An efficient system for the generation of marked genetic mutants in members of the genus Burkholderia.
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.
Department of Infection, Immunity and Cardiovascular Disease, The Medical School, The University of Sheffield, Beech Hill Road, Sheffield S10 2RX, UK.
Sravanthi
S
Shastri
Department of Infection, Immunity and Cardiovascular Disease, The Medical School, The University of Sheffield, Beech Hill Road, Sheffield S10 2RX, UK.
Helena L
HL
Spiewak
Department of Infection, Immunity and Cardiovascular Disease, The Medical School, The University of Sheffield, Beech Hill Road, Sheffield S10 2RX, UK.
Aderonke
A
Sofoluwe
Department of Infection, Immunity and Cardiovascular Disease, The Medical School, The University of Sheffield, Beech Hill Road, Sheffield S10 2RX, UK.
Vigdis A
VA
Eidsvaag
Department of Infection, Immunity and Cardiovascular Disease, The Medical School, The University of Sheffield, Beech Hill Road, Sheffield S10 2RX, UK.
Atif H
AH
Asghar
Department of Infection, Immunity and Cardiovascular Disease, The Medical School, The University of Sheffield, Beech Hill Road, Sheffield S10 2RX, UK.
Tyrone
T
Pereira
Department of Infection, Immunity and Cardiovascular Disease, The Medical School, The University of Sheffield, Beech Hill Road, Sheffield S10 2RX, UK.
Edward H
EH
Bull
Department of Infection, Immunity and Cardiovascular Disease, The Medical School, The University of Sheffield, Beech Hill Road, Sheffield S10 2RX, UK.
Aaron T
AT
Butt
Department of Infection, Immunity and Cardiovascular Disease, The Medical School, The University of Sheffield, Beech Hill Road, Sheffield S10 2RX, UK,. Electronic address: m.s.thomas@sheffield.ac.uk.
Mark S
MS
Thomas
D000900:Anti-Bacterial Agents / Q000494:pharmacology; D019117:Burkholderia / Q000187:drug effects / Q000235:genetics*; D004269:DNA, Bacterial; D024881:Drug Resistance, Bacterial; D023061:Gene Order; D006801:Humans; D009154:Mutation; D010957:Plasmids / Q000235:genetics*
D000900:Anti-Bacterial Agents; D004269:DNA, Bacterial
Antibiotic-resistance; Burkholderia cepacia complex; Gene inactivation; Marked mutation; Suicide vector