Measuring the transcriptional effect of the Caulobacter cckA(ts) mutation and genetic suppression of the cckA(ts) phenotype by loss-of-function mutations in ntrC.

We have discovered an unusual two-tiered pattern of suppression in Caulobacter whereby temperature sensitive mutations in the "essential" cell cycle regulatory histidine kinase, CckA, are variably rescued by structurally distinct loss-of-function mutations in ntrC. NtrC regulates nitrogen assimilation and contains structural features that indicate interaction with s70 RNA polymerase to control transcription. Considering that NtrC is a transcription factor, we hypothesized several mechanisms through which loss-of-function mutations in ntrC could rescue cckA(ts) conditional lethality through its effect on transcription: (a) ntrC mutations in a cckA(ts) background broadly restore Caulobacter gene expression to wild-type levels, (b) ntrC mutations shift gene expression into a state entirely distinct from both wild-type and the primary mutant, or (c) a combination of these two effects. To assess these models, we used RNA sequencing (RNA-seq) to measure the effect of shifting the cckA(ts) strain to the restrictive temperature and the global transcriptional impact of combining loss-of-function ntrC mutations with cckA(ts) at the restrictive temperature. Overall design: We cultivated the cckA(ts) strain and three additional strains in which we combing ntrC mutations (L242P, A446P, and in-frame deletion of ntrC) with the cckA(ts) mutations. These strains were grown at 37 degrees C to mid-log phase and total RNA was isolated by Trizol extraction followed by alcohol precipitation. RNA-sequencing libraries were prepared utilizing the Illumina TruSeq Stranded RNA Kit and sequenced on the Illumina NextSeq 500 platform at SeqCenter (Pittsburgh, PA).