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.

The foodborne pathogen Listeria monocytogenes has the ability to develop biofilm in food-processing environment, which becomes a major concern for the food safety. The biofilm formation is strongly influenced by the availability of nutrients and environmental conditions, and particularly enhanced in poor minimal essential medium (MEM) containing glucose rather than in rich brain heart infusion (BHI) broth. To gain better insight into the conserved protein expression profile in these biofilms, the proteomes from biofilm- and planktonic-grown cells from MEM with 50 mM glucose or BHI were compared using two-dimensional polyacrylamide gel electrophoresis followed by MALDI-TOF/TOF analysis. 47 proteins were successfully identified to be either up (19 proteins) or down (28 proteins) regulated in the biofilm states. Most (30 proteins) of them were assigned to the metabolism functional category in cluster of orthologous groups of proteins. Among them, up-regulated proteins were mainly associated with the pentose phosphate pathway and glycolysis, whereas a key enzyme CitC involved in tricarboxylic acid cycle was down-regulated in biofilms compared to the planktonic states. These data implicate the importance of carbon catabolite control for L. monocytogenes biofilm formation in response to nutrient availability.

The foodborne pathogen Listeria monocytogenes has the ability to develop biofilm in the food-processing environment, which becomes a major concern for food safety. PrfA, a key transcriptional activator that regulates most of the known listerial virulence gene expression, has been shown to promote L. monocytogenes biofilm formation. In this study, the whole-genome microarray was used to identify differentially expressed genes associated with the putative interaction between biofilm formation and PrfA in L. monocytogenes. Comparative transcriptome analyses indicated that over 21.9 % of the L. monocytogenes EGDe genes (627 out of 2,857 predicted) were altered in their expression of biofilm compared to the planktonic phase. These genes were classified into different functional categories which cover most of the biochemical functions encountered in bacterial cells, indicating that L. monocytogenes biofilm formation is probably controlled by a complex regulation network involved in variable genes required for the different biological pathways. Further comparison of gene expression profiles of biofilms between L. monocytogenes EGDe and its PrfA deletion mutant revealed 185 genes associated with PrfA and biofilm formation. Except for 10 genes, transcription levels of 175 genes were completely opposite between ΔprfA and wild-type during the biofilm formation, i.e., up-regulated genes in ΔprfA were down-regulated in the wild-type strain, and vice versa, indicating that loss of PrfA dramatically altered gene expression patterns in L. monocytogenes biofilm and resulted in reduced ability of the biofilm formation.

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 σ (B) 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 σ (B)-dependent genes that are necessary for tolerance to rifampin stress and therefore plays a role in overcoming the antibiotic-triggered growth arrest.

We aimed to construct an effective tumor targeting vaccine delivery plasmid that can express antigen protein in attenuated Listeria monocytogenes (LM). The produced fusion proteins were supposed to induce the antitumor immune response strongly. In our study, the gene of a truncated non-cytolytic form of listeriolysin O (LLO) was cloned in the plasmid pERL3 under the control of a hemolysin promoter (Phly). To check if the genes of interest were successfully inserted into pERL3, recombinant pERL3 was transferred into E. coli strain DH5α ， and positive clones were obtained from LuriaBertani agar plate. Here we collected 46 positive clones and analyzed by PCR. Finally, we found a clear electrophoretic band in electrophoregram that was identified as a 726-bp fragment of hly. In conclusion, we've constructed a listeria-based vaccine delivery plasmid: pERL3-Phly-hly (441aa), with which we can express exogenous proteins in host cells, we planned to use it in delivering MIA (melanoma inhibitory activity) antigens to host cells to induce cellular immune response in further research.