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

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

#### **Figure 5.**

*Bacterial Biofilms*

[60]4

between independently isolated WS [38, 43, 47, 49], suggesting multiple lineages may develop in these populations and compete with one another as Red Queens

produce cellulose [61] and do not contribute to biofilm-formation [62–64].

and further competition occurs with resident cheater lineages which no longer

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

<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

*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* 

**334**

**Figure 4.**

who appears in an earlier story by Lewis Carrol.)

*just visible in (c)). Photographs: (a) A. Spiers, (b and c) O. Moshynets.*

*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).*

microcosms in which we had added low concentrations of agar or polyethylene glycol to increase viscosity, as diffusion is inversely dependent on liquid viscosity [52]. Both wild-type and WS cell localization improved with increasing viscosity and, furthermore, WS competitive fitness was found to decrease with increasing viscosity (**Figure 5**) [52]. This indicates that WS biofilm-formation is a better strategy allowing the colonization of the high-O2 zone and more specifically, of the A-L interface, than constant aerotaxis.

We argue that the need to remain in place at the top of the liquid column efficiently in order to make use of greater O2 availability is the fundamental explanation for the success of A-L interface biofilm-formation in static microcosms by motile aerobic such as the pseudomonads [36] where growth is limited by O2-availability rather than by nutrients [53]. The success is determined by a cost-benefit trade-off, in which resource costs required for biofilm-formation by the community or constant aerotaxis by individual cells are balanced against population gains.
