Chromosome-Encoded Narrow-Spectrum Ambler Class A β-Lactamase GIL-1 from Citrobacter gillenii

Abstract

A novel ␤-lactamase gene was cloned from the whole-cell DNA of an enterobacterial Citrobacter gillenii reference strain that displayed a weak narrow-spectrum ␤-lactam-resistant phenotype and was expressed in Escherichia coli. It encoded a clavulanic acid-inhibited Ambler class A ␤-lactamase, GIL-1, with a pI value of 7.5 and a molecular mass of ca. 29 kDa. GIL-1 had the highest percent amino acid sequence identity with TEM-1 and SHV-1, 77%, and 67%, respectively, and only 46%, 31%, and 32% amino acid sequence identity with CKO-1 (C. koseri), CdiA1 (C. diversus), and SED-1 (C. sedlaki), respectively. The substrate profile of the purified GIL-1 was similar to that of ␤-lactamases TEM-1 and SHV-1. The bla GIL-1 gene was chromosomally located, as revealed by I-CeuI experiments, and was constitutively expressed at a low level in C. gillenii. No gene homologous to the regulatory ampR genes of chromosomal class C ␤-lactamases was found upstream of the bla GIL-1 gene, which fits the noninducibility of ␤-lactamase expression in C. gillenii. Rapid amplification of DNA 5 ends analysis of the promoter region revealed putative promoter sequences that diverge from what has been identified as the consensus sequence in E. coli. The bla GIL-1 gene was part of a 5.5-kb DNA fragment bracketed by a 9-bp duplication and inserted between the D-lactate dehydrogenase gene and the ydbH genes; this DNA fragment was absent in other Citrobacter species. This work further illustrates the heterogeneity of ␤-lactamases in Citrobacter spp., which may indicate that the variability of Citrobacter species is greater than expected.

CTX-M-93, a CTX-M Variant Lacking Penicillin Hydrolytic Activity

“…It was filtrated onto a polyethersulfone membrane (Vivaspin, 50,000 molecular weight [MW]; Sartorius Stedim, Aubagne, France) that removes molecules with a molecular mass higher than 50 kDa, prior to a 10-fold concentration (Vivaspin, 10,000 MW; Sartorius Stedim), according to instructions provided by the manufacturer. The protein content was measured by using the Bio-Rad DC protein assay, and the specific activities of the crude extract and of the purified ␤-lactamase were determined as previously reported (20), with 100 M cephalothin as the substrate. One unit of enzyme activity was defined as the amount of enzyme that hydrolyzes 1 mol of substrate per min.…”

“…One unit of enzyme activity was defined as the amount of enzyme that hydrolyzes 1 mol of substrate per min. The purity of the enzymes was estimated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) (20).…”

“…Crude ␤-lactamase extracts obtained from 10-ml cultures of clinical isolate E. coli Bre-1, electroporants, and recombinant clones were subjected to analytical isoelectrofocusing (IEF) as previously described (20,25).…”

“…A 2-liter culture of E. coli BL21(pET-M93) was induced by isopropyl-␤-D-thiogalactopyranoside (IPTG) (1 mg/ml) as previously described (20). The ␤-lactamase extract was obtained after sonication as described previously (20), was dialyzed overnight against 50 mM diethanolamine (DEA) buffer (pH 9.7), and was then loaded onto a preequilibrated Q-Sepharose column (Amersham Pharmacia Biotech) in 50 mM DEA buffer (pH 9.7). The ␤-lactamase activities, as determined qualitatively for each fraction using nitrocefin hydrolysis (Oxoid), were detected in the flowthrough fraction.…”

AmpC β-Lactamases

“…Klebsiella pneumoniae, Klebsiella oxytoca, Proteus mirabilis, and Salmonella spp. (31) lack a chromosomal bla AmpC gene, as do Citrobacter amalonaticus (328), Citrobacter farmeri, Citrobacter gillenii (224), Citrobacter koseri (formerly Citrobacter diversus and Levinea malonatica), Citrobacter rodentium, Citrobacter sedlakii (252), Edwardsiella hoshinae, Edwardsiella ictaluri (312), Kluyvera ascorbata (138,305), Kluyvera cryocrescens (72), Plesiomonas shigelloides (9), Proteus penneri (175), Proteus vulgaris (60), Rahnella aquatilis (30,308), Yersinia pestis, and Yersinia pseudotuberculosis (313) as well as, probably, Escherichia hermannii (91), Francisella tularensis (27), Shewanella algae (123), and Stenotrophomonas maltophilia (111). However, since bla AmpC genes occur on transmissible plasmids, the clinical microbiologist needs to consider this resistance mechanism whatever the identification of an organism.…”