Sequence and molecular characterization of the ROB-1 beta-lactamase gene from Pasteurella haemolytica

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Abstract
The ROB-1 beta-lactamase-encoding plasmids from eight Pasteurella and two Haemophilus strains were compared by restriction endonuclease and hybridization analyses. Two types of ROB-1-encoding plasmids, which differed in size, were detected. One (4.1 kb) was found only in Pasteurella strains. The other (4.4 kb) was found in both Haemophilus influenzae and in one of the eight Pasteurella strains examined. These two plasmids shared multiple homologous fragments, suggesting that one was derived from the other. The ROB-1-encoding gene from Pasteurella haemolytica LNPB 51 was cloned and sequenced. An open reading frame of 915 nucleotides was found; it encoded a 305-amino-acid protein. Analysis of this amino acid sequence confirmed that the enzyme was found; it encoded a 305-amino-acid protein. Analysis of this amino acid sequence confirmed that the enzyme is a class A beta-lactamase. It had 32 to 48% homology with other class A enzymes and exhibited several common features of the gram-positive beta-lactamases. The ROB-1 mature protein, however, contained only one cysteine residue at position 123. These results suggest that ROB-1 is a link between beta-lactamases of gram-negative and gram-positive bacteria. An internal 230-bp DraI fragment from ROB-1 hybridized only with plasmid DNA from ROB-1-producing strains. This specific probe could be useful in epidemiological studies.