Colony variation in Burkholderia pseudomallei: a strategy for hypoxic growth and persistent gastric colonization

Abstract

Burkholderia pseudomallei is well known for its diverse colony morphologies when recovered from infected patients. We have observed that inhibition of aerobic respiration shifted populations of B. pseudomallei from the canonical white colony morphotype toward two distinct, reversible, yet relatively stable yellow colony variants that are referred to here as YA and YB. The yellow variants contrasted dramatically with the white form (WHT). They exhibited alternative cellular physiology in stationary phase, including gross cellular physiology, divergent gene expression profiles, production of unique excreted compounds, and excretion of a toxic substance. Most importantly, yellow variants exhibited a competitive advantage under hypoxic conditions and alkalized cultures without production of ammonia. Since hypoxia and acidity are key characteristics of the gastric niche environment, we tested the ability of yellow variants to colonize the stomach using a B. pseudomallei persistent gastric colonization model. Virulence studies demonstrated that the YB variant, although attenuated in acute virulence, was the only form that colonized and persisted in the stomach. DNA staining of gastric B. pseudomallei YB colonies demonstrated the bacteria were associated with extracellular DNA. Furthermore, we developed an in vitro model that mimicked the gastric microenvironment. Investigation of YB in this in vitro stomach model demonstrated that the variant forms, but not the parental form produced large amounts of extracellular DNA without indication of cell lysis. The YA and YB variants were also much more resistant to acid stress in the gastric model than the parental form. These data demonstrate that the YB variant is a unique form specifically adapted to the harsh gastric environment and necessary for persistent colonization. Transposon mutagenesis identified a transcriptional regulator (BPSL1887) that, when overexpressed, produced a yellow colony morphology, as well as the media alkalization and eDNA production phenotypes. Deletion of bpsl1887 stopped the shift of the white form to yellow forms under normally permissive conditions, and ablated expression of the other phenotypes. Overexpression of BPSL1887 in either a wild type or deletion background yielded colonies with yellow morphology as well as expression of the media alkalization and eDNA production phenotypes. In addition to results of the transposon mutagenesis, no genetic mutations were observed in the YA and YB forms as determined by whole genome sequencing. Taken together, these two observations suggest a model for control of colony variation in B. pseudomallei. We propose that BPSL1887 (YelR) is a master regulator of the various phenotypes associated with yellow colony variation and the epigenetic maintenance of the yellow colony forms.