TABLE 1.

Bacterial strains, plasmids, and transposon used in this study

Strain, plasmid, or transposonRelevant characteristicsaReference or source
Mesorhizobium loti strains
    MAFF303099Wild type, ACD+ Fix+ Pmr15
    ML-GUSMAFF303099 labeled with gusA by mTn5SSgusA20, Pmr Spr SmrThis study
    ACD-GUSMAFF303099 labeled with gusA fused with the acdS gene under regulation of acdS promoter, Pmr KmrThis study
    nifA1 mutantnifA1::Gm derivative of ML-GUS, Fix+ Pmr Spr Smr Gmr KmsThis study
    nifA2 mutantΔnifA2::Tc derivative of ML-GUS, Fix Pmr Spr Smr Tcr KmsThis study
Escherichia coli strains
    DH5αCloning strainToyobo Inc., Tokyo, Japan
    S17-1Strain used for conjugation and gene disruption30
Plasmids
    pBluescriptII KS(−)Cloning vector, AprTakara Shuzo Co., Kusatsu, Japan
    pBS-GUSpBluescriptII KS(−) carrying gusA amplified from pKW107, AprThis study
    pMS246Plasmid carrying 1-kb gentamicin resistance cassette, Gmr Apr1
    p34S-TcPlasmid carrying 1.5-kb tetracycline resistance cassette, Tcr Apr3
    pmTn5SSgusA20Plasmid used for transposon mTn5SSgusA20 insertion, Apr Spr35
    pK18mobPlasmid used for cloning and mating, mob, Kmr29
    pPD1pK18mob carrying 6.6-kb BamHI fragment containing acdS, Kmr33
    pPD3gusA inserted into acdS gene StuI site of pPD1, KmrThis study
    C229M. loti ordered cosmid clone no. 229 including mll5837 (nifA2), Tcr11
    C230M. loti ordered cosmid clone no. 230 including mll5857 (nifA1), Tcr11
    C232M. loti ordered cosmid clone no. 232 including mlr5905 (nifH), Tcr11
    pKnifA1pK18mob carrying 4.8-kb EcoRI fragment containing mll5857 (nifA1) from C230, KmrThis study
    pKnifA1::GmpK18mob carrying 5.7-kb nifA1::Gm, Kmr GmrThis study
    pKnifA2pK18mob carrying 6.7-kb SmaI fragment containing mll5837 (nifA2) from C229, KmrThis study
    pKnifA2::TcpK18mob carrying 7.2-kb ΔnifA2::Tc, Kmr TcrThis study
    pFAJ1702Plasmid used for complementation, Apr Tcr6
    pFAJnifA2pFAJ1702 containing 6.7-kb nifA2 fragment from pKnifA2, Apr TcrThis study
Transposon
    mTn5SSgusA20Minitransposon, gusA, Spr Smr35
  • 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.