Abstract
ABSTRACTInsertion sequence (IS) elements are found throughout bacterial genomes and contribute to genome variation by interrupting genes or altering gene expression. Few of the more than 30 IS elements described in Acinetobacter baumannii have been characterized for transposition activity or expression effects. A targeted sequencing method, IS-seq, was developed to efficiently map the locations of new insertion events in A. baumannii genomes and was used to identify novel IS sites following growth in the presence of hydrogen peroxide, which causes oxidative stress. Serial subculture in the presence of subinhibitory concentrations of hydrogen peroxide led to rapid selection of cells carrying an ISAba1 element upstream of the catalase-peroxidase gene katG. Several additional sites for the elements ISAba1, ISAba13, ISAba25, ISAba26, and ISAba125 were found at low abundance after serial subculture, indicating that each element is active and contributes to genetic variation that may be subject to selection. Following hydrogen peroxide exposure, rapid changes in gene expression were observed in genes related to iron homeostasis. The IS insertions adjacent to katG resulted in more than 20-fold overexpression of the gene and increased hydrogen peroxide tolerance.IMPORTANCE Insertion sequences (IS) contribute to genomic and phenotypic variation in many bacterial species, but little is known about how transposition rates vary among elements or how selective pressure influences this process. A new method for identifying new insertion locations that arise under experimental growth conditions in the genome, termed IS-seq, was developed and tested with cells grown in the presence of hydrogen peroxide, which causes oxidative stress. Gene expression changes in response to hydrogen peroxide exposure are similar to those observed in other species and include genes that control free iron concentrations. New IS insertions adjacent to a gene encoding a catalase enzyme confirm that IS elements can rapidly contribute to adaptive variation in the presence of selection.
Highlights
Insertion sequence (IS) elements are found throughout bacterial genomes and contribute to genome variation by interrupting genes or altering gene expression
In the The final cultures at 48 h (T48) samples of cells grown in the presence of hydrogen peroxide, a large number of reads supported distinct insertion events of ISAba1 elements upstream of the katG gene in replicates R1 and R2 (Fig. 2A)
The KatG protein is a bifunctional hydroperoxidase I (HPI), exhibiting both catalase and peroxidase activity involved in detoxifying hydrogen peroxide [19], and was shown to be the major contributor to H2O2 resistance in Acinetobacter species [25]
Summary
Insertion sequence (IS) elements are found throughout bacterial genomes and contribute to genome variation by interrupting genes or altering gene expression. A targeted sequencing method, IS-seq, was developed to efficiently map the locations of new insertion events in A. baumannii genomes and was used to identify novel IS sites following growth in the presence of hydrogen peroxide, which causes oxidative stress. We observed new distinct insertion sites for IS elements that arose since strain divergence in very closely related isolates, including in sets of strains obtained from individual patients over a period of days [2], that can have significant impacts on transcription [3]. Pairs of IS elements can act as composite transposons, mobilizing resistance or other genes, including blaOXA-235, which is flanked by inverted-repeat copies of ISAba1 [7]
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