Role of Sphingomonas sp. Strain Fr1 PhyR-NepR-σ ^EcfG Cascade in General Stress Response and Identification of a Negative Regulator of PhyR

Abstract

The general stress response in Alphaproteobacteria was recently described to depend on the alternative sigma factor EcfG , whose activity is regulated by its anti-sigma factor NepR. The response regulator PhyR, in turn, regulates NepR activity in a partner-switching mechanism according to which phosphorylation of PhyR triggers sequestration of NepR by the sigma factor-like effector domain of PhyR. Although genes encoding predicted histidine kinases can often be found associated with phyR, little is known about their role in modulation of PhyR phosphorylation status. We demonstrate here that the PhyR-NepREcfG cascade is important for multiple stress resistance and competitiveness in the phyllosphere in a naturally abundant plant epiphyte, Sphingomonas sp. strain Fr1, and provide evidence that the partner switching mechanism is conserved. We furthermore identify a gene, designated phyP, encoding a predicted histidine kinase at the phyR locus as essential. Genetic epistasis experiments suggest that PhyP acts upstream of PhyR, keeping PhyR in an unphosphorylated, inactive state in nonstress conditions, strictly depending on the predicted phosphorylatable site of PhyP, His-341. In vitro experiments show that Escherichia coli inner membrane fractions containing PhyP disrupt the PhyR-P/NepR complex. Together with the fact that PhyP lacks an obvious ATPase domain, these results are in agreement with PhyP functioning as a phosphatase of PhyR, rather than a kinase.

Complex two-component signaling regulates the general stress response in Alphaproteobacteria

“…Also in agreement with a role of Paks in the GSR, we were able to delete phyP in the sextuple or septuple kinase mutant backgrounds. Because the PhyR phosphatase PhyP is essential in a WT background preventing lethal hyperphosphorylation of PhyR (10,24), the successful deletion of phyP suggested that a major source of PhyR phosphorylation is absent in these genetic backgrounds. However, because the GSR activity in the sextuple or septuple kinase mutants was still substantially higher than in a phyR mutant ( Fig.…”

“…To identify factors positively regulating the PhyR-NepR-σ EcfG cascade, we conducted a genetic screen, performing transposon mutagenesis on a strain expressing the dominant streptomycin-sensitive rpsL1 allele (25) under control of the σ EcfG -dependent ecfG2 promoter (10,24). This construct would allow selection for mutants with an impaired PhyR-NepR-σ EcfG cascade on medium containing streptomycin (details are provided in SI Materials and Methods).…”

“…Among other evidence, this conclusion is based on the findings that phyP is essential unless in a ΔphyR or ΔecfG background (10), and that PhyP depletion leads to lethal overactivation of the GSR in a manner that requires the phosphorylatable Asp residue (Asp194) of PhyR (24). Assuming that PhyP acts solely as a phosphatase, this notion implies that alternative phosphodonors for PhyR must exist.…”

“…Because PhyR uses a degenerate N-terminal sigma factor-like output domain to compete with σ EcfG for NepR binding, this partner switch was coined "sigma factor mimicry" (1). The importance of the alphaproteobacterial GSR in natural environments is underlined by several studies demonstrating its requirement for survival and competitiveness in the phyllosphere in Sphingomonas melonis (10) and Methylobacterium extorquens (8), the establishment of symbiotic interactions in Bradyrhizobium japonicum (9), and host-pathogen interactions in Brucella (6,11) and Bartonella (3) species.…”

“…In Alphaproteobacteria, the GSR is controlled by an alternative extracytoplasmic function (ECF) sigma factor, usually called σ EcfG (1) or ECF15 sigma factor (2), the activity of which is regulated by a conserved partner-switching mechanism (1,(3)(4)(5)(6)(7)(8)(9)(10)(11)(12)(13)(14). In unstressed conditions, σ EcfG is sequestered by its anti-sigma factor NepR.…”