TABLE 1.
Bacterial strains and plasmids used
| Strain or plasmid | Derivation and/or relevant characteristicsa | Source or reference |
|---|---|---|
| Anabaena sp. strains | ||
| PCC 7120 | Wild type | R. Haselkorn |
| CPB7084 | Bmr Nmr Smr Spr; hcwA::Tn5-1058 hepA::luxAB-aadA, formed by double reciprocal recombination of pRL 1992 with DR1069 | This study |
| CPB8089 and CPB8090 | Bmr Emr Nmr Smr Spr; hcwA::Tn5-1058 hepA::luxAB-aadA, with pRL2431 integrated in the chromosome of HNL3 by single-crossover homologous recombination (Fig. 1 and 6) | This study |
| DR1069b | Smr Spr; luxAB-aadA cassette fused at NruI site of hepA | 4 |
| DR1992b | Bmr Nmr Smr; hcwA::Tn5-1058 | This study |
| HNL3 | Bmr Nmr Smr Spr; hcwA::Tn5-1058 hepA::luxAB-aadA | This study |
| Plasmids | ||
| pBS-SK(+) | Apr; cloning vector | Stratagene |
| pIC19H, pIC19R and pIC20H | Apr; cloning vectors | 23 |
| pJRD184 | Apr Tcr; cloning vector | 15 |
| pRL59HE | Apr; cloning vector | 19 |
| pRL148 | Apr Cmr; cloning vector | 10 |
| pRL453 | Apr Smr; cloning vector | 10 |
| pRL660 | Apr; 3′ end of hcwA, as a ClaI-MfeI fragment of pRL2130, inserted between the ClaI and EcoRI sites of pIC20H | This study |
| pRL663 | Apr; SalI fragment of a PCR product that bears the 5′ terminus of hcwA, inserted in the XhoI site of pRL660 | This study |
| pRL669a | Apr Cmr Emr; Cmr Emr cassette C.CE3 (Y. Cai and C. P. Wolk, unpublished data) in the BamHI site of pRL59HE (Ptac→Pcat→cat→erm) | This study |
| pRL677 | Apr Smr Spr Tcr; aadA from the omega interposon (25) excisable with various enzymes, including BsaBI | This study |
| pRL687 | Apr Smr Spr; BsaBI-aadA-BsaBI transferred from pRL677 to the unique unmethylated BsaBI site in Dam-methylated pRL2130 | This study |
| pRL691 | Apr Smr Spr; pIC20H::(hcwA::aadA) | This study |
| pRL838 | Cmr Emr; BAC vector (GenBank AF403425) based on pBAC108L (27) | Tabata et al., unpublished data |
| pRL1058 | Bmr Kmr Smr; bears transposon Tn5-1058 | 35 |
| pRL1977 | Bmr Kmr Smr; ClaI recovery of Tn5-1058 and contiguous DNA from HNL3 | This study |
| pRL1992 | Bmr Cmr Emr Kmr Smr; pRL1977, cut with ClaI and ligated to the sacB-bearing AsuII fragment of pRL1075 (4) | This study |
| pRL2130 | Apr; clone, in the SalI site of pUC19 (29) of the 12-kb insert, bearing orf1 and the 3′ terminal portion of hcwA, from a λEMBL3 phage | This study |
| pRL2138 | Apr; a 6-kb ClaI fragment of pRL2130 in the ClaI site of pIC19H | This study |
| pRL2168 | Apr; PstI-SpeI fragment of pRL2138 in PstI-XbaI hole of pIC19H | This study |
| pRL2174 | Apr; a 5.1-kb, ClaI-SpeI subclone of the insert of pRL2138, blunted with the Klenow fragment, inserted in the SmaI site of pIC19H | This study |
| pRL2332 | Apr; a 790-bp PstI-EcoRI subclone of pRL2174 in the PstI-EcoRI hole of pBS-SK(+) | This study |
| pRL2333 | Apr; a 573-bp PstI-DraI fragment of pRL2168 cloned between the PstI and SmaI sites of pBS-SK(+) | This study |
| pRL2430 | Cmr Emr Smr Spr; BAC vector::hcwA::aadA (see Materials and Methods) | This study |
| pRL2431 | Cmr Emr; BsaBI deletion product of pRL2430; BAC vector bearing a fragment of DNA that contains hcwA and a region 5′ from that gene, including only the 3′ end of orf2 | This study |
| pRL2509a | Apr Cmr Emr Smr Spr; pRL59HE::(hcwA::aadA), Ptac→amidase | This study |
| pRL2509b | Apr Cmr Emr Smr Spr; pRL59HE::(hcwA::aadA), Ptac→←amidase | This study |
| pRL2510 | Cmr Emr; BAC vector (see Materials and Methods) whose sole BsaBI site is Dam-methylatable | This study |
a
Ap, ampicillin; Bm, bleomycin; Cm, chloramphenicol; Em, erythromycin; Km, kanamycin; Nm, neomycin; superscript r, resistance; Sm, streptomycin; Sp, spectinomycin.
b
DRx refers to a product of double reciprocal recombination of plasmid pRLx with the genome of PCC 7120, as confirmed by Southern hybridization.