TABLE 1.
Strains, plasmids, and primers
| Strain, plasmid, or primer | Genotype, phenotype, use, or sequencea | Source or reference(s) |
|---|---|---|
| Strains | ||
| E. coli | ||
| W3110; ATCC 27325 | ATCC, Manassas, VA | |
| ARC523 | W3110 ΔtolC::Tn10 | AstraZeneca culture collection |
| MG1655; ATCC 700926 | F− λ− IlvG−rfb-50 rph-1 | ATCC, Manassas, VA |
| TOP10 | Invitrogen Corp., Carlsbad, CA | |
| BL21(DE3) | Invitrogen Corp., Carlsbad, CA | |
| B | Used to propagate T4 DNA according to ATCC protocol (11303-B4) | ATCC, Manassas, VA |
| H. influenzae | ATCC, Manassas, VA | |
| Rd KW20; ATCC 51907 | Wild-type parent | |
| ARM158 | Rd KW20 ΔacrB::cat; Cmr | AstraZeneca culture collection |
| HIN100 | Rd KW20 ΔacrB::aph3-A; Kmr | This study |
| HIN101 | Rd KW20 ΔacrB::aph3-A ΔompP1::cat; Kmr Cmr | This study |
| HIN102 | Rd KW20 ΔacrB::aph3-A ΔompP1::ligA + cat; Kmr Cmr | This study |
| S. pneumoniae | ||
| D39; NCTC 7466 | 2 | |
| Rx1 | Unencapsulated transformable derivative of R36 | 34 |
| R6; ATCC BAA-255 | Unencapsulated transformable derivative of R36 | 17 |
| SPN100 | Rx1 ΔgalU::ermR; Ermr | This study |
| SPN101 | Rx1 ΔgalU::T4 lig + ermR; Ermr | This study |
| SPN102 | Rx1 ΔgalU::T4 lig + ermR ΔligA::aph3-A; Ermr Kmr | This study |
| SPN103 | Spontaneous D39 mutant, resistant to compound 4; amino acid substitution in LigA (L75F) | This study |
| S. aureus RN4220 | Restriction-deficient mutant of strain 8325-4 | 20 |
| M. pneumoniae | ||
| M129; ATCC 29342 | ATCC, Manassas, VA | |
| FH; NCTC 10119 | NCTC, Salisbury, United Kingdom | |
| Candida albicans B2630 | 35 | |
| H. sapiens A549; ATCC CCL-185 | Human lung carcinoma cell line | ATCC, Manassas, VA |
| Plasmids and DNA | ||
| E. coli bacteriophage T4 | ATCC 11303-B4 | ATCC, Manassas, VA |
| H. sapiens cDNA | Human Universal QUICK-Clone cDNA; catalog no. 7109-1; lot no. 1050696 | Clontech, Mountain View, CA |
| pGEM-T | TA cloning vector; Apr | Promega Corp., Madison, WI |
| pCR4-TOPO | TOPO TA cloning vector; Kmr | Invitrogen Corp., Carlsbad, CA |
| pET21b | Protein expression vector | EMD4Biosciences, San Diego, CA |
| pET30 | Protein expression vector | EMD4Biosciences, San Diego, CA |
| pET28 | Protein expression vector | EMD4Biosciences, San Diego, CA |
| pUC18K | Source of nonpolar aph3-A gene in pUC18; Apr Kmr | 24 |
| pASK4 | Source of 1,238-bp cat cassette in pGEM-T: Apr Cmr | 36, 40 |
| pASK5 | Plasmid used for site-directed integration and expression of selected genes at H. influenzaeompP1 (HI0401) locus of Rd KW20; Apr Cmr | 36 |
| pBA467 | Plasmid used for site-directed integration and expression of selected genes at galU (spr1903) locus of S. pneumoniae R6; Apr Ermr | This study |
| pWY375 | pGEM-T containing H. influenzaeacrB (HI0895) deletion and insertion of 840 bp of nonpolar aph3-A cassette; Apr Kmr | This study |
| pLP109 | pASK5 containing H. influenzae ligA (HI1100) subcloned as NdeI-SalI fragment from pHIN-LigA; Apr Cmr | This study |
| pWY473 | pBA467 containing T4 lig subcloned as NdeI-SalI fragment from pT4-lig; Apr Ermr | This study |
| pLP112 | pGEM-T containing S. pneumoniaeligA (spr1024) deletion and insertion of 840 bp of nonpolar aph3-A cassette; Apr Kmr | This study |
| pT4-lig | pCR4-TOPO containing T4 lig; Kmr | This study |
| pHIN-LigA | H. influenzae LigA expression plasmid; pET30a; Kmr | This study |
| pSPN-LigA | S. pneumoniae LigA expression plasmid; pET30a; Kmr | This study |
| pECO-LigA | E. coli LigA expression plasmid; pET30a; Kmr | This study |
| pMPN-LigA | M. pneumoniae LigA expression plasmid; pET30a; Kmr | This study |
| pSAU-LigA | S. aureus LigA expression plasmid; pET30a; Kmr | This study |
| pHSA-LIGI | H. sapiens LIGI expression plasmid (N-terminal truncation and T7 tag); pET21b; Apr | This study |
| Primers | ||
| LigA protein expression | ||
| E. coli ligA-F (NdeI) | GCATTGATGGTGCCATATGGAATCAATCG | MG1655 |
| E. coli ligA-R (SalI) | CGTATTGGCTATTTCAGTCGACTGC | MG1655 |
| H. influenzae ligA-F (NdeI) | CCGAGAATCATATGACAAATATTCAAACTCAAC | KW20 |
| H. influenzae ligA-R (SalI) | GCCTGTCGACCCTGTCACACCAAGCGTCGG | KW20 |
| M. pneumoniae ligA-F (NdeI) | CCTACATATGGCAAAGGTCGCACAAATTCG | M129 |
| M. pneumoniae ligA-R (SalI) | GCATGTCGACTTAGGTCCAAATTGGTTC | M129 |
| S. aureus ligA-F (NcoI) | GGATTAAGCCATGGCTGATTTATCG | RN4220 |
| S. aureus ligA-R (SalI) | CGACGTCGACCTCTAACTATTTAATTC | RN4220 |
| S. pneumoniae ligA-F (NdeI) | GGTCTTATGAATAAACATATGAATGAGTTAGTCGCTTTGC | R6 |
| S. pneumoniae ligA-R (SalI) | CTCTGTCGACAAACGATCCATTACAAACTTTCTAGCC | R6 |
| Expression of other ligases | ||
| T4 lig-F (NdeI) | GCGCATATGATTCTTAAAATTCTGAACG | Bacteriophage T4 |
| T4 lig-R (SalI) | CTTGTCGACTCATAGACCAGTTACCTC | Bacteriophage T4 |
| H. sapiens lig-F (SacI) | GAGGAGAGCTCGGCTCCAGGAAAGGAGGGAGCTGCTGAGG | H. sapiens |
| H. sapiens lig-R (SalI) | GATCGTCGACTTAGTAGGTATCTTCAGGGTCAGAGCCTGAGTCC | H. sapiens |
a
Restriction enzyme sites in primer sequences are in boldface italics.