Comparative Genomics of Burkholderia singularis sp. nov., a Low G+C Content, Free-Living Bacterium That Defies Taxonomic Dissection of the Genus Burkholderia

Abstract

Four Burkholderia pseudomallei-like isolates of human clinical origin were examined by a polyphasic taxonomic approach that included comparative whole genome analyses. The results demonstrated that these isolates represent a rare and unusual, novel Burkholderia species for which we propose the name B. singularis. The type strain is LMG 28154^T (=CCUG 65685^T). Its genome sequence has an average mol% G+C content of 64.34%, which is considerably lower than that of other Burkholderia species. The reduced G+C content of strain LMG 28154^T was characterized by a genome wide AT bias that was not due to reduced GC-biased gene conversion or reductive genome evolution, but might have been caused by an altered DNA base excision repair pathway. B. singularis can be differentiated from other Burkholderia species by multilocus sequence analysis, MALDI-TOF mass spectrometry and a distinctive biochemical profile that includes the absence of nitrate reduction, a mucoid appearance on Columbia sheep blood agar, and a slowly positive oxidase reaction. Comparisons with publicly available whole genome sequences demonstrated that strain TSV85, an Australian water isolate, also represents the same species and therefore, to date, B. singularis has been recovered from human or environmental samples on three continents.

Keywords: Burkholderia cepacia complex; Burkholderia pseudomallei complex; Burkholderia singularis; comparative genomics; cystic fibrosis microbiology; whole genome sequence.

FIGURE 1

Phylogenetic tree based on nearly complete 16S rRNA gene sequences of Burkholderia representatives and B. singularis sp. nov. isolates. The optimal tree (highest log likelihood) was constructed using the Maximum Likelihood method and Tamura-Nei model in MEGA7 (Kumar et al., 2016). A discrete Gamma distribution was used to model evolutionary rate differences among sites [5 categories (+G, parameter = 0.3465)] and allowed for some sites to be evolutionarily invariable ([+I], 42.1434% sites). The percentage of replicate trees in which the associated taxa clustered together in the bootstrap test (1000 replicates) are shown next to the branches if higher than 50%. The sequence of Ralstonia solanacearum LMG 2299^T was used as outgroup. The scale bar indicates the number of substitutions per site. Taxonomic type strains are indicated by superscript T in strain numbers.

FIGURE 2

Genomic phylogeny and characteristics of the genus Burkholderia. The phylogenetic tree was built using an alignment of 23 conserved single copy COGs and an approximate maximum-likelihood approach (see Materials and Methods for details). The %G+C distribution was calculated on non-overlapping 1 kb segments for each genome and plotted as box plots with extreme values removed for clarity. Total genome size (bar plot) is approximated by size of the assembly and coding content (dark gray) corresponds to the sum of the length of all annotated CDS features, discounting predicted pseudogenes. Shimodaira-Hasegawa-like local support values as given by the program Fasttree are indicated.

FIGURE 3

Distribution of genes in COG functional categories. Frequencies of genes in COG categories of selected Burkholderia genomes. The values represent the number of genes in COG categories, divided by the total number of genes with a COG assignment (genes without COG assignments are not counted for clarity). (C) = Energy production and conversion, (D) = Cell cycle control, cell division, chromosome partitioning, (E) = Amino acid transport and metabolism, (F) = Nucleotide transport and metabolism, (G) = Carbohydrate transport and metabolism, (H) = Coenzyme transport and metabolism, (I) = Lipid transport and metabolism, (J) = Translation, ribosomal structure and biogenesis, (K) = Transcription, (L) = Replication, recombination and repair, (M) = Cell wall/membrane/envelope biogenesis, (N) = Cell motility, (O) = Posttranslational modification, protein turnover, chaperones, (P) = Inorganic ion transport and metabolism, (Q) = Secondary metabolites biosynthesis, transport and catabolism, (R) = General function prediction only, (S) = Function unknown, (T) = Signal transduction mechanisms, (U) = Intracellular trafficking, secretion, and vesicular transport, (V) = Defense mechanisms, (W) = Extracellular structures, (X) = mobilome.