Terminal truncations in amp C beta-lactamase from a clinical isolate of Pseudomonas aeruginosa.
AmpC beta-lactamases from strains of Pseudomonas aeruginosa have previously been shown to be heterogeneous with respect to their isoelectric point (pI). In order to elucidate the origin of this heterogeneity enzymes were isolated from a clinical isolate of a multiresistant P. aeruginosa strain and biochemically characterized. The purification was accomplished in four chromatographic steps comprising dye-affinity, size-exclusion, hydrophobic interaction chromatography, and chromatofocusing; this resulted in five forms with pI values of 9.1, 8.7, 8.3, 8.2, and 7.6. When analysed by SDS/PAGE and agarose IEF each separated beta-lactamase appeared to be both size- and charge-homogeneous. The specific activities of the variants were very similar. MS of each isolated beta-lactamase form showed minor differences in molecular mass (range 40.0-40.8 kDa). MS of the beta-lactamase with a pI of 8.2 demonstrated the presence of two subforms. The N-terminal sequences of three of the beta-lactamases were identical to the published sequence [Lodge, J.M. , Minchin, S.D., Piddock, L.J.V. & Busby, J.W. (1990) Biochem. J. 272, 627-631], while two variants were truncated by two amino-acid residues, one of which was acidic. The previously published sequence contains an alanine as the ultimate residue, but two of the beta-lactamases showed a substitution of Ala371 for arginine, whereas in the remaining forms C-terminal truncations by one and three residues were found. Our results indicate that the P. aeruginosa strain does not harbour multiple copies of the ampC gene, but rather that the five beta-lactamase isoforms are products of a single structural gene. The combinations of the identified N- and/or C-terminal truncations explained the multiple pI values of the beta-lactamase isoforms.
Walther-Rasmussen
J
J
Department of Clinical Microbiology and Department of Clinical Biochemistry, Rigshospitalet, The National University Hospital, Copenhagen, Denmark.
Johnsen
A H
AH
Høiby
N
N
D001426:Bacterial Proteins; D003550:Cystic Fibrosis / Q000382:microbiology; D006801:Humans; D013058:Mass Spectrometry; D020033:Protein Isoforms; D011550:Pseudomonas aeruginosa / Q000737:chemistry*; D017421:Sequence Analysis; D018440:beta-Lactam Resistance; D001618:beta-Lactamases / Q000737:chemistry* / Q000302:isolation & purification
D001426:Bacterial Proteins; D020033:Protein Isoforms; C048795:AmpC beta-lactamases; D001618:beta-Lactamases