Identification of a phosphotransferase system of Escherichia coli required for growth on N-acetylmuramic acid

Abstract

We report here that wild-type Escherichia coli grows on N-acetylmuramic acid (MurNAc) as the sole source of carbon and energy. Analysis of mutants defective in N-acetylglucosamine (GlcNAc) catabolism revealed that the catabolic pathway for MurNAc merges into the GlcNAc pathway on the level of GlcNAc 6-phosphate. Furthermore, analysis of mutants defective in components of the phosphotransferase system (PTS) revealed that a PTS is essential for growth on MurNAc. However, neither the glucose-, mannose/glucosamine-, nor GlcNAc-specific PTS (PtsG, ManXYZ, and NagE, respectively) was found to be necessary. Instead, we identified a gene at 55 min on the E. coli chromosome that is responsible for MurNAc uptake and growth. It encodes a single polypeptide consisting of the EIIB and C domains of a so-far-uncharacterized PTS that was named murP. MurP lacks an EIIA domain and was found to require the activity of the crr-encoded enzyme IIA-glucose (EIIA(Glc)), a component of the major glucose transport system for growth on MurNAc. murP deletion mutants were unable to grow on MurNAc as the sole source of carbon; however, growth was rescued by providing murP in trans expressed from an isopropylthiogalactopyranoside-inducible plasmid. A functional His(6) fusion of MurP was constructed, isolated from membranes, and identified as a polypeptide with an apparent molecular mass of 37 kDa by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and Western blot analysis. Close homologs of MurP were identified in the genome of several bacteria, and we believe that these organisms might also be able to utilize MurNAc.

FIG. 1.

Proposed MurNAc degradation pathway of E. coli. Convergence with the GlcNAc degradation pathway occurs on the level of GlcNAc 6-phosphate. A hypothetical etherase is required for removal of the lactyl ether substituent of MurNAc.

FIG. 2.

Partial multiple sequence alignment of the N terminus of the MurNAc-PTS (YfeV; renamed MurP) and selected members of the glucose-glucoside PTS family (4.A.1). Dark shading indicates a conserved sequence motif that includes the Cys residue presumably representing the phosphorylation site of the EIIB domain. SwissProt/TrEMBL identification numbers are given in the first column, and abbreviations are defined in the text.

FIG. 3.

Growth curves of wild-type E. coli and mutant strains growing on MMA supplemented with 0.2% MurNAc (6.8 mM) and induced with 0.5 mM IPTG (solid symbols) or without induction (open symbols): wild-type E. coli (MC4100) carrying control plasmid pCS19 with (▾) and without induction (▿); murP (yfeV) deletion strain CM103 carrying plasmid pCS19 with (•) and without (○) induction; murP deletion strain CM103 carrying plasmid pCS19YfeV with (▪) and without (□) induction; and murP deletion strain CM103 carrying plasmid pCS19YfeV-His₆ with induction (▴). Representative data out of three replicates are shown.

FIG. 4.

Western blot of SDS-PAGE with anti-His₆ antibodies. Strain CM103 carrying pCS19YfeV-His₆ was induced for 3 h at the indicated IPTG concentrations (lanes 2 to 4). Size standards are shown in lane 1.

FIG. 5.

Maps of the yfeT-yfeW locus of wild-type E. coli (top) and mutant strains constructed by homologous gene displacement that carry a kanamycin resistance cassette insertion (km) instead of murP (CM100; middle) or a complete deletion of murP (CM103; bottom). In the last mutant strain, the kanamycin resistance cassette insertion flanked by DNA recombinase recognition sites (FRT) was cut out with the yeast Flp recombinase, leaving an 80-bp-long scar sequence in place of murP. A putative divergent promoter was identified in the yfeT-yfeU intergenic region, and an additional promoter was identified within murP.