The field of systems biology is often held back by difficulties in obtaining comprehensive, high-quality, quantitative data sets. In this paper, we undertook an interlaboratory effort to generate ...such a data set for a very large number of cellular components in the yeast Saccharomyces cerevisiae, a widely used model organism that is also used in the production of fuels, chemicals, food ingredients and pharmaceuticals. With the current focus on biofuels and sustainability, there is much interest in harnessing this species as a general cell factory. In this study, we characterized two yeast strains, under two standard growth conditions. We ensured the high quality of the experimental data by evaluating a wide range of sampling and analytical techniques. Here we show significant differences in the maximum specific growth rate and biomass yield between the two strains. On the basis of the integrated analysis of the high-throughput data, we hypothesize that differences in phenotype are due to differences in protein metabolism.
Bacterial genomes contain 250 to 500 essential genes, as suggested by single gene disruptions and theoretical considerations. If this view is correct, the remaining nonessential genes of an organism, ...such as Bacillus subtilis, have been acquired during evolution in its perpetually changing ecological niches. Notably, approximately 47% of the approximately 4,100 genes of B. subtilis belong to paralogous gene families in which several members have overlapping functions. Thus, essential gene functions will outnumber essential genes. To answer the question to what extent the most recently acquired DNA contributes to the life of B. subtilis under standard laboratory growth conditions, we initiated a "reconstruction" of the B. subtilis genome by removing prophages and AT-rich islands. Stepwise deletion of two prophages (SPbeta, PBSX), three prophage-like regions, and the largest operon of B. subtilis (pks) resulted in a genome reduction of 7.7% and elimination of 332 genes. The resulting strain was phenotypically characterized by metabolic flux analysis, proteomics, and specific assays for protein secretion, competence development, sporulation, and cell motility. We show that genome engineering is a feasible strategy for functional analysis of large gene clusters, and that removal of dispensable genomic regions may pave the way toward an optimized Bacillus cell factory.
The metabolic impact of electron rerouting in the respiratory chain of
Bacillus subtilis was quantitatively assessed during batch growth of quinol oxidase mutants by
13C-tracer experiments. While ...disruption of the low-coupling cytochrome
bd oxidase was without any apparent phenotype, deletion of the high-coupling cytochrome
aa
3 oxidase caused a severe reduction of tricarboxylic acid cycle fluxes and increased overflow metabolism. Since the product-corrected biomass yields were identical in mutants and parent, the results show that efficient ATP generation is not overly important for exponential growth of
B. subtilis in batch culture.
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BFBNIB, FZAB, GIS, IJS, KILJ, NLZOH, NUK, OILJ, SBCE, SBMB, UL, UM, UPUK
The aim of this study is to increase our knowledge of short-term (month) and long-term (3 year) trends in communities settled on hard artificial substrata at different depths off Loano (Lat. ...44°07′22″ Long. 8°16′25″). An artificial reef complex was built
in response to protection, mitigation and restoration needs in an area subjected to illegal trawling, destruction of seagrass beds and the discharge of muddy material. The reef complex consisted of large concrete blocks (2 × 2 × 2 m) arranged in pyramids and single small concrete
blocks (1.2 × 1.2 × 1.2 m). Short-term observations were intended to show seasonal changes in the settlement periods for exploitable resources such as oysters (Ostrea edulis). The long-term investigations were intended to show the pattern of sessile biota development, climax
stages and interaction with fishes. To accomplish these objectives, asbestos panels (20 × 30 × 0.4 cm) were immersed for 1, 3, 6, 9 and 12 months at four stations in different depths. Photographic and removal sampling have been used since 1986. Results indicated consistent increases
in biomass, cover and number of sessile species with time of panel immersion. Decreases were observed in relation to depth, particularly from stations at −18 m to −30 m. Similar patterns of community development occurred over all 3 years. After 1 year the community was dominated
by encrusting bryozoans, serpulids, hydroids, barnacles, ascidians, bivalves and algae (also Corallinaceae), although they occurred in different proportions according to depth. Mussels were never dominant, as has been described for other artificial reefs in the Adriatic (Ancona) and Middle
Tyrrhenian Sea (Fregene). The activity of the sea-urchins Paracentrotus lividus and Arbacia lixula in cleaning the substrata is described. Five years after the immersion of concrete blocks a climax has not yet been reached. The community is still changing: in particular large
algae and sponges are increasing.
Despite the importance of the oxidative pentose phosphate (PP) pathway as a major source of reducing power and metabolic intermediates for biosynthetic processes, almost no direct genetic or ...biochemical evidence is available for Bacillus subtilis. Using a combination of knockout mutations in known and putative genes of the oxidative PP pathway and 13C-labeling experiments, we demonstrated that yqjI encodes the NADP+-dependent 6-P-gluconate dehydrogenase, as was hypothesized previously from sequence similarities. Moreover, YqjI was the predominant isoenzyme during glucose and gluconate catabolism, and its role in the oxidative PP pathway could not be played by either of two homologues, GntZ and YqeC. This conclusion is in contrast to the generally held view that GntZ is the relevant isoform; hence, we propose a new designation for yqjI, gndA, the monocistronic gene encoding the principal 6-P-gluconate dehydrogenase. Although we demonstrated the NAD+-dependent 6-P-gluconate dehydrogenase activity of GntZ, gntZ mutants exhibited no detectable phenotype on glucose, and GntZ did not contribute to PP pathway fluxes during growth on glucose. Since gntZ mutants grew normally on gluconate, the functional role of GntZ remains obscure, as does the role of the third homologue, YqeC. Knockout of the glucose-6-P dehydrogenase-encoding zwf gene was primarily compensated for by increased glycolytic fluxes, but about 5% of the catabolic flux was rerouted through the gluconate bypass with glucose dehydrogenase as the key enzyme. PUBLICATION ABSTRACT
We present redirection of electron flow to more efficient proton pumping branches within respiratory chains as a generally applicable metabolic engineering strategy, which tailors microbial ...metabolism to the specific requirements of high cell density processes by improving product and biomass yields. For the example of riboflavin production by
Bacillus subtilis, we reduced the rate of maintenance metabolism by about 40% in a cytochrome
bd oxidase knockout mutant. Since the putative Yth and the
caa
3 oxidases were of minor importance, the most likely explanation for this improvement is translocation of two protons per transported electron via the remaining cytochrome
aa
3 oxidase, instead of only one proton via the
bd oxidase. The reduction of maintenance metabolism, in turn, significantly improved the yield of recombinant riboflavin and
B. subtilis biomass in fed-batch cultures.
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GEOZS, IJS, IMTLJ, KILJ, KISLJ, NUK, OILJ, PNG, SAZU, SBCE, SBJE, UL, UM, UPCLJ, UPUK
Quantitative knowledge of intracellular fluxes is important for a comprehensive characterization of metabolic networks and their functional operation. In contrast to direct assessment of metabolite ...concentrations, in vivo metabolite fluxes must be inferred indirectly from measurable quantities in 13C experiments. The required experience, the complicated network models, large and heterogeneous data sets, and the time-consuming set-up of highly controlled experimental conditions largely restricted metabolic flux analysis to few expert groups. A conceptual simplification of flux analysis is the analytical determination of metabolic flux ratios exclusively from MS data, which can then be used in a second step to estimate absolute in vivo fluxes.
Here we describe the user-friendly software package FiatFlux that supports flux analysis for non-expert users. In the first module, ratios of converging fluxes are automatically calculated from GC-MS-detected 13C-pattern in protein-bound amino acids. Predefined fragmentation patterns are automatically identified and appropriate statistical data treatment is based on the comparison of redundant information in the MS spectra. In the second module, absolute intracellular fluxes may be calculated by a 13C-constrained flux balancing procedure that combines experimentally determined fluxes in and out of the cell and the above flux ratios. The software is preconfigured to derive flux ratios and absolute in vivo fluxes from 1-13C and U-13Cglucose experiments and GC-MS analysis of amino acids for a variety of microorganisms.
FiatFlux is an intuitive tool for quantitative investigations of intracellular metabolism by users that are not familiar with numerical methods or isotopic tracer experiments. The aim of this open source software is to enable non-specialists to adapt the software to their specific scientific interests, including other 13C-substrates, labeling mixtures, and organisms.
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DOBA, IZUM, KILJ, NUK, PILJ, PNG, SAZU, SIK, UILJ, UKNU, UL, UM, UPUK
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