An allosteric inhibitor of the PhoQ histidine kinase with therapeutic potential against Salmonella infection.

BACKGROUND: The upsurge of antimicrobial resistance demands innovative strategies to fight bacterial infections. With traditional antibiotics becoming less effective, anti-virulence agents or pathoblockers, arise as an alternative approach that seeks to disarm pathogens without affecting their viability, thereby reducing selective pressure for the emergence of resistance mechanisms. OBJECTIVES: To elucidate the mechanism of action of compound N'-(thiophen-2-ylmethylene)benzohydrazide (A16B1), a potent synthetic hydrazone inhibitor against the Salmonella PhoP/PhoQ system, essential for virulence. MATERIALS AND METHODS: The measurement of the activity of PhoP/PhoQ-dependent and -independent reporter genes was used to evaluate the specificity of A16B1 to the PhoP regulon. Autokinase activity assays with either the native or truncated versions of PhoQ were used to dissect the A16B1 mechanism of action. The effect of A16B1 on Salmonella intramacrophage replication was assessed using the gentamicin protection assay. The checkerboard assay approach was used to analyse potentiation effects of colistin with the hydrazone. The Galleria mellonella infection model was chosen to evaluate A16B1 as an in vivo therapy against Salmonella. RESULTS: A16B1 repressed the Salmonella PhoP/PhoQ system activity, specifically targeting PhoQ within the second transmembrane region. A16B1 demonstrates synergy with the antimicrobial peptide colistin, reduces the intramacrophage proliferation of Salmonella without being cytotoxic and enhances the survival of G. mellonella larvae systemically infected with Salmonella. CONCLUSIONS: A16B1 selectively inhibits the activity of the Salmonella PhoP/PhoQ system through a novel inhibitory mechanism, representing a promising synthetic hydrazone compound with the potential to function as a Salmonella pathoblocker. This offers innovative prospects for combating Salmonella infections while mitigating the risk of antimicrobial resistance emergence.

Insertion sequence IS6770 modulates potassium symporter kup transcription in Enterococcus faecalis JH2-2 under low pH conditions.

Enterococcus faecalis is a phylogenetically and industrially relevant microorganism associated with Lactic Acid Bacteria. Some strains of this bacterium are employed as probiotics in commercial applications, while others serve as the principal component in starter cultures for artisanal regional cheese production. However, over the last decade, this species has emerged as an opportunistic multiresistant pathogen, raising concerns about its impact on human health. Recently, we identified multiple potassium transporter systems in E. faecalis, including the Ktr systems (KtrAB and KtrAD), Kup, KimA and Kdp complex (KdpFABC). Nevertheless, the physiological significance of these proteins remains not fully understood. In this study, we observed that the kup gene promoter region in the JH2-2 strain was modified due to the insertion of a complete copy of the IS6770 insertion sequence. Consequently, we investigated the influence of IS6770 on the expression of the kup gene. To achieve this, we conducted a mapping of the promoter region of this gene in the E. faecalis JH2-2 strain, employing fluorescence gene reporters. In addition, a transcriptional analysis of the kup gene was executed in a strain derived from E. faecalis V583 that lacks the IS30-related insertion element, facilitating the identification of the transcriptional start site. Next, the expression of the kup gene was evaluated via RT-qPCR under different pH stressful conditions. A strong upregulation of the kup gene was observed at an initial pH of 5.0 in the strain derived from E. faecalis V583. However, the activation of transcription was not observed in the E. faecalis JH2-2 strain due to the hindrance caused by the presence of IS6770. Besides that, our computational analysis of E. faecalis genomes elucidates a plausible association between transposition and the regulation of the kup gene. Remarkably, the ubiquitous presence of IS6770 throughout the phylogenetic tree implies its ancient existence within E. faecalis. Moreover, the recurrent co-occurrence of IS6770 with the kup gene, observed in 30 % of IS6770-positive strains, alludes to the potential involvement of this genomic arrangement in the adaptive strategies of E. faecalis across diverse niches.

Enhancing colistin efficacy against Salmonella infections with a quinazoline-based dual therapeutic strategy.

Colistin remains one of the last-resort therapies for combating infections caused by multidrug-resistant (MDR) Enterobacterales, despite its adverse nephro- and neuro-toxic effects. This study elucidates the mechanism of action of a non-antibiotic 4-anilinoquinazoline-based compound that synergistically enhances the effectiveness of colistin against Salmonella enterica. The quinazoline sensitizes Salmonella by deactivating intrinsic, mutational, and transferable resistance mechanisms that enable Salmonella to counteract the antibiotic impact colistin, together with an induced disruption to the electrochemical balance of the bacterial membrane. The attenuation of colistin resistance via the combined treatment approach also proves efficacious against E. coli, Klebsiella, and Acinetobacter strains. The dual therapy reduces the mortality of Galleria mellonella larvae undergoing a systemic Salmonella infection when compared to individual drug treatments. Overall, our findings unveil the potential of the quinazoline-colistin combined therapy as an innovative strategy against MDR bacteria.

Metagenomic approach to infer rumen microbiome derived traits of cattle.

Ruminants enable the conversion of indigestible plant material into animal consumables, including dairy products, meat, and valuable fibers. Microbiome research is gaining popularity in livestock species because it aids in the knowledge of illnesses and efficiency processes in animals. In this study, we use WGS metagenomic data to thoroughly characterize the ruminal ecosystem of cows to infer positive and negative livestock traits determined by the microbiome. The rumen of cows from Argentina were described by combining different gene biomarkers, pathways composition and taxonomic information. Taxonomic characterization indicated that the two major phyla were Bacteroidetes and Firmicutes; in third place, Proteobacteria was highly represented followed by Actinobacteria; Prevotella, and Bacteroides were the most abundant genera. Functional profiling of carbohydrate-active enzymes indicated that members of the Glycoside Hydrolase (GH) class accounted for 52.2 to 55.6% of the total CAZymes detected, among them the most abundant were the oligosaccharide degrading enzymes. The diversity of GH families found suggested efficient hydrolysis of complex biomass. Genes of multidrug, macrolides, polymyxins, beta-lactams, rifamycins, tetracyclines, and bacitracin resistance were found below 0.12% of relative abundance. Furthermore, the clustering analysis of genera and genes that correlated to methane emissions or feed efficiency, suggested that the cows analysed could be regarded as low methane emitters and clustered with high feed efficiency reference animals. Finally, the combination of bioinformatic analyses used in this study can be applied to assess cattle traits difficult to measure and guide enhanced nutrition and breeding methods.

Intranasal trans-sialidase-based vaccine against Trypanosoma cruzi triggers a mixed cytokine profile in the nasopharynx-associated lymphoid tissue and confers local and systemic immunogenicity.

Trypanosoma cruzi, the agent of Chagas disease, can infect through conjunctive or oral mucosas. Therefore, the induction of mucosal immunity by vaccination is relevant not only to trigger local protection but also to stimulate both humoral and cell-mediated responses in systemic sites to control parasite dissemination. In a previous study, we demonstrated that a nasal vaccine based on a Trans-sialidase (TS) fragment plus the mucosal STING agonist c-di-AMP, was highly immunogenic and elicited prophylactic capacity. However, the immune profile induced by TS-based nasal vaccines at the nasopharyngeal-associated lymphoid tissue (NALT), the target site of nasal immunization, remains unknown. Hence, we analyzed the NALT cytokine expression generated by a TS-based vaccine plus c-di-AMP (TSdA+c-di-AMP) and their association with mucosal and systemic immunogenicity. The vaccine was administered intranasally, in 3 doses separated by 15 days each other. Control groups received TSdA, c-di-AMP, or the vehicle in a similar schedule. We demonstrated that female BALB/c mice immunized intranasally with TSdA+c-di-AMP boosted NALT expression of IFN-γ and IL-6, as well as IFN-ß and TGF-ß. TSdA+c-di-AMP increased TSdA-specific IgA secretion in the nasal passages and also in the distal intestinal mucosa. Moreover, T and B-lymphocytes from NALT-draining cervical lymph nodes and spleen showed an intense proliferation after ex-vivo stimulation with TSdA. Intranasal administration of TSdA+c-di-AMP provokes an enhancement of TSdA-specific IgG2a and IgG1 plasma antibodies, accompanied by an increase IgG2a/IgG1 ratio, indicative of a Th1-biased profile. In addition, immune plasma derived from TSdA+c-di-AMP vaccinated mice exhibit in-vivo and ex-vivo protective capacity. Lastly, TSdA+c-di-AMP nasal vaccine also promotes intense footpad swelling after local TSdA challenge. Our data support that TSdA+c-di-AMP nasal vaccine triggers a NALT mixed pattern of cytokines that were clearly associated with an evident mucosal and systemic immunogenicity. These data are useful for further understanding the immune responses elicited by the NALT following intranasal immunization and the rational design of TS-based vaccination strategies for prophylaxis against T. cruzi.

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 high-affinity 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.

Nasal immunization with a L. lactis-derived trans-sialidase antigen plus c-di-AMP protects against acute oral T. cruzi infection.

The new generation of vaccines for Chagas disease, are focused to induce both humoral and cellular response to effectively control Trypanosoma cruzi parasites. The administration of vaccine formulations intranasally has the advantage over parenteral routes that can induce a specific response at mucosal and systemic levels. This study aimed to evaluate and compare the immunogenicity and prophylactic effectiveness of two Trans-sialidase (TS)-based mucosal vaccines against T. cruzi administered intranasally. Vaccines consisted of a recombinant fragment of TS expressed in Lactococcus lactis formulated in two different adjuvants. The first, was an immunostimulant particle (ISPA, an ISCOMATRIX-like adjuvant), while the second was the dinucleotide c-di-AMP, which have shown immunostimulant properties at the mucosal level. BALB/c mice were immunized intranasally (3 doses, one every two weeks) with each formulation (TS + ISPA or TS + c-di-AMP) and with TS alone or vehicle (saline solution) as controls. Fifteen days after the last immunization, both TS + ISPA or TS + c-di-AMP induced an evident systemic humoral and cellular response, as judged by the increased plasma anti-TS IgG2a titers and IgG2a/IgG1 ratio and enhanced cellular response against TS. Plasma derived antibodies from TS + c-di-AMP also inhibit in vitro the invasion capacity of T. cruzi. Furthermore, specific secretory IgA was more enhanced in TS + c-di-AMP group. Protective efficacy was proved in vaccinated animals by an oral T. cruzi-challenge. Parasitemia control was only achieved by animals vaccinated with TS + c-di-AMP, despite all vaccinates groups showed enhanced CD8+IFN-γ+ T cell numbers. In addition, it was reflected during the acute phase in a significant reduction of tissue parasite load, clinical manifestations and diminished tissue damage. The better prophylactic capacity elicited by TS + c-di-AMP was related to the induction of neutralizing plasma antibodies and augmented levels of mucosal IgA since TS + ISPA and TS + c-di-AMP groups displayed similar immunogenicity and CD8+IFN-γ+ T-cell response. Therefore, TS + c-di-AMP formulation appears as a promising strategy for prophylaxis against T. cruzi.

Genomic analysis revealed conserved acid tolerance mechanisms from native micro-organisms in fermented feed.

AIMS: Fermented feed is an agricultural practice used in many regions of the world to improve the growth performance of farm animals. This study aimed to identify and evaluate the lactic acid bacteria and yeast involved in the production of fermented feed. METHODS AND RESULTS: We isolated and described two micro-organisms from autochthonous microbiota origin present in a regional feed product, Lactobacillus paracasei IBR07 (Lacticaseibacillus paracasei) and Kazachstania unispora IBR014 (Saccharomyces unisporum). Genome sequence analyses were performed to characterize both micro-organisms. Potential pathways involved in the acid response, tolerance and persistence were predicted in both genomes. Although L. paracasei and K. unispora are considered safe for animal feed, we analysed the presence of virulence factors, antibiotic resistance and pathogenicity islands. Furthermore, the Galleria mellonella model was used to support the safety of both isolates. CONCLUSIONS: We conclude that IBR07 and IBR014 strains are good candidates to be used as starter cultures for feed fermentation. SIGNIFICANCE AND IMPACT OF THE STUDY: The data presented here will be helpful to explore other biotechnological aspects and constitute a starting point for further studies to establish the consumption benefit of fermented feed in farm animal production.

Diversity of volatile organic compound production from leucine and citrate in Enterococcus faecium.

Enterococcus faecium is frequently isolated from fermented food; in particular, they positively contribute to the aroma compound generation in traditional cheese. Citrate fermentation is a desirable property in these bacteria, but this feature is not uniformly distributed among E. faecium strains. In the present study, three selected E. faecium strains, IQ110 (cit-), GM70 (cit+ type I), and Com12 (cit+ type II), were analyzed in their production of aroma compounds in milk. End products and volatile organic compounds (VOCs) were determined by solid-phase micro-extraction combined with gas chromatography mass spectrometry (SPME-GC-MS). Principal component analysis (PCA) of aroma compound profiles revealed a different VOC composition for the three strains. In addition, resting cell experiments of E. faecium performed in the presence of leucine, citrate, or pyruvate as aroma compound precursors allowed us to determine metabolic differences between the studied strains. GM70 (cit+ type I) showed an active citrate metabolism, with increased levels of diacetyl and acetoin generation relative to Com12 or to citrate defective IQ110 strains. In addition, in the experimental conditions tested, a defective citrate-fermenting phenotype for the Com12 strain was found, while its leucine degradation and pyruvate metabolism were conserved. In conclusion, rational selection of E. faecium strains could be performed based on genotypic and phenotypic analyses. This would result in a performing strain, such as GM70, that could positively contribute to flavor, with typical notes of diacetyl, acetoin, 3-methyl butanal, and 3-methyl butanol in an adjuvant culture.

Implications of the expression of Enterococcus faecalis citrate fermentation genes during infection.

Citrate is an ubiquitous compound in nature. However, citrate fermentation is present only in a few pathogenic or nonpathogenic microorganisms. The citrate fermentation pathway includes a citrate transporter, a citrate lyase complex, an oxaloacetate decarboxylase and a regulatory system. Enterococcus faecalis is commonly present in the gastro-intestinal microbiota of warm-blooded animals and insect guts. These bacteria can also cause infection and disease in immunocompromised individuals. In the present study, we performed whole genome analysis in Enterococcus strains finding that the complete citrate pathway is present in all of the E. faecalis strains isolated from such diverse habitats as animals, hospitals, water, milk, plants, insects, cheese, etc. These results indicate the importance of this metabolic preservation for persistence and growth of E. faecalis in different niches. We also analyzed the role of citrate metabolism in the E. faecalis pathogenicity. We found that an E. faecalis citrate fermentation-deficient strain was less pathogenic for Galleria mellonella larvae than the wild type. Furthermore, strains with deletions in the oxaloacetate decarboxylase subunits or in the α-acetolactate synthase resulted also less virulent than the wild type strain. We also observed that citrate promoters are induced in blood, urine and also in the hemolymph of G. mellonella. In addition, we showed that citrate fermentation allows E. faecalis to grow better in blood, urine and G. mellonella. The results presented here clearly indicate that citrate fermentation plays an important role in E. faecalis opportunistic pathogenic behavior.

Genetic Engineering of Lactococcus lactis Co-producing Antigen and the Mucosal Adjuvant 3' 5'- cyclic di Adenosine Monophosphate (c-di-AMP) as a Design Strategy to Develop a Mucosal Vaccine Prototype.

Lactococcus lactis is a promising candidate for the development of mucosal vaccines. More than 20 years of experimental research supports this immunization approach. In addition, 3' 5'- cyclic di-adenosine monophosphate (c-di-AMP) is a bacterial second messenger that plays a key role in the regulation of diverse physiological functions (potassium and cellular wall homeostasis, among others). Moreover, recent studies showed that c-di-AMP has a strong mucosal adjuvant activity that promotes both humoral and cellular immune responses. In this study, we report the development of a novel mucosal vaccine prototype based on a genetically engineered L. lactis strain. First, we demonstrate that homologous expression of cdaA gen in L. lactis is able to increase c-di-AMP levels. Thus, we hypothesized that in vivo synthesis of the adjuvant can be combined with production of an antigen of interest in a separate form or jointly in the same strain. Therefore, a specifically designed fragment of the trans-sialidase (TScf) enzyme from the Trypanosoma cruzi parasite, the etiological agent of Chagas disease, was selected to evaluate as proof of concept the immune response triggered by our vaccine prototypes. Consequently, we found that oral administration of a L. lactis strain expressing antigenic TScf combined with another L. lactis strain producing the adjuvant c-di-AMP could elicit a TS-specific immune response. Also, an additional L. lactis strain containing a single plasmid with both cdaA and tscf genes under the Pcit and Pnis promoters, respectively, was also able to elicit a specific immune response. Thus, the current report is the first one to describe an engineered L. lactis strain that simultaneously synthesizes the adjuvant c-di-AMP as well as a heterologous antigen in order to develop a simple and economical system for the formulation of vaccine prototypes using a food grade lactic acid bacterium.

Safety assessment and functional properties of four enterococci strains isolated from regional Argentinean cheese.

The members of the Enterococcus genus are widely distributed in nature. Its strains have been extensively reported to be present in plant surfaces, soil, water and food. In an attempt to assess their potential application in food industry, four Enterococcus faecium group-strains recently isolated from Argentinean regional cheese products were evaluated using a combination of whole genome analyses and in vivo assays. In order to identify these microorganisms at species level, in silico analyses using their newly reported sequences were conducted. The average nucleotide identity (ANI), in silico DNA-DNA hybridization, and phylogenomic trees constructed using core genome data allowed IQ110, GM70 and GM75 strains to be classified as E. faecium while IQ23 strain was identified as E. durans. Besides their common origin, the strains showed differences in their genetic structure and mobile genetic element content. Furthermore, it was possible to determine the absence or presence of specific features related to growth in milk, cheese ripening, probiotic capability and gut adaptation including sugar, amino acid, and peptides utilization, flavor compound production, bile salt tolerance as well as biogenic amine production. Remarkably, all strains encoded for peptide permeases, maltose utilization, bile salt tolerance, diacetyl and tyramine production genes. On the other hand, some variability was observed regarding citrate and lactose utilization, esterase, and cell wall-associated proteinase. In addition, while strains were predicted to be non-human pathogens by the in silico inspection of pathogenicity and virulence factors, only the GM70 strain proved to be non-virulent in Galleria mellonella model. In conclusion, we propose that, in order to improve the rational selection of strains for industrial applications, a holistic approach involving a comparative genomic analysis of positive and negative features as well as in vivo evaluation of virulence behavior should be performed.

Draft Genome Sequences of Four Enterococcus faecium Strains Isolated from Argentine Cheese.

We report the draft genome sequences of four Enterococcus faecium strains isolated from Argentine regional cheeses. These strains were selected based on their technological properties, i.e., their ability to produce aroma compounds (diacetyl, acetoin, and 2,3-butanediol) from citrate. The goal of our study is to provide further genetic evidence for the rational selection of enterococci strains based on their pheno- and genotype in order to be used in cheese production.

Aroma compounds generation in citrate metabolism of Enterococcus faecium: Genetic characterization of type I citrate gene cluster.

Enterococcus is one of the most controversial genera belonging to Lactic Acid Bacteria. Research involving this microorganism reflects its dual behavior as regards its safety. Although it has also been associated to nosocomial infections, natural occurrence of Enterococcus faecium in food contributes to the final quality of cheese. This bacterium is capable of fermenting citrate, which is metabolized to pyruvate and finally derives in the production of the aroma compounds diacetyl, acetoin and 2,3 butanediol. Citrate metabolism was studied in E. faecium but no data about genes related to these pathways have been described. A bioinformatic approach allowed us to differentiate cit(-) (no citrate metabolism genes) from cit(+) strains in E. faecium. Furthermore, we could classify them according to genes encoding for the transcriptional regulator, the oxaloacetate decarboxylase and the citrate transporter. Thus we defined type I organization having CitI regulator (DeoR family), CitM cytoplasmic soluble oxaloacetate decarboxylase (Malic Enzyme family) and CitP citrate transporter (2-hydroxy-carboxylate transporter family) and type II organization with CitO regulator (GntR family), OAD membrane oxaloacetate decarboxylase complex (Na(+)-transport decarboxylase enzyme family) and CitH citrate transporter (CitMHS family). We isolated and identified 17 E. faecium strains from regional cheeses. PCR analyses allowed us to classify them as cit(-) or cit(+). Within the latter classification we could differentiate type I but no type II organization. Remarkably, we came upon E. faecium GM75 strain which carries the insertion sequence IS256, involved in adaptative and evolution processes of bacteria related to Staphylococcus and Enterococcus genera. In this work we describe the differential behavior in citrate transport, metabolism and aroma generation of three strains and we present results that link citrate metabolism and genetic organizations in E. faecium for the first time.

Expression of the agmatine deiminase pathway in Enterococcus faecalis is activated by the AguR regulator and repressed by CcpA and PTS(Man) systems.

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 P-Ser-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 (PTS(Man)) system also contributed to CCR in E. faecalis since a complete relief of the PTS-sugars repressive effect was observed only in a PTS(Man) 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 niches.

Biochemical and genetic characterization of the Enterococcus faecalis oxaloacetate decarboxylase complex.

Enterococcus faecalis encodes a biotin-dependent oxaloacetate decarboxylase (OAD), which is constituted by four subunits: E. faecalis carboxyltransferase subunit OadA (termed Ef-A), membrane pump Ef-B, biotin acceptor protein Ef-D, and the novel subunit Ef-H. Our results show that in E. faecalis, subunits Ef-A, Ef-D, and Ef-H form a cytoplasmic soluble complex (termed Ef-AHD) which is also associated with the membrane. In order to characterize the role of the novel Ef-H subunit, coexpression of oad genes was performed in Escherichia coli, showing that this subunit is vital for Ef-A and Ef-D interaction. Diminished growth of the oadA and oadD single deletion mutants in citrate-supplemented medium indicated that the activity of the complex is essential for citrate utilization. Remarkably, the oadB-deficient strain was still capable of growing to wild-type levels but with a delay during the citrate-consuming phase, suggesting that the soluble Ef-AHD complex is functional in E. faecalis. These results suggest that the Ef-AHD complex is active in its soluble form, and that it is capable of interacting in a dynamic way with the membrane-bound Ef-B subunit to achieve its maximal alkalinization capacity during citrate fermentation.

Fine-tuned transcriptional regulation of malate operons in Enterococcus faecalis.

In Enterococcus faecalis, the mae locus is constituted by two putative divergent operons, maePE and maeKR. The first operon encodes a putative H(+)/malate symporter (MaeP) and a malic enzyme (MaeE) previously shown to be essential for malate utilization in this bacterium. The maeKR operon encodes two putative proteins with significant similarity to two-component systems involved in sensing malate and activating its assimilation in bacteria. Our transcriptional and genetic assays showed that maePE and maeKR are induced in response to malate by the response regulator MaeR. In addition, we observed that both operons were partially repressed in the presence of glucose. Accordingly, the cometabolism of this sugar and malate was detected. The binding of the complex formed by CcpA and its corepressor P-Ser-HPr to a cre site located in the mae region was demonstrated in vitro and explains the carbon catabolite repression (CCR) observed for the maePE operon. However, our results also provide evidence for a CcpA-independent CCR mechanism regulating the expression of both operons. Finally, a biomass increment of 40 or 75% was observed compared to the biomass of cells grown only on glucose or malate, respectively. Cells cometabolizing both carbon sources exhibit a higher rate of glucose consumption and a lower rate of malate utilization. The growth improvement achieved by E. faecalis during glucose-malate cometabolism might explain why this microorganism employs different regulatory systems to tightly control the assimilation of both carbon sources.

Draft genome sequence of Enterococcus mundtii CRL1656.

We report the draft genome sequence of Enterococcus mundtii CRL1656, which was isolated from the stripping milk of a clinically healthy adult Holstein dairy cow from a dairy farm of the northwestern region of Tucumán (Argentina). The 3.10-Mb genome sequence consists of 450 large contigs and contains 2,741 predicted protein-coding genes.

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

BACKGROUND: In Enterococcus faecalis the genes encoding the enzymes involved in citrate metabolism are organized in two divergent operons, citHO and oadHDB-citCDEFX-oadA-citMG (citCL 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 cis-acting sequences located in the cit 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. RESULTS: In this study, we observed that the citHO 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 cit pathway. In fact, we demonstrate that CCR is acting on both promoters. It is partially relieved in a ccpA-deficient E. faecalis strain indicating that a CcpA-independent mechanism is also involved in regulation of the two operons. Furthermore, sequence analysis of the citH/oadH intergenic region revealed the presence of three putative catabolite responsive elements (cre). We found that they are all active and able to bind the CcpA/P-Ser-HPr complex, which downregulates the expression of the cit operons. Systematic mutation of the CcpA/P-Ser-HPr binding sites revealed that cre1 and cre2 contribute to citHO repression, while cre3 is involved in CCR of citCL. CONCLUSION: In conclusion, our study establishes that expression of the cit operons in E. faecalis is controlled by CCR via CcpA-dependent and -independent mechanisms.

Identification of malic and soluble oxaloacetate decarboxylase enzymes in Enterococcus faecalis.

Two paralogous genes, maeE and citM, that encode putative malic enzyme family members were identified in the Enterococcus faecalis genome. MaeE (41 kDa) and CitM (42 kDa) share a high degree of homology between them (47% identities and 68% conservative substitutions). However, the genetic context of each gene suggested that maeE is associated with malate utilization whereas citM is linked to the citrate fermentation pathway. In the present work, we focus on the biochemical characterization and physiological contribution of these enzymes in E. faecalis. With this aim, the recombinant versions of the two proteins were expressed in Escherichia coli, affinity purified and finally their kinetic parameters were determined. This approach allowed us to establish that MaeE is a malate oxidative decarboxylating enzyme and CitM is a soluble oxaloacetate decarboxylase. Moreover, our genetic studies in E. faecalis showed that the citrate fermentation phenotype is not affected by citM deletion. On the other hand, maeE gene disruption resulted in a malate fermentation deficient strain indicating that MaeE is responsible for malate metabolism in E. faecalis. Lastly, it was demonstrated that malate fermentation in E. faecalis is associated with cytoplasmic and extracellular alkalinization which clearly contributes to pH homeostasis in neutral or mild acidic conditions.