The murMN operon: A functional link between antibiotic resistance and antibiotic tolerance in Streptococcus pneumoniae

Abstract

Inactivation of the recently identified murMN operon in penicillinresistant strains of Streptococcus pneumoniae was shown already to cause two major effects: elimination of branched-structured muropeptides from the cell wall and complete loss of penicillin resistance. We now show that cells with inactivated murMN also have a third phenotype: an increased susceptibility to lysis when exposed to low concentrations of fosfomycin, D-cycloserine, vancomycin, and nisin, indicating a wide-spectrum hypersensitivity to inhibitors of both early and late stages of cell wall biosynthesis. Mutants of murMN also lysed faster than the parental strain when treated with the detergent deoxycholate. Several different alleles of murM cloned in plasmid pLS578 and introduced into a murM deletion mutant of the penicillin-resistant strain Pen6 were able to reconstitute each one of the three mutant phenotypes: the highly branched cell wall structure, original high level of penicillin resistance, and normal sensitivity to lysis. In a penicillin-susceptible strain the same experiments caused increased concentration of cell wall branched peptides and suppression of sensitivity to antibiotic induced lysis. The observations suggest that the murMN operon plays a key role in the regulation of a stress-response pathway that can be triggered by perturbation of cell wall biosynthesis in S. pneumoniae. S everal penicillin-resistant clones of Streptococcus pneumoniaeproduce abnormal cell walls in which a high proportion of the stem peptide lysine residues carry short (seryl alanine or alanyl alanine) branches (1). The genetic determinants involved with the branching process, the murMN operon, were identified recently (2). Penicillin-resistant isolates with highly branched cell walls were shown to carry murM alleles that contained divergent sequences as compared with the murM genes of penicillin-susceptible pneumococci (3). The two proteins, MurM (407 amino acids) and MurN (411 amino acids), are involved with the addition of the first and second amino acid residues, respectively, to the cell wall precursor stem peptide at the lipid II stage of biosynthesis (4, 5). Inactivation of murMN by insertion-duplication mutagenesis did not interfere with growth of the bacteria but caused two phenotypes: elimination of branched peptides from the cell wall and complete loss of penicillin resistance (2). The purpose of the studies described in this communication was to follow up these earlier findings by the use of a deletion mutant of murM and complementation experiments. The surprising observation made was that inactivation of murM also caused a third phenotype: increased sensitivity of the bacteria to lysis by cell wall inhibitors. Experimental ProceduresStrains and Growth Conditions. All strains and plasmids used in this study are listed in Table 1. S. pneumoniae strains were grown in a casein-based semisynthetic medium (C ϩ Y) at 37°C without aeration as described (11). S. pneumoniae strains containing pLS578 (10) or its derivatives were grown in the presen...

Lipid II-independent trans Editing of Mischarged tRNAs by the Penicillin Resistance Factor MurM

“…The MurMN proteins are responsible for the synthesis of branched muropeptides and are one of the requirements for high level penicillin resistance within this bacterium (27,28). Therefore, the mechanisms used by pneumococcus to ensure sufficient division of aminoacylated tRNA species between peptidoglycan biosynthesis and protein synthesis are of great interest.…”

Kinetic Characterization of Lipid II-Ala:Alanyl-tRNA Ligase (MurN) from Streptococcus pneumoniae using Semisynthetic Aminoacyl-lipid II Substrates

“…Therefore, the full-length MBP-MurN Pn16 and MBP-MurN 159 have been used to fully characterize MurN activity. (14,16,35) demonstrated that MurN adds an alanine residue to a previously acylated stem peptide. To correlate this in vivo finding with the enzymatic properties of MurN, we assayed MurN activity with lipid II and seryl-tRNA Ser .…”

“…Clinical strains of penicillin-resistant S. pneumoniae require for the high level resistance phenotype 1) the presence of specific murMN sequences, responsible for dipeptide cross-link formation and 2) specific modified penicillin-binding protein sequences (16 -20). However, certain laboratory S. pneumoniae strains containing resistant murMN alleles do not show penicillin resistance, since they lack high affinity penicillin-binding proteins (35).…”

Tolerance to the Glycopeptides Vancomycin and Teicoplanin in Coagulase-Negative Staphylococci

“…Tolerance mechanisms remain elusive to this day, even if recent works on Streptococcus pneumoniae and S. aureus have suggested the involvement of impaired autolysin regulation systems (3,18) or modifications in the cell wall composition (7,17). Studies should be undertaken to explore the mechanism of the phenomenon of S. lugdunensis tolerance to glycopeptides observed in the present work and to evaluate its clinical implications.…”