Study On Listeria Monocytogenes Repressor MogR Controlling The Expression Of Flagellar Genes At Cold Temperatures

Listeria monocytogenes（Lm）is a foodborne pathogenic bacterium that causes the zoonosis-listeriosis,with a mortality rate of 20～30%.Lm can grow and reproduce at cold temperatures,which causes a great threat to the safety of cold temperature refrigerated food.It is imperative to prevent and control the growth of Lm at cold temperatures.There are flagella genes in Lm and flagella of Lm were produced below at 30℃。But flagella of Lm are not produced in the host（37℃）as it would be recognized by the pattern receptors of immune cells,stimulating the body to clear bacteria.The results of early stage in this study showed that the production of flagella in Lm at 4℃were significantly less than the it produced at 28℃or even not produced,indicating that flagella production of Lm was inhibited during cold temperatures.Subsequently,this study conducted a preliminary exploration of the mechanism behind this phenomenon,providing new information for revealing the mechanism of cold temperature growth of Lm.And it will provide a theoretical basis for the development of prevention and control strategies for the growth of Lm at cold temperature.Research has shown that because the repressor MogR（mobility gene repressor）inhibits the transcription of flagella genes,flagella of Lm were not produced at 37℃.Is the production of flagella in Lm less or absence at 4℃also due to the inhibition by MogR?This study answered this question by constructing deletion strains and complementary strains of flagellin repressor protein MogR and flagellin protein Fla A,as well as measuring the motility,flagella production,expression of flagella genes,and growth curves of the constructed strains at 4℃.The results are as follows.1.Flagellar expression level of different serotypes Lm strains at 4℃.4b,1/2a,1/2b,and 1/2c are the prevalent serotypes of Lm in food isolates.In this study,these four serotypes’strains were used and their motility and the expression of flagella gene fla A were detected at 37℃,28℃,and 4℃by Swarming assay and RT-q PCR.Results showed that the motility of strains and the relative expression of fla A were significantly decreased in all Lm strains at 4°C than at 28°C（P<0.05）.And the results at 37℃are consistent with those at 4℃.These indicate that low expression of flagella at cold temperatures of Lm is a common phenomenon and not serotype specific.So,we selected Lm ATCC19115（4b）strain as the research material and as the wild strain to conducted the following research.2.Construction of gene deletion and complement strains of Lm repressor MogR and flagellin protein Fla A.In this study,the suicide temperature-sensitive plasmid pHT304-ts was used as the vector to obtain the gene deletion strainsΔmogR andΔfla A through homologous recombination.The shuttle plasmid p HT304 was used as the expression vector to obtained complementary strains cΔmogR and cΔfla A which include the native promoter.The phenotypic experiment results showed successful the complementary strains is success and could express effectively.3.Determination of motility,flagellar production,and flagella genes relative expression of the deletion and complement strains at 4℃.（1）Bacterial motility measurement（Swarming assay）.The determination of motion circles and the size of it by Swarming assay can indirectly reflect whether bacteria produce flagella and the number of flagella.The results showed that,at 28°C,ΔflaA did not motile,while cΔflaA,ΔmogR and cΔflaA were motile and not different from wild strains.At 4℃,the diameter of motion circles ofΔmogR were significantly larger than that of wild strains（P<0.01）,butΔfla A,cΔmogR and cΔfla A were all non-motile like the wild strain,which consistent with the results at 37°C.It indicates that the motility of Lm at 4℃is related to MogR.（2）Observation of bacterial flagella by transmission electron microscope（TEM）.It was observed through TEM that the number of flagella inΔmogR were significantly more than wild strains at 4°C（P<0.001）,while there was no significant difference in flagella quantity betweenΔflaA,cΔmogR,cΔflaA and their wild strains.Which consistent with the TEM results at 37°C.At 28°C,Δfla A without flagella,whileΔfla A,cΔmogR and cΔfla A not only have flagella but also no significant difference from wild strains.These results indicate that MogR inhibits the production of flagella at 4℃.（3）Determination the expression level of flagellar gene by RT-qPCR.The expression level of the flagellar genes including flaA,flgE,fliN,flhB,fliM,flgK,fliF and fli I,which were selected based on their distribution and functional were detected by RT-q PCR.The results showed that when at 28℃,the expression level of these flagellar genes in both ofΔmogR and cΔmogR were no difference from the wild strains.At 4°C,the relative expression level of these flagellar genes inΔmogR were significantly up-regulated（P<0.01）compared to wild strains,while there was no significantly difference in the expression level of these flagellar genes between cΔmogR and wild strains,which were consistent with at 37°C.These results indicated that MogR has an inhibitory effect on the expression of flagella genes at 4℃.4.Determination of growth characteristics of deletion and complementary strains.ΔmogR,cΔmogR,ΔflaA,cΔflaA and ATCC19115 were cultured with medium BHI,and the growth curve was drawn used OD₆₀₀ value which were measured regularly.The differences of growth ability of strains were compared by calculate the area under the curve（ΔAUC）.The results showed that at 28°C,there was no significant difference in the growth of both the deletion strains and its complementation strains compared to the wild strains.However,at 4°C,the growth ability of theΔmogR and cΔfla A were decreased compared with the wild strains（P<0.05）.The growth of the cΔmogR andΔfla A were significantly grow faster than the wild strains（P<0.01）.The differences of growth ability of strains at37℃were consistent with that at 4℃.These results indicated that the inhibition of flagella production by MogR at 4℃is beneficial for the growth of Lm at cold temperatures.In concluding,at 4℃,the suppressor protein MogR reduces flagella production of Listeria monocytogenes by inhibiting the transcriptional expression of the flagella genes,which affects the growth ability of the cell under cold temperature conditions.