Comparative analysis of environmental DNA extraction and purification methods from different humic acid-rich soils.

AIM: To establish a rapid, improved soil environmental DNA extraction and purification protocol. METHODS AND RESULTS: Three different soil DNA isolation and four purification strategies were compared on different soil samples with variable rates of success. Bead beating extraction gave significantly higher DNA yields than microwave-based and liquid nitrogen grinding DNA extraction methods. The inclusion of soil washing prior to cell lysis decreased the amount of purification steps required. Although these soil types differed, polyvinylpolypyrrolidone (PVPP)-sepharose 2B column elution was sufficient for all three samples, yielding DNA pure enough for successful application in molecular studies. One soil sample retained 80% of the initial DNA after successful purification. CONCLUSIONS: Optimization of a purification protocol confirmed that only a combination of previously described methods proved sufficient in yielding pure environmental DNA from humic-rich soils. Total processing time for DNA extraction and subsequent purification from multiple samples was considerably more rapid than the previously described methods. SIGNIFICANCE AND IMPACT OF THE STUDY: This study developed a new optimized soil DNA extraction and purification protocol that is suitable for different environmental sources that are rich in humic acid content.

A rapid method to quantify pro-oxidant activity in cultures of wood-decaying white-rot fungi.

A new, rapid method for evaluation of lipid peroxidation promoting (pro-oxidant) activity in cultures of wood-decaying fungi was developed. The method is based on measurement of the rate of oxygen consumption in the reaction of linoleic acid peroxidation initiated by fungal culture filtrates. The liquid cultures of the white-rot fungi Bjerkandera adusta and Phanerochaete chrysosporium grown on wheat straw-containing glucose-peptone-corn steep liquor medium possessed significant levels of the pro-oxidant activity. Other white-rot fungi producing manganese peroxidase (MnP) were also found to show the activity. MnP demonstrated a crucial role as the major pro-oxidant agent in the fungal cultures. The total pro-oxidant activity may be considered as net result of the peroxidation by MnP and the inhibition by antioxidant compounds present in the fungal culture fluids.

Purification and characterisation of an alkaline protease used in tannery industry from Bacillus licheniformis.

An extracellular alkaline protease produced by Bacillus licheniformis AP-1 was purified 76-fold, yielding a single 28 kDa band on SDS-PAGE. It was optimally active at pH 11 and at 60 degrees C (assayed over 10 min). The protease was completely inhibited by phenylmethylsulfonyl fluoride and diodopropyl fluorophosphate, with little increase upon Ca2+ and Mg2+ addition.

Cloning and over-expression of an alkaline protease from Bacillus licheniformis.

The alkaline protease gene, apr, from Bacillus licheniformis 2709 was cloned into a Bacillus shuttle expression vector, pHL, to yield the recombinant plasmid pHL-apr. The pHL-apr was expressed in Bacillus subtilis WB600, yielding a high expression strain BW-016. The amount of alkaline protease produced in the recombinant increased by 65% relative to the original strain. SDS-PAGE analysis indicated a Mr of 30.5 kDa. The amino acid sequence deduced from the DNA sequence analysis revealed a 98% identity to that of Bacillus licheniformis 6816.

Quantification of plasma membrane ergosterol of Saccharomyces cerevisiae by direct-injection atmospheric pressure chemical ionization/tandem mass spectrometry.

A method for the quantification of ergosterol by atmospheric pressure chemical ionization (APcI) mass spectrometry with direct injection is described. Ergosterol and squalene were ionizable with methanol as the carrier solvent. Using positive-mode tandem mass spectrometry (MS/MS), ergosterol could be identified unambiguously without interference from structurally related compounds such as lanosterol, cholesterol, and squalene. Molecular ions of ergosterol, lanosterol, and cholesterol were detected as the [M + H - H(2)O](+) ion species, while squalene appeared as the [M + H](+) ion species. Upon fragmentation of the three sterols and squalene, the product ion at m/z 69 was present as one of the major fragments in all four compounds. This product ion was used for the quantification of ergosterol in multiple-reaction-monitoring acquisition mode. The relationship between signal intensity and ergosterol concentration was linear over the concentration range of 0.15 to 5 microg/ml, or 7. 56-252 pmol ergosterol per 20 microl injection. The plasma membrane ergosterol of the yeast Saccharomyces cerevisiae could be quantified reproducibly without the need for prior separation from other lipids or derivatization. Six repeated injections of ergosterol standards at concentrations of 0.95 and 4.25 microg/ml gave standard deviations of 0.031 and 0.084, respectively, and coefficients of variation of 3.33 and 1.98%, respectively. The coefficient of variation for the four independently extracted membrane ergosterol samples was 11.18%. The presence of other lipids in a crude lipid extract did not interfere with the ergosterol determination. Direct injection APcI with multiple reaction monitoring is aconvenient and sensitive method for ergosterol quantification requiring no prior fractionation.

Conservation and release of osmolytes by yeasts during hypo-osmotic stress.

In response to fluctuations in environmental osmolarity, yeast cells adjust their intracellular solute concentrations in order to maintain a constant turgor pressure and ensure continuation of cellular activity. In this study, the effect of hypo-osmotic stress on osmolyte content of osmotolerant yeasts Zygosaccharomyces rouxii and Pichia sorbitophila and the less tolerant Saccharomyes cerevisiae was investigated. All these yeasts released glycerol upon hypo-osmotic shock. However, Z. rouxii also released arabitol, whereas P. sorbitophila released erythritol in addition to arabitol and glycerol. Osmolyte release was very rapid and specific and was neither affected by reduced temperatures nor inhibited by the channel blocker gadolinium or the protonophore carbonyl cyanide m-chlorophenyl hydrazone. Extracellular osmolyte levels increased drastically suggesting that osmolytes were not metabolised but mainly released upon exposure to hypotonic conditions. The export process is well controlled, and the amount of osmolyte released was proportional to the shock intensity. Osmolyte release occurred with little cell lysis and thus the survival as well as the subsequent growth of yeast cells was largely unaffected after hypo-osmotic shock. The kinetics and patterns of osmolyte export suggest the involvement of channel proteins, but the molecular nature of this export pathway in yeasts, with exception of S. cerevisiae, remains to be established.

Implications of FPS1 deletion and membrane ergosterol content for glycerol efflux from Saccharomyces cerevisiae.

The deletion of the gene encoding the glycerol facilitator Fps1p was associated with an altered plasma membrane lipid composition in Saccharomyces cerevisiae. The S. cerevisiae fps1delta strain respectively contained 18 and 26% less ergosterol than the wild-type strain, at the whole-cell level and at the plasma membrane level. Other mutants with deficiencies in glycerol metabolism were studied to investigate any possible link between membrane ergosterol content and intracellular glycerol accumulation. In these mutants a modification in intracellular glycerol concentration, or in intra- to extracellular glycerol ratio was accompanied by a reduction in plasma membrane ergosterol content. However, there was no direct correlation between ergosterol content and intracellular glycerol concentration. Lipid composition influences the membrane permeability for solutes during adaptation of yeast cells to osmotic stress. In this study, ergosterol supplementation was shown to partially suppress the hypo-osmotic sensitivity phenotype of the fps1delta strain, leading to more efficient glycerol efflux, and improved survival. The erg-1 disruption mutant, which is unable to synthesise ergosterol, survived and recovered from the hypo-osmotic shock more successfully when the concentration of exogenously supplied ergosterol was increased. The results obtained suggest that a higher ergosterol content facilitates the flux of glycerol across the plasma membrane of S. cerevisiae cells.

Glycerol production by microbial fermentation: a review.

Microbial production of glycerol has been known for 150 years, and glycerol was produced commercially during World War I. Glycerol production by microbial synthesis subsequently declined since it was unable to compete with chemical synthesis from petrochemical feedstocks due to the low glycerol yields and the difficulty with extraction and purification of glycerol from broth. As the cost of propylene has increased and its availability has decreased especially in developing countries and as glycerol has become an attractive feedstock for production of various chemicals, glycerol production by fermentation has become more attractive as an alternative route. Substantial overproduction of glycerol by yeast from monosaccharides can be obtained by: (1) forming a complex between acetaldehyde and bisulfite ions thereby retarding ethanol production and restoring the redox balance through glycerol synthesis; (2) growing yeast cultures at pH values near 7 or above; or (3) using osmotolerant yeasts. In recent years, significant improvements have been made in the glycerol production using osmotolerant yeasts on a commercial scale in China. The most outstanding achievements include: (1) isolation of novel osmotolerant yeast strains producing up to 130 g/L glycerol with yields up to 63% and the productivities up to 32 g/(L day); (2) glycerol yields, productivities and concentrations in broth up to 58%, 30 g/(L day) and 110-120 g/L, respectively, in an optimized aerobic fermentation process have been attained on a commercial scale; and (3) a carrier distillation technique with a glycerol distillation efficiency greater than 90% has been developed. As glycerol metabolism has become better understood in yeasts, opportunities will arise to construct novel glycerol overproducing microorganisms by metabolic engineering.

The production of hemicellulases by Thermomyces lanuginosus strain SSBP: influence of agitation and dissolved oxygen tension.

Shake-flask cultivation of T. lanuginosus strain SSBP on coarse corn cobs yielded beta-xylanase levels of 56,500 nkat/ml at 50 degrees C, whereas other hemicellulases (beta-xylosidase, beta-glucosidase, and alpha-L-arabinofuranosidase) were produced at levels less than 7 nkat/ml. Cultivation on D-xylose yielded much lower levels of xylanase (350 nkat/ml), although other hemicellulase levels were similar to those produced on corn cobs. The influence of agitation rate and dissolved oxygen tension (DOT) on hemicellulase production was studied further in a bioreactor. On xylose, xylanase activities of 4,330 nkat/ml and 4,900 nkat/ml were obtained at stirrer speeds up to 1,400 rpm to control DOT. At a constant stirrer speed of 400 rpm, xylanase activities of 10,930 nkat/ml and 15,630 nkat/ml were obtained when cultivated on xylose and beechwood xylan respectively, despite DOT levels below 5% for the duration of fermentation. The results indicate that there is an interaction between agitation rate and DOT, impacting on xylanase and accessory enzyme production. Higher agitation rates favoured the production of xylosidase, arabinofuranosidase and glucosidase by T. lanuginosus strain SSBP, whereas the lower agitation rates favoured xylanase production. Rheological difficulties precluded cultivation on corn cobs in the bioreactor. Volumetric xylanase productivities of 1,060,000 nkat/l x h and 589,000 nkat/l x h obtained on beechwood xylan and xylose indicate that T. lanuginosus strain SSBP is a hyperxylanase producer with considerable industrial potential.

A chromogenic substrate for a beta-xylosidase-coupled assay of alpha-glucuronidase.

4-Nitrophenyl 2-(4-O-methyl-alpha-d-glucopyranuronosyl)-beta-d-xylopyranoside obtained on deesterification of 4-nitrophenyl 2-O-(methyl 4-O-methyl-alpha-d-glucopyranosyluronate)-beta-d-xylopyranoside (Hirsch et al., Carbohydr. Res. 310, 145-149, 1998) was found to be an excellent substrate for the measurement of hemicellulolytic alpha-glucuronidase activity. A new precise alpha-glucuronidase assay was developed by coupling the alpha-glucuronidase-catalyzed formation of 4-nitrophenyl beta-d-xylopyranoside with its efficient hydrolysis by beta-xylosidase. A recombinant strain of Saccharomyces cerevisiae, harboring and expressing the beta-xylosidase gene xlnD of Aspergillus niger under control of the alcohol dehydrogenase II promoter on a multicopy plasmid, was used as a source of beta-xylosidase. The activity values of beta-xylosidase in the assay required to achieve a steady-state rate of 4-nitrophenol formation shortly after starting the alpha-glucuronidase reaction were obtained both experimentally and by calculation using the kinetics of coupled enzyme reactions.

Effects of purified endo-beta-1,4-xylanases of family 10 and 11 and acetyl xylan esterases on eucalypt sulfite dissolving pulp.

Sulfite dissolving pulp from Eucalyptus grandis contained approximately 3.8% O-acetyl-4-O-methylglucuronoxylan with a molar ratio of xylose:4-O-methylglucuronic acid:acetyl group close to 13.6:1:6.2. The effects produced by purified endo-xylanases from two different glycosyl hydrolase families (family 10 and 11) as well as acetyl xylan esterases were examined and assessed on pulp in relation to their bleaching abilities. The purified endo-xylanases hydrolyzed only a limited portion (less than 30%) of the acetylglucuronoxylan present in the pulp. The enzymes of family 10 produced acetylated xylobiose and xylotriose whereas acetylated xylobiose was not observed among the products released from the pulp by the family 11 xylanases. The esterases however were not capable of deacetylating the acetylated aldouronic acids generated by the xylanases. Regardless of the different mode of action of the endo-xylanases on dissolving pulp, their effect on pulp bleaching was not related to the amount and nature of sugars generated or the glycosyl hydrolase family. No additional brightness gain was obtained when endo-xylanases were used in conjunction with acetyl xylan esterases, suggesting that the latter do not play an important role in biobleaching of eucalypt sulfite dissolving pulps.

Relatedness of Thermomyces lanuginosus strains producing a thermostable xylanase.

Properties of an endo-beta-xylanase produced by a locally isolated Thermomyces lanuginosus strain SSBP was compared to seven other T. lanuginosus strains isolated from different geographical regions. Strain SSBP produced the highest xylanase activity of 59600 nkat ml(-1) when cultivated on corn cobs (maize) medium, whereas the seven other strains produced xylanase activities ranging from 6000 to 32000 nkat ml(-1). No cellulase activity was produced by the strains. Despite the variability in the production of xylanase, little difference in the other characteristics of the strains could be found. The optimal temperature and pH for xylanase production by the strains was either 40 or 50 degrees C and between pH 6 and 7, respectively. Optimal xylanase activity of the strains was observed at 70 degrees C and at pH 6 or 6.5. Culture supernatant analysis by SDS-PAGE and isoelectric focusing PAGE of all strains revealed the presence of a single 24.7 kDa and pI 3.9 xylanase. Phylogenetic analysis by PCR amplification and sequencing of the internal transcribed spacer of nuclear rRNA repeat units and 5.8S rDNA revealed no strain diversity. However, random amplified polymorphic DNA analysis pointed to greater diversity and with one primer (5'-GCCCGACGCG-3'), a relationship was established between xylanase levels and the RAPD pattern.

Production and properties of hemicellulases by a Thermomyces lanuginosus strain.

Thermophilic fungi producing extremely high beta-xylanase and their associated hemicellulases have attracted considerable attention because of potential industrial applications. Thermomyces lanuginosus strain SSBP isolated from soil, produced beta-xylanase activity of 59 600 nkat ml-1 when cultivated on a medium containing corn cobs as substrate and yeast extract as nitrogen source. Lower beta-xylanase activities were produced after growth on other xylan substrates, sugars and soluble starch. Other hemicellulases were produced extracellularly at significantly lower levels than the beta-xylanase activity produced on corn cobs. No cellulase activity was observed. The optimal conditions for beta-xylanase production were 50 degrees C and pH 6.5, whereas 70 degrees C and between pH 5. 5 and 9.5 were optimal for beta-xylanase activity. The temperature optima for other hemicellulases were less than the xylanase with the exception of beta-mannosidase. The pH optima of the other hemicellulases were between 5.0 and 6.5. Xylanase was stable up to 70 degrees C and between pH 5.5 and 9.0 for 30 min whereas the other hemicellulase were less stable. These results suggest that the most suitable conditions for hydrolysis of hemicellulose by these enzymes would be at 50 degrees C and pH 6.0.

Isolation and characterization of the TIM10 homologue from the yeast Pichia sorbitophila: a putative component of the mitochondrial protein import system.

The Saccharomyces cerevisiae TIM10 gene encodes one of the few essential mitochondrial proteins that are required for the import of nuclear-encoded precursor proteins from the cytosol and their subsequent sorting into the different mitochondrial compartments. We have isolated and characterized a putative homologue of TIM10 from the halotolerant yeast Pichia sorbitophila. The Pichia TIM10 gene encodes a protein of 90 amino acids with 66% identity to S. cerevisiae Tim10p. It was capable of suppressing the temperature sensitivity of tim10-1 mutant in S. cerevisiae, suggesting that Pichia TIM10 is both a functional and structural homologue of S. cerevisiae TIM10. The putative Pichia TIM10 gene product contains all the four conserved cysteine residues and the two CX(3)C motifs typical of the Tim family proteins in the mitochondrial intermembrane space. Using anti-Tim10p serum, Western blots detected a protein of about 10 kDa, suggesting that the Pichia Tim10p is a mitochondrial protein. The results suggest that mitochondrial import and sorting systems might be also strongly conserved in other fungi. The coding sequence of the P. sorbitophila TIM10 has been deposited in the EMBL Nucleotide Sequence Database under Accession No. AJ243940.

Microbial MIP channels.

MIP channels occur in all classes of organism ranging from bacteria to man. There are two major categories of MIP channels, aquaporins and glycerol facilitators, which facilitate the diffusion across biological membranes of water or glycerol and other uncharged compounds, respectively. As a result of their involvement in osmoregulation and metabolism, MIP channels are believed to affect a wide range of biological processes.

Modification of the carbohydrate composition of sulfite pulp by purified and characterized beta-xylanase and beta-xylosidase of Aureobasidium pullulans.

Both beta-xylanase and beta-xylosidase were purified to homogeneity from a xylose-grown culture of Aureobasidium pullulans. Cellular distribution studies of enzyme activities revealed that beta-xylanase was an extracellular enzyme, during both the exponential and stationary phases, whereas beta-xylosidase was mostly periplasmic associated. The beta-xylanase exhibited very high specificity for xylan extracted from Eucalyptus grandis dissolving pulp, whereas the beta-xylosidase was only active on p-nitrophenyl xyloside and xylobiose. Comparison of kcat/Km ratios showed that the beta-xylanase hydrolyzed xylan from dissolving pulp 1.3, 2.1, and 2. 3 times more efficiently than Eucalyptus hemicellulose B, Eucalyptus hemicellulose A, and larchwood xylan, respectively. The beta-xylosidase exhibited a transxylosylation reaction during the hydrolysis of xylobiose. When applied on acid sulfite pulp, both enzymes released xylose and hydrolyzed xylan to a different extent. Although beta-xylosidase (0.4 U/g pulp) liberated more xylose from pulp than beta-xylanase (4.7 U/g pulp), it was responsible for only 3% of xylan solubilization. Treatment of pulp with beta-xylanase liberated 51.7 microgram of xylose/g and hydrolyzed 10% of xylan. The two enzymes acted additively on pulp and removed 12% of pulp xylan. A synergistic effect in terms of release of xylose from pulp was observed when the enzyme mixture of beta-xylanase and beta-xylosidase was supplemented with beta-mannanase. However, this did not result in further enzymatic degradation of pulp xylan. Both beta-xylanase and beta-xylosidase altered the carbohydrate composition of sulfite pulp by increasing the relative cellulose content at the expense of reduced hemicellulose content of pulp.

Fps1p controls the accumulation and release of the compatible solute glycerol in yeast osmoregulation.

The accumulation of compatible solutes, such as glycerol, in the yeast Saccharomyces cerevisiae, is a ubiquitous mechanism in cellular osmoregulation. Here, we demonstrate that yeast cells control glycerol accumulation in part via a regulated, Fps1p-mediated export of glycerol. Fps1p is a member of the MIP family of channel proteins most closely related to the bacterial glycerol facilitators. The protein is localized in the plasma membrane. The physiological role of Fps1p appears to be glycerol export rather than uptake. Fps1 delta mutants are sensitive to hypo-osmotic shock, demonstrating that osmolyte export is required for recovery from a sudden drop in external osmolarity. In wild-type cells, the glycerol transport rate is decreased by hyperosmotic shock and increased by hypo-osmotic shock on a subminute time scale. This regulation seems to be independent of the known yeast osmosensing HOG and PKC signalling pathways. Mutants lacking the unique hydrophilic N-terminal domain of Fps1p, or certain parts thereof, fail to reduce the glycerol transport rate after a hyperosmotic shock. Yeast cells carrying these constructs constitutively release glycerol and show a dominant hyperosmosensitivity, but compensate for glycerol loss after prolonged incubation by glycerol overproduction. Fps1p may be an example of a more widespread class of regulators of osmoadaptation, which control the cellular content and release of compatible solutes.

Physiological and genetic characterisation of osmosensitive mutants of Saccharomyes cerevisiae.

The screening of 20,000 Saccharomyces cerevisiae random mutants to identify genes involved in the osmotic stress response yielded 14 mutants whose growth was poor in the presence of elevated concentrations of NaCl and glucose. Most of the mutant strains were more sensitive to NaCl than to glucose at the equivalent water activity (aw) and were classified as salt-sensitive rather than osmosensitive. These mutants fell into 11 genetic complementation groups and were designated osr1-osr11 (osmotic stress response). All mutations were recessive and showed a clear 2(+) : 2(-) segregation of the salt-stress phenotype upon tetrad analysis when crossed to a wild-type strain. The complementation groups osr1, osr5 and osr11 were allelic to the genes PBS2, GPD1 and KAR3, respectively. Whereas intracellular and extracellular levels of glycerol increased in the wild-type strains when exposed to NaCl, all mutants demonstrated some increase in extracellular glycerol production upon salt stress, but a number of the mutants showed little or no increase in intracellular glycerol concentrations. The mutants had levels of glycerol-3-phosphate dehydrogenase, an enzyme induced by osmotic stress, either lower than or similar to those of the parent wild-type strain in the absence of osmotic stress. In the presence of NaCl, the increase in glycerol-3-phosphate dehydrogenase activity in the mutants did not match that of the parent wild-type strain. None of the mutants had defective ATPases or were sensitive to heat stress. It is evident from this study and from others that a wide spectrum of genes is involved in the osmotic stress response in S. cerevisiae.

Characteristics of Fps1-dependent and -independent glycerol transport in Saccharomyces cerevisiae.

Eadie-Hofstee plots of glycerol uptake in wild-type Saccharomyces cerevisiae W303-1A grown on glucose showed the presence of both saturable transport and simple diffusion, whereas an fps1delta mutant displayed only simple diffusion. Transformation of the fps1delta mutant with the glpF gene, which encodes glycerol transport in Escherichia coli, restored biphasic transport kinetics. Yeast extract-peptone-dextrose-grown wild-type cells had a higher passive diffusion constant than the fps1delta mutant, and ethanol enhanced the rate of proton diffusion to a greater extent in the wild type than in the fps1delta mutant. In addition, the lipid fraction of the fps1delta mutant contained a lower percentage of phospholipids and a higher percentage of glycolipids than that of the wild type. Fps1p, therefore, may be involved in the regulation of lipid metabolism in S. cerevisiae, affecting membrane permeability in addition to fulfilling its specific role in glycerol transport. Simultaneous uptake of glycerol and protons occurred in both glycerol- and ethanol-grown wild-type and fps1delta cells and resulted in the accumulation of glycerol at an inside-to-outside ratio of 12:1 to 15:1. Carbonyl cyanide m-chlorophenylhydrazone prevented glycerol accumulation in both strains and abolished transport in the fps1delta mutant grown on ethanol. Likewise, 2,4-dinitrophenol inhibited transport in glycerol-grown wild-type cells. These results indicate the presence of an Fps1p-dependent facilitated diffusion system in glucose-grown cells and an Fps1p-independent proton symport system in derepressed cells.

A Saccharomyces cerevisiae mutant defective in the kinesin-like protein Kar3 is sensitive to NaCl-stress.

Several mutants of Saccharomyces cerevisiae showing poor growth in the presence of elevated concentrations of NaCl were isolated to identify genes involved in the osmo-stress response. One of these mutants (WAY.5-4A-11; osr11) which showed a clear 2:2 segregation of the salt-stress phenotype upon tetrad analysis when crossed to a wild-type strain has been characterised. The mutation responsible for poor growth under salt-stress was recessive. The corresponding gene was cloned by complementation of the mutant phenotype and a 3.5-kb fragment was isolated. The sequence of this fragment matched that of KAR3, a gene previously identified to be involved in karyogamy and mitosis. Allelism of OSR11 to KAR3 was confirmed by tetrad analysis, and disruption mutants showed the same NaCl-phenotype as the original osr11 mutation. The disruption mutant was more sensitive to high sucrose concentrations than the original mutant was to high glucose concentrations. In a different genetic background (W303-1A), the kar3 disruptants were less sensitive to osmo-stress than the WAY.5-4A strain. Heat-stress, nitrogen-starvation and cultivation on ethanol failed to affect the growth of osr11 and kar3 mutants, pointing to a possible specific involvement of KAR3 in the osmotic-stress response. Microscopic studies showed that cell division of the kar3 mutants was impaired and NaCl-stress conditions aggravated the phenotype.