TABLE 1.
Bacterial strains and plasmidsa
| Strains/plasmids | Description | Reference/source |
|---|---|---|
| Bacterial strains | ||
| Burkholderia multivorans ATCC 17616 | Soil isolate, USA | (27) |
| Burkholderia multivorans P0213-1 | Cystic fibrosis isolate VC7495, Canada | CBCCRRR |
| Burkholderia multivorans BM1 | Cystic fibrosis isolate, Canada | (28) |
| Burkholderia dolosa AU0158 | Cystic fibrosis isolate, USA | CBCCRRR |
| Burkholderia cenocepacia J2315 | Cystic fibrosis isolate, ET12 index strain, Edinburgh, UK | (29) |
| Burkholderia cenocepacia K56-2 | Cystic fibrosis isolate, ET12, Toronto, Canada | (30) |
| B. multivorans P0213-1 ΔrpfR | P0213-1 with genes FEP09_03956 and FEP09_03957 (rpfR) deleted | This work |
| B. multivorans P0213-1 ΔbcsB | P0213-1 with gene FEP09_02916 (bcsB) deleted | This work |
| B. multivorans P0213-1 ΔbceF | P0213-1 with gene FEP09_03744 (bceF) deleted | This work |
| B. multivorans P0213-1 ΔcepR | P0213-1 with gene FEP09_04935 (cepR) deleted | This work |
| B. multivorans ATCC 17616 ΔbceF | ATCC 17616 with gene Bmul_4915 (bceF) deleted | This work |
| B. multivorans ATCC 17616 ΔcepR | ATCC 17616 with gene Bmul_3971 (cepR) deleted | This work |
| B. multivorans ATCC 17616::gfp | ATCC 17616 with gene gfp inserted in the intergenic region between Bmul_4902 and Bmul_4903 | This work |
| Escherichia coli DH5α | Δ(lacZYA-argF)U169 ф80dlacZΔM15recA1 | Gibco BRL |
| Plasmids | ||
| pCasPA | Bacterial expression of Cas9 nuclease and λ-Red recombination system; TcR | (10) |
| pACRISPR | Plasmid for the expression of sgRNA for targeting specific bacterial sequences and cloning of homologous arms; ApR |
(10) |
| pACRISPR-Km | pACRISPR derivative with the bla gene replaced by the aph gene; KmR | This work |
| pACRISPR-Cm | pACRISPR derivative with the bla gene replaced by the cat gene; CmR | This work |
| pACRISPR-Tp | pACRISPR derivative with the bla gene replaced by the dhfr gene; TpR | This work |
| pLM127-13 | pBBR1MCS derivative expressing the bceF gene of B. contaminans IST408 under the control of the bce operon promoter; CmR |
(31) |
| pK19mob | pUC18/19 derivative, lacZα, mob, KmR | (32) |
| pBBR1MCS | 4,717-bp broad host range cloning vector, CmR | (33) |
| pUC-TP | pUC-GM derivative with a 1.1 kb TpR gene cassette, ApR, TpR | (34) |
| pRK2013 | Tra+ Mob+ (RK2) Km::Tn7 ColE1 origin, helper plasmid in triparental mating, KmR | (35) |
| pRK600 | ColE1oriV, RK-2Mob+ RK2-Tra+, helper plasmid in triparental mating, CmR | (36) |
| pIN25 | oripBBR, Δmob, GFP; CmR | (37) |
| pMF21-4 | pACRISPR-Km derivative containing the 20-nt spacer targeting B. multivorans P0213-1 rpfR gene; KmR | This work |
| pMF21-7 | pMF21-4 derivative containing the upstream 779 bp and the downstream 620 bp regions of the rpfR gene; KmR | This work |
| pLM2022-4 | pACRISPR-Km derivative containing the 20-nt spacer targeting B. multivorans P0213-1 bcsB gene; KmR |
This work |
| pLM2022-5 | pLM2022-4 derivative containing the 940 bp region upstream and the 912 bp region downstream of the bcsB gene; KmR |
This work |
| pLM2022-6 | pACRISPR-Km derivative containing the 20-nt spacer targeting B. multivorans P0213-1 bceF gene; KmR | This work |
| pLM2022-7 | pACRISPR-Km derivative containing the 20-nt spacer targeting B. multivorans ATCC 17616 bceF gene; KmR | This work |
| pLM2022-8 | pLM2022-6 derivative containing the 971 bp region upstream and the 1,189 bp region downstream of bceF gene from B. multivorans P0213-1; KmR |
This work |
| pLM2022-9 | pLM2022-7 derivative containing the 971 bp region upstream and the 1,189 bp region downstream of bceF gene from B. multivorans ATCC 17616; KmR | This work |
| pLM2022-11 | pACRISPR-Km derivative containing the 20-nt spacer targeting B. multivorans ATCC 17616 cepR gene; KmR | This work |
| pVQ2022-2 | pLM2022-11 derivative containing the 939 bp region upstream and the 936 bp region downstream of the cepR gene from B. multivorans ATCC 17616; KmR |
This work |
| pLM2022-12 | pACRISPR-Km derivative containing the 20-nt spacer targeting B. multivorans P0213-1 cepR gene; KmR | This work |
| pVQ2022-3 | pLM2022-12 derivative containing the 939 bp region upstream and the 936 bp region downstream of the cepR gene from B. multivorans P0213-1; KmR |
This work |
| pLM2022-10 | pACRISPR-Km derivative containing the 20-nt spacer targeting B. multivorans ATCC 17616 intergenic region (IG) between Bmul_4902 and Bmul_4903 genes; KmR |
This work |
| pVQ2022-1 | pLM2022-10 derivative containing the 709 bp IG region upstream of the Cas9 cutting site, the 797 bp gfp gene from pIN25, and the 770 bp IG region downstream of the Cas9 cutting site, from B. multivorans ATCC 17616; KmR |
This work |
a
KmR, kanamycin resistance; CmR, chloramphenicol resistance; TpR, trimethoprim resistance; TcR, tetracycline resistance; and ApR, ampicillin resistance. CBCCRRR, Canadian Burkholderia cepacia complex research and referral repository.