Suppressor Mutations LinkinggpsBwith the First Committed Step of Peptidoglycan Biosynthesis in Listeria monocytogenes

Abstract

The cell division protein GpsB is a regulator of the penicillin binding protein A1 (PBP A1) in the Gram-positive human pathogen Listeria monocytogenes. Penicillin binding proteins mediate the last two steps of peptidoglycan biosynthesis as they polymerize and cross-link peptidoglycan strands, the main components of the bacterial cell wall. It is not known what other processes are controlled by GpsB. L. monocytogenes gpsB mutants are unable to grow at 42°C, but we observed that spontaneous suppressors correcting this defect arise on agar plates with high frequency. We here describe a first set of gpsB suppressors that mapped to the clpC and murZ genes. While ClpC is the ATPase component of the Clp protease, MurZ is a paralogue of the listerial UDP-N-acetylglucosamine (UDPGlcNAc) 1-carboxyvinyltransferase MurA. Both enzymes catalyze the enolpyruvyl transfer from phosphoenolpyruvate to UDP-GlcNAc, representing the first committed step of peptidoglycan biosynthesis. We confirmed that clean deletion of the clpC or murZ gene suppressed the ΔgpsB phenotype. It turned out that the absence of either gene leads to accumulation of MurA, and we show that artificial overexpression of MurA alone was sufficient for suppression. Inactivation of other UDP-GlcNAcconsuming pathways also suppressed the heat-sensitive growth of the ΔgpsB mutant, suggesting that an increased influx of precursor molecules into peptidoglycan biosynthesis can compensate for the lack of GpsB. Our results support a model according to which PBP A1 becomes misregulated and thus toxic in the absence of GpsB due to unproductive consumption of cell wall precursor molecules. IMPORTANCEThe late cell division protein GpsB is important for cell wall biosynthesis in Gram-positive bacteria. GpsB of the human pathogen L. monocytogenes interacts with one of the key enzymes of this pathway, penicillin binding protein A1 (PBP A1), and influences its activity. PBP A1 catalyzes the last two steps of cell wall biosynthesis, but it is unknown how GpsB controls PBP A1. We observed that a L. monocytogenes gpsB mutant forms spontaneous suppressors and have mapped their mutations to genes mediating and influencing the first step of cell wall biosynthesis, likely stimulating the influx of metabolites into this pathway. We assume that GpsB is important to ensure productive incorporation of cell wall precursors into the peptidoglycan sacculus by PBP A1.KEYWORDS GpsB, MurA, UDP-N-acetylglucosamine, PBP A1, peptidoglycan, UDP-N-acetylglucosamine T he cell wall represents the outmost layer of the bacterial envelope in Gram-positive bacteria. It confers rigidity and shape to their cells and provides a platform for incorporation of many molecules, e.g., proteins and wall teichoic acids, which need to be presented on the bacterial surface (1-3). The Gram-positive cell wall consists of a

Stimulation of PgdA‐dependent peptidoglycan N‐deacetylation by GpsB‐PBP A1 in Listeria monocytogenes

“…Inactivation of clpC (encoding an ATPase subunit of the proteasome), of murZ (encoding an UDP‐GlcNAc 1‐carboxyvinyltransferase) and of genes encoding other UDP‐GlcNAc consuming enzymes (GtcA, Lmo2550) also suppress the phenotype of the Δ gpsB mutant, at least its growth defect at higher temperature (Rismondo et al ., ). It has been assumed that these suppressor mutations compensate the effects of PBP A1 inefficiency by redirection of additional precursor molecules into the peptidoglycan biosynthetic pathway (Rismondo et al ., ). We measured the effect of two such suppressor mutations ( clpCR254S and murZ 1–253 ) on lysozyme resistance of the Δ gpsB mutant and found that resistance against lysozyme is corrected back to wild type level in these strains (Supporting Information Fig.…”

“…S7). It has been shown that MurA, which is the primary UDP‐GlcNAc 1‐carboxyvinyltransferase of L. monocytogenes and catalyzes the first committed step of peptidoglycan biosynthesis, is approximately tenfold overproduced in these suppressor strains, leading to redirection of precursors into cell wall biosynthesis (Rismondo et al ., ). In contrast, pbpA1 inactivation does not affect the MurA protein level at all (Rismondo et al ., ).…”

“…It has been shown that MurA, which is the primary UDP‐GlcNAc 1‐carboxyvinyltransferase of L. monocytogenes and catalyzes the first committed step of peptidoglycan biosynthesis, is approximately tenfold overproduced in these suppressor strains, leading to redirection of precursors into cell wall biosynthesis (Rismondo et al ., ). In contrast, pbpA1 inactivation does not affect the MurA protein level at all (Rismondo et al ., ). We hence assume that suppression of the Δ gpsB mutant's increased lysozyme resistance can occur by two mechanisms: (i) By deletion of pbpA1 , neutralizing uncontrolled and deregulated peptidoglycan biosynthesis through PBP A1 and (ii) by activation of precursor influx into peptidoglycan synthesis.…”

Aurantimycin resistance genes contribute to survival of Listeria monocytogenes during life in the environment

“…Genomic DNA was isolated using the GenElute™ Bacterial Genomic DNA Kit (Sigma-Aldrich) as described (Halbedel et al, 2018). Libraries were prepared using the Nextera XT DNA Library Prep Kit and genomes were sequenced on a MiSeq benchtop sequencer as described recently (Rismondo et al, 2017). SNPs were identified using the Geneious software package and the EGD-e genome sequence (NC_003210.1) (Glaser et al, 2001) as the reference.…”

The GpsB files: the truth is out there

“…Other than the capsular biosynthetic operon that is absent in R6, other directly relevant genotype differences include: dltA , which is responsible for the d ‐alanylation of teichoic acids; murA1 , which catalyses the first step in PG biosynthesis; ftsX , an ABC transporter‐like integral membrane protein essential for normal cell division; pbp1A , a bifunctional PBP and cbpD , a PG hydrolase (Lanie et al ., ). It is perhaps pertinent to note that growth of L. monocytogenes gpsb null strains can be suppressed by spontaneous mutation in murZ , a listerial paralogue to murA (Rismondo et al ., ). Since the R800 strain is a cousin of R6 (Fleurie et al ., ) it is entirely plausible that these genetic differences, and/or some unknown suppressors in R800, could explain the GpsB and DivIVA phenotypic differences between S. pneumoniae strains used in labs around the world.…”