A novel family of integrases associated with prophages and genomic islands integrated within the tRNA-dihydrouridine synthase A (dusA) gene.
Bottom Line: 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.
Affiliation: Department of Chemistry and Biomolecular Sciences, Macquarie University, Sydney, NSW 2109, Australia.Show MeSH
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Mentions: BLAST (26) searches to identify integrase homologues were conducted using the default parameters and the NCBI ‘non-redundant protein sequences’ (nr) database, except the maximum target sequences were set to 20 000 and an e-value cutoff of 1e-20 was used. Integrases that were either below this cutoff, or belonging to non-redundant records lacking genome sequences, were excluded from this analysis. Integrases were considered dusA-associated if the 5′ end of the integrase gene adjoined the 5′ end of the dusA gene (Figure 3). However, integrases found not to be dusA-associated, but greater than the 1e-20 cutoff, were included in this analysis. Multiple alignments of protein and nucleotide sequences were carried out using ClustalW (27) and ClustalOmega (28). Neighbour-joining phylogenetic analysis and protein identity/similarity matrices were generated from multiple alignments using MEGA5 (29) and MatGAT (30), respectively. TreeGraph2 (31) and UGENE (32) were routinely used for phylogenetic tree annotation and genome browsing, respectively. Logo consensus sequences were generated using WebLogo (33). The gene content of numerous dusA-associated GEIs (Supplementary Table S1) were compared reciprocally by BLASTP+ (34) to identify putative orthologues, using an e-value cutoff of 1e-5. Oligonucleotides used in PCR (Supplementary Table S2) were designed with the aid of Primer3 (35) with the oligonucleotide length, melting temperature and GC% parameters set to 20–25 nt, 55–60°C and 30–50%, respectively.
Affiliation: Department of Chemistry and Biomolecular Sciences, Macquarie University, Sydney, NSW 2109, Australia.