scholarly journals Enterobactin- and salmochelin-β-lactam conjugates induce cell morphologies consistent with inhibition of penicillin-binding proteins in uropathogenic Escherichia coli CFT073

2021 ◽  
Author(s):  
Artur Sargun ◽  
Timothy C. Johnstone ◽  
Hui Zhi ◽  
Manuela Raffatellu ◽  
Elizabeth M. Nolan
Keyword(s):  
Escherichia Coli ◽  
Binding Proteins ◽  
Uropathogenic Escherichia Coli ◽  
Penicillin Binding Proteins ◽  
E Coli

Siderophore-β-lactam conjugates based on enterobactin and diglucosylated enterobactin enter the periplasm of uropathogenic E. coli CFT073 via the FepA and IroN transporters, and target penicillin-binding proteins.

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 Affinity of Tomopenem (CS-023) for Penicillin-Binding Proteins in Staphylococcus aureus, Escherichia coli, and Pseudomonas aeruginosa

2008 ◽  
Vol 53 (3) ◽  
pp. 1238-1241 ◽  
Author(s):  
Tetsufumi Koga ◽  
Chika Sugihara ◽  
Masayo Kakuta ◽  
Nobuhisa Masuda ◽  
Eiko Namba ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Staphylococcus Aureus ◽  
Pseudomonas Aeruginosa ◽  
Binding Proteins ◽  
Binding Protein ◽  
Penicillin Binding Protein ◽  
Penicillin Binding Proteins ◽  
E Coli ◽  
High Affinity

ABSTRACT Tomopenem (formerly CS-023), a novel 1β-methylcarbapenem, exhibited high affinity for penicillin-binding protein (PBP) 2 in Staphylococcus aureus, PBP 2 in Escherichia coli, and PBPs 2 and 3 in Pseudomonas aeruginosa, which are considered major lethal targets. Morphologically, tomopenem induced spherical forms in E. coli and short filamentation with bulges in P. aeruginosa, which correlated with the drug's PBP profiles. The potential of resistance of these bacteria to tomopenem was comparable to that to imipenem.

scholarly journals Contributions of PBP 5 anddd-Carboxypeptidase Penicillin Binding Proteins to Maintenance of Cell Shape in Escherichia coli

2001 ◽  
Vol 183 (10) ◽  
pp. 3055-3064 ◽  
Author(s):  
David E. Nelson ◽  
Kevin D. Young
Keyword(s):  
Escherichia Coli ◽  
Cell Shape ◽  
Binding Proteins ◽  
Penicillin Binding Proteins ◽  
E Coli ◽  
Morphological Defects ◽  
Membrane Anchoring ◽  
Binding Sequence

ABSTRACT Escherichia coli has 12 recognized penicillin binding proteins (PBPs), four of which (PBPs 4, 5, and 6 and DacD) havedd-carboxypeptidase activity. Although the enzymology of the dd-carboxypeptidases has been studied extensively, the in vivo functions of these proteins are poorly understood. To explain why E. coli maintains four independent loci encoding enzymes of considerable sequence identity and comparable in vitro activity, it has been proposed that thedd-carboxypeptidases may substitute for one another in vivo. We tested the validity of this equivalent substitution hypothesis by investigating the effects of these proteins on the aberrant morphology of ΔdacA mutants, which produce no PBP 5. Although cloned PBP 5 complemented the morphological phenotype of a ΔdacA mutant lacking a total of seven PBPs, controlled expression of PBP 4, PBP 6, or DacD did not. Also, a truncated PBP 5 protein lacking its amphipathic C-terminal membrane binding sequence did not reverse the morphological defects and was lethal at low levels of expression, implying that membrane anchoring is essential for the proper functioning of PBP 5. By examining a set of mutants from which multiple PBP genes were deleted, we found that significant morphological aberrations required the absence of at least three different PBPs. The greatest defects were observed in cells lacking, at minimum, PBPs 5 and 6 and one of the endopeptidases (either PBP 4 or PBP 7). The results further differentiate the roles of the low-molecular-weight PBPs, suggest a functional significance for the amphipathic membrane anchor of PBP 5 and, when combined with the recently determined crystal structure of PBP 5, suggest possible mechanisms by which these PBPs may contribute to maintenance of a uniform cell shape in E. coli.

scholarly journals Profiling of β-Lactam Selectivity for Penicillin-Binding Proteins in Escherichia coli Strain DC2

2015 ◽  
Vol 59 (5) ◽  
pp. 2785-2790 ◽  
Author(s):  
Ozden Kocaoglu ◽  
Erin E. Carlson
Keyword(s):  
Escherichia Coli ◽  
Binding Proteins ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Coli Strain ◽  
Bacterial Cell Division ◽  
Content Type ◽  
Penicillin Binding Proteins ◽  
Whole Cells ◽  
E Coli

ABSTRACTPenicillin-binding proteins (PBPs) are integral players in bacterial cell division, and their catalytic activities can be monitored with β-lactam-containing chemical probes. Compounds that target a single PBP could provide important information about the specific role(s) of each enzyme, making identification of such molecules important. We evaluated 22 commercially available β-lactams for inhibition of the PBPs in liveEscherichia colistrain DC2. Whole cells were titrated with β-lactam antibiotics and subsequently incubated with a fluorescent penicillin derivative, Bocillin-FL (Boc-FL), to label uninhibited PBPs. Protein visualization was accomplished by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) separation and fluorescent scanning. The examined β-lactams exhibited diverse PBP selectivities, with amdinocillin (mecillinam) showing selectivity for PBP2, aztreonam, piperacillin, cefuroxime, cefotaxime, and ceftriaxone for PBP3, and amoxicillin and cephalexin for PBP4. The remaining β-lactams did not block any PBPs in the DC2 strain ofE. colior inhibited more than one PBP at all examined concentrations in this Gram-negative organism.

scholarly journals Penicillin-Binding Proteins inLeptospira interrogans

2001 ◽  
Vol 45 (3) ◽  
pp. 870-877 ◽  
Author(s):  
Audrey Brenot ◽  
Daren Trott ◽  
Isabelle Saint Girons ◽  
Richard Zuerner
Keyword(s):  
Escherichia Coli ◽  
Binding Proteins ◽  
Sequence Variation ◽  
Protein Sequences ◽  
Leptospira Interrogans ◽  
Homologous Proteins ◽  
Penicillin Binding Proteins ◽  
E Coli

ABSTRACT The Leptospira interrogans ponA andpbpB genes were isolated and characterized.ponA and pbpB encode the penicillin-binding proteins (PBPs) 1 and 3, respectively. There is little sequence variation between the PBP genes from two L. interrogans strains (serovar icterohaemorrhagiae strain Verdun and serovar pomona strain RZ11). The deduced L. interrogans PBP 1 and PBP 3 protein sequences from the two strains shared over 50% similarity to homologous proteins fromEscherichia coli. It was demonstrated for strain Verdun that ponA and pbpB are transcribed individually from their own promoter. The ponA andpbpB genes from both strains are separated by 8 to 10 kb and oriented such that their transcription is convergent. The L. interrogans PBP 1 and PBP 3 proteins were synthesized inE. coli and were modified with ampicillin using a digoxigenin-ampicillin conjugate. These data show that both genes encode functional PBPs.

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