Genetic and phenotypic diversity in Burkholderia: contributions by prophage and phage-like elements

Abstract

Background: Burkholderia species exhibit enormous phenotypic diversity, ranging from the nonpathogenic, soil- and water-inhabiting Burkholderia thailandensis to the virulent, host-adapted mammalian pathogen B. mallei. Genomic diversity is evident within Burkholderia species as well. Individual isolates of Burkholderia pseudomallei and B. thailandensis, for example, carry a variety of strain-specific genomic islands (GIs), including putative pathogenicity and metabolic islands, prophage-like islands, and prophages. These GIs may provide some strains with a competitive advantage in the environment and/or in the host relative to other strains.

Results: Here we present the results of analysis of 37 prophages, putative prophages, and prophage-like elements from six different Burkholderia species. Five of these were spontaneously induced to form bacteriophage particles from B. pseudomallei and B. thailandensis strains and were isolated and fully sequenced; 24 were computationally predicted in sequenced Burkholderia genomes; and eight are previously characterized prophages or prophage-like elements. The results reveal numerous differences in both genome structure and gene content among elements derived from different species as well as from strains within species, due in part to the incorporation of additional DNA, or 'morons' into the prophage genomes. Implications for pathogenicity are also discussed. Lastly, RNAseq analysis of gene expression showed that many of the genes in varphi1026b that appear to contribute to phage and lysogen fitness were expressed independently of the phage structural and replication genes.

Conclusions: This study provides the first estimate of the relative contribution of prophages to the vast phenotypic diversity found among the Burkholderiae.

Figure 1

Transmission electron micrographs (TEM) of the Burkholderia bacteriophages analyzed in this project and schematic illustrations of their genomes. (A) TEM of bacteriophages negatively stained with 1% phosphotungstic acid. (B) Schematic illustrations of the P2-like Myoviridae genomes of ϕ52237, ϕE202, and ϕE12-2. Cyan shading represents sequences that are conserved in the subgroup A Myoviridae ϕ52237, ϕE202, and ϕK96243 and lime shading represents sequences that are conserved in the subgroup B Myoviridae ϕE12-2, GI15, and PI-E264-2. Gray shading represents sequences that are variably present in Myoviridae subgroups A and B. (C) Schematic illustration of the lambda-like Siphoviridae genome of ϕ644-2. Gray shading represents sequences that are unique to ϕ644-2. (D) Schematic illustration of the Mu-like Myoviridae genome of ϕE255. Gray shading represents sequences that are unique to ϕE255 and orange shading represents packaged host DNA. The 23-bp imperfect direct repeats at the left and right ends of the ϕE255 genome are shown and sequence differences with the repeat sequences of BcepMu are underlined. Genomic illustrations were obtained from the Integrated Microbial Genomes website http://img.jgi.doe.gov/cgi-bin/pub/main.cgi. Genes are shown as arrows that are pointing in their relative direction of transcription and are color coded based on their % GC composition (see scale at bottom). Individual genes with functional annotations are labeled and designated with an asterisk (*) while groups of genes with a common function are labeled and designated with a line. The locations of att sites are shown as red oblong circles. Nucleotide sequence numbering is shown above each genome.

Figure 2

Unrooted radial phylogenetic tree of the Burkholderia bacteriophages, putative prophages, and prophage-like regions analyzed in this study. The tree was constructed from BLASTP distance matrix (cutoff E^-10) using FITCH with the global and jumble options.

Figure 3

Comparative alignment of B. pseudomallei phage and prophage-like regions. Colors indicate local collinear blocks (LCB or synteny blocks) between phages and PI, the level of similarity between sequences is directly proportional to the height of the colored bars within the LCB. Rectangles highlight hotspots where different morons integrated in each of the genomes. Ovals highlight the same moron which integrates at the same location in each genome, but is differentially present among genomes within the same groups and is present in different phylogenetic groups. Genome comparisons and LCB calculations were performed using Mauve software [34].

Figure 4

Regional sequence alignments of Siphoviridae-like prophages. Comparative genomic analysis of siphoviridae-like prophages and PIs detailing morons encoding DNA methylase RsrI, PAPS reductase/sulfotransferase, and putative chromosome partitioning factor. Gray shading represents conservation at greater than 90% identity among all genomes. Mauve or orange shading represents conservation at 90% identity in a subset of genomes.