A survey of bacterial insertion sequences using IScan

Abstract

Bacterial insertion sequences (ISs) are the simplest kinds of bacterial mobile DNA. Evolutionary studies need consistent IS annotation across many different genomes. We have developed an open-source software package, IScan, to identify bacterial ISs and their sequence elements--inverted and target direct repeats--in multiple genomes using multiple flexible search parameters. We applied IScan to 438 completely sequenced bacterial genomes and 20 IS families. The resulting data show that ISs within a genome are extremely similar, with a mean synonymous divergence of K(s) = 0.033. Our analysis substantially extends previously available information, and suggests that most ISs have entered bacterial genomes recently. By implication, their population persistence may depend on horizontal transfer. We also used IScan's ability to analyze the statistical significance of sequence similarity among many IS inverted repeats. Although the inverted repeats of insertion sequences are evolutionarily highly flexible parts of ISs, we show that this ability can be used to enrich a dataset for ISs that are likely to be functional. Applied to the thousands of genomes that will soon be available, IScan could be used for many purposes, such as mapping the evolutionary history and horizontal transfer patterns of different ISs.

Figure 1.

Illustration of the different parameters IScan uses to identify ISs. The thin horizontal line at the bottom of the panel indicates the queried DNA sequence (genome). Thick arrows indicate matches to IS ORFs. Open bars indicate inverted repeats (middle) and direct target repeats (upper). See main text for explanation of parameters.

Figure 2.

(a) Length distribution of ISs with more than 35% amino acid sequence identity relative to a curated reference sequence. (b) Distribution of the number of IS copies per genome for four abundant IS families studied here. Note the logarithmic scale on the vertical axis, and the skewed distribution.

Figure 3.

Within-genome distribution of (a) overall nucleotide divergence (in fraction of pairwise nucleotide differences), (b) non-synonymous divergence per non-synonymous site K_a (38), (c) synonymous divergence at synonymous sites K_s (38), of IS pairs in the same family, for all ISs where there exist genomes with more than one IS of the same family. Note the logarithmic scale and the peak at identical ISs, which shows that many ISs from the same family within a genome are identical to each other. Here, 1.42% of IS pairs in a genome have a K_s > 1 and are not shown in (b).

Figure 4.

Means and SEs of (a) within-genome K_a and (b) within-genome K_s for those IS families where more than one family member occurred in at least one genome.

Figure 5.

Illustration of the different alignment strategies pursued to assess statistical significance of inverted repeat alignments. The candidate IS is the sequence match produced by IScan to a reference IS from a given family. See text for details.

Figure 6.

Distribution of five test statistics (see text) indicating inverted repeat quality for all ISs studied here. (a) P_LR; (b) P₃; (c) P_L × P_R. The value 0.000023 is the Bonferroni-corrected P-value of 0.05.