TABLE 2

Strains, plasmids, and primers used in this study

Strain, plasmid, or primerRelevant characteristic(s)aReference or source
Strains
    A. vinelandii
        DJ ΔhoxKDJ with hoxK knockout, utilizes Mo nitrogenase in presence of molybdenum, KanrThis study
        DJ ΔhoxK ΔphbCDJ ΔhoxK with phbC knockout, AmprThis study
        CA6Tungsten-tolerant, natural hydrogen-evolving mutant (42-kb deletion including entire hox operon), utilizes Mo nitrogenase in presence of molybdenum40
        CA6 ΔphbCCA6 with phbC knockout, AmprThis study
        CA11.6CA11 (ΔnifHDK, knockout of Mo nitrogenase) with CA6 mutation (42-kb deletion), utilizes V nitrogenase in presence of vanadium and Fe nitrogenase in presence of iron only (no vanadium or molybdenum present in medium)34
        CA11.6 ΔphbCCA11.6 with phbC knockout, AmprThis study
    E. coli
        TOP10F mcrA Δ(mrr-hsdRMS-mcrBC) ϕ80lacZΔM15 ΔlacX74 recA1 araD139 Δ(ara leu)7697 galU galK rpsL (Strr) endA1 nupGInvitrogen
        Thunderbolt GC10F mcrA Δ(mrr-hsdRMS-mcrBC) ϕ80lacZΔM15 ΔlacX74 endA1 recA1 Δ(ara leu)7697 araD139 galU galK nupG rpsL λ T1RGeneChoice
        DH5αsupE44 ΔlacU169 hsdR17 endA1 recA1 gyrA96 thi-1 relA1Invitrogen
Plasmids
    pUC19Ampr, lacZ α-subunit, pMB1 originNew England Biolabs
    pUC-phbpUC19 + A. vinelandii inserted at multicloning siteThis study
    pUC-phb+amppUC-phb with Ampr cassette inserted in A. vinelandii phbC regionThis study
Primers
    pUCphbCF (HindIII site)AGATGT-AAGCTT-TATGGTCCTTGCTGCCTTGAGThis study
    pUCphbCR (EcoRI site)TGAACA-GAATTC-GCCTTTCGCCCTATGTTCTTCThis study
    pUCampF (PstI site)AGTAGT-CTGCAG-TGGTCTGACAGTTACCAATGThis study
    pUCampR (SacI site)AGTACA-GAGCTC-GCCTCGTGATACGCCTATTTThis study
    ampTestFGTCAGCTCAGACGCCCTTCGTAGThis study
    ampTestRTATGCAGCCAGCGAGCCGAACAGThis study
    phbIntLFCGGCTGGTGTCTGTTCATTGThis study
    phbIntLRACGCTCGTCGTTTGGTATGGThis study
    phbIntDFTCGCCGCATACACTATTCTCThis study
    phbIntDRAGGGTCCTTGGCAAGTATTCThis study
    nifDelFTGGCTGAGATGGGCAAGAAGThis study
    nifDelRAGCGGTTACTGGGTGAAACGThis study
    gapFGCGCCGACGACCGGCTCCATGTThis study
    bothgapsRCCGCCGCTGCGTGGCCTGTTCTThis study
    hoxK2FCGGCTTGTCGGTGATGACCTTThis study
    hoxK2RCGGTATGGAGCGGGCATTTCThis study
    kanR5GGGATCGCAGTGGTGAGTAACThis study
  • a Primers are shown with added restriction sites: boldface sequence indicates the added restriction site, and underlined sequence indicates homology to the target.