scholarly journals Mutational Analysis of Class A and Class B Penicillin-Binding Proteins in Streptococcus gordonii

2006 ◽  
Vol 50 (12) ◽  
pp. 4062-4069 ◽  
Author(s):  
Marisa Haenni ◽  
Paul A. Majcherczyk ◽  
Jean-Luc Barblan ◽  
Philippe Moreillon
Keyword(s):  
Binding Proteins ◽  
Mutational Analysis ◽  
A Priori ◽  
Detectable Effect ◽  
Streptococcus Gordonii ◽  
Penicillin Binding Proteins ◽  
Morphological Alterations ◽  
Class A ◽  
Double Deletion ◽  
Class B

ABSTRACT High-molecular-weight (HMW) penicillin-binding proteins (PBPs) are divided into class A and class B PBPs, which are bifunctional transpeptidases/transglycosylases and monofunctional transpeptidases, respectively. We determined the sequences for the HMW PBP genes of Streptococcus gordonii, a gingivo-dental commensal related to Streptococcus pneumoniae. Five HMW PBPs were identified, including three class A (PBPs 1A, 1B, and 2A) and two class B (PBPs 2B and 2X) PBPs, by homology with those of S. pneumoniae and by radiolabeling with [3H]penicillin. Single and double deletions of each of them were achieved by allelic replacement. All could be deleted, except for PBP 2X, which was essential. Morphological alterations occurred after deletion of PBP 1A (lozenge shape), PBP 2A (separation defect and chaining), and PBP 2B (aberrant septation and premature lysis) but not PBP 1B. The muropeptide cross-link patterns remained similar in all strains, indicating that cross-linkage for one missing PBP could be replaced by others. However, PBP 1A mutants presented shorter glycan chains (by 30%) and a relative decrease (25%) in one monomer stem peptide. Growth rate and viability under aeration, hyperosmolarity, and penicillin exposure were affected primarily in PBP 2B-deleted mutants. In contrast, chain-forming PBP 2A-deleted mutants withstood better aeration, probably because they formed clusters that impaired oxygen diffusion. Double deletion could be generated with any PBP combination and resulted in more-altered mutants. Thus, single deletion of four of the five HMW genes had a detectable effect on the bacterial morphology and/or physiology, and only PBP 1B seemed redundant a priori.

scholarly journals Mutational Analysis of the Streptococcus pneumoniae Bimodular Class A Penicillin-Binding Proteins

1999 ◽  
Vol 181 (12) ◽  
pp. 3852-3856 ◽  
Author(s):  
Johanna Paik ◽  
Iza Kern ◽  
Rudi Lurz ◽  
Regine Hakenbeck
Keyword(s):  
Molecular Weight ◽  
Streptococcus Pneumoniae ◽  
Double Mutant ◽  
Binding Proteins ◽  
Mutational Analysis ◽  
Cellular Growth ◽  
Primary Target ◽  
Penicillin Binding Proteins ◽  
Class A ◽  
Higher Sensitivity

ABSTRACT One group of penicillin target enzymes, the class A high-molecular-weight penicillin-binding proteins (PBPs), are bimodular enzymes. In addition to a central penicillin-binding–transpeptidase domain, they contain an N-terminal putative glycosyltransferase domain. Mutations in the genes for each of the three Streptococcus pneumoniae class A PBPs, PBP1a, PBP1b, and PBP2a, were isolated by insertion duplication mutagenesis within the glycosyltransferase domain, documenting that their function is not essential for cellular growth in the laboratory. PBP1b PBP2a and PBP1a PBP1b double mutants could also be isolated, and both showed defects in positioning of the septum. Attempts to obtain a PBP2a PBP1a double mutant failed. All mutants with a disrupted pbp2a gene showed higher sensitivity to moenomycin, an antibiotic known to inhibit PBP-associated glycosyltransferase activity, indicating that PBP2a is the primary target for glycosyltransferase inhibitors in S. pneumoniae.

scholarly journals Role of Class A Penicillin-Binding Proteins in PBP5-Mediated β-Lactam Resistance in Enterococcus faecalis

2004 ◽  
Vol 186 (5) ◽  
pp. 1221-1228 ◽  
Author(s):  
Ana Arbeloa ◽  
Heidi Segal ◽  
Jean-Emmanuel Hugonnet ◽  
Nathalie Josseaume ◽  
Lionnel Dubost ◽  
...  
Keyword(s):  
Enterococcus Faecalis ◽  
Binding Proteins ◽  
Site Directed Mutagenesis ◽  
Intrinsic Resistance ◽  
Triple Mutant ◽  
Penicillin Binding Proteins ◽  
Class A ◽  
Class B ◽  
Chain Polymerization ◽  
High Level

ABSTRACT Peptidoglycan polymerization complexes contain multimodular penicillin-binding proteins (PBP) of classes A and B that associate a conserved C-terminal transpeptidase module to an N-terminal glycosyltransferase or morphogenesis module, respectively. In Enterococcus faecalis, class B PBP5 mediates intrinsic resistance to the cephalosporin class of β-lactam antibiotics, such as ceftriaxone. To identify the glycosyltransferase partner(s) of PBP5, combinations of deletions were introduced in all three class A PBP genes of E. faecalis JH2-2 (ponA, pbpF, and pbpZ). Among mutants with single or double deletions, only JH2-2 ΔponA ΔpbpF was susceptible to ceftriaxone. Ceftriaxone resistance was restored by heterologous expression of pbpF from Enterococcus faecium but not by mgt encoding the monofunctional glycosyltransferase of Staphylococcus aureus. Thus, PBP5 partners essential for peptidoglycan polymerization in the presence of β-lactams formed a subset of the class A PBPs of E. faecalis, and heterospecific complementation was observed with an ortholog from E. faecium. Site-directed mutagenesis of pbpF confirmed that the catalytic serine residue of the transpeptidase module was not required for resistance. None of the three class A PBP genes was essential for viability, although deletion of the three genes led to an increase in the generation time and to a decrease in peptidoglycan cross-linking. As the E. faecalis chromosome does not contain any additional glycosyltransferase-related genes, these observations indicate that glycan chain polymerization in the triple mutant is performed by a novel type of glycosyltransferase. The latter enzyme was not inhibited by moenomycin, since deletion of the three class A PBP genes led to high-level resistance to this glycosyltransferase inhibitor.

scholarly journals Role of Class A Penicillin-Binding Proteins in the Expression of β-Lactam Resistance in Enterococcus faecium

2009 ◽  
Vol 191 (11) ◽  
pp. 3649-3656 ◽  
Author(s):  
Louis B. Rice ◽  
Lenore L. Carias ◽  
Susan Rudin ◽  
Rebecca Hutton ◽  
Steven Marshall ◽  
...  
Keyword(s):  
Enterococcus Faecium ◽  
Binding Proteins ◽  
Mass Spectrometry Analysis ◽  
Cross Linking ◽  
Triple Mutant ◽  
Penicillin Binding Proteins ◽  
Spectrometry Analysis ◽  
Class A ◽  
Class B ◽  
Genes Encoding

ABSTRACT Peptidoglycan is polymerized by monofunctional d,d-transpeptidases belonging to class B penicillin-binding proteins (PBPs) and monofunctional glycosyltransferases and by bifunctional enzymes that combine both activities (class A PBPs). Three genes encoding putative class A PBPs (pbpF, pbpZ, and ponA) were deleted from the chromosome of Enterococcus faecium D344R in all possible combinations in order to identify the glycosyltransferases that cooperate with low-affinity class B Pbp5 for synthesis of peptidoglycan in the presence of β-lactam antibiotics. The viability of the triple mutant indicated that glycan strands can be polymerized independently from class A PBPs by an unknown glycosyltranferase. The susceptibility of the ΔpbpF ΔponA mutant and triple mutants to extended spectrum cephalosporins (ceftriaxone and cefepime) identified either PbpF or PonA as essential partners of Pbp5 for peptidoglycan polymerization in the presence of the drugs. Mass spectrometry analysis of peptidoglycan structure showed that loss of PonA and PbpF activity led to a minor decrease in the extent of peptidoglycan cross-linking by the remaining PBPs without any detectable compensatory increase in the participation of the l,d-transpeptidase in peptidoglycan synthesis. Optical density measurements and electron microscopy analyses showed that the ΔpbpF ΔponA mutant underwent increased stationary-phase autolysis compared to the parental strain. Unexpectedly, deletion of the class A pbp genes revealed dissociation between the expression of resistance to cephalosporins and penicillins, although the production of Pbp5 was required for resistance to both classes of drugs. Thus, susceptibility of Pbp5-mediated peptidoglycan cross-linking to different β-lactam antibiotics differed as a function of its partner glycosyltransferase.

scholarly journals Characterization of the Synechocystis Strain PCC 6803 Penicillin-Binding Proteins and Cytokinetic Proteins FtsQ and FtsW and Their Network of Interactions with ZipN

2009 ◽  
Vol 191 (16) ◽  
pp. 5123-5133 ◽  
Author(s):  
Martial Marbouty ◽  
Khalil Mazouni ◽  
Cyril Saguez ◽  
Corinne Cassier-Chauvat ◽  
Franck Chauvat
Keyword(s):  
Cell Size ◽  
Normal Cell ◽  
Binding Proteins ◽  
Penicillin Binding Proteins ◽  
Class A ◽  
Class B ◽  
Interaction Web ◽  
Two Hybrid ◽  
Pcc 6803

ABSTRACT Because very little is known about cell division in noncylindrical bacteria and cyanobacteria, we investigated 10 putative cytokinetic proteins in the unicellular spherical cyanobacterium Synechocystis strain PCC 6803. Concerning the eight penicillin-binding proteins (PBPs), which define three classes, we found that Synechocystis can survive in the absence of one but not two PBPs of either class A or class C, whereas the unique class B PBP (also termed FtsI) is indispensable. Furthermore, we showed that all three classes of PBPs are required for normal cell size. Similarly, the putative FtsQ and FtsW proteins appeared to be required for viability and normal cell size. We also used a suitable bacterial two-hybrid system to characterize the interaction web among the eight PBPs, FtsQ, and FtsW, as well as ZipN, the crucial FtsZ partner that occurs only in cyanobacteria and plant chloroplasts. We showed that FtsI, FtsQ, and ZipN are self-interacting proteins and that both FtsI and FtsQ interact with class A PBPs, as well as with ZipN. Collectively, these findings indicate that ZipN, in interacting with FtsZ and both FtsI and FtQ, plays a similar role to the Escherichia coli FtsA protein, which is missing in cyanobacteria and chloroplasts.

scholarly journals Real time monitoring of peptidoglycan synthesis by membrane-reconstituted penicillin binding proteins

eLife ◽  
2021 ◽  
Vol 10 ◽  
Author(s):  
Victor M Hernández-Rocamora ◽  
Natalia Baranova ◽  
Katharina Peters ◽  
Eefjan Breukink ◽  
Martin Loose ◽  
...  
Keyword(s):  
Real Time ◽  
Lipid Bilayers ◽  
High Throughput Screening ◽  
Binding Proteins ◽  
Cell Envelope ◽  
Penicillin Binding Proteins ◽  
Peptidoglycan Biosynthesis ◽  
Class A ◽  
Peptidoglycan Synthesis ◽  
Osmotic Lysis

Peptidoglycan is an essential component of the bacterial cell envelope that surrounds the cytoplasmic membrane to protect the cell from osmotic lysis. Important antibiotics such as β-lactams and glycopeptides target peptidoglycan biosynthesis. Class A penicillin binding proteins are bifunctional membrane-bound peptidoglycan synthases that polymerize glycan chains and connect adjacent stem peptides by transpeptidation. How these enzymes work in their physiological membrane environment is poorly understood. Here we developed a novel FRET-based assay to follow in real time both reactions of class A PBPs reconstituted in liposomes or supported lipid bilayers and we applied this assay with PBP1B homologues from Escherichia coli, Pseudomonas aeruginosa and Acinetobacter baumannii in the presence or absence of their cognate lipoprotein activator. Our assay will allow unravelling the mechanisms of peptidoglycan synthesis in a lipid-bilayer environment and can be further developed to be used for high throughput screening for new antimicrobials.

scholarly journals Class A PBPs have a distinct and unique role in the construction of the pneumococcal cell wall

2020 ◽  
Vol 117 (11) ◽  
pp. 6129-6138 ◽  
Author(s):  
Daniel Straume ◽  
Katarzyna Wiaroslawa Piechowiak ◽  
Silje Olsen ◽  
Gro Anita Stamsås ◽  
Kari Helene Berg ◽  
...  
Keyword(s):  
Cell Wall ◽  
Peptidoglycan Hydrolase ◽  
Unique Role ◽  
Penicillin Binding Proteins ◽  
The Core ◽  
Class A ◽  
Peptidoglycan Synthesis ◽  
Resistant Form ◽  
Class B ◽  
Open Question

In oval-shapedStreptococcus pneumoniae, septal and longitudinal peptidoglycan syntheses are performed by independent functional complexes: the divisome and the elongasome. Penicillin-binding proteins (PBPs) were long considered the key peptidoglycan-synthesizing enzymes in these complexes. Among these were the bifunctional class A PBPs, which are both glycosyltransferases and transpeptidases, and monofunctional class B PBPs with only transpeptidase activity. Recently, however, it was established that the monofunctional class B PBPs work together with transmembrane glycosyltransferases (FtsW and RodA) from the shape, elongation, division, and sporulation (SEDS) family to make up the core peptidoglycan-synthesizing machineries within the pneumococcal divisome (FtsW/PBP2x) and elongasome (RodA/PBP2b). The function of class A PBPs is therefore now an open question. Here we utilize the peptidoglycan hydrolase CbpD that targets the septum ofS. pneumoniaecells to show that class A PBPs have an autonomous role during pneumococcal cell wall synthesis. Using assays to specifically inhibit the function of PBP2x and FtsW, we demonstrate that CbpD attacks nascent peptidoglycan synthesized by the divisome. Notably, class A PBPs could process this nascent peptidoglycan from a CbpD-sensitive to a CbpD-resistant form. The class A PBP-mediated processing was independent of divisome and elongasome activities. Class A PBPs thus constitute an autonomous functional entity which processes recently formed peptidoglycan synthesized by FtsW/PBP2×. Our results support a model in which mature pneumococcal peptidoglycan is synthesized by three functional entities, the divisome, the elongasome, and bifunctional PBPs. The latter modify existing peptidoglycan but are probably not involved in primary peptidoglycan synthesis.

scholarly journals The function of the transmembrane and cytoplasmic domains of pneumococcal penicillin-binding proteins 2x and 2b extends beyond that of simple anchoring devices

Microbiology ◽  
2014 ◽  
Vol 160 (8) ◽  
pp. 1585-1598 ◽  
Author(s):  
Kari Helene Berg ◽  
Daniel Straume ◽  
Leiv Sigve Håvarstein
Keyword(s):  
Binding Proteins ◽  
Transmembrane Domain ◽  
Mutational Analysis ◽  
Cytoplasmic Domain ◽  
Loss Of Function ◽  
Penicillin Binding Proteins ◽  
Catalytic Domains ◽  
Complex Process ◽  
Cytoplasmic Tails ◽  
Cross Links

The biosynthesis of cell-wall peptidoglycan is a complex process that involves six different penicillin-binding proteins (PBPs) in Streptococcus pneumoniae. Two of these, PBP2x and PBP2b, are monofunctional transpeptidases that catalyse the formation of peptide cross-links between adjacent glycan strands. Both of them are bitopic membrane proteins with a small cytoplasmic and a large extracellular domain. PBP2x and PBP2b are essential for septal and peripheral peptidoglycan synthesis, respectively. Although several studies have investigated the properties of their extracellular catalytic domains, it is not known whether the role of their N-terminal non-catalytic domains extends beyond that of being simple anchoring devices. We therefore decided to use reciprocal domain swapping and mutational analysis to gain more information about the biological function of the membrane anchors and cytoplasmic tails of PBP2x and PBP2b. In the case of PBP2x both domains are essential, but neither the membrane anchor nor the cytoplasmic domain of PBP2x appear to serve as major localization signals. Instead, our results suggest that they are involved in interactions with other components of the divisome. Mutations of conserved amino acids in the cytoplasmic domain of PBP2x resulted in loss of function, underlining the importance of this region. The cytoplasmic domain of PBP2b could be swapped with the corresponding domain from PBP2x, whereas replacement of the PBP2b transmembrane domain with the corresponding PBP2x domain gave rise to slow-growing cells with grossly abnormal morphology. When both domains were exchanged simultaneously the cells were no longer viable.

scholarly journals The Importance of a Critical Protonation State and the Fate of the Catalytic Steps in Class A β-Lactamases and Penicillin-binding Proteins

2004 ◽  
Vol 279 (33) ◽  
pp. 34665-34673 ◽  
Author(s):  
Dasantila Golemi-Kotra ◽  
Samy O. Meroueh ◽  
Choonkeun Kim ◽  
Sergei B. Vakulenko ◽  
Alexey Bulychev ◽  
...  
Keyword(s):  
Binding Proteins ◽  
Protonation State ◽  
Penicillin Binding Proteins ◽  
Class A

scholarly journals Structural Basis of the Inhibition of Class A β-Lactamases and Penicillin-Binding Proteins by 6-β-Iodopenicillanate

2009 ◽  
Vol 131 (42) ◽  
pp. 15262-15269 ◽  
Author(s):  
Eric Sauvage ◽  
Astrid Zervosen ◽  
Georges Dive ◽  
Raphael Herman ◽  
Ana Amoroso ◽  
...  
Keyword(s):  
Binding Proteins ◽  
Structural Basis ◽  
Penicillin Binding Proteins ◽  
Class A

scholarly journals Author response: Class-A penicillin binding proteins do not contribute to cell shape but repair cell-wall defects

2020 ◽  
Author(s):  
Antoine Vigouroux ◽  
Baptiste Cordier ◽  
Andrey Aristov ◽  
Laura Alvarez ◽  
Gizem Özbaykal ◽  
...  
Keyword(s):  
Cell Wall ◽  
Cell Shape ◽  
Binding Proteins ◽  
Author Response ◽  
Response Class ◽  
Penicillin Binding Proteins ◽  
Class A
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