RESUMO
We investigated in Serratia marcescens the functions of the flhDC operon, which controls motility and cell division in enteric bacteria. Included in our evaluations were investigation of cell division, flagellar synthesis and regulation of the expression of nuclease (encoded by the nucA(Sm) gene, one of the virulence factors). Interruption of the chromosomal flhDC operon in S. marcescens CH-1 resulted in aberrant cell division and loss of nuclease and flagella. Expression of nucA(Sm) and other mutated phenotypes was restored in the flhDC mutant by the induction of overexpression of flhDC in a multicopy plasmid. Multicopied flhDC also induced the formation of differentiated cells (polyploid aseptate cells with oversynthesis of peritrichous flagella) in broth culture using minimal growth medium. Expression of the flhDC operon showed positive autoregulation, and was growth phase dependent (upregulated in early log phase). In addition, flhDC expression was inhibited when the temperature increased from 30 to 37 degrees C, and when osmolarity was increased, but was not influenced by glucose catabolite repression. These results show that FlhD/FlhC is a multifunctional transcriptional activator involved in the process of cell differentiation, swarming and virulence factor expression.
Assuntos
Proteínas de Bactérias/genética , Proteínas de Ligação a DNA/genética , Desoxirribonucleases/genética , Serratia marcescens/enzimologia , Serratia marcescens/genética , Transativadores/genética , Sequência de Aminoácidos , Sequência de Bases , Divisão Celular , Movimento Celular , Primers do DNA/genética , DNA Bacteriano/genética , Proteínas de Escherichia coli , Flagelos/metabolismo , Expressão Gênica , Genes Bacterianos , Dados de Sequência Molecular , Mutação , Óperon , Recombinação Genética , Serratia marcescens/crescimento & desenvolvimentoRESUMO
A fluorescein labelled antibody (Ab)/Fluorescence Activated Cell Sorter (FACS)-based assay was developed for detection of a wide range of mycobacterial species directly from bacterial culture and sputum specimens. The whole process could be completed within 3 hours and had a high specificity and sensitivity for cultured bacteria. The method was also shown to be applicable for direct identification from clinical specimens. This study showed that pretesting of clinical specimens for mycobacteria to the genus level with an antibody to Mycobacterium species offers the routine clinical laboratory a single convenient test for the detection of tuberculous and nontuberculous mycobacteria. Depending on the availability of species-specific antibody, the identification of Mycobacterium to the species level can be achieved.
Assuntos
Técnicas de Tipagem Bacteriana , Mycobacterium/classificação , Anticorpos Antibacterianos , Citometria de Fluxo , Mycobacterium/imunologiaRESUMO
Swarming motility is a multicellular phenomenon comprising population migration across surfaces by specially differentiated cells. In Serratia marcescens, a network exists in which the flhDC flagellar regulatory master operon, temperature, nutrient status, and quorum sensing all contribute to the regulation of swarming motility. In this study, the rsmA (repressor of secondary metabolites) gene (hereafter rsmA(Sm)) was cloned from S. marcescens. The presence of multicopy, plasmid-encoded rsmA(Sm) expressed from its native promoter in S. marcescens inhibits swarming. Synthesis of N-acylhomoserine lactones, presumably by the product of smaI (a luxI homolog isolated from S. marcescens), was also inhibited. Knockout of rsmA(Sm) on the S. marcescens chromosome shortens the time before swarming motility begins after inoculation to an agar surface. A single copy of the chromosomal PrsmA(Sm)::luxAB reporter of rsmA(Sm) transcription was constructed. Using this reporter, the roles of the flhDC flagellar regulatory master operon, temperature and autoregulation in the control of rsmA(Sm) expression were determined. Our findings indicate that RsmA(Sm) is a component of the complex regulatory network that controls swarming.