Team members: Yunhao Liu
Collaborators: Dr. Xue-Xian Zhang, Dr. Rob Jackson
Key publications: Rainey 1999 Env Microbiol, Giddens et al 2007 PNAS, Silby et al 2009 Genome Biol, Zhang et al 2012 Env Microbiol
The bacterium that is (and has been) the focus of much of this research is Pseudomonas fluorescens SBW25. It is a plant colonizing bacterium that was originally isolated from the phyllosphere of a sugar beet plant, but it is equally at home in the rhizosphere. In fact it has a remarkable ability to colonize pretty much any part of a plant: 10 cells applied to a non-sterile (clay coated) sugar beet seed placed in non-sterile soil manages to multilpy to ~10^8 cells within 1 week.
In order to understand the mechanistic basis of SBW25's ecological performance a promoter trapping strategy known as IVET was devised and applied (the acronym stands for in vivoexpression technology and was coined by Mike Mahan and colleagues (see Science 1993 259, 6860). This strategy -- initially based on pantothenate auxotrophy (panB), but later (and more powerfully) based on diaminopimelate and lysine auxotrophy (dapB) -- has led to the identification of genes displaying elevated levels of expression in the plant environment (see Rainey 1999 Env Microbiol 1, 243 and Gal et al 2003 Mol Ecol 12, 3109).
A good deal of work by a number of people over the last 10 years has provided much insight into the biological function and ecological significance of some of these plant / rhizoshere / phyllosphere-specific genes (see papers listed under 'ecological genetics' in publications). Indeed, for several of these loci we have gained some quite considerable insight, but it has been a slog. A particular challenge has been dealing with potentially interesting genes (for example the set that encodes an atypical type three secretion system) for which we have had little by way of concrete laboratory phenotype to get our teeth into. This is of course the downside -- or at least the highly challenging side -- of any attempt to understand the function of traits expressed in the wild: by definition, these traits are silent in the lab. A very staunch example of the effort required to make progress when phenotypes are subtle is in Jackson et al (2005) J Bacteriol 187, 8477; Zhang et al (2004) Microbiol150, 2889 provides a further example.
More recently we (largely Steve Giddens, Rob Jackson and Christina Moon: see PNAS 104, 18247) described a genetic approach involving suppressor analyses for which we coined the term SPyVET (it is an extension of the IVET approach) that allows direct insight into phenotypes and regulatory networks that operate to control the expression of various traits expressed in the environment where the organism naturally resides. This has already allowed a good deal more progress than had previously been possible. Examples from on-going work include the cellulose-encoding wss operon and its regulatory ties with flagella mediated motility (with Rob Jackson) and the genes involved in copper homeostasis, pyoverdine production, histidine uptake and utilization and regulatory co-ordination of cellular carbon and nitrogen metabolism ((see Xue-Xian Zhang and the genetics of hut below).
In addition to our IVET-dependent approach to ecological genetics we have long wanted to understand one particular locus to the extent that we could duplicate the efforts of Tony Dean and Dan Dyhuizen who examined the ecological significance of allelic variants in the lac operon. We are now in such a position with regard to our understanding of the histidine uptake and utilization locus. Hao Chang has begun to document and unravel some quite remarkable polymorphism (particularly in transporters) at the population level and this stands to develop into a significant area of our investigations.
The genetics of histidine utilization
I would never have thought that the genetics of food acquisition could be interesting, but daily it becomes more fascinating. Of course, with hindsight, it is obvious that it should be interesting: nothing is more important than getting food.
Xue-Xian Zhang and PBR began this work some years ago as an antidote to years of trying to do genetics with minimal phenotypes. Put another way, the histidine uptake and utilization (hut) operon of SBW25 is active in the plant environment and inactive in minimal medium in the laboratory, but it can be activated simply by the addition of histidine (or its immediate breakdown product, urocanate).
Actually there was another reason for getting interested in this locus: an examination of the locus from different genome-sequenced Pseudomonas strains showed much polymorphism in the predicted transporters. The hut locus of SBW25 is particular complex with a histidine permease, a urocanate permease, an ABC system and various other transporters elsewhere in the genome -- some of which are tied to to response regulators. The causes of the differences among strains (in the uptake systems) suggested to us that this was where the action lay: that selection might work primarily on the uptake system. The population genetics of this is now something that we are heavily involved in.
Equally complex is the regulation of hut. It contains at least two levels of regulation: one specific and one general. At the specific level expression is controlled by the HutC repressor whose activity is modulated by urocanate (when urocanate is available repression is relieved). Also operating at the specific level is a newly identified regulator (HutD) that we suggest is a governor of hut that prevents the locus exceeding a critical upper level of activity. The need for the governor (we suggest) stems from the liberation of two molecules of ammonia for each molecule of histidine: too much ammonia is not good. And the postiive feed forward regulation of hut means that expression could run unchecked (and to dangerous levels) if there was no means of controlling the upper level of expression (see Zhang & Rainey (2007) Genetics 176, 2165). The possibility that HutD might be a therapeutically useful target is currently being investigated by Yunhao Liu.
At the level of general regulation hut is controlled by CbrAB: CbrB is a sigma 54 enhancer binding protein that can activate hut transcription in response to both carbon and nitrogen starvation conditions. The hut locus is also regulated by the NtrBC two-component regulatorry system that ensures expression when cells are nitrogen starved (see Zhang & Rainey (2008) 178, 185). The ability of CbrAB to regulate expression of hut across a wide range of C:N ensures that histidine can be utilized whatever the carbon to nitrogen ratio. In essence, we show that SBW25 is a glutton; that its regulation is geared toward gaining maximum food as quickly as possible: "an eat now, pay later and sod the neighbour" approach to life.
Our recent work focusses largely on CbrA -- and takes place in collaboration with Greg Cook -- but also tackles the evolutionary causes of regulatory complexity. We're also beginning to recognize the challenges that organisms face when they are presented with a food source (such as histidine) that delivers both C and N; and when the organism has to deal with extracting C under conditions when it doesn't require N, and vice versa. It is fascinating stuff and motivates some interesting selection experiments.
And then there is the mystery of urocanate: where does it come from (apart from dog urine)?