Heterologous expression of genes in the anaerobic bacterium Streptococcus bovis
The main objective of this work was to investigate the expression of xylanase and cellulose genes from Ruminococcus flavefaciens when introduced into related Gram-positive bacteria including the rumen bacterium Streptococcus bovis. In addition the discovery, and isolation of the β(1,3-1,4)-glucanase gene of S. bovis in the course of this work may provide new possibilities to express foreign genes in S. bovis or in other Gram-positive bacteria. The main findings of this work are summarised below: 1. Genes encoding polysaccharidase activity from the strictly anaerobic rumen bacterium R. flavefaciens can be transferred by electroporation into the ruminal bacterium S. bovis and into other Gram-positive bacteria from different habitats, including Lactococcus lactis, Enterococcus faecalis and Streptococcus sanguis. 2. XynD and endA genes of R. flavefaciens can be expressed from their own promoters in these species. Among the bacteria used as hosts for gene expression, S. bovis gave the highest yields of active enzyme. The expression levels of both gene products were found to be higher in S. bovis than in E. coli. 3. The R. flavefaciens enzymes were mainly secreted in the culture medium of S. bovis; however in E. coli they were mainly cell-associated. 4. The full-length enzyme of xynD was detected in several Gram-positive bacteria, suggesting the effect of proteases may be less than in E. coli. 5. The general rearrangement of the introduced plasmid and genes were not found in Gram-positive bacteria and the genes seem to be stable in these organisms. However rearrangement of xynD was observed in some transformants of S. bovis JB1, although non-rearranged transformants were also obtained. 6. Expression of endA and xynD activity was affected by energy sources supplied to S. bovis cultures, reflecting the accumulation of lactic acid.