Table 2.

Plasmid constructions

PlasmidDescriptionaConstructionb
pAH16 Plac-phoR + bla oriR MB1 lacI+ Replaced 0.4-kbp EcoRI-to-BamHI vanS fragment of pSLF13 with similar 1.3-kbp phoR + fragment generated by using P109 and P110 with pBC35 as the template
pAH21c ParaB-phoR + cat oriR 15A araC + Replaced 0.4-kbpNdeI-to-SacI vanS fragment of pSLF22 with similar 1.3-kbp phoR + fragment from pAH16
pAH31 ParaB-lacZ tetAR bla oriR R6Kγ Replaced 0.5-kbpXhoI-to-PstI kan fragment of pWJ18 with 0.5-kbp ParaB PCR fragment generated by using P125 and P126 with pBAD33 as the template
pAH33d ParaB -′araDtetAR bla oriR R6Kγ Replaced 3.0-kbpSalI-to-BsiWI lacZ fragment of pAH31 with 0.6-kbp ′araD′ PCR fragment generated by using P127 and P128 with BW13711 chromosomal DNA as the template
pAH35 ParaB-phoR +-′araDtetAR bla oriR R6Kγ Subcloned 1.4-kbpBamHI phoR + fragment from pAH21 into pAH33
pAH54Like pAH33 except lackingNdeI2 Digested pAH33 with BsiWI and partially with NdeI followed by filling-in with T4 DNA polymerase and religation
pLD68 PrhaB-lacZ tetAR bla oriR R6Kγ Replaced 1.3-kbpXhoI-to-SalI kan fragment of pWJ18 with similar 0.3-kbp PrhaSB fragment generated by using P119 and P120 with pLD1 (3) as the template
pLD69 PrhaS-lacZ tetAR bla oriR R6Kγ Same as pLD68, except fragment is in opposite orientation
pLD70rhaD tetAR bla oriR R6Kγ Replaced 3.0-kbpBamHI-to-BsiWI lacZ fragment of pWJ17 with 0.6-kbp ′rhaD fragment generated by using P121 and P122 with BW13711 chromosomal DNA as the template
pLD71 PrhaB-′rhaD tetAR bla oriR R6Kγ Subcloned 0.7-kbpXhoI-to-BamHI PrhaB fragment from pLD68 into pLD70
pLD77Like pWJ17 except lackingNdeI2 Digested pWJ17 partially withNdeI followed by filling-in with T4 DNA polymerase and religation
pLD78Like PLD71 except lackingNdeI2 Replaced 4.3-kbpXhoI-to-BsiWI lacZ fragment of pLD77 with similar 0.8-kbp PrhaB-′rhaD fragment from pLD71
pLD79 PrhaB-phoB+-′rhaD tetAR bla oriR R6Kγ Subcloned 0.7-kbpNdeI-to-BamHI phoB +fragment from pSK2 (9) into pLD78 digested withBamHI and partially with NdeI
pLD82ΔphoB578 kan oriR R6Kγ Digested pSK47 with NdeI andBamHI followed by filling-in with T4 DNA polymerase and religation
pLD83ΔphoB578 tetAR bla oriR R6Kγ Subcloned 3.4-kbp NcoI fragment from pLD82 that was made blunt with T4 DNA polymerase intoSmaI-cut pLD55 (17)
  • a The replication origins of pMB1 and p15A are denoted oriR MB1 andoriR 15A, respectively.

  • b Additional information is given in the text. Oligonucleotide primers are shown in Table 3.

  • c pAH21 has a unique NdeI site at the phoR start codon and unique downstream SacI,SphI, PstI, and HindIII sites.

  • d Restriction analysis showed that theSphI and BglII sites in P127 were absent from the cloned fragment.