| The gram-positive foodborne bacteria Listeria monocytogenes is a facultative intracellular pathogen. It is ubiquitous in nature world and always exposed to kinds of changes in growth conditions. These changes may include temperature, salt concentration, oxygen pressure or supply of essential nutrients. To respond to these challenges, Listeria has developed several mechanisms, always including adjusting its life style or modulating its metabolism, to allow them to keep alive. Intracellular L. monocytogenes must also develop a set of metabolism mechanisms to adapt to the conditions in host cells or even coordinate themselves with the life cycle of their host cells. Interestingly, it was observed that glycerol and6-p-glucose (that is, PTS-independent carbon substrates) are preferentially used as carbon source by intracellular L. monocytogenes. In addition, a high level of PrfA activity is observed upon the growth of L.monocytogenes in a defined minimal medium (MM) in the presence of PTS-independent carbohydrates. Based on these information, we attempt to find if PrfA, one of the most essential factors that regulate the expression of virulence genes in L. monocytogenes, involved in the regulation of glycerol metabolism, and whether the SigB, which also plays a key role in L. monocytogenes, would affect the glycerol metabolism or not.To explore the role SigB and PrfA played in glycerol metabolism in L. mono cytogenes, comparative proteomics was performed to analyze the whole protein ext racts from the cells of L. monocytogenes EGDe, prfA deletion mutant EGDeΔprfA, sigB deletion mutant EGDeAsigB and double deletion mutant EGDe AprfAΔAsigB which are cultured in defined minimum medium with the addition of glycerol or glucose. Based on the results of comparative proteomics, we next used RT-PCR (Reverse Transcription-Polymerase Chain Reaction) to detect the role of PrfA in gl ycerol metabolism in L. monocytogenes. The transcriptional levels of desired genes in EGDe, EGDeAprfA, EGDeAprfApPrfA(a recombinant strains that can constitut ively express wild PrfA in EGDeΔprfA) and EGDeΔprfApPrfA*(a recombinant str ains that can constitutively express highly active PrfA*in EGDeAprfA) that grown in defined minimal medium with glycerol or glucose were determined and compa red.The results show that1) Twenty one proteins were found to be regulated by PrfA when we compare the protein profiles of EGDe and AprfA that grow to exponential phase in defined minimum medium with glycerol. The expression of Imo1154, lmo1155, lmoll65, lmo1293, which seem to be involved in utilization of glycerol, are modulated by PrfA. In contrast, eight proteins were detected to be modulated by SigB through comparing the protein profiles of EGDe and AsigB that grow to exponential phase in defined minimum medium with glycerol, and all of them do not have the direct relevance to the utilization of glycerol.2) The transcriptional levels of target genes, including lmo1154, lmo1155, lmo1156, lmo1165, lmo0348, lmo1538and lmo1293were analyzed. The transcriptional levels of Imo1538and Imo1293which encoded glycerol kinase and3-p-glycerol dehydrogenase, respectively, were markedly higher in EGDe that grew in MM with glycerol than that of grew in MM with glucose. This finding indicates that glycerol kinase and3-p-glycerol dehydrogenase are quite active in glycerol metabolism of L. monocytogenes. Appropriate concentration of PrfA would positively affect glycerol metabolism while high concentration of PrfA or PrfA*would have the opposite effect.Our results suggest that PrfA has played an important role in glycerol metabolism in L. monocytogenes, and the concentration of PrfA will also be a crucial factor contributed to the efficiency of glycerol metabolism. In contrast, we found that there is no significant link between SigB and glycerol metabolism. |
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