bluuebunny/crossref_metadata_2025 huggingface dataset:
Purification of Thermotoga maritima enzymes for the degradation of cellulosic materials
Authors: Bronnenmeier K , Kern A , Liebl W , Staudenbauer W L
Published: January 2020

A separation procedure for the analysis of the enzyme components of the hyperthermophilic bacterium Thermotoga maritima involved in cellulose and xylan degradation was developed. Resolution of the enzymes was achieved by a combination of fast protein liquid chromatography anion exchange and hydrophobic interaction chromatography. Enzyme fractions were assayed for hydrolysis of Avicel, carboxymethylcellulose (CMC), beta-glucan, laminarin, xylan, p-nitrophenyl-beta-D-glucoside, p-nitrophenyl-beta-D-cellobioside, p-nitrophenyl-beta-D-xyloside, p-nitrophenyl-alpha-L-arabinofuranoside, and 4-O-methyl-glucuronosyl-xylotriose. The activities of two cellulases, one laminarinase, one xylanase, two putative beta-D-xylosidases, alpha-D-glucuronidase, and alpha-L-arabinosidase were identified. Because of their selective retardation on a Superdex gel filtration column, the two cellulases could be purified to homogeneity. According to sodium dodecyl sulfate-polyacrylamide gel electrophoresis, molecular masses of 27 and 29 kDa, respectively, were determined for cellulase I and cellulase II. Maximal activities of both enzymes were observed at 95 degree C between pH 6.0 and 7.5. In the presence of 2.5 M NaC1 the purified enzymes retained about 90% of their initial activities after a 6-h incubation at 80 degree C. On the basis of its activity towards CMC, cellulase I was classified as endo-beta-1,4-glucanase. Cellulase II was able to attack Avicel in addition to CMC, beta-glucan, and p-nitrophenyl-beta-D-cellobioside. It releases cellobiose and cellotriose from Avicel. The latter product is further cleaved into glucose and cellobiose. Cellulase II may therefore be classified as exo-beta-1,4-glucanase.
The lysostaphin endopeptidase resistance gene (epr) specifies modification of peptidoglycan cross bridges in Staphylococcus simulans and Staphylococcus aureus
Authors: DeHart H P , Heath H E , Heath L S , LeBlanc P A , Sloan G L
Published: January 2020

Staphylococcus simulans biovar staphylolyticus produces an extracellular glycylglycine endopeptidase (lysostaphin) that lyses other staphylococci by hydrolyzing the cross bridges in their cell wall peptidoglycans. The genes for endopeptidase (end) and endopeptidase resistance (epr) reside on plasmid pACK1. An 8.4-kb fragment containing end was cloned into shuttle vector pL150 and was then introduced into Staphylococcus aureus RN4220. The recombinant S. aureus cells produced endopeptidase and were resistant to lysis by the enzyme, which indicated that the cloned fragment also contained epr. Treatments to remove accessory wall polymers (proteins, teichoic acids, and lipoteichoic acids) did not change the endopeptidase sensitivity of walls from strains of S. simulans biovar staphylolyticus or of S. aureus with and without epr. Immunological analyses of various wall fractions showed that there were epitopes associated with endopeptidase resistance and that these epitopes were found only on the peptidoglycans of epr+ strains of both species. Treatment of purified peptidoglycans with endopeptidase confirmed that resistance or susceptibility of both species was a property of the peptidoglycan itself. A comparison of the chemical compositions of these peptidoglycans revealed that cross bridges in the epr+ cells contained more serine and fewer glycine residues than those of cells without epr. The presence of the 8.4-kb fragment from pACK1 also increased the susceptibility of both species to methicillin.
Characterization of uptake and hydrolysis of fluorescein diacetate and carboxyfluorescein diacetate by intracellular esterases in Saccharomyces cerevisiae, which result in accumulation of fluorescent product
Authors: Breeuwer P , Drocourt J L , Bunschoten N , Zwietering M H , Rombouts F M , Abee T
Published: January 2020

Flow cytometry is a rapid and sensitive method which may be used for the detection of microorganisms in foods and drinks. A key requirement for this method is a sufficient fluorescence staining of the target cells. The mechanism of staining of the yeast Saccharomyces cerevisiae by fluorescein diacetate (FDA) and 5- (and 6-)carboxyfluorescein diacetate (cFDA) was studied in detail. The uptake rate of the prefluorochromes increased in direct proportion to the concentration and was not saturable, which suggests that transport occurs via a passive diffusion process. The permeability coefficient for cFDA was 1.3 x 10(-8) m s-1. Once inside the cell, the esters were hydrolyzed by intracellular esterases and their fluorescent products accumulated. FDA hydrolysis (at 40 degrees C) in cell extracts could be described by first-order reaction kinetics, and a rate constant (K) of 0.33 s-1 was calculated. Hydrolysis of cFDA (at 40 degrees C) in cell extracts was described by Michaelis-Menten kinetics with an apparent Vmax and Km of 12.3 nmol.min-1.mg of protein-1 and 0.29 mM, respectively. Accumulation of fluorescein was most likely limited by the esterase activity, since transport of FDA was faster than the hydrolysis rate. In contrast, accumulation of carboxyfluorescein was limited by the much slower transport of cFDA through the cell envelope. A simple mathematical model was developed to describe the fluorescence staining. The implications for optimal staining of yeast cells with FDA and cFDA are discussed.
Characterization of diverse 2,4-dichlorophenoxyacetic acid-degradative plasmids isolated from soil by complementation
Authors: Top E M , Holben W E , Forney L J
Published: January 2020

The diversity of 2,4-dichlorophenoxyacetic acid (2,4-D)-degradative plasmids in the microbial community of an agricultural soil was examined by complementation. This technique involved mixing a suitable Alcaligenes eutrophus (Rifr) recipient strain with the indigenous microbial populations extracted from soil. After incubation of this mixture, Rifr recipient strains which grow with 2,4-D as the only C source were selected. Two A. eutrophus strains were used as recipients: JMP228 (2,4-D-), which was previously derived from A. eutrophus JMP134 by curing of the 2,4-D-degradative plasmid pJP4, and JMP228 carrying pBH501aE (a plasmid derived from pJP4 by deletion of a large part of the tfdA gene which encodes the first step in the mineralization of 2,4-D). By using agricultural soil that had been treated with 2,4-D for several years, transconjugants were obtained with both recipients. However, when untreated control soil was used, no transconjugants were isolated. The various transconjugants had plasmids with seven different EcoRI restriction patterns. The corresponding plasmids are designated pEMT1 to pEMT7. Unlike pJP4, pEMT1 appeared not to be an IncP1 plasmid, but all the others (pEMT2 to pEMT7) belong to the IncP1 group. Hybridization with individual probes for the tfdA to tfdF genes of pJP4 demonstrated that all plasmids showed high degrees of homology to the tfdA gene. Only pEMT1 showed a high degree of homology to tfdB, tfdC, tfdD, tfdE, and tfdF, while the others showed only moderate degrees of homology to tfdB and low degrees of homology to tfdC.(ABSTRACT TRUNCATED AT 250 WORDS)
Resource islands predict the distribution of heterotrophic bacteria in chihuahuan desert soils
Authors: Herman R P , Provencio K R , Herrera-Matos J , Torrez R J
Published: January 2020

The resource island hypothesis predicts that soil resources such as nitrogen, phosphorus, and water will be distributed evenly in grasslands but have a patchy distribution focused around plants in shrublands. This hypothesis predicts that microorganism numbers will follow resources and be (i) evenly distributed in grasslands, (ii) concentrated around individual plants in shrublands, and (iii) higher where resources are higher when comparing the same vegetation type. This study enumerated total heterotrophic bacteria and a subset of these, the nitrogen-efficient guild (NEG), in three shrublands (playa fringe mesquite, tar bush, and creosote) and two grasslands (playa and bajada). Both heterotrophs and NEG members followed the distribution pattern predicted by the resource island hypothesis. There were no significant differences in heterotroph or NEG numbers comparing at-plant and interplant samples for both the playa and bajada grasslands. Furthermore, populations were generally higher in nutrient-rich playa grasslands than nutrient-poor bajada grasslands. In contrast, both heterotroph and NEG numbers were higher at shrubs than between shrubs in all three shrub sites. These results suggest that resource abundance in resource islands predicts the distribution of heterotrophic bacterial numbers in desert soils.
Activity and purification of linenscin OC2, an antibacterial substance produced by Brevibacterium linens OC2, an orange cheese coryneform bacterium
Authors: Maisnier-Patin S , Richard J
Published: January 2020

An orange cheese coryneform bacterium isolated from the surface of Gruyère of Comté and identified as Brevibacterium linens produces an antimicrobial substance designated linenscin OC2. This compound inhibits gram-positive food-borne pathogens including Staphylococcus aureus and Listeria monocytogenes but is not active against gram-negative bacteria. Linenscin OC2 caused viability loss and lysis of the test organism, Listeria innocua. Electron microscopy showed that linenscin OC2 induces protoplast formation and cell lysis. The native substance is resistant to proteolytic enzymes, heat, and organic solvents and stable over a wide range of pH. The molecular weight of the native linenscin OC2 was estimated by gel chromatography to be over 285,000. Linenscin OC2 was purified by ammonium sulfate precipitation, 2-propanol extraction, and reverse-phase chromatography. Direct detection of antimicrobial activity on a sodium dodecyl sulfate-polyacrylamide gel suggested an apparent molecular mass under 2,412 Da. Molecular mass was determined to be 1,196.7 Da by mass spectrometry. Amino acid composition analysis indicated that linenscin OC2 may contain 12 residues.
Influence of the RpoS (KatF) sigma factor on maintenance of viability and culturability of Escherichia coli and Salmonella typhimurium in seawater
Authors: Munro P M , Flatau G N , Clément R L , Gauthier M J
Published: January 2020

The sigma factor RpoS is essential for stationary-phase-specific, multiple-stress resistance. We compared the viabilities (direct viable counts) and culturabilities (colony counts) in seawater of Escherichia coli and Salmonella typhimurium strains and those in which rpoS was deleted or which were deficient in guanosine 3',5'-bispyrophosphate (ppGpp) synthesis (relA spoT). RpoS, possibly via ppGpp regulation, positively influenced the culturability of these bacteria in oligotrophic seawater. This influence closely depended, however, upon the growth state of the cells and the conditions under which they were grown prior to their transfer to seawater. The protective effect of RpoS was observed only in stationary-phase cells grown at low osmolarity. A previous exposure of cells to high osmolarity (0.5 M NaCl) also had a strong influence on the effect of RpoS on cell culturability in seawater. Both E. coli and S. typhimurium RpoS mutants lost the ability to acquire a high resistance to seawater, as observed in both logarithmic-phase and stationary-phase RpoS+ cells grown at high osmolarity. A previous growth of S. typhimurium cells under anoxic conditions also modulated the incidence of RpoS on their culturability. When grown anaerobically at high osmolarity, logarithmic-phase S. typhimurium RpoS+ cells partly lost their resistance to seawater through preadaptation to high osmolarity. When grown anaerobically at high osmolarity until stationary phase, both RpoS+ and RpoS- cells retained very high levels of both viability and culturability and then did not enter the viable but nonculturable state for over 8 days in seawater because of an RpoS-independent, unknown mechanism.
Survival of fecal microorganisms in marine and freshwater sediments
Authors: Davies C M , Long J A , Donald M , Ashbolt N J
Published: January 2020

The survival of culturable fecal coliforms, fecal streptococci, and Clostridium perfringens spores in freshwater and marine sediments from sites near sewage outfalls was studied. In laboratory studies, the inhibition of protozoan predators with cycloheximide allowed the fecal coliforms to grow in the sediment whereas the presence of predators resulted in a net die-off. C. perfringens spores did not appear either to be affected by predators or to die off throughout the duration of the experiments (28 days). Studies using in situ membrane diffusion chambers showed that, with the exception of C. perfringens, die-off of the test organisms to 10% of their initial numbers occurred in both marine and freshwater sediments within 85 days. The usual exponential decay model could not be applied to the sediment survival data, with the exception of the data for fecal streptococci. It was concluded that application of the usual decay model to the fecal coliform data was confounded by the complex relationship between growth and predation. The survival of seeded Escherichia coli in marine sediment was studied by using an enumeration method which detected viable but nonculturable bacteria. Throughout the duration of the experiment (68 days), the same proportion of E. coli organisms remained culturable, suggesting that sediment provides a favorable, nonstarvation environment for the bacteria.
Melanin production by a filamentous soil fungus in response to copper and localization of copper sulfide by sulfide-silver staining
Authors: Caesar-Tonthat T , Van Ommen K F , Geesey G G , Henson J M
Published: January 2020

Gaeumannomyces graminis var. graminis, a filamentous soil ascomycete, exhibited enhanced cell wall melanin accumulation when exposed to as little as 0.01 mM CuSO(inf4) in minimal broth culture. Because its synthesis was inhibited by tricyclazole, the melanin produced in response to copper was dihydroxynaphthalene melanin. An additional hyphal cell wall layer was visualized by electron microscopy when hyphae were grown in the presence of copper and fixed by cryotechniques. This electron-dense layer was between the outer cell wall and the inner chitin layer and doubled the total wall thickness. In copper-grown cells that were also treated with tricyclazole, this electron-dense layer was absent. Atomic absorption spectroscopy demonstrated that up to 3.5 mg of Cu per g of fungal mycelium was adsorbed or taken up by hyphae grown in 0.06 mM CuSO(inf4). A method for silver enhancement was developed to determine the cellular location of CuS. CuS was present in cell walls and septa of copper-grown hyphae. Electron microscopy of silver-stained cells suggested that CuS was associated with the melanin layer of cell walls.
Growth of Mycoplasma hyorhinis cultivar alpha on semisynthetic medium
Authors: Gardella R S , Del Giudice R A
Published: January 2020

Serial passage of Mycoplasma hyorhinis cultivar alpha (formerly noncultivable strains) has been accomplished in modified CMRL-1066 medium with fetal bovine serum. In modified CMRL-1066 liquid medium, cultivar alpha strains grow at a similar rate and to equivalent titers when compared with BTS-7, the type strain of the species. Further experiments with BTS-7 demonstrate that the extent of growth obtained in the semidefined medium was comparable to growth in conventional mycoplasma medium. M. hyorhinis strains, including cultivar alpha strains, grow in serial passage when fetal bovine serum is replaced with bovine serum albumin, palmitic acid, and cholesterol. The results of these studies show that M. hyorhinis cultivar alpha strains are not nutritionally more fastidious than other mycoplasmas but that they are noncultivable on standard mycoplasma media because they are sensitive to high levels of inhibition activity by medium components.
Spectrofluorometric assay for rapid detection of total and fecal coliforms from surface water
Authors: Park S J , Lee E J , Lee D H , Lee S H , Kim S J
Published: January 2020

With a spectrofluorometer, the length of the incubation time required in the fluorogenic assay was reduced to 12 h. The threshold emissions for reading the fluorogenic reaction by the spectrofluorometer were 5 and 10 U for lauryl tryptose broth media containing 4-methylumbelliferyl-beta-D-galactoside and 4-methylumbelliferyl-beta-D-glucuronide, respectively. These two kinds of threshold units were equivalent to known concentrations of free 4-methylumbelliferone of 2.5 and 6 microM, respectively, in lauryl tryptose broth media.
Characterization of genetically transformed Saccharomyces cerevisiae baker's yeasts able to metabolize melibiose
Authors: Gasent-Ramírez J M , Codón A C , Benítez T
Published: January 2020

Three transformant (Mel+) Saccharomyces cerevisiae baker's yeast strains, CT-Mel, VS-Mel, and DADI-Mel, have been characterized. The strains, which originally lacked alpha-galactosidase activity (Mel-), had been transformed with a DNA fragment which possessed an ILV1-SMR1 allele of the ILV2 gene and a MEL1 gene. The three transformed strains showed growth rates similar to those of the untransformed controls in both minimal and semi-industrial (molasses) media. The alpha-galactosidase specific activity of strain CT-Mel was twice that of VS-Mel and DADI-Mel. The yield, YX/S (milligrams of protein per milligram of substrate), in minimal medium with raffinose as the carbon source was 2.5 times higher in the transformed strains than in the controls and was 1.5 times higher in CT-Mel than in VS-Mel and DADI-Mel. When molasses was used, YX/S (milligrams of protein per milliliter of culture) increased 8% when the transformed strains CT-Mel and DADI-Mel were used instead of the controls. Whereas no viable spores were recovered from either DADI-Mel or VS-Mel tetrads, genetic analysis carried out with CT-Mel indicated that the MEL1 gene has been integrated in two of three homologous loci. Analysis of the DNA content by flow cytometry indicated that strain CT-Mel was 3n, whereas VS-Mel was 2n and DADI-Mel was 1.5n. Electrophoretic karyotype and Southern blot analyses of the transformed strains showed that the MEL1 gene has been integrated in the same chromosomic band, probably chromosome XIII, in the three strains.(ABSTRACT TRUNCATED AT 250 WORDS)
Genetic diversity of Desulfovibrio spp. in environmental samples analyzed by denaturing gradient gel electrophoresis of [NiFe] hydrogenase gene fragments
Authors: Wawer C , Muyzer G
Published: January 2020

The genetic diversity of Desulfovibrio species in environmental samples was determined by denaturing gradient gel electrophoresis (DGGE) of PCR-amplified [NiFe] hydrogenase gene fragments. Five different PCR primers were designed after comparative analysis of [NiFe] hydrogenase gene sequences from three Desulfovibrio species. These primers were tested in different combinations on the genomic DNAs of a variety of hydrogenase-containing and hydrogenase-lacking bacteria. One primer pair was found to be specific for Desulfovibrio species only, while the others gave positive results with other bacteria also. By using this specific primer pair, we were able to amplify the [NiFe] hydrogenase genes of DNAs isolated from environmental samples and to detect the presence of Desulfovibrio species in these samples. However, only after DGGE analysis of these PCR products could the number of different Desulfovibrio species within the samples be determined. DGGE analysis of PCR products from different bioreactors demonstrated up to two bands, while at least five distinguishable bands were detected in a microbial mat sample. Because these bands most likely represent as many Desulfovibrio species present in these samples, we conclude that the genetic diversity of Desulfovibrio species in the natural microbial mat is far greater than that in the experimental bioreactors.
Occurrence of fecal indicator bacteria in surface waters and the subsurface aquifer in Key Largo, Florida
Authors: Paul J H , Rose J B , Jiang S , Kellogg C , Shinn E A
Published: January 2020

Sewage waste disposal facilities in the Florida Keys include septic tanks and individual package plants in place of municipal collection facilities in most locations. In Key Largo, both facilities discharge into the extremely porous Key Largo limestone. To determine whether there was potential contamination of the subsurface aquifer and nearby coastal surface waters by such waste disposal practices, we examined the presence of microbial indicators commonly found in sewage (fecal coliforms, Clostridium perfringens, and enterococci) and aquatic microbial parameters (viral direct counts, bacterial direct counts, chlorophyll a, and marine vibriophage) in injection well effluent, monitoring wells that followed a transect from onshore to offshore, and surface waters above these wells in two separate locations in Key Largo in August 1993 and March 1994. Effluent and waters from onshore shallow monitoring wells (1.8- to 3.7-m depth) contained two or all three of the fecal indicators in all three samples taken, whereas deeper wells (10.7- to 12.2-m depth) at these same sites contained few or none. The presence of fecal indicators was found in two of five nearshore wells (i.e., those that were < or = 1.8 miles [< or = 2.9 km] from shore), whereas offshore wells (> or = 2.1 to 5.7 miles [< or = 3.4 to 9.2 km] from shore) showed little sign of contamination. Indicators were also found in surface waters in a canal in Key Largo and in offshore surface waters in March but not in August. Collectively, these results suggest that fecal contamination of the shallow onshore aquifer, parts of the nearshore aquifer, and certain surface waters has occurred.(ABSTRACT TRUNCATED AT 250 WORDS)
Effect of Rhamnolipid (Biosurfactant) Structure on Solubilization and Biodegradation of n-Alkanes
Authors: Zhang Y , Miller R M
Published: January 2020

A study to quantify the effect of rhamnolipid biosurfactant structure on the degradation of alkanes by a variety of Pseudomonas isolates was conducted. Two dirhamnolipids were studied, a methyl ester form (dR-Me) and an acid form (dR-A). These rhamnolipids have different properties with respect to interfacial tension, solubility, and charge. For example, the interfacial tension between hexadecane and water was decreased to <0.1 dyne/cm by the dR-Me but was only decreased to 5 dyne/cm by the dR-A. Solubilization and biodegradation of two alkanes in different physical states, liquid and solid, were determined at dirhamnolipid concentrations ranging from 0.01 to 0.1 mM (7 to 70 mg/liter). The dR-Me markedly enhanced hexadecane (liquid) and octadecane (solid) degradation by seven different Pseudomonas strains. For an eighth strain tested, which exhibited extremely high cell surface hydrophobicity, hexadecane degradation was enhanced but octadecane degradation was inhibited. The dR-A also enhanced hexadecane degradation by all degraders but did so more modestly than the dR-Me. For octadecane, the dR-A only enhanced degradation by strains with low cell surface hydrophobicity.
Kinetics and Thermostability of NADP-Isocitrate Dehydrogenase from Cephalosporium acremonium
Authors: Olano J , de Arriaga D , Busto F , Soler J
Published: January 2020

NADP-isocitrate dehydrogenase [isocitrate:NADP(sup+) oxidoreductase (decarboxylating); EC 1.1.1.42] was purified from Cephalosporium acremonium as a single species. The enzyme is a dimer of 140 kDa with identical subunits of 75 kDa. The existence of a monomer-dimer equilibrium is apparent as revealed by an enzyme dilution approach. The chelate complex of the tribasic form of isocitrate and Mg(sup2+) is the true substrate. The V(infmax) depends on a basic form of an ionizable group of the enzyme-substrate complex with a pK(infes) (pK of the enzyme-substrate complex) of 6.9 and a (Delta)H(infion) (activation enthalpy) of -2 (plusmn) 0.4 kcal mol(sup-1) (ca. 8 (plusmn) 2 kJ mol(sup-1)). The enzyme showed maximum activity at 60(deg)C, an unusually high temperature for a nonthermophilic fungus. The thermodynamic parameters for isocitrate oxidative decarboxylation and for the binding of isocitrate and NADP(sup+) were calculated. We analyzed the kinetic thermal stability of the enzyme at pH 6.5 and 7.6. It was inactivated above 40(deg)C following a first-order kinetics. The presence of 12 mM Mg(sup2+) plus 10 mM dl-isocitrate led to 100% protection of enzyme activity against inactivation at 60(deg)C for 120 min. Removal of either or both compounds led to activity loss. A greater stabilizing role for Mg(sup2+) was seen at pH 6.5 than at pH 7.6, whereas the stabilizing effect of isocitrate was not dependent on pH.
Compatible Solutes in the Thermophilic Bacteria Rhodothermus marinus and "Thermus thermophilus"
Authors: Nunes O C , Manaia C M , Da Costa M S , Santos H
Published: January 2020

(sup13)C nuclear magnetic resonance spectroscopy and (sup1)H nuclear magnetic resonance spectroscopy were used to identify and quantify the organic solutes of several strains of halophilic or halotolerant thermophilic bacteria. Two strains of Rhodothermus marinus and four strains of "Thermus thermophilus" grown in complex medium containing NaCl were examined. 2-O-Mannosylglycerate was a major compatible solute in all strains: the Thermus strains accumulated the (beta)-anomer only, whereas both anomers were found in R. marinus. 2-O-(beta)-mannosylglycerate and 2-O-(alpha)-mannosylglycerate were the major compatible solutes in R. marinus. The former was the predominant solute in cells grown in 2.0 and 4.0% NaCl-containing medium, while the latter was the predominant compatible solute at higher salinities. Glutamate, trehalose, and glucose were also present as minor components. The intracellular K(sup+) concentration, as determined by (sup39)K nuclear magnetic resonance spectroscopy, in R. marinus increased with salinity and was sufficient to balance the negative charges of the mannosylglycerate. In addition to 2-O-(beta)-mannosylglycerate, trehalose was a major compatible solute of "T. thermophilus." 2-O-(beta)-Mannosylglycerate was the main solute in medium containing 1.0 or 2.0% NaCl, while trehalose predominated in cells grown in medium supplemented with 3.0 or 4.0% NaCl. Glycine betaine, in lower concentrations, was also detected in two "T. thermophilus" strains. This is the first report of mannosylglycerate as a compatible solute in bacteria.
PCR-mediated analysis of lignocellulolytic gene transcription by Phanerochaete chrysosporium: substrate-dependent differential expression within gene families
Authors: Broda P , Birch P R , Brooks P R , Sims P F
Published: January 2020

We compare the kinetics of appearance of supernatant enzyme activities (lignin peroxidase, manganese peroxidase, and cellulase) and gene expression (LIG, mnp, and cbhI gene families and the unique cbhII gene) in Phanerochaete chrysosporium ME446 when grown on four different carbon sources: ball-milled straw, representing the natural substrate lignocellulose; Avicel as a crystalline cellulose; and high and low concentrations of glucose, in all cases with limiting nitrogen. PCR-based technology utilizing pairs of primers specific for particular genes showed that there is differential expression between and within the families. There were a number of instances of mRNA species being present only on a single day, implying tight regulation of lignocellulose degradation at the mRNA level. The patterns of extracellular enzyme activities and mnp and cbh gene expression are similar whereas LIG gene expression can be detected when no corresponding enzyme activity is observed in the extracellular supernatant. The enzyme produced under these conditions is presumably sequestered by the mycelium and is likely to be functionally significant. Another striking result is that cellulose, in the form of Avicel, elicits the expression of three LIG gene for which there is no expression under the same conditions with the other carbon sources.
Effect of corn and peanut cultivation on soil populations of Aspergillus flavus and A. parasiticus in southwestern Georgia
Authors: Horn B W , Greene R L , Dorner J W
Published: January 2020

The effect of corn and peanut cultivation on the proportion of Aspergillus flavus to A. parasiticus in soil was examined. Soil populations were monitored in three fields during three different years in southwestern Georgia. Each field was planted in both peanuts and corn, and soil was sampled within plots for each crop. A. flavus and A. parasiticus were present in similar proportions in plots from all fields at the beginning of the growing season. A. terreus, A. niger, and A. fumigatus were the other dominant aspergilli in soil. Fields A and B did not show drought stress in peanut or corn plants, and soil populations of A. flavus and A. parasiticus remained stable during the course of the year. In field C, drought stress in corn plants with associated A. flavus infection and aflatoxin contamination greatly increased soil populations of A. flavus relative to A. parasiticus upon dispersal of corn debris to the soil surface by a combine harvester. Colonization of organic debris after it has been added to the soil may maintain soil populations of A. parasiticus despite lower crop infection.
Synthetic Lignin Mineralization by Ceriporiopsis subvermispora Is Inhibited by an Increase in the pH of the Cultures Resulting from Fungal Growth
Authors: Tapia J , Vicuna R
Published: January 2020

(sup14)C-synthetic lignin mineralization by the basidiomycete Ceriporiopsis subvermispora occurs at the highest rate (about 30% after 29 days) in liquid cultures containing 1% glucose and a growth-limiting amount (1 mM) of ammonium tartrate. The titers of manganese peroxidase (MnP) and laccase are lower in these cultures than in cultures containing 1% glucose and 10 mM ammonium tartrate, where the extent of lignin mineralization in the same period is only about 15%. The inverse correlation between enzyme activity and lignin mineralization is also observed when ammonium tartrate is replaced by ammonium chloride or Casamino Acids as the source of nitrogen. This phenomenon can be explained by a gradual increase in the pH of the medium that takes place only in the cultures with high nitrogen concentrations. Supporting this finding, when cultures with 1 mM ammonium tartrate were grown at different pHs, (sup14)CO(inf2) evolved more rapidly from those with pH values near the optimum for MnP activity. On the other hand, (sup14)CO(inf2) evolution from cultures containing 1% glucose supplemented with 1 mM ammonium tartrate plus 9 mM sodium tartrate was as low as that from cultures with a high ammonium tartrate concentration. Since the changes in the pH of these cultures were not as pronounced as those in cultures containing high nitrogen concentrations, tartrate itself may also be contributing to limit the extent of lignin mineralization. Considering that pH instability seems to constitute a common feature of fungal cultures, precautions must be taken to avoid underestimation of their ligninolytic efficiencies.
Analysis of antigens present in the extracellular products and cell surface of Vibrio anguillarum serotypes O1, O2, and O3
Authors: Santos Y , Pazos F , Band�n I , Toranzo A E
Published: January 2020

Antigens present in the extracellular products (ECP) and cell walls of strains of Vibrio anguillarum of serotypes O1, O2, and O3 isolated from different fish species in distinct geographic areas were characterized. The usefulness of slide agglutination, dot blot assay, and quantitative agglutination for subtyping V. anguillarum serovars was also evaluated. The three serological assays used to establish the serogroups within V. anguillarum isolates demonstrated that serotype O1 constitutes a homogeneous group, whereas within serotypes O2 and O3, two different patterns of serological reactions were detected. Among the three serological methods used, only dot blot and quantitative agglutination assays differentiated subgroups within serotypes O2 and O3 with unabsorbed sera. Electrophoretic analysis and immunoblot assays of cell envelope and ECP components showed that strains belonging to serotype O1 possessed immunologically related lipopolysaccharide (LPS) and proteins, while V. anguillarum isolates grouped in serotypes O2 and O3 exhibited internal heterogeneity in their LPS and protein banding patterns. On the other hand, although the LPS present in the ECP and those obtained from cell envelopes of V. anguillarum strains showed apparently different gel patterns, a strong relationship between both types of LPS was seen by immunoblot assay. From these results, it can be concluded that V. anguillarum strains representative of each of the antigenic groups (O1, O2 alpha, O2 beta, O3A, and O3B) and their ECPs should be included in the formulation of vaccines against vibriosis in areas where the three serotypes coexist.
Occurrence of Propionibacterium freudenreichii bacteriophages in swiss cheese
Authors: Gautier M , Rouault A , Sommer P , Briandet R
Published: January 2020

We isolated bacteriophages active against Propionibacterium freudenreichii from 16 of 32 swiss cheese samples. Bacteriophage concentrations ranged from 14 to 7 x 10(5) PFU/g, depending on the sample and the sensitive strain used for detection. Only a few strains, 8 of the 44 strains of P. freudenreichii in our collection, were sensitive. We observed that multiplication of bacteriophages occurred in the cheese loaf during multiplication of propionibacteria in a warm curing room, but it seems that these bacteriophages have no adverse effect on the development of the propionic flora. We also found that sensitive cells, originating from either the starter or the cheese-making milk, were present at a high level (10(9) CFU/g) in the cheese.
Entry into, and resuscitation from, the viable but nonculturable state by Vibrio vulnificus in an estuarine environment
Authors: Oliver J D , Hite F , McDougald D , Andon N L , Simpson L M
Published: January 2020

Using plate counts, total cell counts, and direct viable counts, we examined the fate of cells of Vibrio vulnificus placed into natural estuarine waters during both winter and summer months. Cells inoculated into membrane diffusion chambers and placed into estuarine waters entered into a viable but nonculturable (VBNC) state in January and February, when the water temperatures were low (average, < 15 degrees C). In contrast, when cells in the VBNC state were placed into the same waters in the warmer months of August through November (average water temperature of ca. 21 degrees C), the cells appeared to undergo a rapid (typically, within 24 h) resuscitation to the fully culturable state. These results were independent of whether the cells were in the logarithmic or stationary phase and whether they were encapsulated or not. This study indicates that the inability to isolate V. vulnificus from cold estuarine sites may be accounted for by entrance of the cells into a VBNC state and that recovery from this state in natural environments may result from a temperature upshift.
Isolation and characterization of pediocin L50, a new bacteriocin from Pediococcus acidilactici with a broad inhibitory spectrum
Authors: Cintas L M , Rodriguez J M , Fernandez M F , Sletten K , Nes I F , Hernandez P E , Holo H
Published: January 2020

Lactic acid bacteria were isolated from Spanish dry-fermented sausages and screened for bacteriocin production. About 10% of the isolates produced antimicrobial substances when grown on solid media, but only 2% produced detectable activity in liquid media. Strain L50, identified as Pediococcus acidilactici, showed the strongest inhibitory activity and was active against members of all of the gram-positive genera tested. The strain produced a heat-stable bacteriocin when grown at 8 to 32 degrees C but not at 45 degrees C. The bacteriocin was purified to homogeneity. Its mass was determined to be 5,250.11 +/- 0.30 by electrospray mass spectrometry. The N terminus of the bacteriocin was blocked for sequencing by Edman degradation, but a partial sequence of 42 amino acids was obtained after cleavage of the peptide by cyanogen bromide. The sequence showed no similarity to those of other bacteriocins. Pediocin L50 appears to contain modified amino acids but not lanthionine or methyl-lanthionine.
Temperature-dependent membrane fatty acid and cell physiology changes in coccoid forms of Campylobacter jejuni
Authors: Hazeleger W C , Janse J D , Koenraad P M , Beumer R R , Rombouts F M , Abee T
Published: January 2020

The effect of temperature and the availability of nutrients on the transition of spiral Campylobacter jejuni cells to coccoid forms was investigated. Ageing of spiral C. jejuni cells in either nutrient-poor or nutrient-rich environments resulted in the formation of nonculturable coccoid cells at 4, 12, and 25 degrees C after different periods, with the cells incubated at 4 degrees C in nutrient-deficient media remaining culturable the longest. To study the phenomenon, ATP levels, protein profiles, and fatty acid compositions were monitored under conditions where the transition from spiral to coccoid cells occurred. During storage, the levels of intracellular ATP were highest in cells incubated at low temperatures (4 and 12 degrees C) and remained constant after a small initial decrease. During the transformation from spiral to coccoid forms, no alteration in protein profiles could be detected; indeed, inhibition of protein synthesis by chloramphenicol did not influence the transition. Furthermore, DNA damage by gamma irradiation had no effect on the process. Membrane fatty acid composition of cocci formed at low temperatures was found to be almost identical to that of spiral cells, whereas that of cocci formed at 25 degrees C was clearly different. Combining these results, it is concluded that the formation of cocci is not an active process. However, distinctions between cocci formed at different temperatures were observed. Cocci formed at 4 degrees C show characteristics comparable to those of spirals, and these cocci may well play a role in the contamination cycle of C. jejuni.(ABSTRACT TRUNCATED AT 250 WORDS)
Glucosyltransferase from Streptococcus sobrinus Catalyzes Glucosylation of Catechin
Authors: Nakahara K , Kontani M , Ono H , Kodama T , Tanaka T , Ooshima T , Hamada S
Published: January 2020

We previously showed that the polymeric forms of polyphenols present in oolong tea extract exhibited strong inhibitory activities against glucosyltransferases (GTases) of mutans streptococci, while green tea extract, which is rich in catechins, did not show such GTase-inhibitory activities. In this study, (+)-catechin [2-(3,4-dihydroxyphenyl)-3,4-dihydro-2H-1-benzopyran-3,5,7-triol] was found to be glucosylated by the GTase of Streptococcus sobrinus 6715 with sucrose as the substrate, and the product was identified as 4(prm1)-O-(alpha)-d-glucopyranosyl-(+)-catechin, with a molecular weight of 452. The (alpha)-glucosylated (+)-catechin did not exhibit significant inhibition of glucan synthesis from sucrose by the GTase, which is in contrast to the polymeric polyphenols isolated from oolong tea leaves.
Spatial heterogeneity in the distribution of denitrifying bacteria associated with denitrification activity zones
Authors: Murray R E , Feig Y S , Tiedje J M
Published: January 2020

Denitrifying enzyme activity (DEA) in artificially established denitrification activity zones was, on average, 3.5-fold greater than in bulk soil. Denitrifier biomass was between 1 and 4 orders of magnitude greater in activity zones. Denitrifier specific activity was lower in activity zones and more similar to specific activity values reported for pure cultures of denitrifying bacteria added to soil. Measurements of DEA and biomass carried out on a homogeneous subsample derived from a soil core containing a denitrification activity zone will overestimate the DEA and biomass of the bulk soil and underestimate the DEA and biomass of the activity zone.
Minimizing Ergosterol Loss during Preanalytical Handling and Shipping of Samples of Plant Litter
Authors: Newell S Y
Published: January 2020

Common preliminary treatments of samples of decaying material can involve changes in water content (e.g., via storage in relatively dry air or rinsing) that could conceivably result in loss or gain of fungal membranes and, consequently, ergosterol. A related problem is that collecting of ergosterol content data from widely distributed locales by shipment of samples ideally requires an inexpensive, safe alternative to submerging the samples in methanol for prevention of ergosterol loss. Experimental testing showed that fungal occupants of decaying salt marsh grass leaves did not exhibit loss or gain of ergosterol during air drying (to a water potential of <-8 MPa) or rewetting (to -0.8 MPa). Wet leaves of one grass species (Juncus roemerianus, black needlerush) could be fixed and dried for shipment by microwaving, or by fully drying after alcoholic or pentane fixation, without ergosterol loss, but those of smooth cordgrass (Spartina alterniflora) lost about 40% of their ergosterol content by all three of these drying methods. Ergosterol content of wet leaves of cordgrass could be maintained by alcoholic fixation and subsequent drying down to a thin film of alcohol.
Bacteriolytic activity caused by the presence of a novel lactococcal plasmid encoding lactococcins A, B, and M
Authors: Morgan S , Ross R P , Hill C
Published: January 2020

Lactococcus lactis subsp. lactis biovar diacetylactis DPC938 was identified as a bacteriocin-producing strain which exhibited a bacteriolytic effect on other lactococci. Lysis of such target strains was associated with decreases in optical density and release of the intracellular enzyme lactate dehydrogenase. DPC938 exhibits cross-immunity to L. lactis subsp. cremoris 9B4 (M.J. van Belkum, B.J. Hayema, A. Geis, J. Kok, and G. Venema, Appl. Environ. Microbiol. 55:1187-1191, 1989), a strain which produces the bacteriocins lactococcins A, B, and M. Genetic analyses revealed that a 15.5-kb region of DNA encoding these bacteriocins is highly conserved in 9B4, DPC938, and DPC3286, an overproducing derivative of DPC938. This region is located on a 72- and a 78-kb nonmobilizable plasmid in DPC938 and DPC3286, respectively. The bacteriolytic effect exhibited by DPC938 and DPC3286 on sensitive cultures is most probably due to the concerted action of all three bacteriocins. Since these cultures exhibit a lytic effect on lactococci, they have a potential application in the dairy industry as accelerators of starter lysis and hence accelerators of cheese ripening.
Purification and Characterization of a 1,4-Benzoquinone Reductase from the Basidiomycete Phanerochaete chrysosporium
Authors: Brock B J , Rieble S , Gold M H
Published: January 2020

An intracellular, soluble 1,4-benzoquinone reductase was purified from agitated cultures of Phanerochaete chrysosporium and characterized. The quinone reductase was expressed in cultures grown under both nitrogen-sufficient and nitrogen-limiting (12 and 1.2 mM ammonium tartrate) conditions. The protein was purified to homogeneity by using ammonium sulfate fractionation, hydrophobic interaction, and ion-exchange and blue-agarose affinity chromatographies. The native flavin mononucleotide-containing protein, pI 4.3, has a molecular mass of 44 kDa as determined by gel filtration. The protein has a subunit molecular mass of ;sim22 kDa as determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The quinone reductase exhibits a broad pH optimum between 5.0 and 6.5 and a temperature optimum of 30(deg)C. The enzyme catalyzes the two-electron reduction of several quinones and other electron acceptors utilizing either NADH or NADPH as an electron donor. The apparent K(infm) for 2-methoxy-1,4-benzoquinone is 2.4 (mu)M, and the apparent k(infcat) is 4.4 x 10(sup5) s(sup-1). Enzyme activity is strongly inhibited by Cibacron blue 3GA and by dicumarol.
Phylogenetic comparison of two polycyclic aromatic hydrocarbon-degrading mycobacteria
Authors: Govindaswami M , Feldhake D J , Kinkle B K , Mindell D P , Loper J C
Published: January 2020

Two mycobacterial strains previously isolated from fossil-fuel-contaminated environments and shown to degrade four- and/or five-ring polycyclic aromatic hydrocarbons were further characterized. The two strains, PYR-I and RJGII-135, had similar growth characteristics, colony morphologies, and scotochromogenic pigmentations. DNA amplification fingerprints obtained with total genomic DNA indicated some strain similarities but with several distinctly different bands. Moreover, phylogenetic analysis based upon essentially full-length 16S rRNA gene sequences separates the two strains as distinct species within the fast-growing group of mycobacteria. Although both strains are thermosensitive, strain PYR-I has the bulged U between positions 184 and 193 characteristic of thermotolerant mycobacteria. Both strains are of potential use for reintroduction into and bioremediation of polycyclic aromatic hydrocarbon-contaminated soils.
2,4-Dichlorophenoxyacetic acid-degrading bacteria contain mosaics of catabolic genes
Authors: Fulthorpe R R , McGowan C , Maltseva O V , Holben W E , Tiedje J M
Published: January 2020

DNA from 32 2,4-dichlorophenoxyacetic acid (2,4-D)-degrading bacteria from diverse locations was probed with the first three genes of the well-known 2,4-D degradation pathway found in Alcaligenes eutrophus JMP134(pJP4). The majority of strains did not show high levels of homology to the first three genes of the 2,4-D degradation pathway, tfdA, -B, and -C. Most strains showed combinations of tfdA-, B-, and C-like elements that exhibited various degrees of homology to the gene probes. Strains having the same genomic fingerprints (as determined by repetitive extragenic palindromic PCR) exhibited the same hybridization pattern regardless of the geographic origin of the strain, with the exception of a strain isolated from Puerto Rico. This strain had the same genomic fingerprint as that of numerous other strains in the collection but differed in its hybridization against the tfdA gene probe. Members of the beta subdivision of the Proteobacteria class, specifically Alcaligenes, Burkholderia, and Rhodoferax species, carried DNA fragments with 60% or more sequence similarity to tfdA of pJP4, and most carried fragments showing at least 60% homology to tfdB. However, many strains did not hybridize with tfdC, although they exhibited chlorocatechol dioxygenase activity. Members of the alpha subdivision of the Proteobacteria class, mostly of the genus Sphingomonas, did not hybridize to either tfdA or tfdC, but some hybridized at low stringency to tfdB. The data suggest that extensive interspecies transfer of a variety of homologous degradative genes has been involved in the evolution of 2,4-D-degrading bacteria.
Comparison of phenotypical and molecular methods for the identification of bacterial strains isolated from a deep subsurface environment
Authors: Boivin-Jahns V , Bianchi A , Ruimy R , Garcin J , Daumas S , Christen R
Published: January 2020

Seventy-four bacterial strains were freshly isolated from a mine gallery. Using these bacteria, we have investigated how a molecular identification based on the analysis of small subunit rDNA sequences would compare in terms of precision and reliability to a more classical comparison of phenotypical descriptions (100 morphological and physiological traits). Our data clearly showed that a phylogenetic analysis of small subunit rDNA sequences is more efficient than classical phenotypic methods for the identification of bacterial strains freshly isolated from a natural environment, because occurrences of misidentification are very much decreased by this method. The lack of rDNA sequences for many described species is probably the major cause of a few failures in molecular identification, as the completeness of the database of small subunit rDNA sequences holds much importance in the degree of uncertainty in such identifications.