Agricultural Microbes Genome 2 Conference

Poster: Applied Functional Genomics
P30

RAPID DISCOVERY OF OVER THREE THOUSAND GENES IN THE MAIZE ENDOPHYTE, KLEBSIELLA PNEUMONIAE 342, BY MICROARRAY HYBRIDIZATION WITH E. COLI K12 OPEN READING FRAMES

¹ Department of Agronomy, 1575 Linden Drive, University of Wisconsin-Madison, Madison, WI 53706 USA

² Department of Genetics, Wisconsin Gene Expression Center, 445 Henry Mall, University of Wisconsin-Madison, Madison, WI 53706 USA

In an effort to efficiently discover genes in the diazotrophic endophyte of maize, Klebsiella pneumoniae 342, we have hybridized DNA from strain 342 to a microarray containing 96% (4,098) of the annotated open reading frames from E. coli K12. Using a criterion of 55% homology or greater, 3,020 (70%) of the E. coli K12 open reading frames are also present in strain 342. Approximately 24% (1,010) of the E. coli K12 open reading frames are absent in strain 342. For 1.6% (68) of the open reading frames, the signal was too low to make a determination on the presence or absence of the gene. Genes that are highly homologous between the two organisms are those involved in energy metabolism, amino acid metabolism, fatty acid metabolism, cofactor synthesis, cell division, DNA replication, transcription, translation, transport, and regulatory proteins. Functions that were less highly conserved included carbon compound metabolism, membrane proteins, structural proteins, putative transport proteins, cell processes such as adaptation and protection, and central intermediary metabolism. Open reading frames of E. coli K12 with little or no homology in strain 342 included putative regulatory proteins, putative chaperones, surface structure proteins, mobility proteins, putative enzymes, hypothetical proteins, and proteins of unknown function as well as those genes obtained by lateral transfer from sources such as phage, plasmids, or transposons. The results were in agreement with the physiological properties of the two strains. Our microarray results were also compared to a 3x pass of the genome sequence of Kp MGH78578, a clinical isolated. A list of the genes in common between Ec K12 and Kp MGH78578 was obtained from the Washington University Genome Center and compared with our microarray results. Of the 4,290 ORFs in Ec K12, 3.053 (71%) were found in Kp MGH78578 and 1,045 Ec K12 genes were missing in this clinical isolate. Just 183 (4.3%) of the Ec K12 genes missing in Kp MGH78578 are present in Kp 342 and 113 (2.6%) of the Ec K12 genes missing in Kp 342 are present in Kp MGH78578. The functions of these sets of genes are now being characterized. Nitrogen fixation genes are present in Kp 342 but missing in Kp MGH78578 and Ec K12. An economical approach to rapidly characterize a bacterial genome is presented. This approach is useful for those organisms with a fully sequenced close relative. The cost of this approach is less than $8 per gene including labor and will become increasingly useful as more full sequences become available.