Two MarR-Type Repressors Balance Precursor Uptake and Glycine Betaine Synthesis in Bacillus subtilis to Provide Cytoprotection Against Sustained Osmotic Stress.

Bianca Warmbold,Andreas Seubert,Sven-Andreas Freibert,Erhard Bremer,Tamara Hoffmann,Stefanie Ronzheimer

doi:10.3389/fmicb.2020.01700

Bianca Warmbold, Andreas Seubert + Show 4 more

Open Access

https://doi.org/10.3389/fmicb.2020.01700

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Abstract

Bacillus subtilis adjusts to high osmolarity surroundings through the amassing of compatible solutes. It synthesizes the compatible solute glycine betaine from prior imported choline and scavenges many pre-formed osmostress protectants, including glycine betaine, from environmental sources. Choline is imported through the substrate-restricted ABC transporter OpuB and the closely related, but promiscuous, OpuC system, followed by its GbsAB-mediated oxidation to glycine betaine. We have investigated the impact of two MarR-type regulators, GbsR and OpcR, on gbsAB, opuB, and opuC expression. Judging by the position of the previously identified OpcR operator in the regulatory regions of opuB and opuC [Lee et al. (2013) Microbiology 159, 2087−2096], and that of the GbsR operator identified in the current study, we found that the closely related GbsR and OpcR repressors use different molecular mechanisms to control transcription. OpcR functions by sterically hindering access of RNA-polymerase to the opuB and opuC promoters, while GbsR operates through a roadblock mechanism to control gbsAB and opuB transcription. Loss of GbsR or OpcR de-represses opuB and opuC transcription, respectively. With respect to the osmotic control of opuB and opuC expression, we found that this environmental cue operates independently of the OpcR and GbsR regulators. When assessed over a wide range of salinities, opuB and opuC exhibit a surprisingly different transcriptional profile. Expression of opuB increases monotonously in response to incrementally increase in salinity, while opuC transcription levels decrease after an initial up-regulation at moderate salinities. Transcription of the gbsR and opcR regulatory genes is up-regulated in response to salt stress, and is also affected through auto-regulatory processes. The opuB and opuC operons have evolved through a gene duplication event. However, evolution has shaped their mode of genetic regulation, their osmotic-stress dependent transcriptional profile, and the substrate specificity of the OpuB and OpuC ABC transporters in a distinctive fashion.

Highlights

The soil dwelling Gram-positive bacterium Bacillus subtilis is frequently exposed to fluctuations in the environmental osmolarity, a process caused by flooding and drying (MandicMulec et al, 2015; Hoffmann and Bremer, 2016)
To deepen our understanding of the regulatory circuits and environmental cues contributing to the setting the compatible solute pool(s) to physiologically adequate levels (Hoffmann et al, 2013), we focus here on the systems that permit the import of choline via the OpuB and OpuC transporters (Kappes et al, 1999), its subsequent oxidation to glycine betaine by the GbsAB enzymes (Boch et al, 1996), and the scavenging of osmostress protectants via the promiscuous OpuC system (Hoffmann and Bremer, 2017; Teichmann et al, 2017; Figure 1A)
The cellular pools derived from imported glycine betaine (Hoffmann et al, 2013) and those resulting from the import of choline and its subsequent GbsAB-dependent enzymatic conversion into glycine betaine should be similar

Summary

Introduction

The soil dwelling Gram-positive bacterium Bacillus subtilis is frequently exposed to fluctuations in the environmental osmolarity, a process caused by flooding and drying (MandicMulec et al, 2015; Hoffmann and Bremer, 2016). Like many other microorganisms (Kempf and Bremer, 1998; Roeßler and Müller, 2001; Gunde-Cimerman et al, 2018), B. subtilis accumulates osmostress-relieving organic osmolytes, the compatible solutes This counteracts water efflux and thereby prevents a drop in turgor, when B. subtilis faces hyperosmotic conditions (Hoffmann and Bremer, 2016, 2017). Accumulation of compatible solutes optimizes the solvent properties and ionic composition of the cytoplasm as it prevents the buildup of a long-lasting high ionic strength cytoplasm Such ionic unfavorable conditions of the cells’ interior would otherwise result from the massive uptake of potassium, the initial stress reaction of the cell when it faces high osmolarity surroundings (Dinnbier et al, 1988; Whatmore et al, 1990; Wood, 2011; Bremer and Krämer, 2019). The amassing of compatible solutes protects the native structure of proteins and cellular sub-structures, and preserves the functionality of key biochemical reactions (Record et al, 1998; Barth et al, 2000; Bourot et al, 2000; Bolen and Baskakov, 2001; Ignatova and Gierasch, 2006; Wood, 2011; de Lima Alves et al, 2015; Stadmiller et al, 2017; Bremer and Krämer, 2019)

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