Several clostridia and fusobacteria ferment alpha-amino acids via (R)-2-hydroxyacyl-CoA, which is dehydrated to enoyl-CoA by syn-elimination. This reaction is of great mechanistic interest, since the beta-hydrogen, to be eliminated as proton, is not activated (pK 40-50). A mechanism has been proposed, in which one high-energy electron acts as cofactor and transiently reduces the electrophilic thiol ester carbonyl to a nucleophilic ketyl radical anion. The 2-hydroxyacyl-CoA dehydratases are two-component systems composed of an extremely oxygen-sensitive component A, an activator, and component D, the actual dehydratase. Component A, a homodimer with one [4Fe-4S]cluster, transfers an electron to component D, a heterodimer with 1-2 [4Fe-4S]clusters and FMN, concomitant with hydrolysis of two ATP. From component D the electron is further transferred to the substrate, where it facilitates elimination of the hydroxyl group. In the resulting enoxyradical the beta-hydrogen is activated (pK14). After elimination the electron is handed-over to the next incoming substrate without further hydrolysis of ATP. The helix-cluster-helix architecture of component A forms an angle of 105 degrees, which probably opens to 180 degrees upon binding of ATP resembling an archer shooting arrows. Therefore we designated component A as 'Archerase'. Here, we describe 2-hydroxyglutaryl-CoA dehydratase from Acidaminococcus fermentans, Clostridium symbiosum and Fusobacterium nucleatum, 2-phenyllactate dehydratase from Clostridium sporogenes, 2-hydroxyisocaproyl-CoA dehydratase from Clostridium difficile, and lactyl-CoA dehydratase from Clostridium propionicum. A relative of the 2-hydroxyacyl-CoA dehydratases is benzoyl-CoA reductase from Thauera aromatica. Analogous but unrelated archerases are the iron proteins of nitrogenase and bacterial protochlorophyllide reductase. In anaerobic organisms, which do not oxidize 2-oxo acids, a second energy-driven electron transfer from NADH to ferredoxin, the electron donor of component A, has been established. The transfer is catalysed by a membrane-bound NADH-ferredoxin oxidoreductase driven by an electrochemical Na(+)-gradient. This enzyme is related to the Rnf proteins involved in Rhodobacter capsulatus nitrogen fixation.

Mechanosensitive channels are thought to function as safety valves for the release of cytoplasmic solutes from cells that have to manage a rapid transition from high-to low-osmolarity environments. Subsequent to an osmotic down-shock of cells grown at high osmolarity, Bacillus subtilis rapidly releases the previously accumulated compatible solute glycine betaine in accordance with the degree of the osmotic downshift. Database searches suggest that B. subtilis possesses one copy of a gene for a mechanosensitive channel of large conductance (mscL) and three copies of genes encoding proteins that putatively form mechanosensitive channels of small conductance (yhdY, yfkC, and ykuT). Detailed mutational analysis of all potential channelforming genes revealed that a quadruple mutant (mscL yhdY yfkC ykuT) has no growth disadvantage in high-osmolarity media in comparison to the wild type. Osmotic down-shock experiments demonstrated that the MscL channel is the principal solute release system of B. subtilis, and strains with a gene disruption in mscL exhibited a severe survival defect upon an osmotic down-shock. We also detected a minor contribution of the SigB-controlled putative MscS-type channel-forming protein YkuT to cellular survival in an mscL mutant. Taken together, our data revealed that mechanosensitive channels of both the MscL and MscS types play pivotal roles in managing the transition of B. subtilis from hyper-to hypo-osmotic environments.The gram-positive soil bacterium Bacillus subtilis has to frequently manage the transition from high-to low-osmolarity surroundings due to drying and flooding of the upper layers of the soil (7, 35). To fend off the negative effects of high osmolarity on cellular water content and cell physiology, B. subtilis accumulates compatible solutes through either synthesis or uptake from the environment (7,20). Compatible solutes are highly soluble organic osmolytes (e.g., proline and glycine betaine) that can be amassed by the cell to exceedingly high levels without interfering with cell physiology (12). In this way, the water content of the cell is balanced with the prevalent osmolarity of the environment and cell turgor is stabilized. Consequently, cell growth can occur under osmotically unfavorable conditions.The massive accumulation of compatible solutes permits cell survival under hypertonic circumstances. However, the very same compounds become a threat to the integrity of the cell when B. subtilis is suddenly exposed to low-osmolarity surroundings via rainfall or washout into fresh water sources (8,35). The compatible solutes accumulated by the cell increase the osmotic potential of the cytoplasm and thereby instigate an osmotically controlled water influx (36). This water influx drives up turgor to nonphysiological high values and in extreme cases can lead to cell lysis (4, 6). Various researchers have observed that a number of cytoplasmic solutes, including proline, glycine betaine, potassium, glutamate, trehalose, and ATP, are rapidly released from osmotically downshifte...

Anaerobic bacteria ferment glutamate via two different pathways to ammonia, carbon dioxide, acetate, butyrate and molecular hydrogen. The coenzyme B₁₂-dependent pathway in Clostridium tetanomorphum via 3-methylaspartate involves pyruvate:ferredoxin oxidoreductase and a novel enzyme, a membrane-bound NADH:ferredoxin oxidoreductase. The flavin- and iron-sulfur-containing enzyme probably uses the energy difference between reduced ferredoxin and NADH to generate an electrochemical Na⁺ gradient, which drives transport processes. The other pathway via 2-hydroxyglutarate in Acidaminococcus fermentans and Fusobacterium nucleatum involves glutaconyl-CoA decarboxylase, which uses the free energy of decarboxylation to generate also an electrochemical Na⁺ gradient. In the latter two organisms, similar membrane-bound NADH:ferredoxin oxidoreductases have been characterized. We propose that in the hydroxyglutarate pathway these oxidoreductases work in the reverse direction, whereby the reduction of ferredoxin by NADH is driven by the Na⁺ gradient. The reduced ferredoxin is required for hydrogen production and the activation of radical enzymes. Further examples show that reduced ferredoxin is an agent, whose reducing energy is about 1 ATP ‘richer’ than that of NADH.

SummaryErwinia chrysanthemi insertion mutants were isolated that grew poorly specifically in the presence of glycine betaine (GB) or its analogues in high-salt media. Transposon insertions were found to affect the bspA gene, which forms an operon including the psd locus coding for phosphatidylserine decarboxylase. Initial GB uptake is not affected by the bspA mutation. However, in high-salt medium, its initial accumulation is followed by a reduced glucose uptake and a release of GB but not a loss of viability. BspA is homologous to the widespread MscS channel, YggB, but does not seem to constitute a mechanosensitive channel. We suggest that BspA is a protein sensing both intracellular GB and the extracellular salt content of the medium, the hypothesis being built on the observation that BspA is necessary to maintain the GB pool during osmoadaptation in high-salt media containing this osmoprotectant.

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