PAG-X  Plant, Animal & Microbe Genomes X Conference

January 12-16, 2002
Town & Country Convention Center
San Diego, CA

Poster: Functional Genomics for Microorganisms
            

A GENOMICS APPROACH TO IDENTIFY CODING SEQUENCES UNIQUE TO MYCOBACTERIUM AVIUM SUBSPECIES PARATUBERCULOSIS

John P Bannantine1 , Emily Baechler2 , Qing Zhang3 , Lingling Li3 , Judith R Stabel1 , Vivek Kapur3

1 National Animal Disease Center, USDA-ARS, Ames, IA, USA
2 Department of Medicine, University of Minnesota, St. Paul, MN, USA
3 Biomedical Genomics Center, University of Minnesota, St. Paul, MN, USA

The genetic similarity between Mycobacterium avium subspecies paratuberculosis (M. paratuberculosis) and other mycobacterial species has confounded the development of M. paratuberculosis-specific diagnostic reagents. Random shotgun sequencing of the M. paratuberculosis genome in our laboratories has shown greater than 98% sequence identity with M. avium subsp. avium (M. avium) in some regions. However, an in silico comparison of the largest annotated M. paratuberculosis contigs, totaling 2,658,271 bp with the unfinished M. avium genome has revealed 27 predicted M. paratuberculosis coding sequences that do not align with M. avium sequences. BLASTP analysis of the 27 predicted coding sequences (genes) shows 23 do not match anything in public sequence databases such as Genbank. One region of the M. paratuberculosis genome, designated #481, is 9.4 kilobases in size and contains a cluster of eight genes that are absent in M. avium. All 23 genes were examined by PCR amplification with eight mycobacterial species and eight independent isolates of M. paratuberculosis. From these analyses, 19 of 23 genes were found to be present in all of the M. paratuberculosis isolates and absent from all other mycobacterial species tested. Selected M. paratuberculosis-specific genes were further characterized by cloning into an E. coli expression vector. Expressed recombinant M. paratuberculosis proteins were affinity purified and used in immunoblotting studies with sera from rabbits immunized with whole cell preparations of M. paratuberculosis. These studies show that a few of the gene products are produced in M. paratuberculosis, thus confirming the coding predictions in these novel sequences. Collectively, these studies have used a genomic approach to identify novel M. paratuberculosis antigens that are not present in any other mycobacteria.

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