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Enclosure experiments are frequently used to investigate the impact of changing environmental conditions on microbial assemblages. Yet, how the incubation itself challenges complex bacterial communities is thus far unknown. In this study, metaproteomic profiling, 16S rRNA gene analyses, and cell counts were combined to evaluate bacterial communities derived from marine, mesohaline, and oligohaline conditions after long-term batch incubations. Early in the experiment, the three bacterial communities were highly diverse and differed significantly in their compositions. Manipulation of the enclosures with terrigenous dissolved organic carbon resulted in notable differences compared to the control enclosures at this early phase of the experiment. However, after 55 days, bacterial communities in the manipulated and the control enclosures under marine and mesohaline conditions were all dominated by gammaproteobacterium Spongiibacter. In the oligohaline enclosures, actinobacterial cluster I of the hgc group (hgc-I) remained abundant in the late phase of the incubation. Metaproteome analyses suggested that the ability to use outer membrane-based internal energy stores, in addition to the previously described grazing resistance, may enable the gammaproteobacterium Spongiibacter to prevail in long-time incubations. Under oligohaline conditions, the utilization of external recalcitrant carbon appeared to be more important (hgc-I). Enclosure experiments with complex natural microbial communities are important tools to investigate the effects of manipulations. However, species-specific properties, such as individual carbon storage strategies, can cause manipulation-independent effects and need to be considered when interpreting results from enclosures. IMPORTANCE In microbial ecology, enclosure studies are often used to investigate the effect of single environmental factors on complex bacterial communities. However, in addition to the manipulation, unintended effects ("bottle ef, QC 20191025

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There are strong propositions in the literature that abiotic factors override biotic drivers of diversity on time scales of the fossil record. In order to study the interaction of biotic and abiotic forces on long term changes, we devise a spatio-temporal discrete-time Markov process model of macroevolution featuring population formation, speciation, migration and extinction, where populations are free to migrate. In our model, the extinction probability of these populations is controlled by latitudinally and temporally varying environment (temperature) and competition. Although our model is general enough to be applicable to disparate taxa, we explicitly address planktic organisms, which are assumed to disperse freely without barriers over the Earth's oceans. While rapid and drastic environmental changes tend to eliminate many species, generalists preferentially survive and hence leave generalist descendants. In other words, environmental fluctuations result in generalist descendants which are resilient to future environmental changes. Periods of stable or slow environmental changes lead to more specialist species and higher population numbers. Simulating Cenozoic diversity dynamics with both competition and the environmental component of our model produces diversity curves that reflect current empirical knowledge, which cannot be obtained with just one component. Our model predicts that the average temperature optimum at which planktic species thrive best has declined over the Neogene, following the trend of global average temperatures.

Contains fulltext : 127684.pdf (publisher's version ) (Open Access), Bacillus pumilus is characterized by a higher oxidative stress resistance than other comparable industrially relevant Bacilli such as B. subtilis or B. licheniformis. In this study the response of B. pumilus to oxidative stress was investigated during a treatment with high concentrations of hydrogen peroxide at the proteome, transcriptome and metabolome level. Genes/proteins belonging to regulons, which are known to have important functions in the oxidative stress response of other organisms, were found to be upregulated, such as the Fur, Spx, SOS or CtsR regulon. Strikingly, parts of the fundamental PerR regulon responding to peroxide stress in B. subtilis are not encoded in the B. pumilus genome. Thus, B. pumilus misses the catalase KatA, the DNA-protection protein MrgA or the alkyl hydroperoxide reductase AhpCF. Data of this study suggests that the catalase KatX2 takes over the function of the missing KatA in the oxidative stress response of B. pumilus. The genome-wide expression analysis revealed an induction of bacillithiol (Cys-GlcN-malate, BSH) relevant genes. An analysis of the intracellular metabolites detected high intracellular levels of this protective metabolite, which indicates the importance of bacillithiol in the peroxide stress resistance of B. pumilus.

The bacterial endosymbiont of the deep-sea tube worm Riftia pachyptila has never been successfully cultivated outside its host. In the absence of cultivation data, we have taken a proteomic approach based on the metagenome sequence to study the metabolism of this peculiar microorganism in detail. As one result, we found that three major sulfide oxidation proteins constitute ∼12% of the total cytosolic proteome, which highlights the essential role of these enzymes for the symbiont's energy metabolism. Unexpectedly, the symbiont uses the reductive tricarboxylic acid cycle in addition to the previously identified Calvin cycle for CO2 fixation.

A silicon chip-based electric detector coupled to bead-based sandwich hybridization (BBSH) is presented as an approach to perform rapid analysis of specific nucleic acids. A microfluidic platform incorporating paramagnetic beads with immobilized capture probes is used for the biorecognition steps. The protocol involves simultaneous sandwich hybridization of a single-stranded nucleic acid target with the capture probe on the beads and with a detection probe in the reaction solution, followed by enzyme labeling of the detection probe, enzymatic reaction, and finally, potentiometric measurement of the enzyme product at the chip surface. Anti-DIG-alkaline phosphatase conjugate was used for the enzyme labeling of the DIG-labeled detection probe. p-Aminophenol phosphate (pAPP) was used as a substrate. The enzyme reaction product, p-aminophenol (pAP), is oxidized at the anode of the chip to quinoneimine that is reduced back to pAP at the cathode. The cycling oxidation and reduction of these compounds result in a current producing a characteristic signal that can be related to the concentration of the analyte. The performance of the different steps in the assay was characterized using in vitro synthesized RNA oligonucleotides and then the instrument was used for analysis of 16S rRNA in Escherichia coli extract. The assay time depends on the sensitivity required. Artificial RNA target and 16S rRNA, in amounts ranging from 10(11) to 10(10) molecules, were assayed within 25 min and 4 h, respectively., QC 20100525 QC 20111031

Escherichia coli fed-batch cultivations at 22 m(3) scale were compared to corresponding laboratory scale processes and cultivations using a scale-down reactor furnished with a high-glucose concentration zone to mimic the conditions in a feed zone of the large bioreactor. Formate accumulated in the large reactor, indicating the existence of oxygen limitation zones. It is suggested that the reduced biomass yield at large scale partly is due to repeated production/reassimilation of acetate from overflow metabolism and mixed acid fermentation products due to local moving zones with oxygen limitation. The conditions that generated mixed-acid fermentation in the scale-down reactor also induced a number of stress responses, monitored by analysis of mRNA of selected stress induced genes. The stress responses were relaxed when the cells returned to the substrate limited and oxygen sufficient compartment of the reactor. Corresponding analysis in the large reactor showed that the concentration of mRNA of four stress induced genes was lowest at the sampling port most distant from the feed zone. It is assumed that repeated induction/relaxation of stress responses in a large bioreactor may contribute to altered physiological properties of the cells grown in large-scale bioreactor. Flow cytometric analysis revealed reduced damage with respect to cytoplasmic membrane potential and integrity in cells grown in the dynamic environments of the large scale reactor and the scale-down reactor., QC 20100525

A marine Antarctic psychrotolerant bacterium(strain ANT/505), isolated from sea ice-covered surfacewater from the Southern Ocean, showed pectinolytic activ-ity on citrus pectin agar. The sequencing of the 16S rRNAof isolate ANT/505 indicates a taxonomic affiliation toPseudoalteromonas haloplanktis. The supernatant of thisstrain showed three different pectinolytic activities aftergrowth on citrus pectin. By activity screening of a genomicDNA library of isolate ANT/505 in Escherichia coli, two dif-ferent pectinolytic clones could be isolated. Subcloning andsequencing revealed two open reading frames (ORF) of1,671 and 1,968 nt, corresponding to proteins of 68 and75 kDa, respectively. The deduced amino acid sequence ofthe two ORFs showed homology to pectate lyases fromErwinia chrysanthemi and Aspergillus nidulans. The pectatelyases contain signal peptides of 17 and 26 amino acids thatwere correctly processed after overexpression in E. coliBL21. Both enzymes were purified by anionic exchangechromatography. Maximal enzymatic activities for bothpectate lyases were observed at 30°C and a pH range of 9to 10. The Km values of both lyases for pectate and citruspectin were 1 g l 1 and 5 g l 1 , respectively. Calcium wasrequired for activity on pectic substrates, whereas theaddition of 1 mM ethylenediaminetetraacetic acid (EDTA)resulted in complete inhibition of the enzymes. These twoenzymes represent the first pectate lyases isolated and char-acterized from a cold-adapted marine bacterium.

A marine psychrotolerant bacterium from the Antarctic Ocean showing high chitinolytic activity on chitin agar at 5°C was isolated. The sequencing of the 16S rRNA indicates taxonomic affiliation of the isolate Fi:7 to the genus Vibrio. By chitinase activity screening of a genomic DNA library of Vibrio sp. strain Fi:7 in Escherichia coli, three chitinolytic clones could be isolated. Sequencing revealed, for two of these clones, the same open reading frame of 2,189 nt corresponding to a protein of 79.4 kDa. The deduced amino acid sequence of the open reading frame showed homology of 82% to the chitinase ChiA from Vibrio harveyi. The chitinase of isolate Fi:7 contains a signal peptide of 26 amino acids. Sequence alignment with known chitinases showed that the enzyme has a chitin-binding domain and a catalytic domain typical of other bacterial chitinases. The chitinase ChiA of isolate Fi:7 was overexpressed in E. coli BL21(DE3) and purified by anion-exchange and hydrophobic interaction chromatography. Maximal enzymatic activity was observed at a temperature of 35°C and pH 8. Activity of the chitinase at 5°C was 40% of that observed at 35°C. Among the main cations contained in seawater, i.e., Na+, K+, Ca2+, and Mg2+, the enzymatic activity of ChiA could be enhanced twofold by the addition of Ca2+.

A method to quantify the impact of proteolysis on accumulation of recombinant proteins in E. coli is described. A much smaller intracellular concentration of staphylococcal protein A (SpA) (14.7 mg.g(-1)) compared to the fusion protein SpA-beta galactosidase (138 mg.g(-1)) is explained by a very high proteolysis rate constant of SpA. The SpA synthesis rate reached a maximum one hour after induction and gradually decreased to half of this value at the end of the cultivation. The decrease of the synthesis rate and the Ist order kinetics of proteolysis lead to an equilibrium between synthesis and degradation of SpA from 2 h after induction. This resulted in no further SpA accumulation in cells, though synthesis continued for at least 10 h. Similar experiments with recombinant protein ZZT2 also revealed that most of the synthesized product was degraded. The order of proteolysis kinetics depended on the concentration of the recombinant protein: at low concentrations both SpA and ZZT2 were degraded according to first order kinetics, while at high concentrations ZZT2 was degraded according to zero order kinetics. In a protease Clp mutant the degradation rate decreased and intracellular concentration of ZZT2 increased from 50 mg.g(-1) to 120 mg.g(-1). The measurements of proteolysis rate throughout the cultivation enabled calculation of a hypothetical accumulation of the product assuming complete stabilization. In this case the concentration would have increased from 50 to 280 mg.g(-1) in II h. Thus, this method reveals the potential to increase the productivity by eliminating proteolysis., QC 20100525