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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 (PakFREC) 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 PakFREC 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.Hybrid 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.

SUBMITTER: Kaczmarczyk A

PROVIDER: S-EPMC4372748 | biostudies-literature | 2015 Apr

REPOSITORIES: biostudies-literature

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Two-tiered histidine kinase pathway involved in heat shock and salt sensing in the general stress response of Sphingomonas melonis Fr1.

Kaczmarczyk Andreas A Hochstrasser Ramon R Vorholt Julia A JA Francez-Charlot Anne A

Journal of bacteriology 20150209 8

<h4>Unlabelled</h4>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 sens ...[more]

PMID: 25666137

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Project description:Bacteria are constantly exposed to a variety of environmental stresses. Temperature is considered one of the most important environmental factors affecting microbial growth and survival. As ubiquitous environmental microorganisms, Sphingomonas species play essential roles in the biodegradation of organic contaminants, plant protection, and environmental remediation. Understanding the mechanism by which they respond to heat shock will help further improve cell resistance by applying synthetic biological strategies. Here, we assessed the transcriptomic and proteomic responses of Sphingomonas melonis TY to heat shock and found that stressful conditions caused significant changes in functional genes related to protein synthesis at the transcriptional level. The most notable changes observed were increases in the transcription (1,857-fold) and protein expression (11-fold) of Hsp17, which belongs to the small heat shock protein family, and the function of Hsp17 in heat stress was further investigated in this study. We found that the deletion of hsp17 reduced the capacity of the cells to tolerate high temperatures, whereas the overexpression of hsp17 significantly enhanced the ability of the cells to withstand high temperatures. Moreover, the heterologous expression of hsp17 in Escherichia coli DH5α conferred to the bacterium the ability to resist heat stress. Interestingly, its cells were elongated and formed connected cells following the increase in temperature, while hsp17 overexpression restored their normal morphology under high temperature. In general, these results indicate that the novel small heat shock protein Hsp17 greatly contributes to maintaining cell viability and morphology under stress conditions. IMPORTANCE Temperature is generally considered the most important factor affecting metabolic functions and the survival of microbes. As molecular chaperones, small heat shock proteins can prevent damaged protein aggregation during abiotic stress, especially heat stress. Sphingomonas species are widely distributed in nature, and they can frequently be found in various extreme environments. However, the role of small heat shock proteins in Sphingomonas under high-temperature stress has not been elucidated. This study greatly enhances our understanding of a novel identified protein, Hsp17, in S. melonis TY in terms of its ability to resist heat stress and maintain cell morphology under high temperature, leading to a broader understanding of how microbes adapt to environmental extremes. Furthermore, our study will provide potential heat resistance elements for further enhancing cellular resistance as well as the synthetic biological applications of Sphingomonas.

Project description:Drought and salinity are ubiquitous environmental factors that pose hyperosmotic threats to microorganisms and impair their efficiency in performing environmental functions. However, bacteria have developed various responses and regulatory systems to cope with these abiotic challenges. Posttranscriptional regulation plays vital roles in regulating gene expression and cellular homeostasis, as hyperosmotic stress conditions can lead to the induction of specific small RNA molecules (sRNAs) that participate in stress response regulation. Here, we report a candidate functional sRNA landscape of Sphingomonas melonis TY under hyperosmotic stress, and 18 sRNAs were found with a clear response to hyperosmotic stress. These findings will help in the comprehensive analysis of sRNA regulation in Sphingomonas species. Weighted correlation network analysis revealed a 263 nucleotide sRNA, SNC251, which was transcribed from its own promoter and showed the most significant correlation with hyperosmotic response factors. Deletion of snc251 affected biofilm formation and multiple cellular processes, including ribosome-related pathways, aromatic compound degradation, and the nicotine degradation capacity of S. melonis TY, while overexpression of SNC251 facilitated biofilm formation by TY under hyperosmotic stress. Two genes involved in the TonB system were further verified to be activated by SNC251, which also indicated that SNC251 is a trans-acting sRNA. Briefly, this research reports a landscape of sRNAs participating in the hyperosmotic stress response in S. melonis and reveals a novel sRNA, SNC251, which contributes to the S. melonis TY biofilm formation and thus enhances its hyperosmotic stress response ability.IMPORTANCESphingomonas species play a vital role in plant defense and pollutant degradation and survive extensively under drought or salinity. Previous studies have focused on the transcriptional and translational responses of Sphingomonas under hyperosmotic stress, but the posttranscriptional regulation of small RNA molecules (sRNAs) is also crucial for quickly modulating cellular processes to adapt dynamically to osmotic environments. In addition, the current knowledge of sRNAs in Sphingomonas is extremely scarce. This research revealed a novel sRNA landscape of Sphingomonas melonis and will greatly enhance our understanding of sRNAs' acting mechanisms in the hyperosmotic stress response.

Dataset Information

Two-tiered histidine kinase pathway involved in heat shock and salt sensing in the general stress response of Sphingomonas melonis Fr1.

Publications

Two-tiered histidine kinase pathway involved in heat shock and salt sensing in the general stress response of Sphingomonas melonis Fr1.

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