TABLE 2.
Primers used in this study
| Primer by function | Sequence (5′–3′)a | Comment |
|---|---|---|
| Construction of deletion strains | ||
| 50K-L1 | TAGAGGCGAGTTTCGGACC | Upstream of fusion fragment for Δ50K mutation |
| 50K-L2 | GAACACTATTATACCTTCAATCCGCTAAG | |
| 50K-M1 | GAAGGTATAATAGTGTTCGTGAATACATGTT | Spectinomycin resistance gene |
| 50K-M2 | CGTCACTATTTTCTAAAATCTGATTACCA | |
| 50K-R1 | TTTAGAAAATAGTGACGAGTCAAAAGCAG | Downstream of fusion fragment for Δ50K mutation |
| 50K-R2 | GGGCTAGGACATCAAGTAAG | |
| 58K-L1 | CCTGAGTTGTCATTCGCTGTG | Upstream of fusion fragment for Δ58K mutation |
| 58K-L2 | GAACACTAATTTATCGTCACTTTCGGATT | |
| 58K-M1 | GACGATAAATTAGTGTTCGTGAATACATGTT | Spectinomycin resistance gene |
| 58K-M2 | CTGATAATTTTTTCTAAAATCTGATTACCA | |
| 58K-R1 | TTTAGAAAAAATTATCAGACTGCCGTTAG | Downstream of fusion fragment for Δ58K mutation |
| 58K-R2 | ATTGCCCTTGGAAGAGTTAG | |
| P1-L1 | CATGCTATTGGCCTAGATCAT | Upstream of fusion fragment for ΔP1 mutant |
| P1-L2 | CGAACACTAGTCACTACTCTGACTGTTGGA | |
| P1-M1 | GAGTAGTGACTAGTGTTCGTGAATACATGTT | Spectinomycin resistance gene |
| P1-M2 | TGCGAAGTAATTTTCTAAAATCTGATTACCA | |
| P1-R1 | TTAGAAAATTACTTCGCAAATCCTGTCTC | Downstream of fusion fragment for ΔP1 mutation |
| P1-R2 | TCGTTTACGTCTCATAGAACT | |
| P2-L1 | TTTCTTGCTTCGAGGTTCA | Upstream of fusion fragment for ΔP2 mutation |
| P2-L2 | GAACACTATAAATACAGACGCATATACGAT | |
| P2-M1 | TGTATTTATAGTGTTCGTGAATACATGTT | Spectinomycin resistance gene |
| P2-M2 | ATTCGTCCATTTTCTAAAATCTGATTACCA | |
| P2-R1 | TTAGAAAATGGACGAATTATCCAAGAAGAT | Downstream of fusion fragment for ΔP2 mutation |
| P2-R2 | TATGGCACCAATTTCTTATCAT | |
| P3-L1 | TGAAAACAACGCTCACCAACAC | Upstream of fusion fragment for ΔP3 mutation |
| P3-L2 | CGAACACTACACGCCTCCCGATTCCGGAGTA | |
| P3-M1 | GGAGGCGTGTAGTGTTCGTGAATACATGTT | Spectinomycin resistance gene |
| P3-M2 | CTATACAGTTTTCTAAAATCTGATTACCA | |
| P3-R1 | TTAGAAAACTGTATAGTCGAAGCAGTGAAC | Downstream of fusion fragment for ΔP3 mutation |
| P3-R2 | GAACTACGTTCGCTCTATCACC | |
| P4-L1 | TTCAAACCTAATGCGTTGGAT | Upstream of fusion fragment for ΔP4 mutation |
| P4-L2 | CGAACACTATTCCCTGCGAAACAAATCTGAT | |
| P4-M1 | CGCAGGGAATAGTGTTCGTGAATACATGTT | Spectinomycin resistance gene |
| P4-M2 | ATTTCCTATTTTTCTAAAATCTGATTACCA | |
| P4-R1 | TTAGAAAAATAGGAAATTGCTTGTTTCTTT | Downstream of fusion fragment for ΔP4 mutation |
| P4-R2 | GCCAACACAAACATTAAGCTAT | |
| P5-L1 | TTAGAAAAGCAAGTACG | Upstream of fusion fragment for ΔP5 mutation |
| P5-L2 | GAACACTACTACTCACTGCTATTTGATTA | |
| P5-M1 | CAGTGAGTAGTAGTGTTCGTGAATACATGTT | Spectinomycin resistance gene |
| P5-M2 | CGTACTTGCTTTTCTAAAATCTGATTACCA | |
| P5-R1 | TTAGAAAAGCAAGTACGTCCGCATCGACT | Downstream of fusion fragment for ΔP5 mutation |
| P5-R2 | TTGGTACCAACATACGGCATA | |
| P6-L1 | TGGAGCTCGTTGACGGAGGAT | Upstream of fusion fragment for ΔP6 mutation |
| P6-L2 | GAACACTAATCTCTAACTACAGAACTGCT | |
| P6-M1 | GTTAGAGATTAGTGTTCGTGAATACATGTT | Spectinomycin resistance gene |
| P6-M2 | ATATCTTATTTTCTAAAATCTGATTACCA | |
| P6-R1 | TAGAAAATAAGATATTGAAAGCGAGTTA | Downstream of fusion fragment for ΔP6 mutation |
| P6-R2 | GTGACCGCTAAGAGCCGTTCC | |
| sec-L1 | CATAGAGGCAATGATTGTCGTT | Upstream of fusion fragment for SecY2/A2 system gene deletion |
| sec-L2 | GAACACTAATCTCTTCATAAGGTGTATCCT | |
| sec-M1 | ATGAAGAGATTAGTGTTCGTGAATACATGTT | Spectinomycin resistance gene |
| sec-M2 | ATATCTTTCTTTTCTAAAATCTGATTACCA | |
| sec-R1 | TTTAGAAAAGAAAGATATCCATGACGGCTCC | Downstream of fusion fragment for SecY2/A2 system gene deletion |
| sec-R2 | TGCAAGATAATTAAGCCGAGA | |
| sssP1-L1 | TTCCACTATTCAAGGCAATC | Upstream of fusion fragment for ΔsssP1 mutation (step 1) |
| sssP1-L2 | CAGCATTATCCGCATCTTGACACGGCTCTTAC | |
| sssP1-M1 | TGTCAAGATGCGGATAATGCTGAAAACTCCTTG | Spectinomycin resistance and negative selection marker (step 1) |
| sssP1-M2 | GGTTGTGGACAATCTGATTACCAATTAGAATG | |
| sssP1-R1 | GTAATCAGATTGTCCACAACCGGGTGAAC | Downstream of fusion fragment for ΔsssP1 mutation (step 1) |
| sssP1-R2 | GAACGCCCACCATATAGTA | |
| sssP1-LU | ATATATGTACTGCCGAATGTT | Upstream of fusion fragment for ΔsssP1 mutation (step 2) |
| sssP1-LD | ACGCTATTACTCCTATGCAAAGAATGAGTCACA | |
| sssP1-RU | TTCTTTGCATAGGAGTAATAGCGTTGGTACAGG | Downstream of fusion fragment for ΔsssP1 mutation (step 2) |
| sssP1-RD | GACTACTTCGCCAGGAAGGGT | |
| sssP1-T182A-L1 | ATCTTGATAAGCTCCATAGAG | Upstream of fusion fragment for ΔsssP1-T182A mutation (step 1) |
| sssP1-T182A-L2 | GTTTTCAGCATTATCCACTGTTTCCTGCCCTTTCCCT | |
| sssP1-T182A-M1 | GGATAATGCTGAAAACTCCTTG | Spectinomycin resistance and negative selection marker (step 1) |
| sssP1-T182A-M2 | AATCTGATTACCAATTAGAATG | |
| sssP1-T182A-R1 | ATTGGTAATCAGATTGTGATTCTTCCAGCCAGTCTG | Downstream of fusion fragment for ΔsssP1-T182A mutation (step 1) |
| sssP1-T182A-R1 | GCGTGATTCTCCACGTAATCG | |
| sssP1-T182A-LU | CGTCTTTAGTCGTCGCGTGAC | Upstream of fusion fragment for ΔsssP1-T182A mutation (step 2) |
| sssP1-T182A-LD | CTAACATAGCTAACGTTGACTTTCTTATCGC | |
| sssP1-T182A-RU | GTCAACGTTAGCTATGTTAGCTTCAGGAGCG | Downstream of fusion fragment for ΔsssP1-T182A mutation (step 2) |
| sssP1-T182A-RD | ACCTGTTAGTCTGCCATTGTT | |
| Construction of expression vectors | ||
| SssP1NR216–781-L | CgagctcCTAGGGTCTGTTGATGCTTCT | Construction of SssP1NR216–781 |
| SssP1NR216–781-R | CCGctcgagGTAATCACTAGCATTCCCAAA | |
| SssP1NR1295–1446-L | CgagctcGGTCAAGCTGCTTCTATTGAC | Construction of SssP1Ig-like-1 |
| SssP1NR1295–1446-R | CCGctcgagACTGATACTTGTCGTGGTCGG | |
| SssP1NR2071–2212-L | CgagctcGGTGATTCTTTACCTGTTGAT | Construction of SssP1Ig-like-2 |
| SssP1NR2071–2212-R | CCGctcgagATTCCTGTCAGTAACCTTTAA | |
| Cotranscription test | ||
| secY2/asp1-L | GGCATTCCGATGTTGGCTAGT | Cotranscription test spanning ORFs of secY2-asp1 |
| secY2/asp1-R | CGATTTGGGCATACCGTTCTC | |
| asp1/asp2-L | TTCCTCAGATTAACCGAATAG | Cotranscription test spanning ORFs of asp1-asp2 |
| asp1/asp2-R | TTACTCGTTCTCAAGCGTTCC | |
| asp2/asp3-L | GCCAAACGTCTATGAATCGAC | Cotranscription test spanning ORFs of asp2-asp3 |
| asp2/asp3-R | CCCGCATTCAATAAGGCAATA | |
| asp3/secA2-L | ACGACGCCTCTACTTATCATA | Cotranscription test spanning ORFs of asp3-secA2 |
| asp3/secA2-R | CTTGGACCTTGTACGGAAACA | |
| secA2/gtfA-L | ATCTACAACAACTCCGCGTCA | Cotranscription test spanning ORFs of secA2-gtfA |
| secA2/gtfA-R | TGAAACCGATATTCCGTGTCA | |
| gtfA/gtfB-L | ATCTCTTGCCATTCCCTACAG | Cotranscription test spanning ORFs of gtfA-gtfB |
| gtfA/gtfB-R | CGCTTAGTTGCATAATCATAG | |
| gtfB/asp4-L | CTGCATTTTCATATCGGAGCG | Cotranscription test spanning ORFs of gtfB-asp4 |
| gtfB/asp4-R | TCGGAATAACATAGCGTTCTT | |
aUnderlined nucleotides denote reverse complement; lowercase nucleotides denote restriction enzyme sites.