bluuebunny/crossref_metadata_2025 huggingface dataset:
Assessment of natural mycorrhizal potential in a desertified semiarid ecosystem
Authors: Requena N , Jeffries P , Barea J M
Published: January 2020

A survey of the natural mycorrhizal potential has been carried out in a representative area of a desertified semiarid ecosystem in the southeast of Spain. Many indigenous plants from the field site were mycorrhizal, including the dominant Anthyllis cytisoides, which had high levels of colonization by arbuscular mycorrhizal fungi (AMF). Low numbers of AMF spores were present in the soil, although a range of species, including Scutellospora calospora, Glomus coronatum, Glomus constrictum, and several Acaulospora species, was represented. Soil infectivities, as determined by a soil dilution method, were similar for most plants tested but were significantly lower for Anthyllis cytisoides. Nevertheless, when a less disruptive method to determine soil infectivity was used, the importance of the mycelial network in maintaining the infectivity of soil under perennial shrubs, such as Anthyllis cytisoides, was highlighted. Seasonal variations in the mycorrhizal infectivity showed that it was higher towards the end of the summer period than in midwinter. In screening trials in a greenhouse, the indigenous AMF did not significantly improve the growth of plants compared with that of noninoculated controls. Augmentation of the soil with an inoculum of Glomus intraradices resulted in improved growth of Anthyllis cytisoides in both sterile and nonsterile conditions, in contrast to results obtained following inoculation with Glomus mosseae or another Glomus sp. Our findings suggest that the indigenous inoculum levels of AMF are inadequate to support an extensive revegetation program in the absence of an additional mycorrhizal inoculum.
Hydrogen Peroxide Production as a Limiting Factor in Xenobiotic Compound Oxidation by Nitrogen-Sufficient Cultures of Bjerkandera sp. Strain BOS55 Overproducing Peroxidases
Authors: Kotterman M , Wasseveld R A , Field J A
Published: January 2020

The overproduction of ligninolytic peroxidase by the N-deregulated white rot fungus Bjerkandera sp. strain BOS55 under nitrogen-sufficient conditions had no noteworthy effect on the oxidation of anthracene or the decolorization of the polymeric aromatic dye Poly R-478 in 6-day-old cultures. Only when the endogenous production of H(inf2)O(inf2) was increased by the addition of extra oxygen and glucose could a 2.5-fold increase in the anthracene oxidation rate and a 6-fold increase in the Poly R-478 decolorization rate be observed in high-N cultures with 10- to 35-fold higher peroxidase activities than N-limited cultures. Further increase of the H(inf2)O(inf2) generation rate in high-N cultures with glucose oxidase led to an additional 3.5-fold increase in the anthracene oxidation rate (350 mg liter(sup-1) day(sup-1)) and a 10-fold increase in the Poly R-478 decolorization rate. These results indicate that xenobiotic compound oxidation by white rot fungi cannot be improved by overproducing peroxidases without increasing the endogenous production of H(inf2)O(inf2). The absence of Mn, which decreased the manganese peroxidase titers and increased the lignin peroxidase titers, was associated with up to 95% improvements in the anthracene oxidation rate. The simultaneous presence of Mn and veratryl alcohol was observed to have a synergistic negative effect on the oxidation of anthracene and the decolorization of Poly R-478.
Isolation, partial characterization, and mode of action of Acidocin J1132, a two-component bacteriocin produced by Lactobacillus acidophilus JCM 1132
Authors: Tahara T , Oshimura M , Umezawa C , Kanatani K
Published: January 2020

Lactobacillus acidophilus JCM 1132 produces a heat-stable, two-component bacteriocin designated acidocin J1132 that has a narrow inhibitory spectrum. Maximum production of acidocin J1132 in MRS broth was detected at pH 5.0. Acidocin J1132 was purified by ammonium sulfate precipitation and sequential cation exchange and reversed-phase chromatographies. Acidocin J1132 activity was associated with two components, termed alpha and beta. On the basis of N-terminal amino acid sequencing and the molecular masses of the alpha and beta components, it is interpreted that the compounds differ by an additional glycine residue in the beta component. Both alpha and beta had inhibitory activity, and an increase in activity by the complementary action of the two components was observed. Acidocin J1132 is bactericidal and dissipates the membrane potential and the pH gradient in sensitive cells, which affect such proton motive force-dependent processes as amino acid transport. Acidocin J1132 also caused efflux of preaccumulated amino acid taken up via a unidirectional ATP-driven transport system. Secondary structure prediction revealed the presence of an amphiphilic alpha-helix region that could form hydrophilic pores. These results suggest that acidocin J1132 is a pore-forming bacteriocin that creates cell membrane channels through the "barrel-stave" mechanism.
Siderophore-mediated iron acquisition mechanisms in Vibrio vulnificus biotype 2
Authors: Biosca E G , Fouz B , Alcaide E , Amaro C
Published: January 2020

Vibrio vulnificus biotype 2 is a primary pathogen for eels and, as has recently been suggested, an opportunistic pathogen for humans. In this study we have investigated the ability of V. vulnificus biotype 2 to obtain iron by siderophore-mediated mechanisms and evaluated the importance of free iron in vibriosis. The virulence degree for eels was dependent on iron availability from host fluids, as was revealed by a reduction in the 50% lethal dose for iron-overloaded eels. This biotype produced both phenolate- and hydroxamate-type siderophores of an unknown nature and two new outer membrane proteins of around 84 and 72 kDa in response to iron starvation. No alterations in lipopolysaccharide patterns were detected in response to iron stress. Finally, our data suggest that V. vulnificus biotype 2 uses the hydroxamate-type siderophore for removal of iron from transferrin rather than relying on a receptor for this iron-binding protein.
Natural variability and diurnal fluctuations within the bacteriophage population of the rumen
Authors: Swain R A , Nolan J V , Klieve A V
Published: January 2020

To investigate the impact of nutritional and environmental factors on bacteriophage activity in the rumen, it is first valuable to determine the extent of natural variations and fluctuations in phage populations from different animal species, and from animals located together and separately, and variation in animals over time. Differences in phage populations between sheep on different diets, between sheep and goats, and within the rumen over time were investigated by using pulsed-field gel electrophoresis and comparing total phage DNA in ruminal fluid. It was found that no two individuals had similar DNA banding patterns, even when similarly fed and penned together, indicating there is considerable individual diversity in phage populations between animals. Despite these individual differences, the quantities, but not the banding patterns, of phage DNA were similar for animals within groups but varied between groups, suggesting that nutritional factors may influence overall phage activity in the rumen. In sheep fed once daily, a distinct diurnal variation in the phage population was observed. Two hours postfeeding, total phage DNA dropped to its lowest level. The phage population then increased, reaching a maximal level 8 to 10 h postfeeding before declining over the next 4 h to reach a stable concentration for the rest of the cycle. The general trend in phage DNA concentration appeared similar to previously recorded diurnal fluctuations in ruminal bacterial populations in cattle fed once daily.
Detection of Dekkera-Brettanomyces strains in sherry by a nested PCR method
Authors: Ibeas J I , Lozano I , Perdigones F , Jimenez J
Published: January 2020

Brettanomyces sp. and its ascosporogenous sexual state, Dekkera sp., have been well documented as spoilage microorganisms, usually associated with barrel-aged red wines. In this report, we describe the genetic characterization, on the basis of DNA content per cell, electrophoretic karyotyping, and mitochondrial DNA restriction patterns, of a Dekkera yeast strain isolated from sherries and of a number of other Brettanomyces and Dekkera strains. By using a genomic DNA fragment of the isolated Dekkera strain, we developed a two-step PCR method which directs the specific amplification of target DNA from this strain and from other Brettanomyces-Dekkera strains. The method efficiently amplified the target DNA from intact cells, obviating DNA isolation, and yielded a detection limit of fewer than 10 yeast cells in contaminated samples of sherry.
Duckling toxicity and the production of fumonisin and moniliformin by isolates in the A and E mating populations of Gibberella fujikuroi (Fusarium moniliforme)
Authors: Leslie J F , Marasas W F , Shephard G S , Sydenham E W , Stockenström S , Thiel P G
Published: January 2020

Two biological species of Gibberella fujikuroi (A and F mating populations) share the Fusarium moniliforme anamorph. Twenty strains of each of these biological species were tested for the ability to produce fumonisins B1, B2, and B3 and moniliformin and for toxicity to 1-day-old ducklings. Most of the members of the A mating population (19 of 20 strains) produced more than 60 micrograms of total fumonisins per g, whereas only 3 of 20 members of the F mating population produced more than trace levels of these toxins and none produced more than 40 micrograms of total fumonisins per g. In addition, only 3 of 20 members of the A mating population produced more than 1 microgram of moniliformin per g (and none produced more than 175 micrograms/g), while all 20 strains of the F mating population produced more than 85 micrograms of this toxin per g and 1 strain produced 10,345 micrograms/g. The duckling toxicity profiles of the strains of the two mating populations were similar, however, and the level of either toxin by itself was not strongly correlated with duckling toxicity. On the basis of our data we think that it is likely that the members of both of these mating populations produce additional toxins that have yet to be chemically identified. These toxins may act singly or synergistically with other compounds to induce the observed duckling toxicity.
Geosmin and Related Volatiles in Bioreactor-Cultured Streptomyces citreus CBS 109.60
Authors: Pollak F C , Berger R G
Published: January 2020

Streptomyces citreus CBS 109.60 produced geosmin and a complex pattern of other volatile compounds during cultivation in a 2.5-liter laboratory bioreactor. Volatiles were isolated from disrupted cells, from the culture medium, and from the waste air of the bioreactor by adsorption on Lewatit OC 1064MD. Quantitative and qualitative analyses were carried out using capillary gas chromatography and coupled gas chromatography-mass spectroscopy. S. citreus produced 56 volatile compounds, which were mainly terpenoids but also included aliphatic ketones, alcohols, esters, pyrazines, furan(on)es, and aromatic types during the growth phase. The major components were geosmin and a germacradienol. A biosynthetic pathway for geosmin including eudesmanolides is proposed.
Detection of Vibrio vulnificus biotypes 1 and 2 in eels and oysters by PCR amplification
Authors: Coleman S S , Melanson D M , Biosca E G , Oliver J D
Published: January 2020

DNA extraction procedures and PCR conditions to detect Vibrio vulnificus cells naturally occurring in oysters were developed. In addition, PCR amplification of V. vulnificus from oysters seeded with biotype 1 cells was demonstrated. By the methods described, V. vulnificus cells on a medium (colistin-polymyxin B-cellobiose agar) selective for this pathogen were detectable in oysters harvested in January and March, containing no culturable cells (< 67 CFU/g), as well as in oysters harvested in May and June, containing culturable cells. It was possible to complete DNA extraction, PCR, and gel electrophoresis within 10 h by using the protocol described for oysters. V. vulnificus biotype 2 cells were also detected in eel tissues that had been infected with this strain and subsequently preserved in formalin. The protocol used for detection of V. vulnificus cells in eels required less than 5 h to complete. Optimum MgCl2 concentrations for the PCR of V. vulnificus from oysters and eels were different, although the same primer pair was used for both. This is the first report on the detection of cells of V. vulnificus naturally present in shellfish and represents a potentially powerful method for monitoring this important human and eel pathogen.
Pathways of acetate, propionate, and butyrate formation by the human fecal microbial flora
Authors: Miller T L , Wolin M J
Published: January 2020

The pathways of short-chain fatty acid (SCFA; acetate, propionate, and butyrate) formation from glucose were determined for the human fecal microbial communities of two subjects. The pathways were identified by radioisotope analysis of the SCFA and CO2 obtained after incubation of fecal suspensions with glucose under 20% CO2 with [1-14C]glucose, [3,4-14C]glucose, or 14CO2. Acetate was chemically degraded to learn the labeling of the methyl and carboxyl carbons. The labeling of CO2 and acetate showed that the major route of glucose catabolism was the Embden-Meyerhof-Parnas pathway, with production of CO2 from pyruvate carboxyl carbon. Labeling of the methyl and carboxyl carbons of acetate by 14CO2 or [3,4-14C]glucose proved that acetate was formed from CO2 by the Wood-Ljungdahl pathway. CO2 reduction accounted for about one-third of the acetate formed by suspensions from subject 1 and about one-fourth of the acetate formed by suspensions from subject 2. Propionate was formed by a CO2 fixation pathway, and butyrate was formed by classical routes of acetyl-S coenzyme A condensation. The amount of CO2 formed from [1-14C] glucose and acetate labeling patterns obtained with the other 14C precursors indicated that the Entner-Doudoroff, transketolase-transaldolase, and heterolactic pathways were not significant. Fermentation of cabbage cellulose by subject 1 followed the same pathways as were used for glucose. The results with suspensions from subject 2 suggested that some radioactive acetate was formed from the C-3 of glucose by the Bifidobacterium pathway.
Cloning and expression of a gene encoding a bacterial enzyme for decontamination of organophosphorus nerve agents and nucleotide sequence of the enzyme
Authors: Cheng T C , Harvey S P , Chen G L
Published: January 2020

Organophosphorus acid (OPA) anhydrolase enzymes have been found in a wide variety of prokaryotic and eukaryotic organisms. Interest in these enzymes has been prompted by their ability to catalyze the hydrolysis of toxic organophosphorus cholinesterase-inhibiting compounds, including pesticides and chemical nerve agents. The natural substrates for these enzymes are unknown. The gene (opaA) which encodes an OPA anhydrolase (OPAA-2) was isolated from an Alteromonas sp. strain JD6.5 EcoRI-lambda ZAPII chromosomal library expressed in Escherichia coli and identified by immunodetection with anti-OPAA-2 serum. OPA anhydrolase activity expressed by the immunopositive recombinant clones was demonstrated by using diisopropylfluorophosphate (DFP) as a substrate. A comparison of the recombinant enzyme with native, purified OPAA-2 showed they had the same apparent molecular mass (60 kDa), antigenic properties, and enzyme activity against DFP and the chemical nerve agents sarin, soman, and O-cyclohexyl methylphosphonofluoridate. The gene expressing this activity was found in a 1.74-kb PstI-HindIII fragment of the original 6.1-kb EcoRI DNA insert. The nucleotide sequence of this PstI-HindIII fragment revealed an open reading frame of 1,551 nucleotides, coding for a protein of 517 amino acid residues. Amino acid sequence comparison of OPAA-2 with the protein database showed that OPAA-2 is similar to a 647-amino-acid sequence produced by an open reading frame which appears to be the E. coli pepQ gene. Further comparison of OPAA-2, the E. coli PepQ protein sequence, E. coli aminopeptidase P, and human prolidase showed regions of different degrees of similarity or functionally conserved amino acid substitutions. These findings, along with preliminary data confirming the presence of prolidase activity expressed by OPAA-2, suggest that the OPAA-2 enzyme may, in nature, be used in peptide metabolism.
Direct detection of Brucella spp. in raw milk by PCR and reverse hybridization with 16S-23S rRNA spacer probes
Authors: Rijpens N P , Jannes G , Van Asbroeck M , Rossau R , Herman L M
Published: January 2020

The 16S-23S rRNA spacer regions of Brucella abortus, B. melitensis, and B. suis were cloned and subcloned after PCR amplification. Sequence analysis of the inserts revealed a spacer of about 800 bp with very high ( > 99%) homology among the three species examined. Two genus-specific primer pairs, BRU-P5-BRU-P8 and BRU-P6-BRU-P7, that could be used in a nested PCR format and three genus-specific DNA probes, BRU-ICG2, BRU-ICG3, and BRU-ICG4, were deduced from this spacer. The specificity and sensitivity of both primer sets and probes were examined by testing them against a collection of 18 Brucella strains and 56 strains from other relevant taxa by using PCR and the Line Probe Assay (LiPA), respectively. A method for direct detection of Brucella spp. in 1 ml of raw milk was developed on the basis of enzymatic treatment of the milk components and subsequent PCR and LiPA hybridization. After a single PCR, sensitivities of 2.8 x 10(5) and 2.8 x 10(4) CFU/ml were obtained for detection by agarose gel electrophoresis and LiPA, respectively. Nested PCR yielded a sensitivity of 2.8 x 10(2) CFU/ml for both methods.
Distribution of thermophilic marine sulfate reducers in north sea oil field waters and oil reservoirs
Authors: Nilsen R K , Beeder J , Thorstenson T , Torsvik T
Published: January 2020

The distribution of thermophilic marine sulfate reducers in produced oil reservoir waters from the Gullfaks oil field in the Norwegian sector of the North Sea was investigated by using enrichment cultures and genus-specific fluorescent antibodies produced against the genera Archaeoglobus, Desulfotomaculum, and Thermodesulforhabdus. The thermophilic marine sulfate reducers in this environment could mainly be classified as species belonging to the genera Archaeoglobus and Thermodesulforhabdus. In addition, some unidentified sulfate reducers were present. Culturable thermophilic Desulfotomaculum strains were not detected. Specific strains of thermophilic sulfate reducers inhabited different parts of the oil reservoir. No correlation between the duration of seawater injection and the numbers of thermophilic sulfate reducers in the produced waters was observed. Neither was there any correlation between the concentration of hydrogen sulfide and the numbers of thermophilic sulfate reducers. The results indicate that thermophilic and hyperthermophilic sulfate reducers are indigenous to North Sea oil field reservoirs and that they belong to a deep subterranean biosphere.
Differential susceptibility of aeromonads and coliforms to cefsulodin
Authors: Alonso J L , Amoros I , Alonso M A
Published: January 2020

Cefsulodin was evaluated as a potential selective agent for aeromonads. Resistance of Aeromonas and coliform isolates was determined by using a standard disk diffusion technique. A total of 119 Aeromonas and 78 coliform strains were isolated. For 102 of 130 [corrected] Aeromonas isolates (environmental and reference strains), the MIC of cefsulodin was < 8 micrograms/ml. Results of MIC tests by the agar dilution method showed that a concentration of cefsulodin of 10 micrograms/ml or less inhibited the growth of 96% of isolates. In comparison, for 81 of 94 coliform isolates (environmental and reference strains), the MIC of cefsulodin was > 32 micrograms/ml. Because cefsulodin suppresses growth of Aeromonas and other oxidase-positive organisms, total coliform (TC) and Escherichia coli counts on Chromocult Coliform agar (CC agar) without cefsulodin and on CC agar with 10 mg of cefsulodin per liter (CC-CFS) were compared. Variance analysis of data from 14 sewage-polluted irrigation water specimens did not demonstrate any statistically significant difference in the enumeration of E. coli with CC and CC-CFS media. On average, the CC agar recovered 2.46 times as many TCs as CC-CFS. However, Aeromonas colonies made up an average of 58.6% of the TC counts on CC agar. Because no Aeromonas spp. were recovered on CC-CFS, background interference was eliminated and the counts that were obtained reflected more accurately the number of TCs. Results of this study suggest that cefsulodin may be a useful selective agent against Aeromonas spp. which should be included in coliform chromogenic media when high levels of accompanying flora are expected.
Analysis of Lignin-Polysaccharide Complexes Formed during Grass Lignin Degradation by Cultures of Pleurotus Species
Authors: Gutierrez A , Bocchini P , Galletti G C , Martinez A T
Published: January 2020

A brown material, precipitable with ethanol, was formed during wheat straw and lignin degradation by liquid cultures of different species of Pleurotus. Fourier transform infrared spectroscopy and cross-polarization and magic-angle-spinning (sup13)C nuclear magnetic resonance spectroscopy showed that most of the precipitable material was formed from exopolysaccharide secreted by the fungus but it also contained an aromatic fraction. The results of acid hydrolysis, methylation analysis, and Smith degradation indicated that the major exopolysaccharide produced by these fungi is a (1(symbl)3)-(beta)-glucan branched at C-6 every two or three residues along the main chain. The presence of lignin or straw in the culture medium had little effect on the composition and structure of the extracellular polysaccharide. Cross-polarization and magic-angle-spinning (sup13)C nuclear magnetic resonance spectroscopy provided an estimation of the aromatic content of the lignin-polysaccharide complexes, assigning 20% of the total (sup13)C signal in the material recovered from cultures of Pleurotus eryngii in lignin medium to aromatic carbon. Analytical pyrolysis indicated that the aromatic fractions of the lignin-polysaccharide complexes were derived from lignin, since products characteristic of pyrolytic breakdown of H (p-hydroxyphenylpropane), G (guaiacylpropane), and S (syringylpropane) lignin units were identified. These complexes cannot be fractionated by treatment with polyvinylpyrrolidone or extraction with lignin solvents, suggesting that the two polymers were chemically linked. Moreover, differences in composition with respect to the original lignin indicated that this macromolecule was modified by the fungi during the process of formation of the lignin-polysaccharide complexes.
Molecular microbial diversity of an agricultural soil in Wisconsin
Authors: Borneman J , Skroch P W , O'Sullivan K M , Palus J A , Rumjanek N G , Jansen J L , Nienhuis J , Triplett E W
Published: January 2020

A culture-independent survey of the soil microbial diversity in a clover-grass pasture in southern Wisconsin was conducted by sequence analysis of a universal clone library of genes coding for small-subunit rRNA (rDNA). A rapid and efficient method for extraction of DNA from soils which resulted in highly purified DNA with minimal shearing was developed. Universal small-subunit-rRNA primers were used to amplify DNA extracted from the pasture soil. The PCR products were cloned into pGEM-T, and either hypervariable or conserved regions were sequenced. The relationships of 124 sequences to those of cultured organisms of known phylogeny were determined. Of the 124 clones sequenced, 98.4% were from the domain Bacteria. Two of the rDNA sequences were derived from eukaryotic organelles. Two of the 124 sequences were of nuclear origin, one being fungal and the other a plant sequence. No sequences of the domain Archaea were found. Within the domain, Bacteria, three kingdoms were highly represented: the Proteobacteria (16.1%), the Cytophaga-Flexibacter-Bacteroides group (21.8%), and the low G+C-content gram-positive group (21.8%). Some kingdoms, such as the Thermotogales, the green nonsulfur group, Fusobacteria, and the Spirochaetes, were absent. A large number of the sequences (39.4%) were distributed among several clades that are not among the major taxa described by Olsen et al. (G.J. Olsen, C.R. Woese, and R. Overbeek, J. Bacteriol., 176:1-6, 1994). From the alignments of the sequence data, distance matrices were calculated to display the enormous microbial diversity found in this soil in two ways, as phylogenetic trees and as multidimensional-scaling plots.
Increased inactivation of Saccharomyces cerevisiae by protraction of UV irradiation
Authors: Sommer R , Haider T , Cabaj A , Heidenreich E , Kundi M
Published: January 2020

The principle of equi-effectivity of the product of intensity and exposure time (principle of Bunsen-Roscoe) of UV irradiation has been assumed to be valid for the inactivation of microorganisms in general. Earlier studies claimed higher survival of Escherichia coli B/r with fractionated irradiation compared with single-exposure survival. However, data on the inactivation effect of protraction of UV irradiation are not available. By means of a specially designed UV irradiation apparatus which secured absolute UV dose measurements throughout the experiments, the effects of variation of UV irradiation intensities (253.7 nm) and exposure times were tested on the inactivation of a bacterial virus (Staphylococcus aureus phage A994), a vegetative bacterial strain (E. coli ATCC 25922), and bacterial spores (Bacillus subtilis ATCC 6633) as well as three haploid laboratory strains (RC43a, YNN281, and YNN282) and two diploid strains (commercial bakery yeast strain and laboratory strain YNN281 x YNN282) or yeast (Saccharomyces cerevisiae) and spores of the latter diploid yeast strain. Each test organism was exposed to three UV intensities (0.02, 0.2, and 2 W/m2), with corresponding exposure times resulting in three dose levels for each intensity. Differences in inactivation rates were tested by analyses of variance and Newman-Keuls tests. Virus and bacteria showed no differences in inactivation rates by variation of intensities and exposure times within selected UV doses; hence, the principle of Bunsen-Roscoe could not be rejected for these strains. However, in the eukaryotic test strains of S. cerevisiae longer exposure times with lower intensities led to enhanced inactivation in both haploid and diploid strains, with a more pronounced effect in the diploid yeast strains, whereas in yeast spores in this dose rate effect could not be observed.
Phylogenetic probes for analyzing abundance and spatial organization of nitrifying bacteria
Authors: Mobarry B K , Wagner M , Urbain V , Rittmann B E , Stahl D A
Published: January 2020

A hierarchical set of five 16S rRNA-targeted oligonucleotide DNA probes for phylogenetically defined groups of autotrophic ammonia- and nitrite-oxidizing bacteria was developed for environmental and determinative studies. Hybridization conditions were established for each probe by using temperature dissociation profiles of target and closely related nontarget organisms to document specificity. Environmental application was demonstrated by quantitative slot blot hybridization and whole-cell hybridization of nitrifying activated sludge and biofilm samples. Results obtained with both techniques suggested the occurrence of novel populations of ammonia oxidizers. In situ hybridization experiments revealed that Nitrobacter and Nitrosomonas species occurred in clusters and frequently were in contact with each other within sludge flocs.
A fast method for the confirmation of fecal streptococci from M-enterococcus medium
Authors: Figueras M J , Inza I , Polo F L , Feliu M T , Guarro J
Published: January 2020

M-Enterococcus medium, used for the detection and enumeration of fecal streptococci from different types of water, demonstrated a very low specificity. We propose the transfer of membrane filters to bile-esculin medium as a confirmation technique. It has proved to be more reliable, faster to perform, and more effective than the random selection of colonies, which is recommended for confirmation in Standard Methods for the Examination of Water and Wastewater (A.E. Greenberg, L.S. Clesceri, and A.D. Eaton, ed., 1992).
Comparison of Fatty Acid Methyl Ester Analysis with the Use of API 20E and NFT Strips for Identification of Aquatic Bacteria
Authors: Brown B J , Leff L G
Published: January 2020

Aquatic bacteria grown on MacConkey agar and modified nutrient agar were identified by using API 20E and NFT strips and fatty acid methyl ester (FAME) analysis. Identifications agreed at the species level 35.7% of the time when API 20E strips and FAME analysis were used and in 4.3% of the cases when API NFT strips and FAME analysis were used. These techniques require further development before extended use in ecological studies.
Isolation of Alcaligenes sp. strain L6 at low oxygen concentrations and degradation of 3-chlorobenzoate via a pathway not involving (chloro)catechols
Authors: Krooneman J , Wieringa E B , Moore E R , Gerritse J , Prins R A , Gottschal J C
Published: January 2020

Isolations of 3-chlorobenzoate (3CBA)-degrading aerobic bacteria under reduced O2 partial pressures yielded organisms which metabolized 3CBA via the gentisate or the protocatechuate pathway rather than via the catechol route. The 3CBA metabolism of one of these isolates, L6, which was identified as an Alcaligenes species, was studied in more detail. Resting-cell suspensions of L6 pregrown on 3CBA oxidized all known aromatic intermediates of both the gentisate and the protocatechuate pathways. Neither growth on nor respiration of catechol could be detected. Chloride production from 3CBA by L6 was strictly oxygen dependent. Cell-free extracts of 3CBA-grown L6 cells exhibited no catechol dioxygenase activity but possessed protocatechuate 3,4-dioxygenase, gentisate dioxygenase, and maleylpyruvate isomerase activities instead. In continuous culture with 3CBA as the sole growth substrate, strain L6 demonstrated an increased oxygen affinity with decreasing steady-state oxygen concentrations.
Characterization of a highly repeated DNA sequence (SC1) from the arbuscular mycorrhizal fungus Scutellospora castanea and its detection in planta
Authors: Zeze A , Hosny M , Gianinazzi-Pearson V , Dulieu H
Published: January 2020

A highly repeated DNA sequence from the genome of an arbuscular mycorrhizal fungus has been isolated and characterized. This 1,202-bp sequence (SC1) represents about 0.24% of the Scutellospora castanea genome, estimated to be 1 pg by flow cytometry. The sequence was shown to be a Scutellospora-specific probe in Southern blots and dot blot hybridizations. After complete sequencing of SC1, PCR primers were generated and used to amplify a 907-bp fragment from spores of S. castanea or from colonized Allium porrum roots. No amplification products were obtained with DNA from either spores or mycorrhizal root of other species of arbuscular mycorrhizal fungi. These primers were sufficiently specific for unequivocal detection of S. castanea in planta.
Purification and characterization of a fibrinolytic enzyme produced from Bacillus sp. strain CK 11-4 screened from Chungkook-Jang
Authors: Kim W , Choi K , Kim Y , Park H , Choi J , Lee Y , Oh H , Kwon I , Lee S
Published: January 2020

Bacillus sp. strain CK 11-4, which produces a strongly fibrinolytic enzyme, was screened from Chungkook-Jang, a traditional Korean fermented-soybean sauce. The fibrinolytic enzyme (CK) was purified from supernatant of Bacillus sp. strain CK 11-4 culture broth and showed thermophilic, hydrophilic, and strong fibrinolytic activity. The optimum temperature and pH were 70 degrees C and 10.5, respectively, and the molecular weight was 28,200 as determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The first 14 amino acids of the N-terminal sequence of CK are Ala-Gin-Thr-Val-Pro-Tyr-Gly-Ile-Pro-Leu-Ile-Lys-Ala-Asp. This sequence is identical to that of subtilisin Carlsberg and different from that of nattokinase, but CK showed a level of fibrinolytic activity that was about eight times higher than that of subtilisin Carlsberg. The amidolytic activity of CK increased about twofold at the initial state of the reaction when CK enzyme was added to a mixture of plasminogen and substrate (H-D-Val-Leu-Lys-pNA). A similar result was also obtained from fibrin plate analysis.
A simple filtration method to remove plankton-associated Vibrio cholerae in raw water supplies in developing countries
Authors: Huq A , Xu B , Chowdhury M A , Islam M S , Montilla R , Colwell R R
Published: January 2020

Plankton to which cells of Vibrio cholerae O1 and/or O139 were attached was introduced into 0.5% Instant Ocean microcosms maintained at 25 degrees C. The bulk of the plankton and associated particulates was removed with a filter constructed from either nylon net and one of several different types of sari material, the latter being very inexpensive and readily available in villages in Bangladesh, where V. cholerae is endemic. V. cholerae was enumerated before and after filtration to evaluate the efficiency of the filtration procedure. The results obtained indicate that 99% of V. cholerae, i.e., those cells attached to plankton, were removed from the water samples. Epidemic strains of V. cholerae O1 and O139 from various geographical sources, including Bangladesh, Brazil, India, and Mexico, were included in the experiments. Removal of vibrios from water by this simple filtration method was found to yield consistent results with all strains examined in this study. Thus, it is concluded that a simple filtration procedure involving the use of domestic sari material can reduce the number of cholera vibrios attached to plankton in raw water from ponds and rivers commonly used for drinking. Since untreated water from such sources serves as drinking water for millions of people living in developing countries (e.g., Bangladesh), filtration should prove effective at reducing the incidence and severity of outbreaks, especially in places that lack fuel wood for boiling water and/or municipal water treatment plants. The results of this study provide the basis for determining such reductions, which are to be carried out in the near future.
Bioconversion of 7-Aminocephalosporanic Acid by Intact Rhodotorula glutinis Cells
Authors: Sakai Y , Abe T , Ohbayashi Y , Isaka K , Yamamoto K , Tani Y , Kato N
Published: January 2020

When 7-aminocephalosporanic acid (7-ACA) was used as a single carbon source in the enrichment culture medium for screening 7-ACA-degrading microorganisms, pink yeast colonies appeared frequently, and these were identified as Rhodotorula glutinis. These intact R. glutinis cells converted (i) 7-ACA to deacetyl-7-ACA (7-ADACA) and (ii) monochloroacetyl-7-ACA to monochloroacetyl-7-ADACA at sufficiently high levels to be of commercial interest. Acetylation of 7-ADACA to 7-ACA, the reverse reaction of hydrolysis in an organic medium with methyl acetate as an acetyl donor, was also demonstrated.
Culture variability associated with the U.S. Environmental Protection Agency Tuberculocidal Activity Test Method
Authors: Robison R A , Osguthorpe R J , Carroll S J , Leavitt R W , Schaalje G B , Ascenzi J M
Published: January 2020

Tuberculosis continues to be a major world health threat. The etiologic agent is among the vegetative organisms most resistant to chemical disinfection. Tuberculocidal efficacy testing for regulatory approval of chemical germicides has evolved considerably over the past decade. A method currently in use is the Environmental Protection Agency Tuberculocidal Activity Test Method, a suspension test using a Mycobacterium bovis culture grown under specific conditions and stored frozen until used. Differing tuberculocidal label claims on products with similar formulations have raised questions concerning the equivalence of test suspensions prepared by different laboratories. Five M. bovis suspensions from laboratories currently performing this test were compared against a battery of three disinfectants at a single test site. A significant difference between test cultures was found, with two of the five exhibiting a significant difference from the other three and also from each other. There was a significant culture-by-disinfectant interaction, indicating that the five cultures did not respond in a consistent manner across the different disinfectants used. However, these differences were due to cultures that were not prepared in accordance with the standard procedure or otherwise did not meet the test suspension criteria. In addition, a 0.55% sodium hypochlorite solution was found to be a sensitive indicator of culture variability. These data reinforce the need to adhere to published procedures and guidelines when growing and preparing a tuberculocidal test suspension and shed light on the variables associated with this type of testing.
Bactericidal mode of action of plantaricin C
Authors: Gonzalez B , Glaasker E , Kunji E , Driessen A , Suarez J E , Konings W N
Published: January 2020

Plantaricin C is a bacteriocin produced by Lactobacillus plantarum LL441 that kills sensitive cells by acting on the cytoplasmic membrane. In contrast to its lack of impact on immune cells, plantaricin C dissipates the proton motive force and inhibits amino acid transport in sensitive cells. In proteoliposomes, plantaricin C dissipates the transmembrane electrical potential, and in liposomes, it elicits efflux of entrapped carboxy-fluorescein. It is concluded that plantaricin C is a pore-forming bacteriocin that functions in a voltage-independent manner and does not require a specific protein receptor in the target membrane.
Diversity of Nitrogen Fixation Genes in the Symbiotic Intestinal Microflora of the Termite Reticulitermes speratus
Authors: Ohkuma M , Noda S , Usami R , Horikoshi K , Kudo T
Published: January 2020

The diversity of nitrogen-fixing organisms in the symbiotic intestinal microflora of a lower termite, Reticulitermes speratus, was investigated without culturing the resident microorganisms. Fragments of the nifH gene, which encodes the dinitrogenase reductase, were directly amplified from the DNA of the mixed microbial population in the termite gut and were clonally isolated. The phylogenetic analysis of the nifH product amino acid sequences showed that there was a remarkable diversity of nitrogenase genes in the termite gut. A large number of the termite nifH sequences were most closely related to those of a firmicute, Clostridium pasteurianum, with a few being most closely related to either the (gamma) subclass of the proteobacteria or a sequence of Desulfovibrio gigas. Some of the others were distantly related to those of the bacteria and were seemingly derived from the domain Archaea. The phylogenetic positions of these nifH sequences corresponded to those of genera found during a previous determination of rRNA-based phylogeny of the termite intestinal microbial community, of which a majority consisted of new, yet-uncultivated species. The results revealed that we have little knowledge of the organisms responsible for nitrogen fixation in termites.
Production, Identification, and Toxicity of (gamma)-Decalactone and 4-Hydroxydecanoic Acid from Sporidiobolus spp
Authors: Feron G , Dufosse L , Pierard E , Bonnarme P , Quere J L , Spinnler H
Published: January 2020

During the bioconversion of ricinoleic acid to (gamma)-decalactone under controlled pH conditions, Sporidiobolus salmonicolor produced only the lactone form, while Sporidiobolus ruinenii produced both the lactone form and a precursor. By using gas chromatography-mass spectrometry and gas chromatography-Fourier transform infrared analysis techniques, the precursor was identified as 4-hydroxydecanoic acid. The levels of production in the presence of high concentrations of ricinoleic acid methyl ester differed in the two Sporidiobolus species. This difference was due on the one hand to the high sensitivity of S. salmonicolor to the lactone and on the other hand to the high level of 4-hydroxydecanoic acid produced by S. ruinenii. 4-Hydroxydecanoic acid is much less toxic to the microorganisms than the lactone. In contrast to S. ruinenii, S. salmonicolor is not able to catabolize 4-hydroxydecanoic acid.
Phosphorus-31 and carbon-13 nuclear magnetic resonance studies of glucose and xylose metabolism in cell suspensions and agarose-immobilized cultures of Pichia stipitis and Saccharomyces cerevisiae
Authors: Lohmeier-Vogel E M , McIntyre D D , Vogel H J
Published: January 2020

The metabolism of glucose and xylose as a function of oxygenation in Pichia stipitis and Saccharomyces cerevisiae cell suspensions was studied by 31P and 13C nuclear magnetic resonance spectroscopy. The rate of both glucose and xylose metabolism was slightly higher and the production of ethanol was slightly lower in aerobic than in anoxic cell suspensions of P. stipitis. As well, the cytoplasmic pH of oxygenated cells was more alkaline than that of nonoxygenated cells. In contrast, in S. cerevisiae, the intracellular pH and the rate of glucose metabolism and ethanol production were the same under aerobic and anoxic conditions. Agarose-immobilized Pichia stipitis was able to metabolize xylose or glucose for 24 to 60 h at rates and with theoretical yields of ethanol similar to those obtained with anoxic cell suspensions. Cell growth within the beads, however, was severely compromised. The intracellular pH [pH(int)] of the entrapped cells fell to more acidic pH values in the course of the perfusions relative to corresponding cell suspensions. Of importance was the observation that no enhancement in the rate of carbohydrate metabolism occurred in response to changes in the pH(int) value. In contrast to P. stipitis, agarose-immobilized Saccharomyces cerevisiae showed a dramatic twofold increase in its ability to metabolize glucose in the immobilized state relative to cell suspensions. This strain was also able to grow within the beads, although the doubling time for the entrapped cells was longer, by a factor of 2, than the value obtained for log-phase batch cultures. Initially, the pH(int) of the immobilized cells was more alkaline than was observed with the corresponding S. cerevisiae cell suspensions; however, over time, the intracellular pH became increasingly acidic. As with immobilized P. stipitis, however, the pH(int) did not play a key role in controlling the rate of glucose metabolism.
Keratin Degradation by Fervidobacterium pennavorans, a Novel Thermophilic Anaerobic Species of the Order Thermotogales
Authors: Friedrich A B , Antranikian G
Published: January 2020

From a hot spring of the Azores islands a novel thermophilic bacterium belonging to the Thermotogales order was isolated. This strain, which grows optimally at 70(deg)C and pH 6.5, is the first known extreme thermophile that is able to degrade native feathers at high temperatures. The enzyme system converts feather meal to amino acids and peptides. On the basis of physiological, morphological, and 16S rDNA studies the new isolate was found to be a member of the Thermotogales order and was identified as Fervidobacterium pennavorans. The strain was highly related to Fervidobacterium islandicum and Fervidobacterium pullulanolyticum. The cell-bound keratinolytic enzyme system was purified 32-fold by detergent treatment with CHAPS (3-[(3-cholamidopropyl)-dimethyl-ammonio]-1-propanesulfonate) and sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The enzyme was characterized as a serine protease with a molecular mass of 130 kDa and an isoelectric point of 3.8. Optimal activity was measured at 80(deg)C and pH 10.0. Furthermore, 19 anaerobic thermophilic archaea and bacteria belonging to the orders Thermococcales, Thermoproteales, Thermotogales, and Clostridiales (growth temperatures between 60 and 105(deg)C) were tested for their abilities to grow on feathers and produce heat-stable keratinolytic enzymes. None of the tested extremophilic microorganisms was able to attack the substrate in a native form.
Role of osmolytes in adaptation of osmotically stressed and chill-stressed Listeria monocytogenes grown in liquid media and on processed meat surfaces
Authors: Smith L T
Published: January 2020

Listeria monocytogenes is a food-borne pathogen that is widely distributed in nature and is found in many kinds of fresh and processed foods. The pervasiveness of this organism is due, in part, to its ability to tolerate environments with elevated osmolarity and reduced temperatures. Previously, we showed that L. monocytogenes adapts to osmotic and chill stress by transporting the osmolyte glycine betaine from the environment and accumulating it intracellularly (R. Ko, L. T. Smith, and G. M. Smith, J. Bacteriol. 176:426-431, 1994). In the present study, the influence of various environmental conditions on the accumulation of glycine betaine and another osmolyte, carnitine, was investigated. Carnitine was shown to confer both chill and osmotic tolerance to the pathogen but was less effective than glycine betaine. The absolute amount of each osmolyte accumulated by the cell was dependent on the temperature, the osmolarity of the medium, and the phase of growth of the culture. L. monocytogenes also accumulated high levels of osmolytes when grown on a variety of processed meats at reduced temperatures. However, the contribution of carnitine to the total intracellular osmolyte concentration was much greater in samples grown on meat than in those grown in liquid media. While the amount of each osmolyte in meat was less than 1 nmol/mg (fresh weight), the overall levels of osmolytes in L. monocytogenes grown on meat were about the same as those in liquid samples, from about 200 to 1,000 nmol/mg of cell protein for each osmolyte. This finding suggests that the accumulation of osmolytes is as important in the survival of L. monocytogenes in meat as it is in liquid media.
Toluene and ethylbenzene oxidation by purified naphthalene dioxygenase from Pseudomonas sp. strain NCIB 9816-4
Authors: Lee K , Gibson D T
Published: January 2020

Purified naphthalene dioxygenase (NDO) from Pseudomonas sp. strain NCIB 9816-4 oxidized toluene to benzyl alcohol and benzaldehyde by reactions involving benzylic monooxygenation and dioxygen-dependent alcohol oxidation, respectively. Xylene and nitrotoluene isomers were also oxidized to substituted benzyl alcohol and benzaldehyde derivatives. NDO oxidized ethylbenzene sequentially through (S)-1-phenethyl alcohol (77% enantiomeric excess) and acetophenone to 2-hydroxyacetophenone. In addition, NDO also oxidized ethylbenzene through styrene to (R)-1-phenyl-1,2-ethanediol (74% enantiomeric excess) by reactions involving desaturation and dihydroxylation, respectively. Isotope experiments with 18O2, H2 18O, and D2O suggest that 1-phenethyl alcohol is oxidized to acetophenone by a minor reaction involving desaturation followed by tautomerization. The major reaction in the conversion of 1-phenethyl alcohol and benzyl alcohol to acetophenone and benzaldehyde, respectively, probably involves monohydroxylation to form a gem-diol intermediate which stereospecifically loses the incoming hydroxyl group to leave the carbonyl product. These results are compared with similar reactions catalyzed by cytochrome P-450.