A procedure for anaerobic column chromatography employing an anaerobic freter-type chamber

A technique is described for the anaerobic fractionation of oxygen-sensitive material. A Freter-type chamber in which the oxygen concentration is maintained at 2 to 5 μl/liter is used in conjunction with anaerobic chromatographic columns that are exterior to the chamber. The column inlet and outlet are connected via thick-walled polyethylene tubing to access ports in the chamber wall. Anaerobic buffer inside the chamber is pumped from the chamber to equilibrate the column. The oxygen-labile sample then is pumped onto the anaerobic column followed by the elution gradient buffer. Column eluate is returned to a fraction collector inside the chamber. At no stage is the sample exposed to air. This technique has been used effectively for fractionation of highly oxygen-sensitive enzymes from methanogenic bacteria where use of other methods failed.     

Thermococcus profundus 2-ketoisovalerate ferredoxin oxidoreductase, a key enzyme in the archaeal energy-producing amino acid metabolic pathway

2-Ketoisovalerate ferredoxin oxidoreductase (VOR) is a key enzyme in hyperthermophiles catalyzing the coenzyme A-dependent oxidative decarboxylation of mainly aliphatic amino acid-derived 2-keto acids. The very oxygen-labile enzyme purified under anaerobic conditions from a hyperthermophilic archaeon, Thermococcus profundus, is a hetero-octamer (alphabetagammadelta)(2) consisting of four different subunits, alpha = 45,000, beta = 31,000, gamma = 22,000 and delta = 13,000, respectively. Electron paramagnetic resonance and resonance Raman spectra of the purified enzyme indicate the presence of approximately three [4Fe-4S] clusters per alphabetagammadelta-protomer, although one of the clusters has a tendency to be converted to a [3Fe-4S] form during purification. The optimal temperature for the enzyme activity is 93 +/- 2 degrees C and the cognate [4Fe-4S] ferredoxin serves as an electron acceptor of the enzyme. The purified enzyme is highly oxygen-labile (t(1/2), approximately 5 min at 25 degrees C), and is partly protected in the presence of magnesium ions, thiamine pyrophosphate and sodium chloride (t(1/2), approximately 25 min at 25 degrees C).     

Differential gene expression in Giardia lamblia under oxidative stress: Significance in eukaryotic evolution

Giardia lamblia is a unicellular, early branching eukaryote causing giardiasis, one of the most common human enteric diseases. Giardia, a microaerophilic protozoan parasite has to build up mechanisms to protect themselves against oxidative stress within the human gut (oxygen concentration 60 μM) to establish its pathogenesis. G. lamblia is devoid of the conventional mechanisms of the oxidative stress management system, including superoxide dismutase, catalase, peroxidase, and glutathione cycling, which are present in most eukaryotes. NADH oxidase is a major component of the electron transport chain of G. lamblia, which in concurrence with disulfide reductase, protects oxygen-labile proteins such as pyruvate: ferredoxin oxidoreductase against oxidative stress by sustaining a reduced intracellular environment. It also contains the arginine dihydrolase pathway, which occurs in a number of anaerobic prokaryotes, includes substrate level phosphorylation and adequately active to make a major contribution to ATP production.     

Development of a cell-free system reveals an oxygen-labile step in the maturation of [NiFe]-hydrogenase 2 of Escherichia coli

By combining extracts from strains lacking genes encoding either the maturation enzymes or the large subunits of hydrogenases 1, 2 and 3 we could reconstitute in vitro under strictly anaerobic conditions 10-15% of the hydrogenase activity present in wild type Escherichia coli extracts. Purified, unprocessed Strep-tagged variants of the hydrogenase 2 large subunit, HybC, isolated from either a ΔhybD (encoding the hydrogenase 2-specific protease) mutant or a strain deficient in HypF could also be matured to active, processed enzyme using this system. These studies reveal that minimally one step early on the hydrogenase maturation pathway is oxygen-labile.     

Paenibacillus curdlanolyticus strain B-6 xylanolytic-cellulolytic enzyme system that degrades insoluble polysaccharides

A facultatively anaerobic bacterium, Paenibacillus curdlanolyticus B-6, isolated from an anaerobic digester produces an extracellular xylanolytic-cellulolytic enzyme system containing xylanase, beta-xylosidase, arabinofuranosidase, acetyl esterase, mannanase, carboxymethyl cellulase (CMCase), avicelase, cellobiohydrolase, beta-glucosidase, amylase, and chitinase when grown on xylan under aerobic conditions. During growth on xylan, the bacterial cells were found to adhere to xylan from the early exponential growth phase to the late stationary growth phase. Scanning electron microscopic analysis revealed the adhesion of cells to xylan. The crude enzyme preparation was found to be capable of binding to insoluble xylan and Avicel. The xylanolytic-cellulolytic enzyme system efficiently hydrolyzed insoluble xylan, Avicel, and corn hulls to soluble sugars that were exclusively xylose and glucose. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) of a crude enzyme preparation exhibited at least 17 proteins, and zymograms revealed multiple xylanases and cellulases containing 12 xylanases and 9 CMCases. The cellulose-binding proteins, which are mainly in a multienzyme complex, were isolated from the crude enzyme preparation by affinity purification on cellulose. This showed nine proteins by SDS-PAGE and eight xylanases and six CMCases on zymograms. Sephacryl S-300 gel filtration showed that the cellulose-binding proteins consisted of two multienzyme complexes with molecular masses of 1,450 and 400 kDa. The results indicated that the xylanolytic-cellulolytic enzyme system of this bacterium exists as multienzyme complexes.     

Paenibacillus curdlanolyticus Strain B-6 Xylanolytic-Cellulolytic Enzyme System That Degrades Insoluble Polysaccharides

A facultatively anaerobic bacterium, Paenibacillus curdlanolyticus B-6, isolated from an anaerobic digester produces an extracellular xylanolytic-cellulolytic enzyme system containing xylanase, β-xylosidase, arabinofuranosidase, acetyl esterase, mannanase, carboxymethyl cellulase (CMCase), avicelase, cellobiohydrolase, β-glucosidase, amylase, and chitinase when grown on xylan under aerobic conditions. During growth on xylan, the bacterial cells were found to adhere to xylan from the early exponential growth phase to the late stationary growth phase. Scanning electron microscopic analysis revealed the adhesion of cells to xylan. The crude enzyme preparation was found to be capable of binding to insoluble xylan and Avicel. The xylanolytic-cellulolytic enzyme system efficiently hydrolyzed insoluble xylan, Avicel, and corn hulls to soluble sugars that were exclusively xylose and glucose. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) of a crude enzyme preparation exhibited at least 17 proteins, and zymograms revealed multiple xylanases and cellulases containing 12 xylanases and 9 CMCases. The cellulose-binding proteins, which are mainly in a multienzyme complex, were isolated from the crude enzyme preparation by affinity purification on cellulose. This showed nine proteins by SDS-PAGE and eight xylanases and six CMCases on zymograms. Sephacryl S-300 gel filtration showed that the cellulose-binding proteins consisted of two multienzyme complexes with molecular masses of 1,450 and 400 kDa. The results indicated that the xylanolytic-cellulolytic enzyme system of this bacterium exists as multienzyme complexes.     

A method for the study of fungal growth inhibition by plant proteins

A bioassay is described for the study of inhibitory activity of plant proteins on fungal growth. Fungal spores were germinated in liquid growth medium and pipetted into wells of a microtitre plate. Fungal growth was followed spectrophotometrically. The bioassay was tested using crude protein extracts from plant tissues known to have high activities of chitinase and beta-1,3-glucanase, and with purified enzymes. Crude protein preparations and combinations of the purified enzymes produced a temporary reduction of growth but no permanent growth inhibition.     

Purification of the nitrogenase proteins from Clostridium pasteurianum

A method is described for the purification of the nitrogenase proteins from Clostridium pasteurianum by two polyethylene glycol precipitations and chromatography on columns of DEAE-cellulose, Sephadex G-100 and Sephadex G-200. The Mo-Fe protein and the Fe protein have been purified 70–80-fold from the crude extract, and they appear essentially pure when tested by anaerobic polyacrylamide gel electrophoresis.     

MINIATURIZED ANAEROBIC CULTIVATION METHODS FOR RECOVERY OF CLOSTRIDIUM SPOROGENES FROM MEAT

Two new miniaturized methods (sandwiched microtiter plate [SMP] and mini-tube) for recovery of Clostridium sporogenes from food samples were developed and evaluated. One hundred microliter of SFP (Shahidi Ferguson Perfringens) agar and 10 üL of diluted food sample were used in the SMP anaerobic system. The samples were sandwiched between two sterile microtiter plates to create an anaerobic environment. Black colonies in the sandwiched wells were counted as C. sporogenes. In case of mini-tube system, 1 mL of SFP agar (45C) containing diluted food sample was aspirated into a 1.2 mL sterile pipet and allowed to solidify in place. The two ends are sealed to create an anaerobic environment. Black colonies were counted directly through the plastic tube. C. sporogenes was inoculated into ground beef samples for recovery study. The recovery rates in SFP agar, using the SMP and mini-tube method were compared with the double-tube method. Organisms were recovered more in the double-tube than with SMP method and mini-tube method after 24–30 h incubation. However, the final counts at 48 h were similar in all methods from the food samples. These new simple methods have potential use for recovery of Clostridium spp.     

Properties of a Soluble Nitrogenase in Azotobacter

A nitrogenase system that remains in the supernatant fluid after centrifuging for 3 hr at 180,000 x g can be extracted from Azotobacter vinelandii by osmotic lysis of the bacteria. This nitrogenase preparation is oxygen-labile and appears to be similar, though not identical, to that obtained from Clostridium pasteurianum. The particulate characteristic and oxygen stability of previously described preparations are likely due to the method of cell disruption, e.g., in the French pressure cell. The data support a nitrogenase model system in the intact cell in which oxygen-labile enzymes are protected from oxygen by the extensive internal membranous system which Azotobacter synthesize only when they fix nitrogen.     

Enrichment analysis of phosphorylated proteins as a tool for probing the phosphoproteome

The current progression from genomics to proteomics is fueled by the realization that many properties of proteins (e.g., interactions, post-translational modifications) cannot be predicted from DNA sequence. Although it has become feasible to rapidly identify proteins from crude cell extracts using mass spectrometry after two-dimensional electrophoretic separation, it can be difficult to elucidate low-abundance proteins of interest in the presence of a large excess of relatively abundant proteins. Therefore, for effective proteome analysis it becomes critical to enrich the sample to be analyzed in subfractions of interest. For example, the analysis of protein kinase substrates can be greatly enhanced by enriching the sample of phosphorylated proteins. Although enrichment of phosphotyrosine-containing proteins has been achieved through the use of high-affinity anti-phosphotyrosine antibodies, the enrichment of phosphoserine/threonine-containing proteins has not been routinely possible. Here, we describe a method for enriching phosphoserine/threonine-containing proteins from crude cell extracts, and for subsequently identifying the phosphoproteins and sites of phosphorylation. The method, which involves chemical replacement of the phosphate moieties by affinity tags, should be of widespread utility for defining signaling pathways and control mechanisms that involve phosphorylation or dephosphorylation of serine/threonine residues.     

Characterization of low abundant membrane proteins using the protein sequence tag technology

About 25% of open reading frames in fully sequenced genomes are estimated to encode transmembrane proteins that represent valuable targets for drugs. However, the global analysis of membrane proteins has been proven to be problematic, e.g., because of their very amphiphilic nature. In this paper, we show that the recently published Protein Sequence Tag (PST) technology combined with an efficient sample preparation is a powerful method to perform protein analysis of highly enriched membrane fractions. The PST approach is a gel-free proteomics tool for the analysis of proteins, which relies on a "sampling" strategy by isolating N-terminal protein sequence tags from cyanogen bromide cleaved proteins. The identification of these N-terminal PST peptides is based on LC-MS/MS. The effectiveness of the technology is demonstrated for a membrane fraction, which was isolated from crude mitochondria of yeast after alkaline sodium carbonate treatment. The PST approach performed on this fraction analyzed 148 proteins, whereas 84% are identified as membrane proteins. More interestingly, among these membrane proteins 56% are predicted to be of low abundance. These encouraging results are an important step toward the development of a quantitative PST approach (qPST) for the differential display of membrane protein analysis.     

StreptoTag: a novel method for the isolation of RNA-binding proteins

We describe a fast and simple one-step affinity-purification method for the isolation of specific RNA-binding proteins. An in vitro-transcribed hybrid RNA consisting of an aptamer sequence with high binding specificity to the antibiotic streptomycin and a putative protein-binding RNA sequence is incubated with crude extract. After complex formation, the sample is applied to an affinity column containing streptomycin immobilized to Sepharose. The binding of the in vitro-assembled RNA-protein complex to streptomycin-Sepharose is mediated by the aptamer RNA and the specifically bound proteins are recovered from the affinity matrix by elution with the antibiotic. Employing two well-characterized RNA-protein interactions, we tested the performance of this new method. The spliceosomal U1A protein and the bacteriophage MS2 coat protein could be isolated via their appropriate RNA motif containing hybrid RNA from crude yeast extracts in high yield and purity after only one round of affinity purification. As the purification principle is independent of the extract source, this new affinity chromatography strategy that makes use of an in vitro-selected antibiotic-binding RNA as a tag, "StreptoTag," should be applicable to extracts from other organisms as well. Therefore, we propose StreptoTag to be a versatile tool for the isolation of unknown RNA-binding proteins.     

Microheterogeneity of the mammalian high mobility group (HMG) proteins 1 and 2 investigated by reverse-phase high performance liquid chromatography

Microheterogeneity within the high mobility group (HMG)-1 and HMG-2 groups of nonhistone chromatin proteins has been investigated using reverse-phase high-performance liquid chromatography (RP-HPLC) under conditions (acetonitrile elution with 0.1% trifluoroacetic acid (TFA) as the counter ion) which separate proteins primarily on the basis of differences in their overall hydrophobicity. RP-HPLC proved to be a fast and efficient means for separating multiple subspecies of both the HMG-1 and HMG-2 proteins from both crude nuclear extracts and from ion-exchange column "purified" protein samples obtained from different types of mammalian cell nuclei. In crude nuclear extracts at least eight different HMG-2 protein species (two major and six minor), but only one major HMG-1 species, could be resolved by RP-HPLC. Three of the minor HMG-2 protein species could be isolated in "pure" form from crude extracts in one RP-HPLC step whereas under the same conditions the two major HMG-2 peaks (as well as the other minor species) were contaminated with either HMG-1 or HMG-3 (a degradation product of HMG-1). In crude extracts the major HMG-1 fraction always seems to be contaminated with one of the HMG-2 subfractions. RP-HPLC analysis of apparently "pure" protein preparations isolated by ion-exchange chromatography techniques revealed that "pure" HMG-1 can be resolved into at least three different protein species and "pure" HMG-2 into at least four different species. Amino acid analyses of different resolvable forms of the HMG proteins were not inconsistent with the suggestion that at least some of these may be primary sequence variants of the individual proteins, but other possibilities also exist.     

Coproporphyrinogenase activities in extracts of Rhodopseudomonas spheroides and Chromatium strain D

1. The anaerobic coproporphyrinogenase activity in an extract of Rhodopseudomonas spheroides is inhibited by 1,10-phenanthroline, alphaalpha'-bipyridyl, flavins, 2,4-dinitrophenol and 1,4-naphthaquinone. These compounds have no effect on the aerobic coproporphyrinogenase activity. 2. On removal of small-molecular-weight material from a crude extract, the anaerobic system becomes very unstable; it can be stabilized by adding succinate. Now nicotinamide nucleotides, in addition to Mg(2+), ATP and methionine, are required for protoporphyrin to be formed. 3. A mechanism for the anaerobic reaction is proposed, based on the cofactor requirements and the effect of inhibitors. 4. The enzyme responsible for aerobic activity has been partially purified and some of its properties are reported. 5. A crude extract of Chromatium strain D also exhibits coproporphyrinogenase activity under anaerobic conditions in the presence of S-adenosylmethionine or ATP plus methionine. The requirement for other cofactors is variable.     

Phylogenetic Diversity of Aerobic Saprotrophic Bacteria Isolated from the Daqing Oil Field

A diverse and active microbial community in the stratal waters of the Daqing oil field (China), which is exploited with the use of water-flooding, was found to contain aerobic chemoheterotrophic bacteria (including hydrocarbon-oxidizing ones) and anaerobic fermentative, sulfate-reducing, and methanogenic bacteria. The aerobic bacteria were most abundant in the near-bottom zones of injection wells. Twenty pure cultures of aerobic saprotrophic bacteria were isolated from the stratal waters. Under laboratory conditions, they grew at temperatures, pH, and salinity values typical of the stratal water from which they were isolated. These isolates were found to be able to utilize crude oil and a wide range of hydrocarbons, fatty acids, and alcohols. Phylogenetic analysis carried out with the use of complete 16S rRNA sequences showed that the isolates could be divided into three major groups: gram-positive bacteria with a high and a low G+C content of DNA and gram-negative bacteria of the -subclass of the Proteobacteria. Gram-positive isolates belonged to the genera Bacillus, Brevibacillus, Rhodococcus, Dietzia, Kocuria, Gordonia, Cellulomonas, and Clavibacter. Gram-negative isolates belonged to the genera Pseudomonas and Acinetobacter. In their 16S rRNA sequences, many isolates were similar to the known microbial species and some probably represented new species.     

Micromethod for Identification of Anaerobic Bacteria: Design and Operation of Apparatus

A replicator is described for transferring 48 bacterial cultures into separate wells of microtiter plates. The device was designed for determination of carbohydrate fermentation patterns of anaerobic bacteria but should be useful for other applications. A simple device for filling microtiter wells with media is also described.     

Development of a Micromethod for Identification of Anaerobic Bacteria

A microprocedure was described for determining the carbohydrate fermentation patterns of 48 anaerobic bacteria at one time in microtiter plates. The cultures were transferred into agar-filled wells of microtiter plates with a replicator inside an anaerobic glove box. Fermentation was measured both with a colorimetric indicator and with a small pH electrode. The method was approximately 97% accurate. It would be most useful for laboratories that need to identify large numbers of anaerobes at one time.