The aminoglycoside 6'-N-acetyltransferase type Ib encoded by Tn1331 is evenly distributed within the cell's cytoplasm

Abstract

The multiresistance transposon Tn1331, which mediates resistance to several aminoglycosides and beta-lactams, includes the aac(6')-Ib, aadA1, bla(OXA-9), and bla(TEM-1) genes. The nucleotide sequence of aac(6')-Ib includes a region identical to that of the bla(TEM-1) gene. This region encompasses the promoter and the initiation codon followed by 15 nucleotides. Since there were three possible translation initiation sites, the amino acid sequence at the N terminus of the aminoglycoside 6'-N-acetyltransferase type Ib [AAC(6')-Ib] was determined and was found to be SIQHF. This result indicated that aac(6')-Ib includes a translational fusion: the first five amino acids of the leader peptide of the TEM beta-lactamase are fused to the rest of the AAC(6')-Ib protein. This gene fusion could have formed during the genesis of Tn1331 as a consequence of the generation of a 520-nucleotide duplication (M. E. Tolmasky, Plasmid 24:218-226, 1990). An identical gene isolated from a Serratia marcescens strain has been previously described (G. Tran van Nhieu and E. Collatz, J. Bacteriol. 169:5708-5714, 1987). Extraction of the periplasmic proteins of E. coli harboring aac(6')-Ib by spheroplast formation showed that most of the AAC(6')-Ib protein is present in the cytoplasm. A genetic fusion to phoA confirmed these results. AAC(6')-Ib was shown to be evenly distributed inside the cell's cytoplasm by fluorescent microscopy with an AAC(6')-Ib-cyan fluorescent protein fusion.

FIG. 1.

Genetic map of Tn1331. The genetic map on top shows the relevant genes with the coordinates as defined in the sequence deposited in GenBank (accession number AF479774). The gray boxes encompassing nucleotides 6801 to 7320 and 9846 to 10365 represent the 520-bp direct repeats. The arrows indicate the locations of some genes [tnpA, tnpR, aac(6′)-Ib, and bla_TEM-1]. The translation initiation codon of aac(6′)-Ib is at coordinate 7302, and that of bla_TEM-1 is at coordinate 10347. The nucleotide sequence shown below the genetic map depicts the end of the direct repeat (open box), which is identical to the corresponding region in the bla_TEM-1 gene. The amino acid sequence is shown below the nucleotide sequence. Gray circles show the amino acids identified by sequencing of the N terminus of AAC(6′)-Ib.

FIG. 2.

Immunological detection of AAC(6′)-Ib, β-lactamase, and β-galactosidase in periplasmic and cytoplasmic fractions. The cytoplasmic and periplasmic protein content of E. coli(pJHCMW1) cells was obtained as described in Materials and Methods. Equal amounts of proteins were separated by SDS-polyacrylamide gel electrophoresis and subjected to immunoblotting with anti-AAC(6′)-Ib serum (AAC), anti-β-lactamase serum (β-lac), or monoclonal anti-β-galactosidase (β-gal). Numbers at left of panels are molecular masses in kilodaltons.

FIG. 3.

Visualization of protein fusions by fluorescence microscopy. Cells containing pAACCFP, pTGS, or pJDT1 were treated as described in Materials and Methods. The membranes of all three strains were stained by incubation in 200 μg of FM5-95/ml at 37°C with shaking for 15 min. Cells were focused (A, E, and I) and examined using filter sets 31044v2 to detect CFP (C), 31019 to detect GFP (G and K), and 31058 to detect FM5-95 (B, F, and J). Overlays were generated by coloring the membranes red and the fusion proteins green (D, H, and L).