RESUMEN
Enterococcus cecorum is an emerging pathogen responsible for osteomyelitis, spondylitis, and femoral head necrosis causing animal suffering and mortality and requiring antimicrobial use in poultry. Paradoxically, E. cecorum is a common inhabitant of the intestinal microbiota of adult chickens. Despite evidence suggesting the existence of clones with pathogenic potential, the genetic and phenotypic relatedness of disease-associated isolates remains little investigated. Here, we sequenced and analyzed the genomes and characterized the phenotypes of more than 100 isolates, the majority of which were collected over the last 10 years from 16 French broiler farms. Comparative genomics, genome-wide association studies, and the measured susceptibility to serum, biofilm-forming capacity, and adhesion to chicken type II collagen were used to identify features associated with clinical isolates. We found that none of the tested phenotypes could discriminate the origin of the isolates or the phylogenetic group. Instead, we found that most clinical isolates are grouped phylogenetically, and our analyses selected six genes that discriminate 94% of isolates associated with disease from those that are not. Analysis of the resistome and the mobilome revealed that multidrug-resistant clones of E. cecorum cluster into a few clades and that integrative conjugative elements and genomic islands are the main carriers of antimicrobial resistance. This comprehensive genomic analysis shows that disease-associated clones of E. cecorum belong mainly to one phylogenetic clade. IMPORTANCE Enterococcus cecorum is an important pathogen of poultry worldwide. It causes a number of locomotor disorders and septicemia, particularly in fast-growing broilers. Animal suffering, antimicrobial use, and associated economic losses require a better understanding of disease-associated E. cecorum isolates. To address this need, we performed whole-genome sequencing and analysis of a large collection of isolates responsible for outbreaks in France. By providing the first data set on the genetic diversity and resistome of E. cecorum strains circulating in France, we pinpoint an epidemic lineage that is probably also circulating elsewhere that should be targeted preferentially by preventive strategies in order to reduce the burden of E. cecorum-related diseases.
Asunto(s)
Antiinfecciosos , Enfermedades de las Aves de Corral , Animales , Aves de Corral , Pollos , Estudio de Asociación del Genoma Completo , FilogeniaRESUMEN
Understanding of the distribution of chemerin and its receptors, Chemokine-like Receptor 1 (CMKLR1), G Protein-coupled Receptor 1 (GPR1) and Chemokine (C-C motif) receptor-like 2 (CCRL2), in the egg and the embryonic annexes is currently lacking, and their role during embryogenesis remains unknown. By immunoblot using monoclonal anti-chicken antibodies and Enzyme Linked Immunosorbent Assays (ELISA), we found that chemerin is expressed 10 times higher in albumen eggs than in blood plasma, and it is also abundant in the perivitelline membrane but undetectable in yolk. Chicken chemerin can inhibit bacterial growth. By Reverse Transcription-quantitative Polymerisation Chain Reaction (RT-qPCR), western-blot, and immunofluorescence, we show that chemerin is locally produced by the oviduct magnum that participates in albumen formation. Using cultures of magnum explants, we demonstrate that progesterone (P4) and oestradiol (E2) treatment increases chemerin secretion into cultured media and expression in magnum. Chemerin and its three receptors are present in amniotic and Chorio Allantoic Membranes (CAM). Only CMKLR1 expression decreased from embryonic day (ED) 7 to ED11 and remained low until ED18. Chemerin concentrations strongly increased in amniotic fluid at D14 when egg albumen crossed the amniotic membrane. In ovo injections of neutralising chemerin and CMKLR1 antibodies (0.01, 0.1 and 1 µg) increased embryo mortality, which occurred mainly at ED12-13, in a dose-dependent manner. Chemerin treatment increased primary CAM viability. Finally, chemerin and CMKLR1 inhibition within the CAM led to a decrease in blood vessel development and associated angiogenic gene expression. Our results show an important function of the chemerin system during embryo development in chickens, suggesting the potential use of this adipokine as a predictive marker for egg fertility or hatchability.
Asunto(s)
Quimiocinas , Pollos , Desarrollo Embrionario , Oviductos , Albúminas , Animales , Quimiocinas/metabolismo , Pollos/metabolismo , Femenino , Oviductos/metabolismo , Óvulo , Receptores CCR/metabolismo , Receptores de Quimiocina/metabolismo , Receptores Acoplados a Proteínas G/metabolismoRESUMEN
This article reports draft genome sequences of 17 Pasteurella multocida strains isolated from naturally infected rabbits. The total lengths of the assembled contigs ranged between 2.21 and 2.48 Mb, and the total number of genes detected on the contigs ranged between 2,088 and 2,416.
RESUMEN
We report here the use of a peptidomic approach to revisit the extracellular proteolysis of Lactococcus lactis. More than 1800 distinct peptides accumulate externally during growth of the plasmid-free protease-negative strain L. lactis IL1403 in a protein- and peptide-free medium. These peptides mainly originate from cell-surface- and cytoplasmic-located proteins, despite the fact that no cell lysis could be evidenced. Positioning each identified peptide on its parental protein sequence demonstrated the involvement of exo- and endopeptidase activities. The endopeptidases responsible for the release of surface and cytoplasmic peptides had distinct specificities. The membrane-anchored protease HtrA was responsible for the release of only a part of the surface peptides, and its preference for branched-chain amino acids in the N-terminal side of the cleaved bond was established in situ. Other yet uncharacterized surface proteases were also involved. Several lines of evidence suggest that surface and cytoplasmic peptides were produced by different routes, at least part of the latter being most likely excreted as peptides from the cells. The mechanism by which these cytoplasmic peptides are excreted remains an open question, as it is still the case for excreted cytoplasmic proteins.
Asunto(s)
Péptidos/metabolismo , Proteolisis , Proteómica/métodos , Aminoácidos de Cadena Ramificada/metabolismo , Proteínas Bacterianas/metabolismo , Membrana Celular/enzimología , Citoplasma/enzimología , Espectrometría de Masas , Péptido Hidrolasas/metabolismo , Péptidos/análisis , Serina Endopeptidasas/metabolismoRESUMEN
We described a quorum-sensing mechanism in the streptococci genus involving a short hydrophobic peptide (SHP), which acts as a pheromone, and a transcriptional regulator belonging to the Rgg family. The shp/rgg genes, found in nearly all streptococcal genomes and in several copies in some, have been classified into three groups. We used a genetic approach to evaluate the functionality of the SHP/Rgg quorum-sensing mechanism, encoded by three selected shp/rgg loci, in pathogenic and non-pathogenic streptococci. We characterized the mature form of each SHP pheromone by mass-spectrometry. We produced synthetic peptides corresponding to these mature forms, and used them to study functional complementation and cross-talk between these different SHP/Rgg systems. We demonstrate that a SHP pheromone of one system can influence the activity of a different system. Interestingly, this does not seem to be dependent on the SHP/Rgg group and cross-talk between pathogenic and non-pathogenic streptococci is observed.
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Proteínas Bacterianas/metabolismo , Péptidos/metabolismo , Streptococcus/metabolismo , Proteínas Bacterianas/genética , Regulación Bacteriana de la Expresión Génica , Péptidos/genética , Feromonas/genética , Feromonas/metabolismo , Percepción de Quorum/genética , Percepción de Quorum/fisiología , Streptococcus/genéticaRESUMEN
In many streptococci, competence for natural DNA transformation is regulated by the Rgg-type regulator ComR and the pheromone ComS, which is sensed intracellularly. We compared the ComRS systems of four model streptococcal species using in vitro and in silico approaches, to determine the mechanism of the ComRS-dependent regulation of competence. In all systems investigated, ComR was shown to be the proximal transcriptional activator of the expression of key competence genes. Efficient binding of ComR to DNA is strictly dependent on the presence of the pheromone (C-terminal ComS octapeptide), in contrast with other streptococcal Rgg-type regulators. The 20 bp palindromic ComR-box is the minimal genetic requirement for binding of ComR, and its sequence directly determines the expression level of genes under its control. Despite the apparent species-specific specialization of the ComR-ComS interaction, mutagenesis of ComS residues from Streptococcus thermophilus highlighted an unexpected permissiveness with respect to its biological activity. In agreement, heterologous ComS, and even primary sequence-unrelated, casein-derived octapeptides, were able to induce competence development in S. thermophilus. The lack of stringency of ComS sequence suggests that competence of a specific Streptococcus species may be modulated by other streptococci or by non-specific nutritive oligopeptides present in its environment.
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Proteínas Bacterianas/metabolismo , Competencia de la Transformación por ADN , Regulación Bacteriana de la Expresión Génica , Streptococcus/genética , Sitios de Unión , Biología Computacional , ADN Bacteriano/metabolismo , Proteínas de Unión al ADN/metabolismo , Feromonas/metabolismo , Unión ProteicaRESUMEN
Water-forming NADH oxidases (encoded by noxE, nox2, or nox) are flavoproteins generally implicated in the aerobic survival of microaerophilic bacteria, such as lactic acid bacteria. However, some natural Lactococcus lactis strains produce an inactive NoxE. We examined the role of NoxE in the oxygen tolerance of L. lactis in the rich synthetic medium GM17. Inactivation of noxE suppressed 95% of NADH oxidase activity but only slightly affected aerobic growth, oxidative stress resistance, and NAD regeneration. However, noxE inactivation strongly impaired oxygen consumption and mixed-acid fermentation. We found that the A303T mutation is responsible for the loss of activity of a naturally occurring variant of NoxE. Replacement of A303 with T or G or of G307 with S or A by site-directed mutagenesis led to NoxE aggregation and the total loss of activity. We demonstrated that L299 is involved in NoxE activity, probably contributing to positioning flavin adenine dinucleotide (FAD) in the active site. These residues are part of the strongly conserved sequence LA(T)XXAXXXG included in an alpha helix that is present in other flavoprotein disulfide reductase (FDR) family flavoproteins that display very similar three-dimensional structures.
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Lactococcus lactis/enzimología , Complejos Multienzimáticos/genética , Complejos Multienzimáticos/metabolismo , NADH NADPH Oxidorreductasas/genética , NADH NADPH Oxidorreductasas/metabolismo , Aerobiosis/genética , Aerobiosis/fisiología , Secuencia de Aminoácidos , Secuencia Conservada , Fermentación , Flavina-Adenina Dinucleótido/metabolismo , Regulación Bacteriana de la Expresión Génica/fisiología , Regulación Enzimológica de la Expresión Génica , Mutación , NAD/genética , NAD/metabolismo , Oxígeno , Consumo de Oxígeno , Unión ProteicaRESUMEN
The reduction of tetrazolium salts to coloured formazans is often used as an indicator of cell metabolism during microbiology studies, although the reduction mechanisms have never clearly been established in bacteria. The objective of the present study was to identify the reduction mechanisms of tetrazolium violet (TV) in Lactococcus lactis using a mutagenesis approach, under two experimental conditions generally applied in microbiology: a plate test with growing cells, and a liquid test with non-growing (resting) cells. The results showed that in both tests, TV reduction resulted from electron transfer from an intracellular donor (mainly NADH) to TV via the electron transport chain (ETC), but the reduction sites in the ETC depended on experimental conditions. Using the plate test, menaquinones were essential for TV reduction and membrane NADH dehydrogenases (NoxA and/or NoxB) were partly involved in electron transfer to menaquinones. In this case, TV reduction mainly occurred outside the cells and in the outer part of the plasma membrane. During the liquid test, TV was directly reduced by NoxA and/or NoxB, probably in the inner part of the membrane, where NoxA and NoxB are localized. In this case, reduction was directly related to the intracellular NADH pool. Based on these findings, new applications for TV tests are proposed, such as NADH pool determination with the liquid test and the screening of mutants affected in menaquinone biosynthesis with the plate test. Preliminary results using other tetrazolium salts in the plate test showed that the reduction sites depended on the salt, suggesting that similar studies should be carried out with other tetrazolium salts so that the outcome of each test can be interpreted correctly.
Asunto(s)
Lactococcus lactis/metabolismo , Sales de Tetrazolio/metabolismo , Medios de Cultivo , ADN Bacteriano/genética , Transporte de Electrón , Genes Bacterianos , Lactococcus lactis/genética , Lactococcus lactis/crecimiento & desarrollo , Mutagénesis Insercional , NAD/química , NAD/metabolismo , NADH Deshidrogenasa/genética , NADH Deshidrogenasa/metabolismo , Oxidación-Reducción , Sales de Tetrazolio/química , Vitamina K 2/química , Vitamina K 2/metabolismoRESUMEN
Hydroxyacid dehydrogenases of lactic acid bacteria, which catalyze the stereospecific reduction of branched-chain 2-keto acids to 2-hydroxyacids, are of interest in a variety of fields, including cheese flavor formation via amino acid catabolism. In this study, we used both targeted and random mutagenesis to identify the genes responsible for the reduction of 2-keto acids derived from amino acids in Lactococcus lactis. The gene panE, whose inactivation suppressed hydroxyisocaproate dehydrogenase activity, was cloned and overexpressed in Escherichia coli, and the recombinant His-tagged fusion protein was purified and characterized. The gene annotated panE was the sole gene responsible for the reduction of the 2-keto acids derived from leucine, isoleucine, and valine, while ldh, encoding L-lactate dehydrogenase, was responsible for the reduction of the 2-keto acids derived from phenylalanine and methionine. The kinetic parameters of the His-tagged PanE showed the highest catalytic efficiencies with 2-ketoisocaproate, 2-ketomethylvalerate, 2-ketoisovalerate, and benzoylformate (V(max)/K(m) ratios of 6,640, 4,180, 3,300, and 2,050 U/mg/mM, respectively), with NADH as the exclusive coenzyme. For the reverse reaction, the enzyme accepted d-2-hydroxyacids but not l-2-hydroxyacids. Although PanE showed the highest degrees of identity to putative NADP-dependent 2-ketopantoate reductases (KPRs), it did not exhibit KPR activity. Sequence homology analysis revealed that, together with the d-mandelate dehydrogenase of Enterococcus faecium and probably other putative KPRs, PanE belongs to a new family of D-2-hydroxyacid dehydrogenases which is unrelated to the well-described D-2-hydroxyisocaproate dehydrogenase family. Its probable physiological role is to regenerate the NAD(+) necessary to catabolize branched-chain amino acids, leading to the production of ATP and aroma compounds.
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Proteínas Bacterianas/metabolismo , Cetoácidos/metabolismo , Lactococcus lactis/metabolismo , Oxidorreductasas/metabolismo , Proteínas Bacterianas/genética , Prueba de Complementación Genética , Cinética , L-Lactato Deshidrogenasa/metabolismo , Lactococcus lactis/genética , Leucina/metabolismo , Modelos Biológicos , Oxidación-Reducción , Oxidorreductasas/genética , Proteínas Recombinantes/metabolismo , Transducción de Señal , Especificidad por SustratoRESUMEN
A novel plasmid named pGdh442 had previously been isolated from a plant Lactococcus lactis strain. This plasmid encodes two interesting properties with applications in the dairy industry: a glutamate dehydrogenase activity that stimulates amino acid conversion to aroma compounds, and cadmium/zinc resistance that can be used as a selectable marker. Moreover, this plasmid can be transferred naturally to other strains, but appears to be incompatible with certain other lactococcal plasmids. During this study, the complete sequence of pGdh442 (68 319 bp) was determined and analysed. This plasmid contains 67 ORFs that include 20 IS elements that may have mediated transfer events between L. lactis and other genera living in the same biotope, such as Streptococcus, Pediococcus and Lactobacillus. Even though it is a low-copy-number plasmid, it is relatively stable due to a theta replication mode and the presence of two genes involved in its maintenance system. However, pGdh442 is incompatible with pSK08-derived protease/lactose plasmids because both possess the same replication and partition system. pGdh442 is not self-transmissible, but can be naturally transmitted via mobilization by conjugative elements carried by the chromosome or by other plasmids, such as the 712-type sex factor, which is widely distributed in L. lactis. In addition to several genes already found on other L. lactis plasmids, such as the oligopeptide transport and utilization genes, pGdh442 also carries several genes not yet identified in L. lactis. Finally, it does not carry genes that would trigger concern over its presence in human food.
Asunto(s)
ADN Bacteriano/química , ADN Bacteriano/genética , Glutamato Deshidrogenasa/genética , Lactococcus lactis/genética , Plásmidos/genética , Aminoácidos/metabolismo , Secuencia de Bases , Conjugación Genética , Replicación del ADN , Elementos Transponibles de ADN/genética , Farmacorresistencia Bacteriana/genética , Orden Génico , Transferencia de Gen Horizontal , Genes Bacterianos , Glutamato Deshidrogenasa/biosíntesis , Humanos , Lactococcus lactis/enzimología , Metales/toxicidad , Datos de Secuencia Molecular , Sistemas de Lectura Abierta , Plásmidos/metabolismo , Análisis de Secuencia de ADN , Homología de SecuenciaRESUMEN
Amino acid conversion to aroma compounds by Lactococcus lactis is limited by the low production of alpha-ketoglutarate that is necessary for the first step of conversion. Recently, glutamate dehydrogenase (GDH) activity that catalyzes the reversible glutamate deamination to alpha-ketoglutarate was detected in L. lactis strains isolated from a vegetal source, and the gene responsible for the activity in L. lactis NCDO1867 was identified and characterized. The gene is located on a 70-kb plasmid also encoding cadmium resistance. In this study, gdh gene inactivation and overexpression confirmed the direct impact of GDH activity of L. lactis on amino acid catabolism in a reaction medium at pH 5.5, the pH of cheese. By using cadmium resistance as a selectable marker, the plasmid carrying gdh was naturally transmitted to another L. lactis strain by a mating procedure. The transfer conferred to the host strain GDH activity and the ability to catabolize amino acids in the presence of glutamate in the reaction medium. However, the plasmid appeared unstable in a strain also containing the protease lactose plasmid pLP712, indicating an incompatibility between these two plasmids.
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Aminoácidos/metabolismo , Glutamato Descarboxilasa/genética , Glutamato Descarboxilasa/metabolismo , Lactococcus lactis/genética , Lactococcus lactis/metabolismo , Secuencia de Bases , Cadmio/farmacología , Queso/microbiología , ADN Bacteriano/genética , Farmacorresistencia Bacteriana/genética , Expresión Génica , Transferencia de Gen Horizontal , Genes Bacterianos , Concentración de Iones de Hidrógeno , Lactococcus lactis/efectos de los fármacos , Lactococcus lactis/enzimología , Odorantes , Fenilalanina/metabolismo , Plásmidos/genética , Zinc/farmacologíaRESUMEN
The gene responsible for the uncommon glutamate dehydrogenase (GDH) activity of Lactococcus lactis was identified and characterized. It encodes a GDH of family I that is mainly active in glutamate biosynthesis, is carried by a large plasmid, and is included, with functional cadmium resistance genes, in a remnant Tn3-like transposon.
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Elementos Transponibles de ADN/genética , Glutamato Deshidrogenasa/genética , Lactococcus lactis/enzimología , Plásmidos/genética , Secuencia de Bases , Mapeo Cromosómico , Clonación Molecular , Cartilla de ADN , Glutamato Deshidrogenasa/metabolismo , Lactococcus lactis/clasificación , Lactococcus lactis/genética , Datos de Secuencia Molecular , FilogeniaRESUMEN
The enzymatic degradation of L-methionine and subsequent formation of volatile sulfur compounds (VSCs) is believed to be essential for flavor development in cheese. L-methionine-gamma-lyase (MGL) can convert L-methionine to methanethiol (MTL), alpha-ketobutyrate, and ammonia. The mgl gene encoding MGL was cloned from the type strain Brevibacterium linens ATCC 9175 known to produce copious amounts of MTL and related VSCs. The disruption of the mgl gene, achieved in strain ATCC 9175, resulted in a 62% decrease in thiol-producing activity and a 97% decrease in total VSC production in the knockout strain. Our work shows that L-methionine degradation via gamma-elimination is a key step in the formation of VSCs in B. linens.
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Brevibacterium/metabolismo , Liasas de Carbono-Azufre/genética , Liasas de Carbono-Azufre/metabolismo , Queso/microbiología , Secuencia de Aminoácidos , Brevibacterium/genética , Brevibacterium/crecimiento & desarrollo , Liasas de Carbono-Azufre/química , Eliminación de Gen , Metionina/metabolismo , Datos de Secuencia Molecular , Análisis de Secuencia de ADN , Compuestos de Sulfhidrilo/metabolismo , Compuestos de Azufre/metabolismo , VolatilizaciónRESUMEN
Volatile sulphur compounds (VSCs) production from L-methionine was studied in Lactococcus lactis. In vitro studies with radiolabelled L-methionine and resting cells of L. lactis revealed that L-methionine was initially converted to alpha-keto-gamma-methylthiobutyrate (KMBA) by a transamination reaction. A part of KMBA was subsequently chemically converted to methylthioacetaldehyde, methanethiol and dimethylsulphides. Chemical conversion of KMBA to methylthioacetaldehyde was dependent on pH, Mn(II) and oxygen. Since methanethiol and dimethylsulphide production was highly related to that of methylthioacetaldehyde, the latter compound was proposed as being an intermediate in VSCs production by L. lactis.
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Acetaldehído/metabolismo , Queso/microbiología , Lactococcus lactis/metabolismo , Metionina/metabolismo , Sulfuros/metabolismo , Cromatografía de Gases y Espectrometría de Masas , Compuestos de Sulfhidrilo/metabolismoRESUMEN
Aminotransferases, which catalyze the last step of biosynthesis of most amino acids and the first step of their catabolism, may be involved in the growth of Lactococcus lactis in milk. Previously, we isolated two aminotransferases from L. lactis, AraT and BcaT, which are responsible for the transamination of aromatic amino acids, branched-chain amino acids, and methionine. In this study, we demonstrated that double inactivation of AraT and BcaT strongly reduced the growth of L. lactis in milk. Supplementation of milk with amino acids and keto acids that are substrates of both aminotransferases did not improve the growth of the double mutant. On the contrary, supplementation of milk with isoleucine or a dipeptide containing isoleucine almost totally inhibited the growth of the double mutant, while it did not affect or only slightly affected the growth of the wild-type strain. These results suggest that AraT and BcaT play a major role in the growth of L. lactis in milk by degrading the intracellular excess isoleucine, which is responsible for the growth inhibition. The growth inhibition by isoleucine is likely to be due to CodY repression of the proteolytic system, which is necessary for maximal growth of L. lactis in milk, since the growth of the CodY mutant was not affected by addition of isoleucine to milk. Moreover, we demonstrated that AraT and BcaT are part of the CodY regulon and therefore are regulated by nutritional factors, such as the carbohydrate and nitrogen sources.
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Regulación Bacteriana de la Expresión Génica , Lactococcus lactis/crecimiento & desarrollo , Leche/microbiología , Proteínas Represoras/metabolismo , Transaminasas/metabolismo , Aminoácidos Aromáticos/metabolismo , Aminoácidos de Cadena Ramificada/metabolismo , Animales , Proteínas Bacterianas/genética , Proteínas Bacterianas/metabolismo , Medios de Cultivo , Isoleucina/metabolismo , Lactococcus lactis/enzimología , Mutación , Proteínas Represoras/genéticaRESUMEN
Lactic acid bacteria (LAB) have the enzyme potential to transform amino acids into aroma compounds that contribute greatly to cheese flavour. Generally, amino acid conversion by LAB is limited by their low production of alpha-ketoglutarate since this alpha-ketoacid is essential for the first step of the conversion. Indeed, we have demonstrated that adding exogenous alpha-ketoglutarate to cheese curd, as well as using a genetically modified L. lactis strain capable of producing alpha-ketoglutarate from glutamate, greatly increased the conversion of amino acid to potent aroma compounds in cheese. Here we report the presence of glutamate dehydrogenase (GDH) activity required for the conversion of glutamate to alpha-ketoglutarate in several 'natural' LAB strains, commonly used in cheese manufacturing. Moreover, we show that the ability of LAB to produce aroma compounds from amino acids is closely related to their GDH activity. Therefore, GDH activity appears to be a major criterion for the selection of flavour-producing LAB strains, which could be used as a starter or as an adjunct to intensify flavour formation in some cheeses.