TABLE 2.

Plasmids used in this study

PlasmidDescriptionReference
pPW12IncP mobilizable cosmid pLAFRI with EcoRI-BamHI-EcoRI linker, Tcr, 21.6 kbp13
pRK293IncP mobilizable cloning vector, Tcr Kmr, 21.3 kbp12
pUC8ColE1 cloning vector, Apr, 2.7 kbp50
pUC19ColE1 cloning vector, Apr, 2.7 kbp54
pBluescript SK(+)ColE1 cloning vector, Apr, 3.0 kbp1
pUC4KIXXpUC4K-based plasmid carrying the Tn5 Kmr and Blr resistance genes, Apr Kmr Blr, 4.0 kbp5
pUCNCOColE1 cloning vector, pUC8 with ACCATGG sequence inserted between BamHI and HincII sites, Apr, 2.7 kbpThis study
p21R1pPW12 with a 20-kbp insert of G. diazotrophicus SRT4 containing the lsdAB locus and partial type II secretion locus, Tcr, 42 kbp4
pALS55,181-bp SalI-EcoRI fragment from p21R1 inserted into pUC19: carries lsdABX, Apr, 7.9 kbp4
pALS15pRK293 with HindIII-linearized pALS10 carrying the SRT4 lsdA gene in a 2,339-bp BglII fragment, Apr Tcr 27.2 kbp4
pALS307.7-kbp BamHI-partial EcoRI fragment from p21R1 inserted into pUC8, carries region upstream of lsdA, Apr, 10.4 kbpThis study
pALS318.7-kbp BamHI-partial EcoRI fragment from p21R1 inserted into pUC8, carries region upstream of lsdA, Apr, 11.4 kbpThis study
pALS324,614-bp BamHI-partial EcoRI fragment from p21R1 inserted into pUC8, carries lsdBXGO, Apr, 7.3 kbpThis study
pALS33HindIII-linearized pALS30 inserted at the HindIII site of pRK293, Apr Tcr, 31.7 kbpThis study
pALS34HindIII-linearized pALS31 inserted at the HindIII site of pRK293, Apr Tcr, 32.7 kbpThis study
pALS35BamHI-linearized pALS32 inserted at the BamHI site of pPW12, Apr Tcr, 28.9 kbpThis study
pALS60198-bp PCR band amplified from pALS5 inserted at the SmaI site of pUC19, carries N-terminal region of lsdA, Apr, 2.9 kbThis study
pALS89pUCNCO containing a 1,491-bp NcoI-HincII fragment with the nptII-ble cassette from pUC4KIXX inserted at the EcoRI site of lsdG, Apr Kmr Blr, 5.8 kbpaThis study
pALS1131,050-bp PCR band amplified from pALS32 inserted at the SalI-BamHI sites of pUC19, carries wild-type lsdG, Apr, 3.7 kbpaThis study
pALS1141,050-bp PCR band amplified from pALS32 inserted at the SalI-BamHI sites of pUC19, carries mutated lsdG (C162G) Apr, 3.7 kbpaThis study
pALS117HindIII-linearized pALS113 inserted at the HindIII site of pRK293, Apr Tcr, 24.8 kbpThis study
pALS118HindIII-linearized pALS114 inserted at the HindIII site of pRK293, Apr Tcr, 24.8 kbpThis study
pALS1193,527-bp HindIII-SphI fragment of G. diazotrophicus SRT4 genome inserted into pUC19, carries lsdEF, Apr, 6.2 kbpThis study
pALS127pALS119 with the 1.2-kbp SmaI nptII cassette from pUC4KIXX inserted at the EcoRV site of lsdF, Apr Kmr, 7.4 kbpThis study
pALS130pBluescript SK (+) containing a 7,551-bp BamHI-KpnI fragment with the nptII-ble cassette from pUC4KIXX inserted at the HindIII site of lsdO, Apr Kmr Blr, 12.1 kbpaThis study
pALS1411,967-bp SmaI-BglII fragment from pALS5 inserted at SmaI-BamHI sites in pALS60 carries lsdA, Apr 4.8 kbThis study
pALS1431,432-bp SacII-KpnI fragment of G. diazotrophicus SRT4 genome inserted into pBluescript SK (+), carries lsdHIJ, Apr, 4.3 kbpThis study
pALS1523,679-bp SalI-EcoRI fragment of G. diazotrophicus SRT4 genome inserted into pBluescript SK(+), carries lsdJLMN and partial lsdD, Apr, 6.4 kbpThis study
pALS182584-bp PCR band amplified from pALS5 digested with HindIII-NcoI plus 2,114-bp NcoI-NruI fragment from pALS141 inserted at HindIII-SmaI sites in pUCNCO, carries lsdA under control of 552-bp region upstream of lsdX, Apr, 4.8 kbThis study
pALS184HindIII-linearized pALS182 inserted at the HindIII site of pRK293, Apr Tcr, 26.6 kbpThis study