The Genome of the Obligate Intracellular Parasite Trachipleistophora hominis: New Insights into Microsporidian Genome Dynamics and Reductive Evolution

The dynamics of reductive genome evolution for eukaryotes living inside other eukaryotic cells are poorly understood compared to well-studied model systems involving obligate intracellular bacteria. Here we present 8.5 Mb of sequence from the genome of the microsporidian Trachipleistophora hominis, isolated from an HIV/AIDS patient, which is an outgroup to the smaller compacted-genome species that primarily inform ideas of evolutionary mode for these enormously successful obligate intracellular parasites. Our data provide detailed information on the gene content, genome architecture and intergenic regions of a larger microsporidian genome, while comparative analyses allowed us to infer genomic features and metabolism of the common ancestor of the species investigated. Gene length reduction and massive loss of metabolic capacity in the common ancestor was accompanied by the evolution of novel microsporidian-specific protein families, whose conservation among microsporidians, against a background of reductive evolution, suggests they may have important functions in their parasitic lifestyle. The ancestor had already lost many metabolic pathways but retained glycolysis and the pentose phosphate pathway to provide cytosolic ATP and reduced coenzymes, and it had a minimal mitochondrion (mitosome) making Fe-S clusters but not ATP. It possessed bacterial-like nucleotide transport proteins as a key innovation for stealing host-generated ATP, the machinery for RNAi, key elements of the early secretory pathway, canonical eukaryotic as well as microsporidian-specific regulatory elements, a diversity of repetitive and transposable elements, and relatively low average gene density. Microsporidian genome evolution thus appears to have proceeded in at least two major steps: an ancestral remodelling of the proteome upon transition to intracellular parasitism that involved reduction but also selective expansion, followed by a secondary compaction of genome architecture in some, but not all, lineages.     

ISWpi1 from Wolbachia pipientis defines a novel group of insertion sequences within the IS5 family

Insertion sequences are transposable elements that can represent substantial proportions of prokaryotic genomes and play a substantial role in shaping host genome evolution. As such, evaluating and understanding insertion sequence diversity is an important task to fulfill, because it is expected to yield new insight into the evolution of bacterial transposable elements and contribute to improve genome annotations. Here, I characterized an insertion sequence, termed ISWpi1, for which the taxonomic distribution appears to be restricted to the obligate intracellular alpha-Proteobacterium Wolbachia pipientis. ISWpi1 exhibits approximately 46% identity at the amino acid level with members of the IS1031 group of insertion sequences from the IS5 family. However, the IS1031 group is characterized by a transposase gene encoded by a single open reading frame, whereas the ISWpi1 transposase gene consists of two overlapping open reading frames presumably translated as a single protein via programmed translational frameshifting. Such structure suggests that ISWpi1 may instead be related to the IS427 group of insertion sequences from the IS5 family. Altogether, these data indicate that ISWpi1 extends the known spectrum of diversity of the IS5 family, and I propose to define a novel group of insertion sequences within the IS5 family typified by ISWpi1. Probable transpositional activity, relevant insertion site preferences and taxonomic specificity make ISWpi1 a promising tool for experimentally manipulating W. pipientis bacteria, especially in light of the increasing interest in developing these bacteria as tools for controlling insect disease vectors and agricultural pests.     

Local hopping mobile DNA implicated in pseudogene formation and reductive evolution in an obligate cyanobacteria-plant symbiosis

Background

Insertion sequences (ISs) are approximately 1 kbp long “jumping” genes found in prokaryotes. ISs encode the protein Transposase, which facilitates the excision and reinsertion of ISs in genomes, making these sequences a type of class I (“cut-and-paste”) Mobile Genetic Elements. ISs are proposed to be involved in the reductive evolution of symbiotic prokaryotes. Our previous sequencing of the genome of the cyanobacterium ‘Nostoc azollae’ 0708, living in a tight perpetual symbiotic association with a plant (the water fern Azolla), revealed the presence of an eroding genome, with a high number of insertion sequences (ISs) together with an unprecedented large proportion of pseudogenes. To investigate the role of ISs in the reductive evolution of ‘Nostoc azollae’ 0708, and potentially in the formation of pseudogenes, a bioinformatic investigation of the IS identities and positions in 47 cyanobacterial genomes was conducted. To widen the scope, the IS contents were analysed qualitatively and quantitatively in 20 other genomes representing both free-living and symbiotic bacteria.

Results

Insertion Sequences were not randomly distributed in the bacterial genomes and were found to transpose short distances from their original location (“local hopping”) and pseudogenes were enriched in the vicinity of IS elements. In general, symbiotic organisms showed higher densities of IS elements and pseudogenes than non-symbiotic bacteria. A total of 1108 distinct repeated sequences over 500 bp were identified in the 67 genomes investigated. In the genome of ‘Nostoc azollae’ 0708, IS elements were apparent at 970 locations (14.3%), with 428 being full-length. Morphologically complex cyanobacteria with large genomes showed higher frequencies of IS elements, irrespective of life style.

Conclusions

The apparent co-location of IS elements and pseudogenes found in prokaryotic genomes implies earlier IS transpositions into genes. As transpositions tend to be local rather than genome wide this likely explains the proximity between IS elements and pseudogenes. These findings suggest that ISs facilitate the reductive evolution in for instance in the symbiotic cyanobacterium ‘Nostoc azollae’ 0708 and in other obligate prokaryotic symbionts.

Electronic supplementary material

The online version of this article (doi:10.1186/s12864-015-1386-7) contains supplementary material, which is available to authorized users.     

Mobile DNA in obligate intracellular bacteria

The small genomes of obligate intracellular bacteria are often presumed to be impervious to mobile DNA and the fluid genetic processes that drive diversification in free-living bacteria. Categorized by reductive evolution and streamlining, the genomes of some obligate intracellular bacteria manifest striking degrees of stability and gene synteny. However, recent findings from complete genome sequences of obligate intracellular species and their mobile genetic associates favour the abandonment of these wholesale terms for a more complex and tantalizing picture.     

Obligate intracellular parasites: Rickettsia prowazekii and Chlamydia trachomatis.

Transitions to obligate intracellular parasitism have occurred at numerous times in the evolutionary past. The genome sequences of two obligate intracellular parasites, Rickettsia prowazekii and Chlamydia trachomatis, were published last year. A comparative analysis of these two genomes has revealed examples of reductive convergent evolution, such as a massive loss of genes involved in biosynthetic functions. In addition, both genomes were found to encode transport systems for ATP and ADP, not otherwise found in bacteria. Here, we discuss adaptations to intracellular habitats by comparing the information obtained from the recently published genome sequences of R. prowazekii and C. trachomatis.     

The distribution of insertion sequences in the genome of Shigella flexneri strain 2457T

Shigella flexneri, which causes shigellosis in humans, evolved from Escherichia coli. The sequencing of Shigella genomes has revealed that a large number of insertion sequence (IS) elements (over 200 elements) reside in the genome. Although the presence of these elements has been noted previously and summarized, more detailed analyses are required to understand their evolutionary significance. Here, the genome of S. flexneri strain 2457T is used to investigate the spatial distribution of IS copies around the chromosome and the location of elements with respect to genes. It is found that most IS isoforms occur essentially randomly around the genome. Two exceptions are IS91 and IS911, which appear to cluster due to local hopping. The location of IS elements with respect to genes is biased, however, revealing the action of natural selection. The non-coding regions of the genome (no more than 21%) carry disproportionally more IS elements (at least 28%) than the coding regions, implying that selection acts against insertion into genes. Of the genes disrupted by ISs, those involved in signal transduction, intracellular trafficking, and cell motility are most commonly targeted, suggesting selection against genes in these categories.     

Molecular Characterization of the Principal Symbiotic Bacteria of the Weevil Sitophilus oryzae: A Peculiar G + C Content of an Endocytobiotic DNA

The principal intracellular symbiotic bacteria of the cereal weevil Sitophilus oryzae were characterized using the sequence of the 16S rDNA gene (rrs gene) and G + C content analysis. Polymerase chain reaction amplification with universal eubacterial primers of the rrs gene showed a single expected sequence of 1,501 bp. Comparison of this sequence with the available database sequences placed the intracellular bacteria of S. oryzae as members of the Enterobacteriaceae family, closely related to the free-living bacteria, Erwinia herbicola and Escherichia coli, and the endocytobiotic bacteria of the tsetse fly and aphids. Moreover, by high-performance liquid chromatography, we measured the genomic G + C content of the S. oryzae principal endocytobiotes (SOPE) as 54%, while the known genomic G + C content of most intracellular bacteria is about 39.5%. Furthermore, based on the third codon position G + C content and the rrs gene G + C content, we demonstrated that most intracellular bacteria except SOPE are A + T biased irrespective of their phylogenetic position. Finally, using the hsp60 gene sequence, the codon usage of SOPE was compared with that of two phylogenetically closely related bacteria: E. coli, a free-living bacterium, and Buchnera aphidicola, the intracellular symbiotic bacteria of aphids. Taken together, these results show a peculiar and distinctly different DNA composition of SOPE with respect to the other obligate intracellular bacteria, and, combined with biological and biochemical data, they elucidate the evolution of symbiosis in S. oryzae. Received: 8 September 1997 / Accepted: 24 October 1997     

Intense transpositional activity of insertion sequences in an ancient obligate endosymbiont

The streamlined genomes of ancient obligate endosymbionts generally lack transposable elements, such as insertion sequences (IS). Yet, the genome of Wolbachia, one of the most abundant bacterial endosymbionts on Earth, is littered with IS. Such a paradox raises the question as to why there are so many ISs in the genome of this ancient endosymbiont. To address this question, we investigated IS transpositional activity in the unculturable Wolbachia by tracking the evolutionary dynamics and history of ISWpi1 elements. We show that 1) ISWpi1 is widespread in Wolbachia, being present in at least 55% of the 40 sampled strains, 2) ISWpi1 copies exhibit virtually identical nucleotide sequences both within and among Wolbachia genomes and possess an intact transposase gene, 3) individual ISWpi1 copies are differentially inserted among Wolbachia genomes, and 4) ISWpi1 occurs at variable copy numbers among Wolbachia genomes. Collectively, our results provide compelling evidence for intense ISWpi1 transpositional activity and frequent ISWpi1 horizontal transmission among strains during recent Wolbachia evolution. Thus, the genomes of ancient obligate endosymbionts can carry high loads of functional and transpositionally active transposable elements. Our results also indicate that Wolbachia genomes have experienced multiple and temporally distinct ISWpi1 invasions during their evolutionary history. Such recurrent exposition to new IS invasions may explain, at least partly, the unusually high density of transposable elements found in the genomes of Wolbachia endosymbionts.     

An insertion sequence-dependent plasmid rearrangement in Aeromonas salmonicida causes the loss of the type three secretion system

Aeromonas salmonicida, a bacterial fish pathogen, possesses a functional Type Three Secretion System (TTSS), which is essential for its virulence. The genes for this system are mainly located in a single region of the large pAsa5 plasmid. Bacteria lose the TTSS region from this plasmid through rearrangements when grown in stressful growth conditions. The A. salmonicida genome is rich in insertion sequences (ISs), which are mobile DNA elements that can cause DNA rearrangements in other bacterial species. pAsa5 possesses numerous ISs. Three IS11s from the IS256 family encircle the rearranged regions. To confirm that these IS11s are involved in pAsa5 rearrangements, 26 strains derived from strain A449 and two Canadian isolates (01-B526 and 01-B516) with a pAsa5 rearrangement were tested using a PCR approach to determine whether the rearrangements were the result of an IS11-dependent process. Nine out of the 26 strains had a positive PCR result, suggesting that the rearrangement in these strains were IS-dependent. The PCR analysis showed that all the rearrangements in the A449-derived strains were IS11-dependent process while the rearrangements in 01-B526 and 01-B516 could only be partially coupled to the action of IS11. Unidentified elements that affect IS-dependent rearrangements may be present in 01-B526 and 01-B516. Our results suggested that pAsa5 rearrangements involve IS11. This is the first study showing that ISs are involved in plasmid instability in A. salmonicida.     

Correlations Between Bacterial Ecology and Mobile DNA

Several factors can affect the density of mobile DNA in bacterial genomes including rates of exposure to novel gene pools, recombination, and reductive evolution. These traits are difficult to measure across a broad range of bacterial species, but the ecological niches occupied by an organism provide some indication of the relative magnitude of these forces. Here, by analyzing 384 bacterial genomes assigned to three ecological categories (obligate intracellular, facultative intracellular, and extracellular), we address two, related questions: How does the density of mobile DNA vary across the Bacteria? And is there a statistically supported relationship between ecological niche and mobile element gene density? We report three findings. First, the fraction of mobile element genes in bacterial genomes ranges from 0 to 21% and decreases significantly: facultative intracellular > extracellular > obligate intracellular bacteria. Results further show that the obligate intracellular bacteria that host switch have a higher mobile DNA gene density than the obligate intracellular bacteria that are vertically transmitted. Second, while bacteria from the three ecological niches differ in their average mobile DNA contents, the ranges of mobile DNA found in each category overlap a surprising extent, suggesting bacteria with different lifestyles can tolerate similar amounts of mobile DNA. Third, mobile DNA gene densities increase with genome size across the entire dataset, and the significance of this correlation is dependent on the obligate intracellular bacteria. Further, mobile DNA gene densities do not correlate with evolutionary relationships in a 16S rDNA phylogeny. These findings statistically support a compelling link between mobile element evolution and bacterial ecology.     

Evolutionary genomics of a temperate bacteriophage in an obligate intracellular bacteria (Wolbachia)

Genome evolution of bacteria is usually influenced by ecology, such that bacteria with a free-living stage have large genomes and high rates of horizontal gene transfer, while obligate intracellular bacteria have small genomes with typically low amounts of gene exchange. However, recent studies indicate that obligate intracellular species that host-switch frequently harbor agents of horizontal transfer such as mobile elements. For example, the temperate double-stranded DNA bacteriophage WO in Wolbachia persistently transfers between bacterial coinfections in the same host. Here we show that despite the phage's rampant mobility between coinfections, the prophage's genome displays features of constraint related to its intracellular niche. First, there is always at least one intact prophage WO and usually several degenerate, independently-acquired WO prophages in each Wolbachia genome. Second, while the prophage genomes are modular in composition with genes of similar function grouping together, the modules are generally not interchangeable with other unrelated phages and thus do not evolve by the Modular Theory. Third, there is an unusual core genome that strictly consists of head and baseplate genes; other gene modules are frequently deleted. Fourth, the prophage recombinases are diverse and there is no conserved integration sequence. Finally, the molecular evolutionary forces acting on prophage WO are point mutation, intragenic recombination, deletion, and purifying selection. Taken together, these analyses indicate that while lateral transfer of phage WO is pervasive between Wolbachia with occasional new gene uptake, constraints of the intracellular niche obstruct extensive mixture between WO and the global phage population. Although the Modular Theory has long been considered the paradigm of temperate bacteriophage evolution in free-living bacteria, it appears irrelevant in phages of obligate intracellular bacteria.     

The Whole-genome Sequencing of the Obligate Intracellular Bacterium Orientia tsutsugamushi Revealed Massive Gene Amplification During Reductive Genome Evolution

Scrub typhus (‘Tsutsugamushi’ disease in Japanese) is a mite-borne infectious disease. The causative agent is Orientia tsutsugamushi, an obligate intracellular bacterium belonging to the family Rickettsiaceae of the subdivision alpha-Proteobacteria. In this study, we determined the complete genome sequence of O. tsutsugamushi strain Ikeda, which comprises a single chromosome of 2 008 987 bp and contains 1967 protein coding sequences (CDSs). The chromosome is much larger than those of other members of Rickettsiaceae, and 46.7% of the sequence was occupied by repetitive sequences derived from an integrative and conjugative element, 10 types of transposable elements, and seven types of short repeats of unknown origins. The massive amplification and degradation of these elements have generated a huge number of repeated genes (1196 CDSs, categorized into 85 families), many of which are pseudogenes (766 CDSs), and also induced intensive genome shuffling. By comparing the gene content with those of other family members of Rickettsiacea, we identified the core gene set of the family Rickettsiaceae and found that, while much more extensive gene loss has taken place among the housekeeping genes of Orientia than those of Rickettsia, O. tsutsugamushi has acquired a large number of foreign genes. The O. tsutsugamushi genome sequence is thus a prominent example of the high plasticity of bacterial genomes, and provides the genetic basis for a better understanding of the biology of O. tsutsugamushi and the pathogenesis of ‘Tsutsugamushi’ disease.Key words: Orientia tsutsugamushi, genome sequencing, obligate intracellular bacterium, repetitive sequence, IS element, integrative and conjugative element, gene amplification, genome reduction     

Mutations and rearrangements in the genome of Sulfolobus solfataricus P2

The genome of Sulfolobus solfataricus P2 carries a larger number of transposable elements than any other sequenced genome from an archaeon or bacterium and, as a consequence, may be particularly susceptible to rearrangement and change. In order to gain more insight into the natures and frequencies of different types of mutation and possible rearrangements that can occur in the genome, the pyrEF locus was examined for mutations that were isolated after selection with 5-fluoroorotic acid. About two-thirds of the 130 mutations resulted from insertions of mobile elements, including insertion sequence (IS) elements and a single nonautonomous mobile element, SM2. For each of these, the element was identified and shown to be present at its original genomic position, consistent with a progressive increase in the copy numbers of the mobile elements. In addition, several base pair substitutions, as well as small deletions, insertions, and a duplication, were observed, and about one-fifth of the mutations occurred elsewhere in the genome, possibly in an orotate transporter gene. One mutant exhibited a 5-kb genomic rearrangement at the pyrEF locus involving a two-step IS element-dependent reaction, and its boundaries were defined using a specially developed "in vitro library" strategy. Moreover, while searching for the donor mobile elements, evidence was found for two major changes that had occurred in the genome of strain P2, one constituting a single deletion of about 4% of the total genome (124 kb), while the other involved the inversion of a 25-kb region. Both were bordered by IS elements and were inferred to have arisen through recombination events. The results underline the caution required in working experimentally with an organism such as S. solfataricus with a continually changing genome.     

Identification and distribution of new insertion sequences in the genome of alkaliphilic Bacillus halodurans C-125.

Fifteen kinds of new insertion sequences (ISs), IS641 to IS643, IS650 to IS658, IS660, IS662, and IS663, and a group II intron (Bh.Int) were identified in the 4,202,352-bp genome of alkaliphilic Bacillus halodurans C-125. Out of 120 ISs identified in the C-125 genome, 29 were truncated, indicating the occurrence of internal rearrangements of the genome. The ISs other than IS650, IS653, IS660, and IS663 generated a 2- to 9-bp duplication of the target site sequence, and the ISs other than IS650, IS653, and IS657 carry 14- to 64-bp inverted repeats. Sequence analysis revealed that six kinds of ISs (IS642, IS643, IS654, IS655, IS657, and IS658) belong to a separate IS family (IS630, IS21, IS256, IS3, IS200/IS605, and IS30, respectively) as a new member. Also, IS651 and IS652 were characterized as new members of the ISL3 family. Significant similarity was found between the transposase (Tpase) sequences between IS650 and IS653 (78.2%), IS651 and IS652 (56.3%), IS656 and IS662 (71.0%), and IS660 and IS663 (44.5%), but the others showed no similarity to one another. Tpases in 28 members of IS651 in the C-125 genome were found to have become diversified. Most of the IS elements widely distributed throughout the genome were inserted in noncoding regions, although some genes, such as those coding for an ATP-binding cassette transporter/permease, a response regulator, and L-indole 2-dehydrogenase, have been mutated through the insertion of IS elements. It is evident, however, that not all IS elements have transposed and caused rearrangements of the genome in the past 17 years during which strain C-125 was subcultured under neutral and alkaline conditions.     

Competition between Transposable Elements and Mutator Genes in Bacteria

Although both genotypes with elevated mutation rate (mutators) and mobilization of insertion sequence (IS) elements have substantial impact on genome diversification, their potential interactions are unknown. Moreover, the evolutionary forces driving gradual accumulation of these elements are unclear: Do these elements spread in an initially transposon-free bacterial genome as they enable rapid adaptive evolution? To address these issues, we inserted an active IS1 element into a reduced Escherichia coli genome devoid of all other mobile DNA. Evolutionary laboratory experiments revealed that IS elements increase mutational supply and occasionally generate variants with especially large phenotypic effects. However, their impact on adaptive evolution is small compared with mismatch repair mutator alleles, and hence, the latter impede the spread of IS-carrying strains. Given their ubiquity in natural populations, such mutator alleles could limit early phase of IS element evolution in a new bacterial host. More generally, our work demonstrates the existence of an evolutionary conflict between mutation-promoting mechanisms.     

Widespread recombination throughout Wolbachia genomes

Evidence is growing that homologous recombination is a powerful source of genetic variability among closely related free-living bacteria. Here we investigate the extent of recombination among housekeeping genes of the endosymbiotic bacteria Wolbachia. Four housekeeping genes, gltA, dnaA, ftsZ, and groEL, were sequenced from a sample of 22 strains belonging to supergroups A and B. Sequence alignments were searched for recombination within and between genes using phylogenetic inference, analysis of genetic variation, and four recombination detection programs (MaxChi, Chimera, RDP, and Geneconv). Independent analyses indicate no or weak intragenic recombination in ftsZ, dnaA, and groEL. Intragenic recombination affects gltA, with a clear evidence of horizontal DNA transfers within and between divergent Wolbachia supergroups. Intergenic recombination was detected between all pairs of genes, suggesting either a horizontal exchange of a genome portion encompassing several genes or multiple recombination events involving smaller tracts along the genome. Overall, the observed pattern is compatible with pervasive recombination. Such results, combined with previous evidence of recombination in a surface protein, phage, and IS elements, support an unexpected chimeric origin of Wolbachia strains, with important implications for Wolbachia phylogeny and adaptation of these obligate intracellular bacteria in arthropods.     

The "domino theory" of gene death: gradual and mass gene extinction events in three lineages of obligate symbiotic bacterial pathogens

During the adaptation of an organism to a parasitic lifestyle, various gene functions may be rendered superfluous due to the fact that the host may supply these needs. As a consequence, obligate symbiotic bacterial pathogens tend to undergo reductive genomic evolution through gene death (nonfunctionalization or pseudogenization) and deletion. Here, we examine the evolutionary sequence of gene-death events during the process of genome miniaturization in three bacterial species that have experienced extensive genome reduction: Mycobacterium leprae, Shigella flexneri, and Salmonella typhi. We infer that in all three lineages, the distribution of functional categories is similar in pseudogenes and genes but different from that of absent genes. Based on an analysis of evolutionary distances, we propose a two-step "domino effect" model for reductive genome evolution. The process starts with a gradual gene-by-gene-death sequence of events. Eventually, a crucial gene within a complex pathway or network is rendered nonfunctional triggering a "mass gene extinction" of the dependent genes. In contrast to published reports according to which genes belonging to certain functional categories are prone to nonfunctionalization more frequently and earlier than genes belonging to other functional categories, we could discern no characteristic regularity in the temporal order of function loss.     

Transposition of a bacterial insertion sequence in chloroplasts

Bacterial transposable elements (IS elements, transposons) represent an important determinant of genome structure and dynamics, and are a major force driving genome evolution. Here, we have tested whether bacterial insertion sequences (IS elements) can transpose in a prokaryotic compartment of the plant cell, the plastid (chloroplast). Using plastid transformation, we have integrated different versions of the Escherichia coli IS element IS 150 into the plastid genome of tobacco ( Nicotiana tabacum ) plants. We show that IS 150 is faithfully mobilized inside the chloroplast, and that enormous quantities of transposition intermediates accumulate. As synthesis of the IS 150 transposase is dependent upon programmed ribosomal frame shifting, our data indicate that this process also occurs in chloroplasts. Interestingly, all insertion events detected affect a single site in the plastid genome, suggesting that the integration of IS 150 is highly sequence dependent. In contrast, the initiation of the transposition process was found to be independent of the sequence context. Finally, our data also demonstrate that plastids lack the capacity to repair double-strand breaks in their genomes by non-homologous end joining, a finding that has important implications for genome stability, and which may explain the peculiar immunity of the plastid to invading promiscuous DNA sequences of nuclear and mitochondrial origin.     

The control of copy number of IS6110 in Mycobacterium tuberculosis

Insertion sequence (IS) elements are bacterial genes that are able to transpose to different locations in the genome. These elements are often used in molecular epidemiology as genetic markers that track the spread of pathogens. Transposable elements have frequently been described as "selfish DNA" because they facilitate their own transposition, causing damage when they insert into coding regions, while contributing little if anything to the bacterial host. According to this hypothesis, the expansion of copy number of insertion sequences is opposed by negative selection against high copy numbers. From an alternative point of view, we might expect IS elements to intrinsically regulate transposition within cells, thereby limiting damage to their bacterial host. Here, we report evidence that the copy number of IS6110 in Mycobacterium tuberculosis is controlled by selection against the element. We first construct 12 different models of marker change resulting from a combination of possible transposition functions and selective regimes. We then compute the Akaike Information Criterion for each model to identify the models that best explain data consisting of serial isolates of M. tuberculosis genotyped with IS6110. We find that the best performing models all include selection against the accumulation of copies. Specifically, our analysis points to the interaction of separate copies of the element causing lethal effects. We discuss the implications of these findings for genome evolution and molecular epidemiology.