Interaction of Penicillin-Binding Protein 2 with Soluble Lytic Transglycosylase B1 inPseudomonas aeruginosa

Abstract

Soluble lytic transglycosylase B1 from Pseudomonas aeruginosa was coupled to Sepharose and used to immobilize interaction partners from membrane protein extracts. Penicillin-binding protein 2 (PBP2) was identified as a binding partner, suggesting that the two proteins function together in the biosynthesis of peptidoglycan. By use of an engineered truncated derivative, the N-terminal module of PBP2 was found to confer the binding properties.Enlargement and growth of the peptidoglycan (PG) sacculus is effected by the coordination of both synthetic and lytic enzymes. The penicillin-binding proteins (PBPs) are biosynthetic enzymes that catalyze transglycosylation and/or transpeptidation reactions for the incorporation of new material into the PG layer (11). The lytic transglycosylases (LTs), on the other hand, cleave the ␤-1,4-glycosidic bond between MurNAc and GlcNAc and are understood to function as space-makers for the incorporation of new material (reviewed in reference 29). These conflicting enzymatic activities are thought to be controlled by the association of the respective enzymes in multienzyme complexes (14, 29). Support for this hypothesis is confined to only a few studies involving a limited number of bacterial species. With Escherichia coli, a protein-protein interaction network between high-molecular-weight (HMW) PBPs, HMW LTs, and low-molecular-weight PBPs (specifically, D,D-endopeptidases) has been demonstrated by affinity chromatography experiments (3,26,34,35). Affinity chromatography was also used to indicate that membrane-bound LT A (MltA) interacts with PBP2 in

Bulgecin A: The Key to a Broad‐Spectrum Inhibitor That Targets Lytic Transglycosylases

“…In addition, bulgecin A in combination with β-lactams was ineffective against β-lactamase-producing resistant strain 24753 TEM-1, which strongly supports a bactericidal synergism between β-lactams and bulgecin A that may target the Slt-PBP protein complex simultaneously. It is well established that Lts and PBPs form protein complexes [18,25,28,40,41,42]. Most notable are the interaction between Slt70 and PBPs 1b, 1c, 2, and 3 [43].…”

Changes to Its Peptidoglycan-Remodeling Enzyme Repertoire Modulate β-Lactam Resistance in Pseudomonas aeruginosa

“…SltB1 was previously reported to interact with PBP2 (19,20); therefore, we asked whether increased ␤-lactam resistance in the sltB1 deletion mutant was due to loss of its LT activity or the potential disruption of a PBP2-containing complex due to absence of the SltB1 protein. To address the second possibility, the sltB1 gene was replaced at its chromosomal locus (to ensure native expression levels) with mutant versions expressing one of two putative active-site mutants, one replacing the proposed catalytic Glu135 (21) with Ala and the other replacing Ser189, responsible for orientation of PG in the active site (22), with Ala.…”

“…Furthermore, PG-active enzymes can be organized into multienzyme complexes (19,20,38), and loss of one component might destabilize those complexes, affecting PG metabolism. SltB1 interacts with PBP2 (19,20) but could have additional partners (20). Chromosomal expression of putative active-site mutants of SltB1 did not restore susceptibility, suggesting that increased ␤-lactam resistance in sltB1 mutants is due to the loss of SltB1 function, rather than disruption of protein interactions.…”

From the regulation of peptidoglycan synthesis to bacterial growth and morphology

“…Intact complexes have not yet been isolated, possibly because they are dynamic, held together by weak interactions and/or tend to dissociate on breakage of the sacculus during cell lysis. However, protein interaction data 7,99,100 and the fact that each major bifunctional synthase has an outer-membrane protein regulator 101,102 support the existence of such complexes.…”