Identification Of The Genes Involved In The Production Of Extracellular Protease By Screening The Insertion Mutant Library Of Xan Homonas Campestr1s Pv Campestris

A mutant library of Xanthomonas campestris pv. campestris(hereafter Xcc) strain 8004 with 17820 clones was constructed by random transposon Tn5gwsA5 mutagenesis, which cover 1800 predicted ORFs of Xcc genome. 53 extracellular protease mutants were screened from the library. The insertion sites of Tn5gusA5 were located by methods of TAIL-PCR and sequencing. For these 53 protease mutants, the insertion sites were distributed in 14 ORFs. The result of blastx shows that one ORF is extracellular serine protease precursor gene; 3 ORFs function as regulatory genes; 5 ORFs are involved in secretion pathway; 2 ORFs are related to production of LPS; and the annotation functions of the other 3 ORFs are not clearly related to extracellular protease prouduction. Marker exchange method was used to study the relationship between the production of protease and the 3 ORFs. A deletion mutant of xcc₄463 was constructed successfully. The ability of the mutant to degradate the protein in medium and cause symptom on host plant was significantly decreased.