A mechanistic explanation linking adaptive mutation, niche change, and fitness advantage for the Wrinkly Spreader

Experimental evolution studies have investigated adaptive radiation in static liquid microcosms using the environmental bacterium Pseudomonas fluorescens SBW25. In evolving populations a novel adaptive mutant known as the Wrinkly Spreader arises within days having significant fitness advantage over the ancestral strain. A molecular investigation of the Wrinkly Spreader has provided a mechanistic explanation linking mutation with fitness improvement through the production of a cellulose-based biofilm at the air-liquid interface. Colonisation of this niche provides greater access to oxygen, allowing faster growth than that possible for non-biofilm—forming competitors located in the lower anoxic region of the microcosm. Cellulose is probably normally used for attachment to plant and soil aggregate surfaces and to provide protection in dehydrating conditions. However, the evolutionary innovation of the Wrinkly Spreader in static microcosms is the use of cellulose as the matrix of a robust biofilm, and is achieved through mutations that deregulate multiple diguanylate cyclases leading to the over-production of cyclic-di-GMP and the stimulation of cellulose expression. The mechanistic explanation of the Wrinkly Spreader success is an exemplar of the modern evolutionary synthesis, linking molecular biology with evolutionary ecology, and provides an insight into the phenomenal ability of bacteria to adapt to novel environments

Getting Wrinkly Spreaders to demonstrate evolution in schools

Understanding evolution is crucial to modern biology,but most teachers would assume that practical demonstrations of evolution in school laboratories are unfeasible. However, perhaps they have not heard of ‘evolution in a test tube’ and how Wrinkly Spreaders can form the basis for both practical demonstrations of bacterial evolution and further work

New insights into the effects of several environmental parameters on the relative fitness of a numerically dominant class of evolved niche specialist

Adaptive radiation in bacteria has been investigated using Wrinkly Spreaders (WS), a morphotype which colonises the air-liquid (A-L) interface of static microcosms by biofilm formation with a significant fitness advantage over competitors growing lower down in the O2-limited liquid column. Here, we investigate several environmental parameters which impact the ecological opportunity that the Wrinkly Spreaders exploit in this model system. Manipulation of surface area/volume ratios suggests that the size of the WS niche was not as important as the ability to dominate the A-L interface and restrict competitor growth. The value of this niche to the Wrinkly Spreaders, as determined by competitive fitness assays, was found to increase as O2 flux to the A-L interface was reduced, confirming that competition for O2 was the main driver of WS fitness. The effect of O2 on fitness was also found to be dependent on the availability of nutrients, reflecting the need to take up both for optimal growth. Finally, the meniscus trap, a high-O2 region formed by the interaction of the A-L interface with the vial walls, was also important for fitness during the early stages of biofilm formation. These findings reveal the complexity of this seemingly simple model system and illustrate how changes in environmental physicality alter ecological opportunity and the fitness of the adaptive morphotype

Penetrating the air-liquid interface is the key to colonization and wrinkly spreader fitness

In radiating populations of Pseudomonas fluorescens SBW25, adaptive wrinkly spreader (WS) mutants are able to gain access to the air–liquid (A–L) interface of static liquid microcosms and achieve a significant competitive fitness advantage over other non-biofilm-forming competitors. Aerotaxis and flagella-based swimming allows SBW25 cells to move into the high-O2 region located at the top of the liquid column and maintain their position by countering the effects of random cell diffusion, convection and disturbance (i.e. physical displacement). However, wild-type cells showed significantly lower levels of enrichment in this region compared to the archetypal WS, indicating that WS cells employ an additional mechanism to transfer to the A–L interface where displacement is no longer an issue and a biofilm can develop at the top of the liquid column. Preliminary experiments suggest that this might be achieved through the expression of an as yet unidentified surface active agent that is weakly associated with WS cells and alters liquid surface tension, as determined by quantitative tensiometry. The effect of physical displacement on the colonization of the high-O2 region and A–L interface was reduced through the addition of agar or polyethylene glycol to increase liquid viscosity, and under these conditions the competitive fitness of the WS was significantly reduced. These observations suggest that the ability to transfer to the A–L interface from the high-O2 region and remain there without further expenditure of energy (through, for example, the deployment of flagella) is a key evolutionary innovation of the WS, as it allows subsequent biofilm development and significant population increase, thereby affording these adaptive mutants a competitive fitness advantage over non-biofilm-forming competitors located within the liquid column

Parallel compensatory evolution stabilizes plasmids across the parasitism-mutualism continuum

Plasmids drive genomic diversity in bacteria via horizontal gene transfer [1 and 2]; nevertheless, explaining their survival in bacterial populations is challenging [3]. Theory predicts that irrespective of their net fitness effects, plasmids should be lost: when parasitic (costs outweigh benefits), plasmids should decline due to purifying selection [4, 5 and 6], yet under mutualism (benefits outweigh costs), selection favors the capture of beneficial accessory genes by the chromosome and loss of the costly plasmid backbone [4]. While compensatory evolution can enhance plasmid stability within populations [7, 8, 9, 10, 11, 12, 13, 14 and 15], the propensity for this to occur across the parasitism-mutualism continuum is unknown. We experimentally evolved Pseudomonas fluorescens and its mercury resistance mega-plasmid, pQBR103 [ 16], across an environment-mediated parasitism-mutualism continuum. Compensatory evolution stabilized plasmids by rapidly ameliorating the cost of plasmid carriage in all environments. Genomic analysis revealed that, in both parasitic and mutualistic treatments, evolution repeatedly targeted the gacA/gacS bacterial two-component global regulatory system while leaving the plasmid sequence intact. Deletion of either gacA or gacS was sufficient to completely ameliorate the cost of plasmid carriage. Mutation of gacA/gacS downregulated the expression of ∼17% of chromosomal and plasmid genes and appears to have relieved the translational demand imposed by the plasmid. Chromosomal capture of mercury resistance accompanied by plasmid loss occurred throughout the experiment but very rarely invaded to high frequency, suggesting that rapid compensatory evolution can limit this process. Compensatory evolution can explain the widespread occurrence of plasmids and allows bacteria to retain horizontally acquired plasmids even in environments where their accessory genes are not immediately useful

Extending an eco-evolutionary understanding of biofilm-formation at the air-liquid interface to community biofilms

Growing bacterial populations diversify to produce a number of competing lineages. In the Pseudomonas fluorescens SBW25 model system, Wrinkly Spreader mutant lineages, capable of colonising the air-liquid interface of static microcosms by biofilm-formation, rapidly appear in diversifying populations with a fitness advantage over the ancestral wild-type strain. Similarly, a biofilm is rapidly produced by a community containing many biofilm-competent members, and selection by serial transfer of biofilm samples across microcosms results in a gradually changing community structure. Both the adaptive radiation producing Wrinkly Spreaders and the succession of biofilm communities in these static microcosms can be understood through evolutionary ecology in which ecological interactions and evolutionary processes are combined. Such eco-evolutionary dynamics are especially important for bacteria, as rapid growth, high population densities and strong selection in the context of infections can lead to fast changes in disease progression and resistance phenotypes, while similar changes in community function may also affect many microbially-mediated biotechnological and industrial processes. Evolutionary ecology provides an understanding of why bacterial biofilms are so prevalent and why they are such a successful colonisation strategy, and it can be directly linked to molecular analyses to understand the importance of pathways and responses involved in biofilm-formation

Viewing biofilms within the larger context of bacterial aggregations

The ‘Microbial Cities’ vision of bacterial biofilms has dominated our understanding of the development and functioning of bacterial aggregations for the past 20 years, during which active sludge, clumps, colonies, flocs, mats, pellicles, rafts, slimes, zooglea, etc. have been largely forgotten or ignored. Although the medically inspired developmental model of human pathogen biofilms has merits including providing a rationale for the development of anti-biofilm therapeutics, it fails to provide links to other types of bacterial aggregation that are commonly found in a wide range of natural and man-made environments. Possibly as a result, applied and environmental microbiologists tend to avoid the term ‘biofilm’ and use others such as ‘microbial mats’ instead. Here we challenge the simplistic planktonic (independent and free-swimming bacteria)-biofilm (sessile and co-operative bacteria) dichotomy, and consider biofilms within the larger context of bacterial aggregations. By placing biofilms into context, which we see as a continuum of aggregations or communities with varying abiotic and biotic properties, fundamental physical, biological, and evolutionary ecological processes that effect community development and function can no longer be considered unique to biofilms, but may also be important in other aggregations that develop over time and change in nature depending on prevailing conditions. By doing this, we will be better able to distinguish those processes which govern bacterial colonisation and ecological success in a wider sense from those that are unique to particular environments and specialised strategies

Control of pore geometry in soil microcosms and its effect on the growth and spread of <i>Pseudomonas </i>and <i>Bacillus</i> sp.

Simplified experimental systems, often referred to as microcosms, have played a central role in the development of modern ecological thinking on issues ranging from competitive exclusion to examination of spatial resources and competition mechanisms, with important model-driven insights to the field. It is widely recognized that soil architecture is the key driver of biological and physical processes underpinning ecosystem services, and the role of soil architecture and soil physical conditions is receiving growing interest. The difficulty to capture the architectural heterogeneity in microcosms means that we typically disrupt physical architecture when collecting soils. We then use surrogate measures of soil architecture such as aggregate size distribution and bulk-density, in an attempt to recreate conditions encountered in the field. These bulk-measures are too crude and do not describe the heterogeneity at microscopic scales where microorganisms operate. In the current paper we therefore ask the following questions: (i) To what extent can we control the pore geometry at microscopic scales in microcosm studies through manipulation of common variables such as density and aggregate size?; (ii) What is the effect of pore geometry on the growth and spread dynamics of bacteria following introduction into soil? To answer these questions, we focus on Pseudomonas sp. and Bacillus sp. We study the growth of populations introduced in replicated microcosms packed at densities ranging from 1.2 – 1.6 g cm-3, as well as packed with different aggregate sizes at identical bulk-density. We use X-ray CT and show how pore geometrical properties at microbial scales such as connectivity and solid-pore interface area, are affected by the way we prepare microcosms. At a bulk-density of 1.6 g cm-3 the average number of Pseudomonas was 63% lower than at a bulk-density of 1.3 g cm-3. For Bacillus this reduction was 66 %. Depending on the physical conditions, bacteria in half the samples took between 1.62 and 9.22 days to spread 1.5 cm. Bacillus did spread faster than Pseudomonas and both did spread faster at a lower bulk-density. Our results highlight the importance that soil physical properties be considered in greater detail in soil microbiological studies than is currently the case