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Exploration of oxygen-depleted marine environments has consistently revealed novel microbial taxa and metabolic capabilities that expand our understanding of microbial evolution and ecology. Marine blue holes are shallow karst formations characterized by low oxygen and high organic matter content. They are logistically challenging to sample, and thus our understanding of their biogeochemistry and microbial ecology is limited. We present a metagenomic characterization of Amberjack Hole on the Florida continental shelf (Gulf of Mexico). Dissolved oxygen became depleted at the hole’s rim (32 m water depth), remained low but detectable in an intermediate hypoxic zone (40-75 m), and then increased to a secondary peak before falling below detection in the bottom layer (80-110 m), concomitant with increases in nutrients, dissolved iron, and a series of sequentially more reduced sulfur species. Microbial communities in the bottom layer contained heretofore undocumented levels of the recently discovered phylum Woesearchaeota (up to 58% of the community), along with lineages in the bacterial Candidate Phyla Radiation (CPR). Thirty-one high-quality metagenome-assembled genomes (MAGs) showed extensive biochemical capabilities for sulfur and nitrogen cycling, as well as for resisting and respiring arsenic. One uncharacterized gene associated with a CPR lineage differentiated hypoxic from anoxic zone communities. Overall, microbial communities and geochemical profiles were stable across two sampling dates in the spring and fall of 2019. The blue hole habitat is a natural marine laboratory that provides opportunities for sampling taxa with under-characterized but potentially important roles in redox-stratified microbial processes.

With a rising amount of high voltage dc projects to be overlaid to the transmission grid, a thorough understanding of the utilized equipment regarding its impact on system stability is required. To cover this need, a passivity-based stability assessment represents one promising option to investigate the damping behavior of a converter over a wide frequency range. Assuming a converter acts in a passive manner at a certain frequency, it adds damping to the overall system and contributes to mitigate un-wanted interactions e.g. related to pre-existing ac grid resonances. By means of an encompassing analysis of the converter input-admittance, relevant system sensitivities are classified in terms of their impact on passivity characteristics and frequency ranges. Moreover, feasible system delay design criteria are determined.

In order to address challenges caused by an increasing need for power transmission, embedded bipolar HVDC links utilizing Modular Multilevel Converter (MMC-HVDC) technology present a preferred solution as several operational and dynamic advantages compared to conventional ac grid enforcement are provided. In this paper, an approach to quickly detect and handle internal as well as dc-side faults in a full-bridge bipolar MMC-HVDC link is presented. Due to non-technical reasons and environmental constraints, the system is comprised of mixed overheadline-cable (OHL-cable) transmission and single point solid grounding with dedicated metallic return (DMR). To validate and evaluate the proposed concept, detailed electromagnetic transient (EMT) simulations are provided. These highlight effects of mixed transmission system design on transient voltage and current stresses as well as on converter-internal quantities.

Harmful algae blooms (HAB) in coastal marine environments are increasing in number and duration, pressuring local resource managers to implement mitigation solutions to protect human and ecosystem health. However, insufficient spatial and temporal observations create uninformed management decisions. In order to better detect and map blooms, as well as the environmental conditions responsible for their formation, long-term, unattended observation platforms are desired. In this article, we describe a new cost-efficient, autonomous, mobile platform capable of accepting several sensors that can be used to monitor harmful algae blooms in near real-time. The Navocean autonomous sail-powered surface vehicle is deployable by a single person from shore, capable of waypoint navigation in shallow and deep waters, and powered completely by renewable energy. We present results from three surveys of the Florida Red Tide harmful algae bloom (Karenia brevis) of 2017-2018. The vessel made significant progress towards waypoints regardless of wind conditions while underway measurements revealed patches of elevated chl. a likely attributable to the K. brevis blooms as based on ancillary measurements. Measurements of colored dissolved organic matter (CDOM) and turbidity provided an environmental context for the blooms. While the autonomous sailboat directly adds to our phytoplankton/HAB monitoring capabilities, the package may also help to ground-truth satellite measurements of HABs if careful validation measurements are performed. Finally, several other pending and future use cases for coastal and inland monitoring are discussed. To our knowledge, this is the first demonstration of a sail-driven vessel used for coastal HAB monitoring.

The symmetrical monopolar configuration is the prevailing scheme configuration for high voltage direct current (HVDC) interconnectors utilizing the modular multilevel converter (MMC) topology. However, the rigid bipolar configuration is gaining significance, as first projects are currently in planning stage. The purpose of this paper is to analyse both HVDC scheme configurations with regard to cable voltage stresses originating from short-circuit faults. The study focuses on HVDC cables with extruded insulation connected to MMCs equipped with half-bridge (HB) submodules. Basic design aspects in rigid bipolar configuration such as the station grounding concept are discussed. Moreover, various station internal as well as dc faults are examined by means of electromagnetic transient (EMT) simulations. Occurring cable voltage stresses are analyzed with respect to voltage polarity, peak value and wave front-parameters and compared in symmetrical monopolar and rigid bipolar configuration. It is demonstrated that cable overvoltages with same polarity as the dc operating voltage are significantly reduced in schemes operating in rigid bipolar configuration. Voltage reversals caused by the discharge process during a cable fault are nearly independent of the selected scheme configuration. Obtained results are relevant with regard to upcoming projects in rigid bipolar configuration and provide insights to further refine insulation co-ordination aspects related to dc cable systems.

Harmful algae blooms (HAB) in coastal marine environments are increasing in number and duration, pressuring local resource managers to implement mitigation solutions to protect human and ecosystem health. However, insufficient spatial and temporal observations create uninformed management decisions. In order to better detect and map blooms, as well as the environmental conditions responsible for their formation, long-term, unattended observation platforms are desired. In this article, we describe a new cost-efficient, autonomous, mobile platform capable of accepting several sensors that can be used to monitor harmful algae blooms in near real-time. The Navocean autonomous sail-powered surface vehicle is deployable by a single person from shore, capable of waypoint navigation in shallow and deep waters, and powered completely by renewable energy. We present results from three surveys of the Florida Red Tide harmful algae bloom (Karenia brevis) of 2017-2018. The vessel made significant progress towards waypoints regardless of wind conditions while underway chl. a measurements revealed HAB bloom patches and CDOM and turbidity provided environmental contextual information. While the autonomous sailboat directly adds to our HAB monitoring capabilities, the boat can also help to ground-truth and thus improve satellite monitoring of HABs. Finally, several other pending and future use cases for coastal and inland monitoring are discussed. To our knowledge, this is the first demonstration of a sail-driven vessel used for coastal HAB monitoring.

International audience; Pore water and solid phase geochemical profiles of sediment cores collected along two transects on the western and eastern sides of the Mississippi River mouth in the northern Gulf of Mexico were incorporated into a reactive transport model to determine the role of manganese and iron in the remineralization of carbon. Reactive transport model calculations indicate that sedimentation rates control the intensity of anaerobic carbon remineralization and select for the dominant anaerobic carbon remineralization pathways. Although sulfate reduction dominates the shelf station (65 m water depth), denitrification and microbial manganese reduction appear equally significant anaerobic respiration processes along the continental slope the closest to the Mississippi River, whereas microbial iron reduction does not represent an important process in these sediments. These findings suggest that the differential kinetics of manganese and iron redox transformations influence carbon remineralization processes on the continental slope. The fast kinetics of Fe2+ oxidation near the sediment-water interface and high sedimentation rates maintain Fe under the form of Fe(III) oxides and thermodynamically prevent sulfate reduction from dominating carbon remineralization processes on the slope, whereas the much slower Mn2+ oxygenation kinetics allows diffusion of Mn2+ across the sediment-water interface of the shelf station closest to the river mouth. Exposure to oxygenated bottom waters and entrainment within mobile muds typical of deltaic sediments during high riverine discharge likely promote the formation and downslope transport of Mn(III/IV) oxides within the nepheloid layer. This phenomenon appears to form a manganese ‘conveyor belt’ that selectively enriches Mn(III/IV) oxides relative to Fe(III) oxides in the deep sediment. In contrast, the intensity of anaerobic carbon remineralization processes along the eastern continental slope the farthest from the Mississippi River plume is much lower due to the low organic and lithogenic inputs, and denitrification dominates anaerobic respiration. Overall, these findings suggest that manganese cycling and its role in carbon remineralization processes in continental slope sediments exposed to large riverine inputs may be more important than previously considered.

This contribution focuses on high voltage direct current (HVDC) transmission systems comprising modular multilevel converters (MMC) equipped with half-bridge (HB) submodules and analyses cable voltage stresses during various station internal as well as dc side faults. In order to examine relevant overvoltage characteristics affecting HVDC cable systems, a systematic approach to evaluate overvoltage stresses is presented and an extensive set of time-domain simulations is analysed for schemes operating in symmetrical monopolar configuration. Obtained results are relevant for considerations on insulation co-ordination of HVDC cable systems and for a comprehensive definition of high voltage testing requirements.

High voltage direct current transmission schemes equipped with modular multilevel converter technology are considered in several realized and planned bulk power transmission projects. Especially in the case of dc cable links with high system lengths, the rigid bipolar configuration presents a suitable compromise between power rating, cost-efficiency and converter redundancy. Caused by the absence of a metallic return path, system degrees of freedom are reduced in contrast to the bipolar configuration with dedicated metallic return. Therefore, the dc currents in positive and negative bipolar subsystem are not independent. This leads to new challenges and motivates the development of advanced control and balancing concepts for rigid bipolar schemes. This paper focuses on balanced and unbalanced ac grid operating conditions with subsystem-independent active power set points in an ac split-busbar scenario, which has not yet been covered in literature. In order to validate the presented methodology, case studies are performed in electromagnetic transient software.

Despite the undeniable effect of iron on shaping patterns of ocean productivity, the relative importance of the different sources of this limiting nutrient to the ocean is still under debate. Although global estimates indicate that the benthic input of iron to the oceans is significant, most studies have investigated continental margins exposed to either upwelling or large riverine inputs, environments that are not representative of the majority of the oceans. Additionally, the number of studies that report dissolved iron concentrations in continental slope sediments is limited, despite the fact that these regions between the shelf edge and the continental rise make up >5% of the sedimentary surface area of the global ocean. The sedimentary flux of iron has traditionally been considered negligible due to the rapid oxidation of Fe2+ in oxic waters and poor solubility of the Fe(III) product. The recent realization that ferric iron may be stabilized in solution by organic ligands during oxidation near the sediment-water interface suggests that a significant fraction of the dissolved iron pool may be present under the form organic-Fe(III) complexes that could eventually reach the overlying waters. In this study, the speciation and biogeochemical importance of iron was determined in intact sediment cores along a transect across the entire continental margin near Cape Lookout, North Carolina, a region not dominated by upwelling or riverine inputs that is representative of most passive continental margins. Rates of diffusive oxygen uptake (DOU) and maximum diffusive fluxes of both dissolved Fe2+and organic-Fe(III) complexes decreased from the coastal zone to the continental shelf, remained low on the shelf and the upper continental slope, but rebounded to reach a maximum in mid-slope sediments where concentrations of Fe(III) oxides were the highest along the transect. In turn, DOU decreased and dissolved iron was below detection in lower-slope sediments, indicating that mid-slope sediments represent depocenters where Fe(III) oxides and organic matter may accumulate. Pore water sulfate and sulfide concentrations as well as separate sediment incubations confirmed that sulfate reduction does not greatly influence the cycling of iron in these sediments. The production of dissolved organic-Fe(III) in these continental margin sediments is likely regulated by a combination of aerobic oxidation in the presence of natural organic ligands near the sediment-water interface, dissimilatory iron reduction, or chemical oxidation of Fe(II) complexed to natural organic ligands. Fluxes of Fe2+ and organic-Fe(III) complexes across the sediment-water interface were not observed. However, diffusive fluxes of Fe2+ and organic-Fe(III) complexes into the oxic zone of these sediments (

In the near future, HVAC and HVDC systems may be routed on the same towers in some applications. These so-called hybrid transmission systems raise several novel challenges, such as AC/DC intersystem faults. Previous investigations focus on the analysis of these faults in purely overhead line based HVDC systems. However, new HVDC corridors might be built as partially cabled systems to increase public acceptance. Intersystem faults in combination with mixed DC overhead and cable transmission systems are a novel challenge for both AC and DC protection systems and are analyzed within this paper. It is shown that even after DC fault current interruption the HVDC system is exposed to significant AC overvoltages and fault currents, which are caused by a resonant behavior of the system. These cannot be cleared by standard DC protection systems. To protect the DC system against intersystem faults the use of a fast earthing concept is proposed.

The redox chemistry and speciation of Fe in both solid and dissolved phases were characterized in the organic- and Fe-rich sediments of the Satilla River estuary in South-East Georgia (USA) on a series of four cruises between July 2007 and January 2008. Results indicate that dissolved Fe is present in relatively high concentration in the overlying waters at the freshwater end of the estuary and flocculates along the river as the salinity increases downstream. Soluble organic–Fe(III) complexes comprise the majority of dissolved Fe (

The presently active region of the Congo deep-sea fan (around 330,000 km(2)), called the terminal lobes or lobe complex, covers an area of 2500 km(2) at 4700-5100 m water depth and 750-800 km offshore. It is a unique sedimentary area in the world ocean fed by a submarine canyon and a channel-levee system which presently deliver large amounts of organic carbon originating from the Congo River by turbidity currents. This particularity is due to the deep incision of the shelf by the Congo canyon, up to 30 km into the estuary, which funnels the Congo River sediments into the deep-sea. The connection between the river and the canyon is unique for major world rivers. In 2011, two cruises (WACS leg 2 and Congolobe) were conducted to simultaneously investigate the geology, organic and inorganic geochemistry, and micro- and macro-biology of the terminal lobes of the Congo deep-sea fan. Using this multidisciplinary approach, the morpho-sedimentary features of the lobes were characterized along with the origin and reactivity of organic matter, the recycling and burial of biogenic compounds, the diversity and function of bacterial and archaeal communities within the sediment, and the biodiversity and functioning of the faunal assemblages on the seafloor. Six different sites were selected for this study: Four distributed along the active channel from the lobe complex entrance to the outer rim of the sediment deposition zone, and two positioned cross-axis and at increasing distance from the active channel, thus providing a gradient in turbidite particle delivery and sediment age. This paper aims to provide the general context of this multidisciplinary study. It describes the general features of the site and the overall sampling strategy and provides the initial habitat observations to guide the other in-depth investigations presented in this special issue. Detailed bathymetry of each sampling site using 0.1-1 m resolution multibeam obtained with a remotely operated vehicle (ROV) shows progressive widening and smoothing of the channel-levees with increasing depth and reveals a complex morphology with channel bifurcations, erosional features and massive deposits. Dense ecosystems surveyed in the study area gather high density clusters of two large-sized species of symbiotic Vesicomyidae bivalves and microbial mats. These assemblages, which are rarely observed in sedimentary zones, resemble those based on chemosynthesis at cold-seep sites, such as the active pockmarks encountered along the Congo margin, and share with these sites the dominant vesicomyid species Christineconcha regab. Sedimentation rates estimated in the lobe complex range between 0.5 and 10 cm yr(-1), which is 2-3 orders of magnitude higher than values generally encountered at abyssal depths. The bathymetry, faunal assemblages and sedimentation rates make the Congo lobe complex a highly peculiar deep-sea habitat driven by high inputs of terrigenous material delivered by the Congo channel-levee system. (c) 2016 Elsevier Ltd. All rights reserved. ZAIANGO ANR Congolobe (ANR Blanc SIMI5-6) [11 BS56 030] IFREMER CEA through LSCE U.S. National Science Foundation [OCE-0831156] info:eu-repo/semantics/acceptedVersion

WOS:000408783500011; International audience; Deep-sea fans are well known depot centers for organic carbon that should promote sulfate reduction. At the same time, the high rates of deposition of unconsolidated metal oxides from terrigenous origin may also promote metal-reducing microbial activity. To investigate the eventual coupling between the iron and sulfur cycles in these environments, shallow sediment cores ( \textless 50 cm) across various channels and levees in the Congo River deep-sea fan (similar to 5000 m) were profiled using a combination of geochemical methods. Interestingly, metal reduction dominated suboxic carbon remineralization processes in most of these sediments, while dissolved sulfide was absent. In some 'hotspot' patches, however, sulfate reduction produced large sulfide concentrations which supported chemosynthetic-based benthic megafauna. These environments were characterized by sharp geochemical boundaries compared to the iron-rich background environment, suggesting that FeS precipitation efficiently titrated iron and sulfide from the pore waters. A companion study demonstrated that methanogenesis was active in the deep sediment layers of these patchy ecosystems, suggesting that sulfate reduction was promoted by alternative anaerobic processes. These highly reduced habitats could be fueled by discrete, excess inputs of highly labile natural organic matter from Congo River turbidites or by exhumation of buried sulfide during channel flank erosion and slumping. Sulfidic conditions may be maintained by the mineralization of decomposition products from local benthic macrofauna or bacterial symbionts or by the production of more crystalline Fe(III) oxide phases that are less thermodynamically favorable than sulfate reduction in these bio-turbated sediments. Overall, the iron and sulfur biogeochemical cycling in this environment is unique and much more similar to a coastal ecosystem than a deep-sea environment.

Although a number of techniques exist to measure the major and minor inorganic anions Cl-, NO2-, Br-, NO3-, or SO42- in marine waters and sediment porewaters, a single method that can quantify all five anions in a single step with a sufficiently low detection limit has yet to be developed. In this work, a novel ion-chromatographic separation procedure was developed that exploits the UV-absorbing properties of Br-, NO3-, and NO2-, as well as the suppression of eluent absorbance by high concentrations of Cl- and SO42- to quantify all these anions simultaneously in marine waters. If additional sensitivity for the minor anions Br-, NO3-, and NO2- is needed, NaCl can be used as eluent in a matrix elimination method that no longer allows for Cl- and SO42- determination. These methods are isocratic, require no chemical or electronic suppression, are not subject to interferences, and can resolve the anions in less than 18 min. The decrease in analysis time and the number of analyses required compared with conventional methods lowers the costs associated with laboratory analysis of marine samples. Last, the simplicity and good limits of detection make this separation method suitable for in situ analyses of marine waters and porewaters, and an in situ liquid chromatograph is currently under development for these applications.

Iron speciation in the Satilla River Estuary was investigated using competitive ligand equilibration–adsorptive cathodic stripping voltammetry (CLE-ACSV) with the ligand 1-nitroso-2-naphthol (1N2N). The blackwater Satilla River contains high concentrations of dissolved organic matter and iron, suggesting that it could provide a source of dissolved iron to the continental shelf. Total dissolved iron in the water column decreases along the estuary, likely due to flocculation and precipitation processes associated with the increase in salinity. Simultaneously, the percentage of dissolved organic-Fe(III) complexes measured by CLE-ACSV in overlying waters of sediment cores increases with salinity. The speciation of dissolved iron in the pore waters indicates that these complexes originate in the underlying sediments. Diffusive fluxes calculated from depth profiles in the sediments indicate that only 8% of the sedimentary flux of Fe(III) is delivered to the continental shelf during low riverine discharge. During normal flow conditions, however, the sediment flux of Fe(III) represents 63% of the total riverine flux of Fe(III). These findings suggest that the flux of dissolved iron to the continental shelf is controlled by the sedimentation of iron in the estuary and the remobilization of soluble organic-Fe(III) complexes produced either during iron reduction deep in the sediment or oxidation of Fe(II) close to the sediment–water interface. The Satilla River Estuary generates five to eight times higher concentrations of dissolved Fe(III) than the average major world rivers, suggesting that it is, along with other small blackwater rivers, currently underrepresented in world average river flux calculations. The importance of iron for primary productivity in the open ocean is well documented, and iron fertilization experiments have shown that iron additions can enhance primary productivity in high nutrient, low chlorophyll (HNLC) zones (Breitbarth et al. 2010). Iron is considered a limiting nutrient in marine waters, as the low solubility of Fe(III) limits the concentration of dissolved iron (, 0.4 mm)

The optical backscattering from particles in the ocean is an important quantity that has been measured by remote sensing techniques and in situ instruments. In this paper, we compare estimates of this quantity from airborne lidar with those from an in situ instrument on an underwater glider. Both of these technologies allow much denser sampling of backscatter profiles than traditional ship surveys. We found a moderate correlation (R = 0.28, p < 10−5), with differences that are partially explained by spatial and temporal sampling mismatches, variability in particle composition, and lidar retrieval errors. The data suggest that there are two different regimes with different scattering properties. For backscattering coefficients below about 0.001 m−1, the lidar values were generally greater than the glider values. For larger values, the lidar was generally lower than the glider. Overall, the results are promising and suggest that airborne lidar and gliders provide comparable and complementary information on optical particulate backscattering.

All prokaryotic (NiFe)-hydrogenases so far studied at the primary sequence level appear to have evolved from a common ancestral sequence. Highly conserved cysteinyl and histidinyl residues indicate regions likely to be essential for enzyme activity, ligand and co-factor binding. There is a very highly conserved sequence over 100 basepairs (bp) in length within the intergenic region upstream of the methyl-viologen hydrogenase encoding genes in several different strains of Methanobacterium thermoautotrophicum, indicating that a sequence of this length is needed to direct and regulate the expression of these genes.

A 3.3-kilobase-pair region of the Methanothermus fervidus genome encoding part of the small subunit and all of the large subunit of the methyl viologen-reducing hydrogenase and a polyferredoxin was cloned and sequenced. The sequence of this hyperthermophilic hydrogenase conforms to the consensus sequence established for procaryotic [NiFe] hydrogenases. Although the M. fervidus polyferredoxin is the same size as the Methanobacterium thermoautotrophicum ferredoxin, containing six tandemly arranged bacterial ferredoxinlike domains, these two proteins are predicted to be only 64% identical in their primary sequences.

The two most frequently used systems for in vitro translation are the rabbit reticulocyte system and the wheat germ extract. These systems are useful for mRNAs isolated from cells, tissues, and capped or uncapped mRNA produced in vitro by transcription of cDNA. In a combined system, mRNA can be transcribed and translated in a single reaction. In addition these systems can be used for translation reactions with biotinylated amino acids; this allows capture of the newly synthesized protein using streptavidin immobilized on agarose.

The ability to investigate cellular processes in vitro permits detailed analysis of the process and its molecular components. Eukaryotic translation and expression is one system that has been well studied. This overview describes the development of in vitro systems, including such approaches as continuous-flow systems, coupled transcription/translation, and the incorporation of non-natural amino acids. It also discusses molecular and genetic studies to probe translation, including post-translational fate of the synthesized proteins.

Summary An analysis of the structure and presumed regulatory signals in sequenced methanogen genes is presented. The evidence for polycistronic transcriptional units is extended by inclusion of a DNA sequence, cloned from Methanobrevibacter smithii , which precedes the M. smithii pro C gene ( Hamilton and Reeve , 1985a). Comparison of DNA sequences indicates that a consensus core ribosome binding sequence for methanogens would be 5 'AGGTGA and that many of the ribosome binding sequences are, in fact, longer than the core sequence containing 7 bases with the potential to hybridize to 16SrRNA. The majority, but not all, of the sequenced methanogen genes conform to the rule in which RNY codons (R=purine, Y=pyrimidine, N=purine or pyrimidine) occur most frequently in the translated reading frame ( Shepherd 1981,1983). Codon usages by different methanogens reflect the need to accommodate genomes with very different overall %mol G+C contents. Codons such as AUA, AGA, and AGG, rarely used by E. coli are frequently used by methanogens. The dinucleotide, CG, which occurs very infrequently in eucaryotic DNAs ( Subak-Sharpe et al., 1967; Lennon and Faser , 1983; Nussinov , 1984) is also rarely found in methanogen genes. Methylation and demethylation of the cytosine residue in CG sequences has been implicated in regulation of eucaryotic gene expression ( Weisbrod , 1982) and this role used as an argument for the infrequent occurrence of CG-containing sequences. The rarity of CG in the DNA sequences so far obtained from methanogens may indicate a similar regulatory usage of CG-containing sequences in archaebacterial methanogens.

The genes mvhDGA, which encode the subunit polypeptides of the methyl viologen-reducing hydrogenase in Methanobacterium thermoautotrophicum strain delta H, have been cloned and sequenced. These genes, together with a fourth open reading frame designated mvhB, are tightly linked and appear to form an operon that is transcribed starting 42 base pairs upstream of mvhD. The organization and sequences of the mvhG and mvhA genes indicate a common evolutionary ancestry with genes encoding the small and large subunits of hydrogenases in eubacterial species. The product of the mvhB gene is predicted to contain six tandomly repeated bacterial-ferredoxin-like domains and, therefore, is predicted to be a polyferredoxin that could contain as many as 48 iron atoms in 12 Fe4S4 clusters.

A 2.7 kilobase pair (Kb) fragment of DNA, which complements mutations in the hisI locus of Escherichia coli, has been cloned and sequenced from the genome of the methanogenic archaebacterium Methanococcus vannielii. The cloned DNA directs the synthesis of three polypeptides, with molecular weights of 71,000, 29,000 and 15,600 in minicells of E. coli. Subcloning and mutagenesis demonstrates that hisI complementation results from the activity of the 15,600 molecular weight polypeptide. The primary structure of this archaebacterial gene and its gene product have been compared with the functionally equivalent gene and protein from the eubacterium E. coli (hisI) (Chiariotti et al. 1986) and from the eucaryote Saccharomyces cerevisiae (his4A) (Donahue et al. 1982). The DNA sequences of the archaebacterial and eubacterial genes are 40% homologous, the archaebacterial and eucaryotic DNA sequences are 47% homologous and, as previously reported (Bruni et al. 1986) the eubacterial and eucaryotic DNA sequences are 45% homologous. In E. coli the hisI locus is part of a bifunctional gene (hisI/E) within the single his operon. In S. cerevisiae the his4A locus is part of a multifunctional gene (his4) which encodes a protein with at least four enzymatic activities. The his genes of S. cerevisiae do not form an operon and are not physically linked. The M. vannielii hisI gene does not appear to be part of a multifunctional DNA sequence and, although it does appear to be within an operon, the open reading frames (ORFs) 5' and 3' to the M. vannielii hisI gene are not related to any published his sequences.(ABSTRACT TRUNCATED AT 250 WORDS)

Methanogens are archaebacteria [1]. It was therefore somewhat surprising when it was shown that methanogen-derived genes could function in eubacterial species [2]. This has offered the opportunity of using gene cloning in Escherichia coli to investigate the basic structure of methanogen genes [3, 4] and to use E. coli to synthesize methanogen enzymes involved in methanogenesis [5] and nitrogen fixation [6].

The DNA sequences of a region that includes the hisA gene of two related methanogenic archaebacteria, Methanococcus voltae and Methanococcus vannielii, have been compared. Both organisms show a similar genome organization in this region, displaying three open reading frames (ORFs) separated by regions of very high A + T content. Two of the ORFs, including ORFHisA, show significant DNA sequence homology. As might be expected for organisms having a genome that is A + T-rich, there is a high preference for A and U as the third base in codons. Although the regions upstream of the structural genes contain prokaryotic-like promoter sequences, it is not known whether they are recognized as promoters in these archaebacterial cells. A ribosome binding site, G-G-T-G, is located 6 base pairs preceding the ATG translation initiation sequence of both hisA genes. The sequences upstream of the two hisA genes show only limited sequence homology. The M. voltae intergenic region contains four tandemly arranged repetitions of an 11-base-pair sequence, whereas the M. vannielii sequence contains both direct and inverted repetitive sequences. Based on the degree of hisA sequence homology, we conclude that M. voltae and M. vannielii are less closely related taxonomically than are members of the enteric group of eubacteria.

To identify an archaebacterial promoter sequence, nuclease protection studies with the purified RNA polymerase of Methanococcus vannielii were performed. The enzyme binds specifically both at protein-encoding (hisA and methyl CoM reductase, component C) and tRNA–rRNA genes. The binding region of the RNA polymerase extends from 30 base pairs (bp) upstream (−30) to 20 bp downstream (+20) from the in vivo transcription start site. This finding indicates that the archaebacterial enzyme recognizes promoters without transacting traascription factors. The DNA segment protected from nuclease digestion by bound RNA polymerase contains an octanucleotide sequence centered at −25, which is conserved between the protein-encoding and the stable RNA genes. According to the specific binding of the enzyme to only DNA-fragments harbouring this motif, we propose the sequence TTTATATA as the major recognition signal of the Methanococcus RNA polymerase. Comparison of this motif with published archaebacterial DNA sequences revealed the presence of homologous sequences at the same location upstream of 36 genes. We therefore consider the overall consensus [Formula: see text] as a general element of promoters in archaebacteria. In spite of the specific binding of the enzyme, most preparations of the Methanococcus vannielii RNA polymerase are unable to initiate transcription at the correct sites in vitro. Here we present first evidence for the possible existence of a transcription factor conferring the ability to the enzyme to initiate and terminate transcription specifically in vitro.Key words: promoter, footprint, TATA box, RNA polymerase, transcription.

Transcription initiation of the hisA gene in vivo in the archaebacterium Methanococcus vannielii, as determined by nuclease S1 and primer extension analyses, occurs 73 base pairs (bp) upstream of the translation initiation site. Binding of M. vannielii RNA polymerase protects 43 bp of DNA, from 35 bp upstream (-35) to 8 bp downstream (+8) of the hisA mRNA initiation site, from digestion by DNase I and exonuclease III. An A + T rich region, with a sequence which conforms to the consensus sequence for promoters of stable RNA-encoding genes in methanogens, is found at the same location (-25) upstream of the polypeptide-encoding hisA gene. It appears therefore that a TATA-like sequence is also an element of promoters which direct transcription of polypeptide-encoding genes in this archaebacterium.

A restriction fragment of Methanococcus thermolithotrophicus genomic DNA was cloned into pUC8 to produce plasmid pET9301, which complements mutations in the hisA gene of Escherichia coli. Sequencing the DNA (2,155 base pairs) cloned from this thermophilic methanogen demonstrated that the M. thermolithotrophicus hisA gene is located within a cluster of open reading frames (ORFs) and is 68 and 69% homologous at the nucleotide level to the hisA genes of the mesophilic methanococci M. voltae and M. vannielii, respectively. The ORF (ORF 206) immediately 5' to the hisA gene of M. thermolithotrophicus is partially deleted in the genomes of the two mesophilic species, whereas ORF 114, which is 5' to ORF 206, is conserved in all three species.

RAC's participation in the Economic Impact Project, consists of a series of studies designed to determine the impact of the defense program on the economy. The ultimate objective of the project is to improve the capability of the Department of Defense (DOD) in appraising the economic effects of changes in military programs and force structure. This report presents an account of the research effort during the first phase of study, which was completed in December 1964. Concerned primarily with the development of a methodology for measuring industry impact, rough estimates for FY63-69 were prepared, identifying a wide range of industries that participate in the defense effort. The research was organized in terms of major budget categories. In the area of procurement to which primary attention was devoted, a model was developed for specialized military equipment, including techniques for studying a series of prototypes in depth, utilizing component information and contract sources. In this connection information was sought that would allow the quantification of in-house effort by contractors in order to approximate value-added distributions. Generally, the tracing efforts were successful to second- and third-tier suppliers of prime contractors responsible for the delivery of major end-items such as aircraft, missiles, ships, and tanks. The value-added data also served as a basis for estimating employment from Census industry-wide employment/value-added ratios.