TCSLisK/R Plays A Role In Listeria Monocytogenes Growth At Cold Temperatures By Regulating The Expression Of Flagellar Genes

Listeria monocytogenes is a gram-positive food-borne pathogen that causes listeriosis,a zoonotic disease with a fatality rate of 20-30%.Lm is highly adaptable to the environment,especially capable of growing and reproducing at low temperatures.At present,cold temperature is the most important way to preserve food.Lm can grow under refrigeration temperature,which undoubtedly brings great risks to food safety,and the longer the refrigeration time,the higher the food safety risk.Therefore,it is of great significance to control the growth of Lm at cold temperature.Exploring the cold-temperature growth mechanism of Lm will provide a theoretical basis for the formulation of measures to control the cold-temperature growth of Lm and the development of cold-temperature growth inhibitors.Two-component signal transduction systems(TCSs)are important regulatory systems for bacteria to adjust gene expression by sensing external signals to adapt to the environment and survive.It consists of signal-sensing histidine kinase(HK)and It is composed of response regulator(RR)with transcription factor function.The research group analyzed and summarized the TCSs of the Lm food isolate LM201,and found that LM201 has 14 pairs of TCSs(HK/RR)and two orphan RRs.The research group found that one pair of TCS,namely Lis K/R HK,lacks(?lisK,The corresponding deletion strain was named S0192),which led to a significant decrease in the growth rate of Lm at a low temperature of 4?.This study conducted a preliminary study on the mechanism behind this phenomenon.In this study,the Lis K/R RR-deficient strain S2001(?lisR)and the Lis K/R-deficient strain S0192c(?lisKc)and S2001c(?lisRc)were constructed.The 4°C and 37°C growth curves of the missing strain and the complementing strain were measured.RNA-Seq was performed on the deletion strain S0192(?lisK)and the parent strain LM201 growing at a low temperature of 4?.Analyzing the RNA-Seq data,it was found that the expression of S0192(?lisK)flagella gene was significantly up-regulated.Subsequently,the lack of Lis K/R strains and complement strains were tested for motility measurement at a low temperature of 4?,observation of bacterial flagella with transmission electron microscopy,and RT-qPCR measurement.The results show that Lis K/R affects the cold-temperature growth of Lm by regulating the expression of flagellar genes.The results are summarized as follows:1.Construction of the deletion strain S2001(?lisR)and the complement strain S0192c(?lisKc)and S2001c(?lisRc)Using LM201 as the parent strain,the upstream and downstream homology arms of lisR were amplified,and the homology arms were connected with the suicide temperature-sensitive plasmid p HT304-ts to obtain the recombinant suicide plasmid p1001,which was introduced into LM201.The lisR deletion was completed by homologous exchange,and the lisR deletion strain S2001 was obtained.The genes lisK and lisR were amplified separately and connected to the expression plasmid p HT304 respectively.Recombinant plasmids plisKc and plisRc were obtained,and they were electrotransformed into the corresponding deletion strains to obtain the complementing strains S0192c(?lisKc)and S2001c(?lisRc).2.Determination of growth curves of Lis K/R deletion strains and replenishment strainsS0192(?lisK),S2001(?lisR),S0192c(?lisKc),S2001c(?lisRc)and LM201 were cultured with medium BHI at 4°C and 37°C,and the growth curve was drawn by measuring the OD₆₀₀value.The results showed that the bacterial growth rate of the deleted strains S0192 and S2001 was significantly lower than that of the parent strain LM201 in the logarithmic growth phase and the stable phase at 4°C.The growth of the complementing strains S0192c and S2001c was consistent with that of the parent strain.There were no significant differences in the growth phenotypes of the deletion strain,the deletion filling strain and the parent strain at 37°C.The results showed that the two-component system Lis K/R has an effect on the low-temperature growth of Lm.3.Transcriptome sequencing analysis of the deletion strain S0192(?lisK)and the parent strain LM201 at 4?Transcriptome sequencing(RNA-Seq)was performed on the deleted strain S0192(?lisK)and the parent strain LM201 grown at 4?.Analysis of the sequencing results showed that compared with the parent strain LM201,the expression of flagellar motility genes in the deleted strain S0192 was significantly up-regulated,suggesting that the two-component system Lis K/R has a regulatory effect on flagellar gene expression at a low temperature of 4?.4.Determination of motility,flagella growth and flagella gene expression of Lis K/R-deficient strains and replenishment strains at 4?In order to verify the hypothesis that Lis K/R has a regulatory effect on flagellar gene expression at a low temperature of 4?,the following three experiments were done in this study.(1)Bacterial motility measurementS0192(?lisK),S2001(?lisR),S0192c(?lisKc),S2001c(?lisRc)and LM201 were inoculated on low agar(0.25%)BHI plates and placed at 4°C for 4d and 37°C for 48h,respectively.Judge the motility of bacteria by measuring the growth diameter of the inoculation point.The results showed that,at 4?,the growth diameter of the inoculation point of the deletion strains S0192 and S2001 was significantly larger than that of the parent strain LM201(P<0.01).The motility is significantly stronger than the parent strain.The motility of S0192c and S2001c is the same as that of LM201.At 37?,there is no significant difference between the strains.The results showed that the motility of the Lis K/R-deficient strain was significantly stronger than that of the parent strain LM201 at a low temperature of 4?.(2)Transmission electron microscope(TEM)observation of bacterial flagellaSamples of S0192(?lisK),S2001(?lisR),S0192c(?lisKc),S2001c(?lisRc)and LM201 cells cultured to the logarithmic phase at 4°C and 37°C were taken.The samples were negatively stained,a copper mesh was prepared,and the bacterial flagella were observed under TEM.The results of microscopy showed that the missing strains S0192and S2001 had flagella all over the body,and the amount of flagella was much higher than that of the parent strain LM201.No flagella was seen in the complement strain S0192c,and only a small amount of flagella remained around the bacteria of the complement strain S2001c.At 37°C,there was no flagella growth in the cells of the deletion strain,the complement strain and the parent strain.The results showed that the growth of the flagella of the Lis K/R-deficient strain was significantly higher than that of the parent strain LM201 at a low temperature of 4?.(3)RT-qPCR determination of flagellar gene transcript expressionS2001(?lisR),S0192c(?lisKc),S2001c(?lisRc)and LM201 cells cultured to logarithmic phase at 4°C were taken,and the cell RNA was extracted.Reverse transcription real-time quantitative PCR(RT-qPCR)is used to detect flagellar gene expression.The results showed that compared with the parent strain LM201,the expression of flagellar genes flg B,flg D,fli M,fli R,mot A,and mot B of the deleted strains S0192 and S2001 were significantly up-regulated(P<0.05),these flagellar genes of the annulus S0192c and S2001c compared with the corresponding deletion strain,the expression of is significantly down-regulated.The results showed that the expression of flagellar gene transcripts of Lis K/R-deficient strain was significantly higher than that of the parent strain LM201 at a low temperature of 4?.The above three experimental results confirm the results of transcriptome sequencing.The expression and assembly of flagella genes is very energy-consuming.Reducing the expression of flagella genes is an energy saving strategy adopted by Listeria monocytogenes in harsh environments.The results of this study showed that at a cold temperature of 4?,Lis K/R down-regulates or inhibits the expression of flagella genes,and the production of flagella in the bacteria was reduced,and the energy saved was used for the growth and reproduction of the bacteria.After the loss of Lis K/R,this regulatory effect was not,and the bacteria produced a large number of flagella,but the energy used for growth and reproduction was reduced,causing the growth and reproduction of the missing strain to be significantly slow.Therefore,Lis K/R affects the growth and reproduction of Listeria monocytogenes under cold temperature conditions by regulating the expression of flagella genes.