Perspective on sequence evolution of microsatellite locus (CCG)n in Rv0050 gene from Mycobacterium tuberculosis

Abstract

Background: The mycobacterial genome is inclined to polymerase slippage and a high mutation rate in microsatellite regions due to high GC content and absence of a mismatch repair system. However, the exact molecular mechanisms underlying microsatellite variation have not been fully elucidated. Here, we investigated mutation events in the hyper-variable trinucleotide microsatellite locus MML0050 located in the Rv0050 gene of W-Beijing and non-W-Beijing Mycobacterium tuberculosis strains in order to gain insight into the genomic structure and activity of repeated regions.

Results: Size analysis indicated the presence of five alleles that differed in length by three base pairs. Moreover, nucleotide gains occurred more frequently than loses in this trinucleotide microsatellite. Mutation frequency was not completely related with the total length, though the relative frequency in the longest allele was remarkably higher than that in the shortest. Sequence analysis was able to detect seven alleles and revealed that point mutations enhanced the level of locus variation. Introduction of an interruptive motif correlated with the total allele length and genetic lineage, rather than the length of the longest stretch of perfect repeats. Finally, the level of locus variation was drastically different between the two genetic lineages.

Conclusion: The Rv0050 locus encodes the bifunctional penicillin-binding protein ponA1 and is essential to mycobacterial survival. Our investigations of this particularly dynamic genomic region provide insights into the overall mode of microsatellite evolution. Specifically, replication slippage was implicated in the mutational process of this microsatellite and a sequence-based genetic analysis was necessary to determine that point mutation events acted to maintain microsatellite size integrity while providing genomic diversity.

Figure 1

Distribution of the MML0050 alleles in 462 M. tuberculosis strains as determined by PCR-SSCP. The numbers in brackets refer to the repeat number of each allele.

Figure 2

Distributions of alleles from the MML0050 locus in W-Beijing and non-W-Beijing strains. a: Rv0050*05 (CCG)₈TCG; b: Rv0050*06 (CCG)₃CCT (CCG)₄ TCG.

Figure 3

The neighbor-joining tree of MML0050 alleles based upon the aligned sequence using MEGA4.0 software.

Figure 4

Mutation models of trinucleotide microsatellite locus MML0050 located in the Rv0050 gene. *Location of base substitution.