Coherent feedforward regulation of gene expression byCaulobacterσ^Tand GsrN during hyperosmotic stress

Abstract

GsrN is a conserved small RNA that is under transcriptional control of the general stress sigma factor, σT, and that functions as a post-transcriptional regulator of Caulobacter crescentus survival under multiple stress conditions. We have defined features of GsrN structure that determine survival under hyperosmotic stress, and have applied transcriptomic and proteomic methods to identify regulatory targets of GsrN under hyperosmotic conditions. The 5’ end of GsrN, which includes a conserved cytosine-rich stem loop structure, is necessary for cell survival after osmotic upshock. GsrN both activates and represses gene expression in this stress condition. Expression of an uncharacterized open reading frame predicted to encode a glycine-zipper protein, osrP, is strongly activated by GsrN. Our data support a model in which GsrN physically interacts with osrP mRNA through its 5’ C-rich stem loop to enhance OsrP protein expression. We conclude that sigT, gsrN, and osrP form a coherent feedforward loop in which σT activates gsrN and osrP transcription during stress, and GsrN activates OsrP protein expression at the post-transcriptional level. This study delineates transcriptional and post-transcriptional layers of Caulobacter gene expression control during hyperosmotic stress, uncovers a new regulatory target of GsrN, and defines a coherent feedforward motif in the Caulobacter GSR regulatory network.ImportanceBacteria inhabit diverse niches, and must adapt their physiology to constant environmental fluctuations. A major response to environmental perturbation is to change gene expression. Caulobacter and other alphaproteobacteria initiate a complex gene expression program known as the general stress response (GSR) under conditions including oxidative stress, osmotic stress, and nutrient limitation. The GSR enables cell survival in these environments. Understanding how bacteria survive stress requires that we dissect gene expression responses, such as the GSR, at the molecular level. This study is significant as it defines transcriptional and post-transcriptional layers of gene expression regulation in response to hyperosmotic stress. We further provide evidence that coherent feedforward motifs influence the system properties of the Caulobacter GSR pathway.

The two-component system ChvGI maintains cell envelope homeostasis in Caulobacter crescentus

“…Interestingly, hprK is part of the SigT regulon determined upon osmotic shock (Tien et al ., 2018). Given that hprK is part of the same operon as chvIG , we tested whether the entire operon chvIG-hprK could be under the control of SigT.…”

“…In C. crescentus , the general stress response (GSR) sigma factor SigT is also activated upon osmotic imbalance thanks to a complex network comprising the sRNA (GsrN), the anti-SigT regulator (NepR), the histidine phosphotransferases (LovK and PhyK) and the RR (MrrA, LovR and PhyR) (Alvarez-Martinez et al ., 2007; Lourenço et al ., 2011; Foreman et al ., 2012; Lori et al ., 2018; Tien et al ., 2018). In steady-state conditions, NepR impedes SigT-dependent transcription.…”

The ChvG–ChvI and NtrY–NtrX two-component systems coordinately regulate growth ofCaulobacter crescentus

“…Transcriptome Deep Sequencing (RNA-seq) 2 mL of PYE medium was inoculated with ∆chvI EV (∆chvI xylX::pMT585) and ∆chvI chvI(D52E)++ (∆chvI xylX::pMT585-chvI(D52E)) cells and grown overnight. Cultures were diluted to OD660 = 0.001 in 2 mL fresh PYE and grown for 22.5 h. Cultures were diluted to OD660 = 0.075 in 5 mL PYE + 0.15% xylose and grown for 3.5 h before TRIzol extraction and RNA isolation, as described previously (92). RNA-seq libraries were prepared using an Illumina TruSeq stranded RNA kit and sequenced on an Illumina NextSEQ500 instrument at the University of Chicago Functional Genomics Facility.…”