Screening of Selected Transcription Factors Identifies Strategies for Osmotolerance in Caldicellulosiruptor bescii

Through phenotype screening of single-gene deletion mutants of regulatory genes in C. bescii, two strains of C. bescii showing growth phenotypes in elevated osmolarity conditions were isolated. The first strain carried a deletion of the FapR fatty acid biosynthesis and malonyl-CoA metabolism repressor. This strain had a severe growth defect relative to its genetic parent strain when grown in high-osmolarity conditions introduced through the addition of ethanol, NaCl, glycerol, or glucose to growth media. The second strain carried a deletion of the FruR/Cra carbon metabolism regulator, which conversely displayed a growth rate over three times higher than its genetic parent when grown in high-osmolarity media containing NaCl. Similar growth improvements were seen in elevated levels of added ethanol, glycerol, or glucose as well. RNAseq analysis of mutant and parent strains, along previous predictions, were used to characterize the C. bescii FapR and FruR/Cra regulons. We find evidence of both local repression and novel global gene regulation by the FruR/Cra protein in C. bescii, though we are unable to determine if global gene expression differences are due directly or indirectly to the FruR/Cra protein. Genes exhibiting differential expression in a single-gene deletion of the fapR/cra gene include fructose metabolism genes collocated with the regulator, a transposase, sigma factor E, sigma factor G, two hypothetical proteins and a vaguely annotated oxidoreductase. We sought to generate an even more osmotolerant strain of C. bescii through overexpressing the FapR protein in a genetic background deficient of the fapR/cra gene. This strain did not exhibit osmotolerance any different from its genetic parent strain housing an empty expression vector. A genetic complement of a fruR/cra-deficient strain exhibited improved growth over a fruR/cra-deficient strain housing an empty expression vector, suggesting other genomic differences between these strains are responsible for the growth phenotype observed in the Δcra strain of C. bescii. Proteomic determination of unique peptides confirms heterologous expression of the FruR/Cra in the genetic complement of the cra-deficient mutant, suggesting inadequate complementation was not the reason a phenotype reversion was not observed in the cra-deficient mutant. Genome resequencing identified several genomic alterations in this strain which may be responsible for the observed increase in osmolarity tolerance in the cra-deficient mutant, including a substitution mutation in the dnaK, a gene previously implicated in osmoresistance in bacteria.