Cosmid Vector

Abstract: A genomic library was prepared in Escherichia coli from DNA of wild-type Xanthomonas campestris pv. campestris (aetiological agent of crucifer black rot), partially digested with endonuclease EcoRI, using the mobilisable broad host range cosmid vector pLAFR1. Recombinant plasmids contained inserts ranging in size from 19.1 to 32.3 kb (mean 26.6). Certain of the clones complemented E. coli auxotrophic markers. Using the narrow host range plasmid pRK2013 as a helper the pooled recombinant plasmids were transferred conjugally to X. c. campestris mutants, and clones were identified which restored yellow pigmentation to white mutants, prototrophy to amino acid auxotrophs and pathogenicity towards turnip plants to two non-pathogenic mutants. The lesion in one mutant (8288, complemented by the plasmid pIJ3000) is unknown. However mutant 8237 is defective in production of extracellular protease and polygalacturonate lyase and restoration of pathogenicity by complementation with the plasmid pIJ3020 concomitantly restored both enzyme levels to wild-type values.

Abstract: DNA isolated from the wild-type aflatoxin-producing (Afl+) fungus Aspergillus parasiticus NRRL 5862 was used to construct a cosmid genomic DNA library employing the homologous gene (pyrG) encoding orotidine monophosphate decarboxylase for selection of fungal transformants. The cosmid library was transformed into an Afl- mutant, A. parasiticus CS10 (ver-1 wh-1 pyrG), deficient in the conversion of the aflatoxin biosynthetic intermediate versicolorin A to sterigmatocystin. One pyrG+ Afl+ transformant was identified. DNA fragments from this transformant, recovered by marker rescue, contained part of the cosmid vector including the pyrG gene, the ampr gene, and a piece of the original genomic insert DNA. Transformation of these rescued DNA fragments into A. parasiticus CS10 resulted in production of wild-type levels of aflatoxin and abundant formation of sclerotia. The gene responsible for this complementation (ver-1) was identified by Northern RNA analysis and transformation with subcloned DNA fragments. The approximate locations of transcription initiation and polyadenylation sites of ver-1 were determined by an RNase protection assay and cDNA sequence analysis. The predicted amino acid sequence, deduced from the ver-1 genomic and cDNA nucleotide sequences, was compared with the EMBL and GenBank data bases. The search revealed striking similarity with Streptomyces ketoreductases involved in polyketide biosynthesis. Images

Abstract: The gram-positive bacterium Rhodococcus sp. strain IGTS8 is able to remove sulfur from certain aromatic compounds without breaking carbon-carbon bonds. In particular, sulfur is removed from dibenzothiophene (DBT) to give the final product, 2-hydroxybiphenyl. A genomic library of IGTS8 was constructed in the cosmid vector pLAFR5, but no desulfurization phenotype was imparted to Escherichia coli. Therefore, IGTS8 was mutagenized, and a new strain (UV1) was selected that had lost the ability to desulfurize DBT. The genomic library was transferred into UV1, and several colonies that had regained the desulfurization phenotype were isolated, though free plasmid could not be isolated. Instead, vector DNA had integrated into either the chromosome or a large resident plasmid. DNA on either side of the inserted vector sequences was cloned and used to probe the original genomic library in E. coli. This procedure identified individual cosmid clones that, when electroporated into strain UV1, restored desulfurization. When the origin of replication from a Rhodococcus plasmid was inserted, the efficiency with which these clones transformed UV1 increased 20- to 50-fold and they could be retrieved as free plasmids. Restriction mapping and subcloning indicated that the desulfurization genes reside on a 4.0-kb DNA fragment. Finally, the phenotype was transferred to Rhodococcus fascians D188-5, a species normally incapable of desulfurizing DBT. The mutant strain, UV1, and R. fascians produced 2-hydroxybiphenyl from DBT when they contained appropriate clones, indicating that the genes for the entire pathway have been isolated.