TABLE 1.
Bacterial strains and plasmids used in this study
| Strain or plasmid | Relevant characteristicsa | Reference or source |
|---|---|---|
| B. elkanii | ||
| USDA94 | Wild-type strain producing high concn of rhizobitoxine in culture; Tcr Kms | H. H. Keyserb |
| USDA94Δrtx::Ω1 | USDA94, nodPQ rtxA rtxC (ORF1), ORF2, and ORF3::del/ins Ω cassette; Smr, Spr | This study |
| E. coli | ||
| JM109 | recA; cloning strain | Toyobo Inc. |
| DH5α | recA; cloning strain | Toyobo Inc. |
| HB101 | recA hsdR hsdM pro Smr | 3 |
| Plasmids | ||
| pRK2013 | ColE1 replicon carrying RK2 transfer genes; Kmr | 9 |
| pUC118 | Cloning vector for shotgun sequence; Apr | Takara Shuzo Co. |
| pBluescript II SK(+) | Cloning vector; Apr | Takara Shuzo Co. |
| pBS IIΔSacI | pBluescript II SK(+) lacking SacI site; Apr | This study |
| pHP45Ω | Plasmid carrying 2.1-kb Ω cassette; Smr Spr Apr | 8 |
| pSUP202 | pBR325 carrying oriT from RP4; Apr Cmr Tcr | 51 |
| pBS13.4 | pBSII ΔSacI carrying a 13.4-kb ApaI-NotI fragment from pRTF1 | This study |
| pBS3.6::Ω | pBS13.4 derivatives containing 9.8-kb SacI fragment deletion and Ω cassette insertion | This study |
| pSUP3.6::Ω | pSUP202 carrying a 5.7-kb ApaI-NotI fragment from pBS3.6::Ω | This study |
| pLAFR1 | Broad-host-range cosmid; IncP Tcr | 12 |
| pRTB7, pRTE1, pRTF1, pRTF3, pRTL1, pRTN2, pRTS1 | pLAFR1 containing in rtxA and flanking regions from B. elkanii USDA94 (Fig. 1) | This study |
| pGPS1.1 | Transprimer donor plasmid; Kmr Tcr | New England BioLabs |
| pRTF1-C1 | pRTF1 containing KM cassette insertion at 631-bp upstream region of rtxA | This study |
| pRTF1-C3 | pRTF1 containing KM cassette insertion at 1.3-kb upstream region of rtxA | This study |
| pRTF1-D2 | pRTF1 containing KM cassette insertion at N-terminal domain of rtxA | This study |
| pRTF1-D3 | pRTF1 containing KM cassette insertion at ORF1 (rtxC) | This study |
| pRTF1-D5 | pRTF1 containing KM cassette insertion at C-terminal domain of rtxA | This study |
| pRTF1-D8 | pRTF1 containing KM cassette insertion at 224-bp upstream region of rtxA | This study |
| pRTF1-E2 | pRTF1 containing KM cassette insertion at ORF3 | This study |
| pRTF1-E8 | pRTF1 containing KM cassette insertion at ORF3 | This study |
| pRTF1-E9 | pRTF1 containing KM cassette insertion at 183-bp downstream region of ORF2 | This study |
| pRTF1-E10 | pRTF1 containing KM cassette insertion at 337-bp downstream region of ORF2 | This study |
| pRTF1-E26 | pRTF1 containing KM cassette insertion at ORF2 | This study |
| pRTF1-F1 | pRTF1 containing KM cassette insertion at 5.7-kb downstream region of ORF4 | This study |
| pRTF1-F6 | pRTF1 containing KM cassette insertion at ORF4 | This study |
a
Apr, ampicillin resistant; Tcr, tetracycline resistant; Kmr, kanamycin resistant; Kms, kanamycin sensitive; Smr, streptomycin resistant; Spr, spectinomycin resistant; Cmr, chloramphenicol resistant; KM, kanamycin.
b
U.S. Department of Agriculture, Beltsville, Md.