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profundum has previously been performed using conjugal narrow host-range suicide plasmids for single-crossover insertional mutagenesis ( 6) or for double-crossover allelic exchange ( 7). Here we report the development of a set of conjugal plasmids for insertional mutagenesis, complementation, and inducible expression in a wide variety of Gram-negative bacteria and the test of their efficiency using the psychrotolerant γ-proteobacterium Photobacterium profundum strain SS9, for which the whole genome sequence has recently been completed ( 5). While high-throughput genetic strategies have been described for most members of the family Enterobacte-riaceae ( 1, 2), Bacillus subtilis ( 3), and the yeast Saccharomyces cerevisiae ( 4), all of which can be easily trans-formed, there is a need to develop similar approaches for those hosts that cannot be transformed. As a result, there is a need for greater efficiency when applying these procedures to microbial systems. However, detailed gene characterization still requires in vivo genetic manipulation. The ever increasing pace of genome sequence acquisition, annotation, and transcriptional profiling is providing large data sets of possible open reading frame function.