Two-Tiered Histidine Kinase Pathway Involved in Heat Shock and Salt Sensing in the General Stress Response of Sphingomonas melonis Fr1

Abstract

The general stress response (GSR) allows bacteria to monitor and defend against a broad set of unrelated, adverse environmental conditions. In Alphaproteobacteria, the key step in GSR activation is phosphorylation of the response regulator PhyR. In Sphingomonas melonis Fr1, seven PhyR-activating kinases (Paks), PakA to PakG, are thought to directly phosphorylate PhyR under different stress conditions, but the nature of the activating signals remains obscure. PakF, a major sensor of NaCl and heat shock, lacks a putative sensor domain but instead harbors a single receiver (REC) domain (PakF REC ) N-terminal to its kinase catalytic core. Such kinases are called "hybrid response regulators" (HRRs). How HRRs are able to perceive signals in the absence of a true sensor domain has remained largely unexplored. In the present work, we show that stresses are actually sensed by another kinase, KipF (kinase of PakF), which phosphorylates PakF REC and thereby activates PakF. KipF is a predicted transmembrane kinase, harboring a periplasmic CHASE3 domain flanked by two transmembrane helices in addition to its cytoplasmic kinase catalytic core. We demonstrate that KipF senses different salts through its CHASE3 domain but is not a sensor of general osmotic stress. While salt sensing depends on the CHASE3 domain, heat shock sensing does not, suggesting that these stresses are perceived by different mechanisms. In summary, our results establish a two-tiered histidine kinase pathway involved in activation of the GSR in S. melonis Fr1 and provide the first experimental evidence for the so far uncharacterized CHASE3 domain as a salt sensor. IMPORTANCEHybrid response regulators (HRRs) represent a particular class of histidine kinases harboring an N-terminal receiver (REC) domain instead of a true sensor domain. This suggests that the actual input for HRRs may be phosphorylation of the REC domain. In the present study, we addressed this question by using the HRR PakF. Our results suggest that PakF is activated through phosphorylation of its REC domain and that this is achieved by another kinase, KipF. KipF senses heat shock and salt stress, with the latter requiring the periplasmic CHASE3 domain. This work not only suggests that HRRs work in two-tiered histidine kinase pathways but also provides the first experimental evidence for a role of the so far uncharacterized CHASE3 domain in salt sensing. Many bacteria possess a so-called general stress response (GSR) that allows them to monitor a broad range of stressful conditions and to launch an appropriate response to adapt to adverse environments. In Alphaproteobacteria, the GSR is controlled by an alternative sigma factor, EcfG , whose activity is regulated via a partner-switching mechanism (1, 2). Under nonstress conditions, EcfG is inactivated by binding to the anti-sigma factor NepR. Upon stress, NepR is sequestered by the anti-anti-sigma factor PhyR, thereby releasing EcfG and allowing it to activate transcription of stress-related genes. PhyR is a response regulator, and a...

General Stress Signaling in the Alphaproteobacteria

“…The shared ExxHRxxN motif at the site of histidine phosphorylation (41, 66) has led to the collective moniker HRxxN kinases to represent these two groups (64, 65). The HWE superfamily, Cl06527 (previously called the HisKA_2 superfamily), also encompasses these related DHp domains (29).…”

“…Some GSR kinases possess N-terminal two-component REC domains, which permit integration of signals from other sensory kinase(s). An example is the PhyR kinase PakF, which is activated by phosphorylation of its N-terminal receiver domain by the HisKA-family kinase KipF (65). Finally, some HisKA_2 family kinases such as S. melonis PakG lack signal input domains altogether (64).…”

“…A conserved periplasmic cysteine is important for the function of C. crescentus PhyK as a GSR sensor (71), but it is not clear whether this residue has a generic role in protein structure or whether it is directly involved in signal perception. Similarly, Kaczmarczyk and colleagues identified a small motif in the CHASE3 domain of S. melonis KipF that is important for the salt sensing but not temperature sensing functions of this kinase (65). LOV domains, a subset of PAS domains capable of sensing blue light and/or redox (49), are signal input domains for GSR kinases in a number of species, including C. crescentus, B. abortus , and E. litoralis (21, 33, 68).…”

Structure and function of HWE/HisKA2-family sensor histidine kinases

“…Indeed, all histidine kinases demonstrated to phosphorylate PhyR and/or regulate GSR transcription to date are HWE/HisKA2 kinases [7,9–11,36–40]. The only histidine kinase known to signal to the GSR pathway that is not HWE/HisKA2 does so indirectly via an HWE/HisKA2 kinase that directly phosphorylates PhyR [9,41]. …”

“…Studies of GSR regulation in Alphaproteobacteria have also revealed an important role for SDRRs, which interact biochemically and/or genetically with HWE/HisKA2 kinases to modulate GSR transcription [7,9,36,41–43]. Though the mechanistic basis by which SDRRs execute this function remains undefined, they have been hypothesized to function as allosteric regulators of HWE/HisKA2, competitive kinase inhibitors, phospho-sinks, or as components of parallel regulatory pathways.…”