TABLE 1.
Strains, plasmids, and primers used in this study
| Strain or plasmid | Relevant characteristics or sequencea | Source, reference, or gene |
|---|---|---|
| E. coli TOP10 | General cloning and blue/white screening | Life Technologies |
| E. cloni 10G | General cloning and blue/white screening | Lucigen |
| E. coli S17–1 | Mobilizing strain with transfer genes of RP4 integrated on chromosome; Smr Tpr | 58 |
| B. pseudomallei | ||
| MSHR668 | Isolated in 1995 in Darwin, Australia, from the blood of a 53-year-old male patient with severe melioidosis encephalomyelitis (the patient survived); Pmr | 59 |
| 668 ΔgspD | MSHR668 derivative harboring a 1,476-bp gspD in-frame deletion mutation (ΔgspD) | This study |
| 668 ΔgspE | MSHR668 derivative harboring a 744-bp gspE deletion mutation (ΔgspE) | This study |
| Plasmids | ||
| pCR2.1-TOPO | 3,931-bp TA vector; pMB1 oriR; Kmr Apr | Life Technologies |
| pCR2.1-gspD | pCR2.1-TOPO containing 3,231-bp PCR product generated with gspD-FH and gspD-RXb | This study |
| pMo130 | Suicide vector for allelic exchange in Burkholderia; ColE1 ori RK2 oriT xylE sacB Kmr | 25 |
| pMogspD | pMo130 containing gspD amplified with gspD-FH and gspD-RXb and cloned into HindIII/XbaI sites | This study |
| pMoΔgspD | pMogspD digested with PstI and religated, resulting in a 1,476-bp in-frame deletion in gspD | This study |
| pMogspE-5′ | pMo130 containing 5′ gspE PCR product amplified with BpgspE-5FH and BpgspE-5RB and cloned into HindIII/BamHIb sites | This study |
| pMoΔgspE | pMogspE-5′ containing a 3′ gspE PCR product amplified with BpgspE-3FB and BpgspE-3RXb and cloned into BamHI/XbaI sites, resulting in a 744-bp deletion in gspE | This study |
| pMo168 | Broad-host-range plasmid; pBBR1 ori mob xylE Kmr | 25 |
| pMo168-gspD | pMo168 containing a HindIII/XbaI fragment from pCR2.1-gspD | This study |
| pBHR2 | Broad-host-range plasmid; Kmr | 60 |
| pBHR2-virAG | pBHR2 constitutively expressing virAG | 60 |
| pBHR2-tssM | pBHR2 constitutively expressing tssM | 14 |
| Primers (5′-3′)b | ||
| gspD-FH | CATGAAGCTTACGAGCGGATTCACCTTCTG | gspD |
| gspD-RXb | CATGTCTAGATCGAACGGCTCGATGTGGATGTC | gspD |
| BpgspE-5FH | CATGAAGCTTTCCTCGGCGACATTCCGTGGATCG | gspE |
| BpgspE-5RB | CATGGGATCCATCCACACCTCGAGCGTGTCC | gspE |
| BpgspE-3FB | TCGAGGATCCGATCGAATACGATCTGTCC | gspE |
| BpgspE-3RXb | CATGTCTAGAACGCGTCGATCGCTTCGAAACGGAAGG | gspE |
a
r, resistant; s, susceptible; Sm, streptomycin; Tp, trimethoprim; Pm, polymyxin B; Km, kanamycin; Ap, ampicillin.
b
Restriction enzyme sites in the linker regions are underlined.