Rapid turnover of DnaA at replication origin regions contributes to initiation control of DNA replication.

DnaA is a conserved key regulator of replication initiation in bacteria, and is homologous to ORC proteins in archaea and in eukaryotic cells. The ATPase binds to several high affinity binding sites at the origin region and upon an unknown molecular trigger, spreads to several adjacent sites, inducing the formation of a helical super structure leading to initiation of replication. Using FRAP analysis of a functional YFP-DnaA allele in Bacillus subtilis, we show that DnaA is bound to oriC with a half-time of 2.5 seconds. DnaA shows similarly high turnover at the replication machinery, where DnaA is bound to DNA polymerase via YabA. The absence of YabA increases the half time binding of DnaA at oriC, showing that YabA plays a dual role in the regulation of DnaA, as a tether at the replication forks, and as a chaser at origin regions. Likewise, a deletion of soj (encoding a ParA protein) leads to an increase in residence time and to overinitiation, while a mutation in DnaA that leads to lowered initiation frequency, due to a reduced ATPase activity, shows a decreased residence time on binding sites. Finally, our single molecule tracking experiments show that DnaA rapidly moves between chromosomal binding sites, and does not arrest for more than few hundreds of milliseconds. In Escherichia coli, DnaA also shows low residence times in the range of 200 ms and oscillates between spatially opposite chromosome regions in a time frame of one to two seconds, independently of ongoing transcription. Thus, DnaA shows extremely rapid binding turnover on the chromosome including oriC regions in two bacterial species, which is influenced by Soj and YabA proteins in B. subtilis, and is crucial for balanced initiation control, likely preventing fatal premature multimerization and strand opening of DnaA at oriC.

Two DNA translocases synergistically affect chromosome dimer resolution in Bacillus subtilis.

In Bacillus subtilis, chromosome dimers that block complete segregation of sister chromosomes arise in about 15% of exponentially growing cells. Two dedicated recombinases, RipX and CodV, catalyze the resolution of dimers by site-specific recombination at the dif site, which is located close to the terminus region on the chromosome. We show that the two DNA translocases in B. subtilis, SftA and SpoIIIE, synergistically affect dimer resolution, presumably by positioning the dif sites in close proximity, before or after completion of cell division, respectively. Furthermore, we observed that both recombinases, RipX and CodV, assemble on the chromosome at the dif site throughout the cell cycle. The preassembly of recombinases probably ensures that dimer resolution can occur rapidly within a short time window around cell division.

Identification of commensal bacterial strains that modulate Yersinia enterocolitica and dextran sodium sulfate-induced inflammatory responses: implications for the development of probiotics.

An increasing body of evidence suggests that probiotic bacteria are effective in the treatment of enteric infections, although the molecular basis of this activity remains elusive. To identify putative probiotics, we tested commensal bacteria in terms of their toxicity, invasiveness, inhibition of Yersinia-induced inflammation in vitro and in vivo, and modulation of dextran sodium sulfate (DSS)-induced colitis in mice. The commensal bacteria Escherichia coli, Bifidobacterium adolescentis, Bacteroides vulgatus, Bacteroides distasonis, and Streptococcus salivarius were screened for adhesion to, invasion of, and toxicity for host epithelial cells (EC), and the strains were tested for their ability to inhibit Y. enterocolitica-induced NF-kappaB activation. Additionally, B. adolescentis was administered to mice orally infected with Y. enterocolitica and to mice with mucosae impaired by DSS treatment. None of the commensal bacteria tested was toxic for or invaded the EC. B. adolescentis, B. distasonis, B. vulgatus, and S. salivarius inhibited the Y. enterocolitica-induced NF-kappaB activation and interleukin-8 production in EC. In line with these findings, B. adolescentis-fed mice had significantly lower results for mean pathogen burden in the visceral organs, intestinal tumor necrosis factor alpha mRNA expression, and loss of body weight upon oral infection with Y. enterocolitica. In addition, the administration of B. adolescentis decelerated inflammation upon DSS treatment in mice. We suggest that our approach might help to identify new probiotics to be used for the treatment of inflammatory and infectious gastrointestinal disorders.

Lactobacillus fermentum attenuates the proinflammatory effect of Yersinia enterocolitica on human epithelial cells.

BACKGROUND: Lactobacilli represent a major component of the human microbiota. In this study we investigated whether and how Lactobacillus fermentum inhibits the proinflammatory responses of human epithelial cells on Yersinia enterocolitica infection. METHODS: Human epithelial cells were exposed to Y. enterocolitica pYV(-) or L. fermentum or to both strains, combinations of heat-killed L. fermentum or supernatant of L. fermentum cultures and Y. enterocolitica. The modulation of Y. enterocolitica induced IL-8 levels in the culture supernatants was determined and activation of Rac, p38, and NF-kappaB was investigated. RESULTS: Exposure of human epithelial cells to L. fermentum does not induce NF-kappaB activation and subsequent IL-8 secretion in HeLa cells, whereas Y. enterocolitica induces NF-kappaB activation and high levels of IL-8. Viable L. fermentum, supernatant of L. fermentum cultures, but not heat-killed L. fermentum, inhibited IL-8 secretion of HeLa cells triggered by Y. enterocolitica. Lactobacillus fermentum-exposed HeLa cells showed decreased Rac, p38, and NF-kappaB activation after Y. enterocolitica infection. Treatment of L. fermentum supernatants with phospholipase C abolished the inhibitory effect, indicating that a secreted phospholipid mediates the antiinflammatory properties of L. fermentum. Adhesion to or invasion of Y. enterocolitica into epithelial cells was not altered by coincubation with L. fermentum. CONCLUSION: Our results lead to the conclusion that L. fermentum inhibits the Y. enterocolitica-induced IL-8 production by a possibly secreted phospholipid of <10 kDa molecular weight. These data suggest that L. fermentum may have probiotic properties modulating intestinal inflammatory responses and might offer new therapeutic strategies in the treatment of intestinal inflammatory diseases.