**4. Ecosystem engineering, ecological opportunity and niche creation**

Sterile static microcosms have a uniform O2 distribution throughout the liquid column. However, after inoculation the metabolic activity of wild-type SBW25 cells rapidly produces a steep O2 gradient, with less than 0.1% normal levels of dissolved O2 below 1 mm after 5 h [33]. The ecosystem engineering by these early colonists is driven by O2 uptake levels which exceed the O2 flux from the air above into the liquid column, and as a result the initially spatially homogeneous and unstructured environment is divided into an upper high-O2 zone and a lower O2-depleted zone (**Figure 3a**). The transition between the two zones is arbitrary but reflects a significant change in growth by wild-type SBW25. Further growth makes the O2 gradient even more extreme, with less than 1% O2 found below the top 200 μm layer of the liquid column after 5 days [33].

This depletion of O2 is an example on a bacterial scale of the social dilemma known as the tragedy of the commons. In this, O2 is a shared and limiting resource known as the commons, and if used selfishly and without restraint by members of the community it will be depleted and eventually destroyed [55]. Despite the growing difference between high and low-O2 zones, wild-type SBW25 cells remain distributed throughout the liquid column though there is an appreciable accumulation of cells at the top [52]. Growth rates will be higher in this region which we have described as the Goldilocks zone3 of optimal growth [2, 53], rather than lower down, as growth is limited by O2 availability rather than by nutrient levels in this microcosm system [33, 53].

The ecosystem engineering of the initial colonists is also an example of niche creation (niche construction or biogenic habitat formation) [19, 56], as the high-O2 zone now represents an ecological opportunity [5, 25, 26] for any adaptive lineage capable of colonizing this region more successfully than the initial colonists. Adaptive radiation and niche creation are inter-linked [5, 19, 25, 26, 57], and in this system the high-O2 zone is colonized primarily by the Wrinkly Spreaders by biofilm-formation at the A-L interface (**Figure 3b** and **c**). Single-cell confocal Raman spectroscopy has demonstrated that WS cells recovered from within the biofilm have the same spectral profile as those grown under high-O2 conditions, while cells recovered from the liquid column below the biofilm are more similar to those grown under low-O2 conditions [58].

WS cells under high-O2 conditions also grow faster than those under low O2-conditions [33]. However, although WS cells do not grow faster than wild-type SBW25 cells under high O2-conditions [33], their rapid domination of the A-L interface and subsequent population growth displaces the wild-type colonists from this region in a process known as niche exclusion. WS growth at the A-L interface further reduces O2 flux into the lower parts of the liquid column in a density dependent manner, effectively limiting the growth of any non-biofilm-forming competitor and WS biofilms have more impact on niche divergence as populations lacking WS produce shallower O2 gradients [59].

As the WS biofilm population increases, the division between the high and low-O2 zones also moves up into the biofilm [33], allowing further niche differentiation within the biofilm structure itself. Substantial fitness variation has been observed

*Bacterial Biofilms*

**Figure 3.**

The distinctive WS colony morphology allowed an investigation of the genes required for biofilm-formation, as mini-transposon mutants of the archetypal WS with wild-type-like colony morphologies were also defective in biofilmformation [38]. This approach identified the cellulose biosynthesis (*wss*) operon required for the production of partially acetylated cellulose which was the primary biofilm matrix or EPS [38, 41]. However, the WS colony morphology and biofilm also involves an additional EPS, poly-β-1,6-N-acetyl-D-glucosamine (PNAG), as well as lipopolysaccharide (LPS), and interactions between cellulose, PNAG, LPS, and cells are required to maintain biofilm strength and integrity [42, 43]. Mini-transposon analysis also identified a chemotaxis-like (*wsp*) operon with a diguanylate cyclase (DGC) response regulator [38, 44–46]. Subsequent sequence analysis of this operon from the archetypal WS determined the presence of a single nucleotide mutation changing one amino acid residue in the methylesterase subunit [45] which acts as a negative regulatory component of the system. This results in the over-activation of the DCG, leading to increased c-*di*-GMP levels and the activation of the cellulose synthase complex. Mutations in other Wsp subunits, regulators and DGCs activated the WS phenotype in a series of independently

*and (ii) displacement from this region; WS biofilms (iii) are formed at the A-L interface).*

*The success of the Wrinkly Spreader in static microcosms can be understood from an evolutionary ecological perspective. The ecosystem engineering of the initial wild-type SBW25 colonists produces an O2 gradient (dotted curve) which creates an O2-rich upper zone (the Goldilocks zone) and a lower depleted zone (a). Wildtype SBW25 and Wrinkly Spreaders show different niche preferences with the WS colonising the top of the Goldilocks zone at the A-L interface (b). The WS biofilm-forming strategy is a more efficient use of resources than constant aerotaxis (swimming) to counter Brownian motion, microcurrents and vibrations which would move cells away from the optimal growth zone (c) (cell tracks indicate (i) aerotaxis towards the goldilocks zone* 

This understanding of the underlying molecular biology of the WS phenotype allowed a mechanistic link to be made between adaptive mutation and fitness [45] and demonstrated how easily perturbations c-*di*-GMP homeostasis could result in a key innovation through the activation of a system normally repressed in wildtype SBW25 [1, 2]. The relative ease of recovering WS lineages from diversifying populations of wild-type SBW25, demonstrating a change in niche preference and determining the competitive fitness advantage compared to the ancestral strain, also makes the SBW25 system a model for demonstrating evolution in laboratory

The microcosm system has therefore since been used to examine how wild-type colonists modify their environment [33], cells access the A-L interface [52], different environmental conditions drive WS evolution, phenotype and fitness [35, 53], and whether quorum regulation might be involved in biofilm-formation [54]. In the following subsections, we describe how the ecosystem engineering of the colonists provides the ecological opportunity and creates the niche for adaptive WS lineages and explain why biofilm-formation is the better strategy for colonizing this new

**332**

niche.

classes [50, 51].

isolated mutants [35, 43, 45, 47–49].

<sup>3</sup> 'Goldilocks and the Three Bears', written by Robert Southey, is a tale about a girl called Goldilocks who enters the home of a family of bears while they are away. She tests their chairs, beds and breakfast porridge, always choosing the one most favourable for her, before eventually being chased away when the bears return. The 'Goldilocks zone' is also used to refer to the habitable zone around a star where the temperature is just right for liquid water to exist on an orbiting planet. Here we use the term, stricto sensu, to mean the A-L interface plus the high-O2 zone immediately below it.

### *Bacterial Biofilms*

between independently isolated WS [38, 43, 47, 49], suggesting multiple lineages may develop in these populations and compete with one another as Red Queens [60]4 and further competition occurs with resident cheater lineages which no longer produce cellulose [61] and do not contribute to biofilm-formation [62–64].

Fluorescent microscopy suggests WS cells are most active near the top surface of the biofilm [33] and electron micrographs show that it is a very porous structure [65] (**Figure 4**). It is possible that continuous growth near the top progressively limits the growth of cells lower down in a manner known as the Ancestors' inhibition effect [61], though this can also be interpreted as altruistic behaviour by cells which push their descendants up into better O2 conditions and help suffocate neighbouring competitors [19, 61]. Spatial separation caused by the clumping of WS cells

### **Figure 4.**

*The Wrinkly Spreader biofilm is a complex structure with voids and fibres apparent at different levels of magnification. Shown are views of biofilms in situ from above (a) and by electron microscopy (b and c) (scale bars represent 10 μm; the mean wild-type SBW25 bacterial body length is 3 μm [34] and individual cells are just visible in (c)). Photographs: (a) A. Spiers, (b and c) O. Moshynets.*

**335**

**Figure 5.**

*Extending an Eco-Evolutionary Understanding of Biofilm-Formation at the Air-Liquid Interface…*

by the production of cellulose and the exclusion of cheaters, plus the continued development of the O2 gradient within developing biofilms [33] which limits the distance over which the benefits of cooperation act, will help stabilize cooperation in biofilms [19] and allow kin selection to provide a competitive advantage to WS lineages. Biofilm development, including increasing depth and total biomass, as well as lineage and total population levels, ultimately ends with system failure when

**5. Biofilm-formation is the best strategy for colonising the high-O2 zone**

Aerobic motile bacteria such as SBW25 could gain access to the high-O2 zone by aerotaxis [66], using flagella-mediated swimming motility and following the O2 gradient up towards the A-L interface. Aerotaxis could also be used to maintain position against the physical displacement of cells caused by random diffusion, micro-currents and random knocks and vibrations occurring in microcosms during incubation. Although SBW25 is known to be capable of swimming, swarming and twitching motilities, we only recently demonstrated that wild-type and WS cells are aerotaxic [52] and that the average swimming velocity [34] is sufficient to overcome

However, random diffusion still has a significant effect on maintaining position in the high-O2 zone, and we were able to demonstrate this using modified

*WS fitness decreases with increasing liquid viscosity. Agar (light grey circles) and polyethylene glycol (dark grey circles) were used to increase the viscosity of standard microcosms (white square) and the competitive fitness of the archetypal WS determined in comparison with wild-type SBW25 under Fe-limited conditions where it cannot form a biofilm. Means are shown with standard errors. Dotted lines suggest trends and differences between means were investigated by Tukey-Kramer HSD; means sharing the same letters are not significantly* 

*different (α = 0.05). Data are replotted from [52] (Supplementary Information).*

*DOI: http://dx.doi.org/10.5772/intechopen.90955*

it rips and sinks to the bottom of the microcosm vial [2].

the negative effects of random diffusion on cell localization [52].

<sup>4</sup> The Red Queen is a character in 'Through the looking-glass, and what Alice found there', written by Lewis Carrol. In the Red Queen's race, she and Alice were constantly running yet remained in the same spot. The Red Queen has been adopted as an evolutionary hypothesis which states that lineages must constantly adapt and evolve in order to compete successfully against others which are adapting and to a constantly changing environment. (The Red Queen should not to be confused with the Queen of Hearts who appears in an earlier story by Lewis Carrol.)

*Extending an Eco-Evolutionary Understanding of Biofilm-Formation at the Air-Liquid Interface… DOI: http://dx.doi.org/10.5772/intechopen.90955*

by the production of cellulose and the exclusion of cheaters, plus the continued development of the O2 gradient within developing biofilms [33] which limits the distance over which the benefits of cooperation act, will help stabilize cooperation in biofilms [19] and allow kin selection to provide a competitive advantage to WS lineages. Biofilm development, including increasing depth and total biomass, as well as lineage and total population levels, ultimately ends with system failure when it rips and sinks to the bottom of the microcosm vial [2].
