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

Abstract

Bacterial 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.

Identification of MupP as a New Peptidoglycan Recycling Factor and Antibiotic Resistance Determinant in Pseudomonas aeruginosa

“…Because of its biochemical activity and the similar P ampC :: lacZ induction phenotypes displayed by mutants with murU and PA3172 inactivated, we hypothesized that PA3172 may encode the missing recycling phosphatase. Results presented below and those from a parallel study by the Mayer group (44) support this hypothesis. We therefore have renamed the PA3172 gene mupP for Mu rNAc-6 P p hosphatase.…”

“…On the basis of these results, we conclude that MupP is likely to be the missing MurNAc-6P phosphatase enzyme previously predicted to be functioning in the MurU pathway (33). In support of this designation, the Mayer group has biochemically characterized MupP from Pseudomonas putida (44). They report in a parallel study that MupP specifically hydrolyzes MurNAc-6P to MurNAc in vitro .…”

“…We present genetic evidence that PA3172 , renamed mupP , encodes the missing phosphatase enzyme previously predicted (33) to function in the broadly distributed MurU pathway for PG recycling. Biochemical results in a parallel study by the Mayer group support this designation (44). …”

Cell-wall recycling and synthesis in Escherichia coli and Pseudomonas aeruginosa – their role in the development of resistance

“…In P. aeruginosa, an AnmK homologue PA0666 phosphorylates anhMurNAc to form MurNAc-6-P; subsequently, the phosphate is removed by MupP (PA3172) resulting in MurNAc [174][175][176][177]. MurNAc is further processed by two enzymes AmgK (PA0596) and MurU (PA0597) unique to pseudomonads encoded in the same operon [178].…”

Constructing and deconstructing the bacterial cell wall

“…The allosteric control of S. aureus PBP2a (and perhaps of other PBPs) may well be to synchronize loop opening of the active site with protein translocation, but this explanation is almost certainly an oversimplification. The narrative presented here conceptualizes a two‐dimensional pathway for the peptidoglycan construction and deconstruction by P. aeruginosa when the realities of both AmpR regulation and cell‐wall biosynthesis are multidimensional . Yet this reductionism led to the deduction that the allosteric regulation of PBP2a is a solid basis for small molecule antibacterial discovery, and the parsing of the specificity of bulgecin as a selective inhibitor within the lytic transglycosylase family of P. aeruginosa .…”