An efficient system for the generation of marked genetic mutants in members of the genus Burkholderia

Abstract

To elucidate the function of a gene in bacteria it is vital that targeted gene inactivation (allelic replacement) can be achieved. Allelic replacement is often carried out by disruption of the gene of interest by insertion of an antibiotic-resistance marker followed by subsequent transfer of the mutant allele to the genome of the host organism in place of the wild-type gene. However, due to their intrinsic resistance to many antibiotics only selected antibiotic-resistance markers can be used in members of the genus Burkholderia, including the Burkholderia cepacia complex (Bcc). Here we describe the construction of improved antibiotic-resistance cassettes that specify resistance to kanamycin, chloramphenicol or trimethoprim effectively in the Bcc and related species. These were then used in combination with and/or to construct a series enhanced suicide vectors, pSHAFT2, pSHAFT3 and pSHAFT-GFP to facilitate effective allelic replacement in the Bcc. Validation of these improved suicide vectors was demonstrated by the genetic inactivation of selected genes in the Bcc species Burkholderia cenocepacia and B. lata, and in the non-Bcc species, B. thailandensis.

Burkholderia cenocepacia utilizes a type VI secretion system for bacterial competition

“…Burkholderia cenocepacia chromosomal mutants with insertionally inactivated genes were generated by allelic replacement using the suicide vector pSHAFT2, as previously described (Shastri et al, ). Briefly, DNA fragments containing ~1,200 bp of the N‐terminal coding region of tssM (tssM’) and the entire tssK and tagY genes were amplified from B. cenocepacia H111 using primer pairs tssMfor and tssMrev, tssKfor and tssKrev, and tagYfor and tagYrev, respectively.…”

Mutations in Two Paraburkholderia phymatum Type VI Secretion Systems Cause Reduced Fitness in Interbacterial Competition

“…The PCR products were purified with the Qiagen ® PCR purification kit, and digested with Eco RI (NEB). The suicide vector pSHAFT2 ( Shastri et al, 2017 ) was also digested with Eco RI, and used for a ligation with the digested PCR fragments using T4 DNA Ligase from Roche ® . The ligation reaction was transformed in chemically competent E. coli CC118pir cells, and the insertion of the fragments was confirmed by sequencing with the primer pSHAFTseqFor (Supplementary Table S1 ).…”

Copper resistance genes of Burkholderia cenocepacia H111 identified by transposon sequencing

“…Chromosomal integrants were selected on LB agar containing Tp 50 μg ml −1 for strain H111, Pol 50 mg L −1 and 0.5% (w/v) l ‐rhamnose. An insertion mutant in which the sensor kinase BCAL0471 is inactivated was constructed by the aid of the suicide plasmid pSHAFT2 (Shastri et al ., ). To this end, a 400–500 bp of the target gene was amplified by PCR (for primers see Supporting Information Table S2) and cloned into the vector.…”