Analysis of the Bacillus subtilis genome: cloning and nucleotide sequence of a 62 kb region between 275° (rrnB) and 284° (pai)

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Abstract
Summary: In the framework of the international project aimed at the sequencing of the Bacillus subtilis genome, five DNA fragments in the region between rrnB (275° and pai (284°) were cloned by inverse and combinatorial long-range PCR and their nucleotide sequences were determined and analysed. Together these sequences constituted a contig of 62229 bp. On the basis of the position of NotI and SfiI restriction sites, the orientation and order of known genetic markers was determined to be pai (284°) - degQ comQ comP comAA comAB - pbpD - kapB kinB patB - mcpB tlpA mcpA tlpB - rrnB (275°). Fifty-four ORFs were detected. Thirteen of these coincided with known B. subtilis genes, and 41 new ORFs were found. Of the predicted new gene products, 12 showed no significant similarity to other known proteins, whereas ten showed strong similarity to proteins of other organisms with unknown function. Nineteen predicted proteins showed strong similarity to known proteins of other organisms, for instance a Na+/H+ antiporter system of Bacillus alcalophilus, a sugar transport system found in Mycoplasma genitalium, NADH-dependent butanol dehydrogenase of Clostridium acetobutylicum, glucose-6-phosphate isomerase A of B. subtilis, exo-1,4-α-glucosidase activity of Bacillus stearothermophilus and L-rhamnose isomerase of Escherichia coli.