Table 1.
Bacterial strains, plasmids, and primers used in the study
| Strain, plasmid, or primer | Relevant genotype/characteristics/sequence (5′ to 3′)a | Reference/source |
|---|---|---|
| Strains | ||
| E. coli | ||
| DH5α | Strain for general DNA manipulation and monitoring of polymyxin activity | 8 |
| BL21(DE3) | Host strain for overexpression under controlled T7 promoter | 29 |
| B. subtilis | ||
| BSK3S | Strain containing the entire polymyxin synthetase gene cluster on amyE locus, functional sfp gene | 2 |
| BSK4 | srfC::etcB-tet of BSK3S | This study |
| BSK4-0A | spo0A::erm of BSK4 | This study |
| BSK4-rB | abrB::neo of BSK4 | This study |
| BSK4-0A-rB | spo0A::erm of BSK4-rB | This study |
| P. polymyxa E681 | Wild type; produces polymyxin A | 23 |
| Plasmids | ||
| pHPS9 | E. coli-B. subtilis shuttle vector, cm, erm | 6 |
| pGEM-T Easy | Plasmid for cloning of PCR products, Ap | Promega |
| pET22b | Plasmid for expression of His-tagged protein, Ap | Novagen |
| pET22-bsabrB | Plasmid containing abrB gene of B. subtilis 168 | This study |
| Primers | ||
| ectF | CCTGTTGATAGAGTAAACTCC | |
| ectR | ATGGCCCGTTTGTTGATGAGCAACTAGAGCAATCC | |
| tetF | AACAAACGGGCCATATTGTTG | |
| tetR | GATACAAGAGAGGTCTCTCG | |
| srfC1 | AGATATGTATTACCTATCGCC | |
| srfC2 | TTACTCTATCAACAGGGATTGGCGTTCACTGCTTCCTTG | |
| Srf3 | ACGAGAGACCTCTCTTGTAGGATTGGATGAAGGGGCTTCGCT | |
| Srf4 | TCAACGCTTCGACATCACTTTC | |
| mlkneoF | TCGAGATCAGGGAATGAGTTT | |
| mlkneoR | AATAAATACGTAACCAACATG | |
| abrff | CTGTACCAGCCTTCATACTC | |
| abrfr-neo | AAACTCATTCCCTGATCTCGAACACGTCCTAATTCATCAAC | |
| abrrf-neo | CATGTTGGTTACGTATTTATTCATCAGCGAAATCCAAAACC | |
| abrrr | CAAATCGAGCCGAAACGTGTAC | |
| Bsabr-nde | TACATATGTTTATGAAATCTACTGGTATTG | |
| Bsabr-xho | TGCTCGAGTTTAAGGTTTTGAAGCTGG | |
| pmxAF/pmxAF-biotin | AGCATATTGAAGCAGAGGAG | |
| pmxAR/pmxAR-biotin | TCATCGGTAAAAGCAATCCGG | |
| BSF349 | AGGCAGCAGTGGGGAAT | |
| 16S-786R | CTACCAGGGTATCTAATC |
a
Resistance gene or phenotype against antibiotics: tet (tetracycline), erm (erythromycin), neo (neomycin), cm (chloramphenicol), and Ap (ampicillin). The restriction sites of endonucleases in nucleotide sequences are underlined.