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Identification of Open Reading Frames Unique to a Select Agent: Ralstonia solanacearum Race 3 Biovar 2

¹Plant Pathology Department, University of Florida, Gainesville, U.S.A.; ²Department of Plant Pathology, University of Wisconsin, Madison, U.S.A.; ³Department of Microbiology, University of Georgia, Athens, U.S.A.; ⁴Department of Plant Pathology, University of Georgia, Athens, U.S.A.; ⁵Department of Molecular Genetics and Cell Biology, University of Chicago, Chicago; ⁶United States National Arboretum, United States Department of Agriculture-Agricultural Research Service, Beltsville, MD, U.S.A.; ⁷Department of Molecular Bioscience and Bioengineering, University of Hawaii, Honolulu, U.S.A.; ⁸Plant Pathology Department, University of Florida, Apopka, U.S.A.; ⁹Plant Environment and Bacteriology, Central Science Laboratory, Sand Hutton, York, U.K.; ¹⁰Scottish Agriculture and Science Agency, East Craigs, Edinburgh, U.K.; ¹¹ICBR, University of Florida, Gainesville, U.S.A.; ¹²Department of Plant Environmental Science, University of Hawaii, Honolulu, U.S.A.; ¹³Integrated Genomics, Chicago, IL, U.S.A.

An 8× draft genome was obtained and annotated for Ralstonia solanacearum race 3 biovar 2 (R3B2) strain UW551, a United States Department of Agriculture Select Agent isolated from geranium. The draft UW551 genome consisted of 80,169 reads resulting in 582 contigs containing 5,925,491 base pairs, with an average 64.5% GC content. Annotation revealed a predicted 4,454 protein coding open reading frames (ORFs), 43 tRNAs, and 5 rRNAs; 2,793 (or 62%) of the ORFs had a functional assignment. The UW551 genome was compared with the published genome of R. solanacearum race 1 biovar 3 tropical tomato strain GMI1000. The two phylogenetically distinct strains were at least 71% syntenic in gene organization. Most genes encoding known pathogenicity determinants, including predicted type III secreted effectors, appeared to be common to both strains. A total of 402 unique UW551 ORFs were identified, none of which had a best hit or >45% amino acid sequence identity with any R. solanacearum predicted protein; 16 had strong (E < 10^-13) best hits to ORFs found in other bacterial plant pathogens. Many of the 402 unique genes were clustered, including 5 found in the hrp region and 38 contiguous, potential prophage genes. Conservation of some UW551 unique genes among R3B2 strains was examined by polymerase chain reaction among a group of 58 strains from different races and biovars, resulting in the identification of genes that may be potentially useful for diagnostic detection and identification of R3B2 strains. One 22-kb region that appears to be present in GMI1000 as a result of horizontal gene transfer is absent from UW551 and encodes enzymes that likely are essential for utilization of the three sugar alcohols that distinguish biovars 3 and 4 from biovars 1 and 2.

Additional keywords: bacterial wilt, comparative genomics.