Study On Effect Of Transcriptional Factor CodY In The Listeria Monocytogenes

Listeria monocytogenes is an important food borne pathogen,caused disease known as listeriosis.CodY is a global transcriptional regulator that is widely found in Gram-positive bacteria with low G+C levels and can regulate carbon metabolism,nitrogen metabolism,amino acid biosynthesis,transport and degradation of extracellular substances,flagellar molitity,virulence,early spore formation and other metabolic pathways,as well as cell physiological processes.In this paper,the effect of CodY on the physiological characteristics,flagellar molitity,virulence,biofilm formation of L.monocytogenes was studied and relationships between the carbon metabolism key genes(gdhA)and nitrogen metabolism(glnA,glnR)were also inverstigated.1)The EGDe?codY deletion mutant and the EGDe?codY+pERL3-codY complementation mutant were constructed.2)Bacterial morphology,growth and antibiotic resistance were compared between wild-type,CodY-deficient and complementation strains.The data showed that the deletion of CodY had no significant effect on bacterial morphology,growth in brain heart infusion broth(BHI)and typtic soy broth(TSB),as well as antibiotics(ampicillin,penicillin and chloramphenicol)resistance,but the growth rate of L.monocytogenes in minimal essential medium(MEM)was significantly increased and the ability to against gentamicin sulfate was reduced.3)The capacity of flagellum formation and movement were compared by puncture and motility assay between wild-type,CodY-deficient and complementation strains,the transcriptional expression of flagellum formation and molitity-related genes(motB,motA,fliE,flhA and fliP)were detected by RT-qPCR.The results show that:compared with the wild-type strain,once CodY was deleted,the bacteria could form the inverted umbrella shape as same as the wild-type strain on the soft agar by punctured,and there was no significant difference in microscopic examination after flagellum staining.At the same time,the transcriptional expression of fliE was significantly decreased(P<0.01),no significant change of flhA was found,and the expression of fliP was significantly increased in CodY-deficient strain(P<0.01).All those genes are flagellar structure-related genes.Our results suggest that the effect of CodY-deficient on flagellar structure is quite complex,but overall is limited.After CodY was deleted,the diameter of the circle formed by the swimming and the transcriptional expression of motA,motB were significantly decreased(P<0.01).Suggesting that the deletion of CodY leads to decrease in bacterial flagella molitity.4)The transcriptional expression of the major virulence factor LLO(coding gene hly)and the virulence gene regulator PrfA(coding gene prfA),the hemolytic activity and the semi-lethal dose of Helicoverpa armigera(Hiibner)were compared between the wild-type,CodY-deficient and complementation strains.The results show:compared with the wild-type strain,the transcriptional expression of the major virulence genes hly and prfA,as well as the hemolytic activity of the CodY-deficient strain were significantly reduced(P<0.01);while the semi-lethal dose on Helicoverpa armigera(Hiibner)was increased 5.8 fold,suggesting that the deletion of CodY leads to decrease in bacterial virulence.5)The effect of CodY deletion on the formation of Lm biofilm in BHI and TSB were examined by microplate method.The results showed:compared the wild-type strain,the formation ability of biofilm was significantly enhanced in TSB of CodY-deficient strain,while there is no differecce between wild-type and CodY-deficient strains in BHI,suggesting that the deletion of CodY can enhance the ability of bacterial biofilm formation in a relatively weak environment.6)RT-qPCR was used to detect the transcriptional expression of carbon metabolism key genes(gdhA)and nitrogen metabolism key genes(glnA,glnR)between wild-type,CodY-deficient and complementation strains.The data show that:in the nutrient-rich BHI medium,compared with wild-type strain,the expression of glnA were significantly decreased in the early logarithm and stable periods(P<0.05),while increased in logarithmic medium period of CodY-deficient strain(P<0.01);the expression of glnR were significantly increased in the early and medium logarithum periods(P<0.05),while significant decreased in early stable period of CodY-deficient strain(P<0.01),the transcriptional expression of gdhA were significantly increased in the early logarithm and stable periods(P<0.01),as well as no significant difference in the logarithmic medium period between wild-type and CodY-deficient strains.In the nutrition-poor MEM medium,while CodY was deleted,the expression of glnA in the logarithmic medium and early stable periods were significantly lower of CodY-deficient strain(P<0.05),glnR was significantly increased in the early logarithmic period(P<0.05),while decreased in the logarithmic medium and early stable periods of CodY-deficient strain(P<0.05);gdhA was significantly decreased in the early logarithmic period(P<0.01)and there was no difference in other periods between wild-type and CodY-deficient strains.The above results show that:in L.monocytogenes,CodY plays different roles in the regulation of carbon and nitrogen metabolism-related genes under the different nutritional conditions and growth periods.In conclusions,CodY plays a crucial role in virulence,flagellar molitity,biofilm formation and regulation of carbon and nitrogen metabolism in L.monocytogenes.