Analysis And Identification Of Transposition Tendency Of Bacterial Transposon Tn5gusA5 In Xcc 8004

A mutant library of Xanthomonas campestris pv. campestris (hereafter Xcc) strain 8004 was constructed by random transposon Tn5gusA5 mutagenesis. After amplifying genomic sequences flanked with Tn5gusA5 by TAIL PCR and DNA sequencing, then doing BLAST with genomic sequence of Xcc 8004, insertion sites of 10981 mutants were located precisely in Xcc 8004 genome. Analysis of Tn5 insertional tendency in Xcc 8004 genome was carried out using programs developed by our bioinformatics group employing computer language Visual BASIC and SQL Sever 2000 database. Some tendency of Tn5gusA5 transposition were found that all preferred sites of Tn5gusA5 in Xcc 8004 genomic DNA are in AT-rich regions; target sequences of Tn5gusA5 have some features that the probabilities of guanine and cytosine are high respectively at the head and tail base of target sequence; the level of gene transcription does not influence insertion density of Tn5gusA5 significantly. In order to attest the above tendency of Tn5gusA5 transposition, we constructed plasmid pL77 which is a pUC19 derivative containing low and high GC segments of Xcc 8004 genomic DNA .After culturing and screening JM109: Tn5gusA5/pL77, we harvested 55 clonies of JM109/pL77:: Tn5gusA5. DNA sequence analysis indicated that Tn5gusA5 is prone to insert into low GC content regions; guanine is a preferential base at the first place and cytosine at the last site of target sequence. The tendencies of Tn5 transposition observed both in Xcc 8004 genomic DNA and in pL77 are similar.