PrkA controls peptidoglycan biosynthesis through the essential phosphorylation of ReoM

Abstract

30Peptidoglycan (PG) is the main component of bacterial cell walls and the target for many 31 antibiotics. PG biosynthesis is tightly coordinated with cell wall growth and turnover and many 32 of these control activities depend upon PASTA-domain containing eukaryotic-like 33 serine/threonine protein kinases (PASTA-eSTK) that sense PG fragments. However, only a few 34 PG biosynthetic enzymes are actually direct kinase substrates. Here, we identify the conserved 35 ReoM protein as a novel PASTA-eSTK substrate in the Gram-positive pathogen Listeria 36 monocytogenes. Our data show that the phosphorylation of ReoM is essential as it controls 37 ClpCP-dependent proteolytic degradation of the essential enzyme MurA, which catalyses the first 38 committed step in PG biosynthesis. We also identify ReoY as a second novel factor required for 39 degradation of ClpCP substrates. Collectively, our data imply that the first committed step of PG 40 biosynthesis is activated through control of ClpCP protease activity in response to signals of PG 41 homeostasis imbalance.42 43 44 45The cell wall of Gram-positive bacteria is a complicated three-dimensional structure that engulfs 46 the cell as a closed sacculus. The main component of bacterial cell walls is peptidoglycan (PG), a 47 network of glycan strands crosslinked together by short peptides (1). PG biosynthesis starts with 48 the conversion of UDP-GlcNAc into lipid II, a disaccharide pentapeptide that is ligated to a 49 100 positive bacteria, which explains how PG biosynthesis is adjusted in these bacteria to meet PG 101 production and repair needs. 102 103 6 RESULTS 104 105Novel gpsB suppressor mutations in the lmo1503 (reoM) and lmo1921 (reoY) genes 106 A L. monocytogenes ΔgpsB mutant is unable to replicate at 42°C but forms suppressors 107 correcting this defect with high frequency (28). In a preliminary selection of gpsB suppressors, 108 the clpC and murZ genes, important for the stability of the UDP-N-acetylglucosamine 1-109 carboxyvinyltransferase MurA, were found to be mutated (28). We have characterized three more 110 shg (suppression of heat sensitive growth) suppressor mutants (shg8, shg10 and shg12) isolated 111 from a ΔgpsB mutant incubated on a BHI agar plate at 42°C. These three shg strains grew as fast 112 as the wild type when cultivated at 37°C or 42°C, whereas the parental ΔgpsB mutant grew at a 113 reduced rate at 37°C and did not grow at 42°C (Fig. 1A-B), as shown previously (32). 114Illumina sequencing of the shg8, shg10 and shg12 genomes identified one SNP in each strain that 115 was not found in the parental ΔgpsB strain. Strain shg8 carried a mutation in the uncharacterized 116 lmo1921 gene (herein named reoY, see below) that exchanged H87 into tyrosine; the same gene 117 was affected by the introduction of a premature stop codon after the 73 rd codon of reoY to yield 118 strain shg10. Strain shg12 carried a mutation in the ribosomal binding site (RBS) of the lmo1503 119 gene (renamed reoM), encoding another protein of unknown function and conta...

PrkC modulates MreB filament density and cellular growth rate by monitoring cell wall precursors

“…The PrkC mediated acceleration of growth posits a dilemma, as increased growth should require more lipid II, and thus require more mur activity. Recent work discovered a connection between PrkC and MurAA that might give this feedback: MurAA degradation is mediated by YrzL and YpiB, each of which increase the rate of MurAA proteolysis (Wamp, Rutter, Rismondo, Jennings, Moller, et al 2020). Importantly, YrzL mediated degradation of MurAA is inhibited when it is phosphorylated by PrkC.…”

“…4F). Interestingly, PrkC also controls the rate of lipid II production via YrzL controlling MurAA stability (Wamp, Rutter, Rismondo, Jennings, Moller, et al 2020, thus modulating the amount of the same molecular signal that controls its activity. This positive feedback might serve to increase the amount of PG precursors needed for increased Rod Complex activity as nutrients increase.…”

Time-Resolved Proteome Analysis of Listeria monocytogenes during Infection Reveals the Role of the AAA+ Chaperone ClpC for Host Cell Adaptation

“…We anticipated that direct ClpC substrates could be identified based on their increased abundance in the Δ clpC mutant compared to in the wt strain due to substrate accumulation caused by the lack of ClpCP-dependent proteolysis. To test this hypothesis, we analyzed the profiles of old and newly synthesized MurA1, a well-characterized ClpCP substrate in L. monocytogenes ( 40 ), which catalyzes the first committed step in peptidoglycan biosynthesis. The degradation profile of the old protein level revealed a general stabilization of MurA1 in the Δ clpC mutant throughout the experiment ( Fig.…”

The enterococcal PASTA kinase: A sentinel for cell envelope stress

“…Upon binding to an extracellular ligand (hexagon), the kinase dimerizes (b) which results in autophosphorylation on the activation loops of intracellular kinase domains, activating the kinase for robust phosphorylation of downstream substrates (c) to elicit an adaptive biological response. The cognate phosphatase (IreP in E. faecalis) dephosphorylates IreK and phosphorylated substrates, shutting off the signaling cascade (d) [Colour figure can be viewed at wileyonlinelibrary.com] defined, although a recent study indicated that an IreB homolog in Listeria monocytogenes regulates proteolytic degradation of a key enzyme in the PG synthesis pathway (Wamp et al, 2020).…”

“…IreB forms a dimer in solution, and oligomerization appears to be required for its function (Hall et al., 2017). The specific biochemical mechanism by which IreB influences cephalosporin resistance in enterococci has not been defined, although a recent study indicated that an IreB homolog in Listeria monocytogenes regulates proteolytic degradation of a key enzyme in the PG synthesis pathway (Wamp et al., 2020).…”