Heterologous gene expression in the human gut bacteria Eubacterium rectale and Roseburia inulinivorans by means of conjugative plasmids

Commensal butyrate-producing bacteria in the Firmicutes phylum are abundant in the human intestine and are important for maintaining health. However, understanding of the metabolism and host interaction of these bacteria is limited by the lack of genetic modification techniques. Here we establish a protocol enabling the transfer of autonomously-replicating shuttle vectors by conjugative plasmid transfer from an Escherichia coli donor into representatives of an important sub-group of strictly anaerobic human colonic Firmicutes. Five different plasmid shuttle vectors were tested, each carrying a different origin of replication from Gram-positive bacteria. Plasmid pMTL83151 (pCB102 replicon) were successfully transferred into two strains of Eubacterium rectale, while pMTL83151 and pMTL82151 (pBP1 replicon) were transferred into Roseburia inulinivorans A2-194. Plasmids that carried a Streptococcus bovis JB1 glycoside hydrolase family 16 β-(1,3-1,4)-glucanase gene were constructed and conjugated into Roseburia inulinivorans A2-194 and Eubacterium rectale T1-815, resulting in successful heterologous expression of this introduced enzymatic activity in these two strains of butyrate-producing Firmicutes.