A conserved set of regulators control the general stress response in Caulobacter crescentus , including σ T , its anti-σ factor NepR, the anti-anti-σ factor PhyR, and the transmembrane sensor kinase PhyK. We report that the soluble histidine kinase LovK and the single-domain response regulator LovR also function within the C. crescentus general stress pathway. Our genetic data support a model in which LovK-LovR functions upstream of σ T by controlling the phosphorylation state and thus anti-anti-σ activity of PhyR. Transcription of lovK and lovR is independently activated by stress through a mechanism that requires sigT and phyR . Conversely, lovK and lovR function together to repress transcription of the general stress regulon. Concordant with a functional role of the LovK-LovR two-component system as a negative regulator of the general stress pathway, lovK - lovR -null mutants exhibit increased cell survival after osmotic stress, while coordinate overexpression of lovK and lovR attenuates cell survival relative to that of the wild type. Notably, lovK can complement the transcriptional and cell survival defects of a phyK -null mutant when lovR is deleted. Moreover, in this same genetic background, σ T -dependent transcription is activated in response to osmotic stress. This result suggests that flavin-binding LOV (light, oxygen, or voltage) histidine kinases are competent to perceive cytoplasmic signals in addition to the environmental signal blue light. We conclude that the PhyK-PhyR and LovK-LovR two-component signaling systems coordinately regulate stress physiology in C. crescentus .

Background: Virulence of pathogenic bacteria is often determined by their ability to adapt to stress. Results: The Brucella abortus general stress response (GSR) system is required for chronic mammalian infection and is regulated by phosphorylation and proteolysis. Conclusion: The B. abortus GSR signaling pathway has multiple layers of post-translational control and is a determinant of chronic infection. Significance: This study provides new, molecular level insight into chronic Brucella infection.

SummaryPhyR is a hybrid stress regulator conserved in a-proteobacteria that contains an N-terminal s-like (SL) domain and a C-terminal receiver domain. Phosphorylation of the receiver domain is known to promote binding of the SL domain to an anti-s factor. PhyR thus functions as an anti-anti-s factor in its phosphorylated state. We present genetic evidence that Caulobacter crescentus PhyR is a phosphorylation-dependent stress regulator that functions in the same pathway as s T and its anti-s factor, NepR. Additionally, we report the X-ray crystal structure of PhyR at 1.25 Å resolution, which provides insight into the mechanism of anti-anti-s regulation. Direct intramolecular contact between the PhyR receiver and SL domains spans regions s2 and s4, likely serving to stabilize the SL domain in a closed conformation. The molecular surface of the receiver domain contacting the SL domain is the structural equivalent of a4-b5-a5, which is known to undergo dynamic conformational change upon phosphorylation in a diverse range of receiver proteins. We propose a structural model of PhyR regulation in which receiver phosphorylation destabilizes the intramolecular interaction between SL and receiver domains, thereby permitting regions s 2 and s4 in the SL domain to open about a flexible connector loop and bind anti-s factor.

Gene expression variability in mammalian systems plays an important role in physiological and pathophysiological conditions. This variability can come from differential regulation related to cell state (extrinsic) and allele‐specific transcriptional bursting (intrinsic). Yet, the relative contribution of these two distinct sources is unknown. Here, we exploit the qualitative difference in the patterns of covariance between these two sources to quantify their relative contributions to expression variance in mammalian cells. Using multiplexed error robust RNA fluorescent in situ hybridization (MERFISH), we measured the multivariate gene expression distribution of 150 genes related to Ca2+ signaling coupled with the dynamic Ca2+ response of live cells to ATP. We show that after controlling for cellular phenotypic states such as size, cell cycle stage, and Ca2+ response to ATP, the remaining variability is effectively at the Poisson limit for most genes. These findings demonstrate that the majority of expression variability results from cell state differences and that the contribution of transcriptional bursting is relatively minimal.