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Phenylphosphate Carboxylase: a New C-C Lyase Involved in Anaerobic Phenol Metabolism in Thauera aromatica. Karola Schühle, 2004.The anaerobic metabolism of phenol in the beta-proteobacterium Thauera aromatica proceeds via carboxylation to 4-hydroxybenzoate and is initiated by the ATP-dependent conversion of phenol to phenylphosphate . The subsequent para carboxylation of phenylphosphate to 4-hydroxybenzoate is catalyzed by phenylphosphate carboxylase, which was purified and studied . This enzyme consists of four proteins with molecular masses of 54, 53, 18, and 10 kDa, whose genes are located adjacent to each other in the phenol gene cluster which codes for phenol-induced proteins . Three of the subunits (54, 53, and 10 kDa) were sufficient to catalyze the exchange of 14CO2 and the carboxyl group of 4-hydroxybenzoate but not phenylphosphate carboxylation . Phenylphosphate carboxylation was restored when the 18-kDa subunit was added . The following reaction model is proposed . The 14CO2 exchange reaction catalyzed by the three subunits of the core enzyme requires the fully reversible release of CO2 from 4-hydroxybenzoate with formation of a tightly enzyme-bound phenolate intermediate . Carboxylation of phenylphosphate requires in addition the 18-kDa subunit, which is thought to form the same enzyme-bound energized phenolate intermediate from phenylphosphate with virtually irreversible release of phosphate . The 54- and 53-kDa subunits show similarity to UbiD of Escherichia coli, which catalyzes the decarboxylation of a 4-hydroxybenzoate derivative in ubiquinone (ubi) biosynthesis . They also show similarity to components of various decarboxylases acting on aromatic carboxylic acids, such as 4-hydroxybenzoate or vanillate, whereas the 10-kDa subunit is unique . The 18-kDa subunit belongs to a hydratase/phosphatase protein family . Phenylphosphate carboxylase is a member of a new family of carboxylases/decarboxylases that act on phenolic compounds, use CO2 as a substrate, do not contain biotin or thiamine diphosphate, require K+ and a divalent metal cation (Mg2+or Mn2+) for activity, and are strongly inhibited by oxygen . The Gene yjfQ Encodes the Repressor of the yjfR-X Regulon (ula), Which Is Involved in L-Ascorbate Metabolism in Escherichia coli. Evangelina Campos, 2002.Mutations in yjfQ allowed us to identify this gene as the regulator of the operon yjfS-X (ula operon), reported to be involved in L-ascorbate metabolism . Inactivation of this gene renders constitutive the expression of the ula operon, indicating that YjfQ acts as a repressor . We also demonstrate that this repressor regulates the nearby yjfR gene, which in this way constitutes a regulon with the ula operon . Negative Osmoregulation of the Salmonella ompS1 Porin Gene Independently of OmpR in an hns Background. Mario Alberto Flores-Valdez, 2003.The ompS1 gene encodes a quiescent porin in Salmonella enterica serovars Typhi and Typhimurium . By using random mariner transposon mutagenesis, mutations that caused derepression of ompS1 expression were isolated, one in S . enterica serovar Typhi and two in S . enterica serovar Typhimurium . All of them mapped in the hns gene in the region coding for the carboxy terminus of the H-NS nucleoid protein . The derepressed ompS1 expression was subject to negative regulation at high osmolarity, both in the presence and in the absence of OmpR . This observation was possible due to the fact that there are two promoters: P1, which is OmpR dependent, and P2, which does not require OmpR for activation (rather, OmpR represses P2) . The sequences upstream from position -88, a region previously shown to be involved in the negative regulation of ompS1, can form a static bend, and the integrity of this region was required for function and binding of H-NS and for osmoregulation, as determined with gene reporter fusions of different lengths and with a 31-bp deletion mutant . This is consistent with the notion that this region determines a structure required for repression . Hence, ompS1 shares negative regulation by H-NS with other loci, such as the bgl operon and the ade gene . Population Genetics of the Nomenspecies Enterobacter cloacae. Harald Hoffmann, 2003.The genetic heterogeneity of the nomenspecies Enterobacter cloacae is well known . Enterobacter asburiae, Enterobacter cancerogenus, Enterobacter dissolvens, Enterobacter hormaechei, Enterobacter kobei, and Enterobacter nimipressuralis are closely related to it and are subsumed in the so-called E . cloacae complex . DNA-DNA hybridization studies performed previously identified at least five DNA-relatedness groups of this complex . In order to analyze the genetic structure and the phylogenetic relationships between the clusters of the nomenspecies E . cloacae, 206 strains collected from 22 hospitals, a veterinarian, and an agricultural center in 11 countries plus all 13 type strains of the genus and reference strain CDC 1347-71R were examined with a combination of sequence and PCR-restriction fragment length polymorphism (PCR-RFLP) analyses of the three housekeeping genes hsp60, rpoB, and hemB as well as ampC, the gene of a class C ß-lactamase . Based on the neighbor-joining tree of the hsp60 sequences, 12 genetic clusters (I to XII) and an unstable sequence crowd (xiii) were identified . The robustness of the genetic clusters was confirmed by analyses of rpoB and hemB sequences and ampC PCR-RFLPs . Sequence crowd xiii split into two groups after rpoB analysis . Only three strains formed a cluster with the type strain of E . cloacae, indicating that the minority of isolates identified as E . cloacae truly belong to the species; 13% of strains grouped with other type strains of the genus, suggesting that the phenotypes of these species seem to be more heterogeneous than so far believed . Three clusters represented 70% of strains, but none of them included a type or reference strain . The genetic clustering presented in this study might serve as a framework for future studies dealing with taxonomic, evolutionary, epidemiological, or pathogenetic characteristics of bacteria belonging to the E . cloacae complex .
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