Abstract
Virulence genes on mobile DNAs such as genomic islands (GIs) and plasmids promote bacterial pathogen emergence. Excision is an early step in GI mobilization, producing a circular GI and a deletion site in the chromosome; circular forms are also known for some bacterial insertion sequences (ISs). The recombinant sequence at the junctions of such circles and deletions can be detected sensitively in high-throughput sequencing data, using new computational methods that enable empirical discovery of mobile DNAs. For the rich mobilome of a hospital Klebsiella pneumoniae strain, circularization junctions (CJs) were detected for six GIs and seven IS types. Our methods revealed differential biology of multiple mobile DNAs, imprecision of integrases and transposases, and differential activity among identical IS copies for IS26, ISKpn18 and ISKpn21. Using the resistance of circular dsDNA molecules to exonuclease, internally calibrated with the native plasmids, showed that not all molecules bearing GI CJs were circular. Transpositions were also detected, revealing replicon preference (ISKpn18 prefers a conjugative IncA/C2 plasmid), local action (IS26), regional preferences, selection (against capsule synthesis) and IS polarity inversion. Efficient discovery and global characterization of numerous mobile elements per experiment improves accounting for the new gene combinations that arise in emerging pathogens.
Highlights
Bacterial pathogens acquire many of their antibiotic resistance and virulence genes as cargo on mobile elements that transfer between cells, typically through conjugation tubes or phage particles
While characterizing the newly discovered insertion sequence ISKpn21, we had found that some Illumina reads were from the junction region of circularized forms of the insertion sequences (ISs), indicating that NGS data report on DNA mobility events occurring during culture (Figure 1)
Microbiologists worked with phage and bacterial stocks and thought of integration as the forward reaction for integrase
Summary
Bacterial pathogens acquire many of their antibiotic resistance and virulence genes as cargo on mobile elements that transfer between cells, typically through conjugation tubes or phage particles. Other mobile elements that move only intracellularly, such as transposons, are relevant to pathogen emergence because they can insert into, and thereby augment the cargos of, the inter-bacterial mobile DNAs. While plasmids are readily identified in genome projects by their circular assembly maps, GIs can be more challenging to delineate. Phage-encoded integrase catalyzes crossover between the phage attP site and the related attB chromosomal target site, leaving the prophage flanked by the recombinant attL and attR sites [1]. Upon induction of the lysogen, the reverse excision reaction leaves (i) circular lambda DNA whose circularization junction (CJ) is the regenerated attP and (ii) a deletion junction (DJ) in the chromosome, the regenerated attB (Figure 1). Excised phage lambda goes on to replicate intracellularly [5]
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