Submitted to: American Society for Microbiology Annual Meeting
Publication Type: Abstract Only
Publication Acceptance Date: 5/14/2004
Publication Date: 5/14/2004
Citation: Paustian, M., Kapur, V., Bannantine, J.P. 2004. Whole-genome Transcriptional Response of Mycobacterium avium subspecies paratuberculosis to Mycbactin J [abstract]. American Society of Microbiologists 104th General Meeting, May 23-27, 2004, New Orleans, LA. 2004 CDROM.
Technical Abstract: Mycobacterium avium subspecies paratuberculosis (M. paratuberculosis), the causative agent of Johne's disease in cattle, is commonly distinguished from other Mycobacterium avium complex bacteria by its dependence on the siderophore mycobactin J for growth in vitro. Interestingly, the recently completed genome sequence of M. paratuberculosis K10 (L.-L. Li, J. P. Bannantine, Q. Zhang, D. Alt, A. Amonsin, and V. Kapur, unpublished data) revealed that nearly all of the genes required for mycobactin synthesis are present in the genome. Iron is an essential nutrient required by most bacteria and has been implicated as an environmental signal of in vivo conditions due to its unavailability in healthy hosts. We have constructed a whole-genome DNA microarray representing more than 98% of the open reading frames (ORFs) identified from the genomic sequence of M. paratuberculosis. PCR products ' 500 bp representing individual ORFs were arrayed in triplicate onto poly-L-lysine coated glass slides. M. paratuberculosis was grown to log phase, washed with mycobactin-free media, split into two equal volumes, and resuspended in Middlebrook 7H9 media supplemented with or lacking mycobactin J. RNA from both cell populations was extracted at six time points, reverse transcribed, and labeled with a fluorescent dye. The samples were combined and the arrays were hybridized overnight at 65oC, washed, and scanned. Spots in which 80% of the pixels were within two standard deviations of the background were discarded along with any ORFs that were not represented by at least two spots of acceptable quality. Initial analyses revealed that 641 ORFs were well-measured in at least five of the six time points examined. Genes expressed at elevated levels in the absence of mycobactin J encoded stress response proteins as well as transcriptional regulators and cell wall biosynthesis proteins, while ORFs expressed at reduced levels included transport and metabolic proteins. Our study should help elucidate how M. paratuberculosis responds to the absence of mycobactin J and what role this response might play in the regulation of virulence factors.