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Protein Sequences and Cellular Factors Required for Polar Localization of a Histidine Kinase in Caulobacter crescentus. Stephen A. Sciochetti, 2002.The Caulobacter crescentus sensor kinase DivJ is required for an early cell division step and localizes at the base of the newly formed stalk during the G1-to-S-phase transition when the protein is synthesized . To identify sequences within DivJ that are required for polar localization, we examined the ability of mutagenized DivJ sequences to direct localization of the green fluorescent protein . The effects of overlapping C-terminal deletions of DivJ established that the N-terminal 326 residues, which do not contain the kinase catalytic domain, are sufficient for polar localization of the fusion protein . Internal deletions mapped a shorter sequence between residues 251 and 312 of the cytoplasmic linker that are required for efficient localization of this sensor kinase . PleC kinase mutants, which are blocked in the swarmer-to-stalked-cell transition and form flagellated, nonmotile cells, also fail to localize DivJ . To dissect the cellular factors involved in establishing subcellular polarity, we have examined DivJ localization in a pleC mutant suppressed by the sokA301 allele of ctrA and in a pleD mutant, both of which display a supermotile, stalkless phenotype . The observation that these Mot+ strains localize DivJ to a single cell pole indicate that localization may be closely coupled to the gain of motility and that normal stalk formation is not required . We have also observed, however, that filamentous parC mutant cells, which are defective in DNA segregation and the completion of cell separation, are motile and still fail to localize DivJ to the new cell pole . These results suggest that formation of new sites for DivJ localization depends on events associated with the completion of cell separation as well as the gain of motility . Analysis of PleC and PleD mutants also provides insights into the function of the His-Asp proteins in cell cycle regulation . Thus, the ability of the sokA301 allele of ctrA to bypass the nonmotile phenotype of the pleC null mutation provides evidence that the PleC kinase controls cell motility by initiating a signal transduction pathway regulating activity of the global response regulator CtrA . Analysis of the pleD mutant cell cycle demonstrates that disruption of the swarmer-to-stalked-cell developmental sequence does not affect the asymmetric organization of the Caulobacter cell cycle . Targeted Gene Disruption by Homologous Recombination in the Hyperthermophilic Archaeon Thermococcus kodakaraensis KOD1. Takaaki Sato, 2003.In contrast to the high accumulation in sequence data for hyperthermophilic archaea, methodology for genetically manipulating these strains is still at an early stage . This study aimed to develop a gene disruption system for the hyperthermophilic euryarchaeon Thermococcus kodakaraensis KOD1 . Uracil-auxotrophic mutants with mutations in the orotidine-5'-monophosphate decarboxylase gene (pyrF) were isolated by positive selection using 5-fluoroorotic acid (5-FOA) and used as hosts for further transformation experiments . We then attempted targeted disruption of the trpE locus in the host strain by homologous recombination, as disruption of trpE was expected to result in tryptophan auxotrophy, an easily detectable phenotype . A disruption vector harboring the pyrF marker within trpE was constructed for double-crossover recombination . The host cells were transformed with the exogenous DNA using the CaCl2 method, and several transformants could be selected based on genetic complementation . Genotypic and phenotypic analyses of a transformant revealed the unique occurrence of targeted disruption, as well as a phenotypic change of auxotrophy from uracil to tryptophan caused by integration of the wild-type pyrF into the host chromosome at trpE . As with the circular plasmid, gene disruption with linear DNA was also possible when the homologous regions were relatively long . Shortening these regions led to predominant recombination between the pyrF marker in the exogenous DNA and the mutated allele on the host chromosome . In contrast, we could not obtain trpE disruptants by insertional inactivation using a vector designed for single-crossover recombination . The gene targeting system developed in this study provides a long-needed tool in the research on hyperthermophilic archaea and will open the way to a systematic, genetic approach for the elucidation of unknown gene function in these organisms . A Transcriptional Pause Synchronizes Translation with Transcription in the Tryptophanase Operon Leader Region. Feng Gong, 2003.Regulation of transcription of the tryptophanase operon requires that translation of its leader peptide coding region, tnaC, be coupled with its transcription . We show in vitro that a transcription pause site exists at the end of the tnaC coding region and that translation of tnaC releases the paused transcription complex, coupling transcription with translation . Characterization of Poly-  -Glutamate Hydrolase Encoded by a Bacteriophage Genome: Possible Role in Phage Infection of Bacillus subtilis Encapsulated with Poly--Glutamate.Keitarou Kimura, 2003.Some Bacillus subtilis strains, including natto (fermented soybeans) starter strains, produce a capsular polypeptide of glutamate with a -linkage, called poly--glutamate (-PGA) . We identified and purified a monomeric 25-kDa degradation enzyme for
-PGA (designated
-PGA hydrolase, PghP) from bacteriophage
 NIT1 in B . subtilis host cells . The monomeric PghP internally hydrolyzed
-PGA to oligopeptides, which were then specifically converted to tri-, tetra-, and penta--glutamates . Monoiodoacetate and EDTA both inhibited the PghP activity, but Zn2+ or Mn2+ ions fully restored the enzyme activity inhibited by the chelator, suggesting that a cysteine residue(s) and these metal ions participate in the catalytic mechanism of the enzyme . The corresponding pghP gene was cloned and sequenced from the phage genome . The deduced PghP sequence (208 amino acids) with a calculated Mr of 22,939 was not significantly similar to any known enzyme . Thus, PghP is a novel
-glutamyl hydrolase . Whereas phage
NIT1 proliferated in B . subtilis cells encapsulated with
-PGA, phage BS5 lacking PghP did not survive well on such cells . Moreover, all nine phages that contaminated natto during fermentation produced PghP, supporting the notion that PghP is important in the infection of natto starters that produce
-PGA . Analogous to polysaccharide capsules,
-PGA appears to serve as a physical barrier to phage absorption . Phages break down the
-PGA barrier via PghP so that phage progenies can easily establish infection in encapsulated cells .
Diversity of Bacillus thuringiensis Strains from Latin America with Insecticidal Activity against Different Mosquito Species. Jorge E. Ibarra, 2003.The characterization of selected Bacillus thuringiensis strains isolated from different Latin America countries is presented . Characterization was based on their insecticidal activity against Aedes aegypti, Culex quinquefasciatus, and Anopheles albimanus larvae, scanning electron microscopy, sodium dodecyl sulfate-polyacrylamide gel electrophoresis, and plasmid profiles as well as PCR analysis using novel general and specific primers for cry and cyt genes encoding proteins active against mosquitoes (cyt1, cyt2, cry2, cry4A, cry4B, cry10, cry11, cry17, cry19, cry24, cry25, cry27, cry29, cry30, cry32, cry39, and cry40) . Strains LBIT315, LBIT348, and IB604 showed threefold higher mosquitocidal activity against A . aegypti and C . quinquefasciatus larvae than B . thuringiensis subsp . israelensis and displayed high similarities with the B . thuringiensis subsp . israelensis used in this study with regard to protein and plasmid profiles and the presence of cry genes . Strain 147-8906 has activity against A . aegypti similar to that of B . thuringiensis subsp . israelensis but has different protein and plasmid profiles . This strain, harboring cry11, cry30, cyt1, and cyt2 genes, could be relevant for future resistance management interventions . Finally, the PCR screening strategy presented here led us to identify a putative novel cry11B gene .
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