Environmental Microbiology ReportsVolume 5, Issue 1 p. 1-16 Crystal ballFree Access Crystal ball – 2013 First published: 03 February 2013 https://doi.org/10.1111/1758-2229.12021AboutSectionsPDF ToolsRequest permissionExport citationAdd to favoritesTrack citation ShareShare Give accessShare full text accessShare full-text accessPlease review our Terms and Conditions of Use and check box below to share full-text version of article.I have read and accept the Wiley Online Library Terms and Conditions of UseShareable LinkUse the link below to share a full-text version of this article with your friends and colleagues. Learn more.Copy URL Share a linkShare onFacebookTwitterLinkedInRedditWechat In this feature, leading researchers in the field of environmental microbiology speculate on the technical and conceptual developments that will drive innovative research and open new vistas over the next few years. A high-resolution 3D ‘peek’ into microbial community life Manfred Auer, Life Sciences Division, Lawrence Berkeley National Laboratory, 1 Cyclotron Road, MS Donner, Berkeley, CA 94720, USA. Environmental microbiology, like many other biological disciplines, heavily utilizes the powerful high-throughput tools that modern systems biology has to offer. Huge inventory lists are being generated by all kinds of OMICS, including gen-, metagen-, transcript-, prote- and metabol-OMICS. Despite the emergence of such ever growing lists, we still lack a fundamental understanding of microbial community life, in part – I would argue – for a lack of an understanding of the spatio-temporal relationship of the key components, which can be proteins and macromolecular machines or cells, which function in their respective 3D community context. It would seem that spatial vicinity matters at all levels of complexity, and thus that determination of the 3D ultrastructural organization of sufficiently large volumes, as well as precision protein localization via tag- or affinity-based labelling, are key ingredients to a detailed understanding of community life. Such volumes need to be visualized in 3D, and features of interest must be extracted through segmentation, classification and annotation. In order to go beyond the pretty picture we must determine the volumetric/geometrical parameters, such as volume, shape, diameter, distance, curvature, direction of each of the constituents, and establish their spatial relationships, thus revealing correlations and possibly even causalities. Microbial community architecture has long been the domain of scanning electron microscopy (SEM). However, this traditional imaging approach – while yielding stunning and enticing pictures – does not provide a true 3D impression as it can only reveal the very surface of an object, such as a biofilm, and does not allow a look inside, and often does not allow real quantification of any of the observations. Furthermore, unexpected features such as the frequently encountered intercellular connections between bacteria have been dismissed as sample preparation artefacts (Dohnalkova et al., 2011). Transmission electron microscopy, on the other hand, while allowing for exquisite sample preservation (McDonald and Auer, 2006; Palsdottir et al., 2009), can only cover a tiny sliver of the 3D volume, and the attempt to reconstruct a 3D volume from serial sections has its own challenges, including finding the exact same area to image for different sections and for every grid, as well as compensating for the unique mechanical deformations of each section, making it difficult to unambiguously reconstruct a serial section 3D volume. Recently, two techniques, i.e. focused ion beam SEM and serial block face SEM, have entered the arena of intermediate-resolution 3D electron microscopy imaging and show great promise to overcome traditional limitations: these two novel imaging approaches are somewhat related but yet distinctive from one another, each with its own set of advantages and limitations. They allow imaging of currently tens but soon hundreds of microns of biofilms (both in X, Y and also in Z) at a resolution of ∼10 nm in XY and ∼15 or ∼30–50 nm in Z respectively (Fig. 1). What make these approaches so powerful is that sample preparation does not suffer from traditional SEM sample preparation artefacts, and that the overall biofilm organization at the cellular and community level can be visualized while simultaneously allowing the detection of the deployed macromolecular strategies including vesicles, pili or other internally/externally located macromolecular machines. Figure 1Open in figure viewerPowerPoint Focused ion beam scanning electron microscopy (FIB/SEM) of a mixed microbial community reveals distinct ultrastructural features inside and between cells. Upon 3D segmentation and 3D rendering, the 3D organization can be examined in exquisite detail, probing for the presence and 3D organization of macromolecular machines to cellular and community 3D organization. To be sure, plenty of obstacles remain to be tackled, such as sufficient access to the very expensive 3D imaging equipment, the sheer visualization of such large and highly complex volumes, as well as the need to develop user-guided and/or (semi)-automated approaches for extracting features of interest, easy 3D volume annotation and quantitative 3D geometrical analysis, and ultimately the translation of data voxels into semantic information. As biologists and microscopist team up with computer scientists, some of these enormous challenges are beginning to be addressed, and hence routine large-scale high-resolution imaging of large biofilm volumes is in reach. Clearly, we are entering an exciting new era of 3D imaging in microbiology physiology and pathogenesis that will allow us to map the parts list onto the 3D organization in cells and biofilms, and thus we will be able to take a detailed ‘peek’ into microbial community life. Acknowledgements I would like to thank Phil Hugenholtz, Falk Warnecke, Bernhard Knierim, Brandon Van Leer (FEI), Tom Goddard (UCSF), Monica Lin and Mitalee Desai for their help in sample preparation, 3D FIB/SEM imaging, 3D visualization of the depicted mixed microbial community. This work was supported by the Director, Office of Science, Office of Basic Energy Sciences, of the U.S. Department of Energy under Contract No. DE-AC02-05CH11231. References Dohnalkova, A.C., Marshall, M.J., Areay, B.W., Williams, K.H., Buck, E.C., and Fredrickson, J.K. (2011) Imaging hydrated microbial extracellular polymers: comparative analysis by electron microscopy. Appl Environ Microbiol 77: 1254– 1262. McDonald, K.L., and Auer, M. (2006) High-pressure freezing, cellular tomography and structural cell biology. Biotechniques 41: 137– 141. Palsdottir, H., Remis, J.P., Schaudinn, C., O'Toole, E., Lux, R., Shi, W., et al. (2009) Three-dimensional macromolecular organization of cryo-fixed Myxococcus xanthus biofilms, as revealed by electron microscopic tomography. J Bacteriol 191: 2077– 2082. Microbial Earth: the motion picture Edward F. DeLong, Department of Civil and Environmental Engineering and Department of Biological Engineering, Massachusetts Institute of Technology, Cambridge, MA 02139, USA. Imagine you win the lottery and your prize is to travel with Sir David Frederick Attenborough (OM, CH, CVO, CBE, FRS, FZS, FSA), to train in the art of crafting popular Nature documentaries. In your travels with the master, you are awed by the raw violence of great whites devouring sea lions, by the smooth stealth of a hunting lioness, by the speed and grace of the gazelle that evades her, and by the unimaginable diversity of plant and animal life in the rainforests and coral reefs. You are equally awed by Attenborough's uncanny skill and craft in capturing the essence of nature and nurture, and the beauty, savagery, vastness and variety, which connects his audience emotionally to natural history in a deep, intuitive and visceral way. Now it is your turn, a microbial ecologist having just trained with the great Sir David. The BBC gives you mega bucks to produce a 12-part series, ‘Microbial Earth’. So, how are YOU going to connect in the same emotional, visceral and intuitive ways as Attenborough? Will you show the savagery of exoenzyme hydrolysis attacking a dying diatom bloom, the grace and beauty of runs and tumbles in a chemotactic sensory path, the vicious jab of a Type III pilus, or complex food chain dynamics that recycle carbon and energy between microbes and sediments? Do you think you will get Joe Public's rapt attention in these efforts? Hmmm – it is really NOT as easy a task as Attenborough has! Admittedly, there are some relatively straightforward bridges to be built. After all, videos of ciliates feeding on their bacterial prey, food vacuoles bulging as they gorge, portrays a microbial predator–prey dynamic easily translatable into what people see, know and can intuit. But what about those savage exoenzymes, buzzing electron transport chains, vicious Type III secretion systems, intimate symbioses and vast biogeochemical cycles and gradients? These are not so visceral, intuitive or emotionally accessible, nor arguably so easily portrayed to capture the general public's excitement and imagination. Part of the challenge is that humans simply do not have the intuition, instincts or aesthetic appreciation of microscopic and invisible form, function and interactions (Stahl, 2011; Woese, 1994). The majesty, diversity, impact and complexity of the vast microbial world is not so easily visualized, captured and communicated to the general public – even with the best artists and animators on the planet at your disposal. While the task is certainly not hopeless, and there is great progress to be made, do you really think that today, you could easily top Attenborough's appeal for the public's excitement and attention, in your microbial documentary? (More power to you if your answer is yes – please do it!) But I digress. My gaze into the crystal ball today is not really about one-upping Attenborough. Instead, I will prognosticate briefly on how recent trends have influenced our appreciation of microbial natural history today, and where we may be heading towards in the future: namely towards a much more deep and realistic four-dimensional motion picture of microbial natural history in the wild! To understand the present, look to the past. To understand the future, look to the present It goes with saying that over the past 20 years our perspective on microbial natural history has advanced significantly thanks to the emerging cultivation-independent paradigm (Pace, 2012), as well as advances in more traditional microbiological approaches. From microbial genomics and microarrays, to more recently developed ‘next generation’ sequencing techniques, there have been great gains in the scope, scale and economy of microbial ‘omics’ data acquisition, and the molecular readouts of microbial community structure, function and dynamics that they bring. These advances in turn have brought new insights into the nature of microbial genome evolution, the mechanisms of microbial population dynamics, global maps of microbial taxon distribution and abundance, and the distributions of microbial genes, gene expression and proteins in the environment. The Whole Earth Catalogue of microbes, genomes and genes is fleshing out impressively, at levels unimaginable only just a few years ago. Some may still lament the ‘big data’ problem, complain that we are drowning in data, and quip that information is not knowledge. Of course, there are still great challenges, but the future is bright. While we may be swimming in a sea of big data, as we swim we are learning new ‘strokes’, including new and improved sampling techniques, high-density data archiving capabilities, statistical methods and computational modelling approaches. These newfound capabilities are now facilitating unprecedented views into the natural history of microbial communities and ecosystems, at a scope and scale never-before imaginable. So, at this juncture, what can we predict about the trajectory of future new views of the natural microbial world? One thing seems fairly certain: we soon will move beyond static surveys, snapshot modes and simpler models of the past. This in part will be driven by integrated pictures of in situ microbial community interactions and dynamics, obtained by ‘filming’ the minute-by-minute microbial activities at high biological resolution, at more and more realistic and relevant spatial and temporal scales. This likely will involve the integration of many new and developing technologies including scaled down microfluidic and nanoscale technologies; automated sampling and sensing coupled with high biological resolution ‘omic'-based approaches; high-speed microscopic visualization and chemical approaches (for example, miniaturized flow cytometers and mass spectrometers); and quantitative mapping of the multiple (omic) readouts of indigenous microbial ‘biosensors’ (aka, microbial community members), onto other biological environmental variations. Already some of these new motion pictures are beginning to be released, albeit the technologies still need much improvement, and short film clips are only just now becoming available. The autobiographic human gut microbial community drama entitled ‘Our humans and us’ is now being filmed (Caporaso et al., 2011). Instalments of ‘Seasons of our lives: my years in marine picoplankton’ is also being filmed (Gilbert et al., 2009). The ‘Bloom and Bust’ series, documenting phytoplankton successional events, is also being made in several instalments (Rinta-Kanto et al., 1993; Teeling et al., 2012). And again in the sea, a film entitled ‘A day in the lives of marine picoplankton’, shot with automated, Lagrangian sampling and high-resolution community transcriptome profiling (Ottesen et al., 1997), is also being filmed (coming soon to a theatre near you, Ottesen et al., 2012). These four-dimensional movies of the natural microbial world will increasingly employ remote and continuous sampling and sensing at both micro and macro scales. Sometimes they will be achievable in real time, and sometimes not. And it goes without saying they will require advanced computational, statistical and modelling approaches, to fully develop the plot line and story of the microbial motion picture in the wild. The daily drama and natural historical details of the minutes, days, weeks, months and years in the ‘lives’ of microbial communities that remain obscure at present, will soon come into much sharper focus. With these new perspectives future microbial natural historians are likely to have much richer stories to tell. Microbial natural histories will soon rival the stories told by Sir David Attenborough, as high-resolution, four-dimensional microbial motion pictures more clearly reveal the drama, majesty and intimate interactions that occur each day on the microbial Serengeti. References Caporaso, J.G., Lauber, C.L., Costello, E.K., Berg-Lyons, D., Gonzalez, A., Stombaugh, J., et al. (2011) Moving pictures of the human microbiome. Genome Biol 12: R50. Gilbert, J.A., Field, D., Swift, P., Newbold, L., Oliver, A., Smyth, T., et al. (2009) The seasonal structure of microbial communities in the Western English Channel. Environ Microbiol 11: 3132– 3139. Ottesen, E.A., Marin, R., 3rd, Preston, C.M., Young, C.R., Ryan, J.P., Scholin, C.A., and DeLong, E.F. (2011) Metatranscriptomic analysis of autonomously collected and preserved marine bacterioplankton. ISME J 5: 1881– 1895. Ottesen, E.A., Young, C.R., Eppley, J.M., Ryan, J.P., Chavez, F.P., Scholin, C., and DeLong, E.F. (2012) Pattern and synchrony of gene expression among sympatric marine microbial populations. Proc Natl Acad Sci USA (in press). Pace, N.R. (1997) A molecular view of microbial diversity and the biosphere. Science 276: 734– 740. Rinta-Kanto, J.M., Sun, S., Sharma, S., Kiene, R.P., and Moran, M.A. (2012) Bacterial community transcription patterns during a marine phytoplankton bloom. Environ Microbiol 14: 228– 239. Stahl, D.A. (1993) The natural history of microorganisms. ASM News 59: 609– 613. Teeling, H., Fuchs, B.M., Becher, D., Klockow, C., Gardebrecht, A., Bennke, C.M., et al. (2012) Substrate-controlled succession of marine bacterioplankton populations induced by a phytoplankton bloom. Science 336: 608– 611. Woese, C.R. (1994) Microbiology in transition. Proc Natl Acad Sci USA 91: 1601– 1603. The next big thing in cyanobacteria Robert Haselkorn, Department of Molecular Genetics & Cell Biology, The University of Chicago, 920 East 58 Street, Chicago, IL 60637, USA. It is roughly 20 years since the discovery of the transcription factor HetR in Anabaena. In that period it became clear that the HetR protein alone could not be responsible directly for the expression of the 1500 or so genes needed to turn a vegetative cell fixing carbon into an anaerobic factory fixing nitrogen. With the solution of the X-ray structure of the HetR dimer and studies of its binding to a single palindrome in the Anabaena genome and its regulation by the peptide RGSGR, we are on the cusp of understanding the cascade it directs. The urgently needed information now is the catalogue of auxiliary proteins that associate with HetR to direct it to additional DNA sites, the mechanism by which HetR turns on transcription, and the details of the cascade of genes whose expression is unleashed by HetR. A different set of questions has been posed in connection with the study of toxins produced by cyanobacteria. What functions do these compounds carry out? Why are they made in the first place? During the past year or two the number and character of toxins produced by cyanobacteria has expanded significantly. Originally we were concerned with the microcystins, cyclic heptapeptides that bind irreversibly to protein phosphatases. Microcystins are made by very large synthetic complexes containing multiple domains, each of which binds an activated amino acid, modifies it and joins it to another, using thioester chemistry. This system is termed non-ribosomal peptide synthesis (NRPS). Not all the NRPS products are cyclic; some are linear and at least one has a lipid side-chain that promotes attachment to cholesterol-containing membranes. And now, as a result of genome gazing, another large family of peptides has been uncovered, this time made by ordinary ribosomal peptide synthesis (Wang et al., 2011). One strain of Anabaena has enough genes to encode hundreds of protein precursors, which are processed into tetrapeptides, cyclized and exported. Some of these are protease inhibitors. Finally, there is a family of alkaloids called anatoxins, made by a series of three polyketide synthases (Cadel-Six et al., 2009). Anatoxin binds to the mammalian nicotinic acetylcholine receptor, causing paralysis. In the cases of the microcystins and anatoxins, the known targets are eukaryotic, metazoan, even mammalian. The question then arises: what was the original function of these toxins if their contemporary targets arose a billion years later? Could there have been targets among the prokaryotes that occupied related niches when the cyanobacteria were the most advanced organisms on earth? JP Changeux and PJ Corringer asked this question several years ago and found that the cyanobacterium Gloeocapsa contains an acetylcholine receptor. This protein is pentameric and its X-ray structure is almost identical to that of the pentameric AChR from Torpedo. Expressed in Xenopus oocytes, it functions as a proton pump. It remains to be shown that it binds anatoxin (Corringer et al., 2012). These observations lead to the following prediction: the near and mid-term future will see significant attention to the evolutionary significance of the cyanobacterial toxins: are they signalling molecules, do they play a role in niche competition? Can they be tamed and made useful in medicine or, in the case of anatoxin, basic research on the functions of acetylcholine receptors? References Cadel-Six, S., Iteman, I., Peyraud-Thomas, C., Mann, S., Ploux, O., and Méjean, A. (2009) Identification of a polyketide synthase coding sequence specific for anatoxin-a producing Oscillatoria. Appl Environ Microbiol 75: 4909– 4912. Corringer, P.J., Poitevin, F., Prevost, M.S., Sauguet, L., Delarue, M., and Changeux, J.P. (2012) Structure and pharmacology of pentameric receptor channels: from bacteria to brain. Structure 20: 941– 956. Wang, H., Fewer, D.P., and Sivonen, K. (2011) Genome mining demonstrates the widespread occurrence of gene clusters encoding bacteriocins in cyanobacteria. PLoS ONE 6: e22384. Elephants in the room: protists and the importance of morphology and behaviour Patrick J. Keeling, Canadian Institute for Advanced Research, Botany Department, University of British Columbia, 3529-6270 University Boulevard, Vancouver, BC, Canada V6T 1Z4. A couple of years ago I found out I was not a microbiologist after all. I always thought I was, and even told strangers that is what I did, if they ever asked. But at a meeting of the American Society for Microbiology, I learned that my definition of a ‘microbe’ was not particularly representative. This is because I work on protists. Protists are microbial eukaryotes (more or less – we cannot quite decide on a definition), they are found in most of the environments you would expect to find other kinds of microbes (which is to say, everywhere), they are abundant, extraordinarily diverse, and (among my friends, anyway) generally considered to be ecologically important. They do come up sometimes in conversation, or even arguments, such as ‘who is the most important primary producer?’, or ‘are viruses or grazers more important for nutrient cycling?’. But protists are too often excluded from microbial ecosystem models or assessments of their composition; even studies that assess a complete ‘microbiome’ more often than not ignore the microbial eukaryotes. Before I am written off as a whinging specialist who is feeling marginalized, let me state that there are good reasons for this gap in our knowledge; they reflect interesting reasons that go back to fundamental differences in biology. Indeed, the problems associated with a thorough understanding of microbial eukaryotic ecology are so stark, that my prediction for the next year is not that we will solve these problems, or even make progress. My prediction (or perhaps wishful thinking) is that the ‘eukaryotic question’ will increasingly emerge as an elephant in the room, which is an elegant idiom to describe our failure to grasp the role of so many large microbes that are right under our noses. Bigger yes, but also different I would like to discuss two reasons why protists have not entered the mainstream of conventional high-throughput environmental microbiology. The first of these is trivial and well understood: their genomes are bigger and organized differently. We know that new sequencing technologies have had a major impact in our understanding of the diversity and ecological roles of bacteria, archaea and viruses, for example, by allowing whole-community metagenomic surveys. To include protists in these surveys is easy – simply do not filter them out! However, we also know that nuclear genome sizes would require epic sequencing and analysis budgets that are simply not practical. Moreover, we cannot accrue the same benefits for protists, even if we could sequence enough, because their genomes are fragmented, repeat-rich, and lack functionally related gene clustering, all of which limit the inferences we can make about individual genomes and metabolic networks from metagenomics by limiting our ability to link genes to other genes in a genome. But there is another less discussed, but infinitely more interesting problem. Bacterial and archaeal diversity is substantially manifested at the level of metabolism. Accordingly, the sequence of a bacterial or archaeal genome can go a long way to describing what that organism ‘does’ in the community, because we have developed reasonable ways to translate the information in a genome into predictions about that organism's metabolic actions in the environment. This is not the case for eukaryotes: although microbial eukaryotes harbour a sizable metabolic diversity, they are distinguished from other microbial life in that they manifest a great deal more diversity at the levels of morphology and behaviour. Indeed, morphology and behaviour have a much greater effect on what most protists ‘do’ in the environment than do their metabolic capacities (photosynthesis being an obvious exception). Unfortunately, the manifestation of these properties is much more complex than a straightforward gene–protein correspondence, and we are accordingly much worse at translating the information in a genome into predictions about what an organism looks like or how it behaves. To illustrate this problem, imagine four dinoflagellate protists living in the same marine environment: one is a free-living benthic autotroph, one is an intracellular parasite of gastropods, one is an obligate photosynthetic symbiont of cnidarians, and one is a heterotrophic grazer feeding on bacteria and eukaryotic algae. Now imagine we have sequenced whole genomes and whole transcriptomes for all four of these organisms. How easy would it be to reconstruct these interactions? The answer is, it would be virtually impossible, even with these miraculous quantities of molecular data. We could recognize that two were photosynthetic, but this might even mislead us to assume they shared a similar niche, when in reality the two forming intracellular relationships with invertebrates might share more in common. This failure is because the most important characteristics that distinguish these organisms and their activities are derived from poorly understood coordinated actions of thousands of gene products, and worse still, subtleties of regulation and epigenetics relating to thousands of genes. Organisms DO matter – how do we study them? They say that if you have a hammer, everything looks like a nail, and right now our biggest hammer is sequencing. Getting more sequence data from eukaryotes at the environmental level is a technical problem that can, and soon will be, solved. The most revolutionary solution will be the arrival of routine single-cell genomics and transcriptomics. Despite all we have learned through metagenomic approaches, cells do matter in the final analysis because biological activities are compartmentalized and how the metabolism of a community is partitioned makes a difference; a community is not just the sum of its enzymes, and seeing how functions are distributed across a community will change how we interpret them. Single-cell genomics will therefore be a boon to all environmental microbiology. And for eukaryotes, single-cell transcriptomics in particular will give us a first inroad to their otherwise intractable genomes when it can be automated across natural communities. How we interpret environmental sequence data from eukaryotes is another problem altogether. If the predictive power of even genome-wide sequence data is critically limited by our inability to infer characteristics of morphology and behaviour from it, then how do we integrate protists into a detailed picture of a microbial community that is primarily based on such data? Certainly being able to predict what an organism is like based on its close relatives will continue to be important, but requires a lot of ‘model’ systems scattered around the tree of eukaryotes to be truly effective. The real answer likely lies in a re-emergence, and indeed a reinvention, of arts like cultivation, ultrastructural characterization, identification and observation of live cells within their natural community, and field microscopy – some of which are badly under-appreciated at present. Our challenge is therefore not to put away our hammer, but to place more emphasis on the need for other tools too (in fact, I once watched a graduate student hammering a screw, so perhaps there is even greater depth to this need). It is not always obvious how these tools will be as adapted to a high-throughput approach as genomic methods were, but advances in imaging and cell sorting open a host of possibilities. So, to some extent, the way forward involves integrating existing methods rather than inventing new ones (e.g. linking high-throughput imaging with single-cell sorting would allow morphology to be linked with genomic data). In summary then, it is my hope that in the coming years microbial eukaryotes emerge a bit from the shadows of their smaller cousins. Luring them out into the open will require more than protists simply ‘catching up’ with existing methods: we must improve the integration of protists with our understanding of other members of microbial communities by coordination and deliberate efforts to reconstruct entire microbiomes, including all members and their interactions. The genomic revolution has allowed astonishing advances, but perhaps this only means that it needs to be grounded in biology more than ever. Adopting modularity of metabolism as a guiding paradigm may lead to better accounting and understanding of the unseen majority of life: exercised with focus on the nitrogen cycle Martin G. Klotz, Evolutionary and Genomic Microbiology Laboratory, Department of Biology, The University of North Carolina, Charlotte, NC 28