Microarray-based transcriptional analyses of stationary phase-phenomena and stress responses in Clostridium acetobutylicum.

摘要

DNA-microarrays were developed for global transcriptional analysis of Clostridium acetobutylicum. Analysis of the spo0A-knockout strain SKO1 confirmed major solventogenic genes were downregulated in the absence of spo0A as were early sporulation genes sigF, spoVS, and spoIIAB while motility genes were upregulated. The accelerated sporulation phenotype of spo0A-overexpressing strain 824(pMSPOA) upregulated fatty acid biosynthesis- and decreased glycolytic-gene expression, corresponding to decreased glycolytic metabolic flux. Butanol challenge transcriptional data of four strains at various butanol levels revealed genes differentially upregulated by all strains included solvent production and stress response genes, including chaperones and proteases.; To identify global transcriptional changes during the transition from exponential growth and acidogenesis to stationary phase and solventogenesis, microarray analysis was performed with batch fermentation RNA samples. Functional group analysis showed translation- and motility-related genes were downregulated during the switch. Upregulated genes included solventogenic genes, some amino acid-biosynthesis pathways, heat-shock proteins, most pSOLI megaplasmid genes, and iron transport genes. Early sporulation genes like spo0A, the sigF operon, and putative canonical SigH-regulated genes were upregulated within 7.5 h of the onset of solventogenesis and sporulation, confirming patterns observed in Bacillus subtilis. Expression of later sporulation genes was not observed, suggesting sporulation time length in C. acetobutylicum and B. subtilis is significantly different.; Transcriptional stress responses to the metabolites acetate, butyrate, and butanol were compared. Genes upregulated by all three stresses included chaperones and proteases, the solventogenic sol operon, and putative phase-related genes. Acetate stress downregulated butyryl-CoA- and butyrate-formation genes while butyrate stress downregulated acetate-formation genes. spo0A but not other sporulation-related genes was expressed slightly higher after all three stresses, suggesting sporulation is not part of the metabolite stress response. Pyrimidine biosynthesis genes were downregulated by metabolite stress, but purine biosynthesis genes were upregulated by acetate and butyrate. Acetate stress upregulated expression of methionine biosynthesis genes, but butyrate stress downregulated most sulfur-uptake and cysteine- and methionine-biosynthesis genes. Butanol stress upregulated some glycerol metabolism genes, which was also observed in solvent-tolerant Escherichia coli strains. Metabolite stress transcriptional patterns were also observed during batch fermentation after the onset of stationary phase and solventogenesis, suggesting consistency between the experiments.