Enterococcus faecalis mevalonate kinase

Gram-positive pathogens synthesize isopentenyl diphosphate, the five-carbon precursor of isoprenoids, via the mevalonate pathway. The enzymes of this pathway are essential for the survival of these organisms, and thus may represent possible targets for drug design. To extend our investigation of the mevalonate pathway in Enterococcus faecalis, we PCR-amplified and cloned into pET-28b the mvaK1 gene thought to encode mevalonate kinase, the fourth enzyme of the pathway. Following transformation of the construct EFK1-pET28b into Escherichia coli BL21(DE3) cells, the expressed C-terminally hexahistidine-tagged protein was purified on a nickel affinity support to apparent homogeneity. The purified protein catalyzed the divalent ion-dependent phosphorylation of mevalonate to mevalonate 5-phosphate. The specific activity of the purified kinase was 24 micromole/min/mg protein. Based on sedimentation velocity data, E. faecalis mevalonate kinase exists in solution primarily as a monomer with a mass of 32.2 kD. Optimal activity occurred at pH 10 and at 37 degrees C. Delta H(a) was 22 kcal/mole. Kinetic analysis suggested that the reaction proceeds via a sequential mechanism. K(m) values were 0.33 mM (mevalonate), 1.1 mM (ATP), and 3.3 mM (Mg(2+)). Unlike mammalian mevalonate kinases, E. faecalis mevalonate kinase utilized all tested nucleoside triphosphates as phosphoryl donors. ADP, but not AMP, inhibited the reaction with a K(i) of 2.7 mM.     

Comparison of genotypic and phenotypic cluster analyses of virulence determinants and possible role of CRISPR elements towards their incidence in Enterococcus faecalis and Enterococcus faecium

Enterococcus faecalis and Enterococcus faecium are human commensals frequently found in fermented foods or used as probiotics, but also recognized as opportunistic pathogens. We investigated 62 Enterococcus strains isolated from clinical, food and environmental origins towards a rationale for safety evaluation of strains in food or probiotic applications. All isolates were characterised with respect to the presence of the virulence determinants fsrB, sprE, gelE, ace, efaAfs/fm, as, esp, cob and the cytolysin operon. In addition RAPD-PCR was used to obtain genomic fingerprints that were clustered and compared to phenotypic profiles generated by MALDI-TOF-MS. The gelatinase phenotype (GelE) and the haemolytic activity (β-haemolysis) were analysed. E. faecium strains contained esp and efaAfm only, and none of them contained any CRISPR elements. The amenability of E. faecalis strains to acquisition of virulence factors was investigated along the occurrence of CRISPR associated (cas) genes. While distribution of most virulence factors, and RAPD versus MALDI-TOF-MS typing patterns were unrelated, 2 out of 5 RAPD clusters almost exclusively contained clinical E. faecalis isolates, and an occurrence of CRISPR elements versus reduced number of virulence factors was observed. The presence of the cytolysin operon, cob and as encoding pheromone and aggregation substance, respectively, significantly corresponded to absence of cas. As their production promote genetic exchange, their absence limits further gene acquisition and distribution. Thus, absence of the cytolysin operon, cob and as in a cas positive environment suggests itself as promising candidate for E. faecalis evaluation towards their occurrence in food fermentation or use as probiotics.     

Comprehensive molecular,genomic and phenotypic analysis of a major clone of Enterococcus faecalis MLST ST40

Background

Enterococcus faecalis is a multifaceted microorganism known to act as a beneficial intestinal commensal bacterium. It is also a dreaded nosocomial pathogen causing life-threatening infections in hospitalised patients. Isolates of a distinct MLST type ST40 represent the most frequent strain type of this species, distributed worldwide and originating from various sources (animal, human, environmental) and different conditions (colonisation/infection). Since enterococci are known to be highly recombinogenic we determined to analyse the microevolution and niche adaptation of this highly distributed clonal type.

Results

We compared a set of 42 ST40 isolates by assessing key molecular determinants, performing whole genome sequencing (WGS) and a number of phenotypic assays including resistance profiling, formation of biofilm and utilisation of carbon sources. We generated the first circular closed reference genome of an E. faecalis isolate D32 of animal origin and compared it with the genomes of other reference strains. D32 was used as a template for detailed WGS comparisons of high-quality draft genomes of 14 ST40 isolates. Genomic and phylogenetic analyses suggest a high level of similarity regarding the core genome, also demonstrated by similar carbon utilisation patterns. Distribution of known and putative virulence-associated genes did not differentiate between ST40 strains from a commensal and clinical background or an animal or human source. Further analyses of mobile genetic elements (MGE) revealed genomic diversity owed to: (1) a modularly structured pathogenicity island; (2) a site-specifically integrated and previously unknown genomic island of 138 kb in two strains putatively involved in exopolysaccharide synthesis; and (3) isolate-specific plasmid and phage patterns. Moreover, we used different cell-biological and animal experiments to compare the isolate D32 with a closely related ST40 endocarditis isolate whose draft genome sequence was also generated. D32 generally showed a greater capacity of adherence to human cell lines and an increased pathogenic potential in various animal models in combination with an even faster growth in vivo (not in vitro).

Conclusion

Molecular, genomic and phenotypic analysis of representative isolates of a major clone of E. faecalis MLST ST40 revealed new insights into the microbiology of a commensal bacterium which can turn into a conditional pathogen.

Electronic supplementary material

The online version of this article (doi:10.1186/s12864-015-1367-x) contains supplementary material, which is available to authorized users.     

Enterococcus faecalis phosphomevalonate kinase

The six enzymes of the mevalonate pathway of isopentenyl diphosphate biosynthesis represent potential for addressing a pressing human health concern, the development of antibiotics against resistant strains of the Gram-positive streptococci. We previously characterized the first four of the mevalonate pathway enzymes of Enterococcus faecalis, and here characterize the fifth, phosphomevalonate kinase (E.C. 2.7.4.2). E. faecalis genomic DNA and the polymerase chain reaction were used to clone DNA thought to encode phosphomevalonate kinase into pET28b(+). Double-stranded DNA sequencing verified the sequence of the recombinant gene. The encoded N-terminal hexahistidine-tagged protein was expressed in Escherichia coli with induction by isopropylthiogalactoside and purified by Ni(++) affinity chromatography, yield 20 mg protein per liter. Analysis of the purified protein by MALDI-TOF mass spectrometry established it as E. faecalis phosphomevalonate kinase. Analytical ultracentrifugation revealed that the kinase exists in solution primarily as a dimer. Assay for phosphomevalonate kinase activity used pyruvate kinase and lactate dehydrogenase to couple the formation of ADP to the oxidation of NADH. Optimal activity occurred at pH 8.0 and at 37 degrees C. The activation energy was approximately 5.6 kcal/mol. Activity with Mn(++), the preferred cation, was optimal at about 4 mM. Relative rates using different phosphoryl donors were 100 (ATP), 3.6 (GTP), 1.6 (TTP), and 0.4 (CTP). K(m) values were 0.17 mM for ATP and 0.19 mM for (R,S)-5-phosphomevalonate. The specific activity of the purified enzyme was 3.9 micromol substrate converted per minute per milligram protein. Applications to an immobilized enzyme bioreactor and to drug screening and design are discussed.     

Antibiotic resistance and virulence traits in clinical and environmental Enterococcus faecalis and Enterococcus faecium isolates

This study compared virulence and antibiotic resistance traits in clinical and environmental Enterococcus faecalis and Enterococcus faecium isolates. E. faecalis isolates harboured a broader spectrum of virulence determinants compared to E. faecium isolates. The virulence traits Cyl-A, Cyl-B, Cyl-M, gel-E, esp and acm were tested and environmental isolates predominantly harboured gel-E (80% of E. faecalis and 31.9% of E. faecium) whereas esp was more prevalent in clinical isolates (67.8% of E. faecalis and 70.4% of E. faecium). E. faecalis and E. faecium isolated from water had different antibiotic resistance patterns compared to those isolated from clinical samples. Linezolid resistance was not observed in any isolates tested and vancomycin resistance was observed only in clinical isolates. Resistance to other antibiotics (tetracycline, gentamicin, ciprofloxacin and ampicillin) was detected in both clinical and water isolates. Clinical isolates were more resistant to all the antibiotics tested compared to water isolates. Multi-drug resistance was more prevalent in clinical isolates (71.2% of E. faecalis and 70.3% of E. faecium) compared to water isolates (only 5.7% E. faecium). tet L and tet M genes were predominantly identified in tetracycline-resistant isolates. All water and clinical isolates resistant to ciprofloxacin and ampicillin contained mutations in the gyrA, parC and pbp5 genes. A significant correlation was found between the presence of virulence determinants and antibiotic resistance in all the isolates tested in this study (p<0.05). The presence of antibiotic resistant enterococci, together with associated virulence traits, in surface recreational water could be a public health risk.     

Real-time PCR method to detect Enterococcus faecalis in water

A 16S rDNA real-time PCR method was developed to detect Enterococcus faecalis in water samples. The dynamic range for cell detection spanned five logs and the detection limit was determined to be 6 cfu/reaction. The assay was capable of detecting E. faecalis cells added to biofilms from a simulator of a water distribution system and in freshwater samples. Nucleic acid extraction was not required, permitting the detection of E. faecalis cells in less than 3 h.     

The tyrosine decarboxylation test does not differentiate Enterococcus faecalis from Enterococcus faecium

According to the current edition of the Bergey's Manual of Systematic Bacteriology [11] the tyrosine decarboxylation test allows the differentiation of enterococci. Tyrosine is decarboxylated to the biogenic amine tyramine by E. faecalis and not by E. faecium strains. In the present study we sequenced the16S rDNA of two tyramine-producing strains, BIFI-56 and BIFI-58, presumptively classified as E. faecalis. Their 16S rDNA were identical to the same fragment from the E. faecium type strain. Several E. faecium strains were then checked for their ability to decarboxylate tyrosine and also a putative tyrosine decarboxylase-coding gene was PCR amplified from these strains. All the strains confirmed as E. faecium produced tyramine and possessed a DNA fragment coding for a putative tyrosine decarboxylase. The concordance of the two methods allows us to conclude that the tyrosine decarboxylase test cannot be used in the differentiation of E. faecalis from E. faecium since at least some E. faecium strains are tyramine producers.     

Crystal structure of an aerobic FMN-dependent azoreductase (AzoA) from Enterococcus faecalis

The initial critical step of reduction of the azo bond during the metabolism of azo dyes is catalyzed by a group of NAD(P)H dependant enzymes called azoreductases. Although several azoreductases have been identified from microorganisms and partially characterized, very little is known about the structural basis for substrate specificity and the nature of catalysis. Enterococcus faecalis azoreductase A (AzoA) is a highly active azoreductase with a broad spectrum of substrate specificity and is capable of degrading a wide variety of azo dyes. Here, we report the crystal structure of the AzoA from E. faecalis determined at 2.07 A resolution with bound FMN ligand. Phases were obtained by single wavelength anomalous scattering of selenomethionine labeled protein crystals. The asymmetric unit consisted of two dimers with one FMN molecule bound to each monomer. The AzoA monomer takes a typical NAD(P)-binding Rossmann fold with a highly conserved FMN binding pocket. A salt bridge between Arg18 and Asp184 restricts the size of the flavin binding pocket such that only FMN can bind. A putative NADH binding site could be identified and a plausible mechanism for substrate reduction is proposed. Expression studies revealed azoA gene to be expressed constitutively in E. faecalis.     

Identification of new sex pheromone plasmids in Enterococcus faecalis.

Summary We describe the identification of the following new sex pheromone plasmids inEnterococcus faecalis: a haemolysin-bacteriocin plasmid, pIP964; three R plasmids, pIP1017, pIP1438 and pIP1440; and two cryptic conjugative plasmids, pIP1141 and pMV120. The identification was based on the formation of cell aggregates on filter membranes during conjugation, on efficient transfer in broth matings, and on a positive clumping reaction of cells carrying these plasmids. In addition these plasmids hybridized with DNA probes specific for sex pheromone-induced structural genes encoding surface proteins required for conjugative transfer of the plasmids.     

Sensitivity to detergents and plasmid curing in Enterococcus faecalis

This research reports the sensitivity of a clinical isolate of Enterococcus faecalis to sodium N-lauroylsarcosinate (sarkosyl) and sodium dodecyl sulfate (SDS), as well as the efficiency of these detergents in curing the strain. Compared to Escherichia coli, Enterococcus faecalis was very sensitive to both detergents, with minimum inhibitory concentrations (MIC) for the latter being 100 times lower than for Escherichia coli. The clinical isolate of Enterococcus faecalis used in this study exhibited plasmid-borne resistance to kanamycin (MIC 2 mg/ml) and tetracycline (MIC 50 μg/ml); 3% curing was observed after growth in the presence of sarkosyl but no curing was observed after growth in the presence of either SDS or acridine orange. In contrast, 35% curing of plasmid-bearing Escherichia coli was observed after growth in the presence of either SDS or acridine orange, but none was observed after growth in the presence of sarkosyl.     

一株益生性粪肠球菌的安全性评价

目的 研究1株微生态活菌制品生产用粪肠球菌的安全性。 方法 采用目前肠球菌安全性评价主要方法,测定粪肠球菌GMCC 0460.3株的全基因组序列并分析毒力和耐药性相关基因;以生物化学方法测定其耐药性和有毒代谢产物的产生情况;经口灌胃小鼠测定其动物体内毒力。 结果 该株肠球菌对卡那霉素和磺胺类药物耐药,耐药谱窄;基因组存在多种肠球菌毒力基因;生化试验表明其溶血性阴性、氨基脱羧酶活性阴性、硝基还原酶活性较低、细胞表面疏水性较低、生物膜形成能力较弱;动物实验结果表明该株菌在小鼠体内无明显毒性作用。 结论 粪肠球菌GMCC0 460.3株实验评价的结果显示了良好的安全性。     

Molecular cloning, overexpression, purification, and characterization of an aerobic FMN-dependent azoreductase from Enterococcus faecalis

Azo dyes represent a major class of synthetic colorants that are ubiquitous in foods and consumer products. Enterococcus faecalis is a predominant member of the human gastrointestinal microflora. Strain ATCC 19433 grew in the presence of azo dyes and metabolized them to colorless products. A gene encoding a putative FMN-dependent aerobic azoreductase that shares 34% identity with azoreductase (AcpD) of Escherichia coli has been identified in this strain. The gene in E. faecalis, designated as azoA, encoded a protein of 208 amino acids with a calculated isoelectric point of 4.8. AzoA was heterologously overexpressed in E. coli with a strong band of 23 kDa on SDS-PAGE. The purified recombinant enzyme was a homodimer with a molecular weight of 43 kDa, probably containing one molecule of FMN per dimer. AzoA required FMN and NADH, but not NADPH, as a preferred electron donor for its activity. The apparent Km values for both NADH and 2-[4-(dimethylamino)phenylazo]benzoic acid (Methyl red) substrates were 0.14 and 0.024 mM, respectively. The apparent Vmax was 86.2 microM/min/mg protein. The enzyme was not only able to decolorize Methyl red, but was also able to convert sulfonated azo dyes Orange II, Amaranth, Ponceau BS, and Ponceau S. AzoA is the first aerobic azoreductase to be identified and characterized from human intestinal gram-positive bacteria.     

Enterococcus faecalis cytolysin and lactocin S of Lactobacillus sake

Strains of Enterococcus faecalis and Lactobacillus sake have been found to express lantibiotics with unusual properties. The enterococcal lantibiotic is unusual in that it consists of two dissimilar subunits, both putatively containing modifications consistent with those found in other lantibiotics. The enterococal lantibiotic is also unusual in the number of proteolytic steps involved in secretion signal removal and activation. Moreover, it has been observed to contribute to enterococcal disease in humans and in animal models. Structrural studies of lactocin S, expressed by a strain of L. sake highlight unique properties including the presence of D-alanine within its structure, and a protease putatively responsible for lactocin S secretion signal peptide removal which, itself, lacks a signal or propeptide sequence. Despite the unusual properties of each of these lantibiotics, the operons encoding each, and accompanying auxiliary functions, are collinear suggeting a common ancestry. The accretion of interdigitating DNA sequences between genes encoded within the lactocin S determinant are unique to that determinant, however, and are of unknown function.     

Crystal structure of phosphopantetheine adenylyltransferase from <Emphasis Type="Italic">Enterococcus faecalis</Emphasis> in the ligand-unbound state and in complex with ATP and pantetheine

Phosphopantetheine adenylyltransferase (PPAT) catalyzes the reversible transfer of an adenylyl group from ATP to 4'-phosphopantetheine (Ppant) to form dephospho-CoA (dPCoA) and pyrophosphate in the Coenzyme A (CoA) biosynthetic pathway. Importantly, PPATs are the potential target for developing antibiotics because bacterial and mammalian PPATs share little sequence homology. Previous structural studies revealed the mechanism of the recognizing substrates and products. The binding modes of ATP, ADP, Ppant, and dPCoA are highly similar in all known structures, whereas the binding modes of CoA or 3'-phosphoadenosine 5'-phosphosulfate binding are novel. To provide further structural information on ligand binding by PPATs, the crystal structure of PPAT from Enterococcus faecalis was solved in three forms: (i) apo form, (ii) binary complex with ATP, and (iii) binary complex with pantetheine. The substrate analog, pantetheine, binds to the active site in a similar manner to Ppant. The new structural information reported in this study including pantetheine as a potent inhibitor of PPAT will supplement the existing structural data and should be useful for structure-based antibacterial discovery against PPATs.     

A "retrocidal" plasmid in Enterococcus faecalis: passage and protection

Enterococcus faecalis MC4 harbors a 130 kb conjugative, pheromone (cCF10)-responding plasmid, pAMS1, conferring chloramphenicol, streptomycin and tetracycline resistances. A plasmid-borne class IIa bacteriocin (MC4-1) determinant and cognate immunity gene were present, but not expressed in MC4. However, pAMS1 transfer to E. faecalis JH2-2 (but not to the non-isogenic OG1SS) generated the surprising ability to express bacteriocin activity against the plasmid donor, MC4. The bacteriocin target spectrum includes E. faecalis, Enterococcus faecium, Enterococcus gallinarum, Enterococcus hirae, and Listeria monocytogenes. Those donors unable to express bacteriocin or immunity could protect themselves from the "retrocidal" behavior of transconjugants by a switch to bacteriocin resistance at a frequency of approximately 10(-3). Reversion to sensitivity occurred at a relatively high frequency, suggestive of involvement of a phase variation event. These observations concerning a conjugative plasmid with novel "retrocidal" properties, coupled with a defense mechanism independent of plasmid-borne immunity functions, may relate to phenomena exploiting regulatory features with broader ecological and evolutionary implications.     

Genomic comparative analysis of the environmental Enterococcus mundtii against enterococcal representative species

Background

Enterococcus mundtii is a yellow-pigmented microorganism rarely found in human infections. The draft genome sequence of E. mundtii was recently announced. Its genome encodes at least 2,589 genes and 57 RNAs, and 4 putative genomic islands have been detected. The objective of this study was to compare the genetic content of E. mundtii with respect to other enterococcal species and, more specifically, to identify genes coding for putative virulence traits present in enterococcal opportunistic pathogens.

Results

An in-depth mining of the annotated genome was performed in order to uncover the unique properties of this microorganism, which allowed us to detect a gene encoding the antimicrobial peptide mundticin among other relevant features. Moreover, in this study a comparative genomic analysis against commensal and pathogenic enterococcal species, for which genomic sequences have been released, was conducted for the first time. Furthermore, our study reveals significant similarities in gene content between this environmental isolate and the selected enterococci strains (sharing an “enterococcal gene core” of 805 CDS), which contributes to understand the persistence of this genus in different niches and also improves our knowledge about the genetics of this diverse group of microorganisms that includes environmental, commensal and opportunistic pathogens.

Conclusion

Although E. mundtii CRL1656 is phylogenetically closer to E. faecium, frequently responsible of nosocomial infections, this strain does not encode the most relevant relevant virulence factors found in the enterococcal clinical isolates and bioinformatic predictions indicate that it possesses the lowest number of putative pathogenic genes among the most representative enterococcal species. Accordingly, infection assays using the Galleria mellonella model confirmed its low virulence.

Electronic supplementary material

The online version of this article (doi:10.1186/1471-2164-15-489) contains supplementary material, which is available to authorized users.     

Presence of virulence factors in Enterococcus faecalis and Enterococcus faecium susceptible and resistant to vancomycin

Despite the increasing importance of Enterococcus as opportunistic pathogens, their virulence factors are still poorly understood. This study determines the frequency of virulence factors in clinical and commensal Enterococcus isolates from inpatients in Porto Alegre, Brazil. Fifty Enterococcus isolates were analysed and the presence of the gelE, asa1 and esp genes was determined. Gelatinase activity and biofilm formation were also tested. The clonal relationships among the isolates were evaluated using pulsed-field gel electrophoresis. The asa1, gelE and esp genes were identified in 38%, 60% and 76% of all isolates, respectively. The first two genes were more prevalent in Enterococcus faecalis than in Enterococcus faecium, as was biofilm formation, which was associated with gelE and asa1 genes, but not with the esp gene. The presence of gelE and the activity of gelatinase were not fully concordant. No relationship was observed among any virulence factors and specific subclones of E. faecalis or E. faecium resistant to vancomycin. In conclusion, E. faecalis and E. faecium isolates showed significantly different patterns of virulence determinants. Neither the source of isolation nor the clonal relationship or vancomycin resistance influenced their distribution.     

Characterization of Insecticidal Genes of Bacillus thuringiensis Strains Isolated from Arid Environments

This study aimed at characterizing the insecticidal genes of eight Bacillus thuringiensis isolates that were recovered from the local environment of western Saudi Arabia. The screening for the presence of lepidopteran-specific cry1A family and vip3A genes, dipteran-specific cry4 family and coleopteran-specific cry3A, vip1A and vip2A genes, was carried out by PCR. All eight isolates produced PCR products that confirmed the presence of cry1Aa, cry1Ab, cry1Ac, cry4A, cry4B genes, but not cry3A, vip1A and vip2A genes. However, three isolates only were found to carry vip3A genes as revealed by PCR. The observation of cry1 and cry4 genes suggests that these eight isolates may have dual activity against Lepidoptera and Diptera species, while three isolates possessed vip3 genes in addition to cry1 and cry4 which suggests that these three isolates have toxic crystals and vegetative proteins. The results of this study are interesting in the sense that they may help developing new strategies for controlling insects of economic and medical importance in Saudi Arabia, using B. thuringiensis strains that naturally exist in the local environment instead of the current control strategies that are based solely on chemical insecticides.     

Biotechnological production of l(+)-lactic acid from wood hydrolyzate by batch fermentation of Enterococcus faecalis

The inhibition of lactic acid fermentation by wood hydrolyzate was decreased (approx. 20%) by adaptation of Enterococcus faecalis RKY1 to wood hydrolyzate-based medium whereby lactic acid productivity and cell growth were enhanced by 0.5 g l(-1) h(-1) and 2.1 g l(-1), respectively. When the diluted or concentrated wood hydrolyzate (equivalent to 25-100 g glucose l(-1)) was supplemented with 15 g yeast extract l(-1), 24-93 g lactic acid l(-1) was produced at a rate between 1.7 g l(-1) h(-1) and 3.2 g l(-1) h(-1).     

不同分离源粪肠球菌的毒力基因比较

【背景】粪肠球菌作为一种重要的乳酸菌在食品和医药领域应用广泛。由于很多粪肠球菌为条件致病菌,因此充分了解粪肠球菌基因组中毒力基因(Virulence genes)的携带情况对合理利用该菌种有重要的意义,但目前还没有研究专门报道不同分离源粪肠球菌基因组中毒力基因的携带情况。【目的】了解不同分离源粪肠球菌毒力基因的携带情况,评估分离自自然发酵乳制品中的粪肠球菌的安全性。【方法】利用比较基因组学方法确定107株分离自乳源、血液、尿液、粪便和水源中的粪肠球菌携带毒力基因情况,使用主成分分析比较不同分离源菌株毒力基因的差异,通过卡方检验筛查出环境特异性毒力基因。【结果】在107株粪肠球菌基因组共找到88种编码不同功能蛋白的毒力基因,其中与粘附相关的毒力基因最多。同时发现乳源分离株与其他环境分离株所携带的毒力基因没有显著差异。【结论】乳源分离株中携带的毒力基因与其他环境分离株无显著差异,表明分离自自然发酵乳制品中的粪肠球菌可能同样存在致病风险,因此在食品工业中使用粪肠球菌时一定要对菌株的安全性做全面的评估。