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Loss of Catabolite Repression Function of HPr, the Phosphocarrier Protein of the Bacterial Phosphotransferase System, Affects Expression of the cry4A Toxin Gene in Bacillus thuringiensis subsp . israelensis. Sharik R. Khan, 2002.HPr, the phosphocarrier protein of the bacterial phosphotransferase system, mediates catabolite repression of a number of operons in gram-positive bacteria . In order to participate in the regulatory process, HPr is activated by phosphorylation of a conserved serine-46 residue . To study the potential role of HPr in the regulation of Cry4A protoxin synthesis in Bacillus thuringiensis subsp . israelensis, we produced a catabolite repression-negative mutant by replacing the wild-type copy of the ptsH gene with a mutated copy in which the conserved serine residue of HPr was replaced with an alanine . HPr isolated from the mutant strain was not phosphorylated at Ser-45 by HPr kinase, but phosphorylation at His-14 was found to occur normally . The enzyme I and HPr kinase activities of the mutant were not affected . Analysis of the B . thuringiensis subsp . israelensis mutant harboring ptsH-S45A in the chromosome showed that cry4A expression was derepressed from the inhibitory effect of glucose . The mutant strain produced both cry4A and  35 gene transcripts 4 h ahead of the parent strain, but there was no effect on
28 synthesis . In wild-type B . thuringiensis subsp . israelensis cells, cry4A mRNA was observed from 12 h onwards, while in the mutant it appeared at 8 h and was produced for a longer period . The total amount of cry4A transcripts produced by the mutant was higher than by the parent strain . There was a 60 to 70% reduction in the sporulation efficiency of the mutant B . thuringiensis subsp . israelensis strain compared to the wild-type strain .
Analyses of the Roles of the Three cheA Homologs in Chemotaxis of Vibrio cholerae. Khoosheh K. Gosink, 2002.The Vibrio cholerae genome revealed the presence of multiple sets of chemotaxis genes, including three cheA gene homologs . We found that the cheA-2, but not cheA-1 or cheA-3, gene is essential for chemotaxis under standard conditions . Loss of chemotaxis had no effect on virulence factor expression in vitro . Prokaryotic Homologs of the Eukaryotic 3-Hydroxyanthranilate 3,4-Dioxygenase and 2-Amino-3-Carboxymuconate-6-Semialdehyde Decarboxylase in the 2-Nitrobenzoate Degradation Pathway of Pseudomonas fluorescens Strain KU-7. Takamichi Muraki, 2003.The 2-nitrobenzoic acid degradation pathway of Pseudomonas fluorescens strain KU-7 proceeds via a novel 3-hydroxyanthranilate intermediate . In this study, we cloned and sequenced a 19-kb DNA locus of strain KU-7 that encompasses the 3-hydroxyanthranilate meta-cleavage pathway genes . The gene cluster, designated nbaEXHJIGFCDR, is organized tightly and in the same direction . The nbaC and nbaD gene products were found to be novel homologs of the eukaryotic 3-hydroxyanthranilate 3,4-dioxygenase and 2-amino-3-carboxymuconate-6-semialdehyde decarboxylase, respectively . The NbaC enzyme carries out the oxidation of 3-hydroxyanthranilate to 2-amino-3-carboxymuconate-6-semialdehyde, while the NbaD enzyme catalyzes the decarboxylation of the latter compound to 2-aminomuconate-6-semialdehyde . The NbaC and NbaD proteins were overexpressed in Escherichia coli and characterized . The substrate specificity of the 23.8-kDa NbaC protein was found to be restricted to 3-hydroxyanthranilate . In E . coli, this enzyme oxidizes 3-hydroxyanthranilate with a specific activity of 8 U/mg of protein . Site-directed mutagenesis experiments revealed the essential role of two conserved histidine residues (His52 and His96) in the NbaC sequence . The NbaC activity is also dependent on the presence of Fe2+ but is inhibited by other metal ions, such as Zn2+, Cu2+, and Cd2+ . The NbaD protein was overproduced as a 38.7-kDa protein, and its specific activity towards 2-amino-3-carboxymuconate-6-semialdehyde was 195 U/mg of protein . Further processing of 2-aminomuconate-6-semialdehyde to pyruvic acid and acetyl coenzyme A was predicted to proceed via the activities of NbaE, NbaF, NbaG, NbaH, NbaI, and NbaJ . The predicted amino acid sequences of these proteins are highly homologous to those of the corresponding proteins involved in the metabolism of 2-aminophenol (e.g., AmnCDEFGH in Pseudomonas sp . strain AP-3) . The NbaR-encoding gene is predicted to have a regulatory function of the LysR family type . The function of the product of the small open reading frame, NbaX, like the homologous sequences in the nitrobenzene or 2-aminophenol metabolic pathway, remains elusive .
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