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Journal of Bacteriology, April 2005, p. 2406-2415, Vol. 187, No. 7
0021-9193/05/$08.00+0     doi:10.1128/JB.187.7.2406-2415.2005
Copyright © 2005, American Society for Microbiology. All Rights Reserved.

Comparative Genomic Hybridizations Reveal Genetic Regions within the Mycobacterium avium Complex That Are Divergent from Mycobacterium avium subsp. paratuberculosis Isolates{dagger}

Michael L. Paustian,1* Vivek Kapur,2 and John P. Bannantine1

National Animal Disease Center, U.S. Department of Agriculture-Agricultural Research Service, Ames, Iowa,1 Biomedical Genomics Center, University of Minnesota, St. Paul, Minnesota2

Received 5 November 2004/ Accepted 22 December 2004


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ABSTRACT
 
Mycobacterium avium subsp. paratuberculosis is genetically similar to other members of the Mycobacterium avium complex (MAC), some of which are nonpathogenic and widespread in the environment. We have utilized an M. avium subsp. paratuberculosis whole-genome microarray representing over 95% of the predicted coding sequences to examine the genetic conservation among 10 M. avium subsp. paratuberculosis isolates, two isolates each of Mycobacterium avium subsp. silvaticum and Mycobacterium avium subsp. avium, and a single isolate each of both Mycobacterium intracellulare and Mycobacterium smegmatis. Genomic DNA from each isolate was competitively hybridized with DNA from M. avium subsp. paratuberculosis K10, and open reading frames (ORFs) were classified as present, divergent, or intermediate. None of the M. avium subsp. paratuberculosis isolates had ORFs classified as divergent. The two M. avium subsp. avium isolates had 210 and 135 divergent ORFs, while the two M. avium subsp. silvaticum isolates examined had 77 and 103 divergent ORFs. Similarly, 130 divergent ORFs were identified in M. intracellulare. A set of 97 ORFs were classified as divergent or intermediate in all of the nonparatuberculosis MAC isolates tested. Many of these ORFs are clustered together on the genome in regions with relatively low average GC content compared with the entire genome and contain mobile genetic elements. One of these regions of sequence divergence contained genes homologous to a mammalian cell entry (mce) operon. Our results indicate that closely related MAC mycobacteria can be distinguished from M. avium subsp. paratuberculosis by multiple clusters of divergent ORFs.


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INTRODUCTION
 
Mycobacterium avium subsp. paratuberculosis is a gram-positive, acid-fast bacillus that is the causative agent of Johne's disease, a chronic infection primarily of ruminant animals characterized by inflammation of the digestive tract leading to nutrient malabsorption and eventually death. Diagnosis of M. avium subsp. paratuberculosis infections is confounded by the genetic similarity between M. avium subsp. paratuberculosis and nonpathogenic environmental mycobacteria, especially other members of the Mycobacterium avium complex (MAC). MAC bacteria, comprising three M. avium subspecies and Mycobacterium intracellulare, possess a high degree of genetic similarity but are capable of infecting a diverse range of host species. M. avium subsp. paratuberculosis is traditionally distinguished from other mycobacteria by its dependence on exogenous mycobactin, although intermittent mycobactin dependence has been reported among M. avium subsp. paratuberculosis isolates and within other species (6, 17). Previous studies have utilized DNA hybridizations to demonstrate a close genetic relationship between M. avium subsp. paratuberculosis and Mycobacterium avium subsp. avium, although they can normally be separated into two distinct populations (12, 24, 25, 30). Recent work comparing the whole-genome sequences of M. avium subsp. paratuberculosis and M. avium subsp. avium isolates has revealed that they are greater than 97% identical at the nucleotide level within certain segments of genomic DNA (5).

Genetic analyses of conserved M. avium subsp. paratuberculosis sequences have been able to separate clinical isolates into distinct populations that often correlate with the host species from which they were cultured. Motiwala and coworkers examined a large number of M. avium subsp. paratuberculosis isolates and found that those obtained from cattle could generally be separated into a distinct group compared to isolates from other species, including sheep and humans (19). These findings were based primarily on comparisons of the integration loci of the IS900 insertion sequence (IS) and agree with the findings of Whittington and coworkers, who used polymorphisms in IS1311 to separate sheep and cattle isolates into separate populations (29). While the use of conserved ISs has been able to resolve the population structure of M. avium subsp. paratuberculosis isolates, they do not provide information on the conservation of individual protein coding sequences between isolates. A hybridization-based approach will allow us to identify significant variations in the overall sequence of related genetic features but will be insensitive to very small changes such as point mutations.

The objective in our present study was to determine the genetic variability between isolates of M. avium subsp. paratuberculosis, as well as to identify genomic differences between M. avium subsp. paratuberculosis and other MAC mycobacteria. To accomplish this, we have designed and constructed a whole-genome DNA microarray representing more than 95% of the open reading frames (ORFs) identified from the genome sequence of M. avium subsp. paratuberculosis K10 (16).


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MATERIALS AND METHODS
 
Construction of a whole-genome M. avium subsp. paratuberculosis microarray. A library of PCR products representing all coding sequences was constructed with primers designed by Primer3 software (18) to amplify fragments of ≤500 bp from each ORF identified from the genome sequence of M. avium subsp. paratuberculosis K10 (NCBI accession no. AE016958) with the use of purified genomic DNA as a template. PCRs consisted of 1x PCR Buffer II (Perkin-Elmer, Boston, Mass.), 0.2 mM deoxynucleoside triphosphate mix, 3 mM MgCl2, 5% dimethyl sulfoxide, 0.04 U of Taq Gold (Perkin-Elmer)/µl, and 0.6 µM primers. PCR products amplified from genomic DNA were diluted 1:200 in distilled water and used as the template for a subsequent 50-µl PCR in order to minimize genomic DNA carryover. The conditions used for both rounds of PCR were an initial incubation for 7 min at 94°C and then 35 cycles of 94°C for 45 s, 57°C for 60 s, and 72°C for 90 s followed by a final hold for 7 min at 72°C. The PCR products were checked for quality on agarose gels, at which point it was determined that 4,110 (95%) of the total ORFs had been successfully amplified. They were then purified with Montage 96-well filter plates (Millipore, Bedford, Mass.) and resuspended in 3x SSC containing 1.5 M betaine (1x SSC is 150 mM NaCl, 15 mM sodium citrate, pH 7.0). The resulting products were arrayed in triplicate onto homemade poly-L-lysine-coated glass slides with a MicroGrid II Compact robot (Genomic Solutions, Ann Arbor, Mich.) along with control samples that included spotting buffer alone and Arabidopsis thaliana sequences. This resulted in an array containing over 13,000 spots with an average diameter of 170 µm.

Comparative genomic hybridizations. Mycobacteria were cultured in Middlebrook 7H9 broth (pH 6.0) supplemented with oleic acid-albumin-dextrose-catalase (Becton Dickinson Microbiology, Sparks, Md.), and 0.05% Tween 80. Cultures of M. avium subsp. paratuberculosis were further supplemented with ferric mycobactin J (2 mg/liter; Allied Monitor Inc., Fayette, Mo.). Genomic DNA was extracted from M. avium subsp. paratuberculosis K10 and 15 mycobacterial isolates (Table 1) with Genomic-tip 100/G anion-exchange columns (Qiagen, Valencia, Calif.) as previously described (3) with the modification that D-cycloserine was not added as part of the extraction procedure. Purified genomic DNA was then randomly sheared by nebulization on ice at 10 lb/in2 for 1.5 min, resulting in an average fragment size of 800 bp. Aliquots of sheared genomic DNA (4 µg) were labeled by incorporating an aminoallyl-dUTP nucleotide (Sigma, St. Louis, Mo.) into cDNA by using Klenow enzyme (U.S. Biochemicals, Cleveland, Ohio) primed with random hexamers (Amersham Biosciences, Piscataway, N.J.) overnight. The amine-modified cDNA was purified, resuspended in 0.13 M sodium bicarbonate, and labeled by the addition of either Alexa Fluor 555 or Alexa Fluor 647 succinimidyl ester dye (Molecular Probes, Eugene, Oreg.) followed by incubation for 2 h at room temperature. Labeled cDNA from experimental mycobacterial isolates was then purified and mixed with alternately labeled M. avium subsp. paratuberculosis K10 cDNA in a final volume of 45 µl containing 3x SSC, 0.22% sodium dodecyl sulfate (SDS), and 34 µg of salmon sperm DNA (Invitrogen, Carlsbad, Calif.). This hybridization solution was incubated at 100°C for 2 min, applied to the M. avium subsp. paratuberculosis K10 microarray, and allowed to hybridize overnight at 65°C. The arrays were washed sequentially for 3 min at room temperature in 300-ml volumes of 0.5x SSC-0.01% SDS, 0.5x SSC, 0.1x SSC, and 0.01x SSC and then dried by centrifugation and scanned with a ScanArray 4000 confocal laser scanner (Perkin-Elmer). Each mycobacterial isolate was hybridized against M. avium subsp. paratuberculosis K10 at least twice in a dye-flip experimental design.


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  		TABLE 1. Mycobacterial isolates used in this study

Microarray data analysis. Raw intensity measurements for each spot on the microarray were extracted from scanned images by using ScanArray Express software (Perkin-Elmer) and adjusted with local background subtraction and LOWESS normalization (9). Poorly detected spots were removed by filtering out those in which the measured spot pixels did not have a total median intensity of at least 2,800 or in which more than 50% of the pixels from the M. avium subsp. paratuberculosis K10 control sample were within 2 standard deviations of the local background. At this point, any ORFs not represented by at least two acceptable spots on a hybridized microarray were discarded from further analyses. The median intensity of the remaining spots was determined for each ORF, and then ratios of the spot intensities for experimental and control M. avium subsp. paratuberculosis K10 DNA samples were calculated and log transformed for analysis. The software program Genomotyping Analysis (14) was utilized to grade the ORFs on a linear scale from –0.5 (0% estimated probability of being present) to 0.5 (100% estimated probability of being present). Briefly, this software uses the distribution of measured ratios from a microarray hybridization to determine the probability of an individual ORF being present in a genomic DNA sample. Grades from duplicated experiments were combined, and ORFs were classified as divergent (total grade of <–0.75) or present (total grade of >0.75), with the remaining ORFs listed as having intermediate divergence. Results were reported only if data were obtained from both hybridizations.

IS1311 PCR restriction digest. The IS1311 IS was amplified via PCR from genomic DNA under the standard conditions described above and then subjected to restriction digest with HinfI as described by Whittington and coworkers (29). Briefly, a PCR product representing IS1311 was amplified using genomic DNA from the mycobacterial isolates examined in this study under the standard thermocycling conditions described above. Six microliters of each PCR product was then incubated at 37°C for 1.5 h with 5 U of HinfI, followed by a 20-min deactivation at 80°C. The resulting product was resolved on a 1.5% agarose gel and visualized with ethidium bromide staining under UV light.

Comparative sequence analysis with M. avium subsp. avium 104. The complete but unannotated genome sequence of M. avium subsp. avium 104 was obtained from The Institute for Genomic Research (www.tigr.org). The BLASTN algorithm (1) was used to query the M. avium subsp. avium 104 genome sequence with a library of all the sequences present on the M. avium subsp. paratuberculosis K10 microarray. The resulting output was filtered so that only matches of at least 100 bp with a maximum size difference of 10% between the query and target sequences were reported.

Southern blotting. Four micrograms of genomic DNA from isolates MAA104, 6004, 6058, K10, 19698, 6079, 4138, and 6010 was digested with BamHI (New England Biolabs), separated by electrophoresis on an 0.6% agarose gel, and transferred to a nylon membrane (Ambion) by standard procedures (23). Digoxigenin-labeled probes of approximately 500 bp were generated for MAP0005, MAP0854, and MAP3815 by using the DIG High Prime DNA Labeling and Detection Kit II (Roche Applied Science). These probes were individually hybridized to the membrane according to the manufacturer's instructions. Hybridized blots were exposed to Biomax MR film (Kodak) for 30 to 60 min.

Identification of sequences unique to M. avium subsp. paratuberculosis. The 4,350 annotated ORFs present in the M. avium subsp. paratuberculosis genome were compared to the complete genome sequence of M. avium subsp. avium 104 by using the TBLASTX algorithm (1). With the use of expected value cutoffs of 1.0e–10 for M. avium subsp. avium and 1.0e–6 for GenBank, there were 32 ORFs that had no significant matches to any of the sequences currently available in these databases.


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RESULTS
 
Isolate typing. A HinfI restriction digest of the IS1311 IS from each isolate revealed that all of the M. avium subsp. paratuberculosis isolates examined contained a 220-bp product indicating the presence of a previously described polymorphism found exclusively in nonsheep isolates of M. avium subsp. paratuberculosis (29) (Fig. 1). None of the nonparatuberculosis MAC isolates contained the polymorphism.



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  		FIG. 1. Restriction digest of IS1311 PCR products. Lane 1, 100-bp DNA standard; lane 2, M. avium subsp. paratuberculosis K10; lane 3, M. avium subsp. paratuberculosis 19698; lane 4, M. avium subsp. paratuberculosis 2009; lane 5, M. avium subsp. paratuberculosis 4138; lane 6, M. avium subsp. paratuberculosis 10006; lane 7, M. avium subsp. avium 6004; lane 8, M. avium subsp. paratuberculosis 6079; lane 9, M. avium subsp. paratuberculosis 1434; lane 10, M. avium subsp. paratuberculosis Kay; lane 11, M. avium subsp. silvaticum 6058; lane 12, M. avium subsp. paratuberculosis 4090; lane 13, M. avium subsp. paratuberculosis 1091; lane 14, M. intracellulare 6010; lane 15, M. avium subsp. silvaticum 6006; lane 16, M. avium subsp. avium 104; lane 17, no-DNA control; lane 18, M. smegmatis 19420.

Comparative sequence analysis with M. avium subsp. avium 104. The raw genome sequence of M. avium subsp. avium 104 was compared to the M. avium subsp. paratuberculosis K10 probe sequences and matched with comparative genomic hybridization data to evaluate the performance of the M. avium subsp. paratuberculosis K10 microarray. By using the procedures described in Materials and Methods, microarray probe sequence matches to M. avium subsp. avium 104 were obtained for 3,709 of the 4,350 annotated M. avium subsp. paratuberculosis K10 ORFs, 3,497 (94%) of which were also "well-measured" in comparative genomic hybridizations. Of the 210 ORFs classified as divergent in M. avium subsp. avium 104 by the M. avium subsp. paratuberculosis K10 microarray (Table 2; see also Table S1 in the supplemental material), 197 (94%) had no significant matches to the genome sequence of M. avium subsp. avium 104 by BLAST analysis. The remaining 13 ORFs were either lower-quality matches to the M. avium subsp. avium 104 genome (<92% identical; n = 6) or potential false negatives (>98% identical; n = 7). A total of 2,932 ORFs were identified as present in M. avium subsp. avium 104 by use of the M. avium subsp. paratuberculosis K10 microarray, 2,782 (95%) of which were successfully matched to M. avium subsp. avium 104 genomic sequences at greater than 95% identity. Weaker matches of 90 to 93% identity were found for three additional ORFs that had been classified as present. The remaining sequences identified as present via microarray analysis (n = 147) were either not matched or only partially matched (greater than 10% size difference as described in Materials and Methods) to corresponding M. avium subsp. avium 104 sequences. There were 355 M. avium subsp. avium 104 ORFs classified as having intermediate divergence. Of these ORFs, 309 (87%) were at least 95% identical to M. avium subsp. paratuberculosis K10 sequences, while the remaining ORFs were either lower-quality matches to M. avium subsp. avium 104 sequences (n = 12; 4%), not detected, or unmatched to microarray probes (n = 34; 10%). From this result, we can conclude that a majority of the ORFs classified as intermediate by microarray analysis are most likely present in the genomes examined in this study.


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  		TABLE 2. Classification of M. paratuberculosis K10 ORFs compared with other mycobacterial isolates

M. avium subsp. paratuberculosis isolates. Genomic DNA from nine clinical isolates of M. avium subsp. paratuberculosis was competitively hybridized against M. avium subsp. paratuberculosis K10 DNA on a whole-genome DNA microarray (Table 2). Overall, the M. avium subsp. paratuberculosis isolates were very similar to K10. None of the M. avium subsp. paratuberculosis isolates examined had any ORFs classified as divergent. The type strain 19698 was the most similar to K10, with 88% of the total K10 ORFs being detected by the array and 99% of these identified as being present. A core set of 1,230 ORFs were classified as present among all of the M. avium subsp. paratuberculosis isolates. An analysis of the proteins encoded by these conserved ORFs revealed that the distribution of predicted functions was similar to the whole genome, indicating that no functional categories were preferentially represented.

Notably, M. avium subsp. paratuberculosis 10006 and 4090 were isolated from the same dairy herd in Pennsylvania, and, although they lacked divergent sequences, 458 and 398 ORFs of intermediate divergence were identified in their respective genomic sequences. A total of 152 ORFs were classified as intermediate in both isolates (see Table S2 in the supplemental material). These ORFs include hypothetical proteins (n = 52), proteins involved in energy metabolism (n = 22), and cell envelope proteins (n = 17). It is intriguing that two isolates from a common geographic area contained a significantly higher number of ORFs classified as intermediate than did the other M. avium subsp. paratuberculosis isolates examined in this study.

Other mycobacterial isolates. DNA from five MAC isolates was hybridized against M. avium subsp. paratuberculosis K10 DNA to establish the degree of genome variability that exists between these closely related mycobacteria (Table 2). Additionally, the type strain of Mycobacterium smegmatis (ATCC 19420) was hybridized against the M. avium subsp. paratuberculosis K10 microarray to examine the genetic conservation between less closely related mycobacteria.

M. avium subsp. silvaticum. The two M. avium subsp. silvaticum isolates examined, 6006 and 6058, were found to have 77 and 103 divergent genes, respectively (see Tables S3 and S4 in the supplemental material). Of all the nonparatuberculosis MAC isolates, 6006 had the fewest ORFs classified as divergent or intermediate compared to K10. Among the 77 divergent ORFs reported in isolate 6006, 45 encoded hypothetical proteins, while 16 ORFs represented IS elements, including multiple copies of ISMAP02, ISmav2 (AF286339), and IS900. Additionally, two members of a putative cation transport system (MAP3731c and MAP3732c) and cell invasion proteins (MAP2189 and MAP2192) were also found to be divergent. The divergent ORFs identified in M. avium subsp. silvaticum isolate 6058 included eight encoding oxidoreductase or oxygenase enzymes, six nonribosomal peptide synthesis enzymes (including two involved in mycobactin biosynthesis), and four cell invasion proteins (MAP2189, MAP2190, MAP2193, and MAP2194). Twenty-five IS elements or transposases were found to be divergent in 6058; they included IS1311 (U16276), ISmav2, IS900, ISMAP02, and IS1601 (AF060182). There were 17 ORFs found to be divergent in both M. avium subsp. silvaticum isolates, including IS900, ISmav2, ISMAP02, transposases (n = 3), and hypothetical proteins (n = 5).

M. avium subsp. avium. M. avium subsp. avium isolate 6004 had 135 ORFs classified as divergent from K10 (see Table S5 in the supplemental material). Consistent with the other isolates examined, many of the divergent ORFs (n = 57) encode hypothetical proteins. A total of 33 divergent ORFs contained IS-related elements, including IS1311, IS900, ISmav2, IS1601-B, and ISMAP02. Two ORFs encoding proteins involved in mycobactin synthesis were found to be divergent (mbtD and mbtG), as well as five outer membrane proteins. Five adjacent ORFs encoding cell invasion proteins (MAP2189 to MAP2193) were divergent in isolate 6004, as well as the two putative cation transport proteins (MAP3731c and MAP3732c) that were also identified as divergent in M. avium subsp. silvaticum 6006.

As described above, M. avium subsp. avium isolate 104 had 210 ORFs classified as divergent from M. avium subsp. paratuberculosis K10, 110 of which were shared with M. avium subsp. avium 6004. Similar to the other nonparatuberculosis isolates, the largest groups of divergent genes encoded hypothetical proteins (n = 110) and IS elements (n = 29). Other groups of functionally related divergent genes encoded cell envelope proteins (n = 11), transcriptional regulators (n = 8), and PPE family proteins (n = 3). Clusters of genes divergent only in isolate MAA104 included MAP0956 to MAP0965, which encode multiple hypothetical proteins and putative glycosyltransferases. The presence of glycosyltransferases suggests that some of these ORFs may participate in cell membrane biosynthesis.

M. intracellulare. M. intracellulare isolate 6010 was found to have 130 ORFs divergent from K10 (see Table S6 in the supplemental material). Of these divergent ORFs, 27 contained IS elements or transposases, including IS900, ISmav2, IS1601-B, and ISMAP02. Six divergent ORFs encoded outer membrane or secreted proteins, while 71 divergent ORFs encoded hypothetical proteins or proteins with unknown function. Notably, a cluster of ORFs from MAP1231 to MAP1237 were classified as divergent in M. intracellulare 6010 and M. avium subsp. avium 104 and include the region previously identified by Tizard and coworkers as a low-GC genetic island present only in M. avium subsp. paratuberculosis and M. avium subsp. silvaticum (28). This region was classified as present in all of the M. avium subsp. paratuberculosis and M. avium subsp. silvaticum isolates as well as M. avium subsp. avium 6004.

Common divergent regions. A core set of 97 ORFs were classified as divergent or intermediate in all of the nonparatuberculosis MAC genomes (Table 3). These ORFs included 25 IS features, among which were IS900, IS1311, ISmav2, and ISMAP02, as well as sequences similar to IS1110, IS1547, IS1601-B, IS6110, and ISmav2. As observed in the individual analyses, a large number (n = 42) of the shared divergent ORFs encoded hypothetical proteins.


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  		TABLE 3. Ninety-seven ORFs identified as divergent or intermediate in the nonparatuberculosis MAC isolates MAA104, 6004, 6006, 6010, and 6058

Interestingly, many of the common regions of divergence among the nonparatuberculosis MAC isolates were grouped into clusters of adjacent ORFs (Table 3). Notably, four of the seven IS1311 copies present in the M. avium subsp. paratuberculosis genome were found within four independent regions of divergence. Furthermore, four of these seven divergent regions contained at least one ORF encoding a putative phage-related sequence.

The divergent clusters comprised of MAP0849 to MAP0866 (MAP_RD2) and MAP2750 to MAP2768 (MAP_RD5) include ORFs that predominantly encode hypothetical proteins. Additionally, a whole-genome alignment of M. avium subsp. paratuberculosis and M. avium subsp. avium reveals that a majority of these divergent regions are bordered by inversions in the genome sequences of these two isolates.

The average G+C content of the MAP_RD5 gene cluster is similar to the K10 whole-genome level of 69.3%, while the MAP_RD2 cluster is significantly lower at 60.6%. Another region of relatively low average G+C content that correlates with a cluster of divergent genes is present between MAP2148 and MAP2158 (MAP_RD3). This 12,000-bp region has an average G+C content of 60.6% and contains ISs and ORFs of unknown function, including two sequences previously identified as specific to M. avium subsp. paratuberculosis (MAP2149c and MAP2154c) (4, 20). A 9,500-bp region between MAP3749 and MAP3756c has an average G+C content of 60.5% and was also part of a shared divergent ORF cluster (MAP_RD6). This region encodes two putative membrane proteins, two transposases, and two proteins involved in energy metabolism. Additionally, the MAP_RD6 region also contains seven genes encoding putative ABC transport proteins. The presence of mobile genetic elements as well as the low G+C content found within some of these divergent regions suggests that they may have been acquired via horizontal gene transfer.

The MAP_RD4 region contains five adjacent divergent ORFs (MAP2189 to MAP2194) that are homologous to mammalian cell entry (mce) genes previously identified as important for invasion and survival within macrophages (2). None of the other mce gene clusters present in the M. avium subsp. paratuberculosis genome were found to be divergent in any of the isolates. These genes appear to be ideal candidates for future investigations into the pathogenesis of M. avium subsp. paratuberculosis.

M. smegmatis. M. smegmatis is a fast-growing mycobacterium distantly related to the slow-growing MAC mycobacteria. Genomic DNA from M. smegmatis ATCC 19420 was hybridized against the M. avium subsp. paratuberculosis K10 microarray and analyzed with the GACK software program as described for the other mycobacteria. Initially, only six M. smegmatis ORFs were classified as divergent by the Genomotyping Analysis software, while a manual examination of the data indicated that there should be significantly more divergent ORFs. This discrepancy was determined to be due to the wide distribution of hybridization ratios resulting from a large number of divergent sequences. A conservative cutoff of 1.5-fold was therefore used to sort the ORFs based on the average ratio of hybridization intensities. According to this cutoff classification system, 380 ORFs were identified as divergent in M. smegmatis, while 521 ORFs were present and 931 were intermediate (Table 2). Unlike the nonparatuberculosis MAC mycobacteria, the divergent M. smegmatis ORFs were evenly distributed across the entire genome. Among the divergent ORFs were potential virulence factors including nine mce genes, seven PPE family genes, and 29 cell envelope-associated proteins (see Table S7 in the supplemental material). Notably, many of the proteins encoded by these divergent ORFs fall into the same functional categories as genes identified as divergent between Mycobacterium tuberculosis H37Rv and M. tuberculosis BCG Pasteur (reviewed in reference 8). The large number of divergent M. smegmatis ORFs is reflective of the phylogenetic distance separating slow- and fast-growing mycobacteria and may also reveal genomic features that contribute to pathogenicity. Our results also illustrate the limitations of comparing highly divergent genomes with DNA microarrays.

PCR analysis. Genomic DNA from a limited number of mycobacterial isolates was used as the template for PCRs under the conditions described for the construction of the M. avium subsp. paratuberculosis K10 microarray. Primers designed to amplify ORFs from the shared divergence regions as well as ORFs flanking these regions were used to confirm the microarray findings (Table 4). With the exception of MAP0106c and MAP0850, all of the ORFs selected from the shared divergent regions either were not detected by PCR or had a low yield of product when genomic DNA from the nonparatuberculosis ORFs was used as template. The presence of small amounts of PCR products for ORFs from the divergent regions indicates that some of these regions represent nucleotide sequence variations, while the absence of PCR products reveals that some regions classified as divergent may be at least partially due to whole-gene deletions. One of the ORFs immediately flanking MAP_RD6 (MAP3715) was not detected by PCR for several isolates, indicating that this region may be larger than originally determined by microarray analysis.


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  		TABLE 4. PCR amplification of ORFs from shared regions of divergence and adjacent flanking ORFs

Southern blotting. Genomic DNA from four M. avium subsp. paratuberculosis and four nonparatuberculosis isolates was subjected to restriction digestion and used for Southern blotting (Table 5). All eight isolates were detected with a probe for MAP0005, which carries the highly conserved gene gyrB. Only the M. avium subsp. paratuberculosis isolates were detected by probes for MAP0854 and MAP3815, which encode hypothetical proteins and were classified as divergent by microarray analysis for all of the nonparatuberculosis isolates with the exception of 6006, for which no data were reported. These results further support the findings of the microarray-based analyses.


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  		TABLE 5. Southern hybridization of purified mycobacterial genomic DNA with probes derived from M. avium subsp. paratuberculosis K10 genomic DNA

Unique sequences. Several M. avium subsp. paratuberculosis ORFs that have no homology to any sequences currently available in public databases have been previously described (4, 16, 20). We updated this subset of unique sequences by running TBLASTX searches against the complete genome of M. avium subsp. avium 104 and all of the sequences in GenBank as described in Materials and Methods. Of the 32 ORFs identified as unique to M. avium subsp. paratuberculosis by TBLASTX analysis, comparative genomic hybridizations revealed that 30 were classified as divergent in M. avium subsp. avium 104, one ORF was intermediate, and the remaining ORF had no data reported (Fig. 2). In the other nonparatuberculosis isolates examined there were from one to five unique ORFs identified as being present, including MAP1718c and MAP3383, which were both classified as present in multiple nonparatuberculosis isolates. None of the M. avium subsp. paratuberculosis-specific sequences were identified as divergent in the M. avium subsp. paratuberculosis isolates. These data reveal the utility of using DNA hybridization studies to further establish the specificity of sequences identified as unique by database searches.



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  		FIG. 2. Microarray hybridization results for M. avium subsp. paratuberculosis-specific ORFs identified by TBLASTX searches of GenBank and the genome sequence of M. avium subsp. avium 104.


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DISCUSSION
 
MAC represents an intriguing group of mycobacteria whose members are genetically very similar and yet have extremely variable pathogenicity and host range. Improved diagnostic tests for pathogenic isolates are confounded by the abundance of nonpathogenic mycobacteria present in the environment. In order to better understand the genetic conservation among isolates of M. avium subsp. paratuberculosis and between other MAC bacteria, we have utilized a whole-genome cDNA microarray based on the sequence of M. avium subsp. paratuberculosis K10 to compare genomic content at the level of individual genes via competitive DNA hybridization.

The M. avium subsp. paratuberculosis isolates examined in this study showed a high degree of genetic conservation compared to isolate K10, as no ORFs were classified as divergent in the M. avium subsp. paratuberculosis isolates. Although this result is not altogether unexpected, it is notable that the isolates were cultured from several different host species and geographic locations. Our findings have provided a more rigorous confirmation of previous reports indicating that bovine M. avium subsp. paratuberculosis isolates form a distinct subgroup based on conserved genomic features, as it appears that this conservation extends down to the gene level for a majority of the ORFs identified in the genome sequence. Based on these results, future studies examining additional M. avium subsp. paratuberculosis isolates from a variety of host species and geographic locations are warranted to determine if this level of conservation encompasses a larger population.

Many of the ORFs classified as divergent in the nonparatuberculosis MAC group were shared among isolates MAA104, 6004, 6010, 6006, and 6058 and could be grouped into clusters of adjacent ORFs on the genome. In four of these clusters, phage integrase genes were found to be present, indicating that these regions of divergence may have been acquired by M. avium subsp. paratuberculosis K10 via phage-mediated recombination. Additionally, four of these gene clusters contained large regions in which the average GC content was significantly lower than the genome average. The low GC content of these gene clusters and their divergence from other mycobacterial genomes support the theory that the genetic material in these regions may have been acquired via horizontal gene transfer. Although not specifically shown by this study, some of these regions may be deleted from the MAC isolates relative to M. avium subsp. paratuberculosis, possibly giving investigators clues to identifying genes involved in the pathogenicity of M. avium subsp. paratuberculosis. Notably, recent work by Semret and coworkers has revealed that M. avium subsp. avium isolates contain regions of genetic sequence that are not present in M. avium subsp. paratuberculosis, findings that are complementary to those presented here (26).

One of the regions of shared divergence in the nonparatuberculosis MAC isolates included four ORFs (MAP2189, MAP2190, MAP2192, and MAP2193) homologous to the mce gene family originally identified in M. tuberculosis (2). Although the ORFs were present in all of the M. avium subsp. paratuberculosis isolates, they were found to be divergent or intermediate in all of the nonparatuberculosis MAC isolates. Several other clusters of mce genes were classified as present in all of the isolates examined. These findings are similar to those reported by Zumarraga and coworkers, who observed that one of the mce gene clusters present in M. tuberculosis was missing from the Mycobacterium bovis genome (31). It is notable that M. tuberculosis and M. bovis represent another group of mycobacteria with very similar genomic content but varying pathogenicity and host specificity. The divergence of a specific cluster of mce-like genes among MAC isolates and the previously established role of mce genes in macrophage invasion and survival suggest that this group of genes may confer a specific advantage in the infection of bovine macrophages, a hypothesis that remains to be directly tested.

Our analysis of M. avium subsp. silvaticum isolate 6006 yielded several unexpected results. Compared with the other nonparatuberculosis MAC mycobacteria examined, 6006 had nearly half the number genes classified as divergent or intermediate compared to M. avium subsp. paratuberculosis K10. Additionally, five sequences identified as unique to M. avium subsp. paratuberculosis were detected in 6006. Notably, while 6006 was isolated from a roe deer, it had been previously classified as atypical or wood pigeon M. avium subsp. avium (13). This isolate type is commonly cultured from avian species, has been found to share genetic and biochemical properties with both M. avium subsp. avium and M. avium subsp. paratuberculosis (15, 24, 30), and was later reclassified as a novel subspecies of M. avium (27). As Jorgensen and Clausen note in their original description of this isolate, it is intriguing that a member of this subspecies is capable of infecting a mammalian host. Because M. avium subsp. silvaticum isolates appear to share conserved genomic features with both M. avium subsp. avium and M. avium subsp. paratuberculosis, it is tempting to speculate that M. avium subsp. silvaticum may represent an intermediate step in the adaptation of avian species to mammalian hosts or vice versa.

The M. avium subsp. paratuberculosis isolates from a goat, sheep, and mink were not found to be divergent from the K10 bovine isolate. Isolates from cattle and goats have previously been found to be genetically similar, while sheep isolates commonly form a distinct subgroup from other M. avium subsp. paratuberculosis isolates (7, 19, 21). M. avium subsp. paratuberculosis infections of mink have not previously been reported. A HinfI restriction digest of the IS1311 IS from each isolate revealed that all of the M. avium subsp. paratuberculosis isolates examined contained a polymorphism found exclusively in nonsheep isolates of M. avium subsp. paratuberculosis, while none of the nonparatuberculosis MAC isolates contained this polymorphism. Additional isolates from these animals will need to be examined and compared to these findings, as it appears that they were infected from bovine sources that are not necessarily representative of the isolates most commonly cultured from the respective species.

None of the ORFs identified as unique to M. avium subsp. paratuberculosis in this study were classified as divergent in the M. avium subsp. paratuberculosis isolates examined. M. avium subsp. silvaticum isolate 6006 showed the fewest number of ORFs classified as divergent among the nonparatuberculosis MAC isolates, five of which were identified by a TBLASTX search of publicly available sequence data as specific for M. avium subsp. paratuberculosis. With the exception of MAP1718c and MAP3383, the remaining 30 paratuberculosis-specific ORFs were classified as divergent or intermediate in the other nonparatuberculosis isolates. Isolate 6006 in particular may represent a broader group of isolates that resist rigid classification as either M. avium subsp. paratuberculosis or other M. avium subspecies. As genomic DNA from additional mycobacterial isolates becomes available, a microarray-based approach will be useful to further confirm the specificity of these sequences because it can quickly and easily establish the presence or absence of all potentially unique sequences in a single experiment.

It should be noted that, due to the use of M. avium subsp. paratuberculosis K10 as the template for construction of the cDNA microarray, our analyses will not detect genes that are absent in K10 but present in the other isolates examined. Alternate methodologies such as subtractive hybridization will need to be utilized in order to identify divergent sequences in other mycobacteria. Additionally, the presence of small genetic polymorphisms would not be detected by cDNA microarray analysis. The advantage of using cDNA microarrays for this type of comparative genomic analysis is that differences between isolates can be localized to individual genes rather than gross genomic features.

Our comparative genomic analysis of MAC bacteria has revealed that the subset of bovine M. avium subsp. paratuberculosis isolates examined in this study have a high degree of genetic conservation, with 99% of the ORFs examined being classified as intermediate or present. The closely related M. avium subsp. avium, M. intracellulare, and M. avium subsp. silvaticum displayed various amounts of genetic divergence from M. avium subsp. paratuberculosis K10 and contain core groups of ORFs that appear to diverge across all of the nonparatuberculosis MAC isolates examined. These findings are similar to those reported by Dziejman and coworkers, who compared isolates responsible for causing the sixth, seventh, and eighth Vibrio cholerae pandemics by hybridizing genomic DNA from these strains to a whole-genome microarray based on the sequence of the V. cholerae El Tor isolate (10). On this basis, these V. cholerae strains appear remarkably similar, sharing 99% of their genes with the El Tor isolate. However, other pathogens, such as Staphylococcus aureus and Helicobacter pylori, show much greater divergence among clinical isolates, with a 12% maximum difference in genome content seen between individual strains of S. aureus (11) and an 18% difference between isolates of H. pylori (22).

An understanding of the genomic diversity among MAC mycobacteria should provide additional insight into the mechanisms of host specificity. Additionally, it will allow for improvements in diagnostic tests to distinguish between nonpathogenic environmental mycobacteria and pathogenic mycobacteria such as the Johne's disease-causing bacillus.


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ACKNOWLEDGMENTS
 
The expert technical assistance of Janis K. Hansen is greatly appreciated. M. avium subsp. avium 104 was a gift from Luiz Bermudez.

Mention of trade names or commercial products in this article is solely for the purpose of providing specific information and does not imply recommendation or endorsement by the U.S. Department of Agriculture.

This work was supported by the USDA's Agricultural Research Service.


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FOOTNOTES
 
* Corresponding author. Mailing address: National Animal Disease Center, USDA-ARS, 2300 North Dayton Ave., Ames, IA 50010. Phone: (515) 663-7979. Fax: (515) 663-7458. E-mail: mpaustia{at}nadc.ars.usda.gov. Back

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


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Journal of Bacteriology, April 2005, p. 2406-2415, Vol. 187, No. 7
0021-9193/05/$08.00+0     doi:10.1128/JB.187.7.2406-2415.2005
Copyright © 2005, American Society for Microbiology. All Rights Reserved.



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