Low phosphatase activity of LiaS and strong LiaR-DNA affinity explain the unusual LiaS to LiaR in vivo stoichiometry

Jani et al. BMC Microbiology (2020) 20:104 https://doi.org/10.1186/s12866-020-01796-6 RESEARCH ARTICLE Open Access Low phosphatase activity of LiaS and strong LiaR-DNA affinity explain the unusual LiaS to LiaR in vivo stoichiometry Shailee Jani1, Karen Sterzenbach2, Vijaya Adatrao1, Ghazal Tajbakhsh1, Thorsten Mascher2* and Dasantila Golemi-Kotra1* Abstract Background: LiaRS mediates Bacillus subtilis response to cell envelope perturbations. A third protein, LiaF, has an inhibitory role over LiaRS in the absence of stimulus. Together, LiaF and LiaRS form a three-component system characterized by an unusual stoichiometry, a 4:1 ratio between LiaS and LiaR, the significance of which in the signal transduction mechanism of LiaRS is not entirely understood. Results: We measured, for the first time, the kinetics of the phosphorylation-dependent processes of LiaRS, the DNA-binding affinity of LiaR, and characterized the effect of phosphorylation on LiaR oligomerization state. Our study reveals that LiaS is less proficient as a phosphatase. Consequently, unspecific phosphorylation of LiaR by acetyl phosphate may be significant in vivo. This drawback is exacerbated by the strong interaction between LiaR and its own promoter, as it can drive LiaRS into losing grip over its own control in the absence of stimuli. These intrinsic, seemingly ‘disadvantageous”, attributes of LiaRS are likely overcome by the higher concentration of LiaS over LiaR in vivo, and a pro-phosphatase role of LiaF. Conclusions: Overall, our study shows that despite the conservative nature of two-component systems, they are, ultimately, tailored to meet specific cell needs by modulating the dynamics of interactions among their components and the kinetics of phosphorylation-mediated processes. Keywords: Two-component system, LiaRS, Histidine kinase, Response regulator, Bacillus subtilis, Cell envelope stress Background Two-component systems (TCS) represent a fundamental mechanism of bacterial signal transduction that allows microorganisms to perceive external signals and react appropriately with a cytoplasmic response. Typically, a two-component system (TCS) consists of a membrane bound (most of the time) histidine kinase (HK) and an intracellular soluble protein [1, 2]. The HK intercepts an environmental cue and, through an act of * Correspondence: ; 2 Institute for Microbiology, Technische Universität Dresden, Dresden, Germany 1 Department of Biology, York University, Toronto, ON M3J1P3, Canada autophosphorylation, transduces the signal intracellularly [3, 4]. The response to the cue is mediated through a phosphotransfer process in which the second protein, referred to as the response regulator protein (RR), receives the phosphoryl group from the cognate HK at a conserved aspartate residue. However, studies have shown that the intracellular acetyl phosphate can also serve as a phosphodonor to RRs [5]. The phosphorylation of RR marks its activation and it is often associated with its dimerization [2]. Further, the function of RR, either a transcription factor (most of the time) or an enzyme, determines the outcome of the TCS signal-transduction pathway [6]. In the absence of an extracellular stimulus © The Author(s). 2020 Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if changes were made. The images or other third party material in this article are included in the article's Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article's Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this licence, visit http://creativecommons.org/licenses/by/4.0/. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated in a credit line to the data. Jani et al. BMC Microbiology (2020) 20:104 the signal transduction pathway is switched off through the phosphatase activity of HK. Despite the conservation of the HK and RR in TCS, each TCS performs differently. The TCS LiaRS of Bacillus subtilis is involved in sensing cell envelope stress instigated by perturbation of the cytoplasmic membrane, particularly antibiotics that interfere with the Lipid II cycle of cell-wall peptidoglycan biosynthesis such as bacitracin, ramoplanin, vancomycin, and cationic antimicrobial peptides [7–12]. In addition, LiaRS is also induced by molecules that nonspecifically disrupt the cytoplasmic membrane, such as detergents, ethanol, phenol, organic solvents and secretion stress [8, 13]. Gene deletion and mutagenesis studies showed that LiaS is involved in sensing cell envelope perturbation and that this HK is a bifunctional enzyme that possesses phosphatase activity in vivo. Furthermore, these studies showed that LiaR is susceptible to phosphorylation by acetyl phosphate in vivo: in the absence of liaS and at high liaR expression levels, this can lead to the activation of LiaR-dependent gene expression [14]. LiaRS is part of a three-component system, located in the lia locus of B. subtilis, in which a third protein, LiaF, acts as a strong inhibitor of LiaR-dependent gene expression in the absence of stimuli [15]. The trio, LiaRSLiaF, is found in the same genomic context in many Gram-positive bacteria with a low G + C content (Firmicutes) [15]. The homologs of LiaRS TCS in B. licheniformis, Streptococcus pneumonia, S. aureus, E. faecalis, E. faecium, L. monocytogens and S. mutans, are also involved in the cell envelope stress response to bacitracin, vancomycin or cationic peptides [16–22]. The lia locus in B. subtilis is expressed from a strictly LiaRdependent σA-type promoter upstream of the liaI gene (the first of six genes in the lia locus) [8, 15], which represents the only relevant LiaR target in vivo [23]. This LiaR-dependent promoter is referred to as the liaI promoter (PliaI). Page 2 of 17 LiaS and LiaR are both modular proteins composed of a number of domains (Fig. 1). Analysis of the LiaS amino acid sequence by the UniProt server (ID O32198) revealed that this protein is a typical histidine kinase with two membrane-spanning regions, a HAMP domain, and an intracellular conserved histidine kinase region comprised of the dimerization and phosphotransfer domain (DHp; hosting the conserved histidine residue, His157) and the histidine kinase-like ATPase domain. The two membrane-spanning regions in LiaS are connected with a very short extracellular linker that is characteristic of intramembrane-sensing H (...truncated)