Spatiometabolic stratification of Shewanella oneidensis biofilms

Abstract

Biofilms, or surface-attached microbial communities, are both ubiquitous and resilient in the environment. Although much is known about how biofilms form, develop, and detach, very little is understood about how these events are related to metabolism and its dynamics. It is commonly thought that large subpopulations of cells within biofilms are not actively producing proteins or generating energy and are therefore dead. An alternative hypothesis is that within the growth-inactive domains of biofilms, significant populations of living cells persist and retain the capacity to dynamically regulate their metabolism. To test this, we employed unstable fluorescent reporters to measure growth activity and protein synthesis in vivo over the course of biofilm development and created a quantitative routine to compare domains of activity in independently grown biofilms. Here we report that Shewanella oneidensis biofilm structures reproducibly stratify with respect to growth activity and metabolism as a function of size. Within domains of growth-inactive cells, genes typically upregulated under anaerobic conditions are expressed well after growth has ceased. These findings reveal that, far from being dead, the majority of cells in mature S. oneidensis biofilms have actively turned-on metabolic programs appropriate to their local microenvironment and developmental stage.

FIG. 1.

Time course of S. oneidensis biofilm development. Cells expressed a constitutive GFP. The first four panels (30 min and 11, 23, and 44 h) show x-y slices at the base of the developing biofilm structure. The final panel (52 h) shows a z profile through the structure. Scale bar = 10 μm.

FIG. 2.

Live/dead staining in S. oneidensis biofilms. Green indicates the “live” stain, Syto9, and red indicates the “dead” stain, propidium iodide. The grid lines are 10 μm apart. (A) When the diameter is 60 μm, most cells continue to stain “live.” (B) When the diameter is ∼80 μm, cells throughout the biofilm start to stain “dead.” (C) When the diameter is ∼140 μm, almost all cells in the middle of the biofilm stain “dead.”

FIG. 3.

Fluorescence levels and OD₆₀₀ for S. oneidensis MR-1 (filled circles), S. oneidensis DKN308 constitutively expressing GFP (open circles), and S. oneidensis DKN310 expressing GFP(AAV) from a ribosomal promoter, representing growth activity (filled inverted triangles). OD₆₀₀ is indicated by dashed lines, and fluorescence is indicated by solid lines. As cells grow through the exponential phase, the fluorescence levels increase, but when the stationary phase is reached, the fluorescence levels from the constitutively expressed GFP remain high whereas the fluorescence from the growth-active version decreases rapidly. The error bars indicate standard deviations of triplicate cultures; in some cases the error is less than the size of the symbol.

FIG. 4.

Development of a mushroom structure in an S. oneidensis biofilm with a ribosomal (growth) reporter (DKN310) (A to E) and an anaerobic reporter gene (mtrB) (DKN312) (F to J). The grid squares are 10 μm by 10 μm. In the first column the cells are constitutively expressing ecfp and the fluorescence from ecfp is false red. In the second column of panels A to E, the cells expressing the growth-active GFP(AAV) are green. In the second column of panels F to J, the cells expressing the mtrB reporter are green. The third column is an overlay of the red and green channels. (A) 18 h and 8 μm high; (B) 28 h and 18 μm high; (C) 41 h and 52 μm high; (D) 65 h and 92 μm high; (E) 77 h and 112 μm high; (F) 17 h and 8 μm high; (G) 29 h and 31 μm high; (H) 47 h and 58 μm high; (I) 71 h and 104 μm high; (J) 85 h and 118 μm high.

FIG. 5.

Fold changes in expression, as measured by quantitative RT-PCR, of mtrB and eyfp in S. oneidensis DKN312 relative to aerobic conditions (21% O₂). Solid bars, mtrB; gray bars, eyfp. mtrB and eyfp expression is upregulated under anaerobic and microaerobic conditions. The error bars indicate the ranges for duplicate cultures.

FIG. 6.

Quantitative analysis of patterns of growth activity and metabolism in S. oneidensis biofilms. The gray lines show growth activity profiles for strain DKN310 (rrnB P1), and the black lines show mtrB expression of strain DKN312. (A) Biofilm structures approximately 60 μm in diameter. (B) Structures approximately 110 μm in diameter. (C) Structures approximately 140 μm in diameter. Each line represents an average of a minimum of six different structures. The error bars indicate the standard deviations of the binned pixel intensity values for all the images included in the plot. For panels B and C, local minima at the edges of the colonies are regions with no cells, thought to be extracellular polysaccharide. The patterns of expression relative to the size of the biofilm structure are remarkably consistent across multiple structures, and mtrB continues to be expressed in regions where growth activity has decreased.