Molecular Organization and Functional Analysis of a Novel Plasmid-Borne Gene Cluster from YC41.

Capsular polysaccharide (CPS) can tightly attach to bacterial surfaces and plays a critical role in protecting microorganisms from environmental stresses. However, the molecular and functional properties of some plasmid-borne gene clusters are poorly understood. In this study, comparative genomics of the draft genomes of 21 Lactiplantibacillus plantarum strains revealed that the specific gene cluster for CPS biosynthesis was observed only in the 8 strains with a ropy phenotype. Furthermore, the complete genomes showed that the specific gene cluster was located on the novel plasmid pYC41 in L. plantarum YC41. analysis confirmed that the gene cluster contained the dTDP-rhamnose precursor biosynthesis operon, the repeating-unit biosynthesis operon, and the gene. The insertional inactivation of the and genes abolished the ropy phenotype and reduced the CPS yields by 93.79% and 96.62%, respectively, in YC41 mutants. These results revealed that the gene cluster was responsible for CPS biosynthesis. Moreover, the survival rates of the YC41-rmlA and YC41- mutants under acid, NaCl, and HO stresses were decreased by 56.47 to 93.67% compared to that of the control strain. Furthermore, the specific gene cluster was also confirmed to play a vital role in CPS biosynthesis in MC2, PG1, and YD2. These findings enhance our understanding of the genetic organization and gene functions of plasmid-borne gene clusters in . Capsular polysaccharide is well known to protect bacteria against various environmental stresses. The gene cluster for CPS biosynthesis is typically organized in the chromosome in bacteria. It is worth noting that complete genome sequencing showed that a novel plasmid pYC41-borne gene cluster was identified in YC41. The gene cluster included the dTDP-rhamnose precursor biosynthesis operon, the repeating-unit biosynthesis operon, and the gene, which was verified by the significantly decreased CPS yield and the absent ropy phenotype in the corresponding mutants. The gene cluster plays an important role in bacterial survival under environmental stress, and the mutants had decreased fitness under stress conditions. The vital role of this specific gene cluster in CPS biosynthesis was also confirmed in other CPS-producing strains. These results advanced a better understanding of the molecular mechanisms of plasmid-borne gene clusters and the protective functionality of CPS.