Accessing Uncultivated Microorganisms: From the Environment to Organisms and Genomes and Back

Recently several advances have been made to overcome cultivation biases and have spurred renewed interest in classical microbiology as well as in innovative isolation techniques. Researchers have discovered many clades of so far uncultivated microorganisms while analyzing the genetic information obtained from analyzing soil samples by various methods. This chapter focuses on aspects that are often not considered during cultivation, such as salt components of the medium, the choice of gelling agents and glassware, the size of sample and inoculum, the time of incubation, and how colonies are being detected. It provides detailed and resourceful advice for successful cultivation approaches. The goal of studies focusing on expressed gene products, such as RNA profiling and metaproteome, is not only to determine the genetic potential of an environment but to find out which genes are expressed at a certain moment in time under certain conditions. It is likely that not all microbes can be accessible as defined cultures in the laboratory by using current technology. Therefore, the development and combination of innovative techniques to study uncultivated microorganisms, ideally in their natural environment, is essential to advance the understanding of microbial physiology and ecology, and shed light on these most diverse creatures on our planet.

Although microbes are everywhere and, in theory, they all may have an impact on human health, microbial diversity is so enormous that it is hardly possible to describe all of them. This chapter focuses on the microbes residing in gastrointestinal (GI) tract that are collectively termed the microbiota, since these have the most intimate interaction with human body and, therefore, have a prominent impact on human health. It has been observed that the predominant bacterial composition in feces of healthy adult individuals is relatively stable over time. In contrast to healthy adults, however, severely disturbed and/or unstable fecal microbiotas can be correlated with humans with GI tract disorders, such as Crohn’s disease (CD), ulcerative colitis (UC), and intestinal bowel syndrome (IBS). Bacteria related to known butyrate-producing bacteria predominated in a live fraction, while bacteria affiliated with Bacteroides, Ruminococccus, and Eubacterium were more abundant in dead fractions. These are important findings, since they link phylogenetic information of the GI bacteria to activity. Although metagenomic analysis has shown their power for revealing the coding potential of the microbiome, and the genome of the entire microbial ecosystem, further functional studies, such as metaproteomics, have been reported but show limitations in our predictive capacity. Hence, there is a great need to integrate all available reductionist and global, cultivation- and molecular-based approaches to finally describe and understand micro companions throughout the journey on planet Earth.

In the 1980s and 1990s, it was widely recognized that the extent of microbial diversity in any environmental sample had never been experimentally determined, and some commentators believed bacterial diversity to be beyond practical calculation. The assumptions regarding the shape of the underlying community taxon-abundance distribution is undoubtedly open to criticism; however, what this article serves to do is to focus attention on determining what the underlying taxon-abundance distribution is by demonstrating that it is fundamental in determining the extent of prokaryotic diversity. The lognormal taxon-abundance distribution was used to assemble 30,000 communities, because it fitted the data better than the inverse Gaussian. The rationale for studying bacterial diversity given in this chapter is the prospect of the uncharted taxa being a reservoir of new drugs and metabolic processes. This chapter also describes the mathematical diversity estimators and taxon-abundance distributions and provides maps of the microbial world that will help guide future exploration and direct resources.

Molecular surveys of soil fungi have received much less attention despite the fact that fungi dominate decomposition and nutrient cycles in many soils. The ability to sequence fungal DNA from soil samples has opened up a new realm of possibilities for identifying new fungal groups. A clone library approximates a random sample of fungi in the soil, and is likely to be biased toward some groups of fungi over others based on variables in each step of the procedure used. The Assembling the Fungal Tree of Life (AFTOL) initiative has definitively clarified the phylogenetic tree for all known fungi. Three rDNA regions are commonly used to identify fungi: the 18S small subunit (SSU); the internal transcriber spacer sequences ITS-1 and ITS-2; and the 28S large subunit (LSU). This chapter reviews the new fungal lineages that have been discovered in various soil libraries over the past 10 years. Most soil libraries to date have discovered new fungi only at the genus or species level. The chapter talks about two main discoveries, Chytridiomycota and Ascomycota, after reviewing other important but unrelated clades that have also been discovered in soil libraries. It is important to flesh out the fungal family tree as quickly as possible, as it is possible that many of the missing fungi can go extinct before it is known that they ever existed.

The development of improved preservation and staining techniques and particularly the application of electron microscopy provided a wealth of morphological information to improve the taxonomic criteria used for the description and identification of protists. This chapter concerns the "seen and unseen," "cultured and uncultured" protists, with a brief overview of these three categories. Ecological studies of cultured protists have included representatives from most of the major lineages of protists and have detailed the nutritional aspects of these species, their elemental stoichiometries, feeding behaviors and rates, growth rates, and growth efficiencies. The understanding of the biogeochemical significance and activities of these species has been ascertained largely through the manipulation and experimental examination of cultured protists. Using this information, ecologists have generated biogeochemical and food web models that significantly improved our understanding of the activities of photosynthetic and heterotrophic protists in natural communities and thus lend better insight into how aquatic communities function. The alveolates contain three well-known groups of protists: the ciliates, the dinoflagellates, and the apicomplexans. An example of the extensive genetic diversity detected in these very small protists can be found in the picoprasinophyte genus Micromonas. Success in this work obviously requires some general knowledge of the nutrition of the target cells, so these attempts can improve dramatically as the "needs" of the protistan taxa are characterized.

Microbes inhabit a wide range of habitats, from hot springs to the deep subsurface, and it is highly improbable that one will be able to observe similar biogeographical patterns across the full range of possible microbial habitats. This chapter primarily focuses on selected topics that are particularly relevant to researchers studying uncultivated microbes in natural environments in order to illustrate what one do, or do not, currently know about their biogeography. However, it is important to recognize that the "unknown unknowns" and "known unknowns" in microbial biogeography currently outnumber the "known knowns." For this reason, the chapter highlights key topics where the gaps in one's knowledge of microbial biogeography are particularly apparent. A few research topics that may be ripe avenues for future research are also highlighted in this chapter. Although the field of biogeography principally focuses on the spatial distribution of organisms, the temporal aspects of microbial biogeography may be particularly important. The entire chapter emphasizes on how studies in microbial biogeography are more difficult to conduct than comparable studies of plant or animal biogeography, largely due to the problems associated with surveying microbial communities. The study of microbial biogeography will help one to move beyond anecdotal studies and observations to build a predictive understanding of microbial diversity and the factors influencing this diversity across space and time.

Molecular techniques have given microbial ecologists an entirely new dimension for understanding natural ecosystems. This chapter discusses the common terms used by microbial ecologists to describe diversity, and attempts to explain their virtues and pitfalls. The first classification system was devised by Aristotle at least 2,400 years ago who generated the first hierarchy based on creationist and essential tenets. This system was based on only the two categories "species" and "genus," which were motivated by recurrent observations about the world. The current prokaryotic species concept fulfils the requisites of being universally applicable to the cellular nuclei-less microorganisms. In this chapter, the different terms used by microbial ecologists to name the units that they observe, their suitability, and which of them may (from our point of view) have an optimal applicability, are discussed. It is possible that a single observed band contains up to three different sequences or viceversa where an identical sequence can appear in different bands. There is a growing group of scientists who prefer the use of operational taxonomic unit (OTU) to name their units of comparison.

This chapter deals with the molecular, cultivation, and a few other techniques to evaluate microbial diversity in natural systems. It provides an outline of the various approaches that are used to determine diversity of bacteria and archaea in natural habitats and some guidelines regarding which methods may be most appropriate for specific environments and specific scientific questions. Several of the techniques described rely on the ability to identify particular preselected components of the microbial community. The oldest method for obtaining microbial diversity information is to examine the sample with a microscope and characterize the microbes by their morphology. A major benefit is that the sequences can be used to make probes for quantitative composition analyses of microbial communities. This can prove to be a very powerful approach and can be being augmented by inclusion of 23S rRNA data as well. A related and powerful application has been the use of fingerprinting methods that give a snapshot of the entire microbial community at once, with the ability to tentatively identify different components. An important feature of the fingerprinting methods, such as T-RFLP, length heterogeneity PCR (LHPCR), and ARISA, is that the results are in the form of data on the amount of different PCR products of particular fragment lengths. Some authors have developed tools to “recruit” environmental sequences to known microbial genomes, showing how various relatives of previously sequenced organisms are distributed along the transect, based on all genes and not just phylogenetic markers like 16S rRNA.

Most of the viral sequences that were identified from the rumen metagenome study were novel, with cyanophages and a newly discovered clade of single-stranded DNA phages dominating the samples that were obtained from the Sargasso Sea sample. The authors presented data that showed variation in viral assemblages based of different geographic locations and indicated regional diversity of phages can be almost as high as global diversity, possibly as a result of viral migration. Metagenomics is being used to describe the transcriptome of various environmental samples. In the study, the authors clearly demonstrated that suppression subtractive hybridization (SSH) can allow a clear discerning and identification of DNA fragments that are unique to one complex community relative to another. In the case of eukaryotic organisms that contain introns, the transcriptome is usually accessed by reverse transcription. In many cases the genome of a single species cannot give insight into the extent of diversity within a species. The results from the study showed that for the three animals included in this study that were on identical diets, the community structures were markedly different with respect to nutrient utilization. Metagenomics presents the greatest opportunity to revolutionize understanding of the microbial world, and a detailed analysis of carefully chosen microbial communities worldwide is required.

Soils are populated by microbial cells from all three domains of life: Archaea, Bacteria, and Eukarya. Cultivation of soil bacteria that may display the characteristics of true soil K-strategists can now be carried out routinely, albeit with some effort, and future comparisons with the better-known r-strategists will reveal any underlying physiological and genetic bases for these ecological strategies in soil bacteria. This chapter reviews some of the factors that allow successful isolation of a wider phylogenetic representation of soil bacteria than has traditionally been thought to be possible. Addition of cAMP to media results in significant increase in the cultivation efficiency of marine and freshwater bacteria but did not have an effect on the overall culturability of soil bacteria. Microorganisms can interact in a positive, neutral, or negative manner, and denser inocula can increase the likelihood of these interactions, and so potentially increase or decrease culturability. Some soil bacteria isolated in liquid media grow as colonies on the surfaces of glass tubes in which they are cultured, and do not disperse into liquid culture, and soil myxobacteria often grow on the solid surfaces of culture vessels when grown in liquid culture. The size of the inoculum is an important consideration when attempting to isolate soil bacteria.

This chapter describes the concept of symbiosis in a broad sense. It talks about criteria and methods to demonstrate that an organism is symbiotic, and presents general strategies for the cultivation of marine symbiotic microorganisms. Symbiotic microorganisms do not fit easily into Koch’s postulates because the resulting observable fitness of the host is usually less obvious than acute disease. Cultivation of symbiotic microbes that produce medically relevant secondary metabolites might also allow the production of these metabolites by sustainable and economical fermentative technologies as opposed to collection of marine organisms from the environment. Knowledge that soil contains large amounts of humic substrates leads to increased cultivation of soil microorganisms. Addition of cAMP and acyl-homoserine lactone signaling molecules known to be present in some cells growing as colonies, improves the cultivation of marine bacteria. A first approach for the cultivation of aerobic heterotrophic marine symbionts is to use one-half strength Marine Broth 2216 supplemented with one-half strength natural or artificial sea-water, with 1.5% agar added when required. The critical factors of this approach are (i) surface sterilization and aseptic dissection and handling of symbiotic tissue to remove transient and/or opportunistic symbionts, (ii) dilution and plating to extinction to eliminate competition and or antagonism among strains, and (iii) patience and close examination for colonies and microcolonies that grow on the plates. The ability to cultivate marine symbiotic microorganisms is a great advantage for the study of the symbiotic microorganism and the natural products it might produce.

Nowadays many experimental approaches exist that one could not even imagine in Koch’s days. An alternative approach that focuses on dealing with undefined populations of microbes in the laboratory is the scope of this chapter. The approach consists of four steps. The first step is the definition of an ecological niche. In the second step, the complement of the niche is engineered in a laboratory bioreactor, which is to become an enrichment culture. In the third step, the culture is characterized in molecular detail. Finally, the detailed information is used to investigate the importance of the described processes and microbes in nature. In this approach, the target organism remains in continuous culture under chemo-spatio-temporal conditions that define its natural niche, in an open system that allows competition. With this approach and appropriate primers it is possible to specifically quantify a single organism or a clade of microorganisms. For example, the abundance of Crenarchaeota in soils can be shown by real-time PCR of ammonium monooxygenase genes from archaea and bacteria. Epifluorescence microscopy is of great value to visualize relative abundances determined with cloning or denaturing gradient gel electrophoresis (DGGE). The study of microorganisms in undefined mixed cultures could be applied more generally and has great potential for environmental microbiology.

This chapter focuses on heterogeneity phenomena among single-celled organisms. It has generally been considered that phenotypic heterogeneity provides a dynamic source of diversity in addition to that derived from genotypic changes such as genome rearrangements and mutation (i.e., genotypic heterogeneity). Recent studies highlighted in this chapter have substantiated this view. This chapter addresses examples of cell-individualized phenotypes in which the benefits of heterogeneity to population fitness can be readily envisaged. Moreover, the most recent studies have risen to the challenge of demonstrating fitness benefits of heterogeneity experimentally, and these studies are discussed in this chapter. The chapter integrates recent studies to describe the molecular bases and consequences of heterogeneity for several of the key phenotypes characterized by variability in clonal microbial populations. Much of the recent focus of research relevant to the field of cell individuality has been to uncover the drivers of diversity operating at the molecular level, in particular the contributions of stochasticity or noise to the processes of gene transcription and translation. The chapter presents a study which describes that the heterogeneity-specific advantage was particularly striking considering that it was prevalent in mutant populations that are conventionally considered to be disadvantaged. Recent research efforts in the area of phenotypic heterogeneity have served to advance significantly the understanding of its nature. The potential benefits of this form of heterogeneity to microbial cell populations are now widely acknowledged and have recently been demonstrated experimentally.

In recent years, many novel techniques based on scanning probe microscopies have been developed that have myriad applications in studying cells. This chapter introduces scanning probe microscopy and related techniques that can have application in high-resolution imaging of cells and real-time monitoring of multiple cellular components in a multimodal fashion. The techniques described are scanning probe microscopy, atomic force microscope (AFM), cantilever-based spectroscopy, and cantilever-based sensing. These techniques can be effectively extended to sensing, physiological studies, and diagnostics in biological and microbiological analyses. These truly interdisciplinary developments have immense potential to transcend academic and industrial barriers, and are expected to allow significant advancements in nanoscale studies in biological systems. Future probes may be envisioned to be hybrid and multifunctional such that reproducible and robust data may be collected on a large number of biological samples.

Large metagenomic studies demonstrate the central theme, the microbial heterogeneity of individual microbial cells, which will drive single-cell genomics and microbiology. This chapter gives an overview of the current state of the art and highlights the different technological steps necessary to achieve the total genome sequence from a single microbial cell. It addresses (i) methods to isolate single bacterial cells, (ii) DNA isolation and amplification from a single microorganisms, (iii) DNA sequencing, and (iv) applications and future outlook. Many applications of single-cell genomics require careful DNA amplification to overcome several problems and potential limitations. Multiple displacement amplification (MDA) amplifies all DNA present within the sample, including contaminating DNAs, multicopy sequences, and plasmids, thus making sequence annotation more challenging. MDA techniques are also being used in microbial ecology for strategies beyond single-cell genomics applications, which should accelerate their refinement and adoption. The current and potential uses of MDA warrant further work on addressing the potential biases and problems with its application. One of the most promising new sequencing technologies is the 454 sequencing platforms. Continued improvements in DNA sequencing techniques, bioinformatics, and data analysis over the past few years have helped reduce the cost and time associated with sequencing a genome.

This chapter reviews, discusses, and proposes studies aimed at determining how many Mycoplasma genitalium genes are really necessary when cells are grown under ideal laboratory conditions. A list of the 100 M. genitalium protein-coding genes disrupted in the 2006 study is provided in this chapter. Using The Institute for Genomic Research (TIGR) functional classification scheme, the 100 genes can be broken into 15 groups based on their main roles. Seven DNA repair and recombination genes were disrupted, including recA, which is one of the most ubiquitous proteins found in nature. A minimal set of genes cannot be convincingly determined by either comparative genomics or global transposon mutagenesis. However, these studies do identify more than 200 genes for core functions such as DNA replication, transcription, protein translation, energy metabolism, transport, lipid metabolism, nucleotide metabolism, and protein fate, which one has confidence will be represented in the minimal cell. There are three possible approaches to making a minimal cell: (i) cumulative inactivation of genes using mutagens that produce frameshifts, (ii) sequential genome reduction using recombineering methods, and (iii) chemical synthesis of a minimal genome and installation into receptive cell cytoplasm. A minimal genome may not by itself be useful in a practical sense, but one can envision in the not too distance future the ability to design and synthesize microbes for useful purposes such as production of pharmaceutical products, industrial compounds, and fuels. In the future, stripped down organisms outfitted with useful biosynthetic pathways could provide the basis for new industrial processes.