Search

The anaerobic hypersaline sediments of an ephemeral pond from the S’Avall solar salterns constituted an excellent study system because of their easy accessibility, as well as the analogy of their microbial assemblages with some known deep-sea hypersaline anaerobic brines. By means of shotgun metagenomics and 16S rRNA gene amplicon sequencing, the microbial composition of the sediment was shown to be stable in time and space. The communities were formed by prokaryote representatives with a clear inferred anaerobic metabolism, mainly related to the methane, sulfur and nitrate cycles. The most conspicuous finding was the inverted nature of the vertical stratification. Contrarily to what could be expected, a methanogenic archaeal metabolism was found to dominate in the upper layers, whereas Bacteria with fermentative and anaerobic respiration metabolisms increased with depth. We could demonstrate the methanogenic nature of the members of candidate lineages DHVE2 and MSBL1, which were present in high abundance in this system, and described, for the first time, viruses infecting these lineages. Members of the putatively active aerobic genera Salinibacter and Halorubrum were detected especially in the deepest layers for which we hypothesize that either oxygen could be sporadically available, or they could perform anaerobic metabolisms. We also report a novel repertoire of virus species thriving in these sediments, which had special relevance because of their lysogenic lifestyles., This study was funded by the Spanish Ministry of Science, Innovation and Universities projects Salploma CLG2015_66686-C3-1-P, Micromates subprojects PGC2018-096956-B-C41 and PGC2018-096956-B-C44, and Marbiom RTC-2017-6405-1, which were also supported with European Regional Development Fund (FEDER) funds. RRM acknowledges the financial support of a sabbatical stay at Georgia Tech through the grant PRX18/00048 and also FFV’s for the support of her PhD with the fellowship BES-2016-078138, both from the Spanish Ministry of Science, Innovation and Universities.

The specificity of CRISPR–Cas systems for nucleic acid targets is determined by a combination of binding and cleavage. Understanding the mechanisms by which Cas proteins specifically select their targets is critical for the development of CRISPR–Cas systems for biotechnology applications. Moreover, the specificity of CRISPR–Cas systems plays an important role in prokaryote evolution due to its role in distinguishing self from nonself. Here, I describe Library-ChIP, a high-throughput method for measuring Cas protein occupancy at many DNA sequence variants in a native prokaryotic host. Library-ChIP can be used to identify the determinants of specificity for Cas protein binding to nucleic acid targets.

Following the rise of prokaryote life dependent on anaerobic respiration, oxygen was produced as a toxic by-product. This steadily and significantly contributed to the formation of an oxygen-rich environment that resulted in a shift in atmospheric and oceanic redox status. This necessitated the evolution of aerobic respiration, and mitochondria seemingly arose independently of bacterial life before becoming incorporated into eukaryote cells. Aerobic respiration allowed for a significant increase in adenosine triphosphate production, which is critical to meet the energy demands required for complex multicellular life to evolve. This molecular process also results in a leakage of potentially toxic reactive oxygen species (ROS) from the mitochondria. Although ROS have evolved as critical molecular and cellular regulators, excessive ROS damages cellular molecules and components. Therefore, endogenous antioxidants evolved as an important regulator of the redox state within the cellular environment in order to optimize the beneficial roles of redox biology.

Quorum sensing is the response-regulation system of genes expression in accordance with the variation of cell population density. In this communication system, the response-regulation system induces group behaviors via signal molecules, which enabling cell acts actively to external environmental fluctuation. From practical point of view, these group behaviors ubiquitously occurred in a myriad of environments threatening human health and causing unrecoverable deterioration of manufacturing processes. Therefore, the mechanism of quorum sensing involves producing, releasing, detecting, and responding to small hormone-like chemical compounds called as autoinducers. For intraspecies communications, Gram-negative bacteria representatively utilize acyl-homoserine lactones, and Gram-positive bacteria typically use modified peptides as autoinducer. In case of a eukaryote, fungi generate a variety of autoinducers, such as farnesol, tyrosol, and aromatic alcohols. The intraspecies quorum sensing allows bacteria themselves to recognize the same species in the mixed culture system, but it could not use to detect the presence of other species and identify and act on their proportions in the overall environmental system. Thus, bacteria produce furanone-based signal molecules to build an interspecies communication and maintain a balance in the entire ecosystem. This type of bacterial Esperanto, called autoinducer-2, is synthesized by LuxS protein and its associated genes have been found in over 70 different microorganisms to date. Meanwhile, recent studies also identified that interkingdom quorum sensing between eukaryote and prokaryote, such as a bacteria-to-fungi, bacteria-to-plant, and bacteria-to-mammalian cell. This chapter focuses on the history of quorum sensing and the signal molecules chemistry; how response-regulation system in bacteria is developed; how the definition of quorum sensing is determined; how the signal molecule is generated; and the recent studies related to the exploration of new quorum sensing systems.

Endosymbiosis, more than any other process, perhaps, is the leading narrative upon which the history of eukaryotic evolution has been written. Primary endosymbiosis, which is the uptake of a prokaryote by another living cell, has arguably been the driving force for the origins and diversification of complex life on Earth. The genetic integration of, first, a nonphotosynthetic alphaproteobacterium and, later, a photosynthetic cyanobacterium into a eukaryotic cellular framework have shaped and altered the planet's biodiversity and biogeochemistry in countless ways, from the land, to the water, to the atmosphere. If you are alive today and reading these words, it is in no small part because of endosymbiosis. Like all eukaryotes, we are the product of an ancient endosymbiotic love affair, and for plants and algae the endosymbiotic romance was a complicated triangle. Here, I recount my own passions for the topic of endosymbiosis, highlighting past and present breakthroughs as well as some of the controversies and unanswered questions that have plagued the field. I focus on the evolution of primary plastids, their genomes, and the supergroup to which they are found (the Archaeplastida), including members that have lost photosynthetic capabilities but still retain a colourless plastid.

We explored the possibilities of whole-genome duplication (WGD) in prokaryotic species, where we performed statistical analyses of the configurations of the central angles between homologous tandem repeats (TRs) on the circular chromosomes. At first, we detected TRs on their chromosomes and identified equivalent tandem repeat pairs (ETRPs); here, an ETRP is defined as a pair of tandem repeats sequentially similar to each other. Then we carried out statistical analyses of the central angle distributions of the detected ETRPs on each circular chromosome by way of comparisons between the detected distributions and those generated by null models. In the analyses, we estimated a P value by a simulation using the Kullback-Leibler divergence as a distance measure between two distributions. As a result, the central angle distributions for 8 out of the 203 prokaryotic species showed statistically significant deviations (P

What is life? This is a very big question. In the case of a very simple beginning to life, as described in Chapters 2 and 5 Chapter 2 Chapter 5, it is a fatty acid vesicle full of random simple chemical reactions. This is just a very simplistic chemical chamber that is barely life; it is a chemical life. If one considers human cells, by comparison, this is something extremely different, where incredible genetically controlled precise and complex reactions are occurring in a multifaceted incredible human being. This is superlife, or life at is greatest. As illustrated in Fig. 7.1, these are the extremes, and there are at least five levels of life that lie in-between.

16S ribosomal RNA sequences have been used extensively in the classification and identification of Bacteria and Archaea . The comparison of almost complete 16S rRNA gene sequences has been widely used to establish taxonomic relationships between prokaryotic strains, with 98.65% similarity currently recognized as the cutoff for delineating species. The comparison of the 16S rRNA gene sequence of an isolate against sequences of type strains of all prokaryotic species provides an accurate and convenient way to routinely classify and identify prokaryotes. Here, we present the theoretical background to detecting phylogenetic neighbours and for calculating pairwise similarities between 16S rRNA gene sequences, using a process named the “EzTaxon search”. In addition, a workflow procedure is introduced that allows the analyses necessary for the identification of prokaryote strains, starting from raw Sanger sequencing data to the generation of phylogenetic trees, using the EzTaxon server ( http://www.ezbiocloud.net/eztaxon ) and EzEditor software. The application of these procedures will ensure best practice in manual editing of 16S rRNA sequence data and their use for taxonomic purposes.

The aim of this study was to investigate the influence of monospecific colonization of sediment stands by Spartina maritima or Halimione portulacoides on benthic prokaryote assemblages in a salt marsh located in Ria de Aveiro (Portugal). The distribution of Bacteria , Archaea and sulfate-reducing bacteria (SRB) in sediments with monospecific plant stands and in unvegetated sediments was characterized by Fluorescence In Situ Hybridization (FISH). Total prokaryote abundance (0.4 × 10 9 –1.7 × 10 9 cells gdw −1 ) was highest in sediments from the surface layer. The domain Bacteria comprised approximately 40% of total prokaryote communities with the highest percentages occurring in the surface layer. Archaeal cells corresponded to an average of 25% of total prokaryote population, with higher abundance in the vegetation banks, and displaying homogeneous vertical distribution. The relative abundance of SRB represented approximately 3% of total 4′, 6-diamidino-2-phenylindole dihydrochloride (DAPI) stained cells at unvegetated sediment and H. portulacoides stand and 7% at S. maritima stand. Headspace solid-phase microextraction (HS-SPME) combined with Comprehensive Two-Dimensional Gas Chromatography–Time-of-Flight Mass Spectrometry (GC × GC–ToFMS) was used to analyse the volatile and semi-volatile fraction of root exudates. A total of 171 compounds were identified and Principal Component Analysis showed a clear separation between the chemical composition (volatile and semi-volatile organic compounds) of the exudates of the two plants. The patterns of vertical distribution and differences in the proportion of SRB and Archaea in the prokaryote communities developing in sediments colonized by Spartina maritima or Halimione portulacoides suggest the existence of plant-specific interactions between halophyte vegetation and estuarine sediment bacteria in Ria de Aveiro salt marshes, exerted via sediment lithology and root-derived exudates.

Publisher Summary Prokaryote diversity has been the subject of increased attention in the past two decades. The most accepted concept, considered to be pragmatic and useful for species definition, is the so-called phylo-phenetic species concept—a monophyletic and genomically coherent cluster of individual organisms (strains) that show a high degree of overall similarity in many independent characteristics—and is diagnosable by one or more discriminative phenotypic properties. The species concept is based on a polyphasic approach, which includes description of diagnostic phenotypic features combined with genomic properties. Individually, many of the phenotypic and chemotaxonomic characters used as diagnostic properties are insufficient to delineate a species, but together they provide sufficient descriptive information to allow the definition of a species. Different properties have different resolving power; some are species specific, while others are valuable for discriminating genera, families, and orders. Properties tested generally include morphological characters, information on motility, the mode of nutrition and energy generation, the cells' relationship to molecular oxygen, temperature, pH, tolerance toward and requirement for salt and many others. More specific tests such as serotyping or phage typing may be necessary for certain groups of microorganisms to obtain a reliable identification.

MALDI-TOF MS (matrix-assisted laser desorption/ionization time-of-flight mass spectrometry) is a technique that has gained importance over the last few years for the taxonomic characterization of prokaryotes. MALDI-TOF MS records the time of flight of proteins pulled up from the prokaryotic cell in order to derive a protein profile to be compared with database. Pulling up prokaryotic proteins is achieved by bombarding the cells entrapped into a special matrix with a laser beam. While MALDI-TOF MS can be a first step towards a fine, peptide-sequencing-based analysis of every protein based on the analysis of every peak, the MALDI-TOF MS profile can also be used as a barcode for the rapid identification of prokaryotes to assess the phyletic relationships among organisms. This chapter provides an overview of the techniques used to prepare samples and to record MALDI-TOF spectra, and shows examples of the use of the method in prokaryote taxonomy

Publisher Summary This chapter discusses the composition and function of a cell. A cell is the most fundamental and primary unit of life. These membrane-enclosed units are composed of an aqueous solution and endowed with the unique capability of replication. The chapter elaborates the functions of different parts of a cell including plasma membrane, cytoplasm, nucleus, lysosomes, mitochondria, and endosomes. Lower organisms are unicellular, whereas higher organisms are communities of a wide variety of cells working in consortium to perform the essential functions. Although all living cells exhibit a similar basic cellular chemistry, there is enormous variance in the appearance and function of cells. The solitary cell of unicellular organisms can perform all the vital functions, whereas the cell groups in multicellular organisms are differentiated to perform specific functions. Cells have been classified as prokaryote, or primitive, and eukaryote, according to a very basic fundamental feature: the absence or presence of a well-defined nucleus that contains genetic information. Prokaryotes have been synonymous with bacteria. Most prokaryotes live as unicellular organisms, but they also exhibit association in the form of chains and clusters. The chapter describes the functions of genes, chromosomes, DNA, and RNA. The chapter also focuses on cell communication and biosignaling, cell division, and transportation within a cell.

The fatty acid composition is probably the most important chemotaxonomic parameter used for the differentiation of prokaryote taxa at the species level because it correlates very well with the accepted DNA:DNA hybridization-based species concept of many groups of bacteria. However, care must be taken to standardize the conditions of growth as well as those of the gas chromatography. If these standardized conditions are followed, valuable information may be acquired from the cellular fatty acids composition. This chapter describes methods that should be applied and the care that should be taken to obtain uniform values from fatty acid determination. © 2011 Elsevier Ltd.

Genomics generates an exponentially increasing amount of DNA sequence data. Analyses of DNA sequences from bacteria have revealed many genes and gene clusters encoding proteins predicted to be involved in the biosynthesis of natural products. However, in many cases, the metabolic product(s) of these so-called cryptic biosynthetic systems is not known. This chapter describes the nature of the most common biosynthetic systems and the development of bioinformatics tools for analyzing them. These tools enable predictions regarding the structural features of the products of cryptic biosynthetic systems to be made. Finally, the experimental strategies developed for identifying the products of cryptic biosynthetic systems, several of which rely on the predictions derived from bioinformatics, are discussed.

Publisher Summary This chapter discusses the enzymology and molecular genetics of biosynthetic enzymes for (1,3)-β-glucans of prokaryotes. The enzymes involved in the synthesis of prokaryote (1,3)-β-glucans - curdlan, the cyclic (1,3;1,6)-β-glucans and (1,3;1,2)-β-glucan—are all members of the inverting GT family 2. The curdlan [(1,3)-β-glucan] synthase catalytic subunit (72.9 kDa) is the product of the Agrobacterium crdS gene and was identified on the basis of its sequence and structural similarity with various β-glucan synthases. (1,3)- β-glucans and related glucans produced by prokaryotes include the linear (1,3)-β-glucan, curdlan, found in Agrobacterium, Cellulomonas and Bacillus spp., the rhizobial cyclic (1,3;1,6)-β-glucans found in Bradyrhizobium, Azospirillum and Azorhizobium spp., and the Streptococcus pneumoniae side-chain-branched (1,3;1,2)-β-glucan. The enzymes involved in their synthesis all belong to family GT2 glycosyl transferases (http://afmb.cnrs-mrs.fr/CAZY/), which also includes synthases for bacterial and embryophyte cellulose, invertebrate chitin and chito-oligosaccharides, and bacterial and vertebrate hyaluronan.

Publisher Summary This chapter discusses the origin of eukaryote and mitochondria. On the issue of eukaryote origins, biologists are divided into various camps. The camps express and defend fundamentally different views about how eukaryotes are linked to prokaryotes in both the phylogenetic sense and in the sense of evolutionary mechanisms. The chapter examines hypotheses concerning the origin of mitochondria and hydrogenosomes, the kind of host that might have acquired their common ancestral endosymbiont, how “Canfield” oceans fit into the picture at mitochondrial origin, and how the origin of mitochondria might have precipitated the origin of the nucleus, had the host been a bona fide prokaryote. The standard model of how and why the mitochondrion becomes established is discussed. Substantial problems with the standard model are described. The chapter also discusses the same issues from the standpoint of an alternative theory. The standard model is couched in an older view of Earth history that entails an oxygen catastrophe, during which a radical transition from globally anoxic habitats to globally oxic habitats is assumed. There are many suggestions in the literature for the origin of mitochondria besides the standard model and the hydrogen hypothesis, but all of them except one operate with a heterotrophic host for the origin of mitochondria.

Publisher Summary This chapter provides an overview of the taxonomy and characteristics of soil prokaryotes and shows their importance for prokaryote growth and activity and soil function. Prokaryotes are distinguished from eukaryotes by the absence of a unit membrane-bound nucleus and, usually, the lack of other cell organelles. Ribosomes in prokaryotes are smaller (70S) than in eukaryotes (80S) and no eukaryote is able to fix atmospheric N2. Despite the apparent, relative simplicity of prokaryotic cells, as a group they have the greater taxonomic and functional diversity. Globally, organic carbon in prokaryotes is equivalent to that in plants, and they contain 10-fold more nitrogen. They also possess the most efficient dispersal and survival mechanisms. Consequently, prokaryotes are of enormous importance in the creation, maintenance, and functioning of the soil. The focus in this chapter is on phylogeny and general features of prokaryotes such as cell structure, metabolism, physiology, and biodegradation capacity. Basic concepts related to differentiation, secondary metabolism, and antibiotic productions are explained.

Publisher Summary This chapter provides an overview of biological nitrogen fixation (BNF). BNF is a process exclusively restricted to the prokaryotes of the domains archaea and bacteria. However, there are many examples of symbiotic associations that have developed between eukaryotes and prokaryotes that circumvent the eukaryotes' needs for an exogenous supply of fixed nitrogen. Bacteria that use N2 as the sole source of nitrogen are called “diazotrophs.” Although only a limited number of bacterial species fix N2, they represent a wide variety of phylogenetically and physiologically distinct types that occupy different ecological niches. BNF is mediated by an enzyme complex called “nitrogenase,” which is composed of two proteins (dinitrogenase and nitrogenase reductase). There are three different forms of nitrogenase that differ in their requirements for molybdenum (Mo), vanadium (V), or iron (Fe) as a critical metallic component of the cofactor associated with the catalytic site. The chapter explains concepts of biological nitrogen fixation, free-living N2-fixing bacteria, and associative N2-fixing bacteria. Phototrophic bacteria as well as symbiotic N2-fixing associations between legumes and Rhizobia are discussed along with the biotechnology of BNF.

Publisher Summary Prokaryotes demonstrate the most diverse niche occupancies, metabolisms, and geographic distributions of any living organisms. Prokaryote species may be free-living or exist in pathogenic or mutualistic associations with hosts, either intra- or extracellular. They may exhibit aerobic or anaerobic metabolisms, and many acquire their energy from chemical sources that are toxic to other living things. They can be found from the depths of the ocean to high in the atmosphere and everywhere in between, and have played a fundamental role in shaping and maintaining both the biotic and abiotic characteristics of the planet. Their resiliency in the face of environmental change is phenomenal, allowing them to have survived and reproduced in much the same form for nearly four billion years. As Gould noted, “On any possible, reasonable, or fair criterion, bacteria are —and always have been—the dominant forms of life on earth.” Many discussions of prokaryote genomics place an emphasis on the practical benefits that the field may provide such as potential applications in medicine and industry. Comparative prokaryote genomics has already provided a great deal of knowledge regarding bacterial and archaeal genome structure, content, organization, and size, and has highlighted exceptions to long-held assumptions in each of these areas. It has resulted in the discovery of hundreds of thousands of new genes and has shed light on the vast array of metabolic and ecological means by which prokaryotes make their livings.

Probes hybridize with the homologous genes of any mycoplasma and, in essence, with ribosomal RNA (rRNA) genes of any prokaryote. To provide more specific probes, synthetic oligonucleotides, 15 to 40 nucleotides in length, complementary to variable, species-specific regions of mycoplasmal 16S rRNA genes are applied. When labeled, they are used as probes in dot-blot hybridization with rRNA of the clinical specimens suspected of carrying the mycoplasma for which the probe was developed. The ribosome is an essential organelle in all living cells because it constitutes the protein-synthesizing machinery. The expectations from use of rDNA probes in direct filter hybridization of clinical material were high at the beginning. However, despite the high sensitivity of a diagnostic system based on rDNA probes, it is still not sensitive enough for many clinical applications, and it is difficult to analyze the kind of samples that can be collected from live animals.

Publisher Summary This chapter discusses the history and significance of the archaea. A prokaryote had originally been defined as an organism that did not possess certain eukaryotic cellular features—that is, a membrane-bounded nucleus and mitochondria. The early evidence for the existence of the archaea came in the main from an odd collection of organisms that lived in extreme environments. At that time conventional wisdom held that organisms living in extreme environments represent evolutionary adaptations to their environments, and such adaptations required an organism to undergo unusual phenotypic changes. A very few biologists actually did find the initial claim of a third form of life interesting and important. Many of them were microbiologists who worked with one or more of the archaea and found phylogenetic uniqueness a satisfying explanation for the phenotypic uniqueness they were increasingly encountering. The translation apparatus, an incredibly complex molecular aggregate that today involves of the order of 100 different molecular species, had to have evolved in stages from a far more rudimentary and inaccurate mechanism.

An organism of great value to the field of biotechnology is Saccharomyces cerevisiae ( S. cerevisiae ) and its importance is owed to the fact that even though it is a eukaryote, it can be cultivated like a prokaryote because of its microbial features. It can be analyzed genetically because of its special characteristics like small genome and short doubling time. Advances made in molecular biology research are greatly attributed to this yeast. This organism also has some industrial uses and can be used in the production of beer, bread, wine, and recombinant proteins and peptides. Current advances made in recombinant DNA technology are also owed to the use of S. cerevisiae as a host organism for foreign DNA introduced in the form of plasmid DNA vectors. Similar transformation techniques are applied for Escherichia coli ( E. coli ), with the only difference resulting from a few modifications made because of the complexity of the yeast cell wall. Yeast transformation is generally performed either by the formation of spheroplasts, which includes removal of the cell wall, or by a more rapid treatment of intact cells with alkali cations. The alkali cation method that makes the transformation technique simpler is now widely used by researchers.

The use of sodium deoxycholate (DOC) for the solubilization and crossed immunoelectrophoretic analysis of membrane proteins from the helical, wall-less prokaryote Spiroplasma citri offered several distinct advantages over Triton X-100. (i) The yield of extracted membrane protein was very high (up to 90 per cent instead of 30 per cent); (ii) spiralin, the major integral protein, was extracted with a yield approaching 100 per cent, and (iii) most of the proteins were solubilized in an apparently fully dissociated form. In contrast to its reported effect on other plasma membranes, DOC did not notiCEAbly inhibit the antigenicity of the proteins. Furthermore, the electrophoretic separation of the antigens was quite effective. The use of a composite agarose-polyacrylamide gel in the first direction allowed separation of the proteins according to mainly their molecular mass. A preliminary investigation was performed to probe the usefulness of the technique in cross-linking experiments and to assess the effect of disulfide bond reduction on the antigenicity and molecular mass of membrane proteins.

Thermoplasma acidophilum is a thermophilic mycoplasma that is unusual among prokaryotic organisms in that it has a histone-like protein associated with its DNA. The protein is tightly bound to the DNA, and is dissociated only in salt concentrations of 0.6 M NaCl or higher, which is well above the intracellular K + concentration (17-125 mM). The contents of the cells are also unusually acidic: pH 5.5. These conditions, at the normal culture temperature of 59°, come close to thermally denaturing the DNA, and can also damage the DNA by hydrolysis. Thus it might be expected that the histone-like protein would function to protect the DNA, but in contrast, in physiological conditions, the histone-like protein does not appear to have a protective effect. Furthermore, although eukaryotic histones can inhibit RNA synthesis by E. coli RNA polymerase, the T. acidophilum protein does not affect the template activity of the DNA.

Publisher Summary This chapter focuses on the microbial systematics based on electrophoretic whole-cell protein patterns. It can be estimated that some one to two thousand genes in the prokaryote genome are expressed as proteins at one time in the microbial cell. This estimate is partly based on the number of proteins separated by two dimensional polyacrylamide electrophoresis (2D PAGE) from Escherichia coli. This is currently the highest-resolution technique for the separation of proteins utilizing separation by isoelectric point and then by molecular size to give a pattern of protein spots distributed over a gel. It is apparent that electrophoretic methods for the description of all the proteins in the cell used comparatively would have wide application in microbial taxonomy, identification and typing.

Publisher Summary This chapter describes and emphasizes on two methods for the quantitative analysis of antibiotic-binding to ribosomes: equilibrium dialysis, and ethanol precipitation. The assays give estimates for the dissociation constant and number of available binding sites, and are suitable for correlation with resolved assays of the peptidyl transfer reaction and of substrate-binding at the peptidyltransferase center. The methods are applicable not only to a variety of labeled compounds that bind to the ribosome, but also to unlabeled compounds that compete with the labeled compounds for binding. The alcohol precipitation assay is intended primarily for the correlation of studies on the binding of inhibitors with studies on the peptidyl transfer reaction, but it is also useful for examination of competition between different compounds, and for routine assay of ribosome activities. The equilibrium dialysis assay is of more general applicability, and can be used in a wide range of conditions. Both methods are applicable to eukaryote as well as prokaryote ribosomes.