Development of improved versatile broad-host-range vectors for use in methylotrophs and other Gram-negative bacteria

Full exploitation of the information available in bacterial genome sequence s requires the availability of facile tools for rapid genetic manipulation. One bacterium for which new genetic tools are needed is the methylotroph M ethylobacterium extorquens AM1. IncQ and small IncP vectors were shown to b e unsuitable for use in this bacterium, but a spontaneous mutant of a small IncP plasmid was isolated that functioned efficiently in M. extorquens AMI . This plasmid was sequenced and used as a base for developing improved bro ad-host-range cloning vectors. These vectors were found to replicate in a w ide variety of bacterial species and have the following advantages: (1) hig h copy number in Escherichia coli; (2) small size (7.2 and 8.0 kb); (3) com plete sequences; (4) variety of unique restriction sites; (5) blue-white sc reening via lacZ alpha; (6) conjugative mobilization between bacterial spec ies; and (7) readily adaptable into species-specific promoter-probe and exp ression vectors. Two low-background promoter-probe vectors were constructed based on these cloning vectors with either lacZ or xylE as reporter genes; these were shown to report gene expression effectively in M. extorquens AM 1. Specific expression vectors were developed for use in M. extorquens AM1, which were shown to express foreign genes at significant levels, and a sim ple strategy is outlined to develop specific expression vectors for other b acteria. The strong mxaF promoter was used for expression, since E. coli la c-derived promoters were expressed at very low levels. This suite of geneti c tools will enable a more sophisticated analysis of the physiology of M. e xtorquens AM1, and these vectors should also be valuable tools in the study of a variety of bacterial species.