University of Nebraska - Lincoln DigitalCommons@University of Nebraska - Lincoln U.S. Department of Agriculture: Agricultural Publications from USDA-ARS / UNL Faculty Research Service, Lincoln, Nebraska 2003 Genomic homogeneity between Mycobacterium avium subsp. avium and Mycobacterium avium subsp. paratuberculosis belies their divergent growth rates John P. Bannantine ARS-USDA, [email protected] Qing Zhang 2University of Minnesota, Minneapolis, [email protected] Ling-Ling Li University of Minnesota, Minneapolis, [email protected] Vivek Kapur University of Minnesota, Minneapolis, [email protected] Follow this and additional works at: https://digitalcommons.unl.edu/usdaarsfacpub Bannantine, John P.; Zhang, Qing; Li, Ling-Ling; and Kapur, Vivek, "Genomic homogeneity between Mycobacterium avium subsp. avium and Mycobacterium avium subsp. paratuberculosis belies their divergent growth rates" (2003). Publications from USDA-ARS / UNL Faculty. 2372. https://digitalcommons.unl.edu/usdaarsfacpub/2372 This Article is brought to you for free and open access by the U.S. Department of Agriculture: Agricultural Research Service, Lincoln, Nebraska at DigitalCommons@University of Nebraska - Lincoln. It has been accepted for inclusion in Publications from USDA-ARS / UNL Faculty by an authorized administrator of DigitalCommons@University of Nebraska - Lincoln. BMC Microbiology BioMed Central Research article Open Access Genomic homogeneity between Mycobacterium avium subsp. avium and Mycobacterium avium subsp. paratuberculosis belies their divergent growth rates John P Bannantine*1, Qing Zhang2, Ling-Ling Li2 and Vivek Kapur2 Address: 1National Animal Disease Center, USDA-ARS, 2300 N. Dayton Ave., Ames, IA 50010, USA and 2Biomedical Genomics Center and Departments of Microbiology and Veterinary Pathobiology, University of Minnesota, Minneapolis, MN, USA Email: John P Bannantine* - [email protected]; Qing Zhang - [email protected]; Ling-Ling Li - [email protected]; Vivek Kapur - [email protected] * Corresponding author Published: 9 May 2003 Received: 5 December 2002 Accepted: 9 May 2003 BMC Microbiology 2003, 3:10 This article is available from: http://www.biomedcentral.com/1471-2180/3/10 © 2003 Bannantine et al; licensee BioMed Central Ltd. This is an Open Access article: verbatim copying and redistribution of this article are permitted in all media for any purpose, provided this notice is preserved along with the article's original URL. Abstract Background: Mycobacterium avium subspecies avium (M. avium) is frequently encountered in the environment, but also causes infections in animals and immunocompromised patients. In contrast, Mycobacterium avium subspecies paratuberculosis (M. paratuberculosis) is a slow-growing organism that is the causative agent of Johne's disease in cattle and chronic granulomatous infections in a variety of other ruminant hosts. Yet we show that despite their divergent phenotypes and the diseases they present, the genomes of M. avium and M. paratuberculosis share greater than 97% nucleotide identity over large (25 kb) genomic regions analyzed in this study. Results: To characterize genome similarity between these two subspecies as well as attempt to understand their different growth rates, we designed oligonucleotide primers from M. avium sequence to amplify 15 minimally overlapping fragments of M. paratuberculosis genomic DNA encompassing the chromosomal origin of replication. These strategies resulted in the successful amplification and sequencing of a contiguous 11-kb fragment containing the putative Mycobacterium paratuberculosis origin of replication (oriC). This fragment contained 11 predicted open reading frames that showed a conserved gene order in the oriC locus when compared with several other Gram-positive bacteria. In addition, a GC skew analysis identified the origin of chromosomal replication which lies between the genes dnaA and dnaN. The presence of multiple DnaA boxes and the ATP-binding site in dnaA were also found in M. paratuberculosis. The strong nucleotide identity of M. avium and M. paratuberculosis in the region surrounding the origin of chromosomal replication led us to compare other areas of these genomes. A DNA homology matrix of 2 million nucleotides from each genome revealed strong synteny with only a few sequences present in one genome but absent in the other. Finally, the 16s rRNA gene from these two subspecies is 100% identical. Conclusions: We present for the first time, a description of the oriC region in M. paratuberculosis. In addition, genomic comparisons between these two mycobacterial subspecies suggest that differences in the oriC region may not be significant enough to account for the diverse bacterial replication rates. Finally, the few genetic differences present outside the origin of chromosomal replication in each genome may be responsible for the diverse growth rates or phenotypes observed between the avium and paratuberculosis subspecies. Page 1 of 10 (page number not for citation purposes) BMC Microbiology 2003, 3 http://www.biomedcentral.com/1471-2180/3/10 Background initiator protein (DnaA), the beta subunit of DNA Mycobacteria are Gram-positive, acid-fast, pleomorphic, polymerase III (DnaN), the recombination repair protein non-motile rods belonging to the order Actinomycetales. RecF, and the DNA gyrase proteins GyrA and GyrB. The Mycobacterium avium complex organisms consist of the relative gene order in this region is also highly conserved human and animal pathogens M. avium subsp. avium, M. in many bacteria, especially the Gram-positives [11]. Al- avium subsp. paratuberculosis, and M. avium subsp. silvati- though intergenic sequences in this region are conserved cum [1]. DNA-DNA hybridization studies have long ago only among closely related organisms, the DnaA box is established a genetic similarity between M. avium subspe- found in the non-coding regions flanking dnaA in most cies avium (M. avium) and M. avium subspecies paratuber- bacteria studied [12]. DnaA boxes are conserved nucle- culosis (M. paratuberculosis) [2–4]. Now that whole otide sequences (TTGTCCACA) where the DnaA protein genome sequencing technologies are available, investiga- binds to DNA, triggering events that ultimately lead to tors can begin to examine genetic relatedness in greater replication initiation and DNA synthesis [9]. detail through direct nucleotide-nucleotide comparisons. These comparisons are particularly important in instances In an effort to understand the genetic basis for growth rate where two genetically similar bacteria have little or no and other phenotypic differences between M. paratubercu- specific diagnostic tests to distinguish each. losis and M. avium, we have analyzed the genetic similarity of these genomes using two strategies. First, the putative The literature reports genetic similarity between M. paratu- oriC region of M. paratuberculosis was amplified, se- berculosis and M. avium at between 72% and 95% [2,4] de- quenced and compared with M. avium and other bacteria. pending on the region analyzed. However, despite the Second, we examined nucleotide identity outside the oriC reported similarities, these mycobacteria are quite differ- region using DNA homology matrix analysis as well as us- ent phenotypically. M. paratuberculosis is an intracellular ing several hundred M. paratuberculosis sequences from a pathogen that infects ruminant animals, most notably cat- random shotgun library compared with M. avium se- tle and sheep. The site of infection is the gastrointestinal quences present in the unfinished microbial genomes da- tract, where it causes a chronic inflammatory ailment tabase. Our results show that these subspecies not only termed Johne's disease [5]. In contrast, M. avium is com- have a conserved gene order surrounding the origin of mon in the environment, causes tuberculosis in birds, and chromosomal replication, but also have a high synteny disseminated infections in HIV patients [6]. Growth of M. and nucleotide identity throughout both genomes. In ad- paratuberculosis is characterized by its slow rate (doubling dition, this preliminary comparative survey of the genom- time of 22–26 hours, compared to 10–12 hours for M. avi- es of M. avium and M. paratuberculosis show even greater um) and requirement of mycobactin in culture media [5]. similarity (97%) than the literature suggests (72% to With the absence of a well-defined genetic system for M. 95%) [2]. paratuberculosis, a comparative genomic approach holds great potential in addressing the genetic basis for many of Results these phenotypic differences. Identification of predicted ORFs encoding replication-re- lated proteins The genus Mycobacterium contains species that range from An ~11-kb contiguous genomic fragment from M. paratu- fast-growingsaprophytes such as M. smegmatis and M. for- berculosis was amplified and sequenced using 15 primer tuitum to slow-growing pathogens such asM. leprae, M. tu- pairs designed from M. avium genomic sequence in the berculosis and M. paratuberculosis. Although the putative oriC region (Fig. 1). This strategy enabled the suc- chromosomal origin of replication has been studied in cessful amplification of all 15 minimally overlapping frag- some mycobacteria [7,8], the genetic organization of the ments of ~800 bp in length for this region of the M. origin of replication in M. paratuberculosis has been previ- paratuberculosis chromosome. A putative replication ori- ously unknown. Knowledge of the gene organization and gin