Effect of IS900 Gene of Mycobacterium paratuberculosis on Mycobacterium smegmatis Saleh A. Naser, 1 Robert F. Gillespie, 1 Najih A. Naser, 2 Fouad A.K. El-Zaatari 3 1 Department of Molecular Biology and Microbiology, Center for Diagnostics and Drug Development, University of Central Florida, Orlando, FL 32816, USA 2 NovaSence, Orlando, FL, USA 3 Inflammatory Bowel Disease Laboratory, VeteransAffairs Medical Center and Department of Medicine, Baylor College of Medicine, Houston, TX 77030, USA Received: 12 May 1998 / Accepted: 6 July 1998 Abstract. Mycobacterium paratuberculosis is mycobactin dependent and contains multiple copies of the IS900 gene that encodes for p43 (46.5K protein). The correlation between the two characteristics has been investigated. A 3.2-kb BamHI fragment from M. paratuberculosis containing the 1.451 kb IS900 gene was cloned in Escherichia coli and Mycobacterium smegmatis with pcDNA II and pNEZ6.3 plasmids, respectively. Surprisingly, the recombinant M. smegmatis grew poorly and slower in 7H9 broth supple- mented with OADC (12 day) compared with M. smegmatis wild type or to M. smegmatis transformed with pNEZ6.3 (2 day). The growth rate of the recombinant M. smegmatis was restored by the addition of 2.4 μM ferric mycobactin J to the media. There was no effect on the growth rate of E. coli recombinants. Western blot analysis with p43-specific anti-peptide antibodies resulted in the expression of 46.5K and a cleaved form of 33.5K protein bands in the recombinant E. coli. There was no expression in the recombinant M. smegmatis. A lower expression of 33.5K protein band was detected in the native M. paratuberculosis protein. The nucleotide sequence of the 3.2-kb fragment confirmed the presence of p43-encoded ORF. There was no additional encoding sequence in the fragment. This suggests that the IS900 gene and/or its encoding products are involved in mycobactin dependency and possibly the slow growth rate of M. paratuberculosis. Mycobacterium paratuberculosis is the causative agent of paratuberculosis (Johne’s disease) in ruminants, result- ing in the formation of chronic granulomatous intestinal lesions within the host [3]. M. paratuberculosis has also been isolated from intestinal tissues of Crohn’s disease patients, a similar chronic intestinal disease in humans [4, 19]. The ability of a Mycobacterium species to cause disease in a host is, in part, due to the availability of iron [11]. Hosts restrict the amount of free iron available to microorganisms by producing high-affinity, iron-binding compounds such as transferrin and lactoferrin [10]. Most mycobacteria overcome this by producing two types of high-affinity, iron-chelating compounds termed sidero- phores [13, 17]: exochelin, which is secreted extracellu- larly [1], and mycobactin, which is cell wall associated [21]. Siderophores are low-molecular-weight compounds [5] possessing affinities for iron greater than those of the host proteins [2]. M. paratuberculosis is unique among mycobacteria in that it requires exogenous mycobactin for in vitro growth to occur [15]. The need for exogenous mycabactin by M. paratuberculosis can be relieved by the addition of high concentrations of ferric ammonium citrate [13]. However, it remains unclear how and by what mechanism the mycobactin-dependent mycobacte- ria are able to acquire iron in vivo [14]. The discovery of the insertion sequence (IS) IS900 in M. paratuberculosis has invited speculation that M. paratuberculosis arose from a strain of M. avium that acquired IS900 [9]. Owing to the insertion site specificity of this IS, 15–20 copies of IS900 are present in the genome of M. paratuberculosis. Unlike most classical insertion elements, IS900 lacks inverted terminal repeats Correspondence to: S.A. Naser CURRENT MICROBIOLOGY Vol. 37 (1998), pp. 373–379 An International Journal  Springer-Verlag New York Inc. 1998