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

Penicillin-resistant strains of Streptococcus pneumoniae contain low affini ty penicillin-binding proteins and often also produce abnormal indirectly c rosslinked cell walls. However the relationship between cell wall abnormali ty and penicillin resistance has remained obscure. We now show that the gen ome of S, pneumoniae contains an operon composed of two genes (murM and mur N) that encode enzymes involved with the biosynthesis of branched structure d cell wall muropeptides. The sequences of murMN were compared in two strai ns: the penicillin-susceptible strain R36A producing the species-specific p neumococcal cell wall peptidoglycan in which branched stem peptides are rar e, and the highly penicillin-resistant transformant strain Pen6, the cell w all of which is enriched for branched-structured stem peptides, The two str ains carried different murM alleles: murM of the penicillin-resistant strai n Pen6 had a "mosaic" structure encoding a protein that was only 86.5% iden tical to the product of murM identified in the isogenic penicillin-suscepti ble strain R36A, Mutants of R36A and Pen6 in which the murMN operon was int errupted by insertion-duplication mutagenesis produced peptidoglycan from w hich all branched muropeptide components were missing. The insertional muta nt of Pen6 carried a pbp2x gene with the same "mosaic" sequence found in Pe n6, On the other hand, inactivation of murMN in strain Pen6 and other resis tant strains caused a virtually complete loss of penicillin resistance. Our observations indicate that the capacity to produce branched cell wall prec ursors plays a critical role in the expression of penicillin resistance in S, pneumoniae.