Cloning of the citrate permease gene of Lactococcus lactis subsp. lactis biovar diacetylactis and expression in Escherichia coli.

The citrate plasmid (Cit+ plasmid) from Lactococcus lactis subsp. lactis biovar diacetylactis was cloned into the EcoRI site of plasmid pUC18. This recombinant plasmid enabled Escherichia coli K-12 to transport and utilize citrate as a source of energy, indicating expression of the citrate permease from L. lactis biovar diacetylactis. The citrate permease was under the control of the lac promoter of pUC18. Genetic expression of the Cit+ plasmid in maxicells revealed that the plasmid encoded two polypeptides of 47 and 32 kilodaltons, determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis.     

Cloning of the citrate permease gene of Lactococcus lactis subsp. lactis biovar diacetylactis and expression in Escherichia coli

The citrate plasmid (Cit+ plasmid) from Lactococcus lactis subsp. lactis biovar diacetylactis was cloned into the EcoRI site of plasmid pUC18. This recombinant plasmid enabled Escherichia coli K-12 to transport and utilize citrate as a source of energy, indicating expression of the citrate permease from L. lactis biovar diacetylactis. The citrate permease was under the control of the lac promoter of pUC18. Genetic expression of the Cit+ plasmid in maxicells revealed that the plasmid encoded two polypeptides of 47 and 32 kilodaltons, determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis.     

Molecular cloning of invasion plasmid antigen (ipa) genes from Shigella flexneri: analysis of ipa gene products and genetic mapping.

Tn5-tagged invasion plasmid DNA (pWR110) from Shigella flexneri serotype 5 (strain M90T) was cloned into the expression vector lambda gt11. Recombinant phage (lambda gt11Sfl) expressing pWR110-encoded polypeptide antigens were identified by using rabbit antisera directed against S. flexneri M90T invasion plasmid antigens. Antigens encoded by lambda gt11Sfl recombinant phage were characterized by reacting affinity-purified antibodies, eluted from nitrocellulose-bound plaques of lambda gt11Sfl recombinants, with virulent, wild-type S. flexneri M90T polypeptides in Western blot analyses. lambda gt11Sfl clones directing the synthesis of complete, truncated, and beta-galactosidase fusion versions of three previously identified outer membrane polypeptides (57-, 43-, and 39-kilodalton [kDa] antigens) were isolated. A fourth polypeptide, similar in size to the 57-kDa antigen (ca. 58 kDa) but unrelated as determined by DNA homology and serological measurements, was also identified. Southern blot analysis of S. flexneri M90T invasion plasmid DNA hybridized with lambda gt11Sfl insert DNA probes was used to construct a map of invasion plasmid antigen genes (ipa) corresponding to the 57-kDa (ipaB), 43-kDa (ipaC), and 39-kDa (ipaD) polypeptides. Genes ipaB, ipaC and ipaD mapped to contiguous 4.6-kilobase (kb) and 1.0-kb HindIII fragments contained within a larger (23-kb) BamHI fragment. The ipaH gene, which encodes the synthesis of the 58-kDa polypeptide, did not map in or near the ipaBCD gene cluster, suggesting a distinct location of ipaH on the invasion plasmid.     

Regulated expression by readthrough translation from a plasmid-encoded beta-galactosidase.

We have characterized expression of beta-galactosidase from a plasmid cloning vehicle, pBGP120, which carries most of the lacZ gene and contains a single EcoRI site near the end of lacZ. In addition, we have examined expression of heterologous DNA inserted at the position of the EcoRI site. The EcoRI site was shown to be within the sequence coding for beta-galactosidase and its precise location and phase were deduced. Insertion of heterologous EcoRI-generated DNA fragments altered the molecular weight of the plasmid-encoded beta-galactosidase polypeptide. Those insertions that were in the correct phase were expressed at a high level as a fused protein. The different forms of beta-galactosidase polypeptides produced by various hybrid plasmids were all stable proteins. The level of expression of the plasmid-encoded beta-galactosidase was several times higher than maximal expression of chromosome-encoded beta-galactosidase, suggesting that expression is proportional to gene copy number. The expression of the plasmid lacZ gene was controlled by cyclic AMP. When grown in a cya strain (DG74), expression was dependent on exogenous cyclic AMP. Although in normal strains there was insufficient lac repressor to inactivate all copies of the plasmid, repressor regulation was restored when the plasmid was grown in a strain (M96) that overproduces the lac repressor.     

Polypeptides specified by the mercuric resistance (mer) operon of plasmid R100

Overlapping deletion mutations were constructed in chimaeric plasmids carrying the mer operon of plasmid R100. Polypeptides specified by the mutant plasmids in Escherichia coli minicells correlated with the mer genes as follows: merT, 17- and 16-kDa polypeptides; merP, 9.8- and 9.5-kDa polypeptides; merC, a 14-kDa polypeptide; merA, 65- and 62-kDa polypeptides. The products of the merR and merD genes were not identified. The revised nomenclature of the mer genes is explained.     

pHG276: a multiple cloning site pBR322 copy number vector expressing a functional lac alpha peptide from the bacteriophage lambda PR promoter

The bacteriophage lambda early promoter PR has been used to direct the synthesis of lac alpha in a plasmid containing the multiple cloning site of pUC8. The copy number of the plasmid is controlled by the rop(rom) gene and the plasmid incorporates the cI857 gene for temperature regulation of lac alpha expression. Isolation of recombinant derivatives with DNA inserts in the multiple cloning region can be identified by insertional inactivation of lac alpha and consequently, a Lac- phenotype in a host carrying the M15 deletion of lac. The potential of pHG276 as a fully regulated expression vector is examined.