Frequency of the Insertion Sequence IS4Bsu1 among Bacillus subtilis Strains Isolated from Fermented Soybean Foods in Southeast Asia

Among 45 Bacillus subtilis strains isolated from non-salted types of fermented soybeans produced in several Southeast Asian countries, 20 had the insertion sequence IS4Bsu1 in the chromosome. In contrast, none of 49 B. subtilis strains of non-food origin contained IS4Bsu1. Frequent occurrence of this mobile DNA element in the soybean-fermenting B. subtilis would reflect the fact that few strains flourish on soybeans and thereby contribute to soybean fermentation.     

Determination and characterization of IS4Bsu1-insertion loci and identification of a new insertion sequence element of the IS256 family in a natto starter

The insertion sequence IS4Bsu1 frequently causes Bacillus subtilis starters for the production of Japanese fermented soybean pasts (natto) to lose the ability to produce poly-gamma-glutamate, the viscous material characteristic of natto. Bacillus subtilis NAFM5, a derivative of a natto starter, has six IS4Bsu1 copies on its chromosome. In this study, we determined all six insertion loci of the insertion sequence (IS). One was located in the coding region of yktD, a putative gene involved in polyketide synthesis. Four were located in non-coding regions between iolR and iolA, between tuaA and lytC, between rapI and orf1 (a potential gene of unknown function), and between ynaE and orf3 (a putative gene similar to thiF), and one resided in an intergenic region between divergent possible orf4 and orf5 genes of unknown function. Here we describe the structural features of these loci and discuss the effects of the IS4Bsu1 insertions on the functions of the target gene and the expression of the downstream genes. In addition, we found that strain NAFM5 and commercial natto starters possess eight to 10 loci of another IS of the IS256 family (designated IS256Bsu1) on their chromosomes. IS256Bus1 appeared active in transposition, potentially causing phenotypic alterations in natto starters like those induced by IS4Bsu1.     

Transposition of IS1397 in the Family Enterobacteriaceae and First Characterization of ISKpn1, a New Insertion Sequence Associated with Klebsiella pneumoniae Palindromic Units

IS1397 and ISKpn1 are IS3 family members which are specifically inserted into the loop of palindromic units (PUs). IS1397 is shown to transpose into PUs with sequences close or identical to the Escherichia coli consensus, even in other enterobacteria (Salmonella enterica serovar Typhimurium, Klebsiella pneumoniae, and Klebsiella oxytoca). Moreover, we show that homologous intergenic regions containing PUs constitute IS1397 transpositional hot spots, despite bacterial interspersed mosaic element structures that differ among the three species. ISKpn1, described here for the first time, is specific for PUs from K. pneumoniae, in which we discovered it. A sequence comparison between the two insertion sequences allowed us to define a motif possibly accounting for their specificity.     

Biochemical and Spectroscopic Characterization of the Human Mitochondrial Amidoxime Reducing Components hmARC-1 and hmARC-2 Suggests the Existence of a New Molybdenum Enzyme Family in Eukaryotes

The mitochondrial amidoxime reducing component mARC is a newly discovered molybdenum enzyme that is presumed to form the catalytical part of a three-component enzyme system, consisting of mARC, heme/cytochrome b₅, and NADH/FAD-dependent cytochrome b₅ reductase. mARC proteins share a significant degree of homology to the molybdenum cofactor-binding domain of eukaryotic molybdenum cofactor sulfurase proteins, the latter catalyzing the post-translational activation of aldehyde oxidase and xanthine oxidoreductase. The human genome harbors two mARC genes, referred to as hmARC-1/MOSC-1 and hmARC-2/MOSC-2, which are organized in a tandem arrangement on chromosome 1. Recombinant expression of hmARC-1 and hmARC-2 proteins in Escherichia coli reveals that both proteins are monomeric in their active forms, which is in contrast to all other eukaryotic molybdenum enzymes that act as homo- or heterodimers. Both hmARC-1 and hmARC-2 catalyze the N-reduction of a variety of N-hydroxylated substrates such as N-hydroxy-cytosine, albeit with different specificities. Reconstitution of active molybdenum cofactor onto recombinant hmARC-1 and hmARC-2 proteins in the absence of sulfur indicates that mARC proteins do not belong to the xanthine oxidase family of molybdenum enzymes. Moreover, they also appear to be different from the sulfite oxidase family, because no cysteine residue could be identified as a putative ligand of the molybdenum atom. This suggests that the hmARC proteins and sulfurase represent members of a new family of molybdenum enzymes.     

Identification of a Bifunctional UDP-4-keto-pentose/UDP-xylose Synthase in the Plant Pathogenic Bacterium Ralstonia solanacearum Strain GMI1000, a Distinct Member of the 4,6-Dehydratase and Decarboxylase Family

The UDP-sugar interconverting enzymes involved in UDP-GlcA metabolism are well described in eukaryotes but less is known in prokaryotes. Here we identify and characterize a gene (RsU4kpxs) from Ralstonia solanacearum str. GMI1000, which encodes a dual function enzyme not previously described. One activity is to decarboxylate UDP-glucuronic acid to UDP-β-l-threo-pentopyranosyl-4″-ulose in the presence of NAD⁺. The second activity converts UDP-β-l-threo-pentopyranosyl-4″-ulose and NADH to UDP-xylose and NAD⁺, albeit at a lower rate. Our data also suggest that following decarboxylation, there is stereospecific protonation at the C5 pro-R position. The identification of the R. solanacearum enzyme enables us to propose that the ancestral enzyme of UDP-xylose synthase and UDP-apiose/UDP-xylose synthase was diverged to two distinct enzymatic activities in early bacteria. This separation gave rise to the current UDP-xylose synthase in animal, fungus, and plant as well as to the plant Uaxs and bacterial ArnA and U4kpxs homologs.