TABLE 1.
Bacterial strains, plasmids, and transposon used in this study
| Strain, plasmid, or transposon | Relevant characteristicsa | Reference or source |
|---|---|---|
| Mesorhizobium loti strains | ||
| MAFF303099 | Wild type, ACD+ Fix+ Pmr | 15 |
| ML-GUS | MAFF303099 labeled with gusA by mTn5SSgusA20, Pmr Spr Smr | This study |
| ACD-GUS | MAFF303099 labeled with gusA fused with the acdS gene under regulation of acdS promoter, Pmr Kmr | This study |
| nifA1 mutant | nifA1::Gm derivative of ML-GUS, Fix+ Pmr Spr Smr Gmr Kms | This study |
| nifA2 mutant | ΔnifA2::Tc derivative of ML-GUS, Fix− Pmr Spr Smr Tcr Kms | This study |
| Escherichia coli strains | ||
| DH5α | Cloning strain | Toyobo Inc., Tokyo, Japan |
| S17-1 | Strain used for conjugation and gene disruption | 30 |
| Plasmids | ||
| pBluescriptII KS(−) | Cloning vector, Apr | Takara Shuzo Co., Kusatsu, Japan |
| pBS-GUS | pBluescriptII KS(−) carrying gusA amplified from pKW107, Apr | This study |
| pMS246 | Plasmid carrying 1-kb gentamicin resistance cassette, Gmr Apr | 1 |
| p34S-Tc | Plasmid carrying 1.5-kb tetracycline resistance cassette, Tcr Apr | 3 |
| pmTn5SSgusA20 | Plasmid used for transposon mTn5SSgusA20 insertion, Apr Spr | 35 |
| pK18mob | Plasmid used for cloning and mating, mob, Kmr | 29 |
| pPD1 | pK18mob carrying 6.6-kb BamHI fragment containing acdS, Kmr | 33 |
| pPD3 | gusA inserted into acdS gene StuI site of pPD1, Kmr | This study |
| C229 | M. loti ordered cosmid clone no. 229 including mll5837 (nifA2), Tcr | 11 |
| C230 | M. loti ordered cosmid clone no. 230 including mll5857 (nifA1), Tcr | 11 |
| C232 | M. loti ordered cosmid clone no. 232 including mlr5905 (nifH), Tcr | 11 |
| pKnifA1 | pK18mob carrying 4.8-kb EcoRI fragment containing mll5857 (nifA1) from C230, Kmr | This study |
| pKnifA1::Gm | pK18mob carrying 5.7-kb nifA1::Gm, Kmr Gmr | This study |
| pKnifA2 | pK18mob carrying 6.7-kb SmaI fragment containing mll5837 (nifA2) from C229, Kmr | This study |
| pKnifA2::Tc | pK18mob carrying 7.2-kb ΔnifA2::Tc, Kmr Tcr | This study |
| pFAJ1702 | Plasmid used for complementation, Apr Tcr | 6 |
| pFAJnifA2 | pFAJ1702 containing 6.7-kb nifA2 fragment from pKnifA2, Apr Tcr | This study |
| Transposon | ||
| mTn5SSgusA20 | Minitransposon, gusA, Spr Smr | 35 |
a
ACD, ACC deaminase; Apr, ampicillin resistant; Gmr, gentamicin resistant; Kmr, kanamycin resistant; Pmr, phosphomycine resistant; Smr, streptomycin resistant; Spr, spectinomycin resistant; Tcr, tetracycline resistant.