Mycobacterium avium subspecies paratuberculosis(M. ap) causes an enteric infection in cattle, with a great impact on the dairy industry in the United States and worldwide. Characterizing the gene expression profile of M. ap exposed to different stress conditions could improve our understanding of the pathogenesis of M. ap. Recently, we profiled the stress responses of M. ap on a genome-wide level (stressome) using oligonucleotides DNA microarrays. Expression data analysis revealed unique gene groups of M. ap that were regulated under in vitro stressors or in biofilm cultures while additional groups were regulated in fecal samples collected from clinically infected cows. Interestingly, acidic pH induced the regulation of a large number of genes (N=597) suggesting the high sensitivity of M. ap to acidic environments. Generally, responses to heat shock, acidity and oxidative stress were similar in M. ap and M. tuberculosis suggesting common pathways for mycobacterial defense against stressors. Additionally, we analyzed the virulence of 7 M. ap mutants with inactivation of differentially-regulated genes using a murine model of paratuberculosis. Both bacterial and histopathological examinations indicated the attenuation of all gene mutants, especially those selected based on their expression in the cow samples (e.g., lipN). This analysis also indicated the key role played by genes encoding lipases that are induced in clinically infected cows. Overall, the employed approach profiled mycobacterial genes responsive to variable stress conditions including those activated in fecal samples. Also a list of potential virulence genes was characterized. In this communication, we will further analyze the contribution of our findings to the understanding of the molecular pathogenesis of Johne's disease.