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Two genes encoding haloacetate dehalogenases, H-1 and H-2, are closely linked on a plasmid from Moraxellu sp. strain B. H-1 predominantly acts on fluoroacetate, but H-2 does not. To elucidate the molecular relationship between the two enzymes, we compared their structural genes. Two restriction fragments of the plasmid DNA were subcloned on M13 phages and their nucleotide sequences were determined. The sequence of each fragment contained an open reading frame that was identified as the structural gene for each of the two dehalogenases on the basis of the following criteria; N-terminal amino acid sequence, amino acid composition, and molecular mass. The genes for H-1 and H-2, designated &hHl and khH2, respectively, had Merent sizes (885 bp and 675 bp) and G + C contents (58.3% and 53.4%). Sequence analysis revealed no homology between the two genes. We concluded that the dehalogenases H-1 and H-2 have no enzyme-evolutionary relationship. The deduced amino acid sequence of the &hHl gene showed significant similarity to those of three hydrolases of Pseudomonasputida and a haloalkane dehalogenase of Xanthobacter rurtotrophicus. The &hH2 coding region was sandwiched between two repeated sequences about 1.8 kb long, which might play a part in the frequent spontaneous deletion of &hH2 from the plasmid.

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The self-transmissible plasmid pUO1 from Delftia acidovorans strain B carries two haloacetate-catabolic transposons, TnHad1 and TnHad2, and the mer genes for resistance to mercury. The complete 67,066-bp sequence of pUO1 revealed that the mer genes were also carried by two Tn402/Tn5053-like transposons, Tn4671 and Tn4672, and that the pUO1 backbone regions shared 99% identity to those of the archetype IncP-1␤ plasmid R751. Comparison of pUO1 with three other IncP-1␤ plasmids illustrated the importance of transposon insertion in the diversity and evolution of this group of plasmids. Mutational analysis of the four outermost residues in the inverted repeats (IRs) of TnHad2, a Tn21-related transposon, revealed a crucial role of the second residue of its IRs in transposition.It has been demonstrated that bacterial genes for the degradation of various xenobiotic compounds are often loaded on transposable elements that are mainly classified into two major groups: class I and class II transposons (14). The class I transposon is a mobile element in which a short DNA segment is flanked by two copies of an insertion sequence (IS). The class II transposon usually carries transposase (TnpA), resolvase (TnpR), and a resolution (res) site between short terminal inverted repeats (IRs) and transposes by a two-step processcointegration and resolution (3, 9). Broad-host-range and selftransmissible plasmids are well known to contribute to the wide dissemination of these catabolic transposons (13). The successful conjugal transfer of these plasmids facilitates the spread of the catabolic transposons and at times promotes the evolution of novel catabolic pathways by genetic rearrangements (15). A 67-kb self-transmissible and broad-host-range but not-well characterized plasmid, pUO1, from Delftia acidovorans strain B carries, in addition to the mer genes for resistance to mercury, two haloacetate dehalogenase genes (dehH1 and dehH2) on the two transposons, TnHad1 and TnHad2 (Fig. 1A) (10). TnHad1, with a size of 8.9 kb, is a class I transposon that carries dehH2 between two directly repeated copies of IS1071 (8), and dehH1 and TnHad1 are loaded on a 15.6-kb class II transposon, TnHad2, that lacks the tnpA and tnpR genes (Fig. 1A) (10). TnHad2 has the 38-bp terminal IRs, IR1 and IR3, and the res site (Fig. 1A and 2A), each of which shares extensive homology to the corresponding sequence of Tn21, and the supply in trans of the Tn21 transposition genes enables the complete transposition of TnHad2 (10). The sequence of IR2, which is highly homologous to IR1, is also located very close to the res site, although the 5Ј end of IR2 on pUO1 is occupied by the C residue (Fig. 1A and 2A). In our previous study (10), we replaced this C residue with the T residue, and the mini-TnHad2 derivative flanked by this IR2 derivative (5Ј-TGGG end) and IR1 (5Ј-GGGG end) (Fig. 2B) was found to transpose at a high frequency in the presence of the Tn21 tnpA gene. This finding was unexpected, since (i) the outermost four G residues of the IRs are conserved in ...