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The Aspergillus parasiticus estA-Encoded Esterase Converts Versiconal Hemiacetal Acetate to Versiconal and Versiconol Acetate to Versiconol in Aflatoxin Biosynthesis. Perng-Kuang Chang, 2004.In aflatoxin biosynthesis, the pathway for the conversion of 1-hydroxyversicolorone to versiconal hemiacetal acetate (VHA) to versiconal (VHOH) is part of a metabolic grid . In the grid, the steps from VHA to VHOH and from versiconol acetate (VOAc) to versiconol (VOH) may be catalyzed by the same esterase . Several esterase activities are associated with the conversion of VHA to VHOH, but only one esterase gene (estA) is present in the complete aflatoxin gene cluster of Aspergillus parasiticus . We deleted the estA gene from A . parasiticus SRRC 2043, an O-methylsterigmatocystin (OMST)-accumulating strain . The estA-deleted mutants were pigmented and accumulated mainly VHA and versicolorin A (VA) . A small amount of VOAc and other downstream aflatoxin intermediates, including VHOH, versicolorin B, and OMST, also were accumulated . In contrast, a VA-accumulating mutant, NIAH-9, accumulated VA exclusively and neither VHA nor VOAc were produced . Addition of the esterase inhibitor dichlorvos (dimethyl 2,2-dichlorovinylphosphate) to the transformation recipient strain RHN1, an estA-deleted mutant, or NIAH-9 resulted in the accumulation of only VHA and VOAc . In in vitro enzyme assays, the levels of the esterase activities catalyzing the conversion of VHA to VHOH in the cell extracts of two estA-deleted mutants were decreased to approximately 10% of that seen with RHN1 . Similar decreases in the esterase activities catalyzing the conversion of VOAc to VOH were also obtained . Thus, the estA-encoded esterase catalyzes the conversion of both VHA to VHOH and VOAc to VOH during aflatoxin biosynthesis . Recognition of DNA by Fur: a Reinterpretation of the Fur Box Consensus Sequence. Noel Baichoo, 2002.Ferric uptake repressor (Fur) proteins regulate the expression of iron homeostasis genes in response to intracellular iron levels . In general, Fur proteins bind with high affinity to a 19-bp inverted repeat sequence known as the Fur box . An alignment of 19 operator sites recognized by Bacillus subtilis Fur revealed a different conserved 15-bp (7-1-7) inverted repeat present twice within this 19-bp consensus sequence . We demonstrated using electrophoretic mobility shift assays that this 7-1-7 inverted repeat comprises a minimal recognition site for high-affinity binding by Fur . The resulting revised consensus sequence is remarkably similar to a related 7-1-7 inverted repeat sequence recognized by PerR, a Fur paralog . Our analysis of the affinity and stoichiometry of DNA binding by B . subtilis Fur, together with a reinterpretation of previously described studies of Escherichia coli Fur, supports a model in which the 19-bp Fur box represents overlapping recognition sites for two Fur dimers bound to opposite faces of the DNA helix . The resulting recognition complex is reminiscent of that observed for the functionally related protein DtxR . Like Fur, DtxR contains a helix-turn-helix DNA-binding motif, recognizes a 19-bp inverted repeat sequence, and has a typical DNase I footprint of  30 bp . By envisioning a similar mode of DNA recognition for Fur, we can account for the internal symmetries noted previously within the Fur box, the tendency of Fur to extend into adjacent regions of DNA in a sequence-selective manner, and the observed patterns of DNA protection against enzymatic and chemical probes .
Characterization of TsaR, an Oxygen-Sensitive LysR-Type Regulator for the Degradation of p-Toluenesulfonate in Comamonas testosteroni T-2. Tewes Tralau, 2003.TsaR is the putative LysR-type regulator of the tsa operon (tsaMBCD) which encodes the first steps in the degradation of p-toluenesulfonate (TSA) in Comamonas testosteroni T-2 . Transposon mutagenesis was used to knock out tsaR . The resulting mutant lacked the ability to grow with TSA and p-toluenecarboxylate (TCA) . Reintroduction of tsaR in trans on an expression vector reconstituted growth with TSA and TCA . The tsaR gene was cloned into Escherichia coli with a C-terminal His tag and overexpressed as TsaRHis . TsaRHis was subject to reversible inactivation by oxygen, which markedly influenced the experimental approaches used . Gel filtration showed TsaRHis to be a monomer in solution . Overexpressed TsaRHis bound specifically to three regions within the promoter between the divergently transcribed tsaR and tsaMBCD . The dissociation constant (KD) for the whole promoter region was about 0.9 µM, and the interaction was a function of the concentration of the ligand TSA . A regulatory model for this LysR-type regulator is proposed on the basis of these data . Environmental Determinants of Vibrio cholerae Biofilm Development. Katharine Kierek, 2003.Vibrio cholerae is a versatile bacterium that flourishes in diverse environments, including the human intestine, rivers, lakes, estuaries, and the ocean . Surface attachment is believed to be essential for colonization of all of these natural environments . Previous studies have demonstrated that the vps genes, which encode proteins required for exopolysaccharide synthesis and transport, are required for V . cholerae biofilm development in Luria-Bertani broth . In this work, we showed that V . cholerae forms vps-dependent biofilms and vps-independent biofilms . The vps-dependent and -independent biofilms differ in their environmental activators and in architecture . Our results suggest that environmental activators of vps-dependent biofilm development are present in freshwater, while environmental activators of vps-independent biofilm development are present in seawater . The distinct environmental requirements for the two modes of biofilm development suggest that vps-dependent biofilm development and vps-independent biofilm development may play distinct roles in the natural environment .
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