TABLE 1.

Strains and plasmids used in this study

Strain or plasmidRelevant characteristicsaSource or reference
E. coli strains
    TOP10General cloning and blue/white screeningInvitrogen
    BL21(DE3)E. coli B with DE3, a λ prophage carrying the T7 RNA polymerase geneNovagen
    S17-1Mobilizing strain with transfer genes of RP4 integrated on chromosome; Smr Tpr Pms31
B. pseudomallei strains
    K96243Clinical isolate from northeastern Thailand; Gmr Smr Kmr Pmr17
    AIK96243 derivative harboring an 873-bp amrA deletion mutation (ΔamrA); Gms Sms Kms PmrS. Harding
    AI2AI derivative with the ΔamrA mutation repaired; Gmr Smr Kmr PmrThis study
    DDS1498AI derivative harboring a 162-bp in-frame deletion mutation in hcp1 (Δhcp1); Gms Sms Kms PmrThis study
    DDS1498ADDS1498 derivative with the ΔamrA mutation repaired; Gmr Smr Kmr PmrThis study
    DDS0518AI derivative harboring a 303-bp in-frame deletion mutation in hcp2 (Δhcp2); Gms Sms Kms PmrThis study
    DDS0518ADDS0518 derivative with the ΔamrA mutation repaired; Gmr Smr Kmr PmrThis study
    DDS2098AI derivative harboring a 186-bp in-frame deletion mutation in hcp3 (Δhcp3); Gms Sms Kms PmrThis study
    DDS2098ADDS2098 derivative with the ΔamrA mutation repaired; Gmr Smr Kmr PmrThis study
    DDS0171AI derivative harboring a 321-bp in-frame deletion mutation in hcp4 (Δhcp4); Gms Sms Kms PmrThis study
    DDS0171ADDS0171 derivative with the ΔamrA mutation repaired; Gmr Smr Kmr PmrThis study
    DDS0099AI derivative harboring a 192-bp in-frame deletion mutation in hcp5 (Δhcp5); Gms Sms Kms PmrThis study
    DDS0099ADDS0099 derivative with the ΔamrA mutation repaired; Gmr Smr Kmr PmrThis study
    DDL3105AI derivative harboring a 216-bp in-frame deletion mutation in hcp6 (Δhcp6); Gms Sms Kms PmrThis study
    DDL3105ADDL3105 derivative with the ΔamrA mutation repaired; Gmr Smr Kmr PmrThis study
    DDS1503-1AI derivative harboring a deletion of the 743-bp StuI fragment at the 5′ end of vgrG1 (ΔvgrG1561-1303); Gms Sms Kms PmrThis study
    DDS1503-1ADDS1503-1 derivative with the ΔamrA mutation repaired; Gmr Smr Kmr PmrThis study
    DDS1503-2AI derivative harboring a deletion of the 894-bp PstI fragment at the 3′ end of vgrG1 (ΔvgrG11770-2663); Gms Sms Kms PmrThis study
    DDS1503-2ADDS1503-2 derivative with the ΔamrA mutation repaired; Gmr Smr Kmr PmrThis study
Plasmids
    pCR2.1-TOPO3,931-bp TA vector; pMB1 oriR; Kmr AprInvitrogen
    pCR2.1-BPSS1498pCR2.1-TOPO containing 1,396-bp PCR product generated with BPSS1498-1 and BPSS1498-4This study
    pCR2.1-amrApCR2.1-TOPO containing 1,943-bp PCR product generated with amrR-1/amrB-1This study
    pCR2.1-ΔBPSS1498pCR2.1-TOPO containing 1,252-bp fragment generated by joining BPSS1498-1/BPSS1498-2 and BPSS1498-3/BPSS1498-4 PCR products via SOE-PCRThis study
    pCR2.1-ΔBPSS0518pCR2.1-TOPO containing 1,212-bp fragment generated by joining BPSS0518-1/BPSS0518-2 and BPSS0518-3/BPSS0518-4 PCR products via SOE-PCRThis study
    pCR2.1-ΔBPSS2098pCR2.1-TOPO containing 1,325-bp fragment generated by joining BPSS2098-1/BPSS2098-2 and BPSS2098-3/BPSS2098-4 PCR products via SOE-PCRThis study
    pCR2.1-ΔBPSS0171pCR2.1-TOPO containing 1,301-bp fragment generated by joining BPSS0171-1/BPSS0171-2 and BPSS0171-3/BPSS0171-4 PCR products via SOE-PCRThis study
    pCR2.1-ΔBPSL3105pCR2.1-TOPO containing 1,250-bp fragment generated by joining BPSL3105-1/BPSL3105-2 and BPSL3105-3/BPSL3105-4 PCR products via SOE-PCRThis study
    pCR2.1-BPSS1503pCR2.1-TOPO containing 3,048-bp PCR product generated with VgrG1-5′ and VgrG1-3′This study
    pCR2.1-1503pCR2.1-TOPO containing 1,590-bp PCR product generated with 1503-up2 and 1503-dn2This study
    pCR2.1-ΔA0737pCR2.1-TOPO containing deletion of 743-bp StuI fragment at the 5′ end of vgrG1 (ΔvgrG1561-1303)30
    pCR4Blunt-TOPO3,956-bp positive selection cloning vector; pUC ori; Kmr AprInvitrogen
    pCR4-ΔBPSS0099pCR4Blunt-TOPO containing 1,301-bp fragment generated by joining BPSS0099-/BPSS0099-2 and BPSS0099-3/BPSS0099-4 PCR products via SOE-PCRThis study
    pCRT7/CT-TOPO2.7-kb TA vector; allows C-terminal fusions to V5 epitope and 6×His tag under the control of a T7 promoter; pMB1 oriR; Apr ZeorInvitrogen
    pCRT7/CT-BPSS1498pCRT7/CT-TOPO containing PCR product generated with 1498-up and 1498-dnThis study
    pCRT7/CT-BPSS0518pCRT7/CT-TOPO containing PCR product generated with 0518-up and 0518-dnThis study
    pCRT7/CT-BPSS2098pCRT7/CT-TOPO containing PCR product generated with 2098-up and 2098-dnThis study
    pCRT7/CT-BPSS0171pCRT7/CT-TOPO containing PCR product generated with 0171-up and 0171-dnThis study
    pCRT7/CT-BPSS0099pCRT7/CT-TOPO containing PCR product generated with 0099-up and 0099-dnThis study
    pCRT7/CT-BPSL3105pCRT7/CT-TOPO containing PCR product generated with 3105-up and 3105-dnThis study
    pMo130Suicide vector for allelic exchange in Burkholderia; ColE1 ori RK2 oriT xylE sacB; Kmr14
    pMo130ΔNXpMo130 digested with NotI and XbaI, blunt ended, and ligated to eliminate NotI, PstI, BamHI, and XbaI sitesThis study
    pMo130-ΔBPSS1498pMo130ΔNX containing NheI insert from pCR2.1-ΔBPSS1498This study
    pMo130-ΔBPSS0518pMo130ΔNX containing NheI insert from pCR2.1-ΔBPSS0518This study
    pMo130-ΔBPSS2098pMo130ΔNX containing NheI insert from pCR2.1-ΔBPSS2098This study
    pMo130-ΔBPSS0171pMo130ΔNX containing NheI insert from pCR2.1-ΔBPSS0171This study
    pMo130-ΔBPSL3105pMo130ΔNX containing NheI insert from pCR2.1-ΔBPSL3105This study
    pMo130-ΔBPSS0099pMo130ΔNX containing NheI insert from pCR4-ΔBPSS0099This study
    pMo130-1503pMo130ΔNX containing BamHI insert from pCR2.1-1503 cloned into the BglII siteThis study
    pMo130-ΔBPSS1503pMo130-1503 with 894-bp PstI fragment removedThis study
    pBHR2Broad-host-range vector; Kmr28
    pBHR2-BPSS1498pBHR2 containing XbaI-SpeI insert from pCR2.1-BPSS1498This study
    pBHR2-virAGpBHR2 derivative containing B. mallei virAG genes28
    pBHR1-TGBroad-host-range vector containing gfp; Kmr8
    pBHR1TG-BPSS1503pBHR1-TG containing EcoRV insert from pCR2.1-BPSS1503 cloned into the ScaI siteThis study
    pEx18KmpEx18Tc derivative containing the 1,003-bp Kmr PCR product generated with nptF and nptR from pCR2.1-TOPO; Kmr Tcs30
    pΔA0737pEx18Km derivative containing XbaI-SpeI fragment from pCR2.1-ΔA0737 in XbaI site30
  • a Tc, tetracycline.