Bacterial Community Morphogenesis Is Intimately Linked to the Intracellular Redox State

Abstract

Many microbial species form multicellular structures comprising elaborate wrinkles and concentric rings, yet the rules governing their architecture are poorly understood. The opportunistic pathogen Pseudomonas aeruginosa produces phenazines, small molecules that act as alternate electron acceptors to oxygen and nitrate to oxidize the intracellular redox state and that influence biofilm morphogenesis. Here, we show that the depth occupied by cells within colony biofilms correlates well with electron acceptor availability. Perturbations in the environmental provision, endogenous production, and utilization of electron acceptors affect colony development in a manner consistent with redox control. Intracellular NADH levels peak before the induction of colony wrinkling. These results suggest that redox imbalance is a major factor driving the morphogenesis of P. aeruginosa biofilms and that wrinkling itself is an adaptation that maximizes oxygen accessibility and thereby supports metabolic homeostasis. This type of redox-driven morphological change is reminiscent of developmental processes that occur in metazoans.

Morphological optimization for access to dual oxidants in biofilms

“…More recently, we showed that P. aeruginosa colony wrinkling is a strategy to increase oxygen accessibility (28). We have proposed that phenazines attenuate this mechanism because they act as electron acceptors for cells in anoxic regions of the biofilm and shuttle electrons to the well-aerated regions, allowing cells to balance their internal redox state (28,32,33).…”

“…Colony biofilms that form under these conditions exhibit spatial patterning (Fig. 1A) and environmental sensitivity and constitute an ideal model system for exploring morphology as a metabolic adaptation (28).…”

Metabolite transfer with the fermentation product 2,3-butanediol enhances virulence by Pseudomonas aeruginosa

“…The advantage for P. aeruginosa is to maintain redox homeostasis, and thus to survive, under anaerobic conditions within the deeper layers of the biofilm when PYO is present (Dietrich et al, 2013). This extracellular electron transfer with oxygen over a distance can be measured quantitatively and in real time by electric current production through the oxidation of reduced PYO at an electrode as the solid-state electron acceptor in a bioelectrochemical system (Rabaey et al, 2005;Venkataraman et al, 2011).…”

“…On the other hand, P. aeruginosa has three high-affinity terminal oxidases and has a preference to respire with oxygen and nitrate rather than to ferment (Madigan et al, 2002;Alvarez-Ortega and Harwood, 2007), albeit it can ferment with arginine (Vander Wauven et al, 1984). Even when oxygen is not present in the deeper layers of the biofilm and respiration becomes unlikely, P. aeruginosa may be able to survive by producing reduced PYO that eventually can react extracellularly with oxygen at the boundaries of the biofilm (Dietrich et al, 2013). Regardless, bacterial fermenters have a much higher substrate turnover rate than respirers because of inefficient carbon usage, allowing fermenters to predominate when fermentable substrates (including the blood sugar glucose or extracellular polysaccharides) are present (Madigan et al, 2002).…”

“…In general, phenazines have been observed to increase virulence by: (1) distressing airway epithelial cell function via oxidative stress (Wilson et al, 1988;Denning et al, 1998), (2) inhibiting growth of other microbes via oxidative stress (Hassett et al, 1992) and (3) playing physiologically important roles for P. aeruginosa itself (Hunter et al, 2012). For the latter, research has shown specifically for pure cultures of P. aeruginosa that: (i) PCA increases iron availability by reducing Fe(III) to Fe(II) even when bound to human-produced proteins (Wang et al, 2011), (ii) PCA reduces swarming motility and PYO increases biofilm thickness (Ramos et al, 2010), resulting in changes in biofilm formation, (iii) PYO regulates the intracellular redox state under anaerobic conditions by extracellular electron transfer, promoting survival through redox homeostasis and thicker biofilms (Dietrich et al, 2013) and (iv) PYO enables extracellular DNA binding to the surface of the cell to change aggregation characteristics, and thus biofilm formation (Das et al, 2013). Research had already indicated that reduced PCA and PYO were considerably more reactive with Fe(III) and O 2 , respectively, than vice versa (Wang and Newman, 2008).…”