Questo lavoro è stato supportato anche da finanziamenti DOE Bioenergy Great Lakes Research Center (DOE BER Office of Science DE-FC02-07ER64494). Siamo grati al Dr. Zhang Xudong membri del gruppo e dei suoi utili per le discussioni tecniche e commenti preziosi sulla messa a punto del protocollo.

1. Preparazione del campione e l&#39;estrazione degli acidi <ol><li> Liofilizzare campioni di terreno dopo la raccolta campo. </li><li> Macinare e omogeneizzare campioni di suolo mediante un mulino a sfere, smerigliatrice suolo, o un mortaio e pestello. </li><li> Pesare campioni di suolo (contenente&gt; 0,3 mg N) in un pallone da 25 mL idrolisi. </li><li> Aggiungere 10 ml di HCl 6M in ogni pallone idrolisi, compilare N 2 gas nei fiaschi, e tappo. </li><li> Idrolizzano a 105 ° C in un incubatore per 8 ore utilizzando un timer automatico. </li></ol> 2. Purificazione del campione <ol><li> Rimuovere i palloni dall &#39;incubatrice; raffreddare a temperatura ambiente. </li><li> Aggiungere 100 ul di standard interno myo-inositolo (1 mg mL -1 in acqua) in ciascuno dei palloni, mescolare agitando. </li><li> Impostare imbuti di plastica drenanti in 200 ml flaconi a forma di pera con ST / NS 24/40 articolazioni, situato in bicchieri di plastica per la stabilità. </li><li> Fold Whatman # 2 Circles qualitativi (11 cm di diametro) in quarti e set in imbuti. </li&gt; <li> Swirl ogni pallone idrolisi, e versare il liquame nel imbuto per filtrare (si può ulteriormente lavare ogni pallone con ~ 3 ml di acqua). </li><li> Asciugare il filtrato con un evaporatore a ~ 45 ° C, sotto vuoto applicando. </li><li> Risospendere il residuo secco da ciascuna beuta pera con 3 ~ 5 ml di acqua (usare bagno a ultrasuoni se lo si desidera), e versare in una provetta da 40 ml in teflon, lavare il pallone con una seconda aliquota di acqua. </li><li> Regolare il pH a 6,6-6,8 usando 1M soluzione di KOH a ioni metallici precipitare e altre molecole organiche. </li><li> Rimuovere il precipitati tramite centrifugazione a 2000 rcf per 10 minuti. </li><li> Versare il surnatante in un tubo di 40 ml di vetro, coprire l&#39;apertura del tubo con parafilm, congelare a -20 ° C, poi colpire buchi nel parafilm. </li><li> Liofilizzare il surnatante congelata per rimuovere tutto il liquido. </li><li> Sciogliere il residuo con 3 ml di metanolo a secco, vortex accuratamente (usare bagno a ultrasuoni se lo si desidera), tappare la provetta, quindi si centrifuga a 2000 rcf per 10 minuti per risolverei sali. </li><li> Trasferire il surnatante in una fiala da 3 ml di reazione, porta a secco con macchine RapidVap a 45 ° C (o sotto un leggero flusso di gas di azoto secco se lo si desidera). </li><li> Per ogni flacone, aggiungere 100 microlitri di recupero standard N-metilglucammina (1 mg mL -1 in acqua) e 1 mL H 2 O, coprire le bocche flacone con parafilm, congelare a -20 ° C, perforare la parafilm, e poi congelare a secco . </li><li> Fare standard: aggiungere 100 microlitri di acido muramico (0,5 mg mL -1 in metanolo), 100 pl glucosamina (1 mg mL -1 in acqua), 100 pl myo-inositolo (1 mg mL -1 in acqua), 100 N-pl metilglucamina (1 mg mL -1 in acqua), e 1 ml di H 2 O, parafilm ogni flaconcino, congelare a -20 ° C, perforare la parafilm, poi congelare-dry. </li></ol> 3. Derivatizzazione <ol><li> Preparare derivatizzazione reagente contenente 32 mg mL -1 idrossilammina cloridrato e 40 mg mL -1 4-dimetilammino-PYridine in piridina-metanolo (4:1 v / v). </li><li> Aggiungere 300 pl di reagente derivatizzazione a ciascuno dei reactivials, tappo a, e miscelare accuratamente. </li><li> Mettere le provette in bagnomaria 75-80 ° C per 35 minuti (ben sigillate le fiale per evitare che l&#39;acqua per inserire i flaconi). </li><li> Rimuovere le fiale dal bagnomaria, e lasciare raffreddare a temperatura ambiente. </li><li> Aggiungere 1 ml di anidride acetica a ciascuno dei tre reactivials mL, tappo ben, e vortex accuratamente, poi riscaldare 75-80 ° C in bagnomaria per 25 minuti. </li><li> Rimuovere le fiale dal bagnomaria, e lasciare raffreddare a temperatura ambiente. </li></ol> 4. Separazione e Misura <ol><li> Aggiungere 1,5 mL di diclorometano ad ogni tappo del flaconcino, strettamente, e miscelare accuratamente. </li><li> Aggiungere 1 ml di HCl 1M per ogni tappo del flaconcino, con forza e vortice a fondo per permettere alle soluzioni per riposare fino a quando le due frasi separate, aspirare ed eliminare la parte superiore (acquosa) fase con 1000 pipettatore ul. </li><li> Nella stessa fashion, estrarre la fase organica 3 volte, ma con 1 mL H 2 O (con l&#39;ultimo stadio di lavaggio, prestare particolare attenzione per garantire che tutta la fase acquosa superiore è stato rimosso). </li><li> Essiccare la soluzione finale utilizzando RapidVap a 45 ° C (o secco utilizzando gas azoto se desiderato). </li><li> Sciogliere in 300 ul di acetato di etile-esano (1:1 v / v) e trasferimento a 2 ml ambra con tappo a vite fiale con inserto in un piccolo volume e tappo. </li><li> Per la quantificazione, analizzare mediante GC-FID utilizzando un fuso colonna capillare di silice polare: 30 m di lunghezza, 0,25 mm, spessore 0,25 um; fase stazionaria fenil-5%, 95% di metil-polisilossano (DB-5 o equivalente) con idrogeno o elio come gas di trasporto, a 0,5 mL min -1 flusso costante. La cromatografia è migliore su premium &quot;inerti&quot; fasi e carrier gas idrogeno, ma è accettabile sulle varietà più comuni e con elio. Le impostazioni del rivelatore sono 300 ° C, 400 mL min -1 aria e 30 mL min -1 sia per l&#39;azoto etrucco gas idrogeno. L&#39;iniezione e parametri forno sono i seguenti: 1 pl iniezione a separazione (30:1) con l&#39;entrata GC impostata a 250 ° C, temperatura del forno iniziale, 120 ° C; premuto 1 min; aumentare la temperatura del forno a 10 ° C min. -1 a 250 ° C; tenere 2,5 min;. rampa a 270 ° C a 20 ° C min -1; tenere 2 minuti; rampa a 290 ° C a 20 ° C min -1, tenere premuto 5 min. Regolare il flusso del gas vettore in modo che la eluiscono inositolo, glucosammina e derivati ​​dell&#39;acido muramico a 250 ° C, e le N-metilglucamina eluisce a 270 ° C. </li></ol> 5. Derivata di convalida <ol><li> Utilizzare ionizzazione morbida LC-ESI-TOF-MS per identificare lo ione molecolare e determinare il peso molecolare del derivato. </li><li> Utilizzare GC-EI-MS-SIM o GC-EI-MSMS per il miglioramento della sensibilità per indagare ioni mirati del derivato. </li><li> Utilizzare più reagenti marcati con isotopi per la preparazione dei derivati, e quindi utilizzare lo spostamento di massadi questi derivati ​​marcati con isotopi a MS per indagare ione molecolare e frammenti di ioni. </li></ol> 6. Risultati rappresentativi Il protocollo del metodo comprende essenzialmente quattro fasi: digestione acido, purificazione del campione, derivatizzazione e determinazione GC (Figura 1). Un esempio di analisi per glucosammina e acido muramico dalle scorte standard e da un campione di terreno viene mostrato nella Figura 2. Oltre glucosammina e acido muramico, mannosammina e galattosamina (due isomeri di glucosammina) possono anche essere determinati simultaneamente utilizzando il metodo. Sulla base di fattori di risposta standard rispetto alla standard interno myo-inositolo, si può quantificare questi marcatori in campioni di terreno. Lo standard di recupero è stato usato per monitorare qualitativamente il processo di derivatizzazione. I regimi per la formazione del aldononitrile derivatizzata glucosammina e acido muramico acetato sono mostrati in figura 3 </strong&gt;. Le strutture proposte dei derivati ​​sono stati determinati mediante GC-EIMS-SIM per miglioramento della sensibilità, o ionizzazione morbida LC-ESI-TOF-MS 8; le strutture proposte dei frammenti ionici formate sulla ionizzazione elettronica stati studiati confrontando gli spettri ione del I campioni preparati con isotopi vari incorporazioni 11. La figura 4 mostra lo spostamento della massa dominante di ioni m / z 187 di acetato glucosamina aldononitrile derivatizzato in tre scenari, preparati con agenti non etichettati, D-acetico anidrite, e U -13 C-glucosamina. Altre informazioni dettagliate e chiarimenti è possibile fare riferimento alla nostra recente pubblicazioni 8, 11. Questa strategia potrebbe servire da modello per studiare la chimica derivata. <img alt="Figura 1" src="/files/ftp_upload/3767/3767fig1.jpg" /> Figura 1. Flusso tabella di misura del protocollo per l&#39;analisi di glucosammina e acido muramico nel suolocampioni. Il protocollo contiene principalmente 4 fasi: digestione acida, purificazione, derivatizzazione e determinazione GC. <img alt="Figura 2" src="/files/ftp_upload/3767/3767fig2.jpg" /> Figura 2. GC-FID cromatogrammi di acetati aldononitrile di inositolo, glucosamina, muramico acido, mannosammina, galattosamina e metilglucamina per le norme e del suolo. <img alt="Figura 3" src="/files/ftp_upload/3767/3767fig3.jpg" /> Figura 3. Schemi per la formazione del aldononitrile derivatizzata muramico glucosamina e acido acetico. Il numero 1 rappresenta la reazione nitrile. Il numero 2 rappresenta l&#39;acetilazione. <a href="http://www.jove.com/files/ftp_upload/3767/3767fig4_lg.jpg"><img alt="Figura 4" src="/files/ftp_upload/3767/3767fig4.jpg" /></a> Figura 4. Spettri di massa di acetato di glucosamina aldononitrile derivatizzata associato vede una struttura ionica dominantees in modalità EI preparato con (A) Agenti non-etichettati, (B), D-acetico anidrite, (C) U -13 C-glucosamina. Stella rappresenta isotopo pesante atomo o gruppo di isotopo.

<ol>
	<li>Zelles, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Fatty+acid+patterns+of+phospholipids+and+lipopolysaccharides+in+the+characterisation+of+microbial+communities+in+soil:+a+review.">Fatty acid patterns of phospholipids and lipopolysaccharides in the characterisation of microbial communities in soil: a review.</a> Biology and Fertility of Soils. 29, 111-129 (1999).</li><li>Kirk, J. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Methods+of+studying+soil+microbial+diversity.">Methods of studying soil microbial diversity.</a> Journal of Microbiological Methods. 58, 169-188 (2004).</li><li>Joergensen, R. G., Emmerling, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Methods+for+evaluating+human+impact+on+soil+microorganisms+based+on+their+activity,+biomass,+and+diversity+in+agricultural+soils.">Methods for evaluating human impact on soil microorganisms based on their activity, biomass, and diversity in agricultural soils.</a> Journal of Plant Nutrition and Soil Science. 169, 295-309 (2006).</li><li>Guggenberger, G., Frey, S. D., Six, J., Paustian, K., Elliott, E. T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Bacterial+and+fungal+cell-wall+residues+in+conventional+and+no-tillage+agroecosystems.">Bacterial and fungal cell-wall residues in conventional and no-tillage agroecosystems.</a> Soil Science Society of America Journal. 63, 1188-1198 (1999).</li><li>Amelung, W., Lal, R., Kimble, J. M., Follett, R. F., Stewart, B. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Assessment Methods for Soil Carbon. , 233-270 (2001).</li><li>Guerrant, G. O., Moss, C. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Determination+of+monosaccharides+as+aldononitrile,+O-methyloxime,+alditol,+and+cyclitol+acetate+derivatives+by+gas-chromatography.">Determination of monosaccharides as aldononitrile, O-methyloxime, alditol, and cyclitol acetate derivatives by gas-chromatography.</a> Analytical Chemistry. 56, 633-638 (1984).</li><li>Zhang, X., Amelung, W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Gas+chromatographic+determination+of+muramic+acid,+glucosamine,+mannosamine,+and+galactosamine+in+soils.">Gas chromatographic determination of muramic acid, glucosamine, mannosamine, and galactosamine in soils.</a> Soil Biology and Biochemistry. 28, 1201-1206 (1996).</li><li>Liang, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Investigation+of+the+molecular+ion+structure+for+aldononitrile+acetate+derivatized+muramic+acid.">Investigation of the molecular ion structure for aldononitrile acetate derivatized muramic acid.</a> Journal of Microbiological Methods. 86, 224-230 (2011).</li><li>He, H., Xie, H., Zhang, X. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+novel+GC/MS+technique+to+assess+15N+and+13C+incorporation+into+soil+amino+sugars.">A novel GC/MS technique to assess 15N and 13C incorporation into soil amino sugars.</a> Soil Biology and Biochemistry. 38, 1083-1091 (2006).</li><li>Glaser, B., Gross, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Compound-specific+delta+13C+analysis+of+individual+amino+sugars+-+a+tool+to+quantify+timing+and+amount+of+soil+microbial+residue+stabilization.">Compound-specific delta 13C analysis of individual amino sugars - a tool to quantify timing and amount of soil microbial residue stabilization.</a> Rapid Communications in Mass Spectrometry. 19, 1409-1416 (2005).</li><li>Liang, C., Balser, T. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mass+spectrometric+characterization+of+amino+sugar+aldononitrile+acetate+derivatives+used+for+isotope+enrichment+assessment+of+microbial+residues.">Mass spectrometric characterization of amino sugar aldononitrile acetate derivatives used for isotope enrichment assessment of microbial residues.</a> Soil Biology and Biochemistry. 42, 904-909 (2010).</li><li>Liang, C., Pedersen, J. A., Balser, T. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Aminoglycoside+antibiotics+may+interfere+with+microbial+amino+sugar+analysis.">Aminoglycoside antibiotics may interfere with microbial amino sugar analysis.</a> Journal of Chromatography A. 1216, 5296-5301 (2009).</li></ol>

Non abbiamo nulla da rivelare.

Il presentato GC-based metodo per l&#39;analisi di acetato derivatizzata glucosammina e acido muramico aldononitrile fornisce un metodo relativamente rapido per quantificare questi amminoacidi, zuccheri estratti dal suolo. I derivati ​​sono chimicamente stabili, e può essere determinata in analisi. Il metodo non è limitato a campioni di terreno, e può essere semplificata per campioni di suolo non-matrice. La pompa a vuoto utilizzata in questo metodo è costruito per essere resistenti ...

<table border="1"><tbody><tr><td> Nome del reattivo </td><td> Azienda </td><td> Numero di catalogo </td></tr><tr><td> L&#39;acido muramico </td><td> Sigma-Aldrich </td><td> M2503-25mg </td></tr><tr><td> D-(+)-glucosammina cloridrato </td><td> Sigma-Aldrich </td><td> G1514-100G </td></tr><tr><td> N-metil-D-glucammina </td><td> Sigma-Aldrich </td><td> M2004-500G </td></tr><tr><td> Myo-inositolo </td><td> Fisher Scientific </td><td> A307003G025 </td></tr><tr><td> Metanolo (secco) </td><td> Acros organici </td><td> AC326950010 </td></tr><tr><td> 4-dimetilammino-piridina </td><td> Acros organici </td><td> AC148270050 </td></tr><tr><td> Acetato di etile </td><td> VWR International </td><td> BJGC100-4 </td></tr><tr><td> Hydroxlamine cloridrato </td><td> Fisher Scientific </td><td> H330-100 </td></tr><tr><td> Pyridine </td><td> Fisher Scientific </td><td> P368-500 </td></tr><tr><td> Anidride acetica </td> &lt;td&gt; Fisher Scientific </td><td> A10-100 </td></tr><tr><td> Diclorometano (cloruro di metilene) </td><td> Fisher Scientific </td><td> D37-500 </td></tr><tr><td> Esano </td><td> Fisher Scientific </td><td> H303-4 </td></tr><tr><td> Acido cloridrico (6M) </td><td> Fisher Scientific </td><td> S456-4 </td></tr><tr><td> Idrossilammina cloridrato -15 N </td><td> Icona servizi </td><td> IN5280 </td></tr><tr><td> Anidride acetica -2 H (D 6 C 4 O 3) </td><td> Acros organici </td><td> AC174670050 </td></tr><tr><td> D-glucosio-U -13 C </td><td> Cambridge isotopo lab </td><td> CLM-1396-1 </td></tr><tr><td> Ammonio sufate -15 N </td><td> Cambridge isotopo lab </td><td> NLM-713-1 </td></tr><tr><td></td><td></td><td></td></tr><tr><td> Nome dell&#39;apparecchiatura </td><td> Azienda </td><td> Tipo </td></tr><tr><td> Rapid-Vap </td&gt; <td> Labconco </td><td> 790002 </td></tr><tr><td> Vacum pompa </td><td> KNF Neuberger </td><td> D-79.112 </td></tr><tr><td> Idrolisi pallone </td><td> Fisher Scientific </td><td> 06 423A </td></tr><tr><td> Derivatizzazione microprovetta </td><td> Fisher Scientific </td><td> 06-100E </td></tr><tr><td> GC </td><td> Hewlett-Packard </td><td> 6890 </td></tr><tr><td> MS </td><td> Hewlett-Packard </td><td> 5972 </td></tr><tr><td> LC-ESI-TOF-MS </td><td> Agilent </td><td> Un Agilent 1200 Series HPLC accoppiato ad un LC Agilent / MSD-TOF </td></tr></tbody></table>

gc-based detection of aldononitrile acetate derivatized glucosamine and muramic acid for microbial residue determination in soil

Quantitavo dei microrganismi che caratterizzano sono essenziali per una comprensione più ampia dello stato microbica e la funzione all&#39;interno degli ecosistemi. Attuali strategie per l&#39;analisi microbiologica di laboratorio tradizionali comprendono sia la cultura-dipendenti tecniche e quelle basate su estrazione diretta e la determinazione di alcuni biomarcatori 1, 2. Pochi tra la diversità delle specie microbiche abitano suolo possono essere coltivate, in modo da cultura-dipendenti metodi introdurre distorsioni significative, una limitazione assente nelle analisi di biomarker. L&#39;acido glucosamina, mannosammina, galattosamina e muramico sono stati ben servito come misure sia i vivi ei morti di massa microbica, di questi l&#39;acido della glucosamina (più abbondante) e muramico (unicamente dalla cellula batterica) sono costituenti più importanti nei sistemi suolo 3 , 4. Tuttavia, la mancanza di conoscenza sulle analisi limita la divulgazione a livello scientifico tra pari. Tra tutti existing metodi analitici, derivatizzazione di aldononitrile acetati seguita da GC-based analisi è emerso come una buona opzione rispetto a bilanciare in modo ottimale precisione, sensibilità, semplicità, buona separazione cromatografica, e la stabilità durante lo stoccaggio del campione 5. Qui vi presentiamo un protocollo dettagliato per un&#39;analisi affidabile e relativamente semplice di glucosamina e acido muramico dal terreno dopo la loro conversione al acetati aldononitrile. Il protocollo comprende principalmente quattro fasi: digestione acida, purificazione del campione, derivatizzazione e determinazione GC. Il passo-passo procedura viene modificato secondo ex pubblicazioni 6, 7. Inoltre, presentiamo una strategia per convalidare strutturalmente lo ione molecolare del derivato e suoi frammenti ionici formato sulla ionizzazione elettronica. Abbiamo applicato GC-EI-MS-SIM, LC-ESI-TOF-MS e reagenti marcati con isotopi per determinare il peso molecolare di acetato glucosamina aldononitrile derivatizzata e murL&#39;acido Amic, abbiamo usato lo spostamento di massa di isotopi marcati derivati ​​nello spettro di ioni di indagare frammenti di ioni di ogni derivati ​​8. Oltre alla spiegazione teorica, la convalida di ione molecolare del derivato e suoi frammenti ionici sarà utile per ricercatori utilizzando δ 13 C o frammenti ionici di questi marcatori in studi biogeochimici 9, 10.

Watch this Scientific Journal Video about GC-based Detection of Aldononitrile Acetate Derivatized Glucosamine and Muramic Acid for Microbial Residue Determination in Soil at JoVE.com

GC-based Detection of Aldononitrile Acetate Derivatized Glucosamine and Muramic Acid for Microbial Residue Determination in Soil

Noi descriviamo un protocollo di metodo per la GC-analisi, basata su strumenti derivati ​​di acetato di aldonitrile di glucosamina e acido muramico estratti dal suolo. Per chiarire il meccanismo chimico, si presentano anche una strategia per confermare la struttura del derivato ed i frammenti di ioni formate sulla ionizzazione elettronica.

GC-based Detection di acetato derivatizzata Glucosamina e muramico Acid Aldononitrile per la determinazione dei residui microbica nel terreno

Quantitative approaches to characterizing microorganisms are crucial for a broader understanding of the microbial status and function within ecosystems. ...

gc-based-detection-aldononitrile-acetate-derivatized-glucosamine

Research

JoVE Journal

Biology

16.8K Views.  University of Wisconsin, Madison. Noi descriviamo un protocollo di metodo per la GC-analisi, basata su strumenti derivati ​​di acetato di aldonitrile di glucosamina e acido muramico estratti dal suolo. Per chiarire il meccanismo chimico, si presentano anche una strategia per confermare la struttura del derivato ed i frammenti di ioni formate sulla ionizzazione elettronica.

Video: GC-based Detection of Aldononitrile Acetate Derivatized Glucosamine and Muramic Acid for Microbial Residue Determination in Soil

Microbial Communities in Nature and Laboratory - Interview

Comunità microbica in natura e laboratorio - Intervista

Ricerca

Biologia

Direct Detection of the Acetate-forming Activity  of the Enzyme Acetate Kinase

Acetate kinase, a member of the acetate and sugar kinase-Hsp70-actin (ASKHA) enzyme superfamily1-5, is responsible for the reversible phosphorylation of acetate to acetyl phosphate utilizing ATP as a substrate. Acetate kinases are ubiquitous in the Bacteria, found in one genus of Archaea, and are also present in microbes of the Eukarya6. The most well characterized acetate kinase is that from the methane-producing archaeon Methanosarcina thermophila7-14. An acetate kinase which can only utilize PPi but not ATP in the acetyl phosphate-forming direction has been isolated from Entamoeba histolytica, the causative agent of amoebic dysentery, and has thus far only been found in this genus15,16.
In the direction of acetyl phosphate formation, acetate kinase activity is typically measured using the hydroxamate assay, first described by Lipmann17-20, a coupled assay in which conversion of ATP to ADP is coupled to oxidation of NADH to NAD+ by the enzymes pyruvate kinase and lactate dehydrogenase21,22, or an assay measuring release of inorganic phosphate after reaction of the acetyl phosphate product with hydroxylamine23. Activity in the opposite, acetate-forming direction is measured by coupling ATP formation from ADP to the reduction of NADP+ to NADPH by the enzymes hexokinase and glucose 6-phosphate dehydrogenase24.
Here we describe a method for the detection of acetate kinase activity in the direction of acetate formation that does not require coupling enzymes, but is instead based on direct determination of acetyl phosphate consumption. After the enzymatic reaction, remaining acetyl phosphate is converted to a ferric hydroxamate complex that can be measured spectrophotometrically, as for the hydroxamate assay. Thus, unlike the standard coupled assay for this direction that is dependent on the production of ATP from ADP, this direct assay can be used for acetate kinases that produce ATP or PPi.

A method for the determination of acetate kinase activity is described. This assay utilizes a direct reaction for determining enzyme activity and kinetics of acetate kinase in the acetate-forming direction with different phosphoryl acceptors. Furthermore, this method can be utilized for assaying other acetyl phosphate or acetyl-CoA utilizing enzymes.

Rilevazione diretta della acetato di formazione Attività di acetato enzima chinasi

Acetate kinase, a member of the acetate and sugar kinase-Hsp70-actin (ASKHA) enzyme superfamily1-5, is responsible for the reversible phosphorylation of ...

The Use of Chemostats in Microbial Systems Biology

Cells regulate their rate of growth in response to signals from the external world. As the cell grows, diverse cellular processes must be coordinated including macromolecular synthesis, metabolism and ultimately, commitment to the cell division cycle. The chemostat, a method of experimentally controlling cell growth rate, provides a powerful means of systematically studying how growth rate impacts cellular processes - including gene expression and metabolism - and the regulatory networks that control the rate of cell growth. When maintained for hundreds of generations chemostats can be used to study adaptive evolution of microbes in environmental conditions that limit cell growth. We describe the principle of chemostat cultures, demonstrate their operation and provide examples of their various applications. Following a period of disuse after their introduction in the middle of the twentieth century, the convergence of genome-scale methodologies with a renewed interest in the regulation of cell growth and the molecular basis of adaptive evolution is stimulating a renaissance in the use of chemostats in biological research.

Cell growth rate is a regulated process and a primary determinant of cell physiology. Continuous culturing using chemostats enables extrinsic control of cell growth rate by nutrient limitation facilitating the study of molecular networks that control cell growth and how those networks evolve to optimize cell growth.

L&#39;uso di chemostati in Biologia microbica Sistemi

Cells regulate their rate of growth in response to signals from the external world. As the cell grows, diverse cellular processes must be coordinated ...

Isolation of Native Soil Microorganisms with Potential for Breaking Down Biodegradable Plastic Mulch Films Used in Agriculture

Fungi native to agricultural soils that colonized commercially available biodegradable mulch (BDM) films were isolated and assessed for potential to degrade plastics. Typically, when formulations of plastics are known and a source of the feedstock is available, powdered plastic can be suspended in agar-based media and degradation determined by visualization of clearing zones. However, this approach poorly mimics in situ degradation of BDMs. First, BDMs are not dispersed as small particles throughout the soil matrix. Secondly, BDMs are not sold commercially as pure polymers, but rather as films containing additives (e.g. fillers, plasticizers and dyes) that may affect microbial growth. The procedures described herein were used for isolates acquired from soil-buried mulch films. Fungal isolates acquired from excavated BDMs were tested individually for growth on pieces of new, disinfested BDMs laid atop defined medium containing no carbon source except agar. Isolates that grew on BDMs were further tested in liquid medium where BDMs were the sole added carbon source. After approximately ten weeks, fungal colonization and BDM degradation were assessed by scanning electron microscopy. Isolates were identified via analysis of ribosomal RNA gene sequences. This report describes methods for fungal isolation, but bacteria also were isolated using these methods by substituting media appropriate for bacteria. Our methodology should prove useful for studies investigating breakdown of intact plastic films or products for which plastic feedstocks are either unknown or not available. However our approach does not provide a quantitative method for comparing rates of BDM degradation.

Plastic films labeled "biodegradable" are commercially available for agricultural use as mulches. Tillage represents an attractive disposal method, but degradation under field conditions is poorly understood. The purpose of this study was to develop methods for isolating native soil fungi and bacteria that colonize plastic mulch films after field burial.

Isolamento dei nativi microrganismi del suolo con un potenziale di Breaking Down biodegradabili di pacciamatura film plastici utilizzati in agricoltura

Fungi native to agricultural soils that colonized commercially available biodegradable mulch (BDM) films were isolated and assessed for potential to ...

Pyrosequencing for Microbial Identification and Characterization

Pyrosequencing is a versatile technique that facilitates microbial genome sequencing that can be used to identify bacterial species, discriminate bacterial strains and detect genetic mutations that confer resistance to anti-microbial agents. The advantages of pyrosequencing for microbiology applications include rapid and reliable high-throughput screening and accurate identification of microbes and microbial genome mutations. Pyrosequencing involves sequencing of DNA by synthesizing the complementary strand a single base at a time, while determining the specific nucleotide being incorporated during the synthesis reaction. The reaction occurs on immobilized single stranded template DNA where the four deoxyribonucleotides (dNTP) are added sequentially and the unincorporated dNTPs are enzymatically degraded before addition of the next dNTP to the synthesis reaction. Detection of the specific base incorporated into the template is monitored by generation of chemiluminescent signals. The order of dNTPs that produce the chemiluminescent signals determines the DNA sequence of the template. The real-time sequencing capability of pyrosequencing technology enables rapid microbial identification in a single assay. In addition, the pyrosequencing instrument, can analyze the full genetic diversity of anti-microbial drug resistance, including typing of SNPs, point mutations, insertions, and deletions, as well as quantification of multiple gene copies that may occur in some anti-microbial resistance patterns.

Pyrosequencing is a versatile technique that facilitates microbial genome sequencing that can be used to identify bacterial species, discriminate bacterial strains, and detect genetic mutations that confer resistance to anti-microbial agents. In this video, the procedure for microbial amplicon generation, amplicon pyrosequencing, and DNA sequence analysis will be demonstrated.

Pyrosequencing per identificazione microbica e caratterizzazione

Pyrosequencing is a versatile technique that facilitates microbial genome sequencing that can be used to identify bacterial species, discriminate ...

Determination of Fatty Acid Oxidation and Lipogenesis in Mouse Primary Hepatocytes

Lipid metabolism in liver is complex. In addition to importing and exporting lipid via lipoproteins, hepatocytes can oxidize lipid via fatty acid oxidation, or alternatively, synthesize new lipid via de novo lipogenesis. The net sum of these pathways is dictated by a number of factors, which in certain disease states leads to fatty liver disease. Excess hepatic lipid accumulation is associated with whole body insulin resistance and coronary heart disease. Tools to study lipid metabolism in hepatocytes are useful to understand the role of hepatic lipid metabolism in certain metabolic disorders.
In the liver, hepatocytes regulate the breakdown and synthesis of fatty acids via &#946;-fatty oxidation and de novo lipogenesis, respectively. Quantifying metabolism in these pathways provides insight into hepatic lipid handling. Unlike in vitro quantification, using primary hepatocytes, making measurements in vivo is technically challenging and resource intensive. Hence, quantifying &#946;-fatty acid oxidation and de novo lipogenesis in cultured mouse hepatocytes provides a straight forward method to assess hepatocyte lipid handling. 
Here we describe a method for the isolation of primary mouse hepatocytes, and we demonstrate quantification of &#946;-fatty acid oxidation and de novo lipogenesis, using radiolabeled substrates.

De novo lipogenesis and &#946;-fatty acid oxidation constitute key metabolic pathways in hepatocyte, pathways that are perturbed in several metabolic disorders, including fatty liver disease. Here we demonstrate isolation of mouse primary hepatocytes and describe quantification of &#946;-fatty acid oxidation and lipogenesis.

Determinazione della ossidazione degli acidi grassi e la lipogenesi nel topo primaria Epatociti

Lipid metabolism in liver is complex. In addition to importing and exporting lipid via lipoproteins, hepatocytes can oxidize lipid via fatty acid ...

Isolation of Specific Genomic Regions and Identification of Associated Molecules by enChIP

The identification of molecules associated with specific genomic regions of interest is required to understand the mechanisms of regulation of the functions of these regions. To enable the non-biased identification of molecules interacting with a specific genomic region of interest, we recently developed the engineered DNA-binding molecule-mediated chromatin immunoprecipitation (enChIP) technique. Here, we describe how to use enChIP to isolate specific genomic regions and identify the associated proteins and RNAs. First, a genomic region of interest is tagged with a transcription activator-like (TAL) protein or a clustered regularly interspaced short palindromic repeats (CRISPR) complex consisting of a catalytically inactive form of Cas9 and a guide RNA. Subsequently, the chromatin is crosslinked and fragmented by sonication. The tagged locus is then immunoprecipitated and the crosslinking is reversed. Finally, the proteins or RNAs that are associated with the isolated chromatin are subjected to mass spectrometric or RNA sequencing analyses, respectively. This approach allows the successful identification of proteins and RNAs associated with a genomic region of interest.

The identification of molecules associated with specific genomic regions of interest is required to understand the mechanisms of regulation of the functions of these regions. This protocol describes procedures to perform engineered DNA-binding molecule-mediated chromatin imunoprecipitation (enChIP) for identification of proteins and RNAs associated with a specific genomic region.

L&#39;isolamento di regioni genomiche specifici e l&#39;identificazione di molecole da Associated enChIP

The identification of molecules associated with specific genomic regions of interest is required to understand the mechanisms of regulation of the ...

Methods for the Determination of Rates of Glucose and Fatty Acid Oxidation in the Isolated Working Rat Heart

The mammalian heart is a major consumer of ATP and requires a constant supply of energy substrates for contraction. Not surprisingly, alterations of myocardial metabolism have been linked to the development of contractile dysfunction and heart failure. Therefore, unraveling the link between metabolism and contraction should shed light on some of the mechanisms governing cardiac adaptation or maladaptation in disease states. The isolated working rat heart preparation can be used to follow, simultaneously and in real time, cardiac contractile function and flux of energy providing substrates into oxidative metabolic pathways. The present protocol aims to provide a detailed description of the methods used in the preparation and utilization of buffers for the quantitative measurement of the rates of oxidation for glucose and fatty acids, the main energy providing substrates of the heart. The methods used for sample analysis and data interpretation are also discussed. In brief, the technique is based on the supply of 14C- radiolabeled glucose and a 3H- radiolabeled long-chain fatty acid to an ex vivo beating heart via normothermic crystalloid perfusion. 14CO2 and 3H2O, end byproducts of the enzymatic reactions involved in the utilization of these energy providing substrates, are then quantitatively recovered from the coronary effluent. With knowledge of the specific activity of the radiolabeled substrates used, it is then possible to individually quantitate the flux of glucose and fatty acid in the oxidation pathways. Contractile function of the isolated heart can be determined in parallel with the appropriate recording equipment and directly correlated to metabolic flux values. The technique is extremely useful to study the metabolism/contraction relationship in response to various stress conditions such as alterations in pre and after load and ischemia, a drug or a circulating factor, or following the alteration in the expression of a gene product.

The following protocol describes the preparation and utilization of buffers for the quantitative measurement of rates of glucose and fatty acid oxidation in the isolated working rat heart. The methods used for sample analysis and data interpretation are also discussed.

Metodi per la determinazione dei tassi di glucosio e acidi grassi ossidazione nel cuore isolato di lavoro Rat

The mammalian heart is a major consumer of ATP and requires a constant supply of energy substrates for contraction. Not surprisingly, alterations of ...

A Plate Competition Assay As a Quick Preliminary Assessment of Disease Suppression

The goal was to develop and optimize a simple, affordable, and effective bioassay to detect disease suppressive ability of a specific compost against soilborne fungus Rhizoctonia solani. R. solani is a pathogen of a wide range of plant hosts worldwide. The fungus survives in soils as a saprophyte and grows rapidly on simple water agar media. The plate assay is a rapid method to compare composts for their ability to slow the growth of R. solani. The assay also correlates well with suppression of other soilborne fungal pathogens that survive as saprophytes in soils such as Alternaria early blights, Fusarium wilt, Phytophthora root rot, and Pythium root rot.

Presented is a protocol for a plate competition assay to identify whether a specific compost is likely to contain bacteria and fungi that suppress growth of Rhizoctonia solani.

Un test di competizione di piatto come una rapida valutazione preliminare di soppressione di malattia

The goal was to develop and optimize a simple, affordable, and effective bioassay to detect disease suppressive ability of a specific compost against ...

Laser-Capture Microdissection RNA-Sequencing for Spatial and Temporal Tissue-Specific Gene Expression Analysis in Plants

The development of a complex multicellular organism is governed by distinct cell types that have different transcriptional profiles. To identify transcriptional regulatory networks that govern developmental processes it is necessary to measure the spatial and temporal gene expression profiles of these individual cell types. Therefore, insight into the spatio-temporal control of gene expression is essential to gain understanding of how biological and developmental processes are regulated. Here, we describe a laser-capture microdissection (LCM) method to isolate small number of cells from three barley embryo organs over a time-course during germination followed by transcript profiling. The method consists of tissue fixation, tissue processing, paraffin embedding, sectioning, LCM and RNA extraction followed by real-time PCR or RNA-seq. This method has enabled us to obtain spatial and temporal profiles of seed organ transcriptomes from varying numbers of cells (tens to hundreds), providing much greater tissue-specificity than typical bulk-tissue analyses. From these data we were able to define and compare transcriptional regulatory networks as well as predict candidate regulatory transcription factors for individual tissues. The method should be applicable to other plant tissues with minimal optimization.

Presented here is a protocol for laser-capture microdissection (LCM) of plant tissues. LCM is a microscopic technique for isolating areas of tissue in a contamination-free manner. The procedure includes tissue fixation, paraffin embedding, sectioning, LCM and RNA extraction. RNA is used in the downstream tissue-specific, temporally resolved analysis of transcriptomes.