Evolutionary Trends of the Transposase-Encoding Open Reading Frames A and B (orfA and orfB) of the Mycobacterial IS6110 Insertion Sequence

Abstract

BackgroundThe IS6110 insertion sequence, a member of the IS3 family of insertion sequences, was found to be specific to the Mycobacterium tuberculosis complex (MTBC). Although IS6110 has been extensively characterized as a transposable genetic marker, the evolutionary history of its own transposase-encoding sequence has not, to the best of our knowledge, been investigated.Methodology/Principal FindingsHere we explored the evolution of the IS6110 sequence by analysing the genetic variability and the selective forces acting on its transposase-encoding open reading frames (ORFs) A and B (orfA and orfB). For this purpose, we used a strain collection consisting of smooth tubercle bacilli (STB), an early branching lineage of the MTBC, and present-day M. tuberculosis strains representing the full breadth of genetic diversity in Tunisia. In each ORF, we found a major haplotype that dominated over a flat distribution of rare descendent haplotypes, consisting mainly of single- and double-nucleotide variant singletons. The predominant haplotypes consisted of both ancestral and present-day strains, suggesting that IS6110 acquisition predated the emergence of the MTBC. There was no evidence of recombination and both ORFs were subjected to strict purifying selection, as demonstrated by their dN/dS ratios (0.29 and 0.51, respectively), as well as their significantly negative Tajima’s D statistics. Strikingly, the purifying selection acting on orfA proved much more stringent, suggesting its critical role in regulating the transpositional process. Maximum likelihood analyses further excluded any possibility of positive selection acting on single amino acid residues.Conclusions/SignificanceTaken together our data fit with an evolutionary scenario according to which the observed variability pattern of the IS6110 transposase-encoding ORFs is generated mainly through random point mutations that accrued on a functionally optimal IS6110 copy, whose acquisition predated the emergence of the MTBC complex. Background selection acting against deleterious mutations led to an excess of low-frequency variants.

In-depth Analysis of IS6110 Genomic Variability in the Mycobacterium tuberculosis Complex

“…In addition, two other mutations are in the IR, which we do not expect to be involved in the transposase function as they are outside the coding region. Thabet et al (2015) also found more polymorphic sites for orfB than for orfA. They suggested that non-synonymous mutations tend to be eliminated because the ancestral IS6110 copy inherited by the MTBC would be functionally optimal.…”

“…IS 6110 has been used for a long time for diagnosis and genotyping, but very little is known about its genetic variability, mainly due to the difficulties in determining its complete sequence for each copy location in the genome. The limited data that have been published regarding this issue have shown dissimilar results ( Dale et al, 1997 ; Thabet et al, 2015 ).…”

“…We have analysed the sequences of both orfA and orfB genes that constitute the transposase in several strains for which we knew its exact location in the genome using specific primers for the amplification of each copy. So far, the only data reported on the sequence of IS 6110 were obtained using internal primers that interchangeably amplify all the IS copies of the genome ( Thabet et al, 2015 ), or the number of analysed copies was really low ( Dale et al, 1997 ). In addition, as only one copy of each strain was analysed, the intra-strain variability was not studied.…”

“…Nevertheless, they only checked one copy of each strain. Conversely, Thabet et al (2015) found several different haplotypes when they randomly cloned and sequenced only one of the IS 6110 copies of some characteristic strains from their region.…”

“…In this work, we aimed to determine whether IS 6110 is a conserved gene, as Dale et al (1997) suggested or, on the contrary, it is relatively common to find this IS mutated, as Thabet et al (2015) suggested. In addition to the clinical relevance, since it is widely used for TB diagnosis, we thought mutations of the IS 6110 could affect its transposition ability.…”