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Cloning and Characterization of Acetohydroxyacid Synthase from Bacillus stearothermophilus. Iris Porat, 2004.Five genes from the ilv-leu operon from Bacillus stearothermophilus have been sequenced . Acetohydroxyacid synthase (AHAS) and its subunits were separately cloned, purified, and characterized . This thermophilic enzyme resembles AHAS III of Escherichia coli, and regulatory subunits of AHAS III complement the catalytic subunit of the AHAS of B . stearothermophilus, suggesting that AHAS III is functionally and evolutionally related to the single AHAS of gram-positive bacteria . The ATP-Dependent Lon Protease of Salmonella enterica Serovar Typhimurium Regulates Invasion and Expression of Genes Carried on Salmonella Pathogenicity Island 1. Akiko Takaya, 2002.An early step in the pathogenesis of Salmonella enterica serovar Typhimurium infection is bacterial penetration of the intestinal epithelium . Penetration requires the expression of invasion genes found in Salmonella pathogenicity island 1 (SPI1) . These genes are controlled in a complex manner by regulators in SPI1, including HilA and InvF, and those outside SPI1, such as two-component regulatory systems and small DNA-binding proteins . We report here that the expression of invasion genes and the invasive phenotype of S . enterica serovar Typhimurium are negatively regulated by the ATP-dependent Lon protease, which is known to be a major contributor to proteolysis in Escherichia coli . A disrupted mutant of lon was able to efficiently invade cultured epithelial cells and showed increased production and secretion of three identified SPI1 proteins, SipA, SipC, and SipD . The lon mutant also showed a dramatic enhancement in transcription of the SPI1 genes hilA, invF, sipA, and sipC . The increases ranged from 10-fold to almost 40-fold . It is well known that the expression of SPI1 genes is also regulated in response to several environmental conditions . We found that the disruption of lon does not abolish the repression of hilA and sipC expression by high-oxygen or low-osmolarity conditions, suggesting that Lon represses SPI1 gene expression by a regulatory pathway independent of these environmental signals . Since HilA is thought to function as a central regulator of SPI1 gene expression, it is speculated that Lon may regulate SPI1 gene expression by proteolysis of putative factors required for activation of hilA expression . Effect of Different Concentrations of H-NS Protein on Chromosome Replication and the Cell Cycle in Escherichia coli. T. Atlung, 2002.Flow cytometric analysis showed that the hns205 and hns206 mutants, lacking the abundant nucleoid-associated protein H-NS, have decreased origin concentration, as well as a low number of origins per cell (ploidy) . The most striking observation was that the low ploidy was due to a very short replication time, e.g., at 30°C it was halved compared to that of the hns+ strain . The decreased origin concentration was not caused by a decreased dnaA gene expression, and the hns206 mutant had normal DnaA protein concentrations . The replication phenotypes of the hns206 mutant were independent of RpoS . Cells overproducing H-NS from a LacI-controlled plasmid had a normal origin concentration, indicating that H-NS is not controlling initiation . A wild-type H-NS concentration is, however, required to obtain a wild-type origin concentration, since cells with an intermediate H-NS concentration had an intermediate origin concentration . Two lines of evidence point to an indirect effect of H-NS on initiation . First, H-NS did not show high-affinity binding to any part of oriC, and H-NS had no effect on transcription entering oriC from the mioC promoter . Second, in a shift experiment with the hns206 mutant, when H-NS protein was induced to wild-type levels within 10 min, it took more than one generation before the origin concentration started to increase . Biocontrol of Listeria monocytogenes on Fresh-Cut Produce by Treatment with Lytic Bacteriophages and a Bacteriocin. Britta Leverentz, 2003.The fresh-cut produce industry has been the fastest-growing portion of the food retail market during the past 10 years, providing consumers with convenient and nutritious food . However, fresh-cut fruits and vegetables raise food safety concerns, because exposed tissue may be colonized more easily by pathogenic bacteria than intact produce . This is due to the higher availability of nutrients on cut surfaces and the greater potential for contamination because of the increased amount of handling . We found that applied Listeria monocytogenes populations survived and increased only slightly on fresh-cut Red Delicious apples stored at 10°C but increased significantly on fresh-cut honeydew melons stored at 10°C over 7 days . In addition, we examined the effect of lytic, L . monocytogenes-specific phages via two phage application methods, spraying and pipetting, on L . monocytogenes populations in artificially contaminated fresh-cut melons and apples . The phage mixture reduced L . monocytogenes populations by 2.0 to 4.6 log units over the control on honeydew melons . On apples, the reduction was below 0.4 log units . In combination with nisin (a bacteriocin), the phage mixture reduced L . monocytogenes populations by up to 5.7 log units on honeydew melon slices and by up to 2.3 log units on apple slices compared to the control . Nisin alone reduced L . monocytogenes populations by up to 3.2 log units on honeydew melon slices and by up to 2.0 log units on apple slices compared to the control . The phage titer was stable on melon slices, but declined rapidly on apple slices . The spray application of the phage and phage plus nisin reduced the bacterial numbers at least as much as the pipette application . The effectiveness of the phage treatment also depended on the initial concentration of L . monocytogenes .
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