The soil bacterium Burkholderia pseudomallei is the causative agent of melioidosis and a significant cause of human morbidity and mortality in many tropical and subtropical countries. The species notoriously survives harsh environmental conditions but the genetic architecture for these adaptations remains unclear. Here we employed a powerful combination of genome-wide epistasis and co-selection studies (2,011 genomes), condition-wide transcriptome analyses (82 diverse conditions), and a gene knockout assay to uncover signals of “co-selection”—that is a combination of genetic markers that have been repeatedly selected together through B. pseudomallei evolution. These enabled us to identify 13,061 mutation pairs under co-selection in distinct genes and noncoding RNA. Genes under co-selection displayed marked expression correlation when B. pseudomallei was subjected to physical stress conditions, highlighting the conditions as one of the major evolutionary driving forces for this bacterium. We identified a putative adhesin (BPSL1661) as a hub of co-selection signals, experimentally confirmed a BPSL1661 role under nutrient deprivation, and explored the functional basis of co-selection gene network surrounding BPSL1661 in facilitating the bacterial survival under nutrient depletion. Our findings suggest that nutrient-limited conditions have been the common selection pressure acting on this species, and allelic variation of BPSL1661 may have promoted B. pseudomallei survival during harsh environmental conditions by facilitating bacterial adherence to different surfaces, cells, or living hosts.

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The soil bacterium Burkholderia pseudomallei is the causative agent of melioidosis and a significant cause of human morbidity and mortality in many tropical and sub-tropical countries. The species notoriously survives harsh environmental conditions but the genetic architecture for these adaptations remains unclear. Here we employed a powerful combination of genome-wide epistasis and co-selection studies, gene expression analysis, and gene knockout assays to uncover genetic markers that have been repeatedly selected through B. pseudomallei evolution. These enabled us to identify a large number of mutation pairs under co-selection in distinct genes and non-coding RNA, of which a majority (61.5%) of signals detected in the discovery dataset can be replicated in the validation dataset (totalling 2,011 genomes). Genes under co-selection are mostly conditionally expressed with marked expression correlation of gene-gene pairs found in physical stress conditions. We discovered a putative adhesin (BPSL1661) as a hub of co-selection signals and experimentally confirmed the essential role of BPSL1661 under nutrient deprivation. The gene co-selection network surrounding BPSL1661 likely offers B. pseudomallei a selective advantage to survive nutrient limited conditions. We propose that anthropogenic activities such as pre- and post-harvest crop residue burning have accelerated soil nutrient depletion and may have directly contributed to the preferential survival of B. pseudomallei over benign soil microorganisms. This is expected to lead to a consequent increase in the incidence of melioidosis should the slash-and-burn practices continue to expand.

Burkholderia pseudomallei is an etiological agent of melioidosis, a severe community-acquired infectious disease. B. pseudomallei strain K96243 is sensitive to the drug ceftazidime (CAZ), but has been shown to exhibit transient CAZ tolerance when in a biofilm form. To investigate an observed shift in gene expression profile during CAZ tolerance condition and to better understand the mechanistic aspects of this transient tolerance, RNA-sequencing was performed on B. pseudomallei K96243 from the following three states: planktonic, biofilm, and planktonic shedding. Results indicated that the expression of 651 genes (10.97%) were significantly changed in both biofilm (resistant) and planktonic shedding (sensitive) cells in comparison to the planktonic state. The top four highly expressed genes identified in both states are associated with nitrosative stress response (BPSL2368), Fe–S homeostasis (BPSL2369), and nitrate respiration (BPSS1154 and BPSS1158). Additionally, five orthologous genes, BPSL2370–BPSL2374, implicated in Fe–S cluster biogenesis, and another gene, BPSL2863, involved in DNA-binding of the stress protein ferritin, were shown to increase expression by RT-qPCR. The shift in gene expression was especially prominent at the late stages of biofilm growth (72 and 96 h), specifically in the biofilm-challenged CAZ survivor cells. This suggested that in response to stress in a biofilm, differential expression of these genes may support development of the CAZ tolerance in Burkholderia. The application of iron chelator deferoxamine (DFO) to the biofilm caused a significant reduction in biofilm formation and associated CAZ tolerance. Therefore, the shift in Fe–S metabolism when B. pseudomallei is in a biofilm may help stabilize the levels of reactive oxygen species (ROS), thereby limiting tolerance to CAZ.