Stable cosmid vectors that enable the introduction of cloned fragments into a wide range of gram-negative bacteria

Abstract

A cosmid cloning system has been developed which is useful for the construction of genomic libraries and the introduction of clones into a broad range of bacterial species. The cosmids pMMB33 and pMMB34 allow selective cloning into their unique BamHI site of 36-kb DNA fragments generated by BamHI, Sau3A and MboI partial digestion. This selective cloning is achieved by a strategy that avoids formation of polycosmids without a dephosphorylation step. It uses two unique recognition sites within the vectors for endoncleases that generate blunt-ended DNA fragments for the preparation of left and right cosmid "arms". An alternative method that uses the unique EcoRI and SstI sites and dephosphorylation of the cosmid arms prior to BamHI digestion is also outlined and discussed. The DNA is first cloned with either vector into a rec- E. coli strain, where clones can be maintained stably, and can then be introduced by mobilization into a wide range of Gram-negative species to permit the study of gene expression and complementation. Because mobilization is much more efficient than transformation, the vector has the advantage that it can be transferred between bacterial species that specify different restriction systems, where transformation appears to be inefficient. The vectors have been used to generate gene libraries from the chromosomal DNA of several Pseudomonas and a Thiobacillus species. The genes specifying myo-inositol transport from Pseudomonas strain JD34 have been cloned with this system.