Adaptive mutation related to cellulose producibility in Komagataeibacter medellinensis (Gluconacetobacter xylinus) NBRC 3288.

Gluconacetobacter xylinus (formerly Acetobacter xylinum and presently Komagataeibacter medellinensis) is known to produce cellulose as a stable pellicle. However, it is also well known to lose this ability very easily. We investigated the on and off mechanisms of cellulose producibility in two independent cellulose-producing strains, R1 and R2. Both these strains were isolated through a repetitive static culture of a non-cellulose-producing K. medellinensis NBRC 3288 parental strain. Two cellulose synthase operons, types I and II, of this strain are truncated by the frameshift mutation in the bcsBI gene and transposon insertion in the bcsCII gene, respectively. The draft genome sequencing of R1 and R2 strains revealed that in both strains the bcsBI gene was restored by deletion of a nucleotide in its C-rich region. This result suggests that the mutations in the bcsBI gene are responsible for the on and off mechanism of cellulose producibility. When we looked at the genomic DNA sequences of other Komagataeibacter species, several non-cellulose-producing strains were found to contain similar defects in the type I and/or type II cellulose synthase operons. Furthermore, the phylogenetic relationship among cellulose synthase genes conserved in other bacterial species was analyzed. We observed that the cellulose genes in the Komagataeibacter shared sequence similarities with the γ-proteobacterial species but not with the α-proteobacteria and that the type I and type II operons could be diverged from a same ancestor in Komagataeibacter.

Crucial role for CD69 in the pathogenesis of dextran sulphate sodium-induced colitis.

CD69 is a membrane molecule transiently expressed on activated lymphocytes, and its selective expression in inflammatory infiltrates suggests that it plays a role in the pathogenesis of inflammatory diseases. In this study, we used CD69-deficient (CD69 KO) mice to assess the role of CD69 in the pathogenesis of dextran sulphate sodium (DSS)-induced acute and chronic colitis. The severity of colitis was assessed by the survival rate, clinical signs, colon length, histological examination and the expression of cytokines and chemokines in the large intestines. Both acute and chronic colitis were attenuated in the CD69 KO mice, as reflected by the lower lethality, weight loss, clinical signs, and improved histological findings. CD69(+) cells infiltrated extensively into the inflamed mucosa of the colon in WT mice after DSS treatment. Experiments with the transfer of WT CD4 T cells into CD69 KO mice restored the induction of colitis. The administration of an anti-CD69 antibody also inhibited the induction of the DSS-induced colitis. These results indicate that CD69 expressed on CD4 T cells plays an important role in the pathogenesis of DSS-induced acute and chronic colitis, and that CD69 could be a possible therapeutic target for colitis.

Complete genome sequence of NBRC 3288, a unique cellulose-nonproducing strain of Gluconacetobacter xylinus isolated from vinegar.

Gluconacetobacter xylinus is involved in the industrial production of cellulose. We have determined the genome sequence of G. xylinus NBRC 3288, a cellulose-nonproducing strain. Comparative analysis of genomes of G. xylinus NBRC 3288 with those of the cellulose-producing strains clarified the genes important for cellulose production in Gluconacetobacter.

Regulation of quinone oxidoreductase by the redox-sensing transcriptional regulator QorR in Corynebacterium glutamicum.

Corynebacterium glutamicum cgR_1435 (cg1552) encodes a protein of the DUF24 protein family, which is a novel family of transcriptional regulators. CgR1435 (QorR) is a negative regulator of cgR_1436 (qor2), which is located upstream of cgR_1435 (qorR) in the opposite orientation, and its structural gene. QorR binds to the intergenic region between qor2 and qorR to repress their expression, which is induced by the thiol-specific oxidant diamide. The DNA-binding activity of QorR is impaired by oxidants such as diamide, H(2)O(2), and cumene hydroperoxide in vitro, and its lone cysteine residue (Cys-17) is essential for redox-responsive regulation of QorR activity both in vivo and in vitro. Moreover, a disruptant of qor2, which is a homologue of the ytfG gene of Escherichia coli encoding quinone oxidoreductase, shows increased sensitivity to diamide. It is concluded that the redox-sensing transcriptional regulator QorR is involved in disulfide stress response of C. glutamicum by regulating qor2 expression.

Deletion of cgR_1596 and cgR_2070, encoding NlpC/P60 proteins, causes a defect in cell separation in Corynebacterium glutamicum R.

In previous work, random genome deletion mutants of Corynebacterium glutamicum R were generated using the insertion sequence (IS) element IS31831 and the Cre/loxP excision system. One of these mutants, C. glutamicum strain RD41, resulting from the deletion of a 10.1-kb genomic region (DeltacgR_1595 through cgR_1604) from the WT strain, showed cell elongation, and several lines appeared on the cell surface (bamboo shape). The morphological changes were suppressed by overexpression of cgR_1596. Single disruption of cgR_1596 in WT C. glutamicum R resulted in morphological changes similar to those observed in the RD41 strain. CgR_1596 has a predicted secretion signal peptide in the amino-terminal region and a predicted NlpC/P60 domain, which is conserved in cell wall hydrolases, in the carboxyl-terminal region. In C. glutamicum R, CgR_0802, CgR_1596, CgR_2069, and CgR_2070 have the NlpC/P60 domain; however, only simultaneous disruption of cgR_1596 and cgR_2070, and not cgR_2070 single disruption, resulted in cell growth delay and more severe morphological changes than disruption of cgR_1596. Transmission electron microscopy revealed multiple septa within individual cells of cgR_1596 single and cgR_1596-cgR_2070 double disruptants. Taken together, these results suggest that cgR_1596 and cgR_2070 are involved in cell separation and cell growth in C. glutamicum.

DivS, a novel SOS-inducible cell-division suppressor in Corynebacterium glutamicum.

DNA damage-induced SOS response elicits the induction of cell-division suppressor as well as DNA repair genes. In Gram-positive bacteria, cell-division suppressor genes, so far characterized from Bacillus subtilis (yneA) and Mycobacterium tuberculosis (rv2719c), share limited homology, but are both located in the vicinity of lexA on their respective genomes. Using this proximity to lexA, Corynebacterium glutamicum R divS (cgR1759) was identified as an SOS-inducible cell-division suppressor in this study. The amino acid sequence of DivS showed no homology to that of YneA and Rv2719c. divS expression was markedly induced by DNA-damaging mitomycin C treatment in wild-type cells, but not in its DeltarecA mutant cells, which are unable to induce the SOS response. Wild-type C. glutamicum R cells exposed to DNA-damaging mitomycin C exhibited elongated morphology that, using green fluorescent protein-FtsZ fusion protein, was attributed to defects in FtsZ ring assembly. Cells defective in FtsZ ring assembly were subsequently incapable of septum wall synthesis. In the presence of mitomycin C, divS mutant cells did not exhibit this elongated morphology, whereas cells overexpressing divS were elongated and abnormally branched.

FtsZ-dependent localization of GroEL protein at possible division sites.

When Escherichia coli is treated with penicillin, the envelopes bulge at the centre of the cells and the cells then lyse. The bulges expand into vesicle-like structures termed penicillin-induced vesicles. We have developed a method to isolate these structures and have shown that they contain mainly membrane proteins plus a high concentration of a 60 kDa protein. The N-terminal amino acid sequence of the protein is identical to that of GroEL protein. Western blotting analysis using anti-GroEL antibody showed that GroEL is indeed concentrated in the vesicles. Indirect immuno-fluorescence microscopy showed that GroEL protein is localized at the centre of the cells at the site of formation of FtsZ-rings. Localization of GroEL is dependent on FtsZ but not other Fts proteins. GroEL mutants formed elongated cells having no or asymmetrically localized FtsZ-rings at the restrictive temperature. These findings suggest a possible role of the GroEL protein in cell division.