DOE Human Genome Program Contractor-Grantee Workshop VIII
February 27-March 2, 2000  Santa Fe, NM

116. The Pseudomonas putida KT2440 Genome Sequencing Project

Karen E. Nelson, Hoda Khouri, Erik Holtzapple, Jeff Buchoff, Michael Rizzo, Azita Moazzez, Kelly Moffat, Kevin Tran, Hean Koo, P. Chris Lee, Daniel Kosack, Bradley Slaven, Helmut Hilbert, Burkhard Tuemmler, and C.M. Fraser

The Institute for Genomic Research, Rockville, MD 20850

kenelson@tigr.org

Pseudomonas putida is a ubiquitous soil bacterium that has significant potential for bioremediation of numerous compounds. To determine the complete genome sequence of strain KT2440, a genome sequencing project was initiated in January 1999 as a collaboration between The Institute for Genomic Research in Rockville MD, and a German Consortium (see http://www.tigr.org/tdb/mdb/mdb.html), with primary funding from the Department of Energy. The 6.1 Mbp genome is being sequenced by the random shotgun method, and multiple sized large insert libraries (10 kb, 40 kb) are acting as a scaffold for the genome. At the end of random sequencing, there were a total of 392 sequencing gaps, many of which have been closed by editing off the ends of assemblies, or by sequencing clones that span the respective gaps. The high GC content of the genome is highlighted in long stretches of G's and C's encountered in both sequencing and physical gaps, and through which we have had problems sequencing by traditional methods. Sequencing of short reads that point into gaps, dye primer chemistry, transposon mutagenesis on selected spanning clones, as well as ET chemistry were used for most of the difficult areas. Multiplex PCR and micro-library construction, have also assisted in resolving and ordering the RNA operons. Grouper and autoprimer (TIGR softwares) were improved or modified to deal with the size of the genome. Ultimately, the P. putida genome sequence will identify the real potential of this organism in various biotechnological areas including the production of natural compounds, and remediation of polluted habitats.