Essentiality of WalRK for growth in Bacillus subtilis and its role during heat stress

Abstract

WalRK is an essential two-component signal transduction system that plays a central role in coordinating cell wall synthesis and cell growth in Bacillus subtilis. However, the physiological role of WalRK and its essentiality for growth have not been elucidated. We investigated the behaviour of WalRK during heat stress and its essentiality for cell proliferation. We determined that the inactivation of the walHI genes which encode the negative modulator of WalK, resulted in growth defects and eventual cell lysis at high temperatures. Screening of suppressor mutations revealed that the inactivation of LytE, an dl-endopeptidase, restored the growth of the ΔwalHI mutant at high temperatures. Suppressor mutations that reduced heat induction arising from the walRK regulon were also mapped to the walK ORF. Therefore, we hypothesized that overactivation of LytE affects the phenotype of the ΔwalHI mutant. This hypothesis was corroborated by the overexpression of the negative regulator of LytE, IseA and PdaC, which rescued the growth of the ΔwalHI mutant at high temperatures. Elucidating the cause of the temperature sensitivity of the ΔwalHI mutant could explain the essentiality of WalRK. We proved that the constitutive expression of lytE or cwlO using a synthetic promoter uncouples these expressions from WalRK, and renders WalRK nonessential in the pdaC and iseA mutant backgrounds. We propose that the essentiality of WalRK is derived from the coordination of cell wall metabolism with cell growth by regulating dl-endopeptidase activity under various growth conditions.

The WalRK two-component system is essential for proper cell envelope biogenesis in Clostridioides difficile

“…Phenotypic defects depended on how walRK expression was manipulated, but included elongation, loss of rod shape (curved or wavy cells), phase bright cells, enhanced or reduced autolysis, and altered sensitivity to antibiotics that target PG synthesis. Similar phenotypes have been observed upon manipulating Wal signaling in other organisms (2, 6, 7, 9, 10, 12, 17, 18). But there are some intriguing differences.…”

“…There are also 7 genes for S-layer proteins, consistent with the need to coordinate S-layer assembly and remodeling with growth and turnover of the PG sacculus. The PG hydrolases are of particular interest because they are prominent members of the WalR regulon in other Firmicutes (5,(12)(13)(14)(15)(16) and ectopic expression of one or more of these enzymes can render walRK no longer essential regulators (6,17,18). In C. difficile, Wal-ON conditions induced two putative cell wall amidases (cwp16, cwp17) and repressed one potential D,Lendopeptidase (cd630_27680).…”

“…But there are some intriguing differences. In B. subtilis , where phosphorylated WalR promotes elongation, artificial upregulation of the Wal system causes cells to become elongated while artificial downregulation causes cells to become short (10, 18). In C. difficile , however, overexpression of WalR from P xyl had no obvious effect on cell length while CRISPRi knockdown of the wal operon led to elongation.…”

“…However, the essentiality of walRK in B. subtilis and S. aureus is polygenic in nature, resulting from abnormal expression of multiple genes that are not by themselves essential. In all three organisms, it is possible to bypass the walRK requirement by constitutively expressing one or more Wal regulon PG hydrolases, sometimes in combination with deleting genes for hydrolase inhibitors (6, 17, 18).…”

Glucolipids and lipoteichoic acids affect the activity of SigI, an alternative sigma factor, and WalKR, an essential two‐component system, in Bacillus subtilis

“…TM1 and TM2 are transmembrane domains, and PAS-like is a Per-Arnt-Sim-like domain, which is a protein domain found in all kingdoms of life (Henry & Crosson, 2011) (schematic of the WalK variants is shown in Figure 6a). In the ΔTM1 and ΔTM1/ΔPASlike mutants, activation (phosphorylation) of WalR may be accelerated, and the WalR activity is kept at a constant level in the ΔTM1/2/ΔPASlike mutant (Takada et al, 2018). Abnormal morphology was observed in the walK ΔTM1 and ΔTM1/ΔPASlike mutants, which still have the transmembrane region TM2 of WalK.…”