Isolation, characterization, and DNA sequence analysis of an AAC(6')-II gene from Pseudomonas aeruginosa

Abstract

The gene encoding a 6'-N-acetyltransferase, AAC(6')-II, was cloned from Pseudomonas aeruginosa plasmid pSCH884. This gene mediates resistance to gentamicin, tobramycin, and netilmicin but not amikacin or isepamicin. The DNA sequence of the gene and flanking regions was determined. The 5'- and 3'-flanking sequences showed near identity to sequences found abutting a variety of different genes encoding resistance determinants. It is likely that the current structure arose by the integration of the 572-base-pair sequence containing the AAC(6')-II gene into a Tn21-related sequence at the recombinational hot spot, AAAGTT. We have compared the sequence of the AAC(6')-II gene to genes of other 6'-N-acetyltransferases. An AAC(6')-Ib protein (encoded by the aacA4 gene; G. Tran Van Nhieu and E. Collatz, J. Bacteriol. 169:5708-5714, 1987) that results in resistance to amikacin but not gentamicin was found to share 82% sequence similarity with the AAC(6')-II protein. We speculate that these two genes arose from a common ancestor and that the processes of selection and dissemination have led to the observed differences in the spectrum of aminoglycoside resistance.

Genetic analysis of bacterial acetyltransferases: identification of amino acids determining the specificities of the aminoglycoside 6'-N-acetyltransferase Ib and IIa proteins

“…Previous work has shown that the AAC(6')-Ib and AAC(6')-IIa proteins have different aminoglycoside resistance profiles with respect to gentamicin and amikacin (21). These observed differences were presumably due to amino acid substitutions within the AAC(6')-Ib and AAC(6')-IIa proteins.…”

“…Each of the plasmids, pSCH4663 and pSCHB4105, containing the aac(6')-Ib and aac(6')-IIa genes, respectively, is transcribed from the lac promoter present in the Bluescript plasmid. Previously it had been observed that the amino acid similarity between the AAC(6')-Ib and AAC(6')-IIa proteins began at the second codon of AAC(6')-IIa (21,27). Therefore the coding region of aac(6')-IIa, beginning at the second amino acid, has been fused to the region encoding first 18 amino acids of the AAC(6')-Ib protein.…”

“…In each of these hybrid proteins, the donor of the carboxy riously (21). half was largely responsible for determining the specificity of teins.…”

The integrons In0, In2, and In5 are defective transposon derivatives

“…The further extra segment in In2 corresponds to an acquired insertion sequence, IS1353. There are only three differences between In0 and In5, one of which is the presence of an aacA cassette integrated at attI in In5 (40). The remaining differences are both deletions with one endpoint at an IS1326 boundary and can be explained by invoking deletion events originating at the boundaries of IS1326.…”

Characterization of In0 of Pseudomonas aeruginosa plasmid pVS1, an ancestor of integrons of multiresistance plasmids and transposons of gram-negative bacteria

“…similar structure is consistent with its status as a lessevolved integron. The presence of such an element has been reported to be required on at least one of the sequences participating in the Int-mediated crossing over at the GTT sequence at crossover point 1 (28 (46), aacAlb (8), aadAl (49), aadA2 (36,38,53), aadB (43), dhfrII (52,62), dhfrV (49), oxa2 (19), aacAla (56), aacCl (61), cmUA (3), dhfrI (51), oxal (32), pse2 (20), pse4 (7), and merA (9) were from the respective references.…”