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J Bacteriol. Feb 2009; 191(3): 1018–1025.
Published online Nov 21, 2008. doi:  10.1128/JB.01340-08
PMCID: PMC2632067

Insertion and Deletion Events That Define the Pathogen Mycobacterium avium subsp. paratuberculosis[down-pointing small open triangle]

Abstract

Mycobacterium avium comprises genetically related yet phenotypically distinct subspecies. Consistent with their common origin, whole-genome sequence comparisons have revealed extensive synteny among M. avium organisms. However, the sequenced strains also display numerous regions of heterogeneity that likely contribute to the diversity of the individual subspecies. Starting from a phylogenetic framework derived by multilocus sequence analysis, we examined the distribution of 25 large sequence polymorphisms across a panel of genetically defined M. avium strains. This distribution was most variable among M. avium subsp. hominissuis isolates. In contrast, M. avium subsp. paratuberculosis strains exhibited a characteristic profile, with all isolates containing a set of genomic insertions absent from other M. avium strains. The emergence of the pathogen from its putative M. avium subsp. hominissuis ancestor entailed the acquisition of approximately 125 kb of novel genetic material, followed by a second phase, characterized by reductive genomics. One genomic deletion is common to all isolates while additional deletions distinguish two major lineages of M. avium subsp. paratuberculosis. For the average strain, these losses total at least 38 kb (sheep lineage) to 90 kb (cattle lineage). This biphasic pattern of evolution, characterized by chromosomal gene acquisition with subsequent gene loss, describes the emergence of M. avium subsp. paratuberculosis and may serve as a general model for the origin of pathogenic mycobacteria.

Mycobacterium avium organisms are nontuberculous mycobacteria prevalent in environmental and clinical settings. M. avium infections result in diverse diseases, including avian tuberculosis, Johne's disease, and Lady Windermere's syndrome. Isolates are phenotypically different and were historically classified as separate species. However, current taxonomy, based on molecular analyses, recognizes a single species, M. avium, which is divided into distinct subgroups (21, 22).

At present, M. avium subsp. hominissuis denotes environmental organisms associated with opportunistic infections in humans and swine (13, 23). M. avium subsp. avium is the classical agent of tuberculosis in birds and, along with M. avium subsp. silvaticum, represents a distinct lineage of bird pathogens (22). M. avium subsp. paratuberculosis causes Johne's disease (Paratuberculosis), a chronic granulomatous intestinal disease (5). Although primarily associated with livestock, the bacterium may infect a wide range of mammalian hosts. A number of studies, using molecular testing for the M. avium subsp. paratuberculosis-specific insertion element IS900, have found an association between the presence of M. avium subsp. paratuberculosis and Crohn's disease in humans (1, 9).

Previous studies, including bigenomic comparisons of the sequenced strains M. avium subsp. hominissuis 104 and M. avium subsp. paratuberculosis K-10 (11), have revealed inter- and intrasubspecies differences (6, 12, 15, 16, 18, 19, 26). The phenotypic heterogeneity of M. avium strains may stem from genomic differences, but in the absence of a phylogenetic framework it has been difficult to define the key variations associated with the emergence of an individual subspecies. Recently, we proposed a phylogeny for M. avium based on multilocus sequence analysis (MLSA) of 10 genes and 56 M. avium isolates. This phylogeny is consistent with the current taxonomy and indicates that M. avium subsp. paratuberculosis is a distinct, clonal lineage of M. avium (22). To better understand the evolution of this subspecies, we have now examined the distribution of large sequence polymorphisms among a genetically defined panel of M. avium strains. Our findings reveal a characteristic genomic profile for M. avium subsp. paratuberculosis and provide insight into the biphasic evolution of this successful pathogen.

MATERIALS AND METHODS

Bacterial strains.

The panel of 23 strains used in the present study includes 10 strains of M. avium subsp. hominissuis, 5 IS901-positive isolates of M. avium (3 isolates of M. avium subsp. avium and 2 isolates of M. avium subsp. silvaticum), and 8 strains of M. avium subsp. paratuberculosis (4 isolates of the “cattle” lineage and 4 isolates of the “sheep” lineage). Detailed descriptions of these isolates have been published previously (22) and key features of each strain are listed in Table Table1.1. These isolates were selected to ensure that organisms with different multilocus sequence analysis (MLSA) sequence types were represented (shown in Fig. S1 in the supplemental material). Species and subspecies identification were confirmed by PCR- and restriction fragment length polymorphism-based insertion sequence (IS) typing. Strains positive for IS900 were considered M. avium subsp. paratuberculosis; strains positive for IS901 were considered M. avium of the avian lineage (M. avium subsp. avium and M. avium subsp. silvaticum) and strains negative for IS901 but IS1245 positive were considered M. avium subsp. hominissuis (2). Subspecies designations were consistent with MLSA at all 10 gene loci.

TABLE 1.
Strain identification and characterization

DNA extraction and PCR conditions.

Bacterial DNA was extracted by using the CTAB (cetyltrimethylammonium bromide) methodology (25). All PCRs were performed in a final reaction volume of 25 μl and contained 25 ng of DNA template, 2.5 mM MgCl2, 1× Taq buffer with (NH4)2SO4 (Fermentas), 5 μl of 50% acetamide (Sigma), 0.2 mM deoxynucleoside triphosphates, 0.5 μM concentrations of each primer, and 0.75 U of Taq DNA polymerase (Fermentas). PCR was performed by using an Applied Biosystems GeneAmp 2700 PCR system under the following conditions: 94°C for 3 min; 30 to 35 cycles of 94°C (30 s), 55°C (30 s), and 72°C (1.5 min); 72°C for 5 min; and then holding at 4°C. A higher annealing temperature was necessary with some PCRs to minimize nonspecific amplification. Amplification of PCR products was verified by electrophoresis using 1.5% agarose gels. Primers were designed in Primer3 (http://primer3.sourceforge.net/). Oligonucleotide sequences, as well as annealing temperatures and the expected PCR product sizes for each primer combination, are listed in Table S1 in the supplemental material.

Selection of LSPs.

Large sequence polymorphisms (LSPs) are genomic regions present in some M. avium strains and absent from others. Numerous LSPs have been revealed by DNA microarray-based comparison of M. avium isolates, as well as by in silico analysis of whole-genome sequence data from M. avium subsp. paratuberculosis strain K-10 and M. avium subsp. hominissuis strain 104. However, in the present study, only LSPs previously demonstrated to exhibit specificity for at least one lineage of M. avium subsp. paratuberculosis were examined. These 25 LSPs include 16 “LSPPs” (17), suspected to be present in all M. avium subsp. paratuberculosis strains; 5 “LSPAs” (18), known to be absent from at least one strain; the “deletion 2” region, originally identified as a marker of the M. avium subsp. paratuberculosis sheep lineage (12); region VA15, also described as absent from sheep isolates (16); the “MAV-14 island” (26), considered a marker of the cattle lineage; and finally, the “glycopeptidolipid (GPL) biosynthesis cluster” of M. avium (7), shown previously to be heterogeneous among M. avium subsp. paratuberculosis lineages (16). Detailed descriptions of the polymorphic loci found to be specific for M. avium subsp. paratuberculosis are included in Table S2 in the supplemental material.

Distribution of LSPs.

Two complementary approaches, PCR and Southern hybridization, were used to determine the distribution of the LSPs across our panel of isolates. For PCR-based testing, we used a three-primer strategy in a single PCR to test for simple genetic events. In these cases, amplification from a common primer plus an internal primer indicated the presence of a region, whereas amplification from a common primer and a bridging primer beyond the variable region indicated its absence. In cases where an LSPP was replaced by an LSPA (or vice versa), we used a common flanking primer plus two internal primers, one for each LSP, again using a three-primer PCR. For LSPP7, LSPP14, and LSPP15, we used a four-primer strategy, involving primers inside of the region to indicate presence and primers beyond the region to document its absence. With these multiplex methods, an amplicon was always expected and false-negative results were readily detected. To confirm the identity and uniformity of LSP junctions across different isolates, select PCR products were subjected to DNA sequencing, particularly in rare cases, where the amplicon was of an unexpected size. In cases where multiplex PCR failed, the presence of an LSP was confirmed by amplifying an internal target via traditional, two-primer PCR. For these tests, the absence of a PCR product suggests the absence of the LSP but is not definitive, since a single nucleotide polymorphism in the primer binding region could also result in a negative PCR.

Southern hybridization was used to confirm the distribution of seven LSPs that appeared to be M. avium subsp. paratuberculosis-specific by PCR. For each, a target within the region was amplified by PCR and used as the hybridization probe (see Table S1 in the supplemental material). Due to limitations in the amount of available genomic DNA, only 19 strains from the panel were examined; one M. avium subsp. silvaticum isolate and three M. avium subsp. hominissuis strains were omitted.

Directionality of LSPs.

The differential distribution of a specific LSP may represent either its insertion into one lineage or its deletion from another. This directionality was evaluated by criteria that considered both the content of the LSP and features of the flanking DNA (Table (Table2).2). Low G+C content (<65%), the presence of open reading frames (ORFs) associated with mobile elements (e.g., integrase genes and bacteriophage sequences), and a flanking region containing direct repeat sequences, tRNA genes, or a split ORF were considered indicative of an insertion. In contrast, normal G+C content (65 to 71%), the presence of ORFs with homologs in other mycobacteria, and an LSP junction featuring a truncated or chimeric ORF were considered hallmarks of a putative deletion. To support our findings and to help resolve cases for which the directionality could not be established by the criteria delineated above, comparative genomic studies were conducted with the closely related species, M. intracellulare. In one approach, the status of the LSP regions in M. intracellulare strains ATCC 13950T and FCC 1804 was determined by DNA hybridization to our previously described oligonucleotide-based M. avium microarray (18). Briefly, this array consisted of 70-bp oligonucleotide probes, printed in duplicate, representing 93% of the predicted ORFs in the M. avium 104 genome, as well as 98% of those determine in M. avium subsp. paratuberculosis, for a total of 13,400 spots. In a second approach, the draft genome sequence for the M. intracellulare type strain, ATCC 13950T (NCBI accession no. NZ_ABIN00000000), was inspected to confirm the presence or absence of the LSP regions in this outgroup.

TABLE 2.
Criteria for directionality of LSPs

RESULTS

Distribution of LSPs.

The distribution of 25 LSPs was investigated across our panel of 23 M. avium strains (Table (Table3).3). For most LSPs, the presence or absence was successfully confirmed by a three-primer PCR strategy, while a four-primer approach was used for the evaluation of LSPP7, LSPP14, and LSPP15. For some isolates, the presence of individual LSPs required confirmation by a two-primer strategy that amplified one or two internal targets (Table (Table33).

TABLE 3.
Distribution of LSPs across M. aviuma

LSPs shown to be not specific for M. avium subsp. paratuberculosis.

Nine tested LSPs were conserved among all M. avium subsp. paratuberculosis strains but also found in isolates from other M. avium lineages. For many of these LSPs, their distribution was most variable among the group of M. avium subsp. hominissuis strains (Table (Table3).3). With the exception of a single locus (an internal target within VA15, see below), M. avium subsp. avium and M. avium subsp. silvaticum isolates shared an identical and characteristic set of LSPs. Therefore, these LSPs are not specific to M. avium subsp. paratuberculosis and have been excluded from further analysis below.

LSPs specific to M. avium subsp. paratuberculosis.

Sixteen tested LSPs were exclusive to M. avium subsp. paratuberculosis as determined by PCR. Eight of these polymorphisms were common to all isolates of the subspecies. LSPA8 was always absent, whereas LSPP2, LSPP4, LSPP11, LSPP12, LSPP14, LSPP15, and LSPP16 were always present. This distribution was confirmed by Southern hybridization. The eight other LSPs were absent, or exhibited a distinctive conformation, in a subset of M. avium subsp. paratuberculosis isolates. Notably, these lineage-specific LSPs support the existence of two major M. avium subsp. paratuberculosis genovars. The LSPA20 and “deletion 2” regions were absent exclusively from strains of the sheep lineage. Similarly, MAV-14, LSPA18, LSPA4-II, and a region of the GPL cluster were absent from all strains of the cattle lineage. LSPA11 and VA15 were unique to individual strains.

Directionality of LSPs.

The set of criteria outlined in Table Table22 was used to infer directionality for the M. avium subsp. paratuberculosis-specific genomic events. Sequence data for M. intracellulare provided an outgroup for this analysis such that regions conserved in both M. avium and M. intracellulare were considered ancestral. As such, LSPs were called deletions if absent from individual M. avium subsp. paratuberculosis isolates but present in other M. avium strains plus M. intracellulare. Conversely, because it is highly unlikely for identical deletions to occur independently in distantly related organisms, LSPs were considered insertions if present in M. avium subsp. paratuberculosis but absent from other M. avium subspecies and M. intracellulare. We concluded that six of the M. avium subsp. paratuberculosis-specific LSPs represent genomic insertions (Fig. (Fig.1).1). Likewise, we inferred that the common LSPA8 locus and the eight lineage-specific LSPs represent deletion events. Analysis of LSPP2 was inconclusive. However, the sequence is specific to M. avium subsp. paratuberculosis, and the polymorphism may represent a genomic rearrangement.

FIG. 1.
Biphasic evolution of modern M. avium subsp. paratuberculosis strains. (Phase I) Emergence of the original pathogenic clone of M. avium subsp. paratuberculosis (proto-MAP) from a strain of M. avium subsp. hominissuis via acquisition of novel DNA and polymorphisms ...

Insertions specific to M. avium subsp. paratuberculosis.

The six genomic insertions exclusive to M. avium subsp. paratuberculosis are LSPP4, LSPP11, LSPP12, LSPP14, LSPP15, and LSPP16. Together, they comprise ~125 kb. Functions for these regions and their respective ORFs are summarized in Fig. S2 in the supplemental material. Of these, LSPP4 (15.3 kb) and LSPP11 (13 kb) are putative prophages. Both regions are flanked by direct repeat sequences and contain ORFs characteristic of phages, including recombinase and integrase genes. Moreover, LSPP4 is located at a tRNA gene, a common site for prophage insertion (3, 10). Both LSPs also contain transposon sequences, which may disable the prophages and account for their stability across all M. avium subsp. paratuberculosis lineages.

LSPP12 (19.4 kb) contains a pair of divergently transcribed operons. MAP2180 to MAP2188 encode proteins predicted to have general enzymatic properties, including a P450-type cytochrome, a 3-ketoacyl-(acyl carrier protein) reductase, an amidohydrolase, and an aldehyde dehydrogenase. MAP2181 encodes a TetR-type transcriptional regulator. This arrangement is suggestive of a biosynthetic pathway, but no substrates or products have been identified. MAP2189 to MAP2194 encode an unusual mammalian cell entry (mce) operon. Although homologs to mceA to mceF are present, other genes typically associated with mce loci, such as yrbE-type permeases, are absent (4). MAP2195 and MAP2196 may be cotranscribed with this mce operon, but both are uncharacterized and, according to the M. avium subsp. paratuberculosis K-10 genome sequence, contain frameshift mutations. Because mce genes have been associated with virulence (14), LSPP12 may represent a subspecies-specific pathogenicity island.

LSPP14 (65.1 kb) is the largest M. avium subsp. paratuberculosis-specific insertion. It exhibits a mosaic structure such that ORFs are generally found in functional blocks separated by transposon sequences. Several blocks are predicted to mediate metal acquisition, including siderophore uptake (MAP3726 to MAP3729), inorganic metal uptake (MAP3731 to MAP3736), and siderophore biosynthesis (MAP3740 to MAP3746). Functions for the other blocks (MAP3749 to MAP3853, MAP3756 to MAP3758, and MAP3761 to MAP3864) are less defined, but include ORFs encoding various metabolic and transport proteins, plus an AraC-type transcriptional regulator (MAP3758). A novel polymorphism was identified during the evaluation of LSPP14. Analysis of M. avium subsp. paratuberculosis strain 85/14 (MLSA profile ST34) by PCR and Southern hybridization indicated that two internal targets, MAP3726 and MAP3750, were absent from this isolate. Using comparative genomic hybridization analysis by DNA microarray, we determined that isolate 85/14 contained a portion of LSPP14 (MAP3753 to MAP3765) but was missing a 53-kb block of genes. PCR across the deletion followed by DNA sequencing established the precise boundaries of this LSP, called LSP85/14, from bp 4138484 (MAP3718c) to bp 4191500 (MAP3753). Within the MAP3718c gene, and adjacent to the LSP85/14 junction, is an 118-bp inversion but, at present, its significance is unknown. The impact of this deletion remains to be determined, but it encompasses a region of LSPP14 initially identified as a 38-kb pathogenicity island (20). We have screened for LSP85/14 in other M. avium subsp. paratuberculosis isolates (data not shown), but to date this deletion remains unique to strain 85/14.

LSPP15 is separated from LSPP14 by only 4.8 kb. This intervening region contains genes for five ribosomal proteins. All are conserved in M. intracellulare ATCC 13950T, but the ortholog of MAP3771 (rpmE2) is absent from M. avium subsp. hominissuis 104. LSPP15 (5.4 kb) contains another putative metal transport operon and includes a Fur-like metal-dependent transcriptional regulator.

LSPP16 (6.7 kb) includes an intact transposon (ISMAP16/MAP3814), a transposase fragment (MAP3816), and ORFs encoding a putative membrane protein, a P450-type cytochrome, and two hypothetical genes. In the absence of more precise gene descriptions, it is difficult to predict a function for this insertion. Although the polymorphism defined as LSPP16 is specific to M. avium subsp. paratuberculosis strains, genomic comparisons indicate that this region is unstable, such that distinct insertions occur in other M. avium-M. intracellulare isolates. The equivalent region of the M. avium subsp. hominissuis 104 genome contains three genes (LSPA24, including an esxA-esxB-like pair that encode proteins similar to ESAT6-CFP10), while the equivalent site in the M. intracellulare type strain contains a 38-kb insertion.

Deletions specific to M. avium subsp. paratuberculosis.

The 11 genes of LSPA8 (10 kb) are absent from all M. avium subsp. paratuberculosis lineages. The impact of the deletion is unknown, but it includes two transcriptional regulators, and several genes encoding general (peptidase, hydrolase, oxidoreductase, and dehydrogenase) metabolic functions. Genomic comparisons indicate that one block of these genes (equivalent to MAV_4974 to MAV_4977) is widely conserved among mycobacteria. A second block (i.e., MAV_4978 to MAV_4982) is absent from M. intracellulare and so may be specific to certain lineages of M. avium subsp. hominissuis.

The two regions absent from all strains of the sheep lineage were analyzed previously (12, 19). LSPA20 (8 kb, also called “deletion 1”) contains eight ORFs, several of which encode components of a putative pyruvate dehydrogenase complex. Although it is tempting to speculate that loss of this system contributes to the exceedingly slow growth rate of sheep strains, the M. avium subsp. paratuberculosis genome contains two additional pyruvate dehydrogenase loci, such that functions encoded by LSPA20 may be redundant. “Deletion 2” (19.9 kb) comprises 17 ORFs of diverse functions. The region includes two transcriptional regulators, a PPE gene, an MmpS/MmpL-type transporter, and genes encoding various metabolic (e.g., esterase, amidohydrolase, and dehydrogenase) activities. The impact of their loss is not immediately evident.

Cattle strains are characterized by four deletions: LSPA4-II, LSPA18, MAV-14, and a portion of the GPL cluster. Previous analysis of the GPL cluster suggested that it had two conformations, represented by LSPP6 in M. avium subsp. paratuberculosis K-10 and LSPA9 in M. avium subsp. hominissuis 104 (12, 17, 18). However, investigation of additional strains, including those of M. avium subsp. avium and the sheep lineage of M. avium subsp. paratuberculosis, suggested greater complexity (7, 16). LSP distribution data, plus available genomic sequence from strain M. avium subsp. avium TMC 724 (ATCC 25291T; GenBank no. AF125999), support the existence of three primary configurations (Fig. (Fig.2).2). One is found in some M. avium subsp. hominissuis isolates, all M. avium subsp. avium and M. avium subsp. silvaticum strains, and the sheep genovars of M. avium subsp. paratuberculosis (Fig. (Fig.2B).2B). Conservation of this pattern across multiple M. avium subsets suggests that it constitutes the ancestral configuration. It includes all of LSPP6 and a portion of LSPA9, now designated LSPA9-I. A second configuration (Fig. (Fig.2A)2A) is specific to the cattle genovars of M. avium subsp. paratuberculosis and likely stems from a deletion event that eliminated the LSPA9-I region. A third configuration (Fig. (Fig.2C)2C) is found in a subset of M. avium subsp. hominissuis isolates, including the 104 lineage. It appears to have involved a genomic insertion event during which LSPP6 was replaced by the portion of LSPA9 now called LSPA9-II. The GPL cluster is a very dynamic region of the genome and additional variations, including prophage insertions and transposon-mediated changes, have been reported (8). Some of these contribute to the serotype variation observed among M. avium strains.

FIG. 2.
Schematic representation of the three basic genomic configurations of the M. avium GPL cluster region. (A) Truncated configuration present in cattle strains of M. avium subsp. paratuberculosis (MAP-cattle) that arose via deletion of LSPA9-I (black box) ...

Loss of LSPA4-II is notable because it truncates MAP2178, which encodes MbtA, an enzyme required for mycobactin synthesis. Although this deletion may contribute to the mycobactin dependence of cattle strains, mbtA is intact in sheep isolates. However, sequence analysis of the mycobactin biosynthesis operon of cattle strains has also revealed nonsynonymous mutations in the mbtE and mbtF genes. As such, a direct link between mbtA interruption and mycobactin dependence may be difficult to prove.

The cattle-specific deletion of LSPA18 likely stems from a genomic inversion event. Relative to other M. avium strains, the 885-kb region spanning MAP3493 to MAP4280 is inverted in the cattle lineage. MAP3493 appears to be intact, but MAP4280 represents the truncated remnants of the choD gene, putatively encoding a cholesterol oxidase and orthologous to MAV_4354. Alignment with M. avium subsp. hominissuis and M. intracellulare indicates that, in cattle strains, a copy of IS900 replaces the 19 genes adjacent to MAP4280 that comprise the LSPA18 region. At the MAP3493 edge of the inversion, two additional genes are deleted (orthologs of MAV_4351 and MAV_4353).

The distribution of MAV-14 is based on PCR amplification of internal targets. In M. avium subsp. hominissuis strain 104 this region (MAV_2974 to MAV_2999) is flanked by a pair of IS1245 elements which confounded a three-primer PCR-based strategy. The internal targets were present in all strains of M. avium subsp. hominissuis and M. avium subsp. paratuberculosis, sheep lineage. In contrast, the MAV-14 region could be bridged by PCR in all cattle isolates, demonstrating proof of absence. Among strains of the avian group, the bridging and internal PCR primer pairs failed, such that the configuration of MAV-14 could not be determined. This may be due to nucleotide polymorphisms in the primer binding region but could also indicate that the region is missing from M. avium subsp. avium and M. avium subsp. silvaticum. If so, it is expected to be an independent event, with a deletion junction distinct from that found in the cattle lineage.

The remaining M. avium subsp. paratuberculosis-specific deletions were restricted to individual strains or MLSA sequence types. LSPA11 was absent from the ST35 lineage. This deletion impacts part of a mce operon, as well as recD. Although loss of the mce genes may impact the virulence of these strains, the recD mutation may heighten susceptibility to DNA damage. Further characterization is therefore required to identify the effects of LSPA11.

The VA15 region, spanning MAP1432 to MAP1438c (MAV_3032 to MAV_3043), was recently described as a deletion specific to sheep strains of M. avium subsp. paratuberculosis (16). PCR for an internal target indicated that VA15 was present in M. avium subsp. hominissuis isolates, in M. avium subsp. paratuberculosis cattle strains, and in three of our four sheep isolates. Although this polymorphism does not define all sheep strains, the region was deleted from the ST34 lineage represented by isolate 85/14. Despite several attempts, we were unable to bridge this deletion by PCR or characterize the junction. However, the absence of both VA15 and the previously mentioned LSP85/14 was confirmed by DNA microarray analysis of strain 85/14. A larger, overlapping deletion, spanning MAP1424 to MAP1465 has been described as variable within the “avium-silvaticum genomotype” (16). Consistent with this, our internal PCR for VA15 was also negative in both strains of M. avium subsp. silvaticum and one strain of M. avium subsp. avium.

DISCUSSION

In 1895, Johne and Frothingham observed acid-fast bacilli in the intestines of a cow suffering from pseudotuberculous enteritis. In 1912, Twort and Ingram reported cultivation of the responsible organism, naming it “Mycobacterium enteritidis chronicae pseudotuberculosae bovis johne.” Ever since the initial isolation of this organism, now known as M. avium subsp. paratuberculosis, its extremely slow growth rate and apparent requirement for exogenous siderophores has intrigued researchers. Twort and Ingram (24) hypothesized that the “bacillus has lived a pathogenic existence from such remote ages that it has lost the original power of its wild ancestor—whatever bacillus that may have been—and can no longer build up all its necessary foodstuffs outside the animal body.” Through molecular methods, we can now assign the wild ancestor to be a strain of M. avium subsp. hominissuis, from which a series of insertions, deletions and rearrangements led to the emergence of the pathogen M. avium subsp. paratuberculosis.

The 25 polymorphic regions examined in the present study can be divided into three broad groups (Table (Table3):3): (i) 9 are not specific to M. avium subsp. paratuberculosis, (ii) 8 are conserved among all M. avium subsp. paratuberculosis strains, and (iii) 8 are restricted to specific lineages of M. avium subsp. paratuberculosis. This distribution of LSPs is consistent with a previous screen that used DNA from almost 400 M. avium isolates (17), with one important difference. In that study, we were unable to document the occurrence of every LSP across every M. avium subsp. paratuberculosis isolate, leaving the absolute specificity of some polymorphisms uncertain. In the present study, we have rectified several technical issues that may have influenced our earlier work. For PCR, by testing for the presence or absence of an LSP simultaneously via a multiplex approach, false-negative results due to insufficient or degraded DNA template were instantly identified for further study. We also decreased the likelihood of false-negative reactions by increasing the amount of DNA template from 2 to 5 ng to at least 25 ng per 25-μl reaction. Because this consumed a larger amount of template DNA, our panel was primarily composed of viable strains, available in-house, from which a reliable supply of genomic DNA could be extracted. To provide an additional level of support to the PCR-based assignments, we confirmed the distribution of seven M. avium subsp. paratuberculosis-specific LSPs by Southern hybridization. Together, these measures allowed us to confidently establish the distribution of each LSPs and identify eight that are truly M. avium subsp. paratuberculosis specific.

Using the criteria outlined in Table Table2,2, we determined that the M. avium subsp. paratuberculosis-specific LSPs include one common deletion (LSPA8) and six genomic insertions. The directionality of LSPP2 could not be established. The six insertions comprise ~125 kb of DNA and 82 ORFs not found in any other M. avium subspecies. Some of these ORFs have mycobacterial homologues, but most exhibit greater similarity to genes from environmental actinomycetes, including Streptomyces, Nocardia, Corynebacteria, Salinispora, Rhodococcus, and Frankia. The challenge ahead is to demonstrate functions for M. avium subsp. paratuberculosis-specific LSPs and understand how these account for the idiosyncrasies of this pathogen.

Modern strains of M. avium subsp. paratuberculosis are of two major types: sheep (also called type I) and cattle (type II). The lineage-specific deletions support this division. LSPA4-II, MAV-14, LSPA18, and a portion of the GPL cluster are absent from the cattle lineage, but present in sheep strains (Fig. (Fig.11 and and2).2). Conversely, LSPA20 and deletion 2 are absent from sheep isolates but present in cattle strains. Although our panel includes several isolates (i.e., P465, LN20, and 85/14) previously classified as “type III” or “intermediate,” the existence of a third major lineage is not supported by LSP distribution data. Strains P465, LN20, and 85/14 share the characteristic sheep-lineage deletions but are heterogeneous at other loci. LSPA11 is absent exclusively from LN20 and only 85/14 is missing VA15 and the novel LSP85/14 region. These strains are also of distinct MLSA sequence types. As more isolates are subjected to high-resolution analysis methods, including genome resequencing, it is likely that additional, novel strain-specific changes will be identified, especially among the sheep strains.

Identification of LSPs that are—-or are not—specific to M. avium subsp. paratuberculosis provides insight into evolution of this subspecies. Emergence of the original pathogenic clone of M. avium subsp. paratuberculosis involved a series of gene acquisition events and the loss of LSPA8. Subsequent deletion events resulted in the appearance of distinct sheep and cattle lineages, and ongoing reductive evolution has given rise to distinct genovars of these major lineages. This biphasic pattern describes how an innocuous environmental organism has evolved into a host-associated enteric pathogen and may represent a common process for the responsible for the evolution of other pathogenic mycobacteria.

Supplementary Material

[Supplemental material]

Acknowledgments

We are indebted to D. Collins, D. van Soolingen, G. Cangelosi, and D. Cousins for the provision of strains and DNA. We thank C. Nagy, G. Leveque, J. Dias, V. Forgetta, G. Barreau, P. Lepage, and K. Dewar from the McGill University and Génome Québec Innovation Center, who generated the M. intracellulare draft genome sequence. We thank D. Collins for critical review of the manuscript.

C.Y.T. was supported by the Lloyd-Carr Harris McGill Major and F. C. Harrison fellowships. M.A.B. is a Chercheur Boursier Senior of the FRSQ and a William Dawson Scholar of McGill University. This work was funded by an operating grant from the Canadian Institutes for Health Research (MOP-79309).

Footnotes

[down-pointing small open triangle]Published ahead of print on 21 November 2008.

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

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