In-frame deletions were introduced into each of the eight genes of the hyc operon coding for products required for the formation of the formate hydrogenlyase (FHL) system. The deletions were transferred to the chromosome and the resulting mutants were analysed for development of formate dehydrogenase H and hydrogenase 1, 2 and 3 activity. It was found that hycA, the promoter-proximal gene, is a regulatory gene and that it codes for a product counteracting transcriptional activation by FhlA. Deletions within the hycB to hycH genes specifically affected formate dehydrogenase H activity or hydrogenase 3 activity, or both. None of the mutations affected hydrogenase 1 or 2 activity. A model is proposed for the functional interaction of the different hyc operon gene products in the formate hydrogenlyase complex, which is based on the results of the mutational analysis, on the determination of the subcellular localization of the FdhF, HycE, HycF and HycG polypeptides and on the similarity of hyc gene product sequences with those from other hydrogenase systems. HycH, the product of the most promoter-distal gene, does not seem to form part of the functional FHL complex but rather is required for the conversion of a precursor form of the large subunit of hydrogenase 3 into the mature form.

The products of the hyp operon genes are essential for the formation of catalytically active hydrogenases in Escherichia coli. At least one of these auxiliary proteins, HYPB, appears to be involved in nickel liganding to the hydrogenase apoprotein, since mutations in hypB can be phenotypically suppressed by high nickel concentrations in the medium (R. Waugh and D. H. Boxer, Biochimie 68:157-166, 1986). To approach the identification of the specific function of HYPB, we overexpressed the hypB gene and purified and characterized the gene product. HYPB is a homodimer of 31.6-kDa subunits, and it binds guanine nucleotides, with a Kd for GDP of 1.2 ,uM. The protein displays a low level of GTPase activity, with a kcat of 0.17 min-'. The apparent Km for GTP, as measured in the GTP hydrolysis reaction, was determined to be 4 ,uM. A chromatography system was established to measure nickel insertion into hydrogenase 3 from E. coli and to determine the effects of lesions in hypB. Nickel appears to be associated only with the processed large subunit of hydrogenase 3 in the wild type, and hypB mutants accumulate the precursor form of this subunit, which is devoid of nickel. The results are discussed in terms of a model in which HYPB is involved in nickel donation to the hydrogenase apoprotein and in which GTP hydrolysis is thought to reverse the interaction between either HYPB or another nickel-binding protein and the hydrogenase apoprotein after the nickel has been released.A considerable number of structural genes for hydrogenases have been cloned and characterized during the last few years (for a review, see reference 17). A surprising result of this genetic analysis was that these genes are clustered in operons together with a number of other genes whose products are required for the formation of a functional enzyme. There is, however, no detailed information on the biochemical role of most of these auxiliary proteins. Speculations on the putative functions range from metal cofactor liganding to folding and assembly or to membrane integration (13,15,17).Currently, one of the most complex hydrogenase systems is present in Eschenichia coli, which synthesizes at least three isoenzymes. Hydrogenases 1 and 2 are uptake hydrogenases, and their structural genes are located in operons (hya and hyb) comprising six and seven genes, respectively (14, 17). Hydrogenase 3 is a gas-evolving enzyme, and its structural genes are located in an operon (hyc) of eight genes, one of which has a regulatory function, while the other seven are required for the formation of a functional enzyme (3,18).Mutations in the genes of the hya, hyb, and hyc operons specifically affect the development of the activities of hydrogenases 1, 2, and 3, respectively. However, a number of mutants in which the genetic lesion influences the development of all three hydrogenases in E. coli have been isolated (for a review, see reference 13). Most of the mutations map in the 58-to 59-min region of the E. coli chromosome. A sequence analysis of that region revealed the ...

An 8kb segment of DNA from the 58/59 min region of the E. coli chromosome, which complements the defect of a mutant devoid of hydrogenase 3 activity, has been sequenced. Eight open reading frames were identified which are arranged in a transcriptional unit; all open reading frames were transcribed and translated in vivo in a T7 promoter/polymerase system. Analysis of the amino acid sequences derived from the nucleic acid sequences revealed that one of them, open reading frame 5 (ORF5), exhibits significant sequence similarity to conserved regions of the large subunit from Ni/Fe hydrogenases. Two of the open reading frames (orf2, orf6) code for proteins apparently carrying iron-sulphur clusters of the 4Fe/4S ferredoxin type. The product of one of the open reading frames, orf7, displays extensive sequence similarity with protein G from the chloroplast electron transport chain. ORF3 and ORF4, on the other hand, are extremely hydrophobic proteins with nine and six putative transmembrane helices, respectively. Over a limited hydrophilic sequence stretch, bordered by putative transmembrane areas, ORF3 and ORF4 exhibit homology with subunits 4 and 1 of mitochondrial and plastid NADH-ubiquinol oxidoreductases, respectively. The operon described, therefore, appears to comprise genes for redox carriers linking formate oxidation to proton reduction and for a hydrogenase of hitherto unique composition.

[1] This paper is intended to provide insight into the controlling mechanisms of karst genesis based on an advanced modeling approach covering the characteristic hydraulics in karst systems, the dissolution kinetics, and the associated temporal decrease in flow resistance. Karst water hydraulics is strongly governed by the interaction between a highly conductive low storage conduit network and a low-conductive high-storage rock matrix under variable boundary conditions. Only if this coupling of flow mechanisms is considered can an appropriate representation of other relevant processes be achieved, e.g., carbonate dissolution, transport of dissolved solids, and limited groundwater recharge. Here a parameter study performed with the numerical model Carbonate Aquifer Void Evolution (CAVE) is presented, which allows the simulation of the genesis of karst aquifers during geologic time periods. CAVE integrates several important features relevant for different scenarios of karst evolution: (1) the complex hydraulic interplay between flow in the karst conduits and in the small fissures of the rock matrix, (2) laminar as well as turbulent flow conditions, (3) time-dependent and nonuniform recharge to both flow systems, (4) the widening of the conduits accounting for appropriate physicochemical relationships governing calcite dissolution kinetics. This is achieved by predefining an initial network of karst conduits (''protoconduits'') which are allowed to grow according to the amount of aggressive water available due to hydraulic boundary conditions. The increase in conduit transmissivity is associated with an increase in conduit diameters while the conductivity of the fissured system is assumed to be constant in time. The importance of various parameters controlling karst genesis is demonstrated in a parameter study covering the recharge distribution, the upgradient boundary conditions for the conduit system, and the hydraulic coupling between the conduit network and the rock matrix. In particular, it is shown that conduit diameters increase in downgradient or upgradient direction depending on the spatial distribution (local versus uniform) of the recharge component which directly enters the conduit system.INDEX TERMS: 1829 Hydrology: Groundwater hydrology; 1894 Hydrology: Instruments and techniques; KEYWORDS: karst hydrology, aquifer evolution, groundwater flow, pipe flow, calcite dissolution kinetics, numerical modeling Citation: Liedl, R., M. Sauter, D. Hückinghaus, T. Clemens, and G. Teutsch, Simulation of the development of karst aquifers using a coupled continuum pipe flow model, Water Resour.

Due to its extreme salinity and high Mg concentration the Dead Sea is characterized by a very low density of cells most of which are Archaea. We discovered several underwater fresh to brackish water springs in the Dead Sea harboring dense microbial communities. We provide the first characterization of these communities, discuss their possible origin, hydrochemical environment, energetic resources and the putative biogeochemical pathways they are mediating. Pyrosequencing of the 16S rRNA gene and community fingerprinting methods showed that the spring community originates from the Dead Sea sediments and not from the aquifer. Furthermore, it suggested that there is a dense Archaeal community in the shoreline pore water of the lake. Sequences of bacterial sulfate reducers, nitrifiers iron oxidizers and iron reducers were identified as well. Analysis of white and green biofilms suggested that sulfide oxidation through chemolitotrophy and phototrophy is highly significant. Hyperspectral analysis showed a tight association between abundant green sulfur bacteria and cyanobacteria in the green biofilms. Together, our findings show that the Dead Sea floor harbors diverse microbial communities, part of which is not known from other hypersaline environments. Analysis of the water’s chemistry shows evidence of microbial activity along the path and suggests that the springs supply nitrogen, phosphorus and organic matter to the microbial communities in the Dead Sea. The underwater springs are a newly recognized water source for the Dead Sea. Their input of microorganisms and nutrients needs to be considered in the assessment of possible impact of dilution events of the lake surface waters, such as those that will occur in the future due to the intended establishment of the Red Sea−Dead Sea water conduit.