RESUMO
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.
Assuntos
Lacticaseibacillus paracasei , Lactobacillales , Ração Animal , Animais , Fermentação , Microbiologia de Alimentos , GenômicaRESUMO
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.
Assuntos
Carboxiliases/genética , Enterococcus faecalis/enzimologia , Complexos Multienzimáticos/genética , Sequência de Aminoácidos , Proteínas de Bactérias/genética , Proteínas de Bactérias/isolamento & purificação , Proteínas de Bactérias/metabolismo , Carboxiliases/isolamento & purificação , Carboxiliases/metabolismo , Ácido Cítrico/metabolismo , Citoplasma/enzimologia , Enterococcus faecalis/genética , Enterococcus faecalis/fisiologia , Escherichia coli/genética , Escherichia coli/metabolismo , Fermentação , Concentração de Íons de Hidrogênio , Complexos Multienzimáticos/isolamento & purificação , Complexos Multienzimáticos/metabolismo , Ácido Oxaloacético/metabolismo , Subunidades Proteicas , Proteínas Recombinantes , Deleção de Sequência , TransgenesRESUMO
NAD(P)H-glutamate dehydrogenase (GDH, EC 1.4.1.3) contributes to the control of glutamate homeostasis in all living organisms. In bacteria and animals, GDH is a homohexamer allosterically regulated, whereas in plants NADH-GDH (EC 1.4.1.2) is also found as heterohexamer of α- and ß-subunits, but its regulation remains undefined. In tomato (Solanum lycopersicum), GDH activity increases during the fruit ripening along with the content of free glutamate, the most abundant amino acid of ripe fruit involved in conferring the genuine tomato flavour. In this work, novel Slgdh-NAD genes were identified in the recently deciphered tomato genome: three encoding the α-subunit (Slgdh-NAD;A1-3) and one additional gene encoding the ß-subunit of GDH (Slgdh-NAD;B1) isolated from a genomic library. These genes are located in different chromosomes. Slgdh-NAD;A1-3 show conserved structures, whereas Slgdh-NAD;B1 includes a novel 5'-untranslated exon. Slgdh-NAD;A1-3 transcripts were detected in all tomato tissues examined, showing the highest levels in mature green fruits, contrasting with Slgdh-NAD;B1 transcripts which were detected mainly in roots or in mature fruits when treated with glutamate, NaCl or salicylic acid. Analyses of GDH activity and protein distribution in different tissues of the Micro-Tom cultivar showed that only the active homohexamer of GDH ß-subunits was detected in roots while heterohexamers of GDH α- and ß-subunits were found in fruits. These results indicate that GDH ß-subunit could modulate the heteromeric isoforms of GDH in response to the environment and physiology of the tomato fruit. This information is relevant to manipulate glutamate contents in tomato fruits genetically.
Assuntos
Frutas/enzimologia , Glutamato Desidrogenase/genética , Glutamato Desidrogenase/metabolismo , Ácido Glutâmico/metabolismo , Solanum lycopersicum/enzimologia , Solanum lycopersicum/genética , Sequência de Aminoácidos , China , Regulação Enzimológica da Expressão Gênica , Genes de Plantas , Dados de Sequência Molecular , Proteínas de Plantas/metabolismo , Raízes de Plantas/metabolismo , Transcrição Gênica/efeitos dos fármacosRESUMO
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.
Assuntos
Enterococcus faecalis/genética , Enterococcus faecalis/metabolismo , Regulação Bacteriana da Expressão Gênica , Malatos/metabolismo , Óperon , Transcrição Gênica , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Biomassa , Enterococcus faecalis/crescimento & desenvolvimento , Glucose/metabolismoRESUMO
Diacetyl and acetoin are pyruvate-derived metabolites excreted by many micro-organisms, and are important in their physiology. Although generation of these four-carbon (C4) compounds in Enterococcus faecalis is a well-documented phenotype, little is known about the gene regulation of their biosynthetic pathway and the physiological role of the pathway in this bacterium. In this work, we identified the genes involved in C4 compound biosynthesis in Ent. faecalis and report their transcriptional analysis. These genes are part of the alsSD bicistronic operon, which encodes α-acetolactate synthase (AlsS) and α-acetolactate decarboxylase (AlsD). Our studies showed that alsSD operon transcription levels are maximal during the exponential phase of growth, decreasing thereafter. Furthermore, we found that this transcription is enhanced upon addition of pyruvate to the growth medium. In order to study the functional role of the alsSD operon, an isogenic alsSD mutant strain was constructed. This strain lost its capacity to generate C4 compounds, confirming the role of alsSD genes in this metabolic pathway. In contrast to the wild-type strain, the alsSD-deficient strain was unable to grow in LB medium supplemented with pyruvate at an initial pH of 4.5. This dramatic reduction in growth parameters for the mutant strain was simultaneously accompanied by the inability to alkalinize the internal and external medium under these conditions. In sum, these results suggest that the decarboxylation reactions related to the C4 biosynthetic pathway give enterococcal cells a competitive advantage during pyruvate metabolism at low pH.
Assuntos
Acetolactato Sintase/genética , Carboxiliases/genética , Enterococcus faecalis/genética , Enterococcus faecalis/metabolismo , Óperon , Ácido Pirúvico/metabolismo , Acetolactato Sintase/deficiência , Sequência de Bases , Carbono/metabolismo , Carboxiliases/deficiência , Biologia Computacional , Regulação Bacteriana da Expressão Gênica , Ordem dos Genes , Concentração de Íons de Hidrogênio , Dados de Sequência Molecular , Transcrição GênicaRESUMO
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.