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The anaerobic acetogenic bacterium Acetobacterium woodii carries out a unique type of [Na.sup.+]-motive, anaerobic respiration with caffeate as electron acceptor, termed 'caffeate respiration.' Central, and so far the only identified membrane-bound reaction in this respiration pathway, is a ferredoxin:[NAD.sup.+] oxidoreductase (Fno) activity. Here we show that inverted membrane vesicles of A. woodii couple electron transfer from reduced ferredoxin to [NAD.sup.+] with the transport of [Na.sup.+] from the outside into the lumen of the vesicles. [Na.sup.+] transport was electrogenic, and accumulation was inhibited by sodium ionophores but not protonophores, demonstrating a direct coupling of Fno activity to [Na.sup.+] transport. Results from inhibitor studies are consistent with the hypothesis that Fno activity coupled to [Na.sup.+] translocation is catalyzed by the Rnf complex, a membranebound, iron--sulfur and flavin-containing electron transport complex encoded by many bacterial and some archaeal genomes. Fno is a unique type of primary [Na.sup.+] pump and represents an early evolutionary mechanism of energy conservation that expands the redox range known to support life. In addition, it explains the lifestyle of many anaerobic bacteria and gives a mechanistic explanation for the enigma of the energetic driving force for the endergonic reduction of ferredoxin with NADH plus [H.sup.+] as reductant in a number of aerobic bacteria. Acetobacterium | anaerobic respiration | electron transport | [Na.sup.+] pump | Rnf doi/ 10.1073/pnas.1010318107

ATP synthase catalyzes ATP synthesis at the expense of an electrochemical ion gradient across a membrane that can be generated by different exergonic reactions. Sulfur reduction is the main energy-yielding reaction in the hyperthermophilic strictly anaerobic Crenarchaeon Ignicoccus hospitalis. This organism is unusual in having an inner and an outer membrane that are separated by a huge intermembrane compartment. Here we show, on the basis of immuno-EM analyses of ultrathin sections and immunofluorescence experiments with whole I. hospitalis cells, that the ATP synthase and [H.sub.2]:sulfur oxidoreductase complexes of this organism are located in the outer membrane. These two enzyme complexes are mandatory for the generation of an electrochemical gradient and for ATP synthesis. Thus, among all prokaryotes possessing two membranes in their cell envelope (including Planctomycetes, Gram-negative bacteria), I. hospitalis is a unique organism, with an energized outer membrane and ATP synthesis within the periplasmic space. In addition, DAPI staining and EM analyses showed that DNA and ribosomes are localized in the cytoplasm, leading to the conclusion that in I. hospitalis energy conservation is separated from information processing and protein biosynthesis. This raises questions regarding the function of the two membranes, the interaction between these compartments, and the general definition of a cytoplasmic membrane. Archaea | ATP synthase | ATPase | immunolabeling | sulfur reductase www.pnas.org/cgi/doi/10.1073/pnas.0911711107

The anaerobic acetogenic bacterium Acetobacterium woodii couples caffeate reduction with electrons derived from hydrogen to the synthesis of ATP by a chemiosmotic mechanism with sodium ions as coupling ions, a process referred to as caffeate respiration. We addressed the nature of the hitherto unknown enzymatic activities involved in this process and their cellular localization. Cell extract of A. woodii catalyzes [H.sub.2]-dependent caffeate reduction. This reaction is strictly ATP dependent but can be activated also by acetyl coenzyme A (CoA), indicating that there is formation of caffeyl-CoA prior to reduction. Two-dimensional gel electrophoresis revealed proteins present only in caffeate-grown cells. Two proteins were identified by electrospray ionization-mass spectrometry/mass spectrometry, and the encoding genes were cloned. These proteins are very similar to subunits [alpha] (EtfA) and [beta] (EtfB) of electron transfer flavoproteins present in various anaerobic bacteria. Western blot analysis demonstrated that they are induced by caffeate and localized in the cytoplasm. Etf proteins are known electron carriers that shuttle electrons from NADH to different acceptors. Indeed, NADH was used as an electron donor for cytosolic caffeate reduction. Since the hydrogenase was soluble and used ferredoxin as an electron acceptor, the missing link was a ferredoxin:NA[D.sup.+] oxidoreductase. This activity could be determined and, interestingly, was membrane bound. A search for genes that could encode this activity revealed DNA fragments encoding subunits C and D of a membrane-bound Rnf-type NADH dehydrogenase that is a potential [Na.sup.+] pump. These data suggest the following electron transport chain: [H.sub.2] [right arrow] ferredoxin [right arrow] NA[D.sup.+] [right arrow] Etf [right arrow] caffeyl-CoA reductase. They also imply that the sodium motive step in the chain is the ferredoxin-dependent NA[D.sup.+] reduction catalyzed by Rnf.

The moderately halophilic bacterium Halobacillus halophilus copes with the salinity in its environment by the production of compatible solutes. At intermediate salinities of around 1 M NaCl, cells produce glutamate and glutamine in a chloride-dependent manner (S. H. Saum, J. F. Sydow, P. Palm, F. Pfeiffer, D. Oesterhelt, and V. Muller, J. Bacteriol. 188:6808-6815, 2006). Here, we report that H. halophilus switches its osmolyte strategy and produces proline as the dominant solute at higher salinities (2 to 3 M NaCl). The proline biosynthesis genes proH, proJ, and proA were identified. They form a transcriptional unit and encode the pyrroline-5carboxylate reductase, the glutamate-5-kinase, and the glutamate-5-semialdehyde dehydrogenase, respectively, catalyzing proline biosynthesis from glutamate. Expression of the genes was clearly salinity dependent and reached a maximum at 2.5 M NaCl, indicating that the pro operon is involved in salinity-induced proline biosynthesis. To address the role of anions in the process of pro gene activation and proline biosynthesis, we used a cell suspension system. Chloride salts lead to the highest accumulation of proline. Interestingly, chloride could be substituted to a large extent by glutamate salts. This unexpected finding was further analyzed on the transcriptional level. The cellular mRNA levels of all three pro genes were increased up to 90-fold in the presence of glutamate. A titration revealed that a minimal concentration of 0.2 M glutamate already stimulated pro gene expression. These data demonstrate that the solute glutamate is involved in the switch of osmolyte strategy from glutamate to proline as the dominant compatible solute during the transition from moderate to high salinity.

The genomic organization of luxS of Halobacillus halophilus, its regulation and the cellular function of LuxS in the context of the chloride regulon are discussed. LuxS of Halobacillus halophilus is produced only in the presence of chloride and it might be a central player in the chloride regulon of Halobacillus halophilus.

The moderately halophilic, chloride-dependent bacterium Halobacillus halophilus produces glutamate and glutamine as main compatible solutes at external salinities of 1.0 to 1.5 M NaCl. The routes for the biosynthesis of these solutes and their regulation were examined. The genome contains two genes potentially encoding glutamate dehydrogenases and two genes for the small subunit of a glutamate synthase, but only one gene for the large subunit. However, the expression of these genes was not salt dependent, nor were the corresponding enzymatic activities detectable in cell extracts of cells grown at different salinities. In contrast, glutamine synthetase activity was readily detectable in H. halophilus. Induction of glutamine synthetase activity was strictly salt dependent and reached a maximum at 3.0 M NaCl; chloride stimulated the production of active enzyme by about 300%. Two potential genes encoding a glutamine synthetase, glnA1 and glnA2, were identified. The expression of glnA2 but not of glnA1 was increased up to fourfold in cells adapted to high salt, indicating that GlnA2 is the glutamine synthetase involved in the synthesis of the solutes glutamate and glutamine. Furthermore, expression of glnA2 was stimulated twofold by the presence of chloride ions. Chloride exerted an even more pronounced effect on the enzymatic activity of preformed enzyme: in the absence of chloride in the assay buffer, glutamine synthetase activity was decreased by as much as 90%. These data demonstrate for the first time a regulatory role of a component of common salt, chloride, in the biosynthesis of compatible solutes.

Research has been conducted on gram-positive moderately halophilic bacterium Halobacillus halophilus. The biological functions of chloride in this bacterium have been investigated, and the results suggest that chloride can function as a signal molecule in salt adaptation in moderately halophilic bacterium H. halophilus which may sense salinity via chloride concentration of its environment.

Results reveal that the hydrogen-dependent caffeate reduction by Acetobacterium woodii, growing on fructose plus caffeate, is tightly coupled to sodium ion as indicated by the stimulation by sodium ionophore. Data suggest that the caffeate reduction is coupled to the membrane potential buildup caused by sodium extrusion.

Research has been conducted on the A (sub)1 ATPase complex from the archaeon Methanosarcine mazei Go1. The structure of this complex has been analyzed via synchrotron X-ray small-angle scattering in solution.

Research has been conducted on the Halobacillus halophilus motility. The involvement of chloride in flagella synthesis in H. halophilus is discussed.

Studies have revealed the presence of new coenzyme factors in the methanogenesis and new energy-conserving pathways during close scrutiny of the biosynthesis and related processes involving archaea. Novel mechanisms of proton pumping and novel light-induced mechanisms were also discovered during the study of the unusual composition of archaeal respiratory complexes. Proton pumping mechanisms of heterodisulfide reductase other archaeal membrane protein complexes continue to be understood at the molecular level.

A study was conducted to analyze the Cl- dependence of growth of Halobacillus halophilus. The optimal growth rates were determined at Cl- concentrations between 0.5 and 2.0 M. The final yield was then obtained strictly dependent on the concentration. Experimental results indicated a couple of membrane proteins supporting molecular masses of 31 and 16 kDa as possible candidates for a chloride uptake system.

ATP in membrane vesicles of Methanosarcina mazei Go1 is synthesized by the generation of a sodium ion potential (delta-mu(H+)). These ionic gradients activate the synthesis of ATP through Na+ and H+ translocating ATP synthases. Go1 mediates a methyl transfer reaction using the ionic gradients.

The location of the methyl-tetrahydromethanopterin:coenzymeM methyltransferase (methyltransferase) in Methanosarcina strain Go1 was investigated. It was shown that it is membrane-bound and that its activity is sodium dependent. Its methylation was also coupled to Na+ translocation in a corrinoid-dependent reaction. Moreover, the activity of the methyltransferase was stimulated by ATP.

The cloning and sequencing of an Escherichia coli gene encoding an alpha-amylase were reported. A homologous gene was found to exist in Salmonella typhimurium. Minicell experiments identified the product of the E. coli gene, named amyA, as a 56-kDa protein, and the sequence of AmyA showed that it is a cytoplasmic enzyme. Moreover, the results established that AmyA is an alpha-amylase and that the linear alpha-glucan amylose is the most effective substrate.

The flagellar gene systems in Escherichia coli and Salmonella typhimurium are arranged in three major clusters, the flagellar regions I, II and III. Analysis of region III showed that there is a new region between fliD and fliE, most of which can be deleted without visible effects on flagellar activity. However, further analyses showed that this region also contains two genes which appear to be involved in flagellation. Therefore, region III has been subdivided into regions IIIa containing fliABCD and IIIb, containing fliEFGHIJKLMNOPQR. The two newly described genes have been designated fliS and fliT.

Escherichia coli and Salmonella typhimurium flagellar systems are encoded by at least 40 genes presently grouped into regions I, II, IIIa, and IIIb. One of the genes, fliE, which lies at the start of region IIIb, is a monocistronic operon oriented divergently from the large fliF operon. DNA sequencing showed that its product, FliE, is part of the flagellar hook-basal body. Nine subunits of the FliE proteins was estimated to be part of one basal body. Its functional role remains to be elucidated.

The energetics of CO2 reduction and reduction of other electron acceptors in acetogens are highlighted. Although the elucidation of the mechanisms of energy conservation in acetogens is still n its infancy, it turned out that Acetobacterium woodii is one of the rare cases in which the entire energetics is based on an Na(super +) current across the cytoplasmic membrane.

Focuses on the structural homologies and diversities between the stalk regions of A[sub 1]-, F[sub 1]- and V[sub 1]-adenosine triphosphatases (ATPase). Relationship between the regions of ATP hydrolysis and ion transduction; Location of the coupling subunits within the stalk; Regulatory mechanism for the control of V-ATPase activity.

A sodium ion gradient (inside low) across the cytoplasmic membrane of Methanosarcina barkeri was required for methanogenesis from methanol. This could be concluded from the following results. (a) Inhibition of the Na+/H+ antiporter by K+ or amilotide led to an inhibition of methanogenesis from methanol. (b) Upon addition of the sodium ionophore monensin the Na+ gradient was abolished and at the same time methanogenesis from methanol was inhibited. (c) Methanogenesis was impaired when the Na+ gradient had the opposite orientation (inside high). All these inhibitory effects were not observed when H2 was present in addition to methanol indicating that the oxidation of methanol to CO2 was driven by a sodium-motive force. In accordance with this, a methanoldependent influx of Na+ and a corresponding decrease of the membrane potential could be observed, when the Na+/H+ antiporter was inhibited by amiloride. This influx was indicative of the presence of a Na+ transport system which was functional when the oxidation of methanol had to be driven, but was not functional when H2 was present for reduction of methanol to methane. [ABSTRACT FROM AUTHOR]

Investigates whether electrochemical sodium and electrochemical proton ion gradient are used directly for the synthesis of adenosine triphosphate (ATP) in Methanosarcina mazei. Coupling of methyl transfer reaction to ATP synthase activity; ATP synthesis driven by electrochemical sodium ion gradient-translocating methyl transferase reaction and artificial gradients.

We discuss new algorithms for multiplying points on elliptic curves defined over small finite fields of characteristic two. This algorithm is an extension of previous results by Koblitz, Meier, and Staffelbach. Experimental results show that the new methods can give a running time improvement of up to 50% compared with the ordinary binary algorithm for multiplication. Finally, we present a table of elliptic curves, which are well suited for elliptic curve public key cryptosystems, and for which the new algorithm can be used. [ABSTRACT FROM AUTHOR]