Molecular basis of the phenotypic variants arising from a Pseudoalteromonas lipolytica mutator.

Bacterial deficiencies in the DNA repair system can produce mutator strains that promote adaptive microevolution. However, the role of mutator strains in marine Pseudoalteromonas, capable of generating various gain-of-function genetic variants within biofilms, remains largely unknown. In this study, inactivation of mutS in Pseudoalteromonas lipolytica conferred an approximately 100-fold increased resistance to various antibiotics, including ciprofloxacin, rifampicin and aminoglycoside. Furthermore, the mutator of P. lipolytica generated variants that displayed enhanced biofilm formation but reduced swimming motility, indicating a high phenotypic diversity within the ΔmutS population. Additionally, we observed a significant production rate of approximately 50 % for the translucent variants, which play important roles in biofilm formation, when the ΔmutS strain was cultured on agar plates or under shaking conditions. Using whole-genome deep-sequencing combined with genetic manipulation, we demonstrated that point mutations in AT00_17115 within the capsular biosynthesis cluster were responsible for the generation of translucent variants in the ΔmutS subpopulation, while mutations in flagellar genes fliI and flgP led to a decrease in swimming motility. Collectively, this study reveals a specific mutator-driven evolution in P. lipolytica, characterized by substantial genetic and phenotypic diversification, thereby offering a reservoir of genetic attributes associated with microbial fitness.

Molecular Basis of Wrinkled Variants Isolated From Pseudoalteromonas lipolytica Biofilms.

Many Pseudoalteromonas species are dominant biofilm-forming Gammaproteobacteria in the ocean. The formation of Pseudoalteromonas biofilms is often accompanied by the occurrence of variants with different colony morphologies that may exhibit increased marine antifouling or anticorrosion activities. However, the genetic basis of the occurrence of these variants remains largely unexplored. In this study, we identified that wrinkled variants of P. lipolytica mainly arose due to mutations in the AT00_08765, a wspF-like gene, that are associated with decreased swimming motility and increased cellulose production. Moreover, we found that the spontaneous mutation in flhA, encoding a flagellar biosynthesis protein, also caused a wrinkled colony morphology that is associated with cellulose overproduction, indicating that flhA plays a dual role in controlling flagellar assembly and polysaccharide production in P. lipolytica. Investigation of wrinkled variants harboring spontaneous mutation in dgcB, encoding a GGDEF domain protein, also demonstrated dgcB plays an important role in regulating cellulose production and swimming motility. In addition, by screening the suppressor of the AT00_08765 variant strain, we also identified that the spontaneous mutation in cheR and bcsC directly abolished the wrinkled phenotype of the AT00_08765 variant strain, suggesting that the chemosensory signaling transduction and cellulose production are crucial for the determination of the wrinkled phenotype in P. lipolytica. Taken together, this study provides insights into the genetic variation within biofilms of P. lipolytica.