scholarly journals Functional insights into the high-molecular-mass penicillin-binding proteins of Streptococcus agalactiae revealed by gene deletion and transposon mutagenesis analysis

2021 ◽  
Author(s):  
Luchang Zhu ◽  
Prasanti Yerramilli ◽  
Layne Pruitt ◽  
Abhishek Mishra ◽  
Randall J. Olsen ◽  
...  
Keyword(s):  
Cell Wall ◽  
Molecular Mass ◽  
Binding Proteins ◽  
Bacterial Pathogen ◽  
High Molecular Mass ◽  
Isogenic Mutant ◽  
Penicillin Binding Proteins ◽  
Cell Wall Peptidoglycan ◽  
Mutant Strains ◽  
Group B

High-molecular-mass penicillin-binding proteins (PBPs) are enzymes that catalyze the biosynthesis of bacterial cell wall peptidoglycan. The Gram-positive bacterial pathogen Streptococcus agalactiae (group B streptococcus , or GBS) produces five high-molecular-mass PBPs, namely, PBP1A, PBP1B, PBP2A, PBP2B, and PBP2X. Among these, only PBP2X is essential for cell viability, whereas the other four PBPs are individually dispensable. The biological function of the four non-essential PBPs is poorly characterized in GBS. We deleted the pbp1a , pbp1b , pbp2a , and pbp2b genes individually from a genetically well-characterized serotype V GBS strain, and studied the phenotypes of the four isogenic mutant strains. Compared to the wild-type parental strain (i) none of the pbp isogenic mutant strains had a significant growth defect in THY rich medium, (ii) isogenic mutant strains Δ pbp1a and Δ pbp1b had significantly increased susceptibility to penicillin and ampicillin, and (iii) isogenic mutant strains Δ pbp1a and Δ pbp2b had significantly longer chain lengths. Using saturated transposon mutagenesis and transposon insertion site sequencing, we determined genes essential for the viability of wild-type GBS strain and each of the four isogenic pbp deletion mutant strains in THY rich medium. The pbp1a gene is essential for cell viability in the pbp2b deletion background. Reciprocally, pbp2b is essential in the pbp1a deletion background. Moreover, the gene encoding RodA, a peptidoglycan polymerase that works in conjunction with PBP2B, is also essential in the pbp1a deletion background. Together, our results suggest functional overlap between PBP1A and PBP2B-RodA complex in GBS cell wall peptidoglycan biosynthesis. IMPORTANCE High-molecular-mass penicillin-binding proteins (HMM-PBPs) are enzymes required for bacterial cell-wall biosynthesis. Bacterial pathogen group B streptococcus (GBS) produces five distinct HMM-PBPs. The biological functions of these proteins are not well characterized in GBS. In this study, we performed a comprehensive deletion analysis of genes encoding HMM-PBPs in GBS. We found that deleting certain PBP-encoding genes altered bacterial susceptibility to beta-lactam antibiotics, cell morphology, and the essentiality of other enzymes involved in cell-wall peptidoglycan synthesis. The results of our study shed new light on the biological functions of PBPs in GBS.

scholarly journals 4-Quinolones as Noncovalent Inhibitors of High Molecular Mass Penicillin-Binding Proteins

2012 ◽  
Vol 3 (7) ◽  
pp. 592-595 ◽  
Author(s):  
Abbas G. Shilabin ◽  
Liudmila Dzhekieva ◽  
Pushpa Misra ◽  
B. Jayaram ◽  
R. F. Pratt
Keyword(s):  
Molecular Mass ◽  
Binding Proteins ◽  
High Molecular Mass ◽  
Penicillin Binding Proteins

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

2001 ◽  
Vol 45 (7) ◽  
pp. 2075-2081 ◽  
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.

scholarly journals Two Class A High-Molecular-Weight Penicillin-Binding Proteins of Bacillus subtilis Play Redundant Roles in Sporulation

2001 ◽  
Vol 183 (20) ◽  
pp. 6046-6053 ◽  
Author(s):  
Derrell C. McPherson ◽  
Adam Driks ◽  
David L. Popham
Keyword(s):  
Cell Wall ◽  
Bacillus Subtilis ◽  
Germ Cell ◽  
Double Mutant ◽  
Binding Proteins ◽  
Penicillin Binding Proteins ◽  
Class A ◽  
Mutant Strains ◽  
Spore Walls ◽  
Multiple Class

ABSTRACT The four class A penicillin-binding proteins (PBPs) ofBacillus subtilis appear to play functionally redundant roles in polymerizing the peptidoglycan (PG) strands of the vegetative-cell and spore walls. The ywhE product was shown to bind penicillin, so the gene and gene product were renamedpbpG and PBP2d, respectively. Construction of mutant strains lacking multiple class A PBPs revealed that, while PBP2d plays no obvious role in vegetative-wall synthesis, it does play a role in spore PG synthesis. A pbpG null mutant produced spore PG structurally similar to that of the wild type; however, electron microscopy revealed that in a significant number of these spores the PG did not completely surround the spore core. In a pbpF pbpG double mutant this spore PG defect was apparent in every spore produced, indicating that these two gene products play partially redundant roles. A normal amount of spore PG was produced in the double mutant, but it was frequently produced in large masses on either side of the forespore. The double-mutant spore PG had structural alterations indicative of improper cortex PG synthesis, including twofold decreases in production of muramic δ-lactam and l-alanine side chains and a slight increase in cross-linking. Sporulation gene expression in the pbpF pbpG double mutant was normal, but the double-mutant spores failed to reach dormancy and subsequently degraded their spore PG. We suggest that these two forespore-synthesized PBPs are required for synthesis of the spore germ cell wall, the first layer of spore PG synthesized on the surface of the inner forespore membrane, and that in the absence of the germ cell wall the cells lack a template needed for proper synthesis of the spore cortex, the outer layers of spore PG, by proteins on the outer forespore membrane.

scholarly journals Acyltransferase activities of the high-molecular-mass essential penicillin-binding proteins

1991 ◽  
Vol 279 (2) ◽  
pp. 601-604 ◽  
Author(s):  
M Adam ◽  
C Damblon ◽  
M Jamin ◽  
W Zorzi ◽  
V Dusart ◽  
...  
Keyword(s):  
Molecular Mass ◽  
Binding Proteins ◽  
Spectroscopic Properties ◽  
High Molecular Mass ◽  
Acyl Donor ◽  
Penicillin Binding Proteins ◽  
Cytoplasmic Membranes ◽  
Shape Determination ◽  
Hydrolysis Of

The high-molecular-mass penicillin-binding proteins (HMM-PBPs), present in the cytoplasmic membranes of all eubacteria, are involved in important physiological events such as cell elongation, septation or shape determination. Up to now it has, however, been very difficult or impossible to study the catalytic properties of the HMM-PBPs in vitro. With simple substrates, we could demonstrate that several of these proteins could catalyse the hydrolysis of some thioesters or the transfer of their acyl moiety on the amino group of a suitable acceptor nucleophile. Many of the acyl-donor substrates were hippuric acid or benzoyl-D-alanine derivatives, and their spectroscopic properties enabled a direct monitoring of the enzymic reaction. In their presence, the binding of radioactive penicillin to the PBPs was also inhibited.

scholarly journals Escherichia coli Mutants Lacking All Possible Combinations of Eight Penicillin Binding Proteins: Viability, Characteristics, and Implications for Peptidoglycan Synthesis

1999 ◽  
Vol 181 (13) ◽  
pp. 3981-3993 ◽  
Author(s):  
Sylvia A. Denome ◽  
Pamela K. Elf ◽  
Thomas A. Henderson ◽  
David E. Nelson ◽  
Kevin D. Young
Keyword(s):  
Escherichia Coli ◽  
Cell Wall ◽  
Binding Proteins ◽  
Kanamycin Resistance ◽  
Open Reading Frames ◽  
Penicillin Binding Proteins ◽  
E Coli ◽  
Peptidoglycan Biosynthesis ◽  
Kanamycin Resistance Cassette ◽  
Genes Encoding

ABSTRACT The penicillin binding proteins (PBPs) synthesize and remodel peptidoglycan, the structural component of the bacterial cell wall. Much is known about the biochemistry of these proteins, but little is known about their biological roles. To better understand the contributions these proteins make to the physiology ofEscherichia coli, we constructed 192 mutants from which eight PBP genes were deleted in every possible combination. The genes encoding PBPs 1a, 1b, 4, 5, 6, and 7, AmpC, and AmpH were cloned, and from each gene an internal coding sequence was removed and replaced with a kanamycin resistance cassette flanked by two ressites from plasmid RP4. Deletion of individual genes was accomplished by transferring each interrupted gene onto the chromosome of E. coli via λ phage transduction and selecting for kanamycin-resistant recombinants. Afterwards, the kanamycin resistance cassette was removed from each mutant strain by supplying ParA resolvase in trans, yielding a strain in which a long segment of the original PBP gene was deleted and replaced by an 8-bpres site. These kanamycin-sensitive mutants were used as recipients in further rounds of replacement mutagenesis, resulting in a set of strains lacking from one to seven PBPs. In addition, thedacD gene was deleted from two septuple mutants, creating strains lacking eight genes. The only deletion combinations not produced were those lacking both PBPs 1a and 1b because such a combination is lethal. Surprisingly, all other deletion mutants were viable even though, at the extreme, 8 of the 12 known PBPs had been eliminated. Furthermore, when both PBPs 2 and 3 were inactivated by the β-lactams mecillinam and aztreonam, respectively, several mutants did not lyse but continued to grow as enlarged spheres, so that one mutant synthesized osmotically resistant peptidoglycan when only 2 of 12 PBPs (PBPs 1b and 1c) remained active. These results have important implications for current models of peptidoglycan biosynthesis, for understanding the evolution of the bacterial sacculus, and for interpreting results derived by mutating unknown open reading frames in genome projects. In addition, members of the set of PBP mutants will provide excellent starting points for answering fundamental questions about other aspects of cell wall metabolism.

scholarly journals FmhA and FmhC of Staphylococcus aureus incorporate serine residues into peptidoglycan cross-bridges

2020 ◽  
Vol 295 (39) ◽  
pp. 13664-13676 ◽  
Author(s):  
Stephanie Willing ◽  
Emma Dyer ◽  
Olaf Schneewind ◽  
Dominique Missiakas
Keyword(s):  
Staphylococcus Aureus ◽  
Cell Wall ◽  
Binding Proteins ◽  
Penicillin Binding Proteins ◽  
Daughter Cells ◽  
Cross Bridge ◽  
Resistant Strains ◽  
The Cross ◽  
Cross Bridges ◽  
Adjacent Wall

Staphylococcal peptidoglycan is characterized by pentaglycine cross-bridges that are cross-linked between adjacent wall peptides by penicillin-binding proteins to confer robustness and flexibility. In Staphylococcus aureus, pentaglycine cross-bridges are synthesized by three proteins: FemX adds the first glycine, and the homodimers FemA and FemB sequentially add two Gly-Gly dipeptides. Occasionally, serine residues are also incorporated into the cross-bridges by enzymes that have heretofore not been identified. Here, we show that the FemA/FemB homologues FmhA and FmhC pair with FemA and FemB to incorporate Gly-Ser dipeptides into cross-bridges and to confer resistance to lysostaphin, a secreted bacteriocin that cleaves the pentaglycine cross-bridge. FmhA incorporates serine residues at positions 3 and 5 of the cross-bridge. In contrast, FmhC incorporates a single serine at position 5. Serine incorporation also lowers resistance toward oxacillin, an antibiotic that targets penicillin-binding proteins, in both methicillin-sensitive and methicillin-resistant strains of S. aureus. FmhC is encoded by a gene immediately adjacent to lytN, which specifies a hydrolase that cleaves the bond between the fifth glycine of cross-bridges and the alanine of the adjacent stem peptide. In this manner, LytN facilitates the separation of daughter cells. Cell wall damage induced upon lytN overexpression can be alleviated by overexpression of fmhC. Together, these observations suggest that FmhA and FmhC generate peptidoglycan cross-bridges with unique serine patterns that provide protection from endogenous murein hydrolases governing cell division and from bacteriocins produced by microbial competitors.

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