We have initiated comparative genomic analysis of Mycobacterium avium subspecies by DNA microarray, uncovering 14 large sequence polymorphisms [LSPs] comprising over 700 kb that distinguishM . avium subsp . avium from M . avium subsp . paratuberculosis. Genes predicted to encode metabolic pathways were overrepresented in the LSPs, and analysis revealed a polymorphism within the mycobactin biosynthesis operon that potentially explains thein vitro mycobactin dependence of M . avium subsp . paratuberculosis.

 
The Mycobacterium avium complex [MAC] comprises a group of closely related organisms responsible for a broad range of diseasesin humans and livestock . M . avium subsp . avium causes cervical lymphadenitis in children and disseminated disease in AIDS patients, while M . avium subsp . paratuberculosis causes an inflammatorybowel disease in ruminants and possibly humans [2, 6] . As MACorganisms are highly prevalent in the environment [12], theirgenomic complement is predicted to also reflect this lifestyle.

Recent work in mycobacterial genomics has revealed that genomic reduction through the loss of large sequence polymorphisms [LSPs]is a major contributor to genetic diversity . Studies of the Mycobacterium tuberculosis complex have used LSPs for inferences of phylogenetics [5, 10] and biological properties such as virulence[9, 13] . Since previous DNA hybridization and sequencing studieshave shown that M . avium subspecies are indistinguishable atthe species level [14] and that they share about 98% sequence identity in coding regions [1], we hypothesized that LSPs wouldbe important sources of genetic variability among MAC organisms.

We have annotated the sequence of M . avium subsp . avium strain 104 [provided by the Institute for Genomic Research [http://www.tigr.org]] in order to assemble a whole-genome DNA microarray representing the predicted coding sequences [details on the annotation are provided at www.molepi.mcgill.ca/MAC.htm] . Seventy-base-pair-long oligonucleotide probes were designed and synthesized [MetaBionGmbH, Martinsried, Germany] for 4,158 of 4,480 predicted openreading frames [ORFs] . Each probe was printed in duplicate ontomicroarray slides [SigmascreenTM; Sigma] by using a microarrayrobot [Virtek Chipwriter model SDDC2] to permit genomic DNAcomparisons of M . avium subsp . avium strain 104 and the followingstrains: [i] M . avium subsp . paratuberculosis K10 [cow strain], [ii] M . avium subsp . paratuberculosis LN20 [sheep strain], and [iii] M . avium subsp . silvaticum 49884 [ATCC strain] . Cohybridizationexperiments were performed by using previously published methodsto screen for regions of six or more contiguous M . avium subsp.avium 104 ORFs absent from the test isolate [3]; these regionswere then confirmed by PCR and sequencing [10] . In a secondstep, primers used to confirm the presence or absence of a regionwere used to test a panel of 43 isolates in order to determinethe distribution of these LSPs across other samples.

Microarray comparisons revealed 14 LSPs [LSP1 to LSP14] rangingin length from 21 to 197 kb [Table 1] and encompassing 572 genes [see Table SA in the supplemental material] . Combined, these LSPs comprise 727 kb and represent 13.5% of the M . avium subsp. avium 104 genome . This remarkable diversity far exceeds the genomic variability described among M . tuberculosis complex isolates, estimated to be 1.7% of the genome [9, 11] . Moreover,the MAC diversity documented here must be considered a minimumestimate, as only very large LSPs uncovered from comparisonsof just four clinical isolates were studied . Through the studyof isolates from broader sampling frames and diverse environments,one would expect even greater genomic variability to be revealed.

 
The exact sizes and locations of the LSPs, the subspecies fromwhich they are missing, and the key features of each LSP areshown in Table 1 . Seven of the LSPs revealed are simple genomic deletions or insertions compared to the reference strain M.avium subsp . avium 104 . The other seven LSPs involve a morecomplex combination of insertion and deletion events . This complexity indicates that the genome of MAC organisms is the product ofboth vertical inheritance, as seen in the M . tuberculosis complex, and horizontal acquisition of DNA . Although plasmids have been described for M . avium isolates, the reference strain M . avium subsp . avium 104 does not contain a plasmid, indicating that the genomic variability described here involves chromosomal DNA.

In terms of predicted gene function based on homology searches, genes encoding proteins involved in information pathways andproteins of the PE/PPE family were highly conserved among testedstrains [0.7 and 0.6% of missing genes, respectively] . Considerablediversity within the latter group has been observed in M . tuberculosis, where PE/PPE elements are proposed to be an important sourceof antigenic variation [4] . The surprising lack of diversity in M . avium subspecies was further confirmed by in silico comparisonsof M . avium subsp . avium 104 to the recently sequenced M . aviumsubsp . paratuberculosis K10 [GenBank accession number NC_002944].At the other extreme, genes of unknown function and those predictedto encode proteins involved in lipid metabolism and intermediarymetabolism were overrepresented in the LSPs [19.3, 18, and 20.1%of missing genes, respectively] . The absence of these genes in the more pathogenic M . avium subsp . paratuberculosis suggestsa greater role for these genes in survival in the environmentthan in the intracellular milieu . Another highly variable groupcomprised genes designated mammalian cell entry [mce] genes,a group of genes thought to be involved in host cell invasion and hence virulence . M . avium subsp . avium contains 66 such genes distributed in nine operonic clusters . Of these, 21 [32%] were polymorphic among tested strains . Specifically, one ofthe two homologs of the mce3 operon of M . avium subsp . avium 104 was missing from M . avium subsp . paratuberculosis and M.avium subsp . silvaticum, and four of the six genes belongingto the single mce2 operon were lost in at least one M . aviumsubsp . paratuberculosis strain [LN20] . The loss of mce2 andmce3 genes in the more pathogenic M . avium subsp . paratuberculosisisolates along with the deletion of mce3 from virulent Mycobacteriumbovis [8] together challenge the assignment of these mce operonsto the category of virulence elements . In contrast, the mce1operon, which in M . tuberculosis has been associated with amore virulent phenotype [15], was conserved in M . avium subsp.paratuberculosis and M . avium subsp . silvaticum.

Orthologs of the mycobactin synthesis operon [mbtABCDEFGHIJ] of M . tuberculosis were found in M . avium subsp . avium 104.In M . avium subsp . avium 104, mbtJ is separated from mbtA bya large sequence of 197 kb, corresponding to LSP4 . In M . aviumsubsp . paratuberculosis K10, LSP4 has been replaced by a 19-kbinsert which truncates the 1,724-bp mbtA gene at position 1081.As MbtA is responsible for an early event in mycobactin synthesis[7], disruption of mbtA would predictably impair mycobactin synthesis at its inception and potentially explains the strict dependence of M . avium subsp . paratuberculosis on this siderophore for in vitro growth.

In conclusion, our results reveal remarkable genomic diversity within the MAC . Further characterization of the LSPs and their distribution across more isolates may suggest reasons for thehost species specificities and pathogenic potentials of theM . avium subspecies and provide further insight into their complex evolutionary history.

 
This work was supported by a grant from the Natural Scienceand Engineering Research Council [grant number GEN2282399].M.S . is a recipient of the CIDS/CIHR/Bayer Healthcare fellowshipaward and is currently funded by the Fonds de la Recherche enSanté du Québec [FRSQ] . M.A.B . is a New Investigatorof the Canadian Institutes of Health Research . None of the authorshave a conflict of interest or any commercial association thatmay pose a conflict of interest.

We acknowledge M . Bernstein, M . Kirtsman, M . Katz-Lavigne, D. Shersher, and D . Livingston-Rosanoff for their contributionsto this project and thank L . Mutharia, J . Bannantine, B . Brooks,C . Inderlied, H . Huchzermeyer, G . de Lisle, and F . Saxegaardfor supplying isolates.

 
* Corresponding author . Mailing address: Division of Infectious Diseases and Medical Microbiology, A5-156, Montreal General Hospital, 1650 Cedar Ave., Montreal, Quebec H3G 1A4, Canada . Phone: [514] 934-1934, ext . 42815 . Fax: [514] 934-8423 . E-mail: marcel.behr@mcgill.ca .

Supplemental material for this article may be found at http://jb.asm.org/.

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