RESUMO
In prokaryotes, operon encoded proteins often form protein-protein complexes. Here, we show that the native structure of operons can be used to efficiently overexpress protein complexes. This study focuses on operons from mycobacteria and the use of Mycobacterium smegmatis as an expression host. We demonstrate robust and correct stoichiometric expression of dimers to higher oligomers. The expression efficacy was found to be largely independent of the intergenic distances. The strategy was successfully extended to express mycobacterial protein complexes in Escherichia coli, showing that the operon structure of gram-positive bacteria is also functional in gram-negative bacteria. The presented strategy could become a general tool for the expression of large quantities of pure prokaryotic protein complexes for biochemical and structural studies.
Assuntos
Proteínas de Bactérias/genética , Escherichia coli/genética , Mycobacterium/genética , Óperon/genética , Proteínas de Bactérias/química , Proteínas de Bactérias/metabolismo , Eletroforese em Gel de Poliacrilamida , Regulação Bacteriana da Expressão Gênica , Bactérias Gram-Negativas/genética , Bactérias Gram-Positivas/genética , Microscopia Eletrônica , Modelos Moleculares , Complexos Multiproteicos/química , Complexos Multiproteicos/metabolismo , Complexos Multiproteicos/ultraestrutura , Mycobacterium/metabolismo , Mycobacterium leprae/genética , Mycobacterium leprae/metabolismo , Mycobacterium smegmatis/genética , Mycobacterium smegmatis/metabolismo , Mycobacterium tuberculosis/genética , Mycobacterium tuberculosis/metabolismo , Multimerização ProteicaRESUMO
Phenotypic and phylogenetic studies were performed on 15 isolates of an unidentified Gram-positive, anaerobic, non-sporulating coccobacillus-shaped bacterium isolated from human faeces. The novel organisms were catalase-negative, indole-negative and produced acetate and succinate as end products of metabolism. Comparative 16S rRNA gene sequencing demonstrated that the 15 isolates were highly related to each other and formed a hitherto unknown subline within the clostridial rRNA cluster XIVa. The novel isolates formed a robust phylogenetic group with a number of organisms which included Clostridium coccoides, Ruminococcus luti, Ruminococcus obeum and a number of other misclassified ruminococci. On the basis of these studies, a novel genus, Blautia gen. nov., is proposed. It is suggested that Clostridium coccoides, Ruminococcus hansenii, Ruminococcus hydrogenotrophicus, Ruminococcus luti, Ruminococcus productus, and Ruminococcus schinkii are transferred to this genus as Blautia coccoides gen. nov., comb. nov., Blautia hansenii comb. nov., Blautia hydrogenotrophica comb. nov., Blautia luti comb. nov., Blautia producta comb. nov. and Blautia schinkii comb. nov. One of the new isolates, the hitherto unknown coccus-shaped bacterial strain WAL 14507T (=ATCC BAA-1564T=DSM 19850T) is proposed as representing the type strain of a novel species, Blautia wexlerae sp. nov.
Assuntos
Clostridium/classificação , Fezes/microbiologia , Bactérias Gram-Positivas/classificação , Ruminococcus/classificação , Acetatos/metabolismo , Técnicas de Tipagem Bacteriana , Criança , Pré-Escolar , Clostridium/genética , Clostridium/fisiologia , DNA Bacteriano/análise , DNA Ribossômico/análise , Genes de RNAr , Bactérias Gram-Positivas/genética , Bactérias Gram-Positivas/isolamento & purificação , Bactérias Gram-Positivas/fisiologia , Humanos , Dados de Sequência Molecular , Fenótipo , Filogenia , RNA Ribossômico 16S/genética , Ruminococcus/genética , Ruminococcus/fisiologia , Análise de Sequência de DNA , Especificidade da Espécie , Succinatos/metabolismoRESUMO
AIMS: To determine the relationships between the major organisms from the cheese-making personnel and environment and the surface of a smear cheese. METHODS AND RESULTS: 360 yeast and 593 bacteria from the cheese surface, the dairy environment and the hands and arms of personnel were collected. Pulsed-field gel electrophoresis, repetitive sequence-based polymerase chain reaction and 16S rDNA sequencing were used for typing and identifying the bacteria, and mitochondrial DNA restriction fragment length polymorphism and Fourier-transform infrared spectroscopy for typing and identifying the yeast. The three most dominant bacteria were Corynebacterium casei, Corynebacterium variabile and Staphylococcus saprophyticus, which were divided into three, five and seven clusters, respectively, by macrorestriction analysis. The same clones from these organisms were isolated on the cheese surface, the dairy environment and the skin of the cheese personnel. Debaryomyces hansenii was the most dominant yeast. CONCLUSIONS: A 'house' microflora exists in the cheese plant. Although the original source of the micro-organisms was not identified, the brines were an important source of S. saprophyticus and D. hansenii and, additionally, the arms and hands of the workers the sources of C. casei and C. variabile. SIGNIFICANCE AND IMPACT OF THE STUDY: This is the first time that the major contribution of the house microflora to the ripening of a smear-ripened cheese has been demonstrated.
Assuntos
Queijo/microbiologia , Microbiologia de Alimentos , Biodiversidade , Contagem de Colônia Microbiana/métodos , Corynebacterium/genética , Corynebacterium/isolamento & purificação , DNA Bacteriano/genética , DNA Mitocondrial/genética , DNA Ribossômico/genética , Eletroforese em Gel de Campo Pulsado/métodos , Manipulação de Alimentos/métodos , Indústria Alimentícia , Bactérias Gram-Positivas/genética , Bactérias Gram-Positivas/isolamento & purificação , Humanos , Fenótipo , Polimorfismo de Fragmento de Restrição , Pele/microbiologia , Staphylococcus/genética , Staphylococcus/isolamento & purificação , Local de Trabalho , Leveduras/genética , Leveduras/isolamento & purificaçãoRESUMO
BACKGROUND: The genome of Mycobacterium tuberculosis H37Rv has five copies of a cluster of genes known as the ESAT-6 loci. These clusters contain members of the CFP-10 (lhp) and ESAT-6 (esat-6) gene families (encoding secreted T-cell antigens that lack detectable secretion signals) as well as genes encoding secreted, cell-wall-associated subtilisin-like serine proteases, putative ABC transporters, ATP-binding proteins and other membrane-associated proteins. These membrane-associated and energy-providing proteins may function to secrete members of the ESAT-6 and CFP-10 protein families, and the proteases may be involved in processing the secreted peptide. RESULTS: Finished and unfinished genome sequencing data of 98 publicly available microbial genomes has been analyzed for the presence of orthologs of the ESAT-6 loci. The multiple duplicates of the ESAT-6 gene cluster found in the genome of M. tuberculosis H37Rv are also conserved in the genomes of other mycobacteria, for example M. tuberculosis CDC1551, M. tuberculosis 210, M. bovis, M. leprae, M. avium, and the avirulent strain M. smegmatis. Phylogenetic analyses of the resulting sequences have established the duplication order of the gene clusters and demonstrated that the gene cluster known as region 4 (Rv3444c-3450c) is ancestral. Region 4 is also the only region for which an ortholog could be found in the genomes of Corynebacterium diphtheriae and Streptomyces coelicolor. CONCLUSIONS: Comparative genomic analysis revealed that the presence of the ESAT-6 gene cluster is a feature of some high-G+C Gram-positive bacteria. Multiple duplications of this cluster have occurred and are maintained only within the genomes of members of the genus Mycobacterium.
Assuntos
Antígenos de Bactérias/genética , Família Multigênica , Mycobacterium tuberculosis/genética , Mycobacterium/genética , Proteínas de Bactérias , Corynebacterium diphtheriae/genética , DNA Bacteriano/genética , Sequência Rica em GC , Genoma Bacteriano , Bactérias Gram-Positivas/genética , Mycobacterium avium/genética , Mycobacterium avium subsp. paratuberculosis/genética , Mycobacterium bovis/genética , Mycobacterium leprae/genética , Mycobacterium smegmatis/genética , Filogenia , Análise de Sequência de DNA , Streptomyces/genéticaRESUMO
Phylogenetic analysis of the genus Ruminococcus based on 16S rDNA sequence data showed the genus to be phylogenetically heterogenous. Ruminococcus species fall within the radiation of the Bacillus/Clostridium subphylum of the Gram-positive line of descent. Two distinct and unrelated clusters are recovered. One group contains R. flavefaciens, R. albus, R. bromii, and R. callidus. The second group constitutes R. gnavus, R. hansenii, R. lactaris, R. obeum, R. productus and R. torques which together with some Clostridium species may constitute a new genus.