Contribution of Citrate Metabolism to the Growth of Lactococcus lactis CRL264 at Low pH

Lactococcus lactis subsp. lactis biovar diacetylactis CRL264 is a natural strain isolated from cheese (F. Sesma, D. Gardiol, A. P. de Ruiz Holgado, and D. de Mendoza, Appl. Environ. Microbiol. 56:2099-2103, 1990). The effect of citrate on the growth parameters at a very acidic pH value was studied with this strain and with derivatives whose citrate uptake capacity was genetically manipulated. The culture pH was maintained at 4.5 to prevent alkalinization of the medium, a well-known effect of citrate metabolism. In the presence of citrate, the maximum specific growth rate and the specific glucose consumption rate were stimulated. Moreover, a more efficient energy metabolism was revealed by analysis of the biomass yields relative to glucose consumption or ATP production. Thus, it was shown that the beneficial effect of citrate on growth under acid stress conditions is not primarily due to the concomitant alkalinization of the medium but stems from less expenditure of ATP, derived from glucose catabolism, to achieve pH homeostasis. After citrate depletion, a deleterious effect on the final biomass was apparent due to organic acid accumulation, particularly acetic acid. On the other hand, citrate metabolism endowed cells with extra ability to counteract lactic and acetic acid toxicity. In vivo ¹³C nuclear magnetic resonance provided strong evidence for the operation of a citrate/lactate exchanger. Interestingly, the greater capacity for citrate transport correlated positively with the final biomass and growth rates of the citrate-utilizing strains. We propose that increasing the citrate transport capacity of CRL264 could be a useful strategy to improve further the ability of this strain to cope with strongly acidic conditions.     

Direct electrochemistry of the Desulfovibrio gigas aldehyde oxidoreductase.

This work reports on the direct electrochemistry of the Desulfovibrio gigas aldehyde oxidoreductase (DgAOR), a molybdenum enzyme of the xanthine oxidase family that contains three redox-active cofactors: two [2Fe-2S] centers and a molybdopterin cytosine dinucleotide cofactor. The voltammetric behavior of the enzyme was analyzed at gold and carbon (pyrolytic graphite and glassy carbon) electrodes. Two different strategies were used: one with the molecules confined to the electrode surface and a second with DgAOR in solution. In all of the cases studied, electron transfer took place, although different redox reactions were responsible for the voltammetric signal. From a thorough analysis of the voltammetric responses and the structural properties of the molecular surface of DgAOR, the redox reaction at the carbon electrodes could be assigned to the reduction of the more exposed iron cluster, [2Fe-2S] II, whereas reduction of the molybdopterin cofactor occurs at the gold electrode. Voltammetric results in the presence of aldehydes are also reported and discussed.     

Gas exchange in the salt marsh species <Emphasis Type="Italic">Atriplex portulacoides</Emphasis> L. and <Emphasis Type="Italic">Limoniastrum monopetalum</Emphasis> L. in Southern Portugal

Salt marshes are ecosystems subjected to a variety of environmental stresses like high salinity, water deficit, intense radiation or high temperatures. Field measurements were conduced in two halophyte species, Atriplex portulacoides L. and Limoniastrum monopetalum L., in the Reserva Natural do Sapal de Castro Marim, to compare their physiological response, i.e., water potential (ψ), net photosynthetic rate (A), stomatal conductance (gs) under natural conditions. Both species demonstrated marked variations in ψ throughout the year, with very low values in the summer, the period of higher salinity, drought and temperature. Deficit water potential (Δψ = ψ_midday − ψ_predawn) was lower in the summer than in other seasons in A. portulacoides but not in L. monopetalum. The highest values for A and gs in L. monopetalum were observed in autumn and for A. portulacoides in winter, presenting both lowest values in spring and summer. A_max was particularly high for L. monopetalum than for A. portulacoides in summer and autumn, despite gs_max was similar in both species. Diurnal pattern of A and gs were similar in both species, with higher values in the morning, decreasing throughout the day.     

Response of carnivores to existing highway culverts and underpasses: implications for road planning and mitigation

Roads with high traffic volumes are a source of animal mortality, can disrupt normal animal movements and dispersal, and may represent a potentially serious threat to wildlife population stability and viability. Retrofitting existing structures built for other purposes (e.g., drainage culverts or small below-grade access roads) to facilitate wildlife crossing by animals and to reduce mortality may be expensive if modifications to the existing structures themselves were involved. However, the environmental context surrounding these structures may influence the willingness of animals to cross, and management of some of these attributes may enhance the attractiveness of these structures. Culverts and underpasses are two common structures along roads in Portugal. We quantified the response of small and medium-sized carnivores to the presence of both types of existing passages by determining: (1) frequency of use; (2) whether use differed by type of passage, and if so; (3) by examining if associated environmental attributes might explain the differences observed. We surveyed 57 different passages along 252 km of highway with a total sampling effort of 2,330 passage trap-days. The mean passage rate for carnivores combined was 0.7 complete passages per crossing structure per day. Crossings by weasel, polecat, otter, and wildcat were infrequent or absent. Red fox, badger, genet and Egyptian mongoose used the crossing structures regularly and without obvious preference; stone marten preferred underpasses. Regression analyses showed the frequency of use by carnivores varied with structural, landscape, road-related features, and human disturbance with 17 of 26 (65%) attributes being significant. Larger passages with vegetation close to the passage entrances, favorable habitat in the surrounding area, and low disturbance by humans were important key features to regular use by the guild of species studied. Mitigation planning in areas with ecological significance for carnivores will be beneficial. Structural attributes and human disturbances are more difficult or expensive to change, even though related significantly to crossing use. Management of vegetation at passage entrances and restricting human use near passages in carnivore suitable areas may substantially improve crossing attractiveness for the guild of carnivore species.     

Purification, crystallization and preliminary crystallographic analysis of a dissimilatory DsrAB sulfite reductase in complex with DsrC

Dissimilatory sulfite reductase (dSiR, DsrAB) is a key protein in dissimilatory sulfur metabolism, one of the earliest types of energy metabolism to be traced on earth. dSirs are large oligomeric proteins around 200kDa forming an alpha(2)beta(2) arrangement and including a unique siroheme-[4Fe-4S] coupled cofactor. Here, we report the purification, crystallization and preliminary X-ray diffraction analysis of dSir isolated from Desulfovibrio vulgaris Hildenborough, also known as desulfoviridin. In this enzyme the DsrAB protein is associated with DsrC, a protein of unknown function that is believed to play an important role in the sulfite reduction. Crystals belong to the monoclinic space group P2(1) with unit-cell parameters a=122.7, b=119.4 and c=146.7A and beta =110.0 degrees , and diffract X-rays to 2.8A on a synchrotron source.     

Pyruvate uptake is inhibited by valproic acid and metabolites in mitochondrial membranes

The pyruvate uptake rate in inverted submitochondrial vesicles prepared from rat liver was optimized and further characterized; the potential inhibitory effects of the anticonvulsive drug valproic acid or 2-n-propyl-pentanoic acid (VPA), Delta4-valproic acid or 2-n-propyl-4-pentenoic acid and the respective coenzyme A (CoA) conjugates were studied in the presence of a proton gradient. All tested VPA metabolites inhibited the pyruvate uptake, but the CoA esters were stronger inhibitors (40% and 60% inhibition, respectively, for valproyl-CoA and Delta4-valproyl-CoA, at 1mM). At the same concentration, the specific inhibitor 2-cyano-4-hydroxycinnamate decreased the pyruvate uptake rate by 70%. The reported inhibition of the mitochondrial pyruvate uptake may explain the significant impairment of the pyruvate-driven oxidative phosphorylation induced by VPA.     

Eucleolatin: A dimeric methylnaphthazarin from Euclea lanceolata

8,8′-Dihydroxy-4,4′-dimethoxy-6,6′-dimethyl-2,2′-binaphthyl-1,1′-quinone, 7-methyljuglone, 8′-hydroxydiospyrin, and eucleolatin have been isolated from the root bark of E. lanceolata. Eucleolatin is the 3,6′or 3,7′- dimer of 2-methylnaphthazarin.