Activity and diversity of methanogens in a petroleum hydrocarbon-contaminated aquifer

Methanogenic activity was investigated in a petroleum hydrocarbon-contaminated aquifer by using a series of four push-pull tests with acetate, formate, H(2) plus CO(2), or methanol to target different groups of methanogenic Archaea. Furthermore, the community composition of methanogens in water and aquifer material was explored by molecular analyses, i.e., fluorescence in situ hybridization (FISH), denaturing gradient gel electrophoresis (DGGE) of 16S rRNA genes amplified with the Archaea-specific primer set ARCH915 and UNI-b-rev, and sequencing of DNA from dominant DGGE bands. Molecular analyses were subsequently compared with push-pull test data. Methane was produced in all tests except for a separate test where 2-bromoethanesulfonate, a specific inhibitor of methanogens, was added. Substrate consumption rates were 0.11 mM day(-1) for methanol, 0.38 mM day(-1) for acetate, 0.90 mM day(-1) for H(2), and 1.85 mM day(-1) for formate. Substrate consumption and CH(4) production during all tests suggested that at least three different physiologic types of methanogens were present: H(2) plus CO(2) or formate, acetate, and methanol utilizers. The presence of 15 to 20 bands in DGGE profiles indicated a diverse archaeal population. High H(2) and formate consumption rates agreed with a high diversity of methanogenic Archaea consuming these substrates (16S rRNA gene sequences related to several members of the Methanomicrobiaceae) and the detection of Methanomicrobiaceae by using FISH (1.4% of total DAPI [4',6-diamidino-2-phenylindole]-stained microorganisms in one water sample; probe MG1200). Considerable acetate consumption agreed with the presence of sequences related to the obligate acetate degrader Methanosaeata concilii and the detection of this species by FISH (5 to 22% of total microorganisms; probe Rotcl1). The results suggest that both aceticlastic and CO(2)-type substrate-consuming methanogens are likely involved in the terminal step of hydrocarbon degradation, while methanogenesis from methanol plays a minor role. DGGE profiles further indicate similar archaeal community compositions in water and aquifer material. The combination of hydrogeological and molecular methods employed in this study provide improved information on the community and the potential activity of methanogens in a petroleum hydrocarbon-contaminated aquifer.     

Transforming growth factor-β1 downregulates beating frequency and remodeling of cultured rat adult cardiomyocytes

We have observed increased levels of transforming growth factor-₁ (TGF-₁) in human hibernating myocardium (HM). Impaired ventricular function in HM is known to be restored to normal following revascularization implying that myocardial structure in HM is to a certain degree preserved. We have therefore tested whether TGF-₁ can imitate features of HM by reducing the number and frequency of beating cells (chronotropism) and structural remodeling of cultured adult rat cardiomyocytes (ARC), thus saving substrate, energy, and oxygen. Parameters measured were cell size, protein synthesis, protein degradation, protein content, myofibrillogenesis, and chronotropism. ARC were stimulated for 6 days with sera from patients with coronary heart disease, as this period led to a maximum response of cells. An increase of 90% in cell surface area following such treatment was reduced to a 20% increase of the original size by TGF-₁. Concomitantly, the rate of protein synthesis dropped from 3.6-fold to 2.4-fold, and myofibrillogenesis was reduced. TGF-₁ downregulated both the number of contracting cells from 81% to 10% and the frequency from 52 to nine beats per minute. However, TGF-₁ treatment did not reduce the augmentation of protein content (1.28-fold versus 1.25-fold) indicating that protein degradation was also inhibited. Similar results were obtained with serum from healthy volunters. The effects of TGF-₁ were reversible. We conclude that TGF-₁ constrains protein turnover and beating activity in underperfused myocardium, thus mediating protection by adapting myocytes to shortages in blood supply.T. Kubin and M. Tomars contributed equally to this work.     

Activity and diversity of sulfate-reducing bacteria in a petroleum hydrocarbon-contaminated aquifer

Microbial sulfate reduction is an important metabolic activity in petroleum hydrocarbon (PHC)-contaminated aquifers. We quantified carbon source-enhanced microbial SO(4)(2-) reduction in a PHC-contaminated aquifer by using single-well push-pull tests and related the consumption of sulfate and added carbon sources to the presence of certain genera of sulfate-reducing bacteria (SRB). We also used molecular methods to assess suspended SRB diversity. In four consecutive tests, we injected anoxic test solutions (1,000 liters) containing bromide as a conservative tracer, sulfate, and either propionate, butyrate, lactate, or acetate as reactants into an existing monitoring well. After an initial incubation period, 1,000 liters of test solution-groundwater mixture was extracted from the same well. Average total test duration was 71 h. We measured concentrations of bromide, sulfate, and carbon sources in native groundwater as well as in injection and extraction phase samples and characterized the SRB population by using fluorescence in situ hybridization (FISH) and denaturing gradient gel electrophoresis (DGGE). Enhanced sulfate reduction concomitant with carbon source degradation was observed in all tests. Computed first-order rate coefficients ranged from 0.19 to 0.32 day(-1) for sulfate reduction and from 0.13 to 0.60 day(-1) for carbon source degradation. Sulfur isotope fractionation in unconsumed sulfate indicated that sulfate reduction was microbially mediated. Enhancement of sulfate reduction due to carbon source additions in all tests and variability of rate coefficients suggested the presence of specific SRB genera and a high diversity of SRB. We confirmed this by using FISH and DGGE. A large fraction of suspended bacteria hybridized with SRB-targeting probes SRB385 plus SRB385-Db (11 to 24% of total cells). FISH results showed that the activity of these bacteria was enhanced by addition of sulfate and carbon sources during push-pull tests. However, DGGE profiles indicated that the bacterial community structure of the dominant species did not change during the tests. Thus, the combination of push-pull tests with molecular methods provided valuable insights into microbial processes, activities, and diversity in the sulfate-reducing zone of a PHC-contaminated aquifer.     

Blocking the tunnel: engineering of Candida rugosa lipase mutants with short chain length specificity

The molecular basis of chain length specificity of Candida rugosa lipase 1 was investigated by molecular modeling and site-directed mutagenesis. The synthetic lip1 gene and the lipase mutants were expressed in Pichia pastoris and assayed for their chain length specificity in single substrate assays using triglycerides as well as in a competitive substrate assay using a randomized oil. Mutation of amino acids at different locations inside the tunnel (P246F, L413F, L410W, L410F/S300E, L410F/S365L) resulted in mutants with a different chain length specificity. Mutants P246F and L413F have a strong preference for short chain lengths whereas substrates longer than C10 are hardly hydrolyzed. Increasing the bulkiness of the amino acid at position 410 led to mutants that show a strong discrimination of chain lengths longer than C14. The results obtained can be explained by a simple mechanical model: the activity for a fatty acid sharply decreases as it becomes long enough to reach the mutated site. In contrast, a mutation at the entrance of the tunnel (L304F) has a strong impact on C4 and C6 substrates. This mutant is nevertheless capable of hydrolyzing chain lengths longer than C8.     

Oxygen-regulated expression of genes for pigment binding proteins in Rhodobacter capsulatus

Oxygen is the major external factor affecting the expression of photosynthesis genes in facultatively photosynthetic bacteria. Many investigations over the last years mainly carried out on the closely related species Rhodobacter capsulatus and Rhodobacter sphaeroides have identified a number of proteins involved in the oxygen-regulated signal pathway, in which the RegB/RegA two component system plays a central role. While the RegB/RegA system activates photosynthesis genes under low oxygen tension other proteins like CrtJ and PPBP have a repressing function under high oxygen tension. Additional DNA binding proteins like the integration host factor can modulate the expression of photosynthesis genes. The role of alternative sigma factors in this signal pathway is still unclear.     

New helicase-primase inhibitors as drug candidates for the treatment of herpes simplex disease

The vast majority of the world population is infected with at least one member of the human herpesvirus family. Herpes simplex virus (HSV) infections are the cause of cold sores and genital herpes as well as life-threatening or sight-impairing disease mainly in immunocompromized patients, pregnant women and newborns. Since the milestone development in the late 1970s of acyclovir (Zovirax), a nucleosidic inhibitor of the herpes DNA polymerase, no new non-nucleosidic anti-herpes drugs have been introduced. Here we report new inhibitors of the HSV helicase-primase with potent in vitro anti-herpes activity, a novel mechanism of action, a low resistance rate and superior efficacy against HSV in animal models. BAY 57-1293 (N-[5-(aminosulfonyl)-4-methyl-1,3-thiazol-2-yl]-N-methyl-2-[4-(2-pyridinyl)phenyl]acetamide), a well-tolerated member of this class of compounds, significantly reduces time to healing, prevents rebound of disease after cessation of treatment and, most importantly, reduces frequency and severity of recurrent disease. Thus, this class of drugs has significant potential for the treatment of HSV disease in humans, including those resistant to current medications.     

FLAGELLAR REGENERATION IN SPERMATOZOPSIS SIMILIS (CHLOROPHYTA)

The unicellular green alga Spermatozopsis similis Preisig et Melkonian bears two flagella of unequal length. After deflagellation, cells first regenerated the longer flagellum to about one third of its original length, before the shorter flagellum started to develop. Growth rates were similar for both flagella. Thus, the length difference between both flagella was restored by a lag-phase during regeneration of the shorter flagellum. To explain the lag-phase, we have considered a gating mechanism near the flagellar base that controls the entry of precursors into the flagellum. This would allow cells to restrict the time of effective flagellar growth and thereby control flagellar length. Our data indicated that cells are capable of individually regulating flagellar assembly onto basal bodies. We discuss a recent model of flagellar length regulation based on a balance of assembly and disassembly and conclude that flagellar length is controlled by additional factors, including the availability of flagellar proteins and the developmental status of basal bodies.     

Indole-3-acetaldehyde reductase in Phycomyces blakesleeanus. Characterization of the enzyme

lndole-3-acetaldehyde reductase (lAAld reductase EC 1.2.3.1) from Phycomyces blakesleeanus Bgff., a 38 kDa polypeptide as determined by gel filtration, is probably localized in the cytoplasm. The formation of indole-3-ethanol (lEt) is dependent on the presence of NAD(P)H. The enzymatic reduction of IAAId shows a pH optimum between 6 and 8 and a temperature optimum at 30°C. Enzyme activity follows Michaelis Menten kinetic (K_m= 200 μ M for IAAId; K_m= 24 μ M for NADPH). The isoelectric point of the IAAId reductase is at pH 5.4. Phenylacetaldehyde and benzaldehyde are competitive substrates. Hydroxymeihylindole promotes the reductive IEt formation, whereas NADP⁺ is a non-competitive inhibitor. Changes in lAAJd reductase activity correlate with certain developmental stages of the fungus.