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ABSTRACT: Unusually among bacteria, actinobacteria possess myo-inositol 1-phosphate synthase (mIPS). In the developmentally complex Streptomyces coelicolor, the mIPS-encoding gene (inoA) is cotranscribed with a putative regulatory gene (inoR). The inoRA transcript was more abundant in an inoR in-frame deletion mutant, and InoR formed different complexes in vitro with an extensive region around the inoRA promoter. Binding was relieved by adding glucose 6-phosphate. Thus, InoR is a metabolite-sensitive autorepressor that influences inoA expression, and hence the level of inositol, by controlling transcription from P(inoRA) . Disruption of inoA resulted in inositol-dependent growth and development, with full phenotypic correction at 0.1 mM inositol: at lower inositol concentrations differentiation was arrested at intermediate stages. This pattern may partly reflect increased demand for membrane phospholipids during sporulation septation. A corresponding sharp upregulation of inoRA transcription coincident with sporulation was dependent on a developmental regulator, WhiI. A truncated form of WhiI could bind two sites downstream of P(inoRA) , and one of the WhiI-binding sites overlapped the InoR-binding site. The combined action of a metabolic regulator and a developmental regulator at the simple P(inoRA) promoter is a previously undescribed strategy for the differential provision of developmentally appropriate levels of a substance required during the formation of spore chains.
Molecular Microbiology 02/2012; 83(6):1178-94. · 5.01 Impact Factor
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ABSTRACT: SCO3900, a putative target gene of the developmental regulator WhiI of Streptomyces coelicolor, encodes a protein similar to PadR from Pediococcus pentosaceus. The SCO3900 disruption mutant exhibited a bald phenotype with sparse aerial hyphae. However, a single copy of SCO3900 under the control of its native promoter could not complement the mutant phenotype. A fragment containing SCO3900 and its downstream three genes completely restored the mutant phenotype to the wild-type. These suggested that the disruption of SCO3900 exerted a polar effect on its downstream genes. The co-transcription of SCO3899, SCO3898, and SCO3897 with SCO3900 was confirmed by the RT-PCR. Moreover, overexpression of SCO3900 in the wild-type strain caused the same phenotype as that of the disruption mutant. The results suggested that SCO3900 encodes a negative regulator participating in morphological differentiation of S. coelicolor by influencing the expression of its downstream genes.
Current Microbiology 12/2009; 60(4):268-73. · 1.82 Impact Factor
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ABSTRACT: Spore germination of Bacillus could be triggered by germinants like L-alanine which is thought to bind to and stimulate specific receptors. The GerA receptor responds to L-alanine in Bacillus subtilis. A homologous gerA operon of B. subtilis was isolated from Bacillus thuringiensis subsp. kurstaki. RT-PCR showed that the transcription of the gerA operon was switched on 3 hours after the initiation of sporulation and this operon was cotranscribed. Disruption of the gerA operon led to blockage of the L-alanine-initiated germination pathway and showed a delayed inosine-induced germination response. The germination rate of the gerA complementary strain spore deriving from introducing gerA operon into the disruption mutant was even faster than that of the wild type strain spore. This is caused by overdose GerA receptors which could enhance the sensibility of complementary spore to L-alanine. The overdose GerA receptors came from introducing multicopy plasmid- pKSV7 (about five copies) which could increase the amount of GerA receptors. After treatment of D-cycloserine which could inactivate alanine racemase irreversibly, L-alanine-induced spore could germinate faster than untreated spore. The combination of 0.1 mmol/L D-cycloserine and 1 mmol/L L-alanine could trigger spore germination induced by L-alanine effectively.
ACTA MICROBIOLOGICA SINICA 04/2008; 48(3):281-6.
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ABSTRACT: Spore cortex-lytic enzymes are essential for germination in Bacilli. A gene-encoding spore cortex-lytic enzyme designated sleB was cloned from Bacillus thuringiensis. Disruption of sleB did not affect vegetative growth of B. thuringiensis, but the fall in optical density at 600 nm in the mutant spores was much slower than in the wild type strain during spore germination induced by L-alanine. Moreover, the mutant spores did not become completely dark, as compared with the wild type strain. These showed that sleB is required for normal spore germination in B. thuringiensis. Reverse transcription polymerase chain reaction analysis indicated that sleB is transcribed during sporulation. Western blot experiment also proved that SleB accumulated in sporulating cells as a precursor protein, and in spores as a mature processed form.
Current Microbiology 05/2007; 54(4):292-5. · 1.82 Impact Factor
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ABSTRACT: One polysaccharide deacetylase gene was cloned from Bacillus thuringiensis and designated pdaA. Disruption of pdaA did not affect vegetative growth and sporulation but obviously affected spore germination. When L-alanine was added into the spore suspension, the spores of the pdaA disruption mutant showed a slow and partial reduction in absorbance at OD600 and became phase pale gray compared with phase dark of the wild-type strain. In contrast with the outgrowing of wild-type spores after germination, the pdaA mutant spores were blocked at the stage of spore germination. Transmission electron micrographs revealed a significant difference between the pdaA mutant and the wild-type strain in the spore cortex. Introduction of the pdaA gene into the pdaA disruption mutant complemented the germination-negative phenotype. Reverse transcription--polymerase chain reaction showed that pdaA was transcribed after incubation for 10 h in CCY medium.
Canadian Journal of Microbiology 11/2006; 52(10):935-41. · 1.36 Impact Factor
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ABSTRACT: An in vivo expression technology (IVET) was applied to screen S. flexneri 2a genes induced after invasion of epithelial cells, and virulence-related genes were further identified by mutational analysis. Thirteen intracellular induced genes were identified with a HeLa cell infection model. Of these, two were identified as alkylation-related genes; one was related to metabolism; one encoded a transcriptional regulator; three were identified as insertion elements; three appeared to be antisense to genes involved in the transmethylation, biosynthesis, and phosphotransferase system; and three were predicted to encode polypeptides with unknown functions. Intracellular survival assays showed that the mutants of alkA, citC and wcaJ genes had lower capability of intracellular replication or survival than the wild-type strain. The results indicated that alkA, citC and wcaJ genes could take part in the intracellular survival or replication of S. flexneri 2a and the capability of intracellular survival or replication could be one of the major virulence elements. However, the yaiC mutant was able to survive in the murine infection assay but almost not in HeLa cell infection assay. Very possibly, yaiC gene was involved in the other mechanism of S. flexneri virulence. This study might lead to a better understanding of the intracellular survival or proliferation process of S. flexneri 2a and perhaps provide insights into the pathogenicity of this pathogen.
Science in China Series C Life Sciences 01/2005; 47(6):494-502. · 1.61 Impact Factor
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ABSTRACT: The technology of gene knockout using lambda Red system in bacteria has been developed lately, but its application being limited to Esherichia coli. In order to get some experiences in other bacteria we select four genes, including alkA, wcaJ, yphF and dam, to test gene knockout using Red system in E. coli and Shigella. As a result, three genes were successfully knocked out in E. coli except dam while only alkA gene was knocked out in Shigella. It showed that the Red system should be improved if it was to be effectively used in Shigella and other bacteria.
ACTA MICROBIOLOGICA SINICA 01/2004; 43(6):740-6.
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ABSTRACT: In vivo induced genes are thought to play an important role during infection of host. AlkA was identified as an in vivo-induced gene by in vivo expression technology (IVET), but its virulence in Shigella flexneri was not reported. The purpose of this study was to identify the role of alkA gene in the pathogenesis of S. flexneri.
PCR was used to amplify alkA gene of S. flexneri 2a and fragment 028pKm. The fragment was then transformed into 2457T05 strain, a S flexneri 2a strain containing Red recombination system, which was constructed with a recombinant suicide plasmid pXLkd46. By in vivo homologous recombination, alkA mutants were obtained and verified by PCR and sequencing. Intracellular survival assay and virulence assay were used to test the intracellular survival ability in HeLa cell model and the virulence in mice lung infection model respectively.
Deletion mutant of S. flexneri 2a alkA was successfully constructed by gamma Red recombination system. The mutant exhibited significant survival defects and much significant virulence defects in mice infection assay.
AlkA gene plays an important role in the infection of epithelial cells and is a virulent gene of Shigella spp.
World Journal of Gastroenterology 01/2004; 9(12):2720-5. · 2.47 Impact Factor
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ABSTRACT: Since many DNA-sequencing projects of varied microorganisms have been completed,studies on their functional genomics become more important. Inactivation of an interesting gene is a direct method to characterize its function. Though the Escherichia coli RecA recombination system can be used to produce gene mutants,it needs a complex manipulation process. Furthermore, its efficiency is very low. Recently a Red recombination system was developed. This recombination system consists of three proteins:alpha protein (gamma exonuclease), beta protein and Gam protein. In this system, the linear targeting DNA which contains a selectable marker flanked with a homologous region as short as only 35 approximately 60 bp can be directly targeted for gene knock-out with a higher efficiency.
Hereditas (Beijing) 10/2003; 25(5):628-32.
