Complete nucleotide sequence of Tn10

Abstract

The complete nucleotide sequence of Tn10 has been determined. The dinucleotide signature and percent G+C of the sequence had no discontinuities, indicating that Tn10 constitutes a homogeneous unit. The new sequence contained three new open reading frames corresponding to a glutamate permease, repressors of heavy metal resistance operons, and a hypothetical protein in Bacillus subtilis. The glutamate permease was fully functional when expressed, but Tn10 did not protect Escherichia coli from the toxic effects of various metals.

FIG. 1

Sequencing strategy and map of Tn10. (A) The sequence of Tn10 was used to construct an ORF map which shows the name of each ORF and the predicted number of amino acids. The direction and location of predicted translational start sites are indicated above the map. The locations of predicted stop codons are indicated below the map. The locations of the ends of the flanking IS elements are indicated by asterisks. Analysis of DNA sequences was carried out using web-based resources at the National Center for Biotechnology Information: ORF Finder, BLAST, GenBank, and SwissProt. Additional searches were performed using OmniBlast at the Sanger Centre. (B) The percent G+C and the percent difference in dinucleotide signature were plotted for Tn10 using a circular permutation of the sequence and a window size of 500 bp by N. J. Saunders, J. S. Mirsky, and S. Jarvis at The Institute of Molecular Medicine, University of Oxford. (C) Tn10 was sequenced using a primer walking strategy with plasmids pNK81, pNK82, and pNK83 (4) as templates. The location, direction, and length of sequence data acquired from each sequencing reaction are indicated by arrows below the scale bar. Regions of Tn10 sequenced previously are indicated by arrows accompanied by GenBank accession numbers. Oligonucleotide primers were from Sigma Genosys, and fluorescent sequencing was carried out by the Department of Biochemistry DNA Sequencing Unit using machines from Applied Biosystems.

FIG. 2

jemA encodes a functional glutamate transporter. E. coli K-12 gltS deletion strain MK416 (ΔgltS Kan^r prototroph) (5) was plated on M9 minimal media. (Left) Plate with 0.5 mM IPTG and 0.5% glutamate as the sole source of carbon and energy. Cells were either untransformed or transformed with pRC163 expressing jemA from the ptac promoter. (Right) Plate with M9 minimal glucose agar containing 100 μg of ampicillin/ml, 0.5 mM IPTG, and 40 μg of α-methylglutamate/ml. Cells were transformed either with the control plasmid pBluescript (Stratagene) or with pRC163 expressing jemA from the ptac promoter.