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Prokaryotic genome sequences can now be determined rapidly, but it is commonly found that about 20 to 25% of open reading frames (ORFs) cannot be assigned a likely function by bioinformatics studies alone. Efficient methods for generating and analyzing mutants are needed to determine gene function, and we have been developing transposons (for a review, see references 2-4) for this purpose in extremely halophilic archaea (halobacteria). Our previous haloarchaeal transposon, ThD28 (6) (Fig. 1), contained the Haloferax volcanii mevinolin resistance marker (Mev^r) between two tandem copies of the Halobacterium salinarum insertion sequence, ISH28. ThD28 mutants of Haloarcula hispanica (9) contained insertions at different positions in the host chromosome, but these were unstable, as the tandem nature of the ISH elements and the presence of active transposase genes allowed further transposition and recombination events (6).

View larger version (18K): [in this window] [in a new window]   FIG. 1.   Maps of plasmids pMDS28, carrying the transposon ThD28 (previously described in reference 6), and pMDS73, carrying the improved transposon ThD73 (described in this study). ThD73 consists of a halobacterial mevinolin-simvastatin resistance gene (Mevr; accession no. M83531) (10) and the E. coli plasmid vector pUC19 (19), which includes parts of the lacZ and lacI genes, a pMB1 replicon, and an ampicillin resistance gene, Apr. These are flanked by ISH28 TIRs. The ISH28 transposase (Tpase) gene is located outside the transposon.

An improved transposon, ThD73, is shown in Fig. 1. Construction details can be obtained from the authors. It consisted of a haloarchaeal resistance marker (Mev^r [10]), plasmid pUC19 (19) to allow recovery of the transposon (and flanking DNA) in Escherichia coli, and the terminal inverted repeat (TIR) sequences of ISH28 (18). The ISH28 transposase gene (without TIRs) was placed outside the transposon. The plasmid was unable to replicate in halobacteria, so simvastatin-resistant transformants should arise only if the transposon integrated into the host genome. Plasmid pMDS73 was introduced into H. hispanica cells with a polyethylene glycol-mediated transformation protocol (5) and simvastatin-resistant transformants were selected on solid medium. The transformation frequency was approximately 3 × 10³ colonies per µg of plasmid DNA, similar to that of the previous transposon, ThD28 (6). A replicating plasmid (pWL102 [10]) routinely gave transformation frequencies of 10⁴ transformants per µg.

To show that the transformants contained a copy of ThD73, 100 simvastatin-resistant colonies were patched onto nylon membranes and grown on selective medium; colony blots were prepared and probed with ³²P-labeled pUC19 DNA (a component of ThD73). All colonies hybridized strongly, while wild-type H. hispanica did not (results not shown). To verify that the transposase gene (which was outside the transposon) had not been inserted, the same colony blot of 100 transformant colonies was probed with an internal NaeI-DraIII fragment of ISH28. This fragment was not present inside the transposon and, as expected, the colonies were all negative (data not shown). A positive control (a H. hispanica::ThD28 mutant, containing a complete ISH28) hybridized strongly.

Twenty randomly selected ThD73 mutants were grown in liquid culture, chromosomal DNA was extracted, and Southern blots were prepared. Hybridizations of these blots confirmed that the insertions were single copies, and that each one was at a different location in the 20 mutants (data not shown). The same DNA preparations were used to recover the transposons (as plasmids) back into E. coli. After digestion with MluI (which cuts outside the transposon), restriction fragments containing the inserted transposons were circularized (by ligation) and electroporated into E. coli DH5. Ampicillin-resistant transformants were readily obtained for all chromosomal DNA preparations and cells were found to possess plasmids. Sequencing outward from the transposon ends into the flanking DNA gave, on average, about 500 bases of DNA sequence for each plasmid (Table 1). These were compared to the GenBank nucleotide sequence database by the BLAST suite of programs (1), and several flanking sequences showed significant nucleotide or predicted amino acid similarity to genes from halobacteria and other organisms (Table 1).

Target site duplications were usually 8 bp long (15) but two were 9 bp. They varied widely in sequence, although three target sites were similar (e.g., plasmids pWW1, -5, and -16) (Table 1). The percentages of G+C content of the flanking sequences also varied, from 41 to 63% (Table 1). Halobacteria have a high average G+C content in their DNA (about 62 to 68%), but in many cases this can be separated into two fractions termed FI (high G+C) and FII (low G+C) DNA, respectively. Haloarcula marismortui, a close relative of H. hispanica, has FI and FII DNA fractions of 62 and 55% G+C, respectively. The latter fraction usually represents 10 to 30% of the halobacterial genome and is a mixture of endogenous plasmids and genomic fragments (13, 14). The high G+C fraction is thought to possess most of the important genes (17) and to be relatively stable. The low G+C fraction is quite variable in size and arrangement and seems to contain the majority of insertion sequences (8, 16, 17). The selection pressure maintaining this peculiar distribution is not fully understood (14, 17). The large halobacterial plasmid pNRC100 shows several regions of relatively low percentages of G+C content that contain few genes or long ORFs but have multiple complete or partial ISH elements, suggesting that these regions probably represent favored target areas for ISH element insertion (12).

Two insertions occurred in or near (unrelated) ISH elements, which could reflect a preference either for low G+C DNA or for ISH elements themselves. In IS elements of Bacteria, target preferences for DNA targets with high or low percentages of G+C content, as well as for other IS elements, have all been observed (7, 11) but the nature of these selection preferences, in terms of DNA structure, is not known. The possible role of histone-like proteins has been discussed recently by Mahillon and Chandler (11).

Previous reports of ISH elements in halobacteria have usually analyzed insertions into specific genes, particularly genes with readily observable phenotypes such as bacteriorhodopsin (bop) or gas vesicle production (gvp) genes (13, 14). ISH28 was originally found as an insertion into the bop gene of Halobacterium salinarum (16). Other more general studies have used Southern blot hybridization to establish the distribution of different ISH elements in halobacteria (14). The present study is the first to follow insertions of a haloarchaeal transposon at the molecular level after its introduction into a halobacterial cell. Assuming that the target specificity of ThD73 is similar to that of ISH28, the results indicate that ISH28 is capable of integrating into a wide variety of target sites.