A Preliminary Study On The Cross-feeding Relationship Between Two Vitamin Auxotrophic Bacterial Strains

Vitamin B(VB)contains a variety of water-soluble vitamins,which can be used as coenzymes in various-biochemical and metabolic processes of organisms.B vitamins are essential for the life activities of microorganisms and play an important role in the formation and succession of microbial communities.However,in recent years,biochemical analysis and genomics evidence indicate that many microorganisms cannot de novo synthesize all the essential vitamins.These auxotrophic microorganisms require exogenous vitamins to maintain normal life activities.In natural environment,auxotrophic microorganisms may obtain vitamins through cross-feeding relationship with prototrophs.Therefore,the vitamin cross-feeding of bacteria plays an important role in the physiological functions and community structure of microorganisms.Microorganisms in lakes play an important role in the balance of the ecosystem and the biogeochemical cycle of elements.The results of our previous microbiome research in Wuhan lakes revealed the composition and diversity of lake microbial communities.It is speculated that there will be specific cross-feeding relationship between vitamin auxotrophic and prototrophic microorganisms,and the microorganism producing vitamins may have an important influence as the key species within the microbial community.However,we still lack understanding of its specific physiological functions and mechanisms.In this study,we screened and isolated vitamin auxotrophic microorganisms in the lake samples in Wuhan,and tested the cross-feeding relationship between species in co-cultivation system.The main contents of this research are as follows:(1)Screening of B vitamin auxotrophic strains and prototrophic bacteria.The water samples of Nan Lake were enriched and used to isolate bacteria.We obtained two auxotrophic bacteria 12-19(VB7-deficient strains)and P1(VB1-deficient strain)and 6 prototrophic bacterial strains.Their classification relationships were preliminarily determined based on 16S rRNA gene sequence analysis.(2)Cross-feeding experiment of strains.B vitamin auxotrophic strains grow slowly when grow alone,and must be supplemented with exogenous vitamins.The results showed that the two auxotrophic bacteria can promote each other's growth when they are co-cultured,and this effect was similar to those with vitamin supplementation.The two auxotrophic bacteria can complement each other with vitamins.When the auxotrophic strains are cultured in a minimal medium with the prototrophic strains,only the prototrophic strain Y1 has a promotion effect on the VB1-deficient strain P1,while the other prototrophic strains have no obvious promotion effect,which imply that vitamin cross-feeding can be in a species-specific manner.(3)Genomics analysis of bacterial strains.Genomic sequencing results indicate bacteria strain 12-19 VB7 biosynthesis pathway is incomplete but has a complete VB1 synthesis pathway;P1 contains VB7 synthesis pathway but do not have the complete synthesis pathway of VB1.These results are consistent with the above physiological phenotypes and provide a genetic information basis for further elucidating the mechanism of the complementary relationship between the two vitamins.(4)Proteomics analysis.Proteomics analysis results showed that the VB1 related synthetic genes and transporters in strains 12-19 in the co-culture system are up-regulated,and a number of proteins involved in cell growth,metabolism and other pathways are differentially regulated.These proteins may play an important role in the cross-feeding between the two strains.In summary,in this study we isolated B vitamin auxotrophic bacteria and tested the species specificity of VB complementation between auxotrophic bacteria and prototrophic bacteria.These data will be helpful for understanding the role of VB cross-feeding in microbial community shaping and its molecular mechanism.