TABLE 1.
X. campestrispv. campestris strains, plasmids, and oligonucleotides used or generated in this study
| Strain, plasmid, or oligonucleotide | Sequence (5′ to 3′) | Features or purpose | Source or reference |
|---|---|---|---|
| Plasmids | |||
| pVO155 | pUC119 derivative containing the promoterless gus (uidA) reporter gene encoding β-glucuronidase; used for insertion mutagenesis; Kmr Ampr | 51 | |
| pFAJ1700 | pTR102-derived expression vector containing a multiple-cloning site and transcriptional terminators in both orientations; Tetr Ampr | 25 | |
| pCZ750 | pFAJ1700 containing the KpnI-AscI lacZ gene from the pCZ367 plasmid; Tetr Ampr | 9 | |
| p917 | pFAJ1700 derivative containing 2,094 bp of pSC150 with lacI, tacp promoter, and T7 terminator; Tetr Ampr | 11 | |
| p917-lacI | p917 derivative containing 1,526 bp of pMF533 with lacI, tacp promoter, ribosome binding site, and T7 terminator; used for protein overexpression; Tetr Ampr | This study | |
| pCZ-rpoEp | pCZ750 derivative; rpoEp-lacZ; Tetr Ampr | This study | |
| pCZ-rseAp | pCZ750 derivative; rseAp-lacZ; Tetr Ampr | This study | |
| pCZ-rpoHp | pCZ750 derivative; rpoHp-lacZ; Tetr Ampr | This study | |
| pCZ-prcp | pCZ750 derivative; prcp-lacZ; Tetr Ampr | This study | |
| pCZ-ompWp | pCZ750 derivative; ompWp-lacZ; Tetr Ampr | This study | |
| pCZ-hrpFp | pCZ750 derivative; hrpFp-lacZ; Tetr Ampr | This study | |
| pCZ-xcc0401p | pCZ750 derivative; xcc0401p-lacZ; Tetr Ampr | This study | |
| p917-rpoE | p917-lacI derivative; tacp-rpoE; Tetr Ampr | This study | |
| pMF533 | pMALc2E (New England Biolabs) containing malE-gadE fusion | 16 | |
| pK18mobsacB | Mobilizable cloning vector containing a modified sacB gene from B. subtilis; used for gene disruption; Kanr | 61 | |
| pK18-rpoEU | pK18 containing ∼1 kb upstream of the rpoE gene of X. campestrispv.campestris (up to 15 nucleotides downstream of the ATG start codon) | This study | |
| pK18-rpoEU+D | pK18 containing ∼1 kb upstream and ∼1 kb downstream of the rpoE gene of X. campestrispv.campestris (from 15 nucleotides upstream of the stop codon) | This study | |
| pK18-rseAU | pK18 containing ∼1 kb upstream of the rseA gene of X. campestrispv.campestris (up to 15 nucleotides downstream of the ATG start codon) | This study | |
| pK18-rseAU+D | pK18 containing ∼1 kb upstream and ∼1 kb downstream of the rseA gene of X. campestrispv.campestris (from 15 nucleotides upstream of the stop codon) | This study | |
| pK18-mucDU | pK18 containing ∼1 kb upstream of the mucD gene of X. campestrispv.campestris (up to 15 nucleotides downstream of the ATG start codon) | This study | |
| pK18-mucDU+D | pK18 containing ∼1 kb upstream and ∼1 kb downstream of the mucD gene of X. campestrispv.campestris (from 15 nucleotides upstream of the stop codon) | This study | |
| pK18-rpoEUrseAD | pK18 containing ∼1 kb upstream of the rpoE gene (up to 15 nucleotides downstream of the ATG start codon) and ∼1 kb downstream of the rseA gene of X. campestrispv.campestris (from 15 nucleotides upstream of the stop codon) | This study | |
| pK18-rpoEUmucDD | pK18 containing ∼1 kb upstream of the rpoE gene (up to 15 nucleotides downstream of the ATG start codon) and ∼1 kb downstream of the rseA gene of X. campestrispv.campestris (from 15 nucleotides upstream of the stop codon) | This study | |
| pK18-degSU | pK18 containing ∼1 kb upstream of the degS gene of X. campestrispv.campestris (up to 15 nucleotides downstream of the ATG start codon) | This study | |
| pK18-degSU+D | pK18 containing ∼1 kb upstream and ∼1 kb downstream of of the degS gene of X. campestrispv.campestris (from 15 nucleotides upstream of the stop codon) | This study | |
| pK18-rsePU | pK18 containing ∼1 kb upstream of the rseP gene of X. campestrispv.campestris (up to 15 nucleotides downstream of the ATG start codon) | This study | |
| pK18-rsePU+D | pK18 containing ∼1 kb upstream and ∼1 kb downstream of the rseP gene of X. campestrispv.campestris (from 15 nucleotides upstream of the stop codon) | This study | |
| pK18-xcc1664U | pK18 containing ∼1 kb upstream of the XCC1664 gene of X. campestrispv.campestris (up to 15 nucleotides downstream of the ATG start codon) | This study | |
| pK18-xcc1664U+D | pK18 containing ∼1 kb upstream and ∼1 kb downstream of the XCC1664 gene of X. campestrispv.campestris (from 15 nucleotides upstream of the stop codon) | This study | |
| Strains | |||
| E. coli | |||
| DH5α | λ− φ80dlacZΔM15 Δ(lacZYA-argF)U169 recA1 endA1 hsdR17(rK− mK−) supE44 thi-1 gyrA relA1 | Laboratory collection | |
| X. campestrispv. campestris | |||
| 568 | Wild-type strain; rifampin-resistant derivative of X. campestrispv.campestris LMG568/ATCC33913 | 9 | |
| XcPB1 | ΔrpoE; Rifr | This study | |
| XcPB2 | ΔrseA; Rifr | This study | |
| XcPB3 | ΔmucD; Rifr | This study | |
| XcPB4 | ΔrpoE-rseA; Rifr | This study | |
| XcPB5 | ΔrpoE-rseA-mucD; Rifr | This study | |
| XcPB6 | Δxcc1664; Rifr | This study | |
| XcPB7 | ΔdegS; Rifr | This study | |
| XcPB8 | ΔrseP; Rifr | This study | |
| rpoE::pVO | XCC1267::pVO155; Rifr Kmr | This study | |
| Oligonucleotidesa | |||
| rpoE F-Sma | TTTCCCGGGCATACGGCGCGGGATAGTGTTC | rpoE gene deletion | |
| rpoE R-Xba | TTTTCTAGAGACTTCGGCCATGTCGGG | ||
| rpoE F-Xba | TTTTCTAGACACCGTGTATGACCAATAACC | ||
| rpoE R-Hind | TTTAAGCTTGGCCGAACATCTGGGTGCGG | ||
| rseA F-Sma | TTTCCCGGGTGACATCGCCCAGTTCGAG | rseA gene deletion | |
| rseA R-Xba | TTTTCTAGAGTTATTGGTCATACACGGTG | ||
| rseA F-Xba | TTTTCTAGACCGCAGGACTGATGTTCTCGCC | ||
| rseA R-Hind | TTTAAGCTTGAAAGCCAGGTCGATCGGGATC | ||
| mucD F-Eco (B) | TTTGAATTCGAAAGCGCTACCCGTGAGCGAC | mucD gene deletion | |
| mucD R-Xba | TTTTCTAGAGCGGGGATTCATCAGGTTG | ||
| mucD F-Xba | TTTTCTAGAGCGGGCGGCTGAGACGCAGGG | ||
| mucD R-Hind | TTTAAGCTTGGGTGTCGCCGACCGGCGCGCC | ||
| rseP F-Sma | TTTCCCGGGACCCAGGCGCATGCCGGTGATC | rseP gene deletion | |
| rseP R-Xba | TTTTCTAGAGAAATCACCCATGGATGCAAC | ||
| rseP F-Xba | TTTTCTAGAGTTCCACGATGAAGCTGCTCC | ||
| rseP R-Hind | TTTAAGCTTGAAGATGTCGCCGGCCTTGGGG | ||
| degS F-Eco | TTTGAATTCAACGCTGTTCTTGGCCACCAC | degS gene deletion | |
| degS R-Xba | TTTTCTAGACAGCGGTCGCATGCAACGGATTC | ||
| degS F-Xba | TTTTCTAGACTCATGCGTTGATCCGGCGTG | ||
| degS R-Hind | TTTAAGCTTCATGGCGCCGAATTTCATGGG | ||
| xcc1664 F-Eco | TTTGAATTCGCCGCCCAGATCGGGCGTG | XCC1664 gene deletion | |
| xcc1664 R-Bam | TTTGGATCCCCCACCGGGCACTGCATGATTTC | ||
| xcc1664 F-Bam | TTTGGATCCGCGCCATGACCACGCCCTG | ||
| xcc1664 R-Hind | TTTAAGCTTGGAAAGCCATCCAGGCGC | ||
| rpoE RT-F (A) | GCGGCGTTCGATGTGTTGG | ||
| rpoE-EXT (I) | CCAGCTCCTGAGGTGTATC | Mapping of rpoEp | |
| mucD RT-F (D) | CCATCACCCGCAAGGACGCC | ||
| mucD-EXT (G) | GCCAACGGCAGGGTCATGG | ||
| mucD(2)-EXT (K) | GAACATCTGGGTGCGGATG | ||
| mucD(3)-EXT (H) | GGAAATGATGAAGCCCGAAC | ||
| rseA-EXT (E) | CTTTCGGTCTCCAGCAGAGG | ||
| rseA(2)-EXT (J) | GGACATGTCAGGGTTATTG | Mapping of rseAp | |
| rseA(3)-EXT (F) | GTTCGCGGGACACGAACAAG | ||
| rseA RT-F (C) | GACGAAGAGTTGGCCGGCTG | ||
| lacI-FseI | TTTGGCCGGCCATCGAATGGTGC | ||
| PtaqNcoBam | TTGGATCCATGGGCTATGGTCCTTGTTG | ||
| P1268-Hind | TTTAAGCTTCGGTGGCAGACAGG | Upstream region of rseA (fusion to lacZ) | |
| P1268-Xba | TTTCTAGATGGCCGACCGGAGTTC | ||
| P1267-Hind | TTTAAGCTTGGGGCAGGGCAGCTCGG | Upstream region of rpoE (fusion to lacZ) | |
| P1267-Xba | TTTTCTAGAAGTCGGGCAATGAGACC | ||
| ORF1267-Nco | TTTCCATGGCCGAAGTCGATACACC | rpoE gene | |
| ORF1267-Hind | TTTAAGCTTCATACACGGTGTCGCTGAC | overexpression | |
| xcc1535-EXT | GTTCACCACGTCCGGTGAGCTC | Mapping of XCC1535p | |
| xcc0964-EXT | CTCGGCCAGCATAATCTTGCC | Mapping of XCC0964p | |
| hrpF-EXT | CCTCGCAGTGACAGAGCAG | Mapping of hrpFp | |
| hpa1-EXT | CTGCAGGTTGATGAAGTTGG | Mapping of hpaIp | |
| ompW-EXT | GGAAATGGAACGCATGAGGG | Mapping of ompWp | |
| pqqB-EXT | CCGATCCCAAAACGATGATG | Mapping of pqqAp | |
| xcc0401-EXT | GTCGGTCTGGATCTGGATCTG | Mapping of XCC0401p | |
| prc-EXT | CCAGCAATGCCATCGGAGTG | Mapping of prcp | |
| xcc4186-EXT | GAAGCTGCGTACTGCGTTGCAG | Mapping of XCC4186p | |
| rpoH-EXT | GATTGTTTGCCACAAGGGCAGTC | Mapping of rpoHp | |
| xcc3227-EXT | GGAAGGAGCGGCGGTGCGTTC | Mapping of XCC3227p | |
| xcc1246-EXT | GGCAAGTCTGATCCTCTTGG | Mapping of XCC1246p | |
| P0539-Hind | TTTAAGCTTGGCCGGGCTGGTCGAGGG | Upstream region | |
| P0539-Xba | TTTTCTAGATTGTTAGGCCCTTGGAGTGG | of ompW (fusion to lacZ) | |
| PhrpF-Hind | TTTAAGCTTCGGGGTCCAATCCAAGCC | Upstream region | |
| PhrpF-Xba | TTTCTAGAGCAGTGGCGGAGCTG | of hrpF (fusion to lacZ) | |
| Pprc-Hind | TTTAAGCTTGTCACCCTGCGCGACCTG | Upstream region | |
| Pprc-Xba | TTTTCTAGATGAAAGAAGGGCGGTGATC | of prc (fusion to lacZ) | |
| P0401-Hind | TTTAAGCTTCGTCCTGCGCCACCGCAG | Upstream region | |
| P0401-Xba | TTTTCTAGATTCGATTCTGCGACAGCC | of XCC0401 (fusion to lacZ) | |
| P3771-Hind | TTTAAGCTTCAATGCGGTGCTGACGGTGG | Upstream region | |
| P3771-Xba | TTTTCTAGAACTATTGGACTGCTGGGTAC | of rpoH (fusion to lacZ) | |
| P1230-Hind | TTTAAGCTTCGGCATCGGCGTCCTCTTC | Upstream region of XCC1230 (fusion to lacZ) | |
| P1230-Xba | TTTTCTAGATTGCTGCACCCCCATTCTG |