Aminoacyl-tRNA synthesis in archaea: different but not unique

Accurate aminoacyl-tRNA synthesis is essential for correct translation of the genetic code in all organisms. Whereas many aspects of this process are conserved, others display a surprisingly high level of divergence from the canonical Escherichia coli model system. These differences are most pronounced in archaea where novel mechanisms have recently been described for aminoacylating tRNAs with asparagine, cysteine, glutamine and lysine. Whereas these mechanisms were initially assumed to be uniquely archaeal, both the alternative asparagine and lysine pathways have subsequently been demonstrated in numerous bacteria. Similarly, studies of the means by which archaea insert the rare amino acid selenocysteine in response to UGA stop codons have helped provide a better understanding of both archaeal and eukaryal selenoprotein synthesis. Most recently a new co-translationally inserted amino acid, pyrrolysine, has been found in archaea although again there is some suggestion that it may also be present in bacteria. Thus, whereas archaea contain a preponderance of non-canonical aminoacyl-tRNA synthesis systems most are also found elsewhere albeit less frequently.     

Genetic tools for <Emphasis Type="Italic">Sulfolobus</Emphasis> spp.: vectors and first applications

Sulfolobus species belong to the best-studied archaeal organisms but have lacked powerful genetic methods. Recently, there has been considerable progress in the field of Sulfolobus genetics. Urgently needed basic genetic tools, such as targeted gene knockout techniques and shuttle vectors are being developed at an increasing pace. For S. solfataricus knockout systems as well as different shuttle vectors are available. For the genetically more stable S. acidocaldarius shuttle vectors have been recently developed. In this review we summarize the currently available genetic tools and methods for the genus Sulfolobus. Different transformation protocols are discussed, as well as all so far developed knockout systems and Sulfolobus-Escherichia coli shuttle vectors are summarized. Special emphasis is put on the important vector components, i.e., selectable markers and Sulfolobus replicons. Additionally, the information gathered on different Sulfolobus strains with respect to their use as recipient strains is reviewed. The advantages and disadvantages of the different systems are discussed and aims for further improvement of genetic systems are identified.     

Archaeal flagellar ATPase motor shows ATP-dependent hexameric assembly and activity stimulation by specific lipid binding

Microbial motility frequently depends on flagella or type?IV pili. Using recently developed archaeal genetic tools, archaeal flagella and its assembly machinery have been identified. Archaeal flagella are functionally similar to bacterial flagella and their assembly systems are homologous with type?IV pili assembly systems of Gram-negative bacteria. Therefore elucidating their biochemistry may result in insights in both archaea and bacteria. FlaI, a critical cytoplasmic component of the archaeal flagella assembly system in Sulfolobus acidocaldarius, is a member of the type?II/IV secretion system ATPase superfamily, and is proposed to be bi-functional in driving flagella assembly and movement. In the present study we show that purified FlaI is a Mn2+-dependent ATPase that binds MANT-ATP [2'-/3'-O-(N'- methylanthraniloyl)adenosine-5'-O-triphosphate] with a high affinity and hydrolyses ATP in a co-operative manner. FlaI has an optimum pH and temperature of 6.5 and 75?°C for ATP hydrolysis. Remarkably, archaeal, but not bacterial, lipids stimulated the ATPase activity of FlaI 3-4-fold. Analytical gel filtration indicated that FlaI undergoes nucleotide-dependent oligomerization. Furthermore, SAXS (small-angle X-ray scattering) analysis revealed an ATP-dependent hexamerization of FlaI in solution. The results of the present study report the first detailed biochemical analyses of the motor protein of an archaeal flagellum.     

Bioenergetics of the Archaea

In the late 1970s, on the basis of rRNA phylogeny, Archaea (archaebacteria) was identified as a distinct domain of life besides Bacteria (eubacteria) and Eucarya. Though forming a separate domain, archaea display an enormous diversity of lifestyles and metabolic capabilities. Many archaeal species are adapted to extreme environments with respect to salinity, temperatures around the boiling point of water, and/or extremely alkaline or acidic pH. This has posed the challenge of studying the molecular and mechanistic bases on which these organisms can cope with such adverse conditions. This review considers our cumulative knowledge on archaeal mechanisms of primary energy conservation, in relationship to those of bacteria and eucarya. Although the universal principle of chemiosmotic energy conservation also holds for Archaea, distinct features have been discovered with respect to novel ion-transducing, membrane-residing protein complexes and the use of novel cofactors in bioenergetics of methanogenesis. From aerobically respiring archaea, unusual electron-transporting supercomplexes could be isolated and functionally resolved, and a proposal on the organization of archaeal electron transport chains has been presented. The unique functions of archaeal rhodopsins as sensory systems and as proton or chloride pumps have been elucidated on the basis of recent structural information on the atomic scale. Whereas components of methanogenesis and of phototrophic energy transduction in halobacteria appear to be unique to archaea, respiratory complexes and the ATP synthase exhibit some chimeric features with respect to their evolutionary origin. Nevertheless, archaeal ATP synthases are to be considered distinct members of this family of secondary energy transducers. A major challenge to future investigations is the development of archaeal genetic transformation systems, in order to gain access to the regulation of bioenergetic systems and to overproducers of archaeal membrane proteins as a prerequisite for their crystallization.     

Bioenergetics of the Archaea.

In the late 1970s, on the basis of rRNA phylogeny, Archaea (archaebacteria) was identified as a distinct domain of life besides Bacteria (eubacteria) and Eucarya. Though forming a separate domain, Archaea display an enormous diversity of lifestyles and metabolic capabilities. Many archaeal species are adapted to extreme environments with respect to salinity, temperatures around the boiling point of water, and/or extremely alkaline or acidic pH. This has posed the challenge of studying the molecular and mechanistic bases on which these organisms can cope with such adverse conditions. This review considers our cumulative knowledge on archaeal mechanisms of primary energy conservation, in relationship to those of bacteria and eucarya. Although the universal principle of chemiosmotic energy conservation also holds for Archaea, distinct features have been discovered with respect to novel ion-transducing, membrane-residing protein complexes and the use of novel cofactors in bioenergetics of methanogenesis. From aerobically respiring Archaea, unusual electron-transporting supercomplexes could be isolated and functionally resolved, and a proposal on the organization of archaeal electron transport chains has been presented. The unique functions of archaeal rhodopsins as sensory systems and as proton or chloride pumps have been elucidated on the basis of recent structural information on the atomic scale. Whereas components of methanogenesis and of phototrophic energy transduction in halobacteria appear to be unique to Archaea, respiratory complexes and the ATP synthase exhibit some chimeric features with respect to their evolutionary origin. Nevertheless, archaeal ATP synthases are to be considered distinct members of this family of secondary energy transducers. A major challenge to future investigations is the development of archaeal genetic transformation systems, in order to gain access to the regulation of bioenergetic systems and to overproducers of archaeal membrane proteins as a prerequisite for their crystallization.     

Genetics of the proteolytic system of lactic acid bacteria

Abstract The proteolytic system of lactic acid bacteria is of eminent importance for the rapid growth of these organisms in protein-rich media. The combined action of proteinases and peptidases provides the cell with small peptides and essential amino acids. The amino acids and peptides thus liberated have to be translocated across the cytoplasmic membrane. To that purpose, the cell contains specific transport proteins. The internalized peptides are further degraded to amino acids by intracellular peptidases. The world-wide economic importance of the lactic acid bacteria and their proteolytic system has led to an intensive research effort in this area and a considerable amount of biochemical data has been collected during the last two decades. Since the development of systems to genetically manipulate lactic acid bacteria, data on the genetics of enzymes and processes involved in proteolysis are rapidly being generated. In this review an overview of the latest genetic data on the proteolytic system of lactic acid bacteria will be presented. As most of the work in this field has been done with lactococi, the emphasis will, inevitably, be on this group of organisms. Where possible, links will be made with other species of lactic acid bacteria.     

Genetics of the proteolytic system of lactic acid bacteria

The proteolytic system of lactic acid bacteria is of eminent importance for the rapid growth of these organisms in protein-rich media. The combined action of proteinases and peptidases provides the cell with small peptides and essential amino acids. The amino acids and peptides thus liberated have to be translocated across the cytoplasmic membrane. To that purpose, the cell contains specific transport proteins. The internalized peptides are further degraded to amino acids by intracellular peptidases. The world-wide economic importance of the lactic acid bacteria and their proteolytic system has led to an intensive research effort in this area and a considerable amount of biochemical data has been collected during the last two decades. Since the development of systems to genetically manipulate lactic acid bacteria, data on the genetics of enzymes and processes involved in proteolysis are rapidly being generated. In this review an overview of the latest genetic data on the proteolytic system of lactic acid bacteria will be presented. As most of the work in this field has been done with lactococci, the emphasis will, inevitably, be on this group of organisms. Where possible, links will be made with other species of lactic acid bacteria.     

Development of additional selectable markers for the halophilic archaeon Haloferax volcanii based on the leuB and trpA genes

Since most archaea are extremophilic and difficult to cultivate, our current knowledge of their biology is confined largely to comparative genomics and biochemistry. Haloferax volcanii offers great promise as a model organism for archaeal genetics, but until now there has been a lack of a wide variety of selectable markers for this organism. We describe here isolation of H. volcanii leuB and trpA genes encoding 3-isopropylmalate dehydrogenase and tryptophan synthase, respectively, and development of these genes as a positive selection system. DeltaleuB and DeltatrpA mutants were constructed in a variety of genetic backgrounds and were shown to be auxotrophic for leucine and tryptophan, respectively. We constructed both integrative and replicative plasmids carrying the leuB or trpA gene under control of a constitutive promoter. The use of these selectable markers in deletion of the lhr gene of H. volcanii is described.     

Genetics and molecular biology of methanotrophs.

Over the last 20 years or so, the obligate methane-oxidizing bacteria (methanotrophs) have attracted considerable interest. As they grow on a relatively cheap and abundant carbon source, they appeared ideal organisms for the production of bulk chemicals, single-cell protein and for use in biotransformations. More recently their cooxidation properties have been investigated for bioremediation, including the removal of chlorinated compounds such as trichloroethylene from polluted groundwaters. These studies have resulted in a great deal of information on the physiology and biochemistry of methanotrophs but sadly the molecular biology and genetic studies of these organisms have lagged behind. This has been in part due to the obligate nature of the methanotrophs and the refractory nature of such organisms to conventional genetic analysis. However, the more recent availability of broad-host range plasmids coupled with improvements in molecular biology methods have allowed the development of molecular genetic techniques for methanotrophs. The purpose of this review is to summarize what is known about the genetics and molecular biology of methanotrophs and how this information can be used to complement previous and current biochemical studies on the unique property of these bacteria, i.e. the ability to oxidize methane to methanol. Recent developments in molecular ecology techniques that may be applied to these apparently ubiquitous organism are also considered.     

An integrated analysis of the genome of the hyperthermophilic archaeon Pyrococcus abyssi

The hyperthermophilic euryarchaeon Pyrococcus abyssi and the related species Pyrococcus furiosus and Pyrococcus horikoshii, whose genomes have been completely sequenced, are presently used as model organisms in different laboratories to study archaeal DNA replication and gene expression and to develop genetic tools for hyperthermophiles. We have performed an extensive re-annotation of the genome of P. abyssi to obtain an integrated view of its phylogeny, molecular biology and physiology. Many new functions are predicted for both informational and operational proteins. Moreover, several candidate genes have been identified that might encode missing links in key metabolic pathways, some of which have unique biochemical features. The great majority of Pyrococcus proteins are typical archaeal proteins and their phylogenetic pattern agrees with its position near the root of the archaeal tree. However, proteins probably from bacterial origin, including some from mesophilic bacteria, are also present in the P. abyssi genome.     

REVIEW: Optimality models in the age of experimental evolution and genomics

Optimality models have been used to predict evolution of many properties of organisms. They typically neglect genetic details, whether by necessity or design. This omission is a common source of criticism, and although this limitation of optimality is widely acknowledged, it has mostly been defended rather than evaluated for its impact. Experimental adaptation of model organisms provides a new arena for testing optimality models and for simultaneously integrating genetics. First, an experimental context with a well‐researched organism allows dissection of the evolutionary process to identify causes of model failure – whether the model is wrong about genetics or selection. Second, optimality models provide a meaningful context for the process and mechanics of evolution, and thus may be used to elicit realistic genetic bases of adaptation – an especially useful augmentation to well‐researched genetic systems. A few studies of microbes have begun to pioneer this new direction. Incompatibility between the assumed and actual genetics has been demonstrated to be the cause of model failure in some cases. More interestingly, evolution at the phenotypic level has sometimes matched prediction even though the adaptive mutations defy mechanisms established by decades of classic genetic studies. Integration of experimental evolutionary tests with genetics heralds a new wave for optimality models and their extensions that does not merely emphasize the forces driving evolution.     

Genetics and molecular biology of methanotrophs

Abstract Over the last 20 years or so, the obligate methane-oxidizing bacteria (methanotrophs) have attracted considerable interest. As they grow on a relatively cheap and abundant carbon source, they appeared ideal organisms for the production of bulk chemicals, single-cell protein and for use in biotransformations. More recently their cooxidation properties have been investigated for bioremediation, including the removal of chlorinated compounds such as trichloroethylene from polluted groundwaters. These studies have resulted in a great deal of information on the physiology and biochemistry of methanotrophs but sadly the molecular biology and genetic studies of these organisms have lagged behind. This has been in part due to the obligate nature of the methanotrophs and the refractory nature of such organisms to conventional genetic analysis. However, the more recent availability of broad-host range plasmids coupled with improvements in molecular biology methods have allowed the development of molecular genetic techniques for methanotrophs. The purpose of this review is to summarize what is known about the genetics and molecular biology of methanotrophs and how this information can be used to complement previous and current biochemical studies on the unique property of these bacteria, i.e. the ability to oxidize methane to methanol. Recent developments in molecular ecology techniques that may be applied to these apparently ubiquitous organism are also considered.     

The Genetic Improvement of Entomopathogenic Nematodes and Their Symbiont Bacteria: Phenotypic Targets, Genetic Limitations and an Assessment of Possible Hazards

The methodologies of classical genetics and genetic engineering can be used for the genetic improvement of entomopathogenic nematodes (EPNs) and their symbiont bacteria. Many of the complex behavioural and physiological traits which are targets for genetic improvement are likely to be controlled polygenically, thus selective breeding for improvements to these traits would be appropriate. Much basic research needs to be carried out before researchers will be able to effect improvements to EPNs and their symbionts by genetic engineering. There is a lack of basic information on the genetics and biochemistry of the characteristics that might be altered by transgenic methods in EPNs, and their bacteria, and existing transformation protocols need to be made more effective.     

Quantitative trait locus mapping in natural populations: progress, caveats and future directions

Over the last 15 years quantitative trait locus (QTL) mapping has become a popular method for understanding the genetic basis of continuous variation in a variety of systems. For example, the technique is now an integral tool in medical genetics, livestock production, plant breeding and population genetics of model organisms. Ten years ago, it was suggested that the method could be used to understand continuous variation in natural populations. In this review I: (i) clarify what is meant by natural population in the QTL context, (ii) discuss whether evolutionary biologists have successfully mapped QTL in natural populations, (iii) highlight some of the questions that have been addressed by QTL mapping in natural populations, (iv) describe how QTL mapping can be conducted in unmanipulated natural populations, (v) highlight some of the limitations of QTL mapping and (vi) try to predict some future directions for QTL mapping in natural populations.     

Genetic technologies for extremely thermophilic microorganisms of Sulfolobus,the only genetically tractable genus of crenarchaea

Archaea represents the third domain of life, with the information-processing machineries more closely resembling those of eukaryotes than the machineries of the bacterial counterparts but sharing metabolic pathways with organisms of Bacteria, the sister prokaryotic phylum. Archaeal organisms also possess unique features as revealed by genomics and genome comparisons and by biochemical characterization of prominent enzymes. Nevertheless, diverse genetic tools are required for in vivo experiments to verify these interesting discoveries. Considerable efforts have been devoted to the development of genetic tools for archaea ever since their discovery, and great progress has been made in the creation of archaeal genetic tools in the past decade. Versatile genetic toolboxes are now available for several archaeal models, among which Sulfolobus microorganisms are the only genus representing Crenarchaeota because all the remaining genera are from Euryarchaeota. Nevertheless, genetic tools developed for Sulfolobus are probably the most versatile among all archaeal models, and these include viral and plasmid shuttle vectors, conventional and novel genetic manipulation methods, CRISPR-based gene deletion and mutagenesis, and gene silencing, among which CRISPR tools have been reported only for Sulfolobus thus far. In this review, we summarize recent developments in all these useful genetic tools and discuss their possible application to research into archaeal biology by means of Sulfolobus models.     

A review of bacterial methyl halide degradation: biochemistry,genetics and molecular ecology

Methyl halide-degrading bacteria are a diverse group of organisms that are found in both terrestrial and marine environments. They potentially play an important role in mitigating ozone depletion resulting from methyl chloride and methyl bromide emissions. The first step in the pathway(s) of methyl halide degradation involves a methyltransferase and, recently, the presence of this pathway has been studied in a number of bacteria. This paper reviews the biochemistry and genetics of methyl halide utilization in the aerobic bacteria Methylobacterium chloromethanicum CM4T, Hyphomicrobium chloromethanicum CM2T, Aminobacter strain IMB-1 and Aminobacter strain CC495. These bacteria are able to use methyl halides as a sole source of carbon and energy, are all members of the alpha-Proteobacteria and were isolated from a variety of polluted and pristine terrestrial environments. An understanding of the genetics of these bacteria identified a unique gene (cmuA) involved in the degradation of methyl halides, which codes for a protein (CmuA) with unique methyltransferase and corrinoid functions. This unique functional gene, cmuA, is being used to develop molecular ecology techniques to examine the diversity and distribution of methyl halide-utilizing bacteria in the environment and hopefully to understand their role in methyl halide degradation in different environments. These techniques will also enable the detection of potentially novel methyl halide-degrading bacteria.     

Population genetics of a transformable bacterium: The influence of horizontal genetic exchange on the biology of Neisseria meningitidis

Abstract Information on the biochemistry and genetics of bacterial species, usually obtained by the study of single isolates, is enhanced by studies of populations of bacteria. Recent advances in molecular technology, particularly polymerase chain reaction-based nucleotide sequence analysis, provide powerful for the study of population genetics. Data obtained by such techniques indicate that, while some bacterial species have a clonal population structure, others are non-clonal or panmictic. Clonal populations are a consequence of asexual reproduction by binary fission; panmictic population structures results from 'horizontal' exchange of genetic material between clones. A consequence of horizontal genetic exchange is mosaic gene structures, recognisable by comparisons of nucleotide sequences. In transformable bacteria, for example the human pathogen Neisseria meningitidis , several different genes, including the gene encoding the class 1 outer membrane protein, a major surface antigen, are mosaics. This genetic process has implications both for vaccine design and in the interpretation of epidemiological data.     

Bacillus subtilis and its relatives: molecular biological and industrial workhorses.

The non-pathogenic bacterium Bacillus subtilis, since its first reported genetic transformation in 1959, has become a model system for the study of many aspects of the biochemistry, genetics and physiology of Gram-positive bacteria, and particularly of sporulation and associated metabolism. Extensive knowledge of the molecular biology of B. subtilis has led to the recent development of this bacterium as a host for the industrial production of heterologous proteins. Although difficulties have been encountered, these are being systematically addressed and overcome.     

Evolutionary origin of RNA editing

The term "RNA editing" encompasses a wide variety of mechanistically and phylogenetically unrelated processes that change the nucleotide sequence of an RNA species relative to that of the encoding DNA. Two general classes of editing, substitution and insertion/deletion, have been described, with all major types of cellular RNA (messenger, ribosomal, and transfer) undergoing editing in different organisms. In cases where RNA editing is required for function (e.g., to generate a translatable open reading frame in a mRNA), editing is an obligatory step in the pathway of genetic information expression. How, when, and why individual RNA editing systems originated are intriguing biochemical and evolutionary questions. Here I review briefly what is known about the biochemistry, genetics, and phylogenetics of several very different RNA editing systems, emphasizing what we can deduce about their origin and evolution from the molecular machinery involved. An evolutionary model, centered on the concept of "constructive neutral evolution", is able to account in a general way for the origin of RNA editing systems. The model posits that the biochemical elements of an RNA editing system must be in place before there is an actual need for editing, and that RNA editing systems are inherently mutagenic because they allow potentially deleterious or lethal mutations to persist at the genome level, whereas they would otherwise be purged by purifying selection.