Final Report Summary - BIOSIGHET (Exploring the biological significance of phenotypic heterogeneity in Pseudomonas) Genetically identical individuals sometimes express different phenotypes, even when exposed to the same environmental conditions. Such cases of “phenotypic heterogeneity” are particularly common in asexual bacterial populations. An example was recently described in Pseudomonas fluorescens SBW25, a bacterial species isolated from the leaf of a sugar beet plant in the United Kingdom [1]. Laboratory-grown P. fluorescens SBW25 populations contain cells both with and without capsules (Cap+ and Cap-, respectively; see Figure 1) [2]. The major aims of this project are: (i) to investigate the biological function of bistable capsule expression, and (ii) to explore the molecular mechanisms underpinning capsule bistability. (i) The biological function of bistable SBW25 capsule expressionThe existence of SBW25 capsule bistability leads one to question why only some cells express a capsule - what advantage, if any, does having a mixture of Cap+ and Cap- cells provide SBW25 populations? At the beginning of this project, we proposed two hypotheses: (a) that capsule heterogeneity is a bet hedging strategy that facilitates survival in the face of unpredictable environmental conditions, and (b) that capsule heterogeneity is an indicator of a division of resources strategy, whereby Cap+ and Cap- cells ultimately make use of different environmental resources. In this project, substantial progress has been made with testing hypothesis (a).First, an exploratory analysis showed that many external factors influence the degree of capsule expression in SBW25. A particularly strong effect was observed for temperature: lower temperatures were shown to give rise to higher proportions of Cap+ cells. This is interesting because temperatures experienced in the natural SBW25 environment (a sugar beet plant in the UK) are likely to be considerably lower than those used in the laboratory (26˚C), indicating that bistable capsule expression may be important in the natural environment. Next, we investigated whether the ability to produce capsules provides an advantage at various temperatures in the laboratory. Competition experiments were set up and performed between a capsule-capable strain (i.e. an SBW25 strain that can generate Cap+/Cap- populations) and a capsule-incapable strain (i.e. an SBW25 strain that generates entirely Cap- populations), under various growth conditions and temperatures. At lower temperatures, the capsule-capable strain “won” these competitions, demonstrating that the ability to produce capsules can be beneficial at low temperatures. These results are consistent with capsule heterogeneity facilitating survival of SBW25 at low temperatures, and have led to ongoing microscopic investigations of population growth in various SBW25 strains. (ii) The molecular mechanisms underpinning capsule switchingThe second major achievement of this project concerned elucidating the molecular bases of capsule switching: what makes some cells express a capsule, and others not, and what is the molecular process by which individuals become Cap+ or Cap-? These questions began to be addressed during previous work by the researcher [2-4]. This work showed that the molecular events underpinning the capsule “decision” were embedded deep within the central metabolic pathways of the cell. Of particular interest is the final segment of the pyrimidine biosynthetic pathway – the pathway that synthesizes pyrimidine nucleotides for use in DNA synthesis and/or polymer synthesis (see Figure 2). Here, in collaboration with the Sauer laboratory at ETH Zurich, a method was developed to isolate and quantify pyrimidine nucleotides in P. fluorescens SBW25. This method was used to measure relative quantities of pyrimidine nucleotides in various SBW25-derived strains, showing a generally inverse relationship between pyrimidine nucleotide UTP and capsulation levels. Together, the results led to the proposition of the “growth-capsulation model” of capsule switching, whereby cells with high pyrimidine levels channel their resources into DNA synthesis and cell division (Cap- phenotype), while cells with lower levels preserve pyrimidines by stalling cell division through capsule synthesis (Cap+ phenotype). This work has recently been published in the open access journal PLoS Biology [5]. The conclusions of this project bring together experimental evidence from evolution, cell biology, biochemistry and mathematics to shed light on a topical and broadly relevant biological question. The results have contributed to understanding how microbial populations survive environmental insults, an area that holds great interest for a wide range of disciplines. In particular, mechanisms that enhance microbial survival present a major challenge in the effective treatment of infectious disease in humans, animals and plants [6], rendering this research of interest to medicine and agriculture.Reference List1. Rainey PB & Bailey MJ. (1996). Physical and genetic map of the Pseudomonas fluorescens SBW25 chromosome. Mol Microbiol 19: 521-533.2. Gallie J. (2010). Evolutionary and molecular origins of a phenotypic switch in Pseudomonas fluorescens SBW25. PhD thesis. New Zealand Institute for Advanced Study, Auckland: Massey University.3. Beaumont HJE, Gallie J, Kost C, Ferguson GC & Rainey PB. (2009). Experimental evolution of bet hedging. Nature 462: 90-93.4. Rainey PB, Beaumont HJE, Ferguson GC, Gallie J, Kost C, Libby E & Zhang X-X. (2011). The evolutionary emergence of stochastic phenotype switching in bacteria. Microb Cell Fact 10(Suppl 1): S14. 5. Gallie J, Libby E, Bertels F, Remigi P, Jendresen CB, Ferguson GC, Desprat N, Buffing MF, Sauer U, Beaumont HJE, Martinussen J, Kilstrup M, Rainey PB. (2015). Bistability in a metabolic network underpins the De Novo Evolution of colony switching in Pseudomonas fluorescens. PLoS Biology 13(3): e1002109.6. Jayaraman R. (2008). Bacterial persistence: some new insights into an old phenomenon. J Biosci 33: 795-805 final1-figure2_biosighet.jpg final1-figure1_biosighet.jpg
