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1.
J Bacteriol ; 183(16): 4796-805, 2001 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-11466283

RESUMO

The virulence mechanisms of the facultative intracellular parasite Rhodococcus equi remain largely unknown. Among the candidate virulence factors of this pathogenic actinomycete is a secreted cholesterol oxidase, a putative membrane-damaging toxin. We identified and characterized the gene encoding this enzyme, the choE monocistron. Its protein product, ChoE, is homologous to other secreted cholesterol oxidases identified in Brevibacterium sterolicum and Streptomyces spp. ChoE also exhibits significant similarities to putative cholesterol oxidases encoded by Mycobacterium tuberculosis and Mycobacterium leprae. Genetic tools for use with R. equi are poorly developed. Here we describe the first targeted mutagenesis system available for this bacterium. It is based on a suicide plasmid, a selectable marker (the aacC4 apramycin resistance gene from Salmonella), and homologous recombination. The choE allele was disrupted by insertion of the aacC4 gene, cloned in pUC19 and introduced by electroporation in R. equi. choE recombinants were isolated at frequencies between 10(-2) and 10(-3). Twelve percent of the recombinants were double-crossover choE mutants. The choE mutation was associated with loss of cooperative (CAMP-like) hemolysis with sphingomyelinase-producing bacteria (Listeria ivanovii). Functional complementation was achieved by expression of choE from pVK173-T, a pAL5000 derivative conferring hygromycin resistance. Our data demonstrate that ChoE is an important cytolytic factor for R. equi. The highly efficient targeted mutagenesis procedure that we used to generate choE isogenic mutants will be a valuable tool for the molecular analysis of R. equi virulence.


Assuntos
Colesterol Oxidase/genética , Colesterol Oxidase/metabolismo , Rhodococcus equi/enzimologia , Rhodococcus equi/genética , Alelos , Sequência de Aminoácidos , Animais , Colesterol Oxidase/química , Mapeamento Cromossômico , Cromossomos Bacterianos/genética , Troca Genética , Escherichia coli/enzimologia , Hemólise , Dados de Sequência Molecular , Mutagênese Insercional , Mycobacterium leprae/enzimologia , Mycobacterium tuberculosis/enzimologia , Plasmídeos , Proteínas Recombinantes/química , Proteínas Recombinantes/metabolismo , Rhodococcus equi/patogenicidade , Alinhamento de Sequência , Homologia de Sequência de Aminoácidos , Ovinos , Streptomyces/enzimologia , Virulência
2.
Microbiology (Reading) ; 145 ( Pt 3): 549-559, 1999 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-10217488

RESUMO

During early stages of growth, Streptomyces reticuli synthesizes a hyphae-associated, haem-containing enzyme which exhibits catalase and peroxidase activities with broad substrate specificity (CpeB). The purified dimeric enzyme (160 kDa) consists of two identical subunits. Using anti-CpeB antibodies and an expression- as well as a mini-library, the corresponding cpeB gene was identified and sequenced. It encodes a protein of 740 aa with a molecular mass of 81.3 kDa. The deduced protein shares the highest level of amino acid identity with KatG from Caulobacter crescentus and Mycobacterium tuberculosis, and PerA from Bacillus stearothermophilus. Streptomyces lividans transformants carrying cpeB and the upstream-located furS gene with its regulatory region on the bifunctional vector pWHM3 produced low or enhanced levels of CpeB in the presence or absence of Fe ions, respectively. An in-frame deletion of the major part of furS induces increased CpeB synthesis. The data imply that FurS regulates the transcription of cpeB. The deduced FurS protein is rich in histidine residues, contains a putative N-terminally situated helix-turn-helix motif and has a molecular mass of 15.1 kDa. It shares only 29% amino acid identity with the Escherichia coli ferric uptake regulator (Fur) protein, but about 64% with FurA deduced from the genomic sequences of several mycobacteria. The predicted secondary structures of FurS and FurA are highly similar and considerably divergent from those of the E. coli Fur. In contrast to some Gram-negative bacteria, within several mycobacteria an intact furA gene or a furA pseudogene is upstream of a catalase-peroxidase (katG) gene predicted to encode a functional or a non-functional (Mycobacterium leprae) enzyme. Thus the data obtained for Streptomyces reticuli are expected to serve as an additional model to elucidate the regulation of mycobacterial catalase-peroxidase genes.


Assuntos
Catalase/genética , Regulação Bacteriana da Expressão Gênica , Genes Bacterianos , Streptomyces/genética , Fatores de Transcrição/metabolismo , Sequência de Aminoácidos , Proteínas de Bactérias/genética , Catalase/biossíntese , Catalase/classificação , Catalase/isolamento & purificação , Clonagem Molecular , Dimerização , Evolução Molecular , Regulação Enzimológica da Expressão Gênica , Dados de Sequência Molecular , Proteínas Recombinantes/biossíntese , Proteínas Repressoras/genética , Análise de Sequência de DNA , Homologia de Sequência de Aminoácidos , Streptomyces/enzimologia
3.
FEMS Microbiol Lett ; 146(1): 129-34, 1997 Jan 01.
Artigo em Inglês | MEDLINE | ID: mdl-8997717

RESUMO

A range of organisms known to contain F420 or to be relatives of mycobacteria were examined for F420-dependent glucose-6-phosphate dehydrogenase (FGD) and NADP-dependent glucose-6-phosphate dehydrogenase (NADP-G6PD) activities. All free-growing Mycobacterium species examined (including a virulent Mycobacterium tuberculosis strain) had FGD activities of 0.014-0.418 mumol min-1 mg protein-1, and NADP-G6PD activities of 0.013-0.636 mumol min-1 mg-1. Armadillo-grown Mycobacterium leprae had FGD activity of 0.008 mumol min-1 mg-1, but no detectable NADP-G6PD activity. Nocardia species also had FGD activity (0.088-0.154 mumol min-1 mg-1). Streptomyces and Corynebacterium species had no FGD, but had NADP-G6PD. Methanogenic Archaea had neither activity.


Assuntos
Glucosefosfato Desidrogenase/metabolismo , Mycobacterium/enzimologia , Nocardia/enzimologia , Riboflavina/análogos & derivados , Corynebacterium/enzimologia , Euryarchaeota/enzimologia , Cinética , Mycobacterium leprae/enzimologia , Mycobacterium tuberculosis/enzimologia , NAD/metabolismo , NADP/metabolismo , Riboflavina/metabolismo , Especificidade da Espécie , Streptomyces/enzimologia
4.
J Biol Chem ; 271(36): 21803-7, 1996 Sep 06.
Artigo em Inglês | MEDLINE | ID: mdl-8702978

RESUMO

The gene for transcription termination factor Rho was isolated from Streptomyces lividans ZX7. It encoded a 77-kDa polypeptide (Rho 77) with considerable homology to known Rho factors. An atypical hydrophilic region of 228 residues was found within the N-terminal RNA-binding domain. Only Rho from Micrococcus luteus and Mycobacterium leprae (closely related GC-rich Gram-positive bacteria) had an analogous sequence. Rho 77 was overexpressed in Escherichia coli and purified using an N-terminal hexahistidine-tag. Rho 77 displayed a broad RNA-dependent ATPase activity, with poly(C) RNA being no more than 4-fold more effective than poly(A). This contrasts with the ATPase activity of Rho from E. coli which is stimulated primarily by poly(C) RNA. Rho 77 was a general RNA-dependent NTPase, apparent Km values for NTPs were: GTP 0.13 mM, ATP 0.17 mM, UTP 1.1 mM, and CTP >2 mM. Rho 77 poly(C)-dependent ATPase activity was inhibited by heparin, unlike the E. coli Rho. The antibiotic bicyclomycin inhibited the in vitro RNA-dependent ATPase activity of Rho 77, did not inhibit growth of streptomycetes but delayed the development of aerial mycelia. N-terminal deletion analysis to express a truncated form of Rho (Rho 72, 72 kDa) indicated that the first 42 residues of Rho 77 were not essential for RNA-dependent NTPase activity and were not the targets of inhibition by heparin or bicyclomycin.


Assuntos
Adenosina Trifosfatases/metabolismo , Fator Rho/genética , Streptomyces/enzimologia , Sequência de Aminoácidos , Bacillus subtilis , Sequência de Bases , Mapeamento Cromossômico , Escherichia coli , Regulação Bacteriana da Expressão Gênica , Heparina/farmacologia , Cinética , Micrococcus luteus , Dados de Sequência Molecular , Peso Molecular , Mycobacterium leprae , Rifampina/farmacologia , Saccharopolyspora , Homologia de Sequência de Aminoácidos , Homologia de Sequência do Ácido Nucleico , Streptomyces/genética , Yersinia pestis
5.
J Bacteriol ; 168(1): 72-80, 1986 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-3020007

RESUMO

The ability of "Streptomyces lividans" to use the expression signals of genes from Mycobacterium bovis BCG was tested in vivo by using gene fusions. Random DNA fragments from M. bovis BCG were inserted into promoter-probe plasmids in Escherichia coli and in "S. lividans." Comparison with promoter activity detected with random DNA fragments from the respective hosts suggested that "S. lividans" efficiently utilizes a high proportion of mycobacterial promoters, whereas a smaller fraction are expressed, and expressed more weakly, in E. coli. M. bovis BCG DNA fragments were also inserted into the specially constructed translational fusion vector (pIJ688) in "S. lividans." pIJ688 contains the kanamycin phosphotransferase gene (neo) from transposon Tn5, truncated at its amino terminus, as the indicator. The results suggested that "S. lividans" uses M. bovis BCG translational signals almost as efficiently as its own signals. Moreover, several hybrid proteins with an M. bovis BCG-derived amino terminus seemed to be reasonably stable in "S. lividans." These experiments indicate that "S. lividans" may be a suitable host for the expression of Mycobacterium leprae and Mycobacterium tuberculosis genes from their own signals. This is a precondition for the expression of entire biosynthetic pathways, which could be valuable in the production of diagnostic and therapeutic agents. The vectors may also have wider applications for the analysis of gene expression in Streptomyces.


Assuntos
Clonagem Molecular , Genes Bacterianos , Mycobacterium bovis/genética , Regiões Promotoras Genéticas , Streptomyces/genética , Elementos de DNA Transponíveis , Resistência Microbiana a Medicamentos , Escherichia coli/genética , Canamicina/farmacologia , Canamicina Quinase , Fosfotransferases/biossíntese , Fosfotransferases/genética , Biossíntese de Proteínas , Streptomyces/efeitos dos fármacos , Streptomyces/enzimologia
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