Norvaline is accumulated after a down-shift of oxygen in Escherichia coli W3110

<p>Abstract</p> <p>Background</p> <p>Norvaline is an unusual non-proteinogenic branched-chain amino acid which has been of interest especially during the early enzymological studies on regulatory mutants of the branched-chain amino acid pathway in <it>Serratia marcescens</it>. Only recently norvaline and other modified amino acids of the branched-chain amino acid synthesis pathway got attention again when they were found to be incorporated in minor amounts in heterologous proteins with a high leucine or methionine content. Earlier experiments have convincingly shown that norvaline and norleucine are formed from pyruvate being an alternative substrate of α-isopropylmalate synthase, however so far norvaline accumulation was not shown to occur in non-recombinant strains of <it>E. coli</it>.</p> <p>Results</p> <p>Here we show that oxygen limitation causes norvaline accumulation in <it>E. coli </it>K-12 W3110 during grow in glucose-based mineral salt medium. Norvaline accumulates immediately after a shift to oxygen limitation at high glucose concentration. On the contrary free norvaline is not accumulated in <it>E. coli </it>W3110 in aerobic cultures. The analysis of medium components, supported by transcriptomic studies proposes a purely metabolic overflow mechanism from pyruvate into the branched chain amino acid synthesis pathway, which is further supported by the significant accumulation of pyruvate after the oxygen downshift. The results indicate overflow metabolism from pyruvate as necessary and sufficient, but deregulation of the branched chain amino acid pathway may be an additional modulating parameter.</p> <p>Conclusion</p> <p>Norvaline synthesis has been so far mainly related to an imbalance of the synthesis of the branched chain amino acids under conditions were pyruvate level is high. Here we show that simply a downshift of oxygen is sufficient to cause norvaline accumulation at a high glucose concentration as a consequence of the accumulation of pyruvate and its direct chain elongation over α-ketobutyrate and α-ketovalerate.</p> <p>Although the flux to norvaline is low, millimolar concentrations are accumulated in the cultivation broth, which is far above the level which has been discussed for being relevant for misincorporation of norvaline into recombinant proteins. Therefore we believe that our finding is relevant for recombinant protein production but also may even have implications for the physiology of <it>E. coli </it>under oxygen limitation in general.</p

A proteomic and transcriptional view of acidogenic and solventogenic steady-state cells of Clostridium acetobutylicum in a chemostat culture

The complex changes in the life cycle of Clostridium acetobutylicum, a promising biofuel producer, are not well understood. During exponential growth, sugars are fermented to acetate and butyrate, and in the transition phase, the metabolism switches to the production of the solvents acetone and butanol accompanied by the initiation of endospore formation. Using phosphate-limited chemostat cultures at pH 5.7, C. acetobutylicum was kept at a steady state of acidogenic metabolism, whereas at pH 4.5, the cells showed stable solvent production without sporulation. Novel proteome reference maps of cytosolic proteins from both acidogenesis and solventogenesis with a high degree of reproducibility were generated. Yielding a 21% coverage, 15 protein spots were specifically assigned to the acidogenic phase, and 29 protein spots exhibited a significantly higher abundance in the solventogenic phase. Besides well-known metabolic proteins, unexpected proteins were also identified. Among these, the two proteins CAP0036 and CAP0037 of unknown function were found as major striking indicator proteins in acidogenic cells. Proteome data were confirmed by genome-wide DNA microarray analyses of the identical cultures. Thus, a first systematic study of acidogenic and solventogenic chemostat cultures is presented, and similarities as well as differences to previous studies of batch cultures are discussed

Adhesion and Survival Tools of the Bacterium Deinococcus geothermalis

The known natural habitats of Deinococcus geothermalis are geothermal springs and deep ocean subsurfaces. The bacterium has also found its way to manmade environments, including paper machines and drinking water distribution systems, from which it is very difficult to remove due to its resistance towards industrial washing procedures, dehydration and even high doses of ionizing radiation. D. geothermalis attaches tightly on industrial, even microbially repellent, surfaces initiating slime accumulation. We show that the adhesion of D. geothermalis is mediated by outercellular appendages identified as glycosylated type IV pili. D. geothermalis is the first taxon member of the ancient phylum Deinococcus-Thermus, where glycosylated pili are reported. We propose that D. geothermalis is an obligate microaerobic bacterium. Proteomic analysis showed that D. geothermalis, when cultivated aerobically in nutrient limiting medium prevailing in nature, suffered from oxidative stress. Cells did not grow or respire, unless divalent manganese was supplied. As a novel finding, we reported that the cells secreted succinic acid to the medium, possibly as an end product of metabolic pathways directed to battle oxidative stress. The data suggests that D. geothermalis can grow in aerobic habitats, such as the splash areas of paper machines, when divalent manganese is available. Generation of biofilms may be a physiological response of D. geothermalis to escape atmospheric oxygen. Under aerobic conditions D. geothermalis was intolerant to sodium ions beyond 90 mM. Sodium may have inhibitory effect on V-type ATPases, which D. geothermalis uses as its sole mechanism of oxidative phosphorylation. The findings in this thesis offer novel antifouling strategies in processes where D. geothermalis is harmful: (1) destroying the pili by antifouling treatments; (2) promoting dispersal of D. geothermalis from the biofilm by environment-compatible procedures, such as providing process waters with manganese (0.5 mg L-1) or sodium ions (0.5 % w/v), or (3) by reducing dissolved oxygen to levels where D. geothermalis chooses to grow in suspended form rather than biofilms. Alternatively, the description of the D. geothermalis proteome, physiology and biofilm formation may be applied in the development of biotechnological applications where this robust, non-pathogenic and easy-to-culture bacterium is utilized for demanding jobs such as bioremediation of nuclear waste lands or as a resistant host to produce industrially relevant substances.Deinococcus geothermalis bakteerin luontaisia kasvuympäristöjä ovat kuumat lähteet ja valtameren syvänteet, mutta tämä bakteeri on löytänyt tiensä myös ihmisen tuottamiin ympäristöihin, kuten paperikoneille ja talousveden jakelujärjestelmiin. Bakteeri sietää hyvin tuotantolaitteiden puhdistukseen käytettyjä keinoja, kuten happohuuhteluja ja desinfioivia kemikaaleja, kuivatusta ja jopa ionisoivaa sädetystä. D. geothermalis tarttuu tiukasti teollisiin, jopa antimikrobiaaliseksi suunniteltuihin pintoihin. Osoitimme, että bakteerin tarttumaeliminä toimivat tyypin IV pilukset sekä solun pinnalla olevat sokerijohdannaiset. D. geothermalis on ikivanhan pääluokan, Deinococcus-Thermus, ensimmäinen laji, josta löydettiin piluksia. Ehdotamme, että D. geothermalis on mikroaerobinen bakteeri. Proteomiikka-analyysi paljasti, että matalaravinteisessa ympäristössä D. geothermalis kärsi happistressistä. Solujen kasvu ja hengitys pysähtyivät, ellei kasvualustaan lisätty mangaania. Solut erittivät ympäristöönsä meripihkahappoa, jota mahdollisesti syntyi jätteenä prosesseista, joilla solut suojautuivat taistelussa happistressiä vastaan. Tulokset indikoivat, että D. geothermalis tulee toimeen hapellisessa ympäristössä, kuten paperikoneiden roiskealueilla, vain jos mangaani-ioneja on saatavilla. Biofilmien tuotto voi D. geothermalis-bakteerille olla keino suojautua ilmakehän hapelta. Hapellisissa oloissa D. geothermalis sieti huonosti natriumsuoloja. Syynä saattoi olla energiakriisi seurauksena siitä, että natriumionit inhiboivat D. geothermaliksen hapelliseen ATP tuottoon käyttämää entsyymiä, V-ATPaasia. Tämän väitöskirjan tulokset avaavat uusia mahdollisuuksia torjua D. geothermalis prosesseissa, joissa siitä on haittaa: (1) antifouling toimenpiteet, joilla tuhotaan tarttumapilukset; (2) D. geothermaliksen biofilmien tuhoaminen ympäristöystävällisillä tavoilla, kuten lisäämällä prosessivesiin mangaania tai natriumia, tai (3) alentamalla prosessiympäristön happipitoisuus tasolle, jossa D. geothermalis suosii planktista kasvua biofilmin sijaan. Toisaalta tässä työssä kerätty D. geothermalis bakteerin proteomin, fysiologian ja biofilmin tuoton säätelyn tuntemus, tarjoaa toimintamalleja tämän kestävän, ei-patogeenin ja helposti viljeltävän bakteerin käyttöönottamiseksi haastaviin tehtäviin kuten ydinjätteillä saastuneiden maiden puhdistamiseen, sekä käyttöön tuotantomikrobina vähähappista mutta korkeaa lämpötilaa vaativiin fermentointeihin, joissa tuotetaan aineita teollisuuden tarpeisiin

A disposable, roll-to-roll hot-embossed inertial microfluidic device for size-based sorting of microbeads and cells

We present a low-cost and disposable inertial microfluidic device fabricated using roll-to-roll hot embossing for size-based sorting of microparticles and cells.</p

Online quality assurance of microchannels in roll-to-roll by optical coherence tomography

Abstract Roll-to-roll (R2R) process is an emerging technology to manufacture printed electronics, microfluidics, biochemical sensors etc. The requirements for high quality and small tolerances at the manufacturing phase are of the essence for such products. To verify the quality and guarantee the high production yield, high speed, non-destructive testing methods are needed. In this paper, optical coherence tomography (OCT) device is used at the R2R-line to measure online hot embossed microchannel structures at speed of 1 m/min, which is typical for the hot embossing process. The channel’s width and shape are determined along the web. The applicability of OCT for topography measurements is demonstrated in an actual R2R environment

Wavelength scanning interferometry for topography of microchannels at roll-to-roll Line with optical coherence tomography

Abstract Roll-to-roll (R2R) process is mass manufacturing method that can produce various products ranging from printed electronics to microfluidics. Microfluidics, used in biosensors, demands high tolerances and quality to guarantee correct functionality of sensors. In this work, optical coherence tomography device was installed at the R2R-line to measure the height and width of the hot embossed microchannel structures at speed of 1m/min. The repeating channel structure on the 10 m long sample web was measured at 1m interval. An algorithm, typically used in wavelength scanning interferometry (WSI), is utilized to recover topography at nanometer scale. The results showed that the R2R hot embossing process was very stable and predictable. If process parameters, for instance pressure at the hot embossing unit was increased, it resulted deeper channels as expected. The OCT, with the WSI algorithm, provides topography of microchannel at nanometer scale being thus an excellent tool for optimization of process parameters and for on-line quality control