CosR Is a Global Regulator of the Osmotic Stress Response with Widespread Distribution among Bacteria

Abstract

Bacteria accumulate small, organic compounds called compatible solutes via uptake from the environment or biosynthesis from available precursors to maintain the turgor pressure of the cell in response to osmotic stress. The halophile Vibrio parahaemolyticus has biosynthesis pathways for the compatible solutes ectoine (encoded by ectABC-asp_ect) and glycine betaine (encoded by betIBA-proXWV), four betaine-carnitine-choline transporters (encoded by bccT1 to bccT4), and a second ProU transporter (encoded by proVWX). All of these systems are osmotically inducible with the exception of bccT2. Previously, it was shown that CosR, a MarR-type regulator, was a direct repressor of ectABC-asp_ect in Vibrio species. In this study, we investigated whether CosR has a broader role in the osmotic stress response. Expression analyses demonstrated that betIBA-proXWV, bccT1, bccT3, bccT4, and proVWX are repressed in low salinity. Examination of an in-frame cosR deletion mutant showed that expression of these systems is derepressed in the mutant at low salinity compared with the wild type. DNA binding assays demonstrated that purified CosR binds directly to the regulatory region of both biosynthesis systems and four transporters. In Escherichia coli green fluorescent protein (GFP) reporter assays, we demonstrated that CosR directly represses transcription of betIBA-proXWV, bccT3, and proVWX. Similar to Vibrio harveyi, we showed betIBA-proXWV was directly activated by the quorum-sensing LuxR homolog OpaR, suggesting a conserved mechanism of regulation among Vibrio species. Phylogenetic analysis demonstrated that CosR is ancestral to the Vibrionaceae family, and bioinformatics analysis showed widespread distribution among Gammaproteobacteria in general. Incidentally, in Aliivibrio fischeri, Aliivibrio finisterrensis, Aliivibrio sifiae, and Aliivibrio wodanis, an unrelated MarR-type regulator gene named ectR was clustered with ectABC-asp, which suggests the presence of another novel ectoine biosynthesis regulator. Overall, these data show that CosR is a global regulator of osmotic stress response that is widespread among bacteria. IMPORTANCE Vibrio parahaemolyticus can accumulate compatible solutes via biosynthesis and transport, which allow the cell to survive in high salinity conditions. There is little need for compatible solutes under low salinity conditions, and biosynthesis and transporter systems need to be repressed. However, the mechanism(s) of this repression is not known. In this study, we showed that CosR played a major role in the regulation of multiple compatible solute systems. Phylogenetic analysis showed that CosR is present in all members of the Vibrionaceae family as well as numerous Gammaproteobacteria. Collectively, these data establish CosR as a global regulator of the osmotic stress response that is widespread in bacteria, controlling many more systems than previously demonstrated.

New insights into hydroxyectoine synthesis and its transcriptional regulation in the broad‐salt growing halophilic bacteriumChromohalobacter salexigens

“…Concerning other transcription factors involved in the control of ectoines synthesis, none of the so far reported transcriptional regulators present in other microorganisms are found in C. salexigens genome. These include GlnR, a major regulator for nitrogen metabolism in many actinomycetes, which functions as a repressor of the ectABCD operon in Streptomyces coelicolor (Shao et␣al ., 2015), and the MarR‐type regulator EctR, from halophilic and halotolerant methylotrophic bacteria (Mustakhimov et␣al ., 2010; Mustakhimov et␣al ., 2012), and CosR from Vibrionaceae (Shikuma et␣al ., 2013; Gregory et␣al ., 2020), which function as negative regulators of the ectABC‐ask operon. Whereas the above regulators function as repressors, in a previous work we showed that the ferric iron uptake regulator Fur mediated the osmoregulated expression of the C. salexigens ectABC genes, linking the salt stress response with iron homeostasis.…”

The MarR-Type Regulator PA3458 Is Involved in Osmoadaptation Control in Pseudomonas aeruginosa

“…In motif A’, more positions tend to be conserved, and the partial palindromic structure within the first 12 base pairs positions of the motif is even more evident, as presented by underlined positions marked by // indicating the plane of symmetry in the consensus sequence TT T CA G//T TG G AA GCA presented in Figure 4 F. The identified motif resembles the MarR-type regulator SlyA binding site TTAGCAAGCTAA [ 43 ]. It is worth mentioning that also non-perfectly palindromic sequence motifs bound by MarR-type transcriptional regulators were found e.g., for MalR from Corynebacterium glutamicum , consensus TTnAAnnnTCAA [ 61 ]; HpaR from Xanthomonas campestris —consensus [G/T]CAACAATT[C/T]TTG [ 62 ] or CosR from Vibrio parahaemolyticus —consensus TTTGA-NN-TCTAA [ 63 ].…”

The ups and downs of ectoine: structural enzymology of a major microbial stress protectant and versatile nutrient

“…This finding points to a role of ectoines in protecting cells encased in biofilms, multi-cellular assemblages strained and architecturally shaped by osmotic forces (Rubinstein et al 2012;Seminara et al 2012;Yan et al 2017). In the pathogens Vibrio cholerae and Vibrio parahaemolyticus (and taxonomically related species) the genetic control of ectoine biosynthesis, compatible solute import, and biofilm formation is embedded in complex regulatory circuits that also involve quorum-sensing-type transcriptional regulators (Gregory et al 2019(Gregory et al , 2020Shikuma et al 2013). Dissecting these regulatory processes will open exciting entire new avenues for research.…”

“…However, in some organisms regulatory proteins have been shown to be involved in the regulation of the ectoine biosynthetic genes, such as the MarR-type regulators EctR from M. alcaliphilum (Mustakhimov et al 2010) or CosR from Vibrio cholera (Gregory et al 2019(Gregory et al , 2020Shikuma et al 2013). Interestingly, for CosR ionic strength has been proposed as the key factor determining its DNA-binding activity (Shikuma et al 2013).…”

“…Contributors to this latter regulatory modus might be changes in DNA-topology under osmotic stress conditions (Higgins et al 1988) and altered interaction of RNA-polymerase with -10 and -35 promoter regions in a crowded cytoplasm with changing solvation attributes when the external osmolarity is raised (Cagliero and Jin 2012;Cayley et al 1991;Gralla and Huo 2008;Record et al 1998;van den Berg et al 2017). The mode of action of transcriptional regulators (e.g., EctR, CosR, GlnR, AphA, OpaR, EupR, and alternative sigma factors) that have been implicated in the control of ect promoter activity in various bacteria certainly deserve future study and scrutiny (Calderon et al 2004;Gregory et al 2019Gregory et al , 2020Kuhlmann et al 2011;Mustakhimov et al 2010;Rodriguez-Moya et al 2010;Schwibbert et al 2011;Shao et al 2015;Shikuma et al 2013).…”