The Listeria monocytogenes PASTA Kinase PrkA and Its Substrate YvcK Are Required for Cell Wall Homeostasis, Metabolism, and Virulence

Pensinger, Daniel A.; Boldon, Kyle; Chen, Grischa Y.; Vincent, William J.B.; Kyle, Sherman; Xiong, Min; Schaenzer, Adam J.; Forster, Emily R.; Coers, Jörn; Striker, Rob; Sauer, John-Demian

doi:10.1371/journal.ppat.1006001

Cited by 62 publications

(121 citation statements)

References 105 publications

(175 reference statements)

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“…To study the contribution of the PrkA/PrkP couple to cell wall biosynthesis in more detail, we aimed to construct prkA and prkP deletion mutants, but failed to delete prkA , as has been reported previously (29). However, prkA could be deleted in the presence of an IPTG-inducible ectopic prkA copy and the resulting strain (LMSW84) required IPTG for growth (Fig.…”

Section: Resultsmentioning

confidence: 99%

“…MurA catalyzes the first committed step of PG biosynthesis by transferring an enoylpyruvate moiety to UDP-Glc N Ac; MurA is essential in M. tuberculosis and in many other bacterial species tested (26–29). Finally, the Listeria monocytogenes PASTA-eSTK, PrkA, phosphorylates YvcK, which is required for cell wall homeostasis in a so far unknown way (30). Numerous additional proteins acting to coordinate cell wall biosynthesis with cell division are substrates of PASTA-eSTKs in other Gram-positive bacteria (15), including the late cell division protein GpsB of Bacillus subtilis (31, 32).…”

Section: Introductionmentioning

confidence: 99%

“…MurA catalyzes the first committed step of PG biosynthesis by transferring an enoylpyruvate moiety to UDP-Glc N Ac; MurA is essential in M. tuberculosis and in all other bacterial species tested (25–28). Finally, the Listeria monocytogenes PASTA-eSTK, PrkA, phosphorylates YvcK, which is required for cell wall homeostasis, and both proteins are important for the survival of this facultative intracellular pathogen inside the cytosol of infected host cells (29).…”

Section: Introductionmentioning

confidence: 99%

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PrkA controls peptidoglycan biosynthesis through the essential phosphorylation of ReoM

Wamp

Rutter

Rismondo

et al. 2019

Preprint

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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...

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Section: Resultsmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

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PrkA controls peptidoglycan biosynthesis through the essential phosphorylation of ReoM

Wamp

Rutter

Rismondo

et al. 2019

Preprint

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“…PASTA kinases are ubiquitous among Actinobacteria and Firmicutes and regulate critical processes, including antibiotic resistance, cell division, toxin production, and virulence, and they are even essential for viability in certain organisms (6,(10)(11)(12)(13)(14)(15)(16)(17)(18)(19)(20)(21)(22)(23)(24)(25)(26)(27)(28)(29). However, the molecular signals that are directly sensed by PASTA kinases and the mechanisms by which those signals are processed, integrated, and converted into coordinated adaptive biological responses in vivo are poorly understood.…”

mentioning

confidence: 99%

Growth- and Stress-Induced PASTA Kinase Phosphorylation in Enterococcus faecalis

Labbe

Kristich

2017

J Bacteriol

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Transmembrane Ser/Thr kinases containing extracellular PASTA domains are ubiquitous among Actinobacteria and Firmicutes. Such PASTA kinases regulate critical processes, including antibiotic resistance, cell division, toxin production, and virulence, and are essential for viability in certain organisms. Based on in vitro studies with purified extracellular and intracellular fragments of PASTA kinases, a model for signaling has been proposed, in which the extracellular PASTA domains bind currently undefined ligands (typically thought to be peptidoglycan, or fragments thereof) to drive kinase dimerization, which leads to enhanced kinase autophosphorylation and enhanced phosphorylation of substrates. However, this model has not been rigorously tested in vivo. Enterococcus faecalis is a Gram-positive intestinal commensal and major antibiotic-resistant opportunistic pathogen. In E. faecalis, the PASTA kinase IreK drives intrinsic resistance to cell wall-active antimicrobials, suggesting that such antimicrobials may trigger IreK signaling. Here we show that IreK responds to cell wall stress in vivo by enhancing its phosphorylation and that of a downstream substrate. This response requires both the extracellular PASTA domains and specific phosphorylatable residues in the kinase domain. Thus, our results provide in vivo evidence, with an intact full-length PASTA kinase in its native physiological environment, that supports the prevailing model of PASTA kinase signaling. In addition, we show that IreK responds to a signal associated with growth and/or cell division, in the absence of cell wall-active antimicrobials. Surprisingly, the ability of IreK to respond to growth and/or division does not require the extracellular PASTA domains, suggesting that IreK monitors multiple parameters for sensory input in vivo.IMPORTANCE Transmembrane Ser/Thr kinases containing extracellular PASTA domains are ubiquitous among Actinobacteria and Firmicutes and regulate critical processes. The prevailing model for signaling by PASTA kinases proposes that the extracellular PASTA domains bind ligands to drive kinase dimerization, enhanced autophosphorylation, and enhanced phosphorylation of substrates. However, this model has not been rigorously tested in vivo. We show that the PASTA kinase IreK of Enterococcus faecalis responds to cell wall stress in vivo by enhancing its phosphorylation and that of a downstream substrate. This response requires the PASTA domains and phosphorylatable residues in the kinase domain. Thus, our results provide in vivo evidence, with an intact full-length PASTA kinase in its native physiological environment, that supports the prevailing model of PASTA kinase signaling.

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“…Another example of a connection between carbon metabolism and cell elongation is demonstrated by studies on YvcK. Homologs exist in a variety of bacteria (44)(45)(46), but YvcK is most extensively studied in B. subtilis (44,47,48). Although yvcK is not essential during growth in LB, cells with a yvcK deletion are round and lyse under gluconeogenic growth regimes, indicating a deficiency in elongasome function (44).…”

Section: Metabolism and Cell Elongationmentioning

confidence: 99%

Metabolism Shapes the Cell

Sperber

Herman

2017

J Bacteriol

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More than 5 decades of work support the idea that cell envelope synthesis, including the inward growth of cell division, is tightly coordinated with DNA replication and protein synthesis through central metabolism. Remarkably, no unifying model exists to account for how these fundamentally disparate processes are functionally coupled. Recent studies demonstrate that proteins involved in carbohydrate and nitrogen metabolism can moonlight as direct regulators of cell division, coordinate cell division and DNA replication, and even suppress defects in DNA replication. In this minireview, we focus on studies illustrating the intimate link between metabolism and regulation of peptidoglycan (PG) synthesis during growth and division, and we identify the following three recurring themes. (i) Nutrient availability, not growth rate, is the primary determinant of cell size. (ii) The degree of gluconeogenic flux is likely to have a profound impact on the metabolites available for cell envelope synthesis, so growth medium selection is a critical consideration when designing and interpreting experiments related to morphogenesis. (iii) Perturbations in pathways relying on commonly shared and limiting metabolites, like undecaprenyl phosphate (Und-P), can lead to pleotropic phenotypes in unrelated pathways.KEYWORDS FtsZ, MreB, UDP-glucose, cell division, gluconeogenesis, metabolism, morphogenesis, peptidoglycan, phosphoenolpyruvate, undecaprenyl phosphate T o accurately partition chromosomes and other cell contents during reproduction, cells must possess mechanisms to organize repeated cycles of cell growth, chromosome replication, and division. Eukaryotes orchestrate this coordination using the cell cycle and separate growth, DNA synthesis, and cytokinesis into distinct, temporally sequestered phases. Bacteria, by contrast, simultaneously increase in cell size and replicate DNA before (or concurrently with) cell division. Elucidating the molecular mechanisms prokaryotes employ to achieve spatiotemporal organization of these intertwined yet functionally disparate processes is of considerable interest to scientists seeking to understand bacterial reproduction, and many outstanding questions remain to be answered. For example, how is DNA replication kept in sync with changing growth and division rates? How are cell dimensions maintained or actively rearranged in response to environmental or developmental cues? What signals do cells sense to switch between increasing in cell size and dividing during the cell cycle? Relatedly, how are these signals transduced to activate/deactivate the distinct machineries required for each process?Perhaps one of the biggest mysteries remaining in bacterial cell biology relates to understanding the regulatory cross talk that must occur to integrate central metabolism with macromolecular biosynthesis. Nutrients are converted into stored energy and precursors used to synthesize macromolecules like DNA and peptidoglycan (PG), so it is no surprise that nutrient availability has a profound impac...

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The Listeria monocytogenes PASTA Kinase PrkA and Its Substrate YvcK Are Required for Cell Wall Homeostasis, Metabolism, and Virulence

Cited by 62 publications

References 105 publications

PrkA controls peptidoglycan biosynthesis through the essential phosphorylation of ReoM

PrkA controls peptidoglycan biosynthesis through the essential phosphorylation of ReoM

Growth- and Stress-Induced PASTA Kinase Phosphorylation in Enterococcus faecalis

Metabolism Shapes the Cell

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