Whole Genome Sequencing And Function Analysis Of CpsWc Gene Cluster Of Lactiplantibacillus Plantarum LTC-113

Probiotic is an ideal substitution for antibiotic,maintaining the integrity of gastrointestinal epithelial barrier and preventing the infection by pathogenic bacteria.Whereas,recent studies revealed that limited tolerance to harsh environmental stresses and weak adhesion to the epithelial cells of the host gastrointestinal tract by probiotic significantly restricted its performance.To tackle this problem,genetic engineering may pave a way to fundamentally reshape the performance of probiotics.The bacterial cell surface-attached exopolysaccharides(EPSs)are often involved in the manifestation of the probiotic property of L.plantarum.We have previously identified a probiotic L.plantarum strain LTC-113 isolated from the intestine of a healthy Tibet local chicken.LTC-113 can protect newly-hatched chicks from intestinal barrier disruption by Salmonella typhimurium infection via regulating the expression of tight junction genes and inflammatory meditators and decreasing the S.typhimurium colonization.Moreover,another study also found LTC-113 significantly decreased chickens' mortality and improved the health status of chickens breeding in high altitude regions with lower oxygen levels.In this study,we sought to further investigate the molecular basis of the probiotic properties of LTC-113.Therefore,we first hypothesized that the L.plantarum EPSs might play an essential role.Here,the EPS-synthetic gene clusters in LTC-113 were comprehensively characterized through whole-genome sequencing and comparative genomic analyses.Intriguingly,we have first identified a cpsWc gene cluster originated from Weissella cibaria was integrated into a large L.plantarum plasmid(p YZ1)in LTC-113 possibly via interspecies horizontal gene transfer.Through biochemical and biological characterizations,we found the foreign-origin cpsWc gene cluster significantly contributed to the probiotic properties of the host strain.These findings lay the groundwork for understanding the effect of EPS on the probiotic activity of L.plantarum,and pave the way for engineering novel probiotic L.plantarum strains.The main results of this study are as following:(1)whole-genome sequencing of LTC-113 was carried out to systematically investigate the molecular basis of EPS synthesis in L.plantarum LTC-113.The complete genome of LTC-113 contains a single circular chromosome of 3.14 Mb with a large plasmid of 68.1kb,designated as p YZ1 in this study.Four cps gene clusters,cps2,cps3,cps4,and cpsWc,responsible for EPS biosynthesis,were identified in LTC-113.Two of them(cps3 and cps4)are located on the chromosome,and another two(cps2 and cpsWc)are present in the plasmid p YZ1.The L.plantarum originated cps gene clusters,cps2,cps3,and cps4,are incomplete by missing Wzy polymerase or Wzx flippase.More importantly,we have first identified a cpsWc gene cluster originated from W.cibaria horizontally acquired by interspecies gene transfer.(2)The newly identified cpsWc gene cluster conferred the host strain enhanced biofilm formation ability and boosted tolerance to harsh environmental stresses,including heat,acid,and bile.The cpsWc also elevated the production of EPS that secreted into the culture supernatant,and reduced the final bacteria yield in culture by consuming more cellular resources.In addition,an LCP family protein was identified in the cpsWc gene cluster,which controlled the fate of synthesized EPS that either attaching to the cell-wall peptidoglycan or releasing to culture supernatant.The LCP family protein also significantly contribute to the protection of the bacteria.(3)The cpsWc gene cluster strengthened the ability of L.plantarum strains to adhere intestine epithelial cells,and exhibiting inhibitory effect on the adhesion of pathogenic bacteria,such as S.typhimurium and E.coli.The LCP family protein also contributed to the performance of LTC-113 strain.(4)Introduction of the cpsWc gene cluster into other L.plantarum strains generally transferred the protective effects that observed in LTC-113 to tolerate heat,acid,and bile.However,the performance in transgenic L.plantarum strains differed that the adhesion to Caco-2 cells and the inhibitory effects to S.typhimurium were boosted in some strains,whereas adverse effects were also observed in other strains,possibly owning to the differed strain-specific adhesion mechanism.In conclusion,we have identified a new EPS-synthetic gene cluster,cpsWc,carrying 13 genes,laid on a large plasmid in a well-characterized probiotic L.plantarum strain LTC-113.The cpsWc gene cluster was proposed to be acquired by interspecies horizontal gene transfer from W.cibaria,conferring the host strain enhanced biofilm formation ability and boosted tolerance to harsh environmental stresses,including heat,acid,and bile.Moreover,the introduction of the cpsWc gene cluster into other L.plantarum also transferred the effects on probiotic properties.Furthermore,the cpsWc gene cluster also strengthened the ability of L.plantarum strains to adhere intestine epithelial cells,and exhibiting inhibitory effect on the adhesion of pathogenic bacteria,such as S.typhimurium and E.coli.These works provided insights into the molecular basis for the EPS-derived probiotic activity of L.plantarum.They also opened an avenue for engineering novel probiotic L.plantarum strains by introducing foreign EPS-synthetic gene clusters.