Butanol tolerance of Clostridium beijerinckii NCIMB 8052 associated with down-regulation of gldA by antisense RNA.

Strain BR54 of Clostridium beijerinckii was derived from the wild type strain, NCIMB 8052, by mutagenesis with Tn1545 and selection for butanol tolerance. It harbours a single copy of Tn 1545 in a 435 bp intergenic region separating two convergently transcribed genes, accC and gldA. The former encodes biotin carboxylase (E.C.6.3.4.14), a subunit of acetyl-CoA carboxylase and the latter encodes glycerol dehydrogenase (E.C.1.1.1.6). Since Tn1545 generates outwardly directed transcripts from its right end, we considered the possibility that the transposon inserted in strain BR54 might affect the expression of the adjacent gldA gene. RT-PCR experiments revealed that the mutant, but not the wild type, contains antisense RNA corresponding to the gldA gene. Correlated with this, the level of glycerol dehydrogenase activity in the mutant was only 25% of that in the wild type when bacteria were grown with either glucose or glycerol as the fermentable substrate. We conclude that transcripts emerging from the right end of the conjugative transposon, Tn1545, can reduce the expression of the adjacent gldA gene by the generation of antisense RNA and that this is associated with a butanol-tolerant phenotype.