Phil Poole
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Molecular physiology of plant bacterial interactionsThe general areas in which my group works are bacterial genetics and molecular biology of plant associated bacteria. Our emphasis is to study the physiology of bacterial growth and survival in the rhizosphere and how bacteria establish symbiotic interactions with plants. A further focus of our work is the physiology and biochemistry of nitrogen fixation in legume nodules. Currently we have five principal research areas, which are:
The first area of investigation is motivated by the enormous importance of nitrogen fixation by legumes to both agriculture and the global nitrogen cycle. Nitrogen fixation by the legume-Rhizobium symbiosis is driven by a supply of carbon from the plant, so by understanding this process we can determine which factors regulate the total amount of nitrogen fixed. The regulation of nutrient exchange between a legume and Rhizobium is largely determined by two membrane systems, one is plant derived and the other is bacterial. Our approach to understanding the interactions within and between these membranes is to mutate and clone the bacterial transport systems important for nutrient exchange. The genetics governing regulation of expression of these transport systems is being investigated and their effects on nitrogen fixation assessed. In the second area of investigation we are studying the genes that enable bacteria to survive and reproduce in the soil and particularly the plant rhizosphere. The rhizosphere is the region immediately surrounding the plant root that is rich in nutrients and supports a large bacterial population. Competition among bacteria for nutrients is fierce and we are using signature tagged mutagenesis, microarray analysis, IVET and high throughput sequencing to understand the strategies that bacteria use to colonise this niche. In our third area we are developing a powerful set of molecular biosensors to monitor environmental conditions both temporally and spatially. Our genome analysis of Sinorhizobium meliloti, Mesorhizobium loti and Rhizobium leguminosarum revealed an explosive growth in the number of ABC (ATP binding cassette) uptake systems in these organisms, with around 160 systems present. The binding proteins of these transporters are highly solute specific and tightly induced in response to appropriate conditions. We were then able to identify the inducing solute for 76 binding proteins. This included a wide range of sugars, amino acids, organic acids, nucleotides and metal ions. We are now developing new FRET biosensors to enable real time monitoring of ligands in the environment. A key aim is to map both temporally and spatially the secretion of metabolites by plant roots. In the fourth area, we are examining the mechanism of uptake of solutes by ABC (ATP Binding Cassette) transport systems. The ABC superfamily is one of the largest of all transport families with over 1100 identified members. While we concentrate on the bacterial uptake systems these transporters are found widely in almost all living organisms. They also have tremendous medical relevance with examples such as the multi-drug resistance protein and the cystic fibrosis chloride channel (CFTR). Our work is focused on the apparent ability of these systems to catalyze uptake and efflux in contradiction to the accepted dogma that they are unidirectional. In the fifth area we are studying how free-living bacteria differentiate into root nodule bacteria. The sequencing of the genome of R. leguminosarum offers us a unique opportunity to use microarrays to address the problems both of the final metabolic state of the bacteroid and the genetic and developmental switches that are crucial to differentiation. By comparing gene expression between free-living bacteria, rhizosphere bacteria and bacteria/bacteroids from various stages of nodule development we can begin to reconstruct the regulatory switches and developmental/ metabolic consequences of these switches. In this area we are studying:
Specifically I hypothesise that the three dominant factors regulating bacteroid metabolism are the O2 tension, provision of C4-dicarboxylates and cellular growth rate, therefore gene expression profiles of rhizosphere bacteria and bacteroids are being compared against free-living bacteria grown in chemostat culture limited by glucose, malate or O2 at intermediate and low dilution rates. The expression data will then be mapped to the KEGG metabolic database using the GeneSpring bionformatic analysis suite to allow us to visualise the key metabolic changes that occur in bacteroid formation. Further details can be found at www.rhizobuim.net.
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