TABLE 2.
Primers used in this study
| Purpose and name | Sequence | |
|---|---|---|
| Construction of deletion vectorsa | ||
| GSU2642 U1 XbaI | ACGTCG TCTAGA CCT CAC CTA TGA CAG CCG GTT C | |
| GSU2642 U2 | GCAGGCGGCGTCAACGAAC CCT CTT CAT TGC CAG CGT GCT | |
| GSU2642 L1 | AGCACGCTGGCAATGAAGAGG GTT CGT TGA CGC CTG C | |
| GSU2642 L2 HindIII | ACGTCG AAGCTT CGC GAA CTG CGA TGG AAA CGT AG | |
| GSU2643 U1 XbaI | ACGTCG TCTAGA CGG TAT CTC GAT GTT CGC TCA TTC G | |
| GSU2643 U2 | CCGATCCGTGAAATCACCGTTAACC GGC GAG AAG CAT GCA CCC | |
| GSU2643 L1 | GGGTGCATGCTTCTCGCC GGT TAA CGG TGA TTT CAC GGA TCG G | |
| GSU2643 L2 HindIII | ACGTCG AAGCTT GAC AGA GAA CGC AGT CGC GTA C | |
| GSU2644 U1 XbaI | ACGTCG TCTAGA CTT CAC CTG TCA AGG CTG TCA C | |
| GSU2644 U2 | TCCATCACGCTCTTACCTGCG GGT CAT CCA GGA ACG C | |
| GSU2644 L1 | GCGTTCCTGGTGGATGACC CGC AGG TAA GAG CGT GAT GGA | |
| GSU2644 L2 HindIII | ACGTCG AAGCTT GAC AGA CCT TGC ACT GGT TGA GG | |
| GSU2645 U1 XbaI | ACGTCG TCTAGA CTT CAA TGT GAG CGA TGG TCA CC | |
| GSU2645 U2 | CATCACAACGGACTGTCAGCG GGC AAC CAT CGC CAC CAA G | |
| GSU2645 L1 | CTTGGTGGCGATGGTTGCC CGC TGA CAG TCC GTT GTG ATG | |
| GSU2645 L2 HindIII | ACGTCG AAGCTT CGG AAC GGT CGT TGA GAT AGT C | |
| Confirmation of gene deletion | ||
| ΔextD (ΔGSU2642) | GAC GCT CAA TCT TCT GAC GGG C | |
| CTG TCG GCA GTG CGC TAC TTG | ||
| ΔextC (ΔGSU2643) | CGG AGC GAG GAG CTT CTG G | |
| GGC GTC AAC GAA CGA TTG TCG | ||
| ΔextB (ΔGSU2644) | CTC CGC GTT TCA GGA CAT CAA G | |
| AGC ACC GAG CAG GTT GGT T | ||
| ΔextA (ΔGSU2645) | GTG GCG TGT ACG GCG ATT G | |
| CGG TCA CCG AGT ACC GTC TG | ||
| Construction of complementation plasmids | ||
| p-PacpP-extABCD (NdeI and SacI) | ACGTCG CATATG GTT GCC CTG TTC GGA TG | |
| ACGTCG GAGCTC TCA ACG AAC GAT TGT CGG ATG ACA G | ||
| p-PextA-extABCD | ||
| GSU2645 PextA U1 AscI | ACGTCG GGCGCGCC CGG CCA TTT CAT TGC TTG ACA GG | |
| GSU2645 PextA U2 | CAATGCATCCCCCTCCTCGTG TCA GCG CTG ACG AAC CGG | |
| GSU2644 L1 | CCGGTTCGTCAGCGCTGA CAC GAG GAG GGG GAT GCA TTG | |
| GSU2642 L2 BglII | ACGTCG AGATCT GCA GGC GGC GTC AAC GAA C | |
a
U1-U2 and L1-L2 primers were used to amplify upstream and downstream ∼750-bp flanking regions of target gene. Overlapping PCR was used to combine both products into the insert, which was then ligated into the multiple-cloning site of pK18mobsacB using the indicated restriction enzymes.