The unexpected discovery of a novel low-oxygen-activated locus for the anoxic persistence of Burkholderia cenocepacia

Abstract

Burkholderia cenocepacia is a Gram-negative aerobic bacterium that belongs to a group of opportunistic pathogens displaying diverse environmental and pathogenic lifestyles. B. cenocepacia is known for its ability to cause lung infections in people with cystic fibrosis and it possesses a large 8 Mb multireplicon genome encoding a wide array of pathogenicity and fitness genes. Transcriptomic profiling across nine growth conditions was performed to identify the global gene expression changes made when B. cenocepacia changes niches from an environmental lifestyle to infection. In comparison to exponential growth, the results demonstrated that B. cenocepacia changes expression of over one-quarter of its genome during conditions of growth arrest, stationary phase and surprisingly, under reduced oxygen concentrations (6% instead of 20.9% normal atmospheric conditions). Multiple virulence factors are upregulated during these growth arrest conditions. A unique discovery from the comparative expression analysis was the identification of a distinct, co-regulated 50-gene cluster that was significantly upregulated during growth under low oxygen conditions. This gene cluster was designated the low-oxygen-activated (lxa) locus and encodes six universal stress proteins and proteins predicted to be involved in metabolism, transport, electron transfer and regulation. Deletion of the lxa locus resulted in B. cenocepacia mutants with aerobic growth deficiencies in minimal medium and compromised viability after prolonged incubation in the absence of oxygen. In summary, transcriptomic profiling of B. cenocepacia revealed an unexpected ability of aerobic Burkholderia to persist in the absence of oxygen and identified the novel lxa locus as key determinant of this important ecophysiological trait.

Figure 1

Comparison of B. cenocepacia transcription at the lxa locus across multiple growth conditions. A plot showing the mean relative fluorescence intensity of microarray probes, as a representation of gene expression across multiple growth conditions for B. cenocepacia. Each black line represents the profile of one of 50 CDSs from the lxa locus (BCAM275a to BCAM323) and demonstrates the unique and acute upregulation of this region under low-oxygen growth conditions.

Figure 2

B. cenocepacia gene expression during stationary phase and low-oxygen growth conditions. The Venn diagrams illustrate the number of upregulated genes (a) and downregulated genes (b) observed during stationary phase and low-oxygen growth conditions.

Figure 3

The B. cenocepacia lxa locus and low-oxygen co-regulated genes. (a) The genomic location of the lxa locus between two genomic islands together with a GC content and GC frame plot. (b) The 50 genes of the lxa cluster with the colour of each gene correlating to the level of upregulation, as provided in the key. (c) Four low-oxygen co-regulated B. cenocepacia loci. The scale bars for each panel are indicated on the left, with the key for gene expression and lxa motif location provided at the bottom. The consensus sequence of the lxa motif is shown with the size of the base representative of its degree of conservation in the 21 input sequences analysed.

Figure 4

Conservation of the B. cenocepacia J2315 lxa locus in other Burkholderia genomes. Seventeen complete Burkholderia species genomes were examined for genes homologous to those in the B. cenocepacia J2315 lxa locus and an illustration of the conservation and synteny of these genes is shown above. The genomes and strains analysed are listed on the left; conservation and synteny with: (a) the lxa locus and (b) other genes co-regulated under low-oxygen growth (see Figure 3) are shown by the continuous grey blocks in the respective panels. The gaps between the blocks represent a lack of a continuous gene order with the lxa as no other Burkholderia genome encoded a complete lxa gene cluster. The identity of each orthologous gene is provided in Supplementary Table S6.

Figure 5

Growth and survival characteristics of B. cenocepacia lxa locus deletion mutants. The growth kinetics in minimal medium with and without yeast extract and casamino acid supplementation is shown to the left in a and b, for strains J2315 and K56-2 respectively, with the mean OD plotted as a growth curve. Each curve is labelled as follows: (a) WT+ (crosses), wild-type J2315 and Δlxa+ (shaded diamonds) J2315 lxa mutant, both with supplementation; WT (open circles) wild-type J2315 and Δlxa (shaded triangles) J2315 lxa mutant, both without supplementation; (b) WT+ (crosses), wild-type K56-2 and Δlxa+ (shaded diamonds) K56-2 lxa mutant, both with supplementation; WT (open circles) wild-type K56-2 and Δlxa (shaded triangles) K56-2 lxa mutant, both without supplementation. The viability of bacteria incubated without oxygen in nutrient-rich medium is shown to the right in c and d, for strains J2315 and K56-2, respectively, with the WT (open circles) and Δlxa (triangles) labelled for each survival plot. Each data point is the mean of three replicate cultures.