Chapter 1: The Astrobiology Primer 3.0.

The Astrobiology Primer 3.0 (ABP3.0) is a concise introduction to the field of astrobiology for students and others who are new to the field of astrobiology. It provides an entry into the broader materials in this supplementary issue of Astrobiology and an overview of the investigations and driving hypotheses that make up this interdisciplinary field. The content of this chapter was adapted from the other 10 articles in this supplementary issue and thus represents the contribution of all the authors who worked on these introductory articles. The content of this chapter is not exhaustive and represents the topics that the authors found to be the most important and compelling in a dynamic and changing field.

Chapter 2: What Is Life?

The question "What is life?" has existed since the beginning of recorded history. However, the scientific and philosophical contexts of this question have changed and been refined as advancements in technology have revealed both fine details and broad connections in the network of life on Earth. Understanding the framework of the question "What is life?" is central to formulating other questions such as "Where else could life be?" and "How do we search for life elsewhere?" While many of these questions are addressed throughout the Astrobiology Primer 3.0, this chapter gives historical context for defining life, highlights conceptual characteristics shared by all life on Earth as well as key features used to describe it, discusses why it matters for astrobiology, and explores both challenges and opportunities for finding an informative operational definition.

Chapter 5: Major Biological Innovations in the History of Life on Earth.

All organisms living on Earth descended from a single, common ancestral population of cells, known as LUCA-the last universal common ancestor. Since its emergence, the diversity and complexity of life have increased dramatically. This chapter focuses on four key biological innovations throughout Earth's history that had a significant impact on the expansion of phylogenetic diversity, organismal complexity, and ecospace habitation. First is the emergence of the last universal common ancestor, LUCA, which laid the foundation for all life-forms on Earth. Second is the evolution of oxygenic photosynthesis, which resulted in global geochemical and biological transformations. Third is the appearance of a new type of cell-the eukaryotic cell-which led to the origin of a new domain of life and the basis for complex multicellularity. Fourth is the multiple independent origins of multicellularity, resulting in the emergence of a new level of complex individuality. A discussion of these four key events will improve our understanding of the intertwined history of our planet and its inhabitants and better inform the extent to which we can expect life at different degrees of diversity and complexity elsewhere.

Multicellular magnetotactic bacterial consortia are metabolically differentiated and not clonal.

Consortia of multicellular magnetotactic bacteria (MMB) are currently the only known example of bacteria without a unicellular stage in their life cycle. Because of their recalcitrance to cultivation, most previous studies of MMB have been limited to microscopic observations. To study the biology of these unique organisms in more detail, we use multiple culture-independent approaches to analyze the genomics and physiology of MMB consortia at single cell resolution. We separately sequenced the metagenomes of 22 individual MMB consortia, representing eight new species, and quantified the genetic diversity within each MMB consortium. This revealed that, counter to conventional views, cells within MMB consortia are not clonal. Single consortia metagenomes were then used to reconstruct the species-specific metabolic potential and infer the physiological capabilities of MMB. To validate genomic predictions, we performed stable isotope probing (SIP) experiments and interrogated MMB consortia using fluorescence in situ hybridization (FISH) combined with nano-scale secondary ion mass spectrometry (NanoSIMS). By coupling FISH with bioorthogonal non-canonical amino acid tagging (BONCAT) we explored their in situ activity as well as variation of protein synthesis within cells. We demonstrate that MMB consortia are mixotrophic sulfate reducers and that they exhibit metabolic differentiation between individual cells, suggesting that MMB consortia are more complex than previously thought. These findings expand our understanding of MMB diversity, ecology, genomics, and physiology, as well as offer insights into the mechanisms underpinning the multicellular nature of their unique lifestyle.

Correlative SIP-FISH-Raman-SEM-NanoSIMS links identity, morphology, biochemistry, and physiology of environmental microbes.

Microscopic and spectroscopic techniques are commonly applied to study microbial cells but are typically used on separate samples, resulting in population-level datasets that are integrated across different cells with little spatial resolution. To address this shortcoming, we developed a workflow that correlates several microscopic and spectroscopic techniques to generate an in-depth analysis of individual cells. By combining stable isotope probing (SIP), fluorescence in situ hybridization (FISH), scanning electron microscopy (SEM), confocal Raman microspectroscopy (Raman), and nano-scale secondary ion mass spectrometry (NanoSIMS), we illustrate how individual cells can be thoroughly interrogated to obtain information about their taxonomic identity, structure, physiology, and metabolic activity. Analysis of an artificial microbial community demonstrated that our correlative approach was able to resolve the activity of single cells using heavy water SIP in conjunction with Raman and/or NanoSIMS and establish their taxonomy and morphology using FISH and SEM. This workflow was then applied to a sample of yet uncultured multicellular magnetotactic bacteria (MMB). In addition to establishing their identity and activity, backscatter electron microscopy (BSE), NanoSIMS, and energy-dispersive X-ray spectroscopy (EDS) were employed to characterize the magnetosomes within the cells. By integrating these techniques, we demonstrate a cohesive approach to thoroughly study environmental microbes on a single-cell level.

The molecular dimension of microbial species: 2. Synechococcus strains representative of putative ecotypes inhabiting different depths in the Mushroom Spring microbial mat exhibit different adaptive and acclimative responses to light.

Closely related strains of thermophilic Synechococcus were cultivated from the microbial mats found in the effluent channels of Mushroom Spring, Yellowstone National Park (YNP). These strains have identical or nearly identical 16S rRNA sequences but are representative of separate, predicted putative ecotype (PE) populations, which were identified by using the more highly resolving psaA locus and which predominate at different vertical positions within the 1-mm-thick upper-green layer of the mat. Pyrosequencing confirmed that each strain contained a single, predominant psaA genotype. Strains differed in growth rate as a function of irradiance. A strain with a psaA genotype corresponding to a predicted PE that predominates near the mat surface grew fastest at high irradiances, whereas strains with psaA genotypes representative of predominant subsurface populations grew faster at low irradiance and exhibited greater sensitivity to abrupt shifts to high light. The high-light-adapted and low-light-adapted strains also exhibited differences in pigment content and the composition of the photosynthetic apparatus (photosystem ratio) when grown under different light intensities. Cells representative of the different strains had similar morphologies under low-light conditions, but under high-light conditions, cells of low-light-adapted strains became elongated and formed short chains of cells. Collectively, the results presented here are consistent with the hypothesis that closely related, but distinct, ecological species of Synechococcus occupy different light niches in the Mushroom Spring microbial mat and acclimate differently to changing light environments.

Characterization of an Endophytic Gloeosporium sp. and Its Novel Bioactivity with "Synergistans".

Gloeosporium sp. (OR-10) was isolated as an endophyte of Tsuga heterophylla (Western hemlock). Both ITS and 18S sequence analyses indicated that the organism best fits either Hypocrea spp. or Trichoderma spp., but neither of these organisms possess conidiophores associated with acervuli, in which case the endophytic isolate OR-10 does. Therefore, the preferred taxonomic assignment was primarily based on the morphological features of the organism as one belonging to the genus Gloeosporium sp. These taxonomic observations clearly point out that limited ITS and 18S sequence information can be misleading when solely used in making taxonomic assignments. The volatile phase of this endophyte was active against a number of plant pathogenic fungi including Phytophthora palmivora, Rhizoctonia solani, Ceratocystis ulmi, Botrytis cinerea, and Verticillium dahliae. Among several terpenes and furans, the most abundantly produced compound in the volatile phase was 6-pentyl-2H-pyran-2-one, a compound possessing antimicrobial activities. When used in conjunction with microliter amounts of any in a series of esters or isobutyric acid, an enhanced inhibitory response occurred with each test fungus that was greater than that exhibited by Gloeosporium sp. or the compounds tested individually. Compounds behaving in this manner are hereby designated "synergistans." An expression of the "median synergistic effect," under prescribed conditions, has been termed the mSE50. This value describes the amount of a potential synergistan that is required to yield an additional median 50% inhibition of a target organism. In this report, the mSE50s are reported for a series of esters and isobutyric acid. The results indicated that isoamyl acetate, allyl acetate, and isobutyric acid generally possessed the lowest mSE50 values. The value and potential importance of these microbial synergistic effects to the microbial environment are also discussed.