Tn5-OT182 should not be used to identify genes involved in biofilm formation in Burkholderia pseudomallei.
Burkholderia pseudomallei, a gram-negative bacterium, is the causative agent of melioidosis. One of the important virulence properties of this bacteria is its ability to form a biofilm. Genes involved in biofilm formation in B. pseudomallei have not been thoroughly studied. In this study, Tn5-OT182 mutagenesis was used to isolate of B. pseudomallei strain A2 mutants unable to produce biofilm. Ten biofilm-defective transposon mutants were isolated and analyzed. Flanking DNA from each transposon mutant were self-cloned and sequenced, then the sequences were analyzed with the BLAST program. To confirm these genes are involved in biofilm formation, we constructed three gene deletion mutants marked with a tetracycline resistance gene. The constructed tet(r)-marked deletion mutants were checked for correct structure and size by polymerase chain reaction. When subjected to biofilm assay, all tested tet(r)-marked deletion mutants were still able to produce biofilm, indicating the three genes are not involved in biofilm formation. These results suggest integration of Tn5-OT182 in genes not involved in biofilm production can render B. pseudomallei unable to produce biofilm by an unknown mechanism. This information demonstrates Tn5-OT182 is not a reliable tool for identifying genes involved in biofilm formation unless a confirmatory experiment is carried out in parallel.
Department of Clinical Immunology, Center for Research and Development in Medical Diagnostic Laboratory (CMDL), Faculty of Associated Medical Sciences, Khon Kaen University, Khon Kaen, Thailand.
Jirarat
J
Songsri
Tanakorn
T
Proungvitaya
Surasak
S
Wongratanacheewin
Preecha
P
Homchampa
D018441:Biofilms; D015139:Blotting, Southern; D016957:Burkholderia pseudomallei / Q000235:genetics* / Q000472:pathogenicity*; D004251:DNA Transposable Elements; D004269:DNA, Bacterial / Q000235:genetics; D017353:Gene Deletion; D015964:Gene Expression Regulation, Bacterial; D005798:Genes, Bacterial / Q000235:genetics; D006801:Humans; D016296:Mutagenesis; D019895:Transposases / Q000235:genetics*; D014774:Virulence / Q000235:genetics
D004251:DNA Transposable Elements; D004269:DNA, Bacterial; C473503:Tn5 transposase; D019895:Transposases