Biochemical-genetic analysis and distribution of FAR-1, a class A beta-lactamase from Nocardia farcinica

Abstract

From genomic DNA of the clinical isolate Nocardia farcinica VIC, a 1. 6-kb Sau3AI fragment was cloned and expressed in Escherichia coli JM109. The recombinant strain expressed a beta-lactamase (pI, 4.6), FAR-1, which conferred high levels of resistance to amoxicillin, piperacillin, ticarcillin, and cephalothin. The hydrolysis constants (kcat, Km, Ki, and 50% inhibitory concentration) confirmed the MIC results and showed that FAR-1 activity is inhibited by clavulanic acid and at a low level by tazobactam and sulbactam. Moreover, FAR-1 beta-lactamase hydrolyzes aztreonam (at a low level) without significant activity against ceftazidime, cefotaxime and imipenem. FAR-1 mature protein of molecular mass ca 32 kDa, has less than 60% amino acid identity with any other class A beta-lactamases, being most closely related to PEN-A from Burkholderia cepacia (52%). A blaFAR-1-like gene was found in all studied N. farcinica strains, underlining the constitutive origin of this gene.

FIG. 1

Restriction endonuclease schematic map of the recombinant plasmid pFAR-1, which codes the β-lactamase FAR-1 from N. farcinica VIC. The thin line represents the cloned insert from N. farcinica VIC; the dotted lines indicate vector pBK-CMV, and the thick line represents the studied β-lactamase gene, with the arrow indicating its translational orientation.

FIG. 2

Nucleotide sequence of the 1,543-bp fragment of pFAR-1 containing the β-lactamase coding region. The deduced amino acid sequence is designated in single-letter code below the nucleotide sequence. The start and stop codons and the five structural elements characteristic of class A β-lactamases are in boldface. Additionally, a putative second start site (GTG) is underlined. Inverted repeat sequences are underlined by convergent arrows. The “∫” symbol indicates the putative cleavage site for the leader peptide. The 1,543 bp are numbered successively, and the amino acid numbering is according to the method of Ambler (1). Important amino acid positions of the deduced protein FAR-1 compared to those of TEM-1 are numbered; Ambler positions 69, 104, 182, 238, 240, 244, and 275.

FIG. 3

Dot blot hybridizations of different strains with the radiolabelled 700-bp HincII-SmaI internal probe for bla_FAR-1. Blots: 1, E. coli JM109 (negative control); 2, N. farcinica 94.0250; 3, N. farcinica 94.0664; 4, N. farcinica 95.0288; 5, N. farcinica 95.0684; 6, N. farcinica 96.0027; 7, N. farcinica 96.0087; 8, N. asteroides; 9, N. farcinica 96.0624; 10, R. equi ATCC 6939 (negative control); 11, N. farcinica 96.0691; 12, N. farcinica 96.0994; 13, N. farcinica 96.1087; 14, N. farcinica 97.0244; 15, N. farcinica VIC; and 16, E. coli JM109(pFAR-1).