TABLE 2.
Strains and plasmids used in this study
| Strain or plasmid | Relevant characteristicsa | Source or reference |
|---|---|---|
| Strains | ||
| FD | Anabaena variabilis ATCC 29413 wild-type parent strain | 10 |
| BP457 | pBP457 integrated into the frtBC region of the chromosome via single crossover | This study |
| BP461 | pBP461 integrated into the frtBC region of the chromosome via single crossover | This study |
| BP462 | pBP462 integrated into the ava4055-vnfH region of the chromosome via single crossover | This study |
| BP469 | pBP462 integrated into the frtBC region of the chromosome via single crossover | This study |
| JU333 | pJU333 integrated into the nifH1 gene via double recombination | This study |
| JU408 | pJU408, with a nifU1-nifH1 deletion (Nmr), integrated into the chromosome of FD via double recombination (Nmr, Nif− after segregation) | This study |
| JU417 | pJU457 integrated into the intergenic nifU1-nifH1 region of the chromosome via single crossover | This study |
| JU425 | pJU402 integrated into the chromosome of strain JU408 via double recombination; resulted in replacement of the nifU1-nifH1 deletion of JU408 with the mutated version of the nifU1-nifH1 intergenic region from pJU402, restoring a Nif+ phenotype | This study |
| JU436 | pJU436 integrated into the nif1 region via double recombination, creating nifB1 deletion mutant | This study |
| JU454 | pJU454 integrated into the frtBC region of the chromosome via single crossover | This study |
| JU466 | pJU466 integrated into the nif1 region via double recombination, creating nifB1 deletion mutant (Nif−) | This study |
| JU467 | pJU467 (containing the promoterless lacZ), integrated into the frtBC region of the chromosome via single crossover | This study |
| JU468 | pJU468 integrated into the nif1 region of the chromosome via single crossover | This study |
| JU469 | pJU469 integrated into the nif1 region of the chromosome via single crossover | This study |
| JU472 | pJU472 integrated into the frtBC region of the chromosome via single crossover | This study |
| JU473 | pJU473 integrated into the frtBC region of the chromosome via single crossover | This study |
| JU476 | pJU476 integrated into the frtBC region of the chromosome via single crossover | This study |
| JU477 | pJU477 integrated into the frtBC region of the chromosome via single crossover | This study |
| JU484 | pJU476 integrated into the nif1 region of the chromosome via single crossover | This study |
| JU485 | pJU477 integrated into the nif1 region of the chromosome via single crossover | This Work |
| Plasmids | ||
| pAAWY3162 | 9-kb library clone of A. variabilis containing ava3910-nifH1 region | JGI |
| pBP285 | Kmr Nmr cassette in a polylinker C.K.3 with a transcriptional terminator at the 3′ end | 52 |
| pBP288 | Cloning vector for integration of transcriptional fusions into the chromosome; Tcr Kmr Nmr Spr Smr Apr | 52 |
| pBP457 | 708-bp promoter fragment containing the ava4055-vnfH intergenic region (primers vnfHBgal-L451 and vnfHBgal-R1) inserted into the BglII/SmaI sites of pJU411 | This study |
| pBP461 | 1.6-kb ava4055-vnfH promoter fragment (primers vnfHBgal-L1380 and vnfHBgal-R1) inserted into the BglII/SmaI sites of pJU411 | This study |
| pBP462 | 2.2-kb ava4055-vnfH PCR fragment (primers vnfHBgal-L1961 and vnfHBgal-R1) inserted into the BglII/SmaI sites of pJU411 | This study |
| pBR322 | Mobilizable plasmid; Apr Tcr | 4 |
| pJU332 | BamHI fragment from pMV2 containing the nifS1-nifD1 region was cloned into the BamHI site of pBR322 | This study |
| pJU333 | KpnI fragment from pPE20 containing lacZ was cloned into the KpnI site of nifH1 in pJU332 | This study |
| pJU362 | 302-bp nifH1 promoter fragment (primers nifH1-R2 and nifH1-302L) inserted into the BglII/SmaI sites of pBP288 | This study |
| pJU375 | pEL1 with the Nmr cassette from pBP285 cloned into the EcoRV site and orientated toward the 5′ end of nifU | This study |
| pJU376 | 4.5-kb EcoRV fragment from pRL2948a cloned into the SmaI site on pJU375 | This study |
| pJU408 | pJU376 with the 400-bp AgeI fragment deleted | This study |
| pJU409 | 5.2-kb HindIII-SphI fragment from pJU362, containing the 302-bp nifH1 promoter driving lacZ, cloned into the HindIII-SphI sites of pBR322 | This study |
| pJU410 | Nmr cassette from pBP285 with a 5′ terminator (primers Nm5′TermL and Nm5′TermR), inserted into KpnI site of pJU409 to inhibit plasmid readthrough | This study |
| pJU411 | 1.8-kb frtBC PCR fragment (primers frtB-L and frtB-R), used as region of homology for recombination, inserted into the HindIII site of pJU410 | This study |
| pJU402 | pEL1 (33) with a mutation in the nifH1 upstream region that abolishes the putative NtcA binding site. | This study |
| pJU429 | 1.6-kb PCR fragment of nifB using the primers nifB1L and nifB1R cloned into the ScaI/SpeI sites on pEL1 | This study |
| pJU436 | 5.4-kb NruI/ScaI fragment containing sacB and Emr from pRL2948a cloned into the ZraI site in pJU429 | This study |
| pJU454 | 1.3-kb nifU1-nifH1 promoter fragment (primers nifUH-L and nifH1-R2) inserted into the BglII/SmaI sites of pJU411 | This study |
| pJU457 | 302-bp intergenic nifU1-nifH1 promoter fragment, inserted into BglII-SmaI sites of pJU411 | This study |
| pJU463 | 3.1-kb BsrGI-MscI fragment of pJU455 self-ligated, creates a nifB1 deletion | This study |
| pJU466 | 5.4-kb BglII fragment containing sacB and Emr from pRL2948a inserted into the BamHI site of pJU463 | This study |
| pJU468 | 1.6-kb nifB1 promoter fragment (primers pnifB-L and pnifB-R) inserted into the BglII/SmaI sites of pJU410 | This study |
| pJU469 | 1.3-kb nifH1-nifH1 promoter fragment (primers nifUH-L and nifH1-R2) inserted into the BglII/SmaI sites of pJU410 | This study |
| pJU472 | 743-bp nifH1-nifH1 promoter fragment (primers nifH1-R2 and nifUH-L4) inserted into the BglII/SmaI sites of pJU411 | This study |
| pJU473 | 544-bp nifH1-nifH1 promoter fragment (primers nifH1-R2 and nifUH-L5) inserted into the BglII/SmaI sites of pJU411 | This study |
| pJU476 | 3.0-kb fdxN-nifH1 promoter fragment (nifSUHL2 and nifH1-R2) inserted into the BglII/SmaI sites of pJU411 | This study |
| pJU477 | 6.0-kb nifB1-nifH1 promoter fragment (primers nifBSUHL and nifH1-R2) inserted into the BglII/SmaI sites of pJU411 | This study |
| pMV2 | nifH1 region with Smr Spr cassette inserted at the AgeI site in nifH1 | 32 |
| pPE20 | Source of lacZ for transcriptional fusions | 48 |
| pRL2948a | Source of mobilization site, oriT, and sacB gene, which confers sucrose sensitivity; Cmr Emr | C. P. Wolk |
| pBP285 | Kmr Nmr cassette in a polylinker C.K.3 with a transcriptional terminator at the 3′ end | 52 |
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.