Gradients and consequences of heterogeneity in biofilms

Abstract

Historically, appreciation for the roles of resource gradients in biology has fluctuated inversely to the popularity of genetic mechanisms. Nevertheless, in microbiology specifically, widespread recognition of the multicellular lifestyle has recently brought new emphasis to the importance of resource gradients. Most microorganisms grow in assemblages such as biofilms or spatially constrained communities with gradients that influence, and are influenced by, metabolism. In this Review, we discuss examples of gradient formation and physiological differentiation in microbial assemblages growing in diverse settings. We highlight consequences of physiological heterogeneity in microbial assemblages, including division of labour and increased resistance to stress. Our impressions of microbial behaviour in various ecosystems are not complete without complementary maps of the chemical and physical geographies that influence cellular activities. A holistic view, incorporating these geographies and the genetically encoded functions that operate within them, will be essential for understanding microbial assemblages in their many roles and potential applications.

Figure 1.. Oxygen gradients influence metabolic differentiation and morphogenesis in multicellular systems.

(a) Schematics of a tumor, a biofilm, and a plant showing regions where O₂ (dark to light shading indicates diminishing oxygen concentration) influences the activity of regulatory cascades that modulate metabolism and/or morphogenesis. Each regulatory protein is part of a pathway that links O₂ sensing to physiological outputs and may play an inductive or inhibitory role. (b) Oxygen (O₂, blue shading) diffuses into a biofilm and subsequent consumption by resident cells leads to O₂ gradient formation. O₂ gradients can result in differential gene expression within a monospecies biofilm (depicted as varying colors; c) and/or stratified growth of species (depicted as varying shapes; d) depending on O₂ preferences. For (b) through (d), the top represents the interface with air or oxygenated liquid.

Figure 2.. Physiological stratification of biofilms along chemical gradients.

(a) The image shows fossilized stromatolites, which are structures formed by microbial mats that trap sediment and promote mineralization in layers (Satka Formation, Southern Urals; the wider columns are ~10 cm in diameter). (b) When growing in aggregates, Pseudomonas aeruginosa shows maximal expression of narG (red), which encodes an anaerobically expressed nitrate reductase, in the center (tagging by a universal probe, for rRNA, is shown in green). (c) A layered biofilm, required for production of kombucha, grows at the air-liquid interface of the tea. (d) Cross-section of a P. aeruginosa colony biofilm (top: interface with air; bottom: interface with agar-solidified medium) showing discrete zones of metabolic activity (red). (e) Cross-section of an E. coli colony biofilm (left panel, top view of whole biofilm shown in inset), false-colored to indicate discrete architectures that are visible at high magnification (right panels, top corresponds to the red colored zone in the cross-section, bottom corresponds to the blue zone) (. (f) Stratified growth of bacterial species in Winogradsky columns. These miniature ecosystems develop over several weeks in standing vessels containing aquatic samples that are incubated in sunlight. The enriched species depend on the nutrient conditions of the initial setup. (g) Cross-section of Humboldt Fog goat milk cheese (Cypress Grove Chevre, Inc.) showing the following layers over depth: mold (microbial fungus) at the cheese-air interface, ash (added during production to increase the pH at the surface of the cheese and promote mold growth), runny cheese, and solid core cheese. (h) Cross-section of a cyanobacterial mat from Guerrero Negro, Baja California Sur, Mexico. Part a provided by T Bosak, part b reproduced with permission from ref. , part c provided by J. Gowans, part d provided by L. Florek, part e reproduced with permission from ref , part f provided by D. Giovanelli, part h provided by J. Spear.

Figure 3.. Redox balancing strategies in Pseudomonas aeruginosa biofilms.

(a) P. aeruginosa can use different metabolic pathways in biofilms. The blue shading represents the O₂ gradient across depth. Yellow shading highlights mechanisms (which are shown in a simplified form) that can be used to reoxidize NADH that is generated through carbon metabolism and the tricarboxylic acid cycle (TCA). The left panel shows aerobic respiration occurring in the upper, oxic zone near the interface with air and mixed-acid fermentation occurring in the lower, anoxic zone at the biofilm base and interface with the growth medium. One of the fermentation products, lactate, can be used as a carbon source and electron donor by cells in the oxic zone. The middle panel depicts mechanisms in which phenazines can contribute to redox balancing. In cells carrying out fermentation, phenazines can reoxidize NADH and potentially promote survival. Mutant analyses have implicated components of the electron transport chain (ETC) in phenazine reduction in biofilms. Reduced phenazines can be reoxidized in the oxic zone. The right panel depicts denitrification as an additional mechanism through which NADH can be reoxidized. (b) The aerobic ETC of P. aeruginosa is modular, such that the final electron transfer to O₂ may be carried out by different terminal oxidases. Electrons from the oxidation of NADH enter the ETC and pass sequentially through the quinone pool and other carriers to the terminal oxidases (yellow), which are encoded by differentially regulated operons. The cox operon is regulated by RpoS, whereas the cco2 operon is regulated by Anr . (c) Opposing gradients of carbon source (orange) and O₂ (blue) lead to microniches within the biofilm. (d) Three-day-old P. aeruginosa biofilms show differential expression of respiratory enzymes across depth. The sections shown contain transcriptional reporters in which GFP production is controlled by the cox or cco2 promoters, respectively. cox expression is visible in the uppermost portion of the biofilm (left panel), furthest from the carbon source, whereas cco2 expression is visible in the lower, hypoxic and anoxic biofilm subzones (right panel) .

Figure 4.. Interplay between resource gradients and biofilm architecture in Pseudomonas aeruginosa and Escherichia coli.

(a) A regulatory pathway, involving the phosphodiesterase (PDE) RmcA and diguanylate cyclase (DGC), links redox cues (in this case, oxidized phenazines) to matrix production in wild type (WT, top) and phenazine-deficient (Δphz, bottom) P. aeruginosa biofilms. Biofilms shown were grown on medium containing 1% tryptone and matrix-staining dyes . (b) Several regulators influence the architecture of E. coli colony biofilms. Sigma factors are shaded pink, and activities that modulate levels of cyclic diguanylate (c-di-GMP) and thereby affect matrix production are shaded cyan. The activities of the regulators are influenced by resource gradients and they control pathways with opposing effects on levels of c-di-GMP. This intracellular signal promotes production of cellulose, a component of the biofilm matrix. RpoS also promotes the synthesis of curli fibers, proteinaceous components of the matrix, via c-di-GMP-independent mechanisms. FlhDC is a transcriptional regulator that controls production of the sigma factor FliA. Both FlhDC and FliA promote production and assembly of flagella, which form a mesh-like structure at the biofilm base (not visible in micrograph on the right). Arrows indicate the overall effects of the indicated regulators and not direct interactions. Additional regulatory steps, some of which have been omitted here for simplicity, are described in detail in . The micrograph at the right shows an E. coli biofilm treated with a fluorescent matrix stain, reproduced with permission from . Part a provided by M. Smiley.

Figure 5.. Gradient formation and examples of metabolite exchange in cyanobacterial mats.

This simplified representation shows some of the metabolisms in cyanobacterial mats. Oxygenic and anoxygenic phototrophs produce light-harvesting pigments that are tuned to utilize light at the wavelengths and intensities associated with their specific depth in the mat. Cyanobacteria oxidize H₂O and fix CO₂, producing O₂ and organic carbon that are consumed by aerobic chemoheterotrophs. Purple and green sulphur bacteria oxidize sulphide and produce sulphate, which is transformed back into sulphide by chemotrophic sulphate-reducers.

Figure 6.. Physiological heterogeneity enhances overall biofilm robustness.

(a) Microcolonies of Yersinia pseudotuberculosis grow in the spleen in a mouse model of infection. Host phagocytes produce nitric oxide (NO), and a specialized subpopulation at the periphery of the microcolony expresses the NO-detoxifying enzyme Hmp. NO exposure regulates hmp expression with protected cells inside the microcolony are not expressing the gene. Microcolonies formed by a hmp-deficient (∆hmp) mutant exhibit hmp expression throughout the structure because NO is not degraded. (b) Polymicrobial biofilm from an extracted, diseased tooth exhibit a characteristic rotund structure (top) and uneven distribution of three different categories of bacteria as shown in the model. Bacterial subpopulations were differentiated using fluorescent in situ hybridization and a fluorescence subtraction method. Co-cultures of cariogenic and commensal species of streptococci form biofilms on enamel in the laboratory, with a rotund structure similar to that observed on teeth. The three panels show a 3D reconstruction of a cluster from a mature biofilm, revealing the segregation of the two species at different heights. (c) Model depicting the cariogenic species (Streptococcus mutans; green) localized at the core of a rotund biofilm structure and surrounded by a shell of the commensal species (such as Streptococcus oralis; red), which provides protection from antimicrobials. The rotund structure is associated with a low-pH microenvironment that promotes demineralization of the enamel. Non-streptococcal bacteria (blue) are situated outside the shell of commensal streptococci. (d) The schematic depicts the orientation of the endothelium (blood vessel containing oxygenated blood) relative to the epithelial lining the intestine (top). On top of host intestinal epithelial cells, stratified populations of gut microorganisms can be found within the O₂ gradient that originates at the endothelium (blood vessel). Microbial growth in the gradient is influenced by relative levels of O₂ tolerance. Micrographs in part b reproduced from .