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  • The genomes of F. nodosum Rt17-B1 (NC_009718) [13, 16], F. pennivorans DSM9070 (NC_017095) [17], F. islandicum AW-1 (NZ_CP014334) [18, 19] and a new isolate [20], Fervidobacterium thailandense strain FC2004 (LWAF01000000) were used in this analysis. Annotated transposases in these four genomes and homology searching (using blast algorithms) [21] resulted in the detection of a number of transposases. They were classified based on their similarity to transposase families as proposed by Siguier et al. [8] at the ISFinder database (http://www-is.biotoul.fr). Blastp was used to determine the closest relatives to the transposase genes detected in Fervidobacterium genomes. Besides the closest relative sequences detected in GenBank, the closest sequences to those transposase families within the Phylum Thermotogae were also incorporated into the analyses as a comparative threshold for the detection of HGT events between different phyla. Sequence alignments were performed by ClustalW [22]. Phylogenetic trees based on the amino acid sequences encoded by the detected genes were constructed using MEGA [23] by the Neighbor-joining method with a bootstrap value of 1000. Multivariate analyses of tetranucleotide frequencies: Non-metric MultiDimensional Scaling (NMDS) analyses were performed to obtain graphical distributions of the transposase gene sequences corresponding to each transposase family detected in the Fervidobacterium genomes and their related genes. The frequencies of tetranucleotides [24] were used in NMDS analyses. NMDS plots were constructed using R with the Vegan Package [25]. The conservation of insertion sequence endings were studied by searching through visual inspection the sequence alignments corresponding to related transposase genes plus additional 1000 nucleotides up- and down-stream. When present, the detection of the inverted repeats or palindromic sequences (depending on the transposase family) was guided by the information available at the ISFinder database. The percentage of identity between aligned, conserved endings and the distance to the annotated start or stop codons were noted. The Kullback-Leiber (K-L) divergence metric [26, 27] was used as an approach to estimate the divergence between related transposase genes clustered in a transposase family. K-L divergence (DKL) was calculated on tetranucleotide frequencies for pairwise comparisons as DKL(g//G)=∑g(i)ln(g(i)/G(i))(1) where g is the parameter used (e.g., frequency of a particular tetranucleotide, i) for the analyzed gene and G the same parameter for a reference transposase gene or whole genome.
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