Recent advances in the analysis of individual microbial cells

Conventionally, microorganisms are studied at the population scale because cultures of microbes are considered to be uniform populations which can be adequately described by average values. However, the availability of tools such as flow cytometry and image analysis which allow measurements to be made on individual cells has changed our perception of microbes within both the laboratory and the natural environment. Through application of these techniques to microbial samples it has become increasingly clear that even in the absence of genetic differences populations are far from homogeneous. To improve our understanding of the physiology and operation of microbes within communities and/or microenvironments knowledge of the structure and function of individuals provides much more valuable information. It is perhaps surprising that this gap in our knowledge has not been more frequently addressed as microorganisms are the largest group of living organisms on the planet, both in biomass and in number. For example, 3.6 × 1029 microbial cells with a total cellular carbon content of about 3 × 1011 tons are documented as living in the oceans (1). Furthermore, bacteria are responsible for 98% of the primary carbon production and are important mediators in all biogeochemical cycles (1). Microbes also successfully occupy the human organism to an extent that it seems appropriate to call a person a mutualistic community. The human intestinal microbiota was recently described as being composed of 1013 to 1014 microorganisms, with a collective genome containing at least 100 times as many genes as the human genome (2). The role of bacteria is often viewed primarily as a negative one through their actions as pathogens causing illness and death in humans, our livestock and our crops. To address these problems, sophisticated techniques such as microfluidic surface plasmon resonance (SPR) are being developed to detect pathogens even at very low concentrations (3, 4). These negative actions, however, should be considered to be one of the lesser impacts that microorganisms have on the existence of human society as microorganisms also have many beneficial effects. Only a small proportion of the diversity of microorganisms has been identified and a smaller proportion still has been characterized through laboratory studies. We know very little of their strategies such as the pathways that they use to provide themselves with carbon and energy, the way that they interact with each other, and how they defend themselves from biotic and abiotic stressors. The reasons for this are simple: microorganisms are small and they are very diverse in structure and function; both of these factors lead to technological and methodological problems in studying them. Microbes cannot be investigated without technological assistance, meaning that methods such as microscopy and flow cytometry with appropriate fluorochomes are essential for the acquisition of both qualitative and quantitative information (5). Although these methods have become conventional tools in microbial cell biology and in the analysis of environmental samples, their use in investigations of bacteria is limited by the physical constraint of optical resolution. Application of cell markers is also a challenge, simply because the cells have only a thousandth of the volume of a normal blood cell and correspondingly small amounts of cellular constituents. This is the reason why multicolor approaches in bacteria with small cell volumes will not work, as the close spatial interaction of the dyes prevents quantitative analysis. Notwithstanding these limitations, the role of flow cytometry in microbiology is increasing steadily (Fig. 1). New techniques are of great importance and are now starting to become available for the microbial world. One of these is the noninvasive Single-Cell-Raman-Spectroscopy and the potential applications of this technique on our understanding of microorganisms have been reviewed (6). Bibliometric analysis of flow cytometric studies. A survey was made of the Web of Science database of the Institute for Scientific Information counting all papers whose ‘topic’ database field contained the words flow and cytometr* (the * indicates any number of additional characters following in the word; dash-dot line). Those papers relating to the flow cytometry of microorganisms required that one or more of the following were also present: bacteri* or microorgansim or pro?aryot* or yeast (where * again represents any characters and ? represents a single character; dotted line). The solid line gives the percentage of flow cytometry papers which on the above criteria deal with microorganisms. Data before 1991 do not include a search of abstracts and data for 2008 are correct as of the survey date (20/11/08). Since 1987, microbial flow cytometry shows a steady growth, and now ∼8% of flow cytometry articles included studies of microbes. [Color figure can be viewed in the online issue, which is available at www. interscience.wiley.com.] It is not only the observation of microorganisms that may be an obstacle to their study but cultivation is often also a challenge. It has been estimated from environmental studies that in excess of 98% of the microbial diversity remains inaccessible to us through culture-based analysis. Obtaining information on noncultivable bacteria is extremely problematic as the availability of axenic biomass for biochemical analysis is limited. Often we cannot even obtain enough cellular material to produce, for example, specific antibodies against surface antigens or important cellular proteins. However, some approaches are in development, although still in their infancy, which will permit characterization of this microbial diversity (7-9). These techniques will allow site-specific capture of individual bacteria, specific labeling via diverse FISH-techniques and/or individual cultivation in gel drops in combination with targeted metagenomic approaches or with whole genome amplification. To obtain more material to work with new approaches to cultivation techniques are obligatory. One of them is the fascinating Pallet-Technology (10) which allows populations to be grown from single cells without losing chemical contact to the other individual cells of the cell community. It has long been hypothesized that the so called noncultivability of some bacteria might be caused by the necessity of interspecies exchange of substrates, intermediates and signal molecules; these phenomena do not occur when cultivation is attempted in axenic culture. The Pallet-Technology has not yet been tested for bacteria but it offers the potential for future breakthroughs in culturing microorganisms that could allow a greater diversity of microorganisms and their products to be exploited in biotechnology as well as furthering our understanding of the microorganisms and their interactions. For millennia microorganisms have been exploited in traditional biotechnologies such as brewing and bread making. A growing range of microorganisms are used in modern biotechnology industries for the synthesis of valuable materials such as pharmaceuticals, antibiotics, enzymes, metabolites, and various other compounds. Maintenance of viable and productive populations within the industrial environment is of paramount importance to the success and efficiency of these industries and these processes can be optimized by the use of flow cytometric analyses (11). Microbes also act as catalysts for the degradation and detoxification of hazardous chemicals. Understanding bacterial physiology, growth behavior and survival strategies will open new ways to use, exploit or overcome bacteria on the basis of their inherent characteristics. One of the many tools to do this involves analysis of their proliferation patterns and linking of this to the basic knowledge of the physiological characteristics of a species (12). Bacteria were shown to employ a complex network of regulatory systems that temporally and spatially organize cellular processes. Localized protein complexes were found to have pivotal roles in driving cell cycle progression and bacterial differentiation. The high degree of internal organization also extends to bacterial chromosomes, which present a conserved arrangement in the cell and to be segregated in an active and orderly fashion (13-15). Bacterial cell cycle regulation and chromosome biology evolve as research areas which can further our understanding of the function of the microbial cell and can probably be used to determine cell states quickly and reliably and thereby predict the metabolic and survival potential of a species. Another matter that is often overlooked when we study microorganisms in the laboratory is the fact that many of them do not normally grow as planktonic single cells in nature. Structured communities known as biofilms are the most common state of many bacteria within the environment and this distinction has important consequences for the physiology and behavior of the individual cells within these communities. Cells respond to local environments by placing stimuli which may arise both from, for example, nutritional local ‘hot spots’ but also from signaling from neighboring cells. Biofilm structures offer protection against extrinsic stressors such as predators or toxic chemicals via the production of impenetrable extracellular matrices. Such multicellular biofilms are ubiquitous and play a well accepted role in stabilizing infection in human disease. Recent work has sought to resolve the interactions between individuals of Pseudomonas spp. to understand infection processes (16). It challenges our preconceptions of the microbial world to learn of the extent to which even genetically identical bacteria are able to differentiate and to assume distinct tasks for efficient functioning of their community. The heterogeneity of cells within a population might be considered as an important factor in maintaining stability of a variety of systems. It is known from phylogenetic diversity analyses that an increase in species numbers leads to a concomitant increase in the stability of a system (17). Additionally, heterogeneity of cells with regard to productivity within populations should be seen not only in terms of its impact on bioprocess yields, but also in terms of the revelation of information about cell response to a complex interaction of environmental stimuli. Currently our understanding of how and why an individual microbial cell reacts to its environment is often poorly understood. A recent approach that will improve our understanding of the mechanisms and triggers involved in heterogeneity of response is a single cell bioreactor (18). Such devices will facilitate the acquisition of functional and structural information about individual cells. In addition it will permit analysis of the single cell fluxome, the quantification of metabolite turnover in the single cell, at least for larger microorganisms such as yeast cells. This sensitive approach will be an important addition to our growing arsenal of Omics-Technologies and will be an important contribution to studies in systems biology. Mining genes and transcriptomic and proteomic approaches are important additions to the concept of cytomics on the microbial scale and will allow in depth analysis of pathway regulation (19) or the concurrence of the microbial cells within populations or complex consortia (20). Of equal importance is the need to develop methods to administer the huge single cell data profiles in a form which can be understood and efficiently accessed by the user community, whether they are academics, clinicians, or industrial scientists. The application of mathematical concepts can provide valuable contributions to the understanding of microbial life. For example, in modeling applications (21) such data must be accessed and used to elucidate the behavior of a cell in a community or population to improve our understanding of populations leading to process optimization and permitting accurate predictions. Mathematical modeling helps to understand the behavior of biochemical reaction networks, or the highly coordinated response of single cell populations to changing environmental conditions. If the environmental triggers responsible for a particular cell reaction are known this can be used in the development of a structural or functional marker (22). This focus issue on microbiology contains a selection of manuscripts based on presentations at the 4th International Conference on the Analysis of Microbial Cells at the Single Cell Level which was organized on behalf of the Microbial Physiology Section of the European Federation of Biotechnology, and held in Bad Schandau, Germany. Abstracts of all papers presented at the conference are available as supporting information (SI1). The conference was attended by 121 delegates from 18 countries worldwide and followed previous successful conferences in Italy (1999), Denmark (2002), and Austria (2005). These works are representative of the quality and diversity of topics discussed and show how the field of microbial single cell research is evolving through a combination of technology and curiosity driven science. We are looking forward to what will evolve within the next 3 years and to the next Conference on the Analysis of Microbial Cells at the Single Cell Level which will take place in Marseille, 2011. Additional Supporting Information may be found in the online version of this article. Please note: The publisher is not responsible for the content or functionality of any supporting information supplied by the authors. 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