Single-cell Gene Expression Analysis Of Transcriptional Heterogeneity In Monoculture And Dual-culture Of Desulfovibrio Vulgaris

Sulfate-reducing bacteria(SRB)are the most common anaerobic microorganisms which play an extremely important role in the global carbon and sulfur cycles.In order to thoroughly research metabolic regulation mechanisms of SRB in two lifestyles,we have developed and optimized a set of relatively mature coculture culture technique and single-cell RT-qPCR analysis.We used a model sulfate-reducing bacteria,Desulfovibrio vulgaris,as a model to conduct three comparative analyses: 1)Comparison of the gene-expression differences between D.vulgaris monoculture(sulfidogenic metabolism)and syntrophic dual-culture with Methanosarcina barkeri;2)Under the sulfidogenic metabolism,comparison of the gene-expression differences between D.vulgaris biofilm and planktonic cells;3)Under the syntrophic metabolism,comparison of the gene-expression differences between the processes from D.vulgaris monoculture metabolism to the formation and maintainance of coculture metabolism with M.barkeri to reveal microevolution of D.vulgaris-Methanosarcina barkeri dualculture.It was confirmed at the single-cell level the existence of the specific genes to be responsible for the maintainence of symbiotic relationship(eg.,gene cluster(DVU0148-DVU0150))in the cells,and obvious gene-expression heterogeneity was also found in D.vulgaris monoculture and syntrophic dual-culture with Methanosarcina barkeri.The results contributed to the understanding of the molecular mechanisms of metabolic regulation of microbial coculture;By comparing the geneexpression levels between the biofilm and planktonic culture cells at single-cell level,significant gene-expression heterogeneity was found existing between single cells in both biofilm and planktonic culture,and the heterogeneity levels of biofilm cells were obviously higher than planktonic cells increased significantly.In addition,to investigate the microevolution of the formation and maintainence of microbial symbiotic systems,firstly,we applied genome re-sequencing technology to the evoluted D vulgaris strains after 38 mM and 50 m M continuous symbiotic growth(E38,E50),respectively,and 12 and 11 genetic mutants were identified in E38 and E50 strains,respectively.Moreover,we used the single-cell based RT-qPCR analysis to quantitatively analyze the variation of 8 muted genes of D vulgaris from the original monoculture to the mature and stable symbiotic system.The results showed the geneexpression heterogeneity of the 8 mutation genes was obviously increased in D vulgaris strains when compared between coculture to monoculture,which may be resulted from the complexity of microflora structures and the enhancement of the communication between cells,while the heterogeneity was decreased with symbiotic transfers,probably because the gene-expression levels of the cells were adjusted to rapidly adapt to the new environment.Moreover,we also found the gene-expression levels of 8 muted genes were obviously upregulated in coculture when compared with monoculture,suggesting the muted genes might play roles in formation and maintainance of the symbiotic metabolism.In summary,using the single-cell analysis combined with high-throughput genome sequencing technology,a comprehensive analysis of transcriptional levels and geneexpression heterogeneity in two lifestyles(sulfidogenic and syntrophic metabolism)of D.vulgaris was conducted.The results showed that some special genes were related to the regulation and metabolism in the symiotic evolution process,and the relationship between some genes and symbiotic or biofilm metabolism was identified.The results contributed to the better understanding of D.vulgaris genetics,physiology and evolution and mutualistic interactions in mix-culture microorganisms from different species.