Transcriptional regulation of central carbon metabolism in Pseudomonas aeruginosa

Abstract

Microbes such as Pseudomonas aeruginosa are often challenged by rapidly changing nutritional environments. In order to adapt to these shifts in nutrient availability, bacteria exert tight transcriptional control over the enzymes of central metabolism. This transcriptional control is orchestrated by a series of transcriptional repressors and activators. Although a number of these transcription factors have been identified, many others remain uncharacterized. Here, we present a simple pipeline to uncover and validate the targets of uncharacterized transcriptional regulators in P. aeruginosa. We use this approach to identify and confirm that an orthologue of the Pseudomonas fluorescens transcriptional regulator (RccR) binds to the upstream region of isocitrate lyase (aceA) in P. aeruginosa, thereby repressing flux through the glyoxylate shunt during growth on non-C2 carbon sources.

Figure 1

Schematic of the workflow pipeline.

Figure 2

aceA:lux expression for the wild‐type and the ΔPA5438 mutant grown in MOPS minimal medium containing tryptone, acetate, glucose or succinate, as indicated. Gene expression was measured as relative light units (RLU) derived from the activity of the expressed lux enzymes. RLU values are normalized to the culture OD ₆₀₀. Data represent mean ± SD from three biological replicates.

Figure 3

Western blot to detect isocitrate lyase (AceA, 58 kDa) expression in wild‐type P. aeruginosa (strain PAO1) and in an otherwise isogenic ΔPA5438 mutant during growth on acetate and glucose (as indicated) sole carbon sources. Molecular mass markers in kDa (red) are indicated. Note how AceA expression is repressed in the wild type during growth in MOPS‐glucose, but not in the ΔPA5438 mutant. Each lane represents an independent biological replicate.