Light/Dark and Temperature Cycling Modulate Metabolic Electron Flow in Pseudomonas aeruginosa Biofilms

Abstract

Sunlight drives phototrophic metabolism, which affects redox conditions and produces substrates for nonphototrophs. These environmental parameters fluctuate daily due to Earth's rotation, and nonphototrophic organisms can therefore benefit from the ability to respond to, or even anticipate, such changes. Circadian rhythms, such as daily changes in body temperature, in host organisms can also affect local conditions for colonizing bacteria. Here, we investigated the effects of light/dark and temperature cycling on biofilms of the opportunistic pathogen Pseudomonas aeruginosa PA14. We grew biofilms in the presence of a respiratory indicator dye and found that enhanced dye reduction occurred in biofilm zones that formed during dark intervals and at lower temperatures. This pattern formation occurred with cycling of blue, red, or far-red light, and a screen of mutants representing potential sensory proteins identified two with defects in pattern formation, specifically under red light cycling. We also found that the physiological states of biofilm subzones formed under specific light and temperature conditions were retained during subsequent condition cycling. Light/dark and temperature cycling affected expression of genes involved in primary metabolic pathways and redox homeostasis, including those encoding electron transport chain components. Consistent with this, we found that cbb₃-type oxidases contribute to dye reduction under light/dark cycling conditions. Together, our results indicate that cyclic changes in light exposure and temperature have lasting effects on redox metabolism in biofilms formed by a nonphototrophic, pathogenic bacterium. IMPORTANCE Organisms that do not obtain energy from light can nevertheless be affected by daily changes in light exposure. Many aspects of animal and fungal physiology fluctuate in response to these changes, including body temperature and the activities of antioxidant and other redox enzymes that play roles in metabolism. Whether redox metabolism is affected by light/dark and temperature cycling in bacteria that colonize such circadian organisms has not been studied in detail. Here, we show that growth under light/dark and temperature cycling lead to rhythmic changes in redox metabolism in Pseudomonas aeruginosa and identify proteins involved in this response. P. aeruginosa is a major cause of health care-associated infections and is designated a serious threat by the CDC due to its recalcitrance during treatments. Our findings have the potential to inform therapeutic strategies that incorporate controlled light exposure or consider P. aeruginosa's responses to conditions in the host.

Keywords: Pseudomonas aeruginosa; biofilms; light-regulated metabolism; metabolic cycling; respiration.

FIG 1

Pseudomonas aeruginosa Δphz colony biofilms grown under light/dark or temperature cycling conditions form patterns of TTC reduction. (A) Schematic showing PA14 electron transport chain (ETC)-dependent reduction of TTC, which produces a red precipitate. (B) Schematic of the experimental setup examining the effect of biofilm growth under constant or cycling conditions (with light or dark conditions indicated by the sun and moon). (C and D) (Left) Δphz biofilm grown under the indicated condition. Right: Quantification of red color intensity (i.e., TTC reduction) at the indicated distance from the biofilm center for a radius of the biofilm. (E to G) Left: Δphz biofilm grown under the indicated conditions. Middle: Quantification of red color intensity (i.e., TTC reduction) at the indicated distance from the biofilm center for a radius of the biofilm. (Right) Results of detrending analysis applied to the linear portion of the data (generated using BioDare2) (132). For all experiments, the concentration of TTC in the growth medium was 0.004%. Experiments were performed with biological triplicates, and representative data are shown. Scale bars = 2.5 mm.

FIG 2

Conditions present during formation of each colony biofilm growth ring affect the total TTC reduced by the ring. (A) Left: Kymograph showing pixel coloration over time across a radius of a biofilm (as depicted in the drawing, top) grown under light/dark and 25°C/23°C cycling conditions on medium containing 0.004% TTC. Bars labeled i to iv indicate the points at which biomass became detectable via image analysis. Middle: Quantification of red color intensity (i.e., TTC reduction) at the indicated distance from the biofilm center for a radius of the biofilm. Right: Analysis of a time-lapse movie of biofilm growth. The average value of all pixels at the colony edge was tracked over time; the average pixel value, 12 h after pixels were first detected, is shown. The graph was detrended using the linear detrend function of BioDare2 (132). (B) Reduced TTC quantified as average pixel value over time for each ring of growth of the biofilm shown in panel A. The i to iv labeling corresponds to pixels of biomass becoming detectable as indicated in panel A. Traces corresponding to biomass appearing during dark intervals are shown in blue; those corresponding to biomass appearing during light intervals are shown in yellow. Traces that initiate in dark or light intervals are shown on separate plots, for clarity, on the right. Bars indicate the spread of values for biomass formed in the dark and the light.

FIG 3

TTC reduction patterns respond to altered periods of light/dark and temperature cycling. (A) Top: Biofilms grown under light/dark and 25°C/23°C cycling conditions with 6-h (left panel), 12-h (middle panel), or 24-h (right panel) alternating intervals. Biofilms were grown for a total of 144 h when using cycling conditions with 6-h and 12-h alternating intervals and for a total of 312 h when using cycling conditions with 24-h alternating intervals. Bottom: Quantification of red color intensity (i.e., TTC reduction) at the indicated distance from the biofilm center for a radius of the biofilm. The concentration of TTC in the growth medium was 0.004%. (B) Analyses of time-lapse movies of biofilm growth. For each biofilm, the average value of all pixels at the colony edge was tracked over time; the average pixel value, 12 h after pixels were first detected, is shown. The graph was detrended using the linear detrend function of BioDare2 (132). Experiments were performed with biological triplicates, and representative data are shown.

FIG 4

Fixed physiological states are maintained after growth under cycling conditions. (A) Model of biofilm development under cycling conditions. The environmental condition (e.g., light or dark) experienced by perceptive cells at the leading edge of the biofilm (blue) sets their physiological status, which then remains fixed through subsequent changes in conditions. (B) Left panels: Biofilm coloration after 144 h of growth under light/dark and 25°C/23°C cycling conditions on medium without TTC (0 min) and then after transfer to medium containing 0.005% TTC and incubation under constant dark and 23°C conditions for 270 min. Right panel: Quantification of pixel intensity across a selected radius, at the indicated time points after TTC exposure, for the biofilm shown on the left. These TTC reduction values were corrected using a pixel quantification of the pretransfer biofilm (0 min).

FIG 5

Biofilms grown under light/dark cycling conditions form TTC reduction patterns in blue, red, or far-red light, and ΔbphP and ΔptsP mutants show defects in TTC ring pattern formation, specifically under red light. (A) Schematics illustrating domain architectures of BphP and PtsP, highlighting (putative) light-sensing domains. (B) TTC reduction patterns in biofilms of the parent strain (Δphz) and indicated mutants grown under light/dark cycling of blue, red, or far-red light with 12-h intervals. Temperature was kept constant at 25°C, and the growth medium contained 0.004% TTC. Experiments were performed with biological triplicates, and representative data are shown.

FIG 6

Biofilms grown with light/dark and temperature cycling show fixed physiological responses, evident as differences in transcription in bands of TTC reduction. (A) Top: Schematic showing sampling sites in a biofilm grown under cycling conditions on medium containing TTC. The indicated biofilm zones were sampled from duplicate biofilms and subjected to RNAseq. Bottom: Heatmaps representing relative transcript levels (Z-scores), shown for each replicate biofilm sample in order of fold change. One sample was omitted because it was determined to be an outlier by principal-component analysis. (B) Volcano plot showing the log₂-transformed average fold change versus statistical significance for each gene, based on normalized transcript levels detected by RNAseq in biofilm zones formed during light versus dark intervals. Genes that showed a >2-fold increase in expression under dark/23°C or light/25°C conditions are represented in blue or yellow, respectively. A total of 5,945 genes are represented. (C) Gene set enrichment analysis using groups of genes based on KEGG-annotated metabolic pathways. Significant enrichment scores above 0 correspond to overrepresented groups of genes in biofilm zones from light intervals, while significant enrichment scores below 0 correspond to overrepresented groups of genes in biofilm zones from dark intervals. The gene ratio corresponds to the fraction of genes per pathway contributing to the enrichment.

FIG 7

Light/dark and temperature cycling affects the expression of PA14 terminal oxidase genes, and cbb₃-type oxidases influence the dynamics of biofilm TTC reduction under this condition. (A) Average relative expression of genes that code for subunits of terminal oxidase complexes (source: RNAseq analysis in Fig. 6). L1 refers to the light 1 ring depicted in Fig. 6A (top), D1 refers to the dark 1 ring, and so on. Error bars indicate the standard deviation of duplicate samples. (B) Schematic of P. aeruginosa’s branched ETC. Cyo, Cio, Cox, Cco2, and Cco1 are terminal oxidase complexes, and Cco4 refers to two orphan terminal oxidase subunits that can replace the corresponding subunits in Cco2 or Cco1 (32, 64). (C) Pixel intensity over time for selected pixels in biofilms of the indicated strains grown under light/dark and temperature cycling on medium containing 0.004% TTC. (D) TTC reduction rate over time for data shown in panel C, with data for biomass that formed under dark conditions on the left and data for biomass that formed under light conditions on the right. For panels C and D, data points for biomass formed under dark conditions are blue and those for biomass formed under light conditions are yellow. Darker shades of blue and yellow are used for biomass that formed at earlier time points within an interval, and lighter shades are used for biomass that formed at later time points. Results are representative of 3 biological replicates.