DNA-array based transcriptional analysis of solvent tolerance and degeneration in Clostridium acetobutylicum: groESL overexpression, butanol challenges and a novel DNA-array normalization and gene identification method.

摘要

DNA-microarray and Western analyses were used to examine the effects of groESL overexpression and host-plasmid interactions on solvent production in Clostridium acetobutylicum ATCC 824. Strain 824(pGROE1) was created to overexpress the groESL operon genes from a clostridial thiolase promoter. 824(pGROE1) produced more solvents than the wild type (WT) and plasmid control strains, had increased expression of the motility and chemotaxis genes and decreased expression of other major stress response genes. It is proposed that groESL modulates the CIRCE regulon, which is shown here to include the hsp90 gene. Growth and glucose metabolism of 824(pGROE1) was less inhibited by butanol challenges which was shown to illicit a dose-dependent increased expression of all major stress-protein genes, major solvent-formation genes, the butyrate formation genes, the butyryl-CoA biosynthesis operon, and a gene with homology to kinA. A dose-dependent decreased expression was observed for the genes of the major fatty-acid synthesis operon, several glycolytic genes, and only a few sporulation genes. It is suggested that the butanol-stress initiated induction of the solventogenic genes was due to increased spo0A expression and enhanced Spo0A phosphorylation mediated by a KinA homolog. A complex host-plasmid interaction was demonstrated, resulting in prolonged, biphasic growth and metabolism, decreased expression of DNA gyrases, differential expression of primary metabolism genes, altered cell division and sporulation patterns, and elevated stress protein expression.; To facilitate large-scale transcriptional analysis, glass DNA arrays containing a select set of 1019 genes (more than 25% genome coverage) were designed, constructed and validated for data reliability. Transcriptional analysis of the degenerate, asporogenous and non-solventogenic Clostridium acetobutylicum strain M5 was utilized in the validation process. Strain M5 lacks the pSOL1 megaplasmid. Analysis of M5 array data using a new DNA-array normalization and gene identification method resulted in proper identification of 99.4% of the pSOL1 genes. Notable among other genes expressed higher in WT compared to M5 were sigF, several two-component histidine kinases, spo0A, cheA, cheC, stress response genes, fts family genes, DNA topoisomerases, and central-carbon metabolism genes. Genes expressed higher in M5 include electron transport genes and motility and chemotaxis genes.