Qin Luo

SigB plays a major role in Listeria monocytogenes tolerance to bile stress

The ability of Listeria monocytogenes to tolerate high levels of bile stress is critical to its successful infection and colonization in the human gastrointestinal tract. L. monocytogenes encodes bile salt hydrolase by a bsh gene which plays a significant role in hydrolyzing high concentrations of bile salt when L. monocytogenes grows under hypoxemic condition. As the bsh promoter contains consensus SigB and PrfA binding sites, we investigated the role of SigB (σ(B)) and PrfA in L. monocytogenes tolerance against bile stress by comparing the survival of isogenic deletion mutants of L. monocytogenes EGD(ΔsigB), EGD(ΔprfA) and EGD(ΔprfAΔsigB) with their parent strain EGD at high levels of bile salt. Our results show that the sigB deletion significantly reduced the MICs of bile salt for EGD(ΔsigB) and EGD(ΔprfAΔsigB) (2.6% and 2.2% vs 3.5% in wild type strain EGD), while the growth rates of these two sigB deletion mutants (EGD(ΔsigB) and EGD(ΔprfAΔsigB)) were affected the most in the presence of 3% bile salt. Pre-exposure to alkali (pH 9.0) and osmotic (0.3M NaCl) stresses for a short period of time (30 min) resulted in improved growth of L. monocytogenes as well as its prfA-sigB isogenic mutants even under sublethal concentrations of bile salt, while pre-exposure to acid pH (pH 4.5) failed to provide cross-protection against subsequent bile stress. Furthermore, the sigB gene had more remarkable influence than that of prfA on bsh expression, as much lower levels of bsh transcription were observed in EGD(ΔsigB) and EGD(ΔprfAΔsigB). Meanwhile, bsh expression in the deletion mutants did not respond to elevated levels of bile salt. These data indicate that σ(B) might play a crucial role in Listeria survival under bile salt environment in the gastrointestinal tract before its successful colonization, invasion and intracellular propagation.

Virulence Regulator PrfA is Essential for Biofilm Formation in Listeria monocytogenes but not in Listeria innocua

The ability of the foodborne pathogen Listeria monocytogenes to develop biofilm in food-processing environment is a major concern for the food safety, because biofilms allow bacteria to better resist environmental stresses. PrfA is a key transcriptional activator that positively regulates most of the known listerial virulence gene expression. In order to explore the role of PrfA on Listeria biofilm development, we compared the abilities of biofilm formation by L. monocytogenes wild type strains (EGD and EGDe) and their prfA deletion mutants (EGD∆prfA and EGDe∆prfA), nonpathogenic Listeria innocua, as well as the recombinant strains that express constitutively active mutant PrfA (PrfA*) in L. innocua (LI-pERL3-prfA*) and in EGDe∆prfA (EGDe∆prfA-pERL3-prfA*) at 37°C in brain heart infusion (BHI) medium using the polyvinyl chloride (PVC) microtiter plate assay and microscopic examination. Our results showed that the wild types of L. monocytogenes had strong abilities to develop biofilm with meshwork of bacterial aggregates, while biofilm with sparse small clumps were observed in L. innocua. The biofilm production of strains EGD∆prfA and EGDe∆prfA that lack funtional PrfA was reduced and could be recovered by the introduction of the PrfA*, however, the PrfA* had no impact on the biofilm forming ability of L. innocua. Our results suggest that PrfA plays a significant role in biofilm formation in L. monocytogenes but not in L. innocua, thus may reflect differences in the molecular mechanisms of biofilm formation by these two closely related species.

SigB-Dependent Tolerance to Protein Synthesis-Inhibiting Antibiotics in Listeria monocytogenes EGDe

The alternative sigma factor SigB in food-borne pathogen Listeria monocytogenes was determined in this study to be required for tolerance to protein synthesis-inhibiting antibiotics. The minimum inhibitory concentrations of tetracycline HCl and gentamicin sulphate against EGDeΔsigB were two- and fourfold less than those for EGDe, respectively. The ability of EGDeΔsigB to overcome the growth arrest caused by erythromycin and rifampin was also weaker than that of EGDe. The transcription analysis of four genetic loci (known to be induced by rifampin in Bacillus subtili) kat, fri, ropB and rsbU in EGDe and EGDeΔsigB in the absence or presence of rifampin revealed that: (1) expression of kat and fri genes is σB dependent, but only the former is inducible by rifampin stress; (2) the transcriptional level of rpoB gene was stable under all the experimental conditions, while that of rsbU in EGDeΔsigB was remarkably higher in the absence of rifampin and significantly increased in EGDe but reduced in EGDeΔsigB after rifampin application, when compared to those in EGDe and EGDeΔsigB control without antibiotic, respectively. These results suggest that complex physiological reactions to tolerance of the antibiotic stress are variably regulated in bacteria, and in contrast to B. subtilis, rsbU in EGDeΔsigB may compensate for the σB-dependent genes that are necessary for tolerance to rifampin stress and therefore plays a role in overcoming the antibiotic-triggered growth arrest.

Study on Oxidative Damage and Genotoxicity of Butyl Benzyl Phthalate on the Hepatic Cells of Rat

In order to study the effect of butyl benzyl phthalatee (BBP) on hepatic cells of rat, hepatic cells of rat were exposed to different dose (5 µmol / L, 20 µmol / L, 80 µmol / L) of BBP in vitro for 1 hour. Thiobarbituric acid (TBA) method , KCl-SDS precipitation method and Dinitrophenylhydrazone (DNPH) colorimetric method were used to detect malondialdehyde (MDA) content, DPC coefficient changes and content, respectively. MDA levels in hepatic cells showed a dose-dependent enhancement after BBP exposure. Significant MDA content was observed in all groups compared to control group. The content of carbonyl was all increased in exposure groups, especially in the group of 5µmol / L. DPC coefficient presented a slight increase compared with the control group but no significant difference in the dose of 5 µmol / L and 20 µmol / L. When the concentration of BBP increased to 80 µmol / L, DPC coefficient increased, and there was a significant difference compared with the control group. The carbonyl content was significantly increased in the dose of 5µmol / L. These results suggest that BBP can cause liver damage in rats, and might have toxic effects on the human body.

A Novel Giant DNA Transpson in Nematostella vectensis

With genome-wide screening of N. vectensis, two new groups of Polinton were identified, beside five Polinton groups deposited in Repbase. The typical N. vectensis Polinton is in length of 15-20kb and has long TIRs with a 6-bp TSDs. it encodes as many as 6 different proteins, including retroviral-like integrase (INT), adenovirus protease (PRO), DNA polymerase B (POLB) and putative ATPase (ATP). These ancient N. vectensis Polinton groups fall to two clades by phylogenetic analysis. One clade evolves evolves to vertebrates together with large scale gene linkage of N. vectensis, another clade following to the evolution of fly and nematode genome.