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Bacillus subtilis LmrA Is a Repressor of the lmrAB and yxaGH Operons: Identification of Its Binding Site and Functional Analysis of lmrB and yxaGH. Ken-ichi Yoshida, 2004.The Bacillus subtilis lmrAB operon is involved in multidrug resistance . LmrA is a repressor of its own operon, while LmrB acts as a multidrug efflux transporter . LmrA was produced in Escherichia coli cells and was shown to bind to the lmr promoter region, in which an LmrA-binding site was identified . Genome-wide screening involving DNA microarray analysis allowed us to conclude that LmrA also repressed yxaGH, which was not likely to contribute to the multidrug resistance . LmrA bound to a putative yxaGH promoter region, in which two tandem LmrA-binding sites were identified . The LmrA regulon was thus determined to comprise lmrAB and yxaGH . All three LmrA-binding sites contained an 18-bp consensus sequence, TAGACCRKTCWMTATAWT, which could play an important role in LmrA binding . Resuscitation of a Defective Prophage in Salmonella Cocultures. Nara Figueroa-Bossi, 2004.Widely studied Salmonella enterica serovar Typhimurium strains ATCC 14028s and SL1344 harbor a cryptic ST64B prophage unable to produce infectious virions . We found that coculturing either strain with an isogenic sibling lacking the prophage leads to the appearance of active forms of the virus . Active phage originates from reversion of a +1 frameshift mutation at a monotonous G:C run in a presumptive tail assembly pseudogene . Identification of the Periplasmic Cobalamin-Binding Protein BtuF of Escherichia coli. Nathalie Cadieux, 2002.Cells of Escherichia coli take up vitamin B12 (cyano-cobalamin [CN-Cbl]) and iron chelates by use of sequential active transport processes . Transport of CN-Cbl across the outer membrane and its accumulation in the periplasm is mediated by the TonB-dependent transporter BtuB . Transport across the cytoplasmic membrane (CM) requires the BtuC and BtuD proteins, which are most related in sequence to the transmembrane and ATP-binding cassette proteins of periplasmic permeases for iron-siderophore transport . Unlike the genetic organization of most periplasmic permeases, a candidate gene for a periplasmic Cbl-binding protein is not linked to the btuCED operon . The open reading frame termed yadT in the E . coli genomic sequence is related in sequence to the periplasmic binding proteins for iron-siderophore complexes and was previously implicated in CN-Cbl uptake in Salmonella . The E . coli yadT product, renamed BtuF, is shown here to participate in CN-Cbl uptake . BtuF protein, expressed with a C-terminal His6 tag, was shown to be translocated to the periplasm concomitant with removal of a signal sequence . CN-Cbl-binding assays using radiolabeled substrate or isothermal titration calorimetry showed that purified BtuF binds CN-Cbl with a binding constant of around 15 nM . A null mutation in btuF, but not in the flanking genes pfs and yadS, strongly decreased CN-Cbl utilization and transport into the cytoplasm . The growth response to CN-Cbl of the btuF mutant was much stronger than the slight impairment previously described for btuC, btuD, or btuF mutants . Hence, null mutations in btuC and btuD were constructed and revealed that the btuC mutant had a strong impairment similar to that of the btuF mutant, whereas the btuD defect was less pronounced . All mutants with defective transport across the CM gave rise to frequent suppressor variants which were able to respond at lower levels of CN-Cbl but were still defective in transport across the CM . These results finally establish the identity of the periplasmic binding protein for Cbl uptake, which is one of few cases where the components of a periplasmic permease are genetically separated .
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