Deletion analysis of the essentiality of penicillin-binding proteins 1A, 2B and 2X of Streptococcus pneumoniae

Abstract

An internal fragment from each of the penicillin-binding protein (PBP) 1A, 2B and 2X genes of Streptococcus pneumoniae, which included the region encoding the active-site serine residue, was replaced by a fragment encoding spectinomycin resistance. The resulting constructs were tested for their ability to transform S. pneumoniae strain R6 to spectinomycin resistance. Spectinomycin-resistant transformants could not be obtained using either the inactivated PBP 2X or 2B genes, suggesting that deletion of either of these genes was a lethal event, but they were readily obtained using the inactivated PBP 1A gene. Analysis using the polymerase chain reaction confirmed that the latter transformants had replaced their chromosomal copy of the PBP 1A gene with the inactivated copy of the gene. Deletion of the PBP 1A gene was therefore tolerated under laboratory conditions and appeared to have little effect on growth or susceptibility to benzylpenicillin.

FemABX Peptidyl Transferases: a Link between Branched-Chain Cell Wall Peptide Formation and β-Lactam Resistance in Gram-Positive Cocci

“…In contrast to methicillin resistance in staphylococci, penicillin resistance arises by modification of endogenous PBPs. The first PBPs to be affected are PBP2b or the essential PBP, PBP2x, depending on the antibiotic used for selection (41,59). Mutations in just three amino acids in the active-site region of PBP2x are sufficient to create low-affinity derivatives (55).…”

Single-Step Capsular Transformation and Acquisition of Penicillin Resistance inStreptococcus pneumoniae

“…For example, the cps locus of penicillin-susceptible strain TIGR4 is a 15.2-kb region encoding 13 open reading frames, flanked by the pbp2x gene 10.9 kb upstream and by the pbp1a gene 11.6 kb downstream, and genes coding for all other PBPs are located at least 450 kbp away from the cps locus (40). There are six PBPs in a pneumococcal cell (PBP1a, -1b, -2a, -2b, -2x, and -3), of which PBP2x and PBP2b have been confirmed to be essential for cell growth (20,21,25). The pbp genes in S. pneumoniae have a mosaic structure and can undergo inter-and intraspecies recombination (5,8,19).…”

Functional Characterization of Penicillin-Binding Protein 1b fromStreptococcus pneumoniae

“…This gram-positive pathogen possesses six PBPs, including three class A (PBP1a, 1b, and 2a) and two class B (PBP2x and 2b) molecules as well as a D,D-carboxypeptidase, PBP 3 (14). Individual deletion of the pbp2x or pbp2b genes is lethal for S. pneumoniae (18). However, neither the pbp1a, pbp1b, nor pbp2a gene is required for growth when deleted individually, but the presence of at least pbp1a or pbp2a is essential for cell viability (16,27).…”