Nucleotide sequence analysis and DNA hybridization studies of the ant(4')-IIa gene from Pseudomonas aeruginosa.
The ant(4')-IIa gene was previously cloned from Pseudomonas aeruginosa on a 1.6-kb DNA fragment (G. A. Jacoby, M. J. Blaser, P. Santanam, H. Hächler, F. H. Kayser, R. S. Hare, and G. H. Miller, Antimicrob. Agents Chemother. 34:2381-2386, 1990). In the current study, the ant(4')-IIa gene was localized by gamma-delta mutagenesis. A region of approximately 600 nucleotides which contained the ant(4')-IIa gene was identified, and DNA sequence analysis revealed two overlapping open reading frames (ORFs) within this region. Northern (RNA) blot analysis demonstrated expression of both ORFs in P. aeruginosa; therefore, site-directed mutagenesis was used to identify the ORF which encodes the ant(4')-IIa gene. No homology was found between ant(4')-IIa and ant(4')-Ia DNA sequences. Hybridization experiments confirmed that the ant(4')-Ia probe hybridized only to gram-positive presumptive ANT(4')-I strains and that the ant(4')-IIa probe hybridized only to gram-negative strains presumed to carry ANT(4')-II. Seven gram-negative strains which had been classified as having ANT(4')-II resistance profiles did not hybridize with probes for either ant(4')-Ia or ant(4')-IIa, suggesting that at least one additional ant(4') gene may exist. The predicted amino-terminal sequences of the ANT(4')-Ia and ANT(4')-IIa proteins showed significant sequence similarity between residues 38 and 63 of the ANT(4')-Ia protein and residues 26 and 51 of the ANT(4')-IIa protein.
Shaw
K J
KJ
Schering-Plough Research Institute, Kenilworth, New Jersey 07033.
Munayyer
H
H
Rather
P N
PN
Hare
R S
RS
Miller
G H
GH
D001483:Base Sequence; D015152:Blotting, Northern; D003001:Cloning, Molecular; D015342:DNA Probes; D004269:DNA, Bacterial / Q000032:analysis*; D004352:Drug Resistance, Microbial; D008969:Molecular Sequence Data; D016296:Mutagenesis; D009693:Nucleic Acid Hybridization; D009841:Oligonucleotides / Q000235:genetics; D016366:Open Reading Frames; D010957:Plasmids; D011550:Pseudomonas aeruginosa / Q000235:genetics*
D015342:DNA Probes; D004269:DNA, Bacterial; D009841:Oligonucleotides