Microbes constitute about a third of the Earth's biomass and exhibit remarkable genetic diversity. In a majority of environments, microbes live as interacting communities. Yet, we know almost nothing about how microbial worlds communicate, evolve, share resources, interact with other organisms in the rhizosphere and in aquatic environments, and ultimately shape the landscape of the Earth. Our recent research on speciation and evolution of thermophilic cyanobacteria that thrive in the microbial mats of hot springs in Yellowstone National Park has set the stage for a move into challenging new territory. We now have full genome sequences of two dominant cyanobacteria (Synechococcus sp.) and a green, non-sulfur photosynthetic bacterium Roseiflexus sp. Analyses of these genomes and metagenomes (i.e. genetic repertoire of the whole community) have given us a first glimpse into the complexity of microbial populations. We also pioneered methods to investigate the pattern of gene expression, protein abundances and activities in these microbes over the day-night cycle and discovered daily changes that describe the key features of the energetics of the mat community over the diel cycle. There appear to be a complex, integrated regulatory network (signaled by light, anoxia, or circadian rhythms) that occurs within these interactive communities. There also appears to be metabolite/energetic exchange among the organisms in the mat, which suggests that hypotheses generated using pure laboratory cultures would require careful in situ analyses in parallel.  This is one of the emerging, challenging and exciting areas in environmental biology.
Our initial physiological findings, coupled with the genetic diversity of microbes, provide a unique and exciting opportunity to explore, in real time, how evolution and adaptation work in the microbial world at temperatures exceeding 50oC. I believe that cyanobacterial interactions in the environment provide an excellent paradigm for dissecting the survival, functional diversity and evolution of microbial communities in a variety of harsh environmental settings and how the diversification within the community reflects the integrated utilization of limiting resources. The biogeochemical and regulatory processes underlying these microbial communities can now be queried by a number of sophisticated tools.

Measures of Diversity in Microbial Communities: One of the surprises of our investigation of microbial communities was the discovery of unexpected diversity at every level, from gene variants to the level of whole genome architecture.  These findings raise fundamental questions that we can begin to tackle:
(i) how and why is bacterial diversity maintained and how do populations evolve?
(ii) how can our study of model organisms, (that are essentially clonal) which is an accepted and powerful paradigm benefit from the idea that in the environment genomic complexity is the norm, rather than the exception? 

We are approaching these questions with the following methodolgies:
Exploiting comparative genomics/metagenomics: Comparative genomics/ metagenomics is a very powerful tool and with the advent of high throughput/low cost sequencing it is possible to explore this at a level that was not feasible even a few years ago. Even with our limited analysis we have seen that genomic variability is extensive in the closely related cyanobacteria that we study.  Taking this to the next logical step requires a deeper focus on specific questions.  One such example which we are currently pursuing is the adaptation of cyanobacteria to high temperatures.  In the microbial mats, there is a gradient of temperature (from ~50C to 70C) that the cyanobacteria have adapted to in various as yet unknown ways.  This natural temperature gradient and moderately simple assemblage of organisms provides an excellent setting for probing this question. 

Ecophysiology of cyanobacteria: Beyond comparative genomics lies the question of how these differences in gene content impact the ecophysiology of cyanobacteria in the environment. For instance, we identified an entire nitrogenase (nif) gene cluster in these organisms suggesting that these unicellular cyanobacteria may be capable of nitrogen fixation. Nitrogen fixation reduces nitrogen gas to biomass, and is of paramount importance in biogeochemical cycling of nitrogen. To address this point, we analyzed nif transcript levels and nitrogenase activity of the Synechococcus ecotypes, over the diel cycle in the microbial mat of an alkaline hot spring in Yellowstone National Park.  Nif transcripts rise in the evening, with a subsequent decline over the course of the night. In contrast, the level of the NifH polypeptide remained stable during the night, and only declined when the mat became oxic in the morning. Nitrogenase activity was low throughout the night; however, it exhibited two peaks, a small one in the evening and a large one in the early morning, when light began to stimulate cyanobacterial photosynthetic activity, but oxygen consumption by respiration still exceeded the rate of oxygen evolution.  Transcripts for proteins associated with energy-producing metabolisms in the cell also followed diel patterns, with fermentation-related transcripts accumulating at night, photosynthesis- and respiration-related transcripts accumulating during the day and late afternoon, respectively. This study, among others, suggests that a multi-faceted approach to understand microbial communities can provide insights into the regulation of metabolism.  Combining eco-physiology with experiments using clean Synechococcus isolates is also an approach we are actively developing. We have recently characterized a number of responses of these isolates to nutrient stress and to high light.

Exploring diversity at the single cell level: Extending from our results from the microbial mats it might not be too much of a stretch to say that bacterial populations in the environment are genetically varied and far from clonal. This raises the obvious question of how we can best assess this variability. A few years ago we initiated a collaboration with Dick Zare’s group in Chemistry to exploit the power of microfluidic cell chambers and single cell capture, followed by capillary electrophoresis to count single molecules (in this case the highly fluorescent phycobiliproteins in cyanobacteria). This led to interesting observations of variability in phycobiliprotein content in single cells and the conclusion that clonality is not necessarily the norm (put in another way, it says that averaging cellular output, although powerful, can conceal these single cell variations). 

1.    Complete genome sequences of two dominant cyanobacteria (Synechococcus sp.) and an abundant green, non-sulfur photosynthetic bacterium (Roseiflexus sp.).
2.    Metagenome dataset (i.e. genetic repertoire of the whole community) of microbial populations in the hotsprings. ~220 Mb of paired Sanger reads
3.    In-house tools developed for comparative genomic analyses
4.    Methods to directly measure expression of specific genes in situ
5.    Measurements of light and oxygen levels directly in the mats (using microsensor technology developed by Michael Kuhl and others). 
6.    Axenic (pure) isolates of Synechococcus sp. grown under defined laboratory conditions

RELEVANT PUBLICATIONS (PDF version available under Publications)

1.    Jensen S., Steunou, A-S, Bhaya, D, Kühl M., Arthur R. Grossman In situ Dynamics of O2, pH and Cyanobacterial Transcripts Associated with Inorganic Carbon Concentration, Photosynthesis and Detoxification of Reactive Oxygen Species in Hot Spring Microbial Mats ISME J  Epub Aug 26, (2010)
2.    Gomez-Garcia M.R., M. Davison, M. B.-Hartnung A. R. Grossman, D Bhaya Alternative Pathways for Phosphonate Metabolism in Thermophilic Cyanobacteria from Microbial Mats ISME J. EPub July 15, (2010)
3.    Adams MM, Gómez-García MR, Grossman AR, Bhaya D. Phosphorus deprivation responses and phosphonate utilization in a thermophilic Synechococcus sp. from microbial mats. J Bacteriol. Dec;190 (24):8171-84 (2008)
4.    Steunou A-S, Jensen S, Brecht E., Becraft E.D., Bateson, Kilian O, Bhaya D. , Ward D.M., Peters J.W., Grossman A.R., Kühl M. (Regulation of nif Gene Expression and the Energetics of N2 Fixation over the Diel Cycle in a Hot Spring Microbial Mat ISME Journal, (2008) 2(4):364-78.
5.    Kilian O, Steunou A-S., Grossman A.R., Bhaya D. A novel two domain-fusion protein in cyanobacteria with similarity to the CAB/ELIP/HLIP superfamily: Evolutionary implications and regulation Molecular Plant 1;155-166 (2008)
6.    Bhaya D., Grossman, A.R., Steunou A.S., N.Khuri, F.M. Cohan, N. Hamamura, M. C. Melendrez, M. M. Bateson, D. M. Ward, J.F. Heidelberg Genomic, metagenomic and functional analyses of cyanobacteria from hot-spring microbial mats reveal an unexpected diversity in nutrient utilization strategies  ISME J: 1(8):703-13 (2007)
7.    Kilian O, Steunou A.S., Fazeli F, Bailey S., Bhaya D., Grossman A.R.: Light Responses of Thermophilic Synechococcus isolates from the Microbial Mats of Octopus Spring Appl. Environ, Microbiol.  73:4268-4278 (2007)
8.    Ward D.M., Cohan, F.M., Bhaya D., Heidelberg J.F., Kühl, M.,and Grossman A.R. Genomics, Environmental Genomics and the Issue of Microbial Species . Nature Heredity 100(2):207-19 (2007)
9.    Huang, B., Wu H., Bhaya D., Grossman A. R., Granier, S., Kobilka B.K. and Zare R. NCounting low-copy number proteins in a single cell. Science 315: 81-84 .(2007)
10.    Labiosa, R.G., Arrigo, K.R. Tu C.J., Bhaya D, Bay S, Grossman A.R., and J Shrager Examination of diel changes in global transcript accumulation in Synechocystis sp. Strain PCC6803: A study in photoacclimation. J Phycol. 42:622-636 (2006)