We report a salvage pathway in Gram-negative bacteria that bypasses de novo biosynthesis of UDP N-acetylmuramic acid (UDP-MurNAc), the first committed peptidoglycan precursor, and thus provides a rationale for intrinsic fosfomycin resistance. The anomeric sugar kinase AmgK and the MurNAc α-1-phosphate uridylyl transferase MurU, defining this new cell wall sugar-recycling route in Pseudomonas putida, were characterized and engineered into Escherichia coli, channeling external MurNAc directly to peptidoglycan biosynthesis.

Gram-negative bacteria recycle as much as half of their cell wall per generation. Here we show that interference with cell wall recycling in Pseudomonas aeruginosa strains results in four-to eight-fold increased susceptibility to the antibiotic fosfomycin, pushing the minimal inhibitory concentration for strains PA14 and PA01 to therapeutically appropriate values of 2-4 and 8-16 mg/L, respectively. A newly discovered metabolic pathway that connects cell wall recycling with peptidoglycan de novo biosynthesis is responsible for the high intrinsic resistance of P. aeruginosa to fosfomycin. The pathway comprises an anomeric cell wall amino sugar kinase (AmgK) and an uridylyl transferase (MurU), which together convert N-acetylmuramic acid (MurNAc) through MurNAc a-1-phosphate to uridine diphosphate (UDP)-MurNAc, thereby bypassing the fosfomycinsensitive de novo synthesis of UDP-MurNAc. Thus, inhibition of peptidoglycan recycling can be applied as a new strategy for the combinatory therapy against multidrug-resistant P. aeruginosa strains.

Molybdenum (Mo) is an important trace element that is toxic at high concentrations. To resolve the mechanisms underlying Mo toxicity, Rhodobacter capsulatus mutants tolerant to high Mo concentrations were isolated by random transposon Tn5 mutagenesis. The insertion sites of six independent isolates mapped within the same gene predicted to code for a permease of unknown function located in the cytoplasmic membrane. During growth under Mo-replete conditions, the wild-type strain accumulated considerably more Mo than the permease mutant. For mutants defective for the permease, the high-affinity molybdate importer ModABC, or both transporters, in vivo Mo-dependent nitrogenase (Mo-nitrogenase) activities at different Mo concentrations suggested that ModABC and the permease import molybdate in nanomolar and micromolar ranges, respectively. Like the permease mutants, a mutant defective for ATP sulfurylase tolerated high Mo concentrations, suggesting that ATP sulfurylase is the main target of Mo inhibition in R. capsulatus. Sulfate-dependent growth of a double mutant defective for the permease and the high-affinity sulfate importer CysTWA was reduced compared to those of the single mutants, implying that the permease plays an important role in sulfate uptake. In addition, permease mutants tolerated higher tungstate and vanadate concentrations than the wild type, suggesting that the permease acts as a general oxyanion importer. We propose to call this permease PerO (for oxyanion permease). It is the first reported bacterial molybdate transporter outside the ABC transporter family.Molybdenum (Mo) is utilized by many bacteria, archaea, and eukaryotes as a cofactor of redox enzymes catalyzing key reactions in the nitrogen, sulfur, and carbon cycles (62). Nitrogenase, which catalyzes the reduction of dinitrogen to ammonia, carries the unique iron-molybdenum cofactor FeMoco. In contrast to nitrogenase, all other molybdoenzymes harbor the molybdenum cofactor Moco, which transfers either an oxo group or two electrons to or from the substrate in a wide variety of transformations at nitrogen, sulfur, and carbon atoms (47).The phototrophic alphaproteobacterium Rhodobacter capsulatus serves as a model organism to study Mo metabolism because it synthesizes several molybdoenzymes, including dimethyl sulfoxide reductase, xanthine dehydrogenase, and nitrogenase (29,30,46). In addition to Mo-dependent nitrogenase (Mo-nitrogenase), R. capsulatus uses an alternative, Mofree nitrogenase when Mo is limiting (55, 57). Two related Mo-responsive regulators, MopA and MopB, control expression of the alternative nitrogenase and molybdate uptake genes (22,57,58).Mo is available for living cells in its oxyanion form, molybdate. The vast majority of Mo-utilizing bacteria is known or predicted to possess ModABC-type high-affinity molybdate uptake systems (62, 63). These importers belong to the family of ATP-binding cassette (ABC) transporters, which couple ATP hydrolysis to substrate translocation across biological membranes (13, 15). ModABC transpor...

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.