Check out our New Publishers' Select for Free Articles

Archives of Microbiology (v.190, #3)

Gene regulation and genome function in Archaea: a progress survey by Harald Huber; Jörg Soppa (pp. 195-196).

Genomics and functional genomics with haloarchaea by J. Soppa; A. Baumann; M. Brenneis; M. Dambeck; O. Hering; C. Lange (pp. 197-215).

The first haloarchaeal genome was published in 2000 and today five genome sequences are available. Transcriptome and proteome analyses have been established for two and three haloarchaeal species, respectively, and more than 20 studies using these functional genomic approaches have been published in the last two years. These studies gave global overviews of metabolic regulation (aerobic and anaerobic respiration, phototrophy, carbon source usage), stress response (UV, X-rays, transition metals, osmotic and temperature stress), cell cycle-dependent transcript level regulation, and transcript half-lives. The only translatome analysis available for any prokaryotic species revealed that 10 and 20% of all transcripts are translationally regulated in Haloferax volcanii and Halobacterium salinarum, respectively. Very effective methods for the construction of in frame deletion mutants have been established recently for haloarchaea and are intensively used to unravel the biological roles of genes in this group. Bioinformatic analyses include both cross-genome comparisons as well as integration of genomic data with experimental results. The first systems biology approaches have been performed that used experimental data to construct predictive models of gene expression and metabolism, respectively. In this contribution the current status of genomics, functional genomics, and molecular genetics of haloarchaea is summarized and selected examples are discussed.

Keywords: Archaea; Haloarchaea; Genomes; Functional genomics; Transcriptome; Proteome; Deletion mutant; Bioinformatics; Systems biology; Translatome

Genetic tools for Sulfolobus spp.: vectors and first applications by Silvia Berkner; Georg Lipps (pp. 217-230).

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.

Keywords: Shuttle vector; Knockouts; Selectable markers; Archaea; Crenarchaea

The central carbohydrate metabolism of the hyperthermophilic crenarchaeote Thermoproteus tenax: pathways and insights into their regulation by Melanie Zaparty; Britta Tjaden; Reinhard Hensel; Bettina Siebers (pp. 231-245).

Although the complexity and modifications of the archaeal central carbohydrate metabolism (CCM) are well established, the knowledge about its regulation is rather limited. The facultatively heterotrophic, hyperthermophilic crenarchaeote Thermoproteus tenax utilizes a modified version of the reversible Embden-Meyerhof-Parnas (EMP) and the catabolic, branched Entner-Doudoroff (ED) pathway for glucose metabolism. Glucose is completely oxidized to carbon dioxide via the oxidative tricarboxylic acid (TCA) cycle, which is supposedly used in the reductive direction for carbon dioxide fixation under autotrophic growth conditions. Elemental sulfur is used as final electron acceptor. The CCM of T. tenax has been well studied on protein level as well as on gene level by performing a focused transcriptional analysis (CCM DNA microarray). In contrast to the classical pathways found in Bacteria and Eucarya allosteric regulation seems to play a minor role, therefore emphasizing the important role of regulation on transcript level in T. tenax. Whereas the EMP pathway and the TCA cycle show a highly coordinated regulation on gene level, the catabolic, branched ED pathway reveals no strong regulation. The CCM pathways in T. tenax and the current understanding of their regulation are presented.

Keywords: Central carbohydrate metabolism; Thermoproteus tenax ; Archaea; Hyperthermophile; Embden-Meyerhof-Parnas pathway; Entner-Doudoroff pathway; Regulation of metabolism

The role of TrmB and TrmB-like transcriptional regulators for sugar transport and metabolism in the hyperthermophilic archaeon Pyrococcus furiosus by Sung-Jae Lee; Melanie Surma; Winfried Hausner; Michael Thomm; Winfried Boos (pp. 247-256).

TrmB of Pyrococcus furiosus was discovered as the trehalose/maltose-specific repressor for the genes encoding the trehalose/maltose high-affinity ABC transporter (the TM system). TrmB also represses the genes encoding the high affinity maltodextrin-specific ABC transporter (the MD system) with maltodextrin and sucrose as inducers. In addition, TrmB binds glucose leading to an increased repression of both, the TM and the MD system. Thus, TrmB recognizes different promoters and depending on the promoter it will be activated or inactivated for promoter binding by different sugar effectors. The TrmB-like protein TrmBL1 of P. furiosus is a global regulator and recognizes preferentially, but not exclusively, the TGM (for Thermococcales–glycolytic motif) sequence that is found upstream of the MD system as well as of genes encoding enzymes involved in the glycolytic and the gluconeogenic pathway. It responds to maltose and maltotriose as inducers and functions as repressor for the genes encoding the MD system and glycolytic enzymes, but as activator for genes encoding gluconeogenic enzymes. The TrmB-like protein TrmBL2 of P. furiosus lacks the sugar-binding domain that has been determined in TrmB. It recognizes the MD promoter, but not all TGM harboring promoters. It is evolutionary the most conserved among the Thermococcales. The regulatory range of TrmBL2 remains unclear.

Keywords: Global gene regulation; Catabolite repression; ABC transport; TGM sequence; Sugar binding motif

Carbon monoxide-dependent energy metabolism in anaerobic bacteria and archaea by Ellen Oelgeschläger; Michael Rother (pp. 257-269).

Despite its toxicity for the majority of living matter on our planet, numerous microorganisms, both aerobic and anaerobic, can use carbon monoxide (CO) as a source of carbon and/or energy for growth. The capacity to employ carboxidotrophic energy metabolism anaerobically is found in phylogenetically diverse members of the Bacteria and the Archaea. The oxidation of CO is coupled to numerous respiratory processes, such as desulfurication, hydrogenogenesis, acetogenesis, and methanogenesis. Although as diverse as the organisms capable of it, any CO-dependent energy metabolism known depends on the presence of carbon monoxide dehydrogenase. This review summarizes recent insights into the CO-dependent physiology of anaerobic microorganisms with a focus on methanogenic archaea. Carboxidotrophic growth of Methanosarcina acetivorans, thought to strictly rely on the process of methanogenesis, also involves formation of methylated thiols, formate, and even acetogenesis, and, thus, exemplifies how the beneficial redox properties of CO can be exploited in unexpected ways by anaerobic microorganisms.

Keywords: Carboxidotrophic; Carbon monoxide dehydrogenase; Acetyl-CoA synthase; Methanosarcina acetivorans

The molecular basis of salt adaptation in Methanosarcina mazei Gö1 by Regina Spanheimer; Volker Müller (pp. 271-279).

The study on the molecular basis of salt adaptation and its regulation in archaea is still in its infancy, but genomics and functional genome analyses combined with classical biochemistry shed light on the processes conferring salt adaptation in the methanogenic archaeon Methanosarcina mazei Gö1. In this article, we will review discoveries made within the last years that will culminate in the description of the overall cellular response of M. mazei Gö1 to elevated salinities. This response includes accumulation of solutes and export of Na⁺ as well as potential uptake/export of K⁺ but also a restructuring of the cell surface.

Keywords: Archaea; Methanogens; Osmoregulation; Salt adaptation; Methanosarcina mazei Gö1

Genome information management and integrated data analysis with HaloLex by Friedhelm Pfeiffer; Alexander Broicher; Thomas Gillich; Kathrin Klee; José Mejía; Markus Rampp; Dieter Oesterhelt (pp. 281-299).

HaloLex is a software system for the central management, integration, curation, and web-based visualization of genomic and other -omics data for any given microorganism. The system has been employed for the manual curation of three haloarchaeal genomes, namely Halobacterium salinarum (strain R1), Natronomonas pharaonis, and Haloquadratum walsbyi. HaloLex, in particular, enables the integrated analysis of genome-wide proteomic results with the underlying genomic data. This has proven indispensable to generate reliable gene predictions for GC-rich genomes, which, due to their characteristically low abundance of stop codons, are known to be hard targets for standard gene finders, especially concerning start codon assignment. The proteomic identification of more than 600 N-terminal peptides has greatly increased the reliability of the start codon assignment for Halobacterium salinarum. Application of homology-based methods to the published genome of Haloarcula marismortui allowed to detect 47 previously unidentified genes (a problem that is particularly serious for short protein sequences) and to correct more than 300 start codon misassignments.

Keywords: Halophilic archaea; Genome information system; Genome browser; Proteomics; Biological data curation; Start codon assignment; Dinucleotide bias

Two archaeal tRNase Z enzymes: similar but different by Bettina Späth; Sylvia Schubert; Annika Lieberoth; Florian Settele; Stefanie Schütz; Susan Fischer; Anita Marchfelder (pp. 301-308).

The endoribonuclease tRNase Z plays an essential role in tRNA metabolism by removal of the 3′ trailer element of precursor RNAs. To investigate tRNA processing in archaea, we identified and expressed the tRNase Z from Haloferax volcanii, a halophilic archaeon. The recombinant enzyme is a homodimer and efficiently processes precursor tRNAs. Although the protein is active in vivo at 2–4 M KCl, it is inhibited by high KCl concentrations in vitro, whereas 2–3 M (NH₄)₂SO₄ do not inhibit tRNA processing. Analysis of the metal content of the metal depleted tRNase Z revealed that it still contains 0.4 Zn²⁺ ions per dimer. In addition tRNase Z requires Mn²⁺ ions for processing activity. We compared the halophilic tRNase Z to the homologous one from Pyrococcus furiosus, a thermophilic archaeon. Although both enzymes have 46% sequence similarity, they differ in their optimal reaction conditions. Both archaeal tRNase Z proteins process mitochondrial pre-tRNAs. Only the thermophilic tRNase Z shows in addition activity toward intron containing pre-tRNAs, 5′ extended precursors, the phosphodiester bis(p-nitrophenyl)phosphate (bpNPP) and the glyoxalase II substrate S-d-lactoylglutathion (SLG).

Keywords: Haloferax volcanii ; Pyrococcus furiosus ; tRNase Z; tRNA Processing; Metallo-β-lactamase

Variations in the multiple tbp genes in different Halobacterium salinarum strains and their expression during growth by Katharina Teufel; Anne Bleiholder; Tim Griesbach; Felicitas Pfeifer (pp. 309-318).

The presence and expression of the multiple tbp genes encoding TATA-box binding proteins (TBPs) was investigated in various strains and mutants of the archaeon Halobacterium salinarum. Six genes, tbpA through tbpF, are present in the genome of Hbt. salinarum NRC-1 and also in the gas vesicle negative mutant strain R1. The only tbp gene located in the chromosome is tbpE, whereas all others are found in the plasmid DNA. Due to the dynamic nature of the plasmids in the Halobacterium strains, the copy numbers of the alternative tbp genes vary significantly. Five tbp genes (tbpA through tbpE) were present in the wild-type strain Hbt. salinarum PHH1. The tbpC gene of Hbt. salinarum PHH1 carried an ISH27-2 insertion element at the start of the reading frame that prevented the expression. All other tbp genes of PHH1 were expressed under aerobic and anaerobic growth conditions and quantitative RT-PCR yielded tbpE as dominant tbp transcript during the exponential growth phase. The plasmid deletion variant Hbt. salinarum PHH4 lacked all of the tbp genes except for tbpE and showed an altered growth behaviour compared to PHH1 wild-type in the stationary growth phase under anaerobic growth conditions.

Keywords: TBP proteins; Plasmid variation; ISH elements; Alternative tbp genes

Insights into the NrpR regulon in Methanosarcina mazei Gö1 by Katrin Weidenbach; Claudia Ehlers; Jutta Kock; Armin Ehrenreich; Ruth A. Schmitz (pp. 319-332).

The methanogenic archaeon Methanosarcina mazei strain Gö1 contains two homologues of NrpR, the transcriptional repressor of nitrogen assimilation genes recently discovered and characterized in Methanococcus maripaludis. Insertion of a puromycin-resistance conferring cassette into MM1085 encoding a single NrpR domain with an N-terminal helix–turn–helix domain (NrpRI) lead to a significant reduction of the lag-phase after a shift from nitrogen sufficiency to nitrogen limitation. Consistent with this finding, loss of NrpRI resulted in significantly increased transcript levels of genes involved in nitrogen fixation or nitrogen assimilation though growing under nitrogen sufficiency as demonstrated by quantitative reverse transcriptional PCR analysis. Genome-wide analysis using DNA-microarrays confirmed that transcript levels of 27 ORFs were significantly elevated in the M. mazei MM1085::pac mutant under nitrogen sufficiency, including genes known to be up-regulated under nitrogen limitation (e.g., nifH, glnA 1, glnK 1), and 17 additional genes involved in metabolism (4), encoding a flagella related protein (1) and genes encoding hypothetical proteins (12). Using cell extracts of Escherichia coli expressing MM1085 fused to the maltose binding protein (MBP–NrpRI) and employing promoter binding studies by DNA-affinity chromatography demonstrated that MBP–NrpRI binds specifically to the nifH-promoter. Deletion of various bases in the promoter region of nifH confirmed that the regulatory element ACC-N₇-GGT is required for specific binding of NrpRI to the promoter.

Keywords: Archaea ; Nitrogen regulation; Global transcription regulation; nif-genes; NrpR Methanosarcina mazei

Regulation of gvp genes encoding gas vesicle proteins in halophilic Archaea by Sandra Scheuch; Larissa Marschaus; Simone Sartorius-Neef; Felicitas Pfeifer (pp. 333-339).

Three gas vesicle gene clusters derived from Halobacterium salinarum (p-vac and c-vac) and Haloferax mediterranei (mc-vac) are used as model systems to study gene regulation in Archaea. An unusual pair of regulatory proteins is involved here, with GvpE acting as transcription activator and GvpD exhibiting a repressing function. Both regulators are able to interact leading to the loss of GvpE and the repression (or turnoff) of the gas vesicle formation. The latter function of GvpD requires a p-loop motif and an arginine-rich region, bR1. Both regulator proteins are differentially expressed from the same gvp transcript in Hfx. mediterranei and Hbt. salinarum PHH4. GvpE appears to recognize a 20-nucleotide activator sequence (UAS) located upstream and adjacent to the TFB-recognition element BRE of the two promoters driving the transcription of the divergently oriented gvpACNO and gvpDEFGHIJKLM gene clusters. The BRE elements of these two promoters are separated by 35 nucleotides only, and the distal portions of the two GvpE-UAS overlap considerably in the center of this region. Mutations here negatively affect the GvpE-induced activities of both gvp promoters, whereas alterations in the proximal UAS portions only affect the activity of the promoter located close by.

Keywords: GvpE activator; TFB and TBP; Gas vesicle genes

Characterization of a Haloferax volcanii member of the enolase superfamily: deletion mutant construction, expression analysis, and transcriptome comparison by Michael Dambeck; Jörg Soppa (pp. 341-353).

The enolase superfamily (COG4948) contains proteins with very different biological functions including regulators like the Escherichia coli RspA and metabolic enzymes like enolase. To unravel the biological function of an archaeal family member, an in frame deletion mutant of a gene encoding a COG4948 protein of Haloferax volcanii was generated. The mutant had a lag phase of 3 days after transition from a richer to a poorer medium, in contrast to the wild-type, and the gene was therefore named “important for transition” (iftA). After inoculation of fresh casamino acids or complex medium with stationary phase wild-type cells, the transcript level of iftA was transiently induced at the onset of growth. In contrast, in minimal (or “poor”) glucose medium, both transcript and protein were present throughout growth, even in late stationary phase. A comparison of the transcriptomes of deletion mutant and wild-type revealed that transcript levels of a very restricted set of genes were differentially regulated, including genes encoding proteins involved in phosphate metabolism, regulators and stress response proteins. Taken together, the results indicate that IftA might have a dual function, i.e., transiently after transition to fresh medium and permanently during growth in glucose medium.

Keywords: Haloferax volcanii ; Enolase superfamily; COG4948; In frame deletion mutant; DNA microarray

A novel trehalose synthesizing pathway in the hyperthermophilic Crenarchaeon Thermoproteus tenax: the unidirectional TreT pathway by Theresa Kouril; Melanie Zaparty; Jeannette Marrero; Henner Brinkmann; Bettina Siebers (pp. 355-369).

In the genome of the hyperthermophilic archaeon Thermoproteus tenax a gene (treS/P) encoding a protein with similarity to annotated trehalose phosphorylase (TreP), trehalose synthase (TreS) and more recently characterized trehalose glycosyltransferring synthase (TreT) was identified. The treS/P gene as well as an upstream located ORF of unknown function (orfY) were cloned, heterologously expressed in E. coli and purified. The enzymatic characterization of the putative TreS/P revealed TreT activity. However, contrary to the previously characterized reversible TreT from Thermococcus litoralis and Pyrococcus horikoshii, the T. tenax enzyme is unidirectional and catalyzes only the formation of trehalose from UDP (ADP)-glucose and glucose. The T. tenax enzyme differs from the reversible TreT of T. litoralis by its preference for UDP-glucose as co-substrate. Phylogenetic and comparative gene context analyses reveal a conserved organization of the unidirectional TreT and OrfY gene cluster that is present in many Archaea and a few Bacteria. In contrast, the reversible TreT pathway seems to be restricted to only a few archaeal (e.g. Thermococcales) and bacterial (Thermotogales) members. Here we present a new pathway exclusively involved in trehalose synthesis––the unidirectional TreT pathway––and discuss its physiological role as well as its phylogenetic distribution.

Keywords: Trehalose metabolism; Unidirectional trehalose glycosyltransferring synthase (TreT); Thermoproteus tenax ; Hyperthermophile; Archaea

An archaeal bi-species biofilm formed by Pyrococcus furiosus and Methanopyrus kandleri by Simone Schopf; Gerhard Wanner; Reinhard Rachel; Reinhard Wirth (pp. 371-377).

Recently it was shown that Pyrococcus furiosus uses its flagella not only for swimming, but also for establishment of cell–cell connections, and for adhesion to abiotic surfaces. Therefore, it was asked here if P. furiosus might be able to adhere also to biotic surfaces. Since Methanopyrus kandleri can be found in habitats similar to those of P. furiosus (seawater close to the boiling point and anaerobic conditions) it was tested if interactions between both archaea occur. Using a standard medium and a gas phase reduced in H₂ (compared with the optimal gas phase for M. kandleri) we were able to grow both species in a stable coculture. Very interestingly, M. kandleri could adhere to glass under such conditions, but not P. furiosus. This latter archaeum, however, was able to adhere onto M. kandleri cells and onto itself, resulting in structured biofilms on glass. These very often appeared as a bottom layer of M. kandleri cells covered by a multitude of P. furiosus cells. Interactions between P. furiosus and M. kandleri were mediated not only by flagella, but also by direct cell–cell contact.

Keywords: Archaea; Biofilm; Pyrococcus furiosus ; Methanopyrus kandleri

Insight into the proteome of the hyperthermophilic Crenarchaeon Ignicoccus hospitalis: the major cytosolic and membrane proteins by Tillmann Burghardt; Manfred Saller; Sonja Gürster; Daniel Müller; Carolin Meyer; Ulrike Jahn; Eduard Hochmuth; Rainer Deutzmann; Frank Siedler; Patrick Babinger; Reinhard Wirth; Harald Huber; Reinhard Rachel (pp. 379-394).

Ignicoccus hospitalis, a hyperthermophilic, chemolithoautotrophic Crenarchaeon, is the host of Nanoarchaeum equitans. Together, they form an intimate association, the first among Archaea. Membranes are of fundamental importance for the interaction of I. hospitalis and N. equitans, as they harbour the proteins necessary for the transport of macromolecules like lipids, amino acids, and cofactors between these organisms. Here, we investigated the protein inventory of I. hospitalis cells, and were able to identify 20 proteins in total. Experimental evidence and predictions let us conclude that 11 are soluble cytosolic proteins, eight membrane or membrane-associated proteins, and a single one extracellular. The quantitatively dominating proteins in the cytoplasm (peroxiredoxin; thermosome) antagonize oxidative and temperature stress which I. hospitalis cells are exposed to at optimal growth conditions. Three abundant membrane protein complexes are found: the major protein of the outer membrane, which might protect the cell against the hostile environment, forms oligomeric complexes with pores of unknown selectivity; two other complexes of the cytoplasmic membrane, the hydrogenase and the ATP synthase, play a key role in energy production and conversion.

Keywords: Crenarchaeota; Chemolithoautotrophy; Proteome; MALDI; Ignicoccus hospitalis

Ignicoccus hospitalis and Nanoarchaeum equitans: ultrastructure, cell–cell interaction, and 3D reconstruction from serial sections of freeze-substituted cells and by electron cryotomography by Benjamin Junglas; Ariane Briegel; Tillmann Burghardt; Paul Walther; Reinhard Wirth; Harald Huber; Reinhard Rachel (pp. 395-408).

Ultrastructure and intercellular interaction of Ignicoccus hospitalis and Nanoarchaeum equitans were investigated using two different electron microscopy approaches, by three-dimensional reconstructions from serial sections, and by electron cryotomography. Serial sections were assembled into 3D reconstructions, for visualizing the unusual complexity of I. hospitalis, its huge periplasmic space, the vesiculating cytoplasmic membrane, and the outer membrane. The cytoplasm contains fibres which are reminiscent to a cytoskeleton. Cell division in I. hospitalis is complex, and different to that in Euryarchaeota or Bacteria. An irregular invagination of the cytoplasmic membrane is followed by separation of the two cytoplasms. Simultaneous constriction of cytoplasmic plus outer membrane is not observed. Cells of N. equitans show a classical mode of cell division, by constriction in the mid-plane. Their cytoplasm exhibits two types of fibres, elongated and ring-shaped. Electron micrographs of contact sites between I. hospitalis and N. equitans exhibit two modes of interaction. One is indirect and mediated by thin fibres; in other cells the two cell surfaces are in direct contact. The two membranes of I. hospitalis cells are frequently seen in direct contact, possibly a prerequisite for transporting metabolites or substrates from the cytoplasm of one cell to the other. Rarely, a transport based on cargo vesicles is observed between I. hospitalis and N. equitans.

Keywords: Crenarchaeota; Ignicoccus hospitalis ; Nanoarchaeum equitans ; Ultrastructure; 3D reconstruction; Serial sections; Cytoskeleton; Cell–cell interaction

Sections

Personal tools

Archives of Microbiology (v.190, #3)

Sections

Personal tools

Local resources

Archives of Microbiology (v.190, #3)