1887

Abstract

Full exploitation of the information available in bacterial genome sequences requires the availability of facile tools for rapid genetic manipulation. One bacterium for which new genetic tools are needed is the methylotroph AM1. IncQ and small IncP vectors were shown to be unsuitable for use in this bacterium, but a spontaneous mutant of a small IncP plasmid was isolated that functioned efficiently in AM1. This plasmid was sequenced and used as a base for developing improved broad-host-range cloning vectors. These vectors were found to replicate in a wide variety of bacterial species and have the following advantages: (1) high copy number in ; (2) small size (72 and 80 kb); (3) complete sequences; (4) variety of unique restriction sites; (5) blue–white screening via α; (6) conjugative mobilization between bacterial species; and (7) readily adaptable into species-specific promoter-probe and expression vectors. Two low-background promoter-probe vectors were constructed based on these cloning vectors with either or as reporter genes; these were shown to report gene expression effectively in AM1. Specific expression vectors were developed for use in AM1, which were shown to express foreign genes at significant levels, and a simple strategy is outlined to develop specific expression vectors for other bacteria. The strong promoter was used for expression, since -derived promoters were expressed at very low levels. This suite of genetic tools will enable a more sophisticated analysis of the physiology of AM1, and these vectors should also be valuable tools in the study of a variety of bacterial species.

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2001-08-01
2020-01-18
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