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NpdR, a Repressor Involved in 2,4,6-Trinitrophenol Degradation in Rhodococcus opacus HL PM-1. Dang P. Nga, 2004.Rhodococcus opacus HL PM-1 utilizes 2,4,6-trinitrophenol (picric acid) as a sole nitrogen source . The initial attack on picric acid occurs through two hydrogenation reactions . Hydride transferase II (encoded by npdI) and hydride transferase I (encoded by npdC) are responsible for the hydride transfers . Database searches with the npd genes have indicated the presence of a putative transcriptional regulator, npdR . Here, the npdR gene was expressed in Escherichia coli, and the protein was purified and shown to form a complex with intergenic regions between open reading frames A and B and between npdH and npdI within the npd gene cluster . A change in DNA-NpdR complex formation occurred in the presence of 2,4-dinitrophenol, picric acid, 2-chloro-4,6-dinitrophenol, and 2-methyl-4,6-dinitrophenol . By constructing a promoter-probe vector, we demonstrated that both intergenic regions caused the expression of reporter gene xylE . Hence, both of these regions contain promoters . A deletion mutant of R . opacus HL PM-1 was constructed in which part of npdR was deleted . The expression of npdI and npdC was induced by 2,4-dinitrophenol in the wild-type strain, while in the mutant these genes were constitutively expressed . Hence, NpdR is a repressor involved in picric acid degradation . Transcription Regulation by Tandem-Bound FNR at Escherichia coli Promoters. Anne M. L. Barnard, 2003.FNR is an Escherichia coli transcription factor that regulates the transcription of many genes in response to anaerobiosis . We have constructed a series of artificial FNR-dependent promoters, based on the melR promoter, in which a consensus FNR binding site was centered at position -41.5 relative to the transcription start site . A second consensus FNR binding site was introduced at different upstream locations, and promoter activity was assayed in vivo . FNR can activate transcription from these promoters when the upstream FNR binding site is located at many different positions . However, sharp repression is observed when the upstream-bound FNR is located near positions -85 or -95 . This repression is relieved by the FNR G74C substitution mutant, previously identified as being defective in transcription repression at the yfiD promoter . A parallel series of artificial FNR-dependent promoters, carrying a consensus FNR binding site at position -61.5 and a second upstream DNA site for FNR, was also constructed . Again, promoter activity was repressed by FNR when the upstream-bound FNR was located at particular positions . Identification of Equine Cecal Bacteria Producing Amines in an In Vitro Model of Carbohydrate Overload. S. R. Bailey, 2003.Acute laminitis has been associated with the overgrowth of gram-positive bacteria within the equine hindgut, causing the release of factor(s) leading to ischemia-reperfusion of the digits . The products of fermentation which trigger acute laminitis are, as yet, unknown; however, vasoactive amines are possible candidates . The objectives of this study were to use an in vitro model of carbohydrate overload to study the change in populations of cecal streptococci and lactobacilli and to establish whether certain species of these bacteria were capable of producing vasoactive amines from amino acids . Cecal contents from 10 horses were divided into aliquots and incubated anaerobically with either corn starch or inulin (fructan; both at 1 g/100 ml) . Samples were taken at 6-h intervals over a 24-h period for enumeration of streptococci, lactobacilli, and gram-negative anaerobes by a dilution method onto standard selective growth media . The effects of the antibiotic virginiamycin (1 mg/100 ml) and calcium hydrogen phosphate (CaHPO4; 0.3 g/100 ml) were also examined . Fermentation of excess carbohydrate was associated with increases in numbers of streptococci and lactobacilli (2- to 3.5-log unit increases; inhibited by virginiamycin) but numbers of gram-negative anaerobes were not significantly affected . A screening agar technique followed by 16S rRNA gene sequence analysis enabled the identification of 26 different bacterial strains capable of producing one or more vasoactive amines . These included members of the species Streptococcus bovis and five different Lactobacillus spp . These data suggest that certain bacteria, whose overgrowth is associated with carbohydrate fermentation, are capable of producing vasoactive amines which may play a role in the pathogenesis of acute laminitis .
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