Project description:The beta-lactamase Toho-1 exhibits a strong tendency to form merohedrally twinned crystals. Here, the crystal quality of Toho-1 was improved by using surface modification to remove a sulfate ion involved in crystal packing. The surface-modified Toho-1 variant (R274N/R276N) was crystallized under similar conditions to those used for wild-type Toho-1. R274N/R276N did not form merohedrally twinned crystals. The crystals diffracted to a significantly higher resolution (approximately 0.97 A) than the wild-type crystals (1.65 A); they belonged to the same space group and had almost identical unit-cell parameters to those of wild-type Toho-1.
Project description:Escherichia coli TUM1083, which is resistant to ampicillin, carbenicillin, cephaloridine, cephalothin, piperacillin, cefuzonam, and aztreonam while being sensitive to cefoxitin, moxalactam, cefmetazole, ceftazidime, and imipenem, was isolated from the urine of a patient treated with beta-lactam antibiotics. The beta-lactamase (Toho-2) purified from the bacteria hydrolyzed beta-lactam antibiotics such as penicillin G, carbenicillin, cephaloridine, cefoxitin, cefotaxime, ceftazidime, and aztreonam and especially had increased relative hydrolysis rates for cephalothin, cephaloridine, cefotaxime, and ceftizoxime. Different from other extended-spectrum beta-lactamases, Toho-2 was inhibited 16-fold better by the beta-lactamase inhibitor tazobactam than by clavulanic acid. Resistance to beta-lactams was transferred by conjugation from E. coli TUM1083 to E. coli ML4909, and the transferred plasmid was about 54.4 kbp, belonging to the incompatibility group IncFII. The cefotaxime resistance gene for Toho-2 was subcloned from the 54.4-kbp plasmid. The sequence of the gene was determined, and the open reading frame of the gene was found to consist of 981 bases. The nucleotide sequence of the gene (DDBJ accession no. D89862) designated as bla(toho) was found to have 76.3% identity to class A beta-lactamase CTX-M-2 and 76.2% identity to Toho-1. It has 55.9% identity to SHV-1 beta-lactamase and 47.5% identity to TEM-1 beta-lactamase. Therefore, the newly isolated beta-lactamase designated as Toho-2 produced by E. coli TUM1083 is categorized as an enzyme similar to Toho-1 group beta-lactamases rather than to mutants of TEM or SHV enzymes. According to the amino acid sequence deduced from the DNA sequence, the precursor consisted of 327 amino acid residues. Comparison of Toho-2 with other beta-lactamase (non-Toho-1 group) suggests that the substitutions of threonine for Arg-244 and arginine for Asn-276 are important for the extension of the substrate specificity.
Project description:We studied the genetic organization of bla(ACC-1) in 14 isolates of Enterobacteriaceae from France, Tunisia, and Germany. In a common ancestor, ISEcp1 was likely involved in the mobilization of this gene from the Hafnia alvei chromosome to a plasmid. Other genetic events involving insertion sequences (particularly IS26), transposons (particularly Tn1696), or sulI-type integrons have occurred, leading to complex genetic environments.
Project description:PER-1 extended-spectrum β-lactamase-producing Gram-negative bacilli are resistant to oxyimino-cephalosporins. However, the bla(PER-1) gene has never been reported in Klebsiella pneumoniae. Here, we studied interspecies dissemination of the bla(PER-1) gene by horizontal transfer of Tn1213 among Acinetobacter baumannii, Pseudomonas aeruginosa, and K. pneumoniae. In a K. pneumoniae clinical isolate, the bla(PER-1) gene was located on a 150-kbp incompatibility group A/C plasmid.
Project description:ImportanceDespite increasing reports, class A β-lactamases of environmental bacteria remain very poorly characterized, with limited understanding of their transmission patterns. To address this knowledge gap, we focused on a recently designated GMA family β-lactamase gene, bla GMA-1, found in marine bacterial genera such as Vibrio. This study shows that gammaproteobacterial mobile class A β-lactamase is specialized for penicillin degradation, and bla GMA-1 is frequently linked to strand-biased circularizing integrative elements (SEs) in sequences in the RefSeq/GenBank database. Evidence for the implication of SEs in β-lactamase environmental transmission provides insights for future surveillance studies of antimicrobial resistance genes in human clinical settings.
Project description:Backbone chemical shift assignments for the Toho-1 β-lactamase (263 amino acids, 28.9 kDa) are reported based on triple resonance solution-state NMR experiments performed on a uniformly 2H,13C,15N-labeled sample. These assignments allow for subsequent site-specific characterization at the chemical, structural, and dynamical levels. At the chemical level, titration with the non-β-lactam β-lactamase inhibitor avibactam is found to give chemical shift perturbations indicative of tight covalent binding that allow for mapping of the inhibitor binding site. At the structural level, protein secondary structure is predicted based on the backbone chemical shifts and protein residue sequence using TALOS-N and found to agree well with structural characterization from X-ray crystallography. At the dynamical level, model-free analysis of 15N relaxation data at a single field of 16.4 T reveals well-ordered structures for the ligand-free and avibactam-bound enzymes with generalized order parameters of ~ 0.85. Complementary relaxation dispersion experiments indicate that there is an escalation in motions on the millisecond timescale in the vicinity of the active site upon substrate binding. The combination of high rigidity on short timescales and active site flexibility on longer timescales is consistent with hypotheses for achieving both high catalytic efficiency and broad substrate specificity: the induced active site dynamics allows variously sized substrates to be accommodated and increases the probability that the optimal conformation for catalysis will be sampled.
Project description:Genetic structures surrounding the carbapenem-hydrolyzing Ambler class A bla KPC gene were characterized in several KPC-positive Klebsiella pneumoniae and Pseudomonas aeruginosa strains isolated from the United States, Colombia, and Greece. The bla KPC genes were associated in all cases with transposon-related structures. In the K. pneumoniae YC isolate from the United States, the beta-lactamase bla KPC-2 gene was located on a novel Tn3-based transposon, Tn4401. Tn4401 was 10 kb in size, was delimited by two 39-bp imperfect inverted repeat sequences, and harbored, in addition to the beta-lactamase bla KPC-2 gene, a transposase gene, a resolvase gene, and two novel insertion sequences, ISKpn6 and ISKpn7. Tn4401 has been identified in all isolates. However, two isoforms of this transposon were found: Tn4401a was found in K. pneumoniae YC and K. pneumoniae GR from the United States and Greece, respectively, and differed by a 100-bp deletion, located just upstream of the bla KPC-2 gene, compared to the sequence of Tn4401b, which was found in the Colombian isolates. In all isolates tested, Tn4401 was flanked by a 5-bp target site duplication, the signature of a recent transposition event, and was inserted in different open reading frames located on plasmids that varied in size and nature. Tn4401 is likely at the origin of carbapenem-hydrolyzing beta-lactamase KPC mobilization to plasmids and its further insertion into various-sized plasmids identified in nonclonally related K. pneumoniae and P. aeruginosa isolates.
Project description:A Pseudomonas aeruginosa isolate highly resistant to carbapenems was collected from a patient with postsurgical cerebrospinal infection in Greece. The isolate carried a class 1 integron that contained as a sole cassette the gene bla(VIM-4), a novel variant of bla(VIM-1), with one nucleotide difference resulting in a Ser-to-Arg change at amino acid position 175 of the VIM-1 enzyme. This is the first detection of a VIM-1 variant after its appearance in Italy.
Project description:The spread of clonally related carbapenem-resistant Pseudomonas aeruginosa producing the metallo-beta-lactamase SPM-1 was found in Recife, Brazil. Upstream of bla(SPM-1), a novel common region (CR4) was identified, comprising an open reading frame, orf495, whose product shares significant identity with putative recombinases, such as Orf513. CR4 may be responsible for mobilization and expression of bla(SPM-1).
Project description:The genetic structures (ca. 10-kb DNA fragment) surrounding the plasmid-borne extended-spectrum beta-lactamase bla(CTX-M-19) gene in a Klebsiella pneumoniae clinical isolate were determined. This beta-lactamase gene was part of a 4,797-bp transposon inserted inside orf1 of Tn1721. Inside this transposon, bla(CTX-M-19) was bracketed upstream and downstream by insertion sequences ISE cp1B and IS903D, respectively, and further downstream by a truncated gene encoding an outer membrane protein for iron transport. The single-copy ISEcp1B element was probably involved alone in the mobilization process that led to a 5-bp duplication at the target site of the transposed fragment. This mobilization event probably involved one inverted repeat of ISE cp1B and a second sequence farther away, resembling its second inverted repeat. Additionally, ISEcp1B provided -35 and -10 promoter sequences, contributing to the high-level expression of the bla(CTX-M-19) gene. Southern blot analysis failed to identify a reservoir of ISEcp1-like sequences among a series of gram-negative and gram-positive bacterial species usually found in the skin and intestinal human floras. The ability of ISEcp1-like elements to mobilize and to promote the expression of beta-lactamase genes may explain, in part, the current spread of CTX-M-type enzymes worldwide.