scholarly journals Characterization of the penA and penR genes of Burkholderia cepacia 249 which encode the chromosomal class A penicillinase and its LysR-type transcriptional regulator.

1997 ◽  
Vol 41 (11) ◽  
pp. 2399-2405 ◽  
Author(s):  
S Trépanier ◽  
A Prince ◽  
A Huletsky
Keyword(s):  
Burkholderia Cepacia ◽  
Klebsiella Oxytoca ◽  
Molecular Size ◽  
Beta Lactamase ◽  
Beta Lactam ◽  
Reading Frame ◽  
Beta Lactamases ◽  
Class A ◽  
High Degree ◽  
Degree Of Similarity

Burkholderia cepacia is recognized as an important pathogen in the lung infections of patients with cystic fibrosis. An inducible beta-lactamase activity has been associated with increased resistance to beta-lactam antibiotics in clinical isolates of B. cepacia. In this study, we report the revised sequence of the penA gene, which encodes the inducible penicillinase of B. cepacia, and show that it belongs to the molecular class A beta-lactamases and exhibits a high degree of similarity to the chromosomal beta-lactamase of Klebsiella oxytoca. Analysis of the nucleotide sequence of the DNA region directly upstream of the penA coding sequence revealed an open reading frame (penR), the transcription of which was oriented opposite to that of penA and whose initiation was 130 bp away from that of penA. Two potential ribosome-binding sites and two overlapping -10 and -35 promoter sequences were identified in the intercistronic region. The predicted translation product of penR was a polypeptide of 301 amino acids with an estimated molecular size of 33.2 kDa. The deduced polypeptide of penR showed a high degree of similarity with AmpR-like transcriptional activators of class A and C beta-lactamases, with identities of 59 and 58.7% with Pseudomonas aeruginosa PAO1 AmpR and Proteus vulgaris B317 CumR, respectively. The N-terminal portion of B. cepacia PenR was predicted to include a helix-turn-helix motif, which may bind the LysR motif identified in the intercistronic region. Induction of PenA by imipenem was shown to be dependent upon the presence of PenR. Expression of the cloned B. cepacia penA and penR genes in Escherichia coli SNO302 (ampD) resulted in a high basal and hyperinducible PenA activity. These results suggest that the regulation of the PenA penicillinase of B. cepacia 249 is similar to that observed in other class A and class C beta-lactamases that are under the control of a divergently transcribed AmpR-like regulator.

scholarly journals Proteolytic interconversion and N-terminal sequences of the Citrobacter diversus major β-lactamases

1991 ◽  
Vol 275 (3) ◽  
pp. 629-633 ◽  
Author(s):  
N Franceschini ◽  
G Amicosante ◽  
M Perilli ◽  
M Maccarrone ◽  
A Oratore ◽  
...  
Keyword(s):  
Klebsiella Oxytoca ◽  
Limited Proteolysis ◽  
Major Product ◽  
Beta Lactamase ◽  
Amino Acid Residues ◽  
Beta Lactamases ◽  
Citrobacter Diversus ◽  
High Degree ◽  
Papain Digestion ◽  
Degree Of Similarity

The N-terminal sequences of the two major beta-lactamases produced by Citrobacter diversus differed only by the absence of the first residue in form II and the loss of five amino acid residues at the C-terminal end. Limited proteolysis of the homogeneous form I protein yielded a variety of enzymatically active products. In the major product obtained after the action of papain, the first three N-terminal residues of form I had been cleaved, whereas at the C-terminal end the treated enzyme lacked five residues. However, this cannot explain the different behaviours of form I, form II and papain digestion product upon chromatofocusing. Form I, which was sequenced up to position 56, exhibited a very high degree of similarity with a Klebsiella oxytoca beta-lactamase. The determined sequence, which contained the active serine residue, demonstrated that the chromosome-encoded beta-lactamase of Citrobacter diversus belong to class A.

scholarly journals Characterization of beta-lactamase gene blaPER-2, which encodes an extended-spectrum class A beta-lactamase.

1996 ◽  
Vol 40 (3) ◽  
pp. 616-620 ◽  
Author(s):  
A Bauernfeind ◽  
I Stemplinger ◽  
R Jungwirth ◽  
P Mangold ◽  
S Amann ◽  
...  
Keyword(s):  
Amino Acid ◽  
Amino Acid Sequence ◽  
Beta Lactamase ◽  
Reading Frame ◽  
Beta Lactamases ◽  
Class A ◽  
Extended Spectrum ◽  
The Family ◽  
Extended Spectrum Beta Lactamases ◽  
Lactamase Gene

Plasmidic extended-spectrum beta-lactamases of Ambler class A are mostly inactive against ceftibuten. Salmonella typhimurium JMC isolated in Argentina harbors a bla gene located on a plasmid (pMVP-5) which confers transferable resistance to oxyiminocephalosporins, aztreonam, and ceftibuten. The beta-lactamase PER-2 (formerly ceftibutenase-1; CTI-1) is highly susceptible to inhibition by clavulanate and is located at a pI of 5.4 after isoelectric focusing. The blaPER-2 gene was cloned and sequenced. The nucleotide sequence of a 2.2-kb insert in vector pBluescript includes an open reading frame of 927 bp. Comparison of the deduced amino acid sequence of PER-2 with those of other beta-lactamases indicates that PER-2 is not closely related to TEM or SHV enzymes (25 to 26% homology). PER-2 is most closely related to PER-1 (86.4% homology), an Ambler class A enzyme first detected in Pseudomonas aeruginosa. An enzyme with an amino acid sequence identical to that of PER-1, meanwhile, was found in various members of the family Enterobacteriaceae isolated from patients in Turkey. Our data indicate that PER-2 and PER-1 represent a new group of Ambler class A extended-spectrum beta-lactamases. PER-2 so far has been detected only in pathogens (S. typhimurium, Escherichia coli, Klebsiella pneumoniae, Proteus mirabilis) isolated from patients in South America, while the incidence of PER-1-producing strains so far has been restricted to Turkey, where it occurs both in members of the family Enterobacteriaceae and in P. aeruginosa.

scholarly journals Sequences of beta-lactamase genes encoding CTX-M-1 (MEN-1) and CTX-M-2 and relationship of their amino acid sequences with those of other beta-lactamases.

1996 ◽  
Vol 40 (2) ◽  
pp. 509-513 ◽  
Author(s):  
A Bauernfeind ◽  
I Stemplinger ◽  
R Jungwirth ◽  
S Ernst ◽  
J M Casellas
Keyword(s):  
Amino Acid ◽  
Klebsiella Oxytoca ◽  
Amino Acid Sequences ◽  
Protein Sequencing ◽  
Beta Lactamase ◽  
Beta Lactamases ◽  
Class A ◽  
Men 1 ◽  
Genes Encoding ◽  
Relationship Of

Amino acid sequences determined either by protein sequencing or by DNA sequencing are identical for cefotaximases CTX-M-1 and MEN-1, whereas CTX-M-2 is 84% identical to CTX-M-1/MEN-1. Both beta-lactamases are distantly related to other plasmidic class A enzymes (homology to TEM-1 is 38.1% for CTX-M-1/MEN-1 and 36.5% for CTX-M-2); the closest relationship was with the chromosomal beta-lactamase of Klebsiella oxytoca E23004 (homologies of 74.5% for CTX-M-1/MEN-1 and 77.9% for CTX-M-2). The cefotaximases CTX-M-1/MEN-1 and CTX-M-2 represent two members of a new subgroup of plasmidic class A beta-lactamases.

scholarly journals Vaborbactam: Spectrum of Beta-Lactamase Inhibition and Impact of Resistance Mechanisms on Activity in Enterobacteriaceae

2017 ◽  
Vol 61 (11) ◽  
Author(s):  
Olga Lomovskaya ◽  
Dongxu Sun ◽  
Debora Rubio-Aparicio ◽  
Kirk Nelson ◽  
Ruslan Tsivkovski ◽  
...  
Keyword(s):  
Efflux Pump ◽  
Beta Lactamase ◽  
Beta Lactam ◽  
Content Type ◽  
Beta Lactamases ◽  
Class A ◽  
Class C ◽  
The Impact ◽  
Isogenic Strains ◽  
Lactamase Inhibitor

ABSTRACT Vaborbactam (formerly RPX7009) is a new beta-lactamase inhibitor based on a cyclic boronic acid pharmacophore. The spectrum of beta-lactamase inhibition by vaborbactam and the impact of bacterial efflux and permeability on its activity were determined using a panel of strains with beta-lactamases cloned from various classes and a panel of Klebsiella pneumoniae carbapenemase 3 (KPC-3)-producing isogenic strains with various combinations of efflux and porin mutations. Vaborbactam is a potent inhibitor of class A carbapenemases, such as KPC, as well as an inhibitor of other class A (CTX-M, SHV, TEM) and class C (P99, MIR, FOX) beta-lactamases. Vaborbactam does not inhibit class D or class B carbapenemases. When combined with meropenem, vaborbactam had the highest potency compared to the potencies of vaborbactam in combination with other antibiotics against strains producing the KPC beta-lactamase. Consistent with broad-spectrum beta-lactamase inhibition, vaborbactam reduced the meropenem MICs for engineered isogenic strains of K. pneumoniae with increased meropenem MICs due to a combination of extended-spectrum beta-lactamase production, class C beta-lactamase production, and reduced permeability due to porin mutations. Vaborbactam crosses the outer membrane of K. pneumoniae using both OmpK35 and OmpK36, but OmpK36 is the preferred porin. Efflux by the multidrug resistance efflux pump AcrAB-TolC had a minimal impact on vaborbactam activity. Investigation of the vaborbactam concentration necessary for restoration of meropenem potency showed that vaborbactam at 8 μg/ml results in meropenem MICs of ≤2 μg/ml in the most resistant engineered strains containing multiple mutations. Vaborbactam is a highly active beta-lactamase inhibitor that restores the activity of meropenem and other beta-lactam antibiotics in beta-lactamase-producing bacteria, particularly KPC-producing carbapenem-resistant Enterobacteriaceae.

Interaction of β-lactamases I and II from Bacillus cereus with semisynthetic cephamycins. Kinetic studies

1991 ◽  
Vol 279 (1) ◽  
pp. 111-114 ◽  
Author(s):  
J Martin Villacorta ◽  
P Arriaga ◽  
J Laynez ◽  
M Menendez
Keyword(s):  
Bacillus Cereus ◽  
Kinetic Studies ◽  
Beta Lactamase ◽  
Beta Lactam ◽  
Beta Lactamases ◽  
Class A ◽  
Rate Determining Step ◽  
Class B ◽  
Lactam Ring ◽  
Beta Lactamase Ii

The influence of C-6 alpha- or C-7 alpha-methoxylation of the beta-lactam ring in the catalytic action of class A and B beta-lactamases has been investigated. For this purpose the kinetic behaviour of beta-lactamases I (class A) and II (class B) from Bacillus cereus was analysed by using several cephamycins, moxalactam, temocillin and related antibiotics. These compounds behaved as poor substrates for beta-lactamase II, with high Km values and very low catalytic efficiencies. In the case of beta-lactamase I, the substitution of a methoxy group for a H atom at C-7 alpha or C-6 alpha decreased the affinity of the substrates for the enzyme. Furthermore, the acylation of cephamycins was completely blocked, whereas that of penicillins was slowed down by a factor of 10(4)-10(5), acylation being the rate-determining step of the process.

scholarly journals The phototrophic bacterium Rhodopseudomonas capsulata sp108 encodes an indigenous class A β-lactamase

1989 ◽  
Vol 260 (3) ◽  
pp. 803-812 ◽  
Author(s):  
J I A Campbell ◽  
S Scahill ◽  
T Gibson ◽  
R P Ambler
Keyword(s):  
Sequence Similarity ◽  
Rhodopseudomonas Capsulata ◽  
Beta Lactamase ◽  
Reading Frame ◽  
Beta Lactamases ◽  
Class A ◽  
Coli Plasmid ◽  
Total Dna ◽  
Lactamase Gene ◽  
Protein Sequence Comparison

The nucleotide sequence of a 2.37 kb DNA fragment derived from cloning a total DNA digest of Rhodopseudomonas capsulata sp108 was determined. The DNA codes for a beta-lactamase, a protein showing sequence similarity to the ampR protein of Enterobacter cloacae and an unidentified open reading frame. Hybridization experiments with a probe carrying DNA from within the beta-lactamase gene suggests a chromosomal location for the coding sequences in strain sp108 and in sp109, a penicillin-sensitive revertant of sp108 in which the enzyme is not inducible. A protein-sequence comparison of the deduced amino acid sequence of the Rps. capsulata beta-lactamase indicates that it is a Class A enzyme and that its sequence can be aligned with those of the characterized beta-lactamases from Staphylococcus aureus, Bacillus licheniformis and the Escherichia coli plasmid (R-TEM enzyme), with only a few insertions or deletions. The corresponding DNA sequence is, however, characteristically rhodopseudomonad, suggesting that it is not a recently transposed gene.

scholarly journals Variability of chromosomally encoded beta-lactamases from Klebsiella oxytoca.

1997 ◽  
Vol 41 (8) ◽  
pp. 1641-1648 ◽  
Author(s):  
B Fournier ◽  
P H Roy
Keyword(s):  
Klebsiella Oxytoca ◽  
Beta Lactamase ◽  
Beta Lactam ◽  
Beta Lactams ◽  
Beta Lactamases ◽  
Main Form ◽  
Extended Spectrum Beta Lactamases ◽  
Lactamase Gene ◽  
Hydrolysis Of ◽  
Substrate Hydrolysis

The beta-lactamase genes of Klebsiella oxytoca were previously divided into two main groups: bla(OXY-1) and bla(OXY-2). The two beta-lactamase groups were each represented by beta-lactamases with four different pIs. In each group, one form of beta-lactamase is more frequent than the others combined. The beta-lactamase gene of each representative beta-lactamase with a different pI that was not yet sequenced (pIs 5.7, 6.8 [OXY-2], 7.1, 8.2, and 8.8 [OXY-1]) was cloned and sequenced. The susceptibility patterns as well as relative rates and kinetic parameters for beta-lactam hydrolysis revealed that OXY-2 enzymes hydrolyzed several of the beta-lactams that were examined (carbenicillin, cephalothin, cefamandole, ceftriaxone, and aztreonam) at a greater rate than the OXY-1 enzymes did. Comparison of K. oxytoca beta-lactamases with plasmid-mediated extended-spectrum beta-lactamases MEN-1 and TOHO-1 implied that the threonine at position 168 present in OXY-2 beta-lactamase instead of the alanine in OXY-1 could be responsible for its modified substrate hydrolysis. In each group, the beta-lactamase with a variant pI differs from the main form of beta-lactamase by one to five amino acid substitutions. The substrate profile and the 50% inhibitory concentrations revealed that all substitutions differing from the main form of beta-lactamase were neutral except one difference in the OXY-1 group. This substitution of an Ala to a Gly at position 237 increases the hydrolysis of some beta-lactams, particularly aztreonam; decreases the hydrolysis of benzylpenicillin, cephaloridine, and cefamandole, and decreases the susceptibility to clavulanic acid (fivefold increase in the 50% inhibitory concentration).

scholarly journals Evolution of an enzyme activity: crystallographic structure at 2-A resolution of cephalosporinase from the ampC gene of Enterobacter cloacae P99 and comparison with a class A penicillinase

1993 ◽  
Vol 90 (23) ◽  
pp. 11257-11261 ◽  
Author(s):  
E Lobkovsky ◽  
P C Moews ◽  
H Liu ◽  
H Zhao ◽  
J M Frere ◽  
...  
Keyword(s):  
Binding Site ◽  
Enterobacter Cloacae ◽  
Crystallographic Structure ◽  
Beta Lactamase ◽  
Beta Lactam ◽  
Beta Lactamases ◽  
X Ray Crystallography ◽  
Class A ◽  
Beta Lactam Antibiotics ◽  
Class C

The structure of the class C ampC beta-lactamase (cephalosporinase) from Enterobacter cloacae strain P99 has been established by x-ray crystallography to 2-A resolution and compared to a class A beta-lactamase (penicillinase) structure. The binding site for beta-lactam (penicillinase) structure. The binding site for beta-lactam antibiotics is generally more open than that in penicillinases, in agreement with the ability of the class C beta-lactamases to better bind third-generation cephalosporins. Four corresponding catalytic residues (Ser-64/70, Lys-67/73, Lys-315/234, and Tyr-150/Ser-130 in class C/A) lie in equivalent positions within 0.4 A. Significant differences in positions and accessibilities of Arg-349/244 may explain the inability of clavulanate-type inhibitors to effectively inactivate the class C beta-lactamases. Glu-166, required for deacylation of the beta-lactamoyl intermediate in class A penicillinases, has no counterpart in this cephalosporinase; the nearest candidate, Asp-217, is 10 A from the reactive Ser-64. A comparison of overall tertiary folding shows that the cephalosporinase, more than the penicillinase, is broadly similar to the ancestral beta-lactam-inhibited enzymes of bacterial cell wall synthesis. On this basis, it is proposed that the cephalosporinase is the older of the two beta-lactamases, and, therefore, that a local refolding in the active site, rather than a simple point mutation, was required for the primordial class C beta-lactamase to evolve to the class A beta-lactamase having an improved ability to catalyze the deacylation step of beta-lactam hydrolysis.

scholarly journals Structural and kinetic studies on β-lactamase K1 from Klebsiella aerogenes

1986 ◽  
Vol 234 (2) ◽  
pp. 343-347 ◽  
Author(s):  
E L Emanuel ◽  
J Gagnon ◽  
S G Waley
Keyword(s):  
Active Site ◽  
Ph Dependence ◽  
Kinetic Studies ◽  
Thiol Group ◽  
Klebsiella Aerogenes ◽  
Beta Lactamase ◽  
Beta Lactam ◽  
Beta Lactamases ◽  
Class A ◽  
Rate Determining Step

beta-Lactamase K1 from Klebsiella aerogenes 1082E hydrolyses both penicillins and cephalosporins comparably and is inhibited by mercurials but not by cloxacillin. These properties distinguish it from those other beta-lactamases that have been allotted to classes on the basis of their amino sequences. beta-Lactamase K1 has been isolated by affinity chromatography; its composition shows resemblances to class A beta-lactamases. Moreover, the N-terminal sequence is similar to those of class A beta-lactamases: there is about 30% identity over the first 32 residues. Furthermore, a putative active-site octapeptide has been isolated and its sequence is similar to the region around the active-site serine residue in class A beta-lactamases. There is one thiol group in beta-lactamase K1; it is not essential for activity. The pH-dependence of kcat. and kcat./Km for the hydrolysis of benzylpenicillin by beta-lactamase K1 were closely similar, suggesting that the rate-determining step is cleavage of the beta-lactam ring.

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