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 virulenceFil: Repizo, Guillermo Daniel. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Rosario. Instituto de Biología Molecular y Celular de Rosario. Universidad Nacional de Rosario. Facultad de Ciencias Bioquímicas y Farmacéuticas. Instituto de Biología Molecular y Celular de Rosario; ArgentinaFil: Espariz, Martin. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Rosario. Instituto de Biología Molecular y Celular de Rosario. Universidad Nacional de Rosario. Facultad de Ciencias Bioquímicas y Farmacéuticas. Instituto de Biología Molecular y Celular de Rosario; ArgentinaFil: Blancato, Victor Sebastian. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Rosario. Instituto de Biología Molecular y Celular de Rosario. Universidad Nacional de Rosario. Facultad de Ciencias Bioquímicas y Farmacéuticas. Instituto de Biología Molecular y Celular de Rosario; ArgentinaFil: Suárez, Cristian Alejandro. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Rosario. Instituto de Biología Molecular y Celular de Rosario. Universidad Nacional de Rosario. Facultad de Ciencias Bioquímicas y Farmacéuticas. Instituto de Biología Molecular y Celular de Rosario; ArgentinaFil: Esteban, Luis. Universidad Nacional de Rosario. Facultad de Cs.médicas. Escuela de Cs.médicas. Cátedra de Fisiología; ArgentinaFil: Magni, Christian. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Rosario. Instituto de Biología Molecular y Celular de Rosario. Universidad Nacional de Rosario. Facultad de Ciencias Bioquímicas y Farmacéuticas. Instituto de Biología Molecular y Celular de Rosario; Argentin

Expression of the Agmatine Deiminase Pathway in Enterococcus faecalis Is Activated by the AguR Regulator and Repressed by CcpA and PTSMan System

Although the agmatine deiminase system (AgDI) has been investigated in Enterococcus faecalis, little information is available with respect to its gene regulation. In this study we demonstrate that the presence of exogenous agmatine induces the expression of agu genes in this bacterium. In contrast to the homologous and extensively characterized AgDI system of S. mutants, the aguBDAC operon in E. faecalis is not induced in response to low pH. In spite of this, agmatine catabolism in this bacterium contributes by neutralizing the external medium while enhancing bacterial growth. Our results indicate that carbon catabolic repression (CCR) operates on the AgDI system via a mechanism that involves interaction of CcpA and PSer- HPr with a cre site found in an unusual position considering the aguB promoter (55 nt upstream the +1 position). In addition, we found that components of the mannose phosphotransferase (PTSMan) system also contributed to CCR in E. faecalis since a complete relief of the PTS-sugars repressive effect was observed only in a PTSMan and CcpA double defective strain. Our gene context analysis revealed that aguR is present in oral and gastrointestinal microorganisms. Thus, regulation of the aguBDAC operon in E. faecalis seems to have evolved to obtain energy and resist low pH conditions in order to persist and colonize gastrointestinal nichesFil: Suárez, Cristian Alejandro. Consejo Nacional de Invest.cientif.y Tecnicas. Centro Cientifico Tecnol.conicet - Rosario. Instituto de Biologia Molecular y Celular de Rosario;Fil: Espariz, Martin. Consejo Nacional de Invest.cientif.y Tecnicas. Centro Cientifico Tecnol.conicet - Rosario. Instituto de Biologia Molecular y Celular de Rosario;Fil: Blancato, Victor Sebastian. Consejo Nacional de Invest.cientif.y Tecnicas. Centro Cientifico Tecnol.conicet - Rosario. Instituto de Biologia Molecular y Celular de Rosario;Fil: Magni, Christian. Consejo Nacional de Invest.cientif.y Tecnicas. Centro Cientifico Tecnol.conicet - Rosario. Instituto de Biologia Molecular y Celular de Rosario

Loading of Cells with Fluorescent Probe to Study Intracellular Acid-base Homeostasis in Lactic Acid Bacteria

Here we describe a protocol which we have used to study the homeostasis intracellular in vivo in lactic acid bacteria (LAB) using a fluorescent probe. This type of probes can be used for determining changes in the pH of cytoplasm with high sensitivity, temporal resolution and technical simplicity as well as accessing the rate of change of intracellular pH in response to a stimulus from kinetic measurements on short time scales (Breeuwer et al., 1996; Molenaar et al., 1991). This protocol has been designed to measure the intracellular pH using the pH-sensitive fluorescent probe 2´,7´-bis-(2-carboxyethyl)-5(and-6)-carboxyfluorescein (BCECF) in LAB, Enterococcus faecalis (E. faecalis), Lactococcus lactis (L. lactis) and Lactobacillus casei (L. casei)Fil: Mortera, Pablo. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Rosario. Instituto de Química Rosario; ArgentinaFil: Zuljan, Federico Alberto. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Rosario. Instituto de Química Rosario; ArgentinaFil: Magni, Christian. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Rosario. Instituto de Biología Molecular y Celular de Rosario; ArgentinaFil: Alarcon, Sergio Hugo. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Rosario. Instituto de Química Rosario; Argentin

CcpA represses the expression of the divergent cit operons of Enterococcus faecalis through multiple cre sites

<p>Abstract</p> <p>Background</p> <p>In <it>Enterococcus faecalis </it>the genes encoding the enzymes involved in citrate metabolism are organized in two divergent operons, <it>citHO </it>and <it>oadHDB-citCDEFX-oadA-citMG </it>(<it>citCL </it>locus). Expression of both operons is specifically activated by adding citrate to the medium. This activation is mediated by binding of the GntR-like transcriptional regulator (CitO) to the <it>cis</it>-acting sequences located in the <it>cit </it>intergenic region. Early studies indicated that citrate and glucose could not be co-metabolized suggesting some form of catabolite repression, however the molecular mechanism remained unknown.</p> <p>Results</p> <p>In this study, we observed that the <it>citHO </it>promoter is repressed in the presence of sugars transported by the Phosphoenolpyruvate:carbohydrate Phosphotranserase System (PTS sugars). This result strongly suggested that Carbon Catabolic Repression (CCR) impedes the expression of the activator CitO and the subsequent induction of the <it>cit </it>pathway. In fact, we demonstrate that CCR is acting on both promoters. It is partially relieved in a <it>ccpA</it>-deficient <it>E. faecalis </it>strain indicating that a CcpA-independent mechanism is also involved in regulation of the two operons. Furthermore, sequence analysis of the <it>citH</it>/<it>oadH </it>intergenic region revealed the presence of three putative catabolite responsive elements (<it>cre</it>). We found that they are all active and able to bind the CcpA/P-Ser-HPr complex, which downregulates the expression of the <it>cit </it>operons. Systematic mutation of the CcpA/P-Ser-HPr binding sites revealed that <it>cre1 </it>and <it>cre2 </it>contribute to <it>citHO </it>repression, while <it>cre3 </it>is involved in CCR of <it>citCL</it></p> <p>Conclusion</p> <p>In conclusion, our study establishes that expression of the <it>cit </it>operons in <it>E. faecalis </it>is controlled by CCR via CcpA-dependent and -independent mechanisms.</p

Multivariate analysis of organic acids in fermented food from reversed-phase high-performance liquid chromatography data

Multivariate calibration coupled to RP-HPLC with diode array detection (HPLC-DAD) was applied to the identification and the quantitative evaluation of the short chain organic acids (malic, oxalic, formic, lactic, acetic, citric, pyruvic, succinic, tartaric, propionic and α-cetoglutaric) in fermented food. The goal of the present study was to get the successful resolution of a system in the combined occurrence of strongly coeluting peaks, of distortions in the time sensors among chromatograms, and of the presence of unexpected compounds not included in the calibration step. Second-order HPLC-DAD data matrices were obtained in a short time (10 min) on a C18 column with a chromatographic system operating in isocratic mode (mobile phase was 20 mmol L−1 phosphate buffer at pH 2.20) and a flow-rate of 1.0 mL min−1 at room temperature. Parallel factor analysis (PARAFAC) and unfolded partial least-squares combined with residual bilinearization (U-PLS/RBL) were the second-order calibration algorithms select for data processing. The performance of the analytical parameters was good with an outstanding limit of detection (LODs) for acids ranging from 0.15 to 10.0 mmol L−1 in the validation samples. The improved method was applied to the analysis of many dairy products (yoghurt, cultured milk and cheese) and wine. The method was shown as an effective means for determining and following acid contents in fermented food and was characterized by reducibility with simple, high resolution and rapid procedure without derivatization of analytes.Fil: Mortera, Pablo. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Rosario. Instituto de Química Rosario. Universidad Nacional de Rosario. Facultad de Ciencias Bioquímicas y Farmacéuticas. Instituto de Química Rosario; ArgentinaFil: Zuljan, Federico Alberto. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Rosario. Instituto de Biología Molecular y Celular de Rosario. Universidad Nacional de Rosario. Facultad de Ciencias Bioquímicas y Farmacéuticas. Instituto de Biología Molecular y Celular de Rosario; ArgentinaFil: Magni, Christian. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Rosario. Instituto de Biología Molecular y Celular de Rosario. Universidad Nacional de Rosario. Facultad de Ciencias Bioquímicas y Farmacéuticas. Instituto de Biología Molecular y Celular de Rosario; ArgentinaFil: Bortolato, Santiago Andres. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Rosario. Instituto de Química Rosario. Universidad Nacional de Rosario. Facultad de Ciencias Bioquímicas y Farmacéuticas. Instituto de Química Rosario; ArgentinaFil: Alarcon, Sergio Hugo. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Rosario. Instituto de Química Rosario. Universidad Nacional de Rosario. Facultad de Ciencias Bioquímicas y Farmacéuticas. Instituto de Química Rosario; Argentin

The assessment of leading traits in the taxonomy of the Bacillus cereus group

Bacillus cereus sensu lato strains (B. cereus group) are widely distributed in nature and have received interest for decades due to their importance in insect pest management, food production and their positive and negative repercussions in human health. Consideration of practical uses such as virulence, physiology, morphology, or ill-defined features have been applied to describe and classify species of the group. However, current comparative studies have exposed inconsistencies between evolutionary relatedness and biological significance among genomospecies of the B. cereus group. Here, the combined analyses of core-based phylogeny and all versus all Average Nucleotide Identity values based on 2116 strains were conducted to update the genomospecies circumscriptions within B. cereus group. These analyses suggested the existence of 57 genomospecies, 37 of which are novel, thus indicating that the taxonomic identities of more than 39% of the analyzed strains should be revised or updated. In addition, we found that whole-genome in silico analyses were suitable to differentiate genomospecies such as B. anthracis, B. cereus and B. thuringiensis. The prevalence of toxin and virulence factors coding genes in each of the genomospecies of the B. cereus group was also examined, using phylogeny-aware methods at wide-genome scale. Remarkably, Cry and emetic toxins, commonly assumed to be associated with B. thuringiensis and emetic B. paranthracis, respectively, did not show a positive correlation with those genomospecies. On the other hand, anthrax-like toxin and capsule-biosynthesis coding genes were positively correlated with B. anthracis genomospecies, despite not being present in all strains, and with presumably non-pathogenic genomospecies. Hence, despite these features have been so far considered relevant for industrial or medical classification of related species of the B. cereus group, they were inappropriate for their circumscription. In this study, genomospecies of the group were accurately affiliated and representative strains defined, generating a rational framework that will allow comparative analysis in epidemiological or ecological studies. Based on this classification the role of specific markers such as Type VII secretion system, cytolysin, bacillolysin, and siderophores such as petrobactin were pointed out for further analysis.Fil: Torres Manno, Mariano Alberto. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Rosario. Instituto de Biología Molecular y Celular de Rosario. Universidad Nacional de Rosario. Facultad de Ciencias Bioquímicas y Farmacéuticas. Instituto de Biología Molecular y Celular de Rosario; ArgentinaFil: Repizo, Guillermo Daniel. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Rosario. Instituto de Biología Molecular y Celular de Rosario. Universidad Nacional de Rosario. Facultad de Ciencias Bioquímicas y Farmacéuticas. Instituto de Biología Molecular y Celular de Rosario; ArgentinaFil: Magni, Christian. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Rosario. Instituto de Biología Molecular y Celular de Rosario. Universidad Nacional de Rosario. Facultad de Ciencias Bioquímicas y Farmacéuticas. Instituto de Biología Molecular y Celular de Rosario; ArgentinaFil: Dunlap, Christopher A.. United States Department of Agriculture; Estados UnidosFil: Espariz, Martin. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Rosario. Instituto de Biología Molecular y Celular de Rosario. Universidad Nacional de Rosario. Facultad de Ciencias Bioquímicas y Farmacéuticas. Instituto de Biología Molecular y Celular de Rosario; Argentin

Redundant potassium transporter systems guarantee the survival of Enterococcus faecalis under stress conditions

Enterococcus is able to grow in media at pH from 5.0 to 9.0 and a high concentration of NaCl (8%). The ability to respond to these extreme conditions requires the rapid movement of three critical ions: proton (H+), sodium (Na+), and potassium (K+). The activity of the proton F0F1 ATPase and the sodium Na+ V0V1 type ATPase under acidic or alkaline conditions, respectively, is well established in these microorganisms. The potassium uptake transporters KtrI and KtrII were described in Enterococcus hirae, which were associated with growth in acidic and alkaline conditions, respectively. In Enterococcus faecalis, the presence of the Kdp (potassium ATPase) system was early established. However, the homeostasis of potassium in this microorganism is not completely explored. In this study, we demonstrate that Kup and KimA are highaffinity potassium transporters, and the inactivation of these genes in E. faecalis JH2-2 (a Kdp laboratory natural deficient strain) had no effect on the growth parameters. However, in KtrA defective strains (ΔktrA, ΔkupΔktrA) an impaired growth was observed under stress conditions, which was restored to wild type levels by external addition of K+ ions. Among the multiplicity of potassium transporters identify in the genus Enterococcus, Ktr channels (KtrAB and KtrAD), and Kup family symporters (Kup and KimA) are present and may contribute to the particular resistance of these microorganisms to different stress conditions. In addition, we found that the presence of the Kdp system in E. faecalis is strain-dependent, and this transporter is enriched in strains of clinical origin as compared to environmental, commensal, or food isolates.Fil: Acciarri, Giuliana. Universidad Nacional de Rosario. Facultad de Ciencias Bioquímicas y Farmacéuticas. Laboratorio de Fisiología y Genética de Bacterias Lácticas. Instituto de Biología Molecular y Celular de Rosario (IBR-CONICET); Argentina.Fil: Gizzi, Fernán O. Universidad Nacional de Rosario. Facultad de Ciencias Bioquímicas y Farmacéuticas. Laboratorio de Fisiología y Genética de Bacterias Lácticas. Instituto de Biología Molecular y Celular de Rosario (IBR-CONICET); Argentina.Fil: Torres Manno, Mariano. Universidad Nacional de Rosario. Facultad de Ciencias Bioquímicas y Farmacéuticas. Laboratorio de Fisiología y Genética de Bacterias Lácticas. Instituto de Biología Molecular y Celular de Rosario (IBR-CONICET); Argentina.Fil: Blancato, Victor. Universidad Nacional de Rosario. Facultad de Ciencias Bioquímicas y Farmacéuticas. Laboratorio de Fisiología y Genética de Bacterias Lácticas. Instituto de Biología Molecular y Celular de Rosario (IBR-CONICET); Argentina.Fil: Magni, Christian. Universidad Nacional de Rosario. Facultad de Ciencias Bioquímicas y Farmacéuticas. Laboratorio de Fisiología y Genética de Bacterias Lácticas. Instituto de Biología Molecular y Celular de Rosario (IBR-CONICET); Argentina.Fil: Torres Manno, Mariano. Universidad Nacional de Rosario. Facultad de Ciencias Bioquímicas y Farmacéuticas. Área Bioinformática. Departamento de Matemática y Estadística; Argentina.Fil: Stülke, Jörg. Georg August University. Department of General Microbiology; Germany.Fil: Blancato, Victor. Universidad Nacional de Rosario. Facultad de Ciencias Bioquímicas y Farmacéuticas. Laboratorio de Biotecnología e Inocuidad de los Alimentos. Área de Biotecnología de los Alimentos; Argentina.Fil: Magni, Christian. Universidad Nacional de Rosario. Facultad de Ciencias Bioquímicas y Farmacéuticas. Laboratorio de Biotecnología e Inocuidad de los Alimentos. Área de Biotecnología de los Alimentos; Argentina

Gaussian pulse dynamics in gain media with Kerr nonlinearity

Using the Kantorovitch method in combination with a Gaussian ansatz, we derive the equations of motion for spatial, temporal and spatiotemporal optical propagation in a dispersive Kerr medium with a general transverse and spectral gain profile. By rewriting the variational equations as differential equations for the temporal and spatial Gaussian q parameters, optical ABCD matrices for the Kerr effect, a general transverse gain profile and nonparabolic spectral gain filtering are obtained. Further effects can easily be taken into account by adding the corresponding ABCD matrices. Applications include the temporal pulse dynamics in gain fibers and the beam propagation or spatiotemporal pulse evolution in bulk gain media. As an example, the steady-state spatiotemporal Gaussian pulse dynamics in a Kerr-lens mode-locked laser resonator is studied