MUTAGENESIS OF BURKHOLDERIA-PSEUDOMALLEI WITH TN5-OT182 - ISOLATION OF MOTILITY MUTANTS AND MOLECULAR CHARACTERIZATION OF THE FLAGELLIN STRUCTURAL GENE

Burkholderia pseudomallei is a human and animal pathogen in tropical r egions, especially Southeast Asia and northern Australia, Currently li ttle is known about the genetics and molecular biology of this organis m, In this report, we describe the mutagenesis of B. pseudomallei with the transposon Tn5-OT182. B. pseudomallei 1026b transposon mutants we re obtained at a frequency of 4.6 x 10(-4) per initial donor cell, and the transposon inserted randomly into the chromosome, We used Tn5-OT1 82 to identify the flagellin structural gene, fliC. We screened 3,500 transposon mutants and identified 28 motility mutants. Tn5-OT182 integ rated into 19 unique genetic loci encoding proteins with homology to E scherichia coli and Salmonella typhimurium flagellar and chemotaxis pr oteins, Two mutants, MM35 and MM36, contained Tn5-OT182 integrations i n fliC. We cloned and sequenced fliC and used it to complement MM35 an d MM36 in trans, The fliC transcriptional start site and a sigma(F)-li ke promoter were identified by primer extension analysis, We observed a significant difference in the expression of two distinct fliC-lacZ t ranscriptional fusions during bacterial growth, suggesting the presenc e of a latent intragenic transcriptional terminator in fliC. There was no significant difference in the virulence of 1026b compared to that of MM36 in diabetic rats or Syrian hamsters, suggesting that flagella and/or motility are probably not virulence determinants in these anima l models of B. pseudomallei infection, A phylogenetic analysis based o n the flagellins from a variety of bacterial species supported the rec ent transfer of B. pseudomallei from the genus Pseudomonas to Burkhold eria.