human gastrointestinal tract; it is also an opportunistic pathogen and one of the leading causes of hospital-acquired infections. E. faecalis produces biofilms that are highly resistant to antibiotics, and it has been previously reported that certain genes of the epa operon contribute to biofilm-associated antibiotic resistance. Despite several studies examining the epa operon, many gene products of this operon remain annotated as hypothetical proteins. Here, we further explore the epa operon; we identified epaQ, currently annotated as encoding a hypothetical membrane protein, as being important for biofilm formation in the presence of the antibiotic daptomycin. Mutants with disruptions of epaQ were more susceptible to daptomycin relative to the wild type, suggesting its importance in biofilm-associated antibiotic resistance. Furthermore, the ΔepaQ mutant exhibited an altered biofilm architectural arrangement and formed small aggregates in liquid cultures. Our cumulative data show that epa mutations result in altered polysaccharide content, increased cell surface hydrophobicity, and decreased membrane potential. Surprisingly, several epa mutations significantly increased resistance to the antibiotic ceftriaxone, indicating that the way in which the epa operon impacts antibiotic resistance is antibiotic dependent. These results further define the key role of epa in antibiotic resistance in biofilms and in biofilm architecture.IMPORTANCEE. faecalis is a common cause of nosocomial infection, has a high level of antibiotic resistance, and forms robust biofilms. Biofilm formation is associated with increased antibiotic resistance. Therefore, a thorough understanding of biofilm-associated antibiotic resistance is important for combating resistance. Several genes from the epa operon have previously been implicated in biofilm-associated antibiotic resistance, pathogenesis, and competitive fitness in the GI tract, but most genes in this locus remain uncharacterized. Here, we examine epaQ, which has not been characterized functionally. We show that the ΔepaQ mutant exhibits reduced biofilm formation in the presence of daptomycin, altered biofilm architecture, and increased resistance to ceftriaxone, further expanding our understanding of the contribution of this operon to intrinsic enterococcal antibiotic resistance and biofilm growth.

Complex Bacterial Consortia Reprogram the Colitogenic Activity of Enterococcus faecalis in a Gnotobiotic Mouse Model of Chronic, Immune-Mediated Colitis.

Inflammatory bowel diseases (IBD) are associated with compositional and functional changes of the intestinal microbiota, but specific contributions of individual bacteria to chronic intestinal inflammation remain unclear. Enterococcus faecalis is a resident member of the human intestinal core microbiota that has been linked to the pathogenesis of IBD and induces chronic colitis in susceptible monoassociated IL-10-deficient (IL-10−/−) mice. In this study, we characterized the colitogenic activity of E. faecalis as part of a simplified human microbial consortium based on seven enteric bacterial strains (SIHUMI). RNA sequencing analysis of E. faecalis isolated from monoassociated wild type and IL-10−/− mice identified 408 genes including 14 genes of the ethanolamine utilization (eut) locus that were significantly up-regulated in response to inflammation. Despite considerable up-regulation of eut genes, deletion of ethanolamine utilization (ΔeutVW) had no impact on E. faecalis colitogenic activity in monoassociated IL-10−/− mice. However, replacement of the E. faecalis wild type bacteria by a ΔeutVW mutant in SIHUMI-colonized IL-10−/− mice resulted in exacerbated colitis, suggesting protective functions of E. faecalis ethanolamine utilization in complex bacterial communities. To better understand E. faecalis gene response in the presence of other microbes, we purified wild type E. faecalis cells from the colon content of SIHUMI-colonized wild type and IL-10−/− mice using immuno-magnetic separation and performed RNA sequencing. Transcriptional profiling revealed that the bacterial environment reprograms E. faecalis gene expression in response to inflammation, with the majority of differentially expressed genes not being shared between monocolonized and SIHUMI conditions. While in E. faecalis monoassociation a general bacterial stress response could be observed, expression of E. faecalis genes in SIHUMI-colonized mice was characterized by up-regulation of genes involved in growth and replication. Interestingly, in mice colonized with SIHUMI lacking E. faecalis enhanced inflammation was observed in comparison to SIHUMI-colonized mice, supporting the hypothesis that E. faecalis ethanolamine metabolism protects against colitis in complex consortia. In conclusion, this study demonstrates that complex bacterial consortia interactions reprogram the gene expression profile and colitogenic activity of the opportunistic pathogen E. faecalis toward a protective function.

Persistence of vancomycin resistance genes and transposable elements in Enterococcus faecalis isolated from groundwater intended for human consumption: A public health implication

Enterococcus species are capable of surviving in different environments such as water, soil, plants and food products for long periods and has been used as an indicator of faecal contamination particularly in water. In the last decade organisms in this genus have gained significant attention due to the ability to cause opportunistic infections especially in immunocompromised individuals, patients receiving broad spectrum antibiotic treatment as well as those with long hospital stays. E. faecalis and E. faecium are the principal species of clinical importance and they have developed both natural and acquired resistance to various antimicrobial agents including beta (β)‑lactams, glycopeptides and aminoglycosides thus presenting severe public health challenges. The present study elucidated the antibiotic resistance profiles and vancomycin resistance gene determinants as well as transposable elements Tn1546 and Tn1547 in Enterococcus faecalis isolated from groundwater intended for human consumption in the North West Province, South Africa. A total of 60 water samples were collected from borehole taps between February 2014 and May 2015. Typical black Enterococcus colonies were isolated on Bile Esculin agar. The identities of E. faecalis isolates were confirmed using Gram staining, catalase test, growth in 6.5% NaCl broth, haemolysis, species-specific 16S rRNA and ddlE. faecalis gene specific PCR. Antimicrobial susceptibility profiles of isolates were determined using the agar disc diffusion assay while vancomycin resistance gene markers (vanA, vanB, Tn1546 and Tn1547) were detected by PCR. A total of 253 presumptive typical black colonies were analysed and 122 E. faecalis isolates were obtained. Large proportions (62%–100%) of the isolates were resistant to vancomycin, penicillin, amoxicillin, ampicillin, tetracycline and erythromycin and displayed multiple antibiotic resistance phenotypes. Both the vanA and vanB resistance gene determinants were detected in 63 and 83 of the isolates respectively. Twenty-seven vanA isolates harbored the Tn1546 transposon while none of the vanB isolates possessed Tn1546.

A novel and ultrasensitive electrochemical DNA biosensor based on an ice crystals-like gold nanostructure for the detection of Enterococcus faecalis gene sequence.

Bacteria, parasites and viruses are found widely in the environment as potential pathogens, and can be the source of infections. Therefore, sensitive and rapid methods for identification of the pathogens are required to achieve a better quality of life. Enterococcus faecalis commonly colonizes and threatens human health. In the present study, we demonstrate the fabrication of a novel electrochemical DNA biosensor based on electrodeposited gold nanostructures as a transducer substrate combined with toluidine blue (TB) as a redox marker. Binding of TB with the single and double stranded DNA (ssDNA and dsDNA) was shortly investigated, and based on the results, TB could discriminate between ssDNA and dsDNA. A specific thiolated ssDNA sequence was immobilized on the transducer substrate, and DNA hybridization was followed by differential pulse voltammetry. The DNA biosensor showed excellent performances with high sensitivity and good selectivity. The DNA biosensor was applied to detect a synthetic target in a linear range of 1.0 × 10-17-1.0 × 10-10 mol L-1 with a limit of detection (LOD) of 4.7 × 10-20 mol L-1. In addition, LOD of the DNA biosensor for the detection of genomic DNA was found to be 30.1 ng μL-1.

Metabolic engineering of mevalonate-producing Escherichia coli strains based on thermodynamic analysis

Thermodynamic states of the central metabolism in a metabolically engineered Escherichia coli strain producing mevalonate (MVA) were studied to identify metabolic reactions with regulatory function for improvement of the specific rate of MVA production. Intracellular concentrations of metabolites were determined for E. coli strains expressing Enterococcus faecalis genes mvaE and mvaS (strain MV) by gas chromatography (GC)- and liquid chromatography (LC)-mass spectrometry (MS). Mixtures of 13C-labeled metabolites served as internal standards were prepared from E. coli cultured in a completely 13C-labeled medium. Based on the concentration data, the change in Gibbs energy (ΔG) and substrate saturation ([S]/KM) were calculated for each metabolic reaction and then compared between the control and MVA-producing strains. The thermodynamic and kinetic analyses showed that further activation of thermodynamically feasible reactions in the upper part of glycolysis and the pentose phosphate pathway seems difficult and that metabolic bypassing to the Entner–Doudoroff pathway was a promising strategy to improve the acetyl coenzyme A (AcCoA) and NADPH supply required for MVA biosynthesis. Strain MV-ΔGndΔGntR was constructed by deletion of the gnd and gntR genes, which respectively encode 6-phosphogluconate dehydrogenase and a negative regulator of the expression of two enzyme genes responsible for the Entner–Doudoroff pathway. Cultivation in the nongrowth phase revealed that the yield and specific production rate of MVA increased to 0.49 ± 0.01 Cmol (Cmol glucose)−1 and 2.61 ± 0.10 mmol (g dry cell weight)−1 h−1, which were 113% and 158% that of the MV strain, respectively.

Characterization of biofilm formation by Enterococcus faecalis isolates derived from urinary tract infections in China.

PurposeThis study explored the prevalence and characteristics of Enterococcus faecalis biofilm formation by urinary tract infection (UTI) isolates in order to identify virulence factors associated with biofilm formation.MethodologyA total of 113 E. faecalis isolates were collected from UTI patients in Shenzhen, China. The isolates were subjected to multilocus sequence typing based on housekeeping genes. Biofilms were detected by crystal violet staining and the expression levels of the E. faecalis genes were detected by quantitative real-time PCR.Results/Key findingsThe main sequence types (STs) were ST16 and ST179 with the ST16 isolates more likely to form strong biofilms than the ST179 isolates (P=0.008). Strong biofilm formation was more frequently detected in aggregation substance (agg)-positive (+) isolates than in negative (−) isolates (P=0.033). Biofilm formation was also more common in isolates containing enterococcal surface protein (esp), or cytolysin A (cylA)-positive (+) isolates than in isolates negative (−) for these virulence factors. Multivariate regression analysis indicated that cylA [odds ratio (OR), 7.143, P=0.012] was associated with weak biofilm formation, and that agg (OR, 4.471, P=0.004) was associated with strong biofilm formation. The expression of cylA was increased (8.75- to 23.05-fold) in weak biofilm, and the expression of agg was greatly elevated (11.99- to 439.10-fold) in strong biofilm isolates when compared to biofilm-negative isolates.ConclusionST16 classification was positively associated with strong biofilm formation in E. faecalis as was agg, while cylA was associated with weak biofilm formation.

Functional studies of E. faecalis RNase J2 and its role in virulence and fitness.

Post-transcriptional control provides bacterial pathogens a method by which they can rapidly adapt to environmental change. Dual exo- and endonucleolytic activities of RNase J enzymes contribute to Gram-positive RNA processing and decay. First discovered in Bacillus subtilis, RNase J1 plays a key role in mRNA maturation and degradation, while the function of the paralogue RNase J2 is largely unknown. Previously, we discovered that deletion of the Enterococcus faecalis rnjB gene significantly attenuates expression of a major virulence factor involved in enterococcal pathogenesis, the Ebp pili. In this work, we demonstrate that E. faecalis rnjB encodes an active RNase J2, and that the ribonuclease activity of RNase J2 is required for regulation of Ebp pili. To further investigate how rnjB affects E. faecalis gene expression on a global scale, we compared transcriptomes of the E. faecalis strain OG1RF with its isogenic rnjB deletion mutant (ΔrnjB). In addition to Ebp pili regulation, previously demonstrated to have a profound effect on the ability of E. faecalis to form biofilm or establish infection, we identified that rnjB regulates the expression of several other genes involved in bacterial virulence and fitness, including gls24 (a virulence factor important in stress response). We further demonstrated that the E. faecalis RNase J2 deletion mutant is more sensitive to bile salt and greatly attenuated in in vivo organ infection as determined by an IV-sublethal challenge infection mouse model, indicating that E. faecalis RNase J2 plays an important role in E. faecalis virulence.

Enterococcus faecalis zinc-responsive proteins mediate bacterial defence against zinc overload, lysozyme and oxidative stress.

Two Enterococcus faecalis genes encoding the P-type ATPase EF1400 and the putative SapB protein EF0759 were previously shown to be strongly upregulated in the presence of high concentrations of zinc. In the present work, we showed that a Zn(2+)-responsive DNA-binding motif (zim) is present in the promoter regions of these genes. Both proteins were further studied with respect to their involvement in zinc homeostasis and invasion of the host. EF0759 contributed to intramacrophage survival by an as-yet unknown mechanism(s). EF1400, here renamed ZntAEf, is an ATPase with specificity for zinc and plays a role in dealing with several host defences, i.e. zinc overload, oxidative stress and lysozyme; it provides E. faecalis cells with the ability to survive inside macrophages. As these three host defence mechanisms are important at several sites in the host, i.e. inside macrophages and in saliva, this work suggested that ZntAEf constitutes a crucial E. faecalis defence mechanism that is likely to contribute to the ability of this bacterium to endure life inside its host.

The surface rhamnopolysaccharide epa of Enterococcus faecalis is a key determinant of intestinal colonization.

Enterococcus faecalis is a commensal bacterium of the human intestine and a major opportunistic pathogen in immunocompromised and elderly patients. The pathogenesis of E. faecalis infection relies in part on its capacity to colonize the gut. Following disruption of intestinal homeostasis, E. faecalis can overgrow, cross the intestinal barrier, and enter the lymph and bloodstream. To identify and characterize E. faecalis genes that are key to intestinal colonization, our strategy consisted in screening mutants for the following phenotypes related to intestinal lifestyle: antibiotic resistance, overgrowth, and competition against microbiota. From the identified colonization genes, epaX encodes a glycosyltransferase located in a variable region of the enterococcal polysaccharide antigen (epa) locus. We demonstrated that EpaX acts on sugar composition, promoting resistance to bile salts and cell wall integrity. Given that EpaX is enriched in hospital-adapted isolates, this study points to the importance of the epa variability as a key determinant for enterococcal intestinal colonization.

Genes Important for Catalase Activity in Enterococcus faecalis

Little in general is known about how heme proteins are assembled from their constituents in cells. The Gram-positive bacterium Enterococcus faecalis cannot synthesize heme and does not depend on it for growth. However, when supplied with heme in the growth medium the cells can synthesize two heme proteins; catalase (KatA) and cytochrome bd (CydAB). To identify novel factors important for catalase biogenesis libraries of E. faecalis gene insertion mutants were generated using two different types of transposons. The libraries of mutants were screened for clones deficient in catalase activity using a colony zymogram staining procedure. Analysis of obtained clones identified, in addition to katA (encoding the catalase enzyme protein), nine genes distributed over five different chromosomal loci. No factors with a dedicated essential role in catalase biogenesis or heme trafficking were revealed, but the results indicate the RNA degradosome (srmB, rnjA), an ABC-type oligopeptide transporter (oppBC), a two-component signal transducer (etaR), and NADH peroxidase (npr) as being important for expression of catalase activity in E. faecalis. It is demonstrated that catalase biogenesis in E. faecalis is independent of the CydABCD proteins and that a conserved proline residue in the N-terminal region of KatA is important for catalase assembly.

Use of recombinase-based in vivo expression technology to characterize Enterococcus faecalis gene expression during infection identifies in vivo-expressed antisense RNAs and implicates the protease Eep in pathogenesis.

Enterococcus faecalis is a member of the mammalian gastrointestinal microflora that has become a leading cause of nosocomial infections over the past several decades. E. faecalis must be able to adapt its physiology based on its surroundings in order to thrive in a mammalian host as both a commensal and a pathogen. We employed recombinase-based in vivo expression technology (RIVET) to identify promoters on the E. faecalis OG1RF chromosome that were specifically activated during the course of infection in a rabbit subdermal abscess model. The RIVET screen identified 249 putative in vivo-activated loci, over one-third of which are predicted to generate antisense transcripts. Three predicted antisense transcripts were detected in in vitro- and in vivo-grown cells, providing the first evidence of in vivo-expressed antisense RNAs in E. faecalis. Deletions in the in vivo-activated genes that encode glutamate 5-kinase (proB [EF0038]), the transcriptional regulator EbrA (ebrA [EF1809]), and the membrane metalloprotease Eep (eep [EF2380]) did not hinder biofilm formation in in vitro assays. In a rabbit model of endocarditis, the ΔebrA strain was fully virulent, the ΔproB strain was slightly attenuated, and the Δeep strain was severely attenuated. The Δeep virulence defect could be complemented by the expression of the wild-type gene in trans. Microscopic analysis of early Δeep biofilms revealed an abundance of small cellular aggregates that were not observed in wild-type biofilms. This work illustrates the use of a RIVET screen to provide information about the temporal activation of genes during infection, resulting in the identification and confirmation of a new virulence determinant in an important pathogen.

Construction of a New Sensitive Molecular Tool for the Study of Gene Expression in Enterococcus faecalis

A promoter-probe vector designated pVEPhoZ-P has been developed to provide a convenient system to analyze transcription activities of promoters. It was constructed on the basis of pVE14218, a plasmid which lacks the replication protein and in which the 5′ part of alkaline phosphatase (AP) phoZ gene of Enterococcus faecalis was inserted. The pVEPhoZ vector was used to clone promoters of interest. The resulting promoter-probe vectors were integrated in the phoZ chromosomal locus by homologous recombination. This procedure generates recombinant clones with a single copy of phoZ functional allele placed under the control of the desired promoter. This system was investigated with different promoters of E. faecalis genes, namely those of sigV encoding an ECF sigma factor, croRS encoding a two-component system and dhaK operon. In all cases, expression data obtained previously for the three promoters were properly reported for this new tool. The pVEPhoZ-P promoter-probe vector is easy to use and it showed higher reporter activity in comparison to systems based on β-galactosidase. Therefore, this system constitutes a useful molecular tool for the study of promoters in E. faecalis or other bacterial species that possess a homologous phoZ gene.

Characterization and Kinetics of Phosphopantothenoylcysteine Synthetase from Enterococcus faecalis

The enzyme phosphopantothenoylcysteine synthetase (PPCS) catalyzes the nucleotide-dependent formation of phosphopantothenoylcysteine from (R)-phosphopantothenate and L-cysteine in the biosynthetic pathway leading to the formation of the essential biomolecule, coenzyme A. The Enterococcus faecalis gene coaB encodes a novel monofunctional PPCS which has been cloned into pET23a and expressed in Escherichia coli BL21 AI. The heterologous expression system yielded 30 mg of purified PPCS per liter of cell culture. The purified enzyme chromatographed as a homodimer of 28 kDa subunits on Superdex HR 200 gel filtration resin. The monofunctional protein displayed a nucleotide specificity for cytidine 5'-triphosphate (CTP) analogous to that seen for bifunctional PPCS expressed by most prokaryotes. Kinetic characterization, utilizing initial velocity and product inhibition studies, found the mechanism of PPCS to be Bi Uni Uni Bi Ping-Pong, with the nucleotide CTP binding first and CMP released last. Michaelis constants were 156, 17, and 86 microM for CTP, (R)-phosphopantothenate, and L-cysteine, respectively, and the k(cat) was 2.9 s(-1). [carboxyl-(18)O]Phosphopantothenate was prepared by hydrolysis of methyl pantothenate with Na(18)OH, followed by enzymatic phosphorylation with E. faecalis pantothenate kinase (PanK). The fate of the carboxylate oxygen of labeled phosphopantothenate, during the course of the PPCS-catalyzed reaction with CTP and L-cysteine, was monitored by (31)P NMR spectroscopy. The results show that the carboxylate oxygen of the phosphopantothenate is recovered with the CMP formed during the reaction, indicative of the formation of a phosphopantothenoyl cytidylate catalytic intermediate, which is consistent with the kinetic mechanism.

The Enterococcus faecalis gene encoding the novel general stress protein Gsp62.

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 beta-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.

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.