glmM operon and methicillin-resistant glmM suppressor mutants in Staphylococcus aureus

Abstract

The Staphylococcus aureus phosphoglucosamine mutase gene glmM was shown to be the last gene of a three-cistron operon, orf1-orf2-glmM. One transcriptional start was identified upstream of orf1, and a second start producing a monocistronic transcript was identified upstream of glmM. Disruption of glmM abolished GlmM production, decreased methicillin resistance, and resulted in teicoplanin hypersusceptibility without affecting the production of the endogenous penicillin-binding proteins and PBP 2'. Complementation of the glmM mutation by the complete glmM operon restored both methicillin resistance and normal teicoplanin susceptibility. In contrast, a highly methicillin-resistant suppressor mutant obtained by selection for growth in the presence of methicillin remained GlmM deficient and teicoplanin hypersusceptible. The suppressor mutation was not linked to the glmM operon but was correlated with decreased autolysis and increased production of a 49-kDa protein, suggesting that there is an alternative pathway for glucosamine-1-phosphate synthesis in S. aureus.

FIG. 1

Genetic and restriction map of the region containing the glmM operon. Restriction sites are indicated on the bottom line by the following abbreviations: P, PstI; A, Asp718; H, HindII; C, ClaI; N, NsiI. Open reading frames are indicated by arrows, and the position and orientation of the Tn551 insert in glmM are shown. The Tn551 insert is not drawn to scale. The small numbered arrows show the positions and orientations of the primers used for RT-PCR. The products generated by RT-PCR are shown in the top rows. The probes used for Northern blotting are shown by heavy dashed lines.

FIG. 2

Population analysis using overnight cultures plated on increasing concentrations of methicillin. Symbols: •, BB255; ■, BB270; □, PG108; ▵, PG100; ▴, PG105; ○, PG79.

FIG. 3

Spontanous autolysis of strains BB270 (■), PG108 (□), and PG100 (▵). OD₆₀₀, optical density at 600 nm.

FIG. 4

Western blot of the phosphoglucosamine mutase in the different strains. Cell extracts were probed with anti-GlmM antibodies. Lane a, PG79; lane b, PG100; lane c, PG108; lane d, BB255; lane e, BB270; and lane f, recombinant GlmM.

FIG. 5

Membrane proteins and PBPs. (A) Coomassie blue-stained membrane proteins of strains BB255 (lane a), BB270 (lane b), BB270 (lane c), PG100 (lane d), and PG108 (lane e). All strains except strain BB270 in lane c were grown in LB medium without antibiotics. Strain BB270 in lane c was grown in the presence of 256 μg of methicillin ml⁻¹. (B) Fluorography of the [³H]penicillin-labelled PBPs in the gel shown in panel A. The sizes of the molecular weight markers and the positions of the high-molecular-mass PBPs and of the 49-kDa band are indicated.

FIG. 6

Transcriptional starts. The transcriptional starts of the glmM operon were determined by primer extension. Lanes G, A, T, and C present the sequence of the corresponding region of the glmM operon. (A) Transcriptional starts of orf1. (B) Transcriptional start of glmM. Transcriptional starts are indicated with an asterisk.

FIG. 7

Northern blot analysis of the glmM region. Strain BB255 (lanes a), strain BB270 (lanes b), strain PG108 (lanes c), and strain PG100 (lanes d) were probed with the internal glmM probe (A) and with the argI fragment (B). The numbers indicate the sizes of the transcripts.