Mobile group II introns are site-specific retroelements that use a novel mobility mechanism in which the excised intron RNA inserts directly into a DNA target site and is then reverse transcribed by the associated intron-encoded protein. Because the DNA target site is recognized primarily by base-pairing of the intron RNA with only a small number of positions recognized by the protein, it has been possible to develop group II introns into a new type of gene targeting vector ("targetron"), which can be reprogrammed to insert into desired DNA targets simply by modifying the intron RNA. Here, we used databases of retargeted Lactococcus lactis Ll.LtrB group II introns and a compilation of nucleotide frequencies at active target sites to develop an algorithm that predicts optimal Ll.LtrB intron-insertion sites and designs primers for modifying the intron to insert into those sites. In a test of the algorithm, we designed one or two targetrons to disrupt each of 28 Escherichia coli genes encoding DExH/D-box and DNA helicase-related proteins and tested for the desired disruptants by PCR screening of 100 colonies. In 21 cases, we obtained disruptions at frequencies of 1-80% without selection, and in six other cases, where disruptants were not identified in the initial PCR screen, we readily obtained specific disruptions by using the same targetrons with a retrotransposition-activated selectable marker. Only one DExH/D-box protein gene, secA, which was known to be essential, did not give viable disruptants. The apparent dispensability of DExH/D-box proteins in E.coli contrasts with the situation in yeast, where the majority of such proteins are essential. The methods developed here should permit the rapid and efficient disruption of any bacterial gene, the computational analysis provides new insight into group II intron target site recognition, and the set of E.coli DExH/D-box protein and DNA helicase disruptants should be useful for analyzing the function of these proteins.