A cholate-inducible, NADH-dependent flavin oxidoreductase from the intestinal bacterium Eubacterium sp. strain VPI 12708 was purified 372-fold to apparent electrophoretic homogeneity. The subunit and native molecular weights were estimated to be 72,000 and 210,000, respectively, suggesting a homotrimeric organization. Three peaks of NADH:flavin oxidoreductase activity (forms I, H, and III) eluted from a DEAE-high-performance liquid chromatography column. Absorption spectra revealed that purified form III, but not form I, contained bound flavin, which dissociated during purification to generate form I. Enzyme activity was inhibited by sulfhydryl-reactive compounds, acriflavine, o-phenanthroline, and EDTA. Activity assays and Western blot (immunoblot) analysis confirmed that expression of the enzyme was cholate inducible. The first 25 N-terminal amino acid residues of purified NADH:flavin oxidoreductase were determined, and a corresponding oligonucleotide probe was synthesized for use in cloning of the associated gene, baiH. Restriction mapping, sequence data, and RNA blot analysis suggested that the baiH gene was located on a previously described, cholate-inducible operon 210 kb long. The baiH gene encoded a 72,006-Da polypeptide containing 661 amino acids. The deduced amino acid sequence of the baiH gene was homologous to that of NADH oxidase from Thermoanaerobium brockii, trimethylamine dehydrogenase from methylotrophic bacterium W3A1, Old Yellow Enzyme from Saccharomyces carisbergensis, and the product of the baiC gene ofEubacterium sp. strain VPI 12708, located upstream from the baiH gene in the cholate-inducible operon. Alignment of these five sequences revealed potential ligands for an iron-sulfur cluster, a putative fiavin adenine dinucleotide-binding domain, and two other well-conserved domains of unknown function.

Repression by vitamin B 12 of the cobalamin transport protein BtuB in the outer membrane of Escherichia coli operates at both the transcriptional and translational levels and is controlled by transcribed sequences within the leader and proximal portion of the btuB coding sequence. The effects of deletions from either end of this region on repression and expression were determined with lac fusions. An element at the 5 end of the transcript and the putative attenuator within the coding sequence were required for transcriptional repression. The presence of either element caused a marked reduction in btuB-lacZ expression which was reversed by the presence of a conserved sequence element in the leader, suggesting the importance of long-range interactions in the btuB leader for expression and regulation.Most genes for biosynthetic and catabolic activities and for nutrient transport systems are regulated in response to the availability of specific substrates or products. Vitamin B 12 (cyanocobalamin [CN-Cbl]) is known to repress expression of the btuB genes of Escherichia coli and Salmonella typhimurium (Salmonella enterica serovar Typhimurium) for the outer membrane Cbl transport protein BtuB (6, 16) and the cob operon for Cbl biosynthesis in S. typhimurium (2). (For a recent review of Cbl biosynthesis, transport, and regulation, see reference 14). Regulation of these genes exhibits several unusual features. Their expression occurs in response to adenosyl-Cbl (Ado-Cbl), not 14), and appears to operate after the stage of transcription initiation. Evidence has been presented for both attenuation control of transcript elongation and control of translation initiation by sequestration of the ribosome-binding site (8,12,13). The btuB and cbiA (the first gene of the cob operon) genes are transcribed with long leader segments (241 and 468 nucleotides [nt], respectively) which are the sites of numerous mutations that decrease repression (8,12,13). These leader segments have few regions of sequence similarity, other than a conserved 25-nt sequence called the B12 box (12). However, extensive portions of these leader sequences might form alternative secondary structures. An RNA secondary structure that can sequester the Shine-Dalgarno sequence is important for regulation, as shown by analysis of compensatory mutations that maintain this structure but not its sequence (11). A potential Rho-independent terminator structure that might serve as a transcriptional attenuator lies in the proximal portion of the coding sequence. Figure 1 shows a schematic representation of the locations of these possible regulatory elements.Transcriptional and translational fusions to btuB or to cbiA show different levels of regulation depending on the position of the fusion junction. Fusions well within the coding regions show control of both types of reporters. Fusions early in the coding region show repression of translational but not of transcriptional fusions, and fusions in the leader region show no regulation (8,(11)(12)(13). Despite the complexit...

Southern blot analysis indicated that the gene encoding the constitutive, NADP-linked bile acid 7a-hydroxysteroid dehydrogenase of Eubacterium sp. strain VPI 12708 was located on a 6.5-kb EcoRI fragment of the chromosomal DNA. This fragment was cloned into bacteriophage lambda gtll, and a 2.9-kb piece of this insert was subcloned into pUC19, yielding the recombinant plasmid pBH51. DNA sequence analysis of the 7a-hydroxysteroid dehydrogenase gene in pBH51 revealed a 798-bp open reading frame, coding for a protein with a calculated molecular weight of 28,500. A putative promoter sequence and ribosome binding site were identified. The 7a-hydroxysteroid dehydrogenase mRNA transcript in Eubacterium sp. strain VPI 12708 was about 0.94 kb in length, suggesting that it is monocistronic. An Escherichia coli DH5a transformant harboring pBH51 had approximately 30-fold greater levels of 7a-hydroxysteroid dehydrogenase mRNA, immunoreactive protein, and specific activity than Eubacterium sp. strain VPI 12708. The 7af-hydroxysteroid dehydrogenase purified from the pBH51 transformant was similar in subunit molecular weight, specific activity, and kinetic properties to that from Eubacterium sp. strain VPI 12708, and it reacted with antiserum raised against the authentic enzyme on Western immunoblots. Alignment of the amino acid sequence of the 7a-hydroxysteroid dehydrogenase with those of 10 other pyridine nucleotide-linked alcohol/polyol dehydrogenases revealed six conserved amino acid residues in the N-terminal regions thought to function in coenzyme binding.Many members of the human intestinal microflora can modify the glycine and taurine conjugates of the primary bile acids, cholic acid and chenodeoxycholic acid, producing up to 20 different bile acid metabolites (21). The predominant modifications include deconjugation of the glycine and taurine moieties by bile salt hydrolase, removal of 7-hydroxy groups (7-dehydroxylation), and dehydrogenation of a-and ,B-hydroxy groups at carbons 3, 7, and 12 by stereospecific bile acid hydroxysteroid dehydrogenases to yield oxo bile acids.Quantitatively, the most important of these transformations is the 7ux-dehydroxylation of cholic and chenodeoxycholic acids, producing deoxycholic and lithocholic acids, respectively (43). The human intestinal anaerobic bacterium, Eubacterium sp. strain VPI 12708, has a multistep 7a-dehydroxylation pathway which is induced by addition of cholic acid to the culture medium (6,9,11, 22,(46)(47)(48). Several bile acid-inducible (bai) genes encoding proteins involved in this pathway have been cloned and sequenced (10,11,18,31,49,50).7a-Hydroxysteroid dehydrogenase is the most common class of bile acid hydroxysteroid dehydrogenases found in nature (3), having been detected in numerous genera of bacteria (3, 14-16, 28, 30, 37) and in mammalian liver (1). 7a-Hydroxysteroid dehydrogenases have been purified from Bacteroides species (23,29,41), Escherichia coli (28,36,37), and rat liver microsomes (1). Recently, two 7a-hydroxysteroid dehydrogenases have been pu...

We previously cloned a genomic DNA fragment from the serogroup O11Pseudomonas aeruginosa strain PA103 that contained all genes necessary for O-antigen synthesis and directed the expression of serogroup O11 antigen on recombinant Escherichia coli andSalmonella. To elucidate the pathway of serogroup O11 antigen synthesis, the nucleotide sequence of the biosynthetic genes was determined. Eleven open reading frames likely to be involved in serogroup O11 O-antigen biosynthesis were identified and are designated in order as wzz PaO111 (wzz fromP. aeruginosa serogroup O11),wzx PaO11, wbjA,wzy PaO11, wbjB to wbjF,wbpL O11 and wbpM O11(wbpL and wbpM from serogroup O11). Consistent with previous descriptions of O-antigen biosynthetic gene loci, the entire region with the exception of wbpM O11 has a markedly reduced G+C content relative to the chromosomal average. WzyPaO11 shows no significant similarity at the protein or DNA sequence level to any database sequence and is very hydrophobic, with 10 to 12 putative transmembrane domains, both typical characteristics of O-antigen polymerases. A nonpolar chromosomal insertion mutation in wzy PaO11 in P. aeruginosa PA103 confirmed the identity of this gene. There is striking similarity between WbjBCDE and Cap(5/8)EFGL, involved in type 5 and type 8 capsule biosynthesis in Staphylococcus aureus. There is nearly total identity between wbpM O11and wbpM O5, previously shown by others to be present in all 20 P. aeruginosa serogroups. Using similarity searches, we have assigned functions to the proteins encoded by the PA103 O-antigen locus and present the potential steps in the pathway for the biosynthesis of P. aeruginosa serogroup O11 O antigen.

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