TABLE 1

Plasmids and strains

Strain or plasmidDescription or relevant characteristic(s)aSource or reference
Strains
    FDA. variabilis ATCC 29413 wild-type parent strain66
    BP703pBP703 integrated into the chromosome of strain FD via double recombination, creating a stem-loop structure at the transcription-processing site in the nifUH intergenic region similar to the WT using a different nucleotide sequenceThis study
    BP706pBP706 integrated into the chromosome of strain FD via double recombination, creating a mutation in the stem-loop structure at the transcription-processing site in the nifUH intergenic regionThis study
    BP712pBP712 integrated into the chromosome of strain FD via double recombination, creating a deletion in the promoter for the antisense transcript in the nifUH intergenic regionThis study
    BP750pBP750 inserted into the frtBC region of FD, creating a wild-type nifU1-lacZ fusion strainThis study
    BP751pBP751 inserted into the frtBC region of FD, creating a wild-type nifU1-lacZ fusion strainThis study
    BP752pBP752 inserted into the frtBC region of FD, creating a wild-type nifUH1-lacZ fusion strainThis study
    BP753pBP753 inserted into the frtBC region of FD, creating a wild-type nifUH1-lacZ fusion strainThis study
    JU408Double recombinant of pJU408 conjugated into FD to yield a deletion of the 5′ end of nifH1This study
    JU420Double recombinant of pJU397 conjugated into JU408This study
    JU422Double recombinant of pJU399 conjugated into JU408This study
    JU423Double recombinant of pJU400 conjugated into JU408This study
    JU472Fusion of lacZ to a 751-bp nifU1-nifH1 fragment, placing lacZ at the start of nifH139
Plasmids
    pBP609Vector with a promoterless lacZ for promoter fusions and an internal frtABC fragment for recombination; KmrThis study
    pBP7002.35-kb nifUH1 intergenic region with the stem-loop structure restored to WT structure using different nucleotides, made with two PCR products (made from primer sets nifHLoop1-L/nifHLoop1-R and nifHResLoop2-L/nifHLoop2-R) and inserted into PstI/KpnI sites of pUC18This study
    pBP7012.35-kb nifUH1 intergenic region with the stem-loop structure mutated, made with two PCR products (made from primer sets nifHLoop1-L/nifHLoop1-R and nifHMutLoop2-L/nifHLoop2-R) and inserted into PstI/KpnI sites of pUC18This study
    pBP7036-kb SacI fragment of pRL2948a cloned into the SacI site on pBP700; Apr Cmr EmrThis study
    pBP7066-kb SacI fragment of pRL2948a cloned into the SacI site on pBP701; Apr Cmr EmrThis study
    pBP7112.9-kb fragment containing the nifUH1 intergenic region with a deletion in the promoter of an antisense nifU1 transcript, made with two PCR products (made from primer sets antiloopmut-1/antiloopmut-2 and antiloopmut-3/antiloopmut-4) and inserted into PstI/KpnI sites of pUC18This study
    pBP7126-kb SacI fragment of pRL2948a cloned into the SacI site on pBP711, with the sacB gene transcribed in the same direction as nifH1; Apr Cmr EmrThis study
    pBP750264-bp nifU1 fragment (made from primers pnifUH-L4 and pnifU-R1) inserted into the BglII/SmaI sites of pBP609This study
    pBP751457-bp nifU1 fragment (made from primers pnifUH-L4 and pnifU-R2) inserted into the BglII/SmaI sites of pBP609This study
    pBP752550-bp nifU1-nifH1 fragment (made from primers pnifUH-L4 and pnifU-R3) inserted into the BglII/SmaI sites of pBP609This study
    pBP753628-bp nifU1-nifH1 fragment (made from primers pnifUH-L4 and pnifU-R4) inserted into the BglII/SmaI sites of pBP609This study
    pEL14.1-kb BamHI region from 33D12 containing nifSUHD1 (67) in pUC118This study
    pJU375Nmr cassette from pBP285 in EcoRV on pBP285This study
    pJU376Fragment of pRL2948 cut with EcoRV, containing sacB and a Cmr cassette inserted into the SmaI site of pJU375This study
    pJU378PCR product made from pEL1 using nifH69L/nifH69R and then ligated to circularizeThis study
    pJU380PCR product made from pEL1 using nifH95L/nifH95R and then ligated to circularizeThis study
    pJU381PCR product made from pEL1 using nifH120L/nifH120R and then ligated to circularizeThis study
    pJU397Insertion of sacB and the Cmr cassette from pRL2948 cut with SacI into the SacI site on pJU378This study
    pJU399Insertion of sacB and the Cmr cassette from pRL2948 cut with SacI into the SacI site on pJU380This study
    pJU400Insertion of sacB and the Cmr cassette from pRL2948 cut with SacI into the SacI site on pJU381This study
    pJU408377-bp 5′ nifH1 fragment removed from pJU376 using partial AgeI and religating of the vectorThis study
    pRL2948aSource of mobilization site, oriT, and sacB gene, which confers sucrose sensitivity; Cmr EmrC. P. Wolk
    pUC18Cloning vector; Apr68
  • a Cmr, chloramphenicol resistance; Tcr, tetracycline resistance; Apr, ampicillin resistance; Smr, streptomycin resistance; Kmr, kanamycin resistance; Spr, spectinomycin resistance; Emr, erythromycin resistance; Nmr, neomycin resistance.