Tjakko Abee

ALCOHOL CONVERSIONS BY DESULFOBULBUS-PROPIONICUS LINDHORST IN THE PRESENCE AND ABSENCE OF SULFATE AND HYDROGEN

Ethanol was rapidly degraded to mainly acetate in anaerobic freshwater sediment slurries. Propionate was produced in small amounts. Desulfovibrio species were the dominant bacteria among the ethanol-degrading organisms. The propionate-producing Desulfobulbus propionicus came to the fore under iron-limited conditions in an ethanol-limited chemostat with excess sulfate inoculated with anaerobic intertidal freshwater sediment. In the absence of sulfate, ethanol was fermented by D. propionicus Lindhorst to propionate and acetate in a molar ratio of 2.0.l-Propanol was intermediately produced during the fermentation of ethanol. In the presence of H2 and CO2, ethanol was quantitatively converted to propionate. H2-plus sulfate-grown cells of D. propionicus Lindhorst were able to oxidize l-propanol and l-butanol to propionate and butyrate respectively with the concomitant reduction of acetate plus CO2 to propionate. Growth was also observed on acetate alone in the presence of H2 and CO2D. propionicus was able to grow mixotrophically on H2 plus an organic compound. Finally, a brief discussion has been given of the ecological niche of D. propionicus in anaerobic freshwater sediments.

THE BIOENERGETICS OF AMMONIA AND HYDROXYLAMINE OXIDATION IN NITROSOMONAS-EUROPAEA AT ACID AND ALKALINE PH

Autotrophic ammonia oxidizers depend on alkaline or neutral conditions for optimal activity. Below pH 7 growth and metabolic activity decrease dramatically. Actively oxidizing cells of Nitrosomonas europaea do not maintain a constant internal pH when the external pH is varied from 5 to 8. Studies of the kinetics and pH-dependency of ammonia and hydroxylamine oxidation by N. europaea revealed that hydroxylamine oxidation is moderately pH-sensitive, while ammonia oxidation decreases strongly with decreasing pH. Oxidation of these oxogenous substrates results in the generation of higher proton motive force which is mainly composed of a ΔΨ. Hydroxylamine, but not ammonia, is oxidized at pH 5, which leads to the generation of a high proton motive force which drives energy-dependent processes such as ATP-synthesis and secondary transport of amino acids.Endogenoussubstrates can be oxidized between pH 5 to 8 and this results in the generation of a considerable proton motive force which is mainly composed of a ΔΨ. Inhibition of ammonia-mono-oxygenase or cytochrome aa3 does not influence the magnitude of this gradient or the oxygen consumption rate, indicating that endogenous respiration and ammonia oxidation are two distinct systems for energytransduction.The results indicate that the first step in ammonia oxidation is acid sensitive while the subsequent steps can take place and generate a proton motive force at acid pH.

SECONDARY TRANSPORT OF AMINO-ACIDS IN NITROSOMONAS-EUROPAEA

Nitrosomonas europaea is capable of incorporating exogenously supplied amino acids. Studies in whole cells revealed that at least eight amino acids are actively accumulated, probably by the action of three different transport systems, each with high affinity (μ molar range) for several amino acids. Evidence for the action of secondary mechanisms of transport was obtained from efflux, counterflow and exchange experiments. More detailed information was obtained from studies in liposomes in which solubilized integral membrane proteins of N. europaea were incorporated. Uptake of l-alanine in these liposomes could be driven by artificially imposed pH gradients and electrical potentials, but not by chemical sodium-ion gradients. These observations indicate that l-alanine is transported by a H+/alanine symport system. The ecological significance of secondary amino acid transport systems in autotrophic ammonium-oxidizing bacteria is discussed.

Lactococcal Bacteriocins: Genetics and Mode of Action

Lactic acid bacteria produce a variety of antimicrobial substances which are important in food fermentation and preservation. In several instances, the inhibitory activity results from metabolic end products such as hydrogen peroxide, diacetyl, and organic acids (Lindgren & Dobrogosz, 1990). In addition, the bactericidal activity of many strains appeared to result from bacteriocin production (Klaenhammer, 1988). Although bacteriocins of lactic acid bacteria have been the subject of many studies, only little is known about their chemical structure, their mode of action, and their genetic determinants. In recent years, the increasing interest in bacteriocins produced by these organisms has resulted in the cloning and genetic characterization of several bacteriocin determinants (Joerger & Klaenhammer, 1990; Marugg, 1991; Muriana & Klaenhammer, 1991). From lactococci, the structural gene for the lantibiotic nisin has been cloned and sequenced by several groups (Buchman et al., 1988; Kaletta & Entian, 1989; Dodd et al., 1990). The genetic determinant for bacteriocin production byLactococcus lactis subsp. lactis WM4 was shown to be associated with the 131-kb plasmid pNP2 (Scherwitz et al., 1983). Cloning experiments inL. lactis identified an 18.4-kb DNA region containing the bacteriocin determinant (Scherwitz-Harmon & McKay, 1987).

A Kdp-like, high-affinity, K+-translocating ATPase is expressed during growth of Rhodobacter sphaeroides in low potassium media

Cells of the purple non-sulphur bacterium Rhodobacter sphaeroides express a high-affinity K+ uptake system when grown in media with low K+ concentrations. Antibodies againts the catalytic KdpB protein or the whole KdpABC complex of Escherichia coli crossreact with a 70.0 kDa R. sphaeroides protein that was expressed only in cells grown in media with low K+ concentrations. In membranes derived from R. sphaeroides cells grown with low K+ concentrations (induced cells), a high ATPase activity could be detected when assayed in Tris-HCl pH 8.0 containing 1 mM MgSO4. This ATPase activity increased upon addition of 1 mM KCl from 166 to 289 μmol ATP hydrolysed x min-1 x g protein-1 (1.7-fold stimulation). The K+-stimulated ATPase activity was inhibited approximately 93% by 0.5 mM vanadate but hardly by N,N′-dicyclohexylcarbo-diimide (DCCD). These results indicate that the inducible K+-ATPase in R. sphaeroides resembles the Kdp K+-translocating ATPase of Escherichia coli. This Kdp-like transport system is also expressed in R. capsulatus and Rhodospirillum rubrum during growth in media with low K+ concentrations suggesting a wide distribution of this transport system among phototrophic bacteria.

THE RELATION BETWEEN ELECTRON-TRANSFER, PROTONMOTIVE FORCE AND LACTOSE TRANSPORT IN MEMBRANE-VESICLES FROM AEROBICALLY GROWN RHODOBACTER-SPHAEROIDES 4P1

Abstract Membrane vesicles were isolated from Rhodobacter sphaeroides strain 4P1, grown aerobically in the dark. This strain is equipped with the lactose transport protein from Escherichia coli . In the membrane vesicles a branched electron-transfer chain is located; one branch contains cytochrome c oxidase and the other the so-called ‘alternative oxidase’. Cytochrome c oxidase is the major energy-coupling device in these membrane vesicles. Several electron donors (succinate, NADH, reduced cytochrome c , etc.) are oxidized via the electron-transfer chain with different contributions of the two branches of the terminal part of the electron-transfer chain. The capacity of these membrane vesicles to generate a protonmotive force correlates well with the activity of the cytochrome c oxidase-containing branch of the electron-transfer chain. This correlation was revealed by the use of electron-transfer chain inhibitors like antimycin A and KCN. The rate and maximal level of lactose accumulation correlated chemiosmotically with the magnitude of the protonmotive force, assuming 1 H + /lactose symport, without the need to invoke additional regulatory mechanisms. Electron donors that can directly reduce cytochrome c oxidase were most efficient in protonmotive force generation and in the energization of lactose transport.