A Highly Conserved Interaction Involving the Middle Residue of the SXN Active-Site Motif Is Crucial for Function of Class B Penicillin-Binding Proteins: Mutational and Computational Analysis of PBP 2 fromN. gonorrhoeae

SEDS (Shape, Elongation, Division and Sporulation) proteins are widely conserved peptidoglycan (PG) glycosyltransferases that form complexes with class B penicillin-binding proteins (bPBPs, with transpeptidase activity) to synthesize PG during bacterial cell growth and division. Because of their crucial roles in bacterial morphogenesis, SEDS proteins are one of the most promising targets for the development of new antibiotics. However, how SEDS proteins recognize their substrate lipid II, the building block of the PG layer, and polymerize it into glycan strands is still not clear. In this study, we isolated and characterized dominant-negative alleles of FtsW, a SEDS protein critical for septal PG synthesis during bacterial cytokinesis. Interestingly, most of the dominant-negative FtsW mutations reside in extracellular loops that are highly conserved in the SEDS family. Moreover, these mutations are scattered around a central cavity in a modeled FtsW structure, which has been proposed to be the active site of SEDS proteins. Consistent with this, we found that these mutations blocked septal PG synthesis but did not affect FtsW localization to the division site, interaction with its partners nor its substrate lipid II. Taken together, these results suggest that the residues corresponding to the dominant-negative mutations likely constitute the active site of FtsW, which may aid in the design of FtsW inhibitors.

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Role of penicillin-binding proteins in the viability, morphology, stress tolerance and pathogenicity of Clavibacter michiganensis

Phytopathology ◽

10.1094/phyto-08-20-0326-r ◽

2020 ◽

Author(s):

Xing Chen ◽

Kaihong Bai ◽

Qingyang Lyu ◽

Na Jiang ◽

Jianqiang Li ◽

...

Keyword(s):

Binding Proteins ◽

Protective Role ◽

Vbnc State ◽

Clavibacter Michiganensis ◽

Penicillin Binding Proteins ◽

Class B ◽

Increased Sensitivity ◽

Type C ◽

Adverse Environmental Conditions

Previous research has shown that penicillin-binding proteins (PBPs), enzymes involved in peptidoglycan (PG) assembly, could play an important role during the induction of viable but non-culturable (VBNC) state, which allows non-spore forming bacteria to survive adverse environmental conditions. The current study found that C. michiganensis has a total of seven PBP proteins. Mutant analysis indicated that deletion of either of the class B PBPs was lethal, and that the class A PBP, PBPC, had an important role in PG synthesis, with the ΔpbpC mutant having an altered cellular morphology that resulted in longer cells that were swollen at one end, and had thinner cell walls. The ΔpbpC mutant was also found to produce mucoid colonies in solid culture and a lower final cell titer in liquid medium, as well as having increased sensitivity to osmotic stress and lysozyme treatment, and surprisingly increased pathogenicity. The double mutant, ΔdacB/ΔpbpE also had a slightly altered phenotype resulting in longer cells. Further analysis revealed that both mutants had increased sensitivity to copper, which resulted in quicker induction into the VBNC state. However, only the ΔpbpC mutant had significantly reduced survivorship in the VBNC state. The study also confirmed the VBNC state significantly improved the survivorship of wild-type C. michiganensis cells in response to environmental stresses, and systemically demonstrated the protective role of the VBNC state in C. michiganensis, which is an important finding regarding its epidemiology, and has serious implications for disease management.

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All Detectable High-Molecular-Mass Penicillin-Binding Proteins Are Modified in a High-Level β-Lactam-Resistant Clinical Isolate of Streptococcus mitis

Antimicrobial Agents and Chemotherapy ◽

10.1128/aac.45.7.2075-2081.2001 ◽

2001 ◽

Vol 45 (7) ◽

pp. 2075-2081 ◽

Cited By ~ 9

Author(s):

Ana Amoroso ◽

Diego Demares ◽

Marta Mollerach ◽

Gabriel Gutkind ◽

Jacques Coyette

Keyword(s):

Molecular Mass ◽

Clinical Isolate ◽

Active Site ◽

Binding Proteins ◽

High Molecular Mass ◽

Mosaic Structure ◽

Kinetic Properties ◽

Streptococcus Mitis ◽

Penicillin Binding Proteins ◽

High Level

ABSTRACT All detectable high-molecular-mass penicillin-binding proteins (HMM PBPs) are altered in a clinical isolate of Streptococcus mitis for which the β-lactam MICs are increased from those previously reported in our region (cefotaxime MIC, 64 μg/ml). These proteins were hardly detected at concentrations that saturate all PBPs in clinical isolates and showed, after densitometric analysis, 50-fold-lower radiotracer binding. Resistance was related to mosaic structure in all HMM PBP-coding genes, where critical region replacement was complemented not only by substitutions already reported for the closely related Streptococcus pneumoniae but also by other specific replacements that are presumably close to the active-site serine. Mosaic structure was also presumed in apbp1a-sensitive strain used for comparison, confirming that these structures do not unambiguously imply, by themselves, detectable critical changes in the kinetic properties of these proteins.

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Rod Shape Determination by the Bacillus subtilis Class B Penicillin-Binding Proteins Encoded by pbpA and pbpH

Journal of Bacteriology ◽

10.1128/jb.185.16.4717-4726.2003 ◽

2003 ◽

Vol 185 (16) ◽

pp. 4717-4726 ◽

Cited By ~ 49

Author(s):

Yuping Wei ◽

Teresa Havasy ◽

Derrell C. McPherson ◽

David L. Popham

Keyword(s):

Cell Wall ◽

Bacillus Subtilis ◽

Binding Proteins ◽

Structural Integrity ◽

Sequence Similarity ◽

Inducible Promoter ◽

Penicillin Binding Proteins ◽

Peptidoglycan Cell Wall ◽

Class B ◽

Shape Determination

ABSTRACT The peptidoglycan cell wall determines the shape and structural integrity of a bacterial cell. Class B penicillin-binding proteins (PBPs) carry a transpeptidase activity that cross-links peptidoglycan strands via their peptide side chains, and some of these proteins are directly involved in cell shape determination. No Bacillus subtilis PBP with a clear role in rod shape maintenance has been identified. However, previous studies showed that during outgrowth of pbpA mutant spores, the cells grew in an ovoid shape for several hours before they recovered and took on a normal rod shape. It was postulated that another PBP, expressed later during outgrowth, was able to compensate for the lack of the pbpA product, PBP2a, and to guide the formation of a rod shape. The B. subtilis pbpH (ykuA) gene product is predicted to be a class B PBP with greatest sequence similarity to PBP2a. We found that a pbpH-lacZ fusion was expressed at very low levels in early log phase and increased in late log phase. A pbpH null mutant was indistinguishable from the wild-type, but a pbpA pbpH double mutant was nonviable. When pbpH was placed under the control of an inducible promoter in a pbpA mutant, viability was dependent on pbpH expression. Growth of this strain in the absence of inducer resulted in conversion of the cells from rods to ovoid/round shapes and lysis. We conclude that PBP2a and PbpH play redundant roles in formation of a rod-shaped peptidoglycan cell wall.

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A Mother Cell-Specific Class B Penicillin-Binding Protein, PBP4b, in Bacillus subtilis

Journal of Bacteriology ◽

10.1128/jb.186.1.258-261.2004 ◽

2004 ◽

Vol 186 (1) ◽

pp. 258-261 ◽

Cited By ~ 10

Author(s):

Yuping Wei ◽

Derrell C. McPherson ◽

David L. Popham

Keyword(s):

Bacillus Subtilis ◽

Rna Polymerase ◽

Mother Cell ◽

Binding Proteins ◽

Binding Protein ◽

Specific Class ◽

Penicillin Binding Protein ◽

Penicillin Binding Proteins ◽

Peptidoglycan Structure ◽

Class B

ABSTRACT The Bacillus subtilis genome encodes 16 penicillin-binding proteins (PBPs), some of which are involved in synthesis of the spore peptidoglycan. The pbpI (yrrR) gene encodes a class B PBP, PBP4b, and is transcribed in the mother cell by RNA polymerase containing σE. Loss of PBP4b, alone and in combination with other sporulation-specific PBPs, had no effect on spore peptidoglycan structure.

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The active-site-serine penicillin-recognizing enzymes as members of the Streptomyces R61 dd-peptidase family

Biochemical Journal ◽

10.1042/bj2500313 ◽

1988 ◽

Vol 250 (2) ◽

pp. 313-324 ◽

Cited By ~ 217

Author(s):

B Joris ◽

J M Ghuysen ◽

G Dive ◽

A Renard ◽

O Dideberg ◽

...

Keyword(s):

Amino Acid ◽

Active Site ◽

Binding Proteins ◽

Site Directed Mutagenesis ◽

Beta Lactamases ◽

Penicillin Binding Proteins ◽

E Coli ◽

X Ray Crystallography ◽

Binding Domains ◽

Class D

Homology searches and amino acid alignments, using the Streptomyces R61 DD-peptidase/penicillin-binding protein as reference, have been applied to the beta-lactamases of classes A and C, the Oxa-2 beta-lactamase (considered as the first known member of an additional class D), the low-Mr DD-peptidases/penicillin-binding proteins (protein no. 5 of Escherichia coli and Bacillus subtilis) and penicillin-binding domains of the high-Mr penicillin-binding proteins (PBP1A, PBP1B, PBP2 and PBP3 of E. coli). Though the evolutionary distance may vary considerably, all these penicillin-interactive proteins and domains appear to be members of a single superfamily of active-site-serine enzymes distinct from the classical trypsin or subtilisin families. The amino acid alignments reveal several conserved boxes that consist of strict identities or homologous amino acids. The significance of these boxes is highlighted by the known results of X-ray crystallography, chemical derivatization and site-directed-mutagenesis experiments.

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Evolution of a family of metazoan active-site-serine enzymes from penicillin-binding proteins: a novel facet of the bacterial legacy

BMC Evolutionary Biology ◽

10.1186/1471-2148-8-26 ◽

2008 ◽

Vol 8 (1) ◽

pp. 26 ◽

Cited By ~ 19

Author(s):

Nina Peitsaro ◽

Zydrune Polianskyte ◽

Jarno Tuimala ◽

Isabella Pörn-Ares ◽

Julius Liobikas ◽

...

Keyword(s):

Active Site ◽

Binding Proteins ◽

Penicillin Binding Proteins

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A Simple Screen for Murein Transglycosylase Inhibitors

Antimicrobial Agents and Chemotherapy ◽

10.1128/aac.44.5.1181-1185.2000 ◽

2000 ◽

Vol 44 (5) ◽

pp. 1181-1185 ◽

Cited By ~ 21

Author(s):

Waldemar Vollmer ◽

Joachim-Volker Höltje

Keyword(s):

High Throughput ◽

Active Site ◽

Binding Proteins ◽

Covalent Binding ◽

Penicillin Binding Proteins ◽

Crude Membrane ◽

Gel Beads ◽

Simple Filtration ◽

High Throughput Screens

ABSTRACT A simple assay for detection of compounds that bind to the active site in the transglycosylation domain of the essential bifunctional transglycosylase and transpeptidase penicillin-binding proteins (PBPs) is reported. The method is based on a competition with the specific transglycosylase inhibitor moenomycin. With moenomycin coupled to Affi-Gel beads, a simple filtration procedure allows the amount of labeled PBPs that bind to moenomycin beads in the presence of test substances to be determined. The PBPs can easily be labeled by the covalent binding of penicillin derivatives. Crude membrane extracts can be used as a source for the PBPs, and different kinds of labels for the penicillin-PBP complexes can be used. The assay can be adapted to high-throughput screens.

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

Antimicrobial Agents and Chemotherapy ◽

10.1128/aac.00677-06 ◽

2006 ◽

Vol 50 (12) ◽

pp. 4062-4069 ◽

Cited By ~ 15

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.

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