Inhibition of the expression of penicillin resistance in Streptococcus pneumoniae by inactivation of cell wall muropeptide branching genes

Abstract

Penicillin-resistant strains of Streptococcus pneumoniae contain low affinity penicillin-binding proteins and often also produce abnormal indirectly crosslinked cell walls. However the relationship between cell wall abnormality and penicillin resistance has remained obscure. We now show that the genome of S. pneumoniae contains an operon composed of two genes (murM and murN) that encode enzymes involved with the biosynthesis of branched structured cell wall muropeptides. The sequences of murMN were compared in two strains: the penicillin-susceptible strain R36A producing the species-specific pneumococcal cell wall peptidoglycan in which branched stem peptides are rare, and the highly penicillin-resistant transformant strain Pen6, the cell wall of which is enriched for branched-structured stem peptides. The two strains carried different murM alleles: murM of the penicillin-resistant strain Pen6 had a "mosaic" structure encoding a protein that was only 86.5% identical to the product of murM identified in the isogenic penicillin-susceptible strain R36A. Mutants of R36A and Pen6 in which the murMN operon was interrupted by insertion-duplication mutagenesis produced peptidoglycan from which all branched muropeptide components were missing. The insertional mutant of Pen6 carried a pbp2x gene with the same "mosaic" sequence found in Pen6. On the other hand, inactivation of murMN in strain Pen6 and other resistant strains caused a virtually complete loss of penicillin resistance. Our observations indicate that the capacity to produce branched cell wall precursors plays a critical role in the expression of penicillin resistance in S. pneumoniae.

Figure 1

Genetic organization of the murMN operon in S. pneumoniae (A) and inactivation of murMN by insertion duplication mutagenesis (B). Erm-r, erythromycin resistance gene from the plasmid pJDC9.

Figure 2

Comparison of the primary structure of the murM protein in penicillin-susceptible and -resistant pneumococcal strains. The murM genes were sequenced from the penicillin-susceptible laboratory strain R36A, the penicillin-resistant South African strain 8249, and its transformant-derivative Pen6. Parts of the murM of the resistant strain showing divergence from the corresponding sequences in strain R36A are shown in black.

Figure 3

HPLC profiles of the stem peptide components of the peptidoglycan from the penicillin- susceptible strain R36A and the penicillin-resistant strain Pen6 and their murMN-inactivated mutant derivatives. Cell wall peptidoglycans were prepared from strains R36A, Pen6, and their mutant derivatives with insertionally inactivated murMN, and the stem peptide composition was determined by HPLC analysis as described in Experimental Procedures.

Figure 4

Effect of the inactivation of murMN on the stem peptide composition of peptidoglycan. The amounts of individual stem peptides in the peptidoglycans isolated from the S. pneumoniae strains analyzed in Fig. 3 were determined by integration of the UV absorbance peaks in the HPLC profiles. Data are expressed as % of total stem peptides.

Figure 5

Structures of the cell wall stem peptides identified in the pneumococcal peptidoglycan of penicillin-susceptible and -resistant strains of pneumococci. Structural assignments were based on methods described earlier (2, 4).

Figure 6

Inhibition of the expression of penicillin resistance of strain Pen6 by interruption of the murMN operon. Cultures of strains R36A, Pen6, and its mutant derivative carrying an inactivated murMN operon were plated at various cell concentrations on agar containing serial dilutions of penicillin, and the population analysis profiles were determined as described in Experimental Procedures. Strain R36A, ◊; Pen6, ■, and mutant Pen6murMN, X.