The N -Acetylmuramic Acid 6-Phosphate Phosphatase MupP Completes the Pseudomonas Peptidoglycan Recycling Pathway Leading to Intrinsic Fosfomycin Resistance

Marina Borisova,Christoph Mayer,Jonathan Gisin,Howard A Shuman

doi:10.1128/mbio.00092-17

Abstract

ABSTRACTBacterial cells are encased in and stabilized by a netlike peptidoglycan (PGN) cell wall that undergoes turnover during bacterial growth. PGN turnover fragments are frequently salvaged by the cells via a pathway referred to as PGN recycling. Two different routes for the recycling of the cell wall sugar N-acetylmuramic acid (MurNAc) have been recognized in bacteria. In Escherichia coli and related enterobacteria, as well as in most Gram-positive bacteria, MurNAc is recovered via a catabolic route requiring a MurNAc 6-phosphate etherase (MurQ in E. coli) enzyme. However, many Gram-negative bacteria, including Pseudomonas species, lack a MurQ ortholog and use an alternative, anabolic recycling route that bypasses the de novo biosynthesis of uridyldiphosphate (UDP)-MurNAc, the first committed precursor of PGN. Bacteria featuring the latter pathway become intrinsically resistant to the antibiotic fosfomycin, which targets the de novo biosynthesis of UDP-MurNAc. We report here the identification and characterization of a phosphatase enzyme, named MupP, that had been predicted to complete the anabolic recycling pathway of Pseudomonas species but has remained unknown so far. It belongs to the large haloacid dehalogenase family of phosphatases and specifically converts MurNAc 6-phosphate to MurNAc. A ΔmupP mutant of Pseudomonas putida was highly susceptible to fosfomycin, accumulated large amounts of MurNAc 6-phosphate, and showed lower levels of UDP-MurNAc than wild-type cells, altogether consistent with a role for MupP in the anabolic PGN recycling route and as a determinant of intrinsic resistance to fosfomycin.

Highlights

IMPORTANCE Many Gram-negative bacteria, but not E. coli, make use of a cell wall salvage pathway that contributes to the pool of UDP-MurNAc, the first committed precursor of cell wall synthesis in bacteria
We showed previously that MurNAc accumulates in a ΔamgK mutant of P. putida, while MurNAc 6P is the product of an AnmK reaction [22]
A very intriguing finding is that a mupP deletion causes a 6-fold drop in UDP-MurNAc levels, which could explain the fosfomycin susceptibility of a ΔmupP mutant

Summary

Introduction

IMPORTANCE Many Gram-negative bacteria, but not E. coli, make use of a cell wall salvage pathway that contributes to the pool of UDP-MurNAc, the first committed precursor of cell wall synthesis in bacteria This salvage pathway is of particular interest because it confers intrinsic resistance to the antibiotic fosfomycin, which blocks de novo UDP-MurNAc biosynthesis. In Gram-negative bacteria, the PGN cell wall is steadily dismantled (PGN turnover) by the action of lytic transglycosylases and endopeptidases [9] These potentially autolytic enzymes (autolysins) release anhydro-muropeptides (GlcNAc–1,6-anhydro-MurNAc [GlcNAc-anhMurNAc-peptides]) from the cell wall (Fig. 1) that contain 1,6-anhydroMurNAc (anhMurNAc), a unique, nonreducing form of the cell wall sugar MurNAc. Anhydro-muropeptides are instantly recovered (PGN recycling) by a set of conserved recycling proteins best studied in the Gram-negative model organism Escherichia coli, which reportedly recycles about 45% of its PGN wall in each generation [9, 10]. We recently revealed an alternative PGN recycling pathway mbio.asm.org 2

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