A novel family of integrases associated with prophages and genomic islands integrated within the tRNA-dihydrouridine synthase A (dusA) gene

Abstract

Genomic islands play a key role in prokaryotic genome plasticity. Genomic islands integrate into chromosomal loci such as transfer RNA genes and protein coding genes, whilst retaining various cargo genes that potentially bestow novel functions on the host organism. A gene encoding a putative integrase was identified at a single site within the 5' end of the dusA gene in the genomes of over 200 bacteria. This integrase was discovered to be a component of numerous genomic islands, which appear to share a target site within the dusA gene. dusA encodes the tRNA-dihydrouridine synthase A enzyme, which catalyses the post-transcriptional reduction of uridine to dihydrouridine in tRNA. Genomic islands encoding homologous dusA-associated integrases were found at a much lower frequency within the related dusB and dusC genes, and non-dus genes. Excision of these dusA-associated islands from the chromosome as circularized intermediates was confirmed by polymerase chain reaction. Analysis of the dusA-associated islands indicated that they were highly diverse, with the integrase gene representing the only universal common feature.

Figure 1.

The inferred phylogenetic relationship of the DAIs in relation to representative tyrosine recombinases of the shufflon-specific DNA recombinase (Rci), phage integrase (Phage), integrative and conjugative element (IntC), integron integrase (IntI), site-specific recombinase (XerDC) and genomic island integrase (IntG) families. Protein accession numbers of sequences used to generate this phylogenetic tree are in brackets. The interior values are the bootstrap probabilities after 1000 replicates.

Figure 2.

PCR detection of excised dus-associated GEIs (X) and restored dusA/dusB chromosomal junctions (J) in Acinetobacter baumannii D1279779 (740 bp and 494 bp), A. baumannii ACICU (534 bp each) and Pseudomonas protegens Pf-5 (472 bp and 485 bp). The faint high molecular weight bands originate from genomic DNA template added to the PCR reactions for A. baumannii ACICU and P. protegens Pf-5 (black arrow). Covalently closed circular DNA (cccDNA) was used as template for A. baumannii D1279779.

Figure 3.

Excision and integration of the Acinetobacter baumannii D1279779 dusA-specific GEI. The dusA-specific GEIs consist of a DAI (plaid) that putatively catalyses excision of the GEI as a circular intermediate, as well as its integration into the 5′ end of the chromosomal tRNA-dihydrouridine synthase A (dusA) gene (black). The 5′ portion of the dusA gene affected by the integration is replaced by a new 5′ end (white) provided by the GEI, with the original dusA 5′ end (dark grey) forming the outer boundary of the island. This particular GEI variant encodes several hypothetical proteins (light grey) as well as proteins putatively involved in metabolism (diamonds), transport (outlined diamonds), transcriptional regulation (dashed lines), and type I restriction-modification (diagonal lines). As is true of other A. baumannii strains, the dusA-associated GEIs are flanked by a gene encoding a putative major facilitator superfamily transporter (MFS) (black). The binding sites of oligonucleotides used to detect excision of the GEI (black triangles) and restoration of the dusA sequence (white triangles) are indicated. This figure is drawn to scale with the exception of the excised island, which is displayed at 60% scale.

Figure 4.

Predicted mechanism of GEI excision in Acinetobacter baumannii D1279779, A. baumannii ACICU and Pseudomonas protegens Pf-5, as a closed circular molecule (2) from the chromosome (1), resulting in the reformation of the native dusA/B sequence (3). The chromosomal gene flanking the island is designated as X (white). The shading of the nucleotide sequences represent their origin as either from the circularized GEI (black and dark grey) or chromosome (white). The underlined sequences represent the putative attachment sites involved in GEI excision and integration (attP and attB), which upon the latter occurring, a pair of imperfect direct repeats (attL and attR) are generated. Nucleotides in bold are identical in the attP/attB and attL/attR pairs of their respective organisms. The shaded (light grey) boxes represent the 21 bp consensus attL/attR sequences present in dusA-specific GEIs, whilst the indicated adenines (arrows) represent the experimentally determined island termini in the sequence of Acinetobacter baumannii D1279779.

Figure 5.

Logo consensus sequence of attL and attR direct repeats (as defined in Figure 4) generated with WebLogo (33), using alignments of flanking sequences from the 94 dusA-specific genome island sequences outlined in Supplementary Table S1. The indicated adenines (arrows) in both sequences represent the experimentally determined island termini in the sequence of Acinetobacter baumannii D1279779.