bluuebunny/crossref_metadata_2025 huggingface dataset:
Detection of specific bacterial cells with 2-hydroxy-3-naphthoic acid-2'-phenylanilide phosphate and fast red TR in situ hybridization
Authors: Yamaguchi N , Inaoka S , Tani K , Kenzaka T , Nasu M
Published: January 2020

An in situ hybridization technique with HNPP (2-hydroxy-3-naphthoic acid-2'-phenylanilide phosphate) and Fast Red TR was used to detect specific bacterial cells at the single-cell level. By this technique, the fluorescent signals of target bacterial cells were up to eight times more intense than those of standard fluorescence in situ hybridization with mono-fluorescein isothiocyanate-labeled oligonucleotide probes. This novel HNPP-Fast Red TR whole-cell hybridization technique is available for the identification of small or low-rRNA-content bacterial cells in the natural environment.
Description of a new polymer-secreting bacterium from a deep-sea hydrothermal vent, Alteromonas macleodii subsp. fijiensis, and preliminary characterization of the polymer
Authors: Raguenes G , Pignet P , Gauthier G , Peres A , Christen R , Rougeaux H , Barbier G , Guezennec J
Published: January 2020

A deep-sea, aerobic, mesophilic, heterotrophic bacterium was isolated from fluid collected near an active hydrothermal vent. On the basis of phenotypic and phylogenetic analyses and DNA-DNA relatedness, strain ST716 could be assigned to the species Alteromonas macleodii as a new subspecies. This bacterium secreted an unusual high-molecular-weight polysaccharide in the presence of glucose in batch cultures. The viscosity of this exopolysaccharide is of the same order of magnitude as that of xanthan, another bacterial polysaccharide of industrial interest. This polysaccharide, produced during the stationary phase, contained glucose, mannose, pyruvated mannose, and galactose along with galacturonic acid and glucuronic acid.
Degradation of cocaine by a mixed culture of Pseudomonas fluorescens MBER and Comamonas acidovorans MBLF
Authors: Lister D L , Sproulé R F , Britt A J , Lowe C R , Bruce N C
Published: January 2020

A mixed culture that could utilize cocaine as the sole source of carbon and energy for growth was isolated by selective enrichment. The individual microorganisms within this mixed culture were identified as Pseudomonas fluorescens (termed MBER) and Comamonas acidovorans (termed MBLF). Each microorganism was shown to be unable to grow to any appreciable extent on 10 mM cocaine in the absence of the other. C. acidovorans MBLF was found to possess an inducible cocaine esterase which catalyzed the hydrolysis of cocaine to ecgonine methyl ester and benzoate. C. acidovorans was capable of growth on benzoate at concentrations below 5 mM but was unable to metabolize ecgonine methyl ester. P. fluorescens MBER was capable of growth on either benzoate as the sole source of carbon or ecgonine methyl ester as the sole source of carbon and nitrogen. P. fluorescens MBER was found to initiate the degradation of ecgonine methyl ester via ecgonine, pseudoecgonine, and pseudoecgonyl-coenzyme A. Subcellular studies resulted in the identification of an ecgonine methyl esterase, an ecgonine epimerase, and a pseudoecgonyl-coenzyme A synthetase which were induced by growth on ecgonine methyl ester or ecgonine. Further metabolism of the ecgonine moiety is postulated to involve nitrogen debridging, with the production of carbonyl-containing intermediates.
Manganese-Dependent Cleavage of Nonphenolic Lignin Structures by Ceriporiopsis subvermispora in the Absence of Lignin Peroxidase
Authors: Jensen K A , Bao W , Kawai S , Srebotnik E , Hammel K E
Published: January 2020

Many ligninolytic fungi appear to lack lignin peroxidase (LiP), the enzyme generally thought to cleave the major, recalcitrant, nonphenolic structures in lignin. At least one such fungus, Ceriporiopsis subvermispora, is nevertheless able to degrade these nonphenolic structures. Experiments showed that wood block cultures and defined liquid medium cultures of C. subvermispora rapidly depolymerized and mineralized a (sup14)C-labeled, polyethylene glycol-linked, high-molecular-weight (beta)-O-4 lignin model compound (model I) that represents the major nonphenolic structure of lignin. The fungus cleaved model I between C(inf(alpha)) and C(inf(beta)) to release benzylic fragments, which were shown in isotope trapping experiments to be major products of model I metabolism. The C(inf(alpha))-C(inf(beta)) cleavage of (beta)-O-4 lignin structures to release benzylic fragments is characteristic of LiP catalysis, but assays of C. subvermispora liquid cultures that were metabolizing model I confirmed that the fungus produced no detectable LiP activity. Three results pointed, instead, to the participation of a different enzyme, manganese peroxidase (MnP), in the degradation of nonphenolic lignin structures by C. subvermispora. (i) The degradation of model I and of exhaustively methylated (nonphenolic), (sup14)C-labeled, synthetic lignin by the fungus in liquid cultures was almost completely inhibited when the Mn concentration of the medium was decreased from 35 (mu)M to approximately 5 (mu)M. (ii) The fungus degraded model I and methylated lignin significantly faster in the presence of Tween 80, a source of unsaturated fatty acids, than it did in the presence of Tween 20, which contains only saturated fatty acids. Previous work has shown that nonphenolic lignin structures are degraded during the MnP-mediated peroxidation of unsaturated lipids. (iii) In experiments with MnP, Mn(II), and unsaturated lipid in vitro, this system mimicked intact C. subvermispora cultures in that it cleaved nonphenolic (beta)-O-4 lignin model compounds between C(inf(alpha)) and C(inf(beta)) to release a benzylic fragment.
Physiology and metabolic fluxes of wild-type and riboflavin-producing Bacillus subtilis
Authors: Sauer U , Hatzimanikatis V , Hohmann H P , Manneberg M , van Loon A P , Bailey J E
Published: January 2020

Continuous cultivation in a glucose-limited chemostat was used to determine the growth parameters of wild-type Bacillus subtilis and of a recombinant, riboflavin-producing strain. Maintenance coefficients of 0.45 and 0.66 mmol of glucose g-1 h-1 were determined for the wild-type and recombinant strains, respectively. However, the maximum molar growth yield of 82 to 85 g (cell dry weight)/mol of glucose was found to be almost identical in both strains. A nonlinear relationship between the specific riboflavin production rate and the dilution rate was observed, revealing a coupling of product formation and growth under strict substrate-limited conditions. Most prominently, riboflavin formation completely ceased at specific growth rates below 0.15 h-1. For molecular characterization of B. subtilis, the total amino acid composition of the wild type was experimentally determined and the complete building block requirements for biomass formation were derived. In particular, the murein sacculus was found to constitute approximately 9% of B. subtilis biomass, three- to fivefold more than in Escherichia coli. Estimation of intracellular metabolic fluxes by a refined mass balance approach revealed a substantial, growth rate-dependent flux through the oxidative branch of the pentose phosphate pathway. Furthermore, this flux is indicated to be increased in the strain engineered for riboflavin formation. Glucose catabolism at low growth rates with reduced biomass yields was supported mainly by the tricarboxylic acid cycle.
Expression of lip genes during growth in soil and oxidation of anthracene by Phanerochaete chrysosporium
Authors: Bogan B W , Schoenike B , Lamar R T , Cullen D
Published: January 2020

mRNA extraction from soil and quantitation by competitive reverse transcription-PCR were combined to study the expression of the 10 known lignin peroxidase (lip) genes in anthracene-transforming soil cultures of Phanerochaete chrysosporium. Levels of extractable lipA transcript and protein (LiP H8) were well correlated, although they were separated by a 2-day lag period. The patterns of transcript abundance over time in soil-grown P. chrysosporium varied among the nine lip mRNAs detected; comparison with lip gene expression under different liquid culture conditions suggested an early phase of carbon limitation for the cultures as a whole, which was followed by a transition to nitrogen starvation. Anthracene transformation occurred throughout the 25-day course of the experiment and, therefore, likely involves mechanisms distinct from those involved in oxidation of non-LiP substrate polycyclic aromatic hydrocarbons.
Novel Metabolic Transformation Pathway for Cyclic Imides in Blastobacter sp. Strain A17p-4
Authors: Ogawa J , Soong C L , Honda M , Shimizu S
Published: January 2020

The metabolic transformation pathway for cyclic imides in microorganisms was studied in Blastobacter sp. strain A17p-4. This novel pathway involves, in turn, hydrolytic ring opening of a cyclic imide to yield a monoamidated dicarboxylate, hydrolytic deamidation of the monoamidated dicarboxylate to yield a dicarboxylate, and dicarboxylate transformation similar to that in the tricarboxylic acid cycle. The initial step is catalyzed by a novel enzyme, imidase. Imidase and subsequent enzymes involved in this metabolic pathway are induced by some cyclic imides, such as succinimide and glutarimide. Induced cells metabolize various cyclic imides.
Phenotypic and genotypic characterization of phenanthrene-degrading fluorescent Pseudomonas biovars
Authors: Johnsen K , Andersen S , Jacobsen C S
Published: January 2020

A total of 41 phenanthrene degraders were isolated from a former coal gasification site by using Pseudomonas-selective Gould's S1 medium. All isolates were found to belong to the fluorescent Pseudomonas group and were subjected to characterization by phenotypic methods, including classical taxonomic tests, API 20NE, and Biolog GN, and the strains were further characterized by the genotypic method repetitive extragenic palindromic PCR (REP-PCR). By using classical tests, the population was found to consist of 38 strains belonging to P. fluorescens, 2 P. putida strains, and 1 Pseudomonas sp. Bacteria in phenograms from Biolog GN and REP-PCR data were divided into groups, which were in good agreement with classical test and API 20NE results. We found a nonfluorescent group of 22 bacteria inconsistent with any Pseudomonas sp. in Bergey's Manual of Systematic Bacteriology. The group showed small differences in the genotypic test, indicating that all 22 isolates were not recent clones of the same isolate. Analyses of the nonfluorescent group indicated that it belonged to Pseudomonas, but the group could not be affiliated with P. fluorescens because of differences in DNA-DNA hybridization. Identifications using classical tests and API 20NE were found to correlate, but Biolog GN identifications after 24-h incubation resulted very often in the distantly related P. corrugata. The reproducibilities of individual tests of each phenotypic method were assessed, and low reproducibilities were mainly found to be associated with specific Biolog GN test wells. Classical tests and API 20NE proved to be the best for identification of isolates, whereas Biolog GN and REP-PCR were found to be the best tests for high resolution among these closely related isolates.
Natural contamination of Manitoba barley by 3,15-diacetyldeoxynivalenol and its detection by immunochromatography
Authors: Usleber E , Abramson D , Gessler R , Smith D M , Clear R M , Märtlbauer E
Published: January 2020

Contamination of Canadian barley samples by 3,15-diacetyldeoxynivalenol was detected by enzyme immunoassays combined with liquid chromatography-mass spectrometry. This is the first reported natural occurrence of this mycotoxin. The barley was infected mainly with Fusarium graminearum. Deoxynivalenol, 3-acetyldeoxynivalenol, and zearalenone were also found.
Expression of virulence-related properties by, and intestinal adhesiveness of, Vibrio mimicus strains isolated from aquatic environments
Authors: Alam M , Miyoshi S , Yamamoto S , Tomochika K , Shinoda S
Published: January 2020

A study of the major pathogenic characteristics of Vibrio mimicus was carried out with 77 strains isolated from aquatic environments in Okayama, Japan. Of the strains tested, 96% demonstrated in vitro adherence to the rabbit intestinal mucosa, of which 36, 20, and 43% belonged to the strongly, moderately, and weakly adhesive groups, respectively. Of the 27 strains which appeared to be enterotoxigenic in the experiments using rabbit ileal loops, 74% belonged to the strongly adhesive group. All strains of V. mimicus at early log phase showed cell-mediated hemagglutination, and 70% of strongly hemagglutinative strains belonged to the strongly adhesive group, implying a possible correlation between cell-mediated hemagglutination and bacterial adherence. However, no significant correlation could be detected in the production of putative exocellular pathogenic factors and bacterial adherence or enterotoxigenicity.
Evaluation of Enterolert for enumeration of enterococci in recreational waters
Authors: Budnick G E , Howard R T , Mayo D R
Published: January 2020

Enterolert (IDEXX Laboratories Inc., Westbrook, Maine), a semiautomated, most probable number method for enumeration of enterococci, was compared with the standard membrane filter method by parallel testing of 138 marine and freshwater recreational bathing water samples. No statistically significant difference and a strong linear correlation were found between methods. Culturing of 501 Enterolert test wells resulted in false-positive and false-negative rates of 5.1 and 0.4%, respectively. Less time for setup, incubation (24 versus 48 h), and reading of Enterolert permits more efficient monitoring of recreational bathing areas.
Demethylation of dimethylsulfoniopropionate to 3-S-methylmercaptopropionate by marine sulfate-reducing bacteria
Authors: van der Maarel M J , Jansen M , Haanstra R , Meijer W G , Hansen T A
Published: January 2020

The initial step in the anaerobic degradation of the algal osmolyte dimethylsulfoniopropionate (DMSP) in anoxic marine sediments involves either a cleavage to dimethylsulfide and acrylate or a demethylation to 3-S-methylmercaptopropionate. Thus far, only one anaerobic bacterial strain has been shown to carry out the demethylation, namely, Desulfobacterium sp. strain PM4. The aims of the present work were to study how common this property is among certain groups of anaerobic bacteria and to obtain information on the affinities for DMSP of DMSP-demethylating strains. Screening of several pure cultures of sulfate-reducing and acetogenic bacteria showed that Desulfobacterium vacuolatum DSM 3385 and Desulfobacterium niacini DSM 2059 are also able to demethylate DMSP; a very slow demethylation of DMSP was observed with a salt-tolerant strain of Eubacterium limosum. From a 10(5) dilution of intertidal sediment a new marine DMSP-demethylating sulfate-reducing bacterium (strain WN) was isolated. Strain WN was a short, gram-negative, nonmotile rod that grew on betaine, sarcosine, palmitate, H2 plus CO2, and several alcohols, organic acids, and amino acids. Extracts of betaine-grown cells had hydrogenase, formate dehydrogenase, and CO dehydrogenase activities but no alpha-ketoglutarate oxidoreductase activity, indicating the presence of the acetyl coenzyme A-CO dehydrogenase pathway. Analysis of the 16S rRNA gene sequence of strain WN revealed a close relationship with Desulfobacter hydrogenophilus, Desulfobacter latus, and Desulfobacula toluolica. Strain PM4 was shown to group with Desulfobacterium niacini. The K(m) of strain WN for DMSP, as derived from substrate progress curves in cell suspensions, was approximately 10 microM. A similar value was found for D. niacini PM4.
Culturing enterohemorrhagic Escherichia coli in the presence and absence of glucose as a simple means of evaluating the acid tolerance of stationary-phase cells
Authors: Buchanan R L , Edelson S G
Published: January 2020

Prior growth of seven enterohemorrhagic and one nonenterohemorrhagic strains of Escherichia coli in tryptic soy broth with (TSB+G) and without (TSB-G) 1% glucose was evaluated for its effect on acid tolerance. The final pHs of 18-h TSB+G and TSB-G cultures were 4.6 to 5.2 and 6.9 to 7.0, respectively. Cells were then transferred to brain heart infusion broth adjusted to pH 2.5 or 3.0 with HCl, incubated at 37 degrees C for up to 7 h, and assayed periodically for viable populations with brain heart infusion and MacConkey agars. All enterohemorrhagic strains were acid resistant (< 0.5 log decline after 7 h) when initially cultured in TSB+G, but substantial differences in acid tolerance were observed among strains cultured in TSB-G (log declines ranged from < 0.3 to > 3.8). The results indicated that prior growth in a medium with and without a fermentable carbohydrate is a convenient way to studying the induction of acid tolerance, that acid inactivation is preceded by a period of acid injury, and that pH-independent and pH-dependent stationary-phase acid tolerance phenotypes may exist among strains of enterohemorrhagic E. coli.
Aflatoxin and cyclopiazonic acid production by a sclerotium-producing Aspergillus tamarii strain
Authors: Goto T , Wicklow D T , Ito Y
Published: January 2020

The production of aflatoxins B1 and B2 by Aspergillus tamarii (subgenus Circumdati section Flavi) is reported for the first time. The fungus was isolated from soil collected from a tea (Camellia sinensis) field in Miyazaki Prefecture, Japan. Three single-spore cultures, NRRL 25517, NRRL 25518, and NRRL 25519, were derived from subcultures of the original isolate 19 (MZ2). Each of these single-spore cultures of A. tamarii produced aflatoxins B1 and B2 and cyclopiazonic acid, as well as black, pear-shaped sclerotia. The demonstration of aflatoxin production by A. tamarii is examined in connection with A. tamarii phylogenetic relationships, chemical ecology, and potential use in food fermentations.
Biocontrol of Rhizoctonia solani Damping-Off of Tomato with Bacillus subtilis RB14
Authors: Asaka O , Shoda M
Published: January 2020

Bacillus subtilis RB14, which showed antibiotic activities against several phytopathogens in vitro by producing the antibiotics iturin A and surfactin, was subjected to a pot test to investigate its ability to suppress damping-off of tomato seedlings caused by Rhizoctonia solani. To facilitate recovery from soil, B. subtilis RB14-C, a spontaneous streptomycin-resistant mutant of RB14, was used. Damping-off was suppressed when the culture broth, cell suspension, or cell-free culture broth of RB14-C was inoculated into soil. Iturin A and surfactin were recovered from the soils inoculated with the cell suspension of RB14-C, confirming that RB14-C produced them in soil. The gene lpa-14, which was cloned from RB14 and required for the production of both antibiotics, was mutated in RB14-C, and a mutant, R(Delta)1, was constructed. The level of disease suppressibility of R(Delta)1 was low, but R(Delta)1(pC115), a transformant of R(Delta)1 with the plasmid pC115 carrying lpa-14, was restored in suppressibility. These results show that the antibiotics iturin A and surfactin produced by RB14 play a major role in the suppression of damping-off caused by R. solani. RB14-C, R(Delta)1, and R(Delta)1(pC115) persisted in soil during the experimental period and were recovered from the soil, mostly as spores.
Death of the Escherichia coli K-12 strain W3110 in soil and water
Authors: Bogosian G , Sammons L E , Morris P J , O'Neil J P , Heitkamp M A , Weber D B
Published: January 2020

Whether Escherichia coli K-12 strain W3110 can enter the "viable but nonculturable" state was studied with sterile and nonsterile water and soil at various temperatures. In nonsterile river water, the plate counts of added E. coli cells dropped to less than 10 CFU/ml in less than 10 days. Acridine orange direct counts, direct viable counts, most-probable-number estimates, and PCR analyses indicated that the added E. coli cells were disappearing from the water in parallel with the number of CFU. Similar results were obtained with nonsterile soil, although the decline of the added E. coli was slower. In sterile water or soil, the added E. coli persisted for much longer, often without any decline in the plate counts even after 50 days. In sterile river water at 37 degrees C and sterile artificial seawater at 20 and 37 degrees C, the plate counts declined by 3 to 5 orders of magnitude, while the acridine orange direct counts remained unchanged. However, direct viable counts and various resuscitation studies all indicated that the nonculturable cells were nonviable. Thus, in either sterile or nonsterile water and soil, the decline in plate counts of E. coli K-12 strain W3110 is not due to the cells entering the viable but nonculturable state, but is simply due to their death.
Direct ribosome isolation from soil to extract bacterial rRNA for community analysis
Authors: Felske A , Engelen B , Nübel U , Backhaus H
Published: January 2020

A simple method that combines an adapted ribosome isolation method and a common RNA extraction step has been developed for selective recovery of intact rRNA from natural microbial communities in soil. After mechanical cell lysis, ribosomes are separated by centrifugation steps, avoiding massive humic acid contamination and RNA degradation. The protocol accommodates the complex composition of soils by blocking adsorbing surfaces and humic acids with polyvinylpyrrolidone and bovine serum albumin. A usual RNA extraction step yields rRNA accessible for hybridization or reverse transcription-PCR. Hybridization with specific oligonucleotide probes was used for group-specific yield comparison. By using a probe hybridizing to the 16S rRNA of the bacterial kingdom, total bacterial rRNA yield was estimated to be in the range of 0.2 microgram per g for different soils. Group-specific probes did not indicate a selectivity of the isolation procedure and differentiated the compositions of different soil microbial communities. The sequence diversity of the isolated RNA population was also revealed by temperature gradient gel electrophoresis of reverse transcription-PCR amplification products by using a region of the 16S rRNA as a target. The pattern obtained by this analysis differed from a similar one resulting from the separation of amplification products of community DNA preparations. This different view of the community composition is attributable to the correlation of ribosome numbers to the metabolic activity of bacteria in the habitat under observation.
Altered binding of the Cry1Ac toxin to larval membranes but not to the toxin-binding protein in Plodia interpunctella selected for resistance to different Bacillus thuringiensis isolates
Authors: Mohammed S I , Johnson D E , Aronson A I
Published: January 2020

Immunoblotting and cytochemical procedures were used to determine whether toxin binding was altered in strains of the Indianmeal moth, Plodia interpunctella, selected for resistance to various strains of Bacillus thuringiensis. Each of these B. thuringiensis subspecies produces a mixture of protoxins, primarily Cry1 types, and the greatest insect resistance is to the Cry1A protoxins. In several cases, however, there was also resistance to toxins not present in the B. thuringiensis strains used for selection. The Cry1Ab and Cry1Ac toxins bound equally well over a range of toxin concentrations and times of incubation to a single protein of ca. 80-kDa in immunoblots of larval membrane extracts from all of the colonies. This binding protein is essential for toxicity since a mutant Cry1Ac toxin known to be defective in binding and thus less toxic bound poorly to the 80-kDa protein. This binding protein differed in size from the major aminopeptidase N antigens implicated in toxin binding in other insects. Binding of fluorescently labeled Cry1Ac or Cry1Ab toxin to larval sections was found at the tips of the brush border membrane prepared from the susceptible but not from any of the resistant P. interpunctella. Accessibility of a major Cry1A-binding protein appears to be altered in resistant larvae and could account for their broad resistance to several B. thuringiensis toxins.
Simultaneous detection of different Rhizobium strains marked with either the Escherichia coli gusA gene or the Pyrococcus furiosus celB gene
Authors: Sessitsch A , Wilson K J , Akkermans A D , de Vos W M
Published: January 2020

A new marker system for gram-negative bacteria was developed on the basis of the celB gene from the hyperthermophilic archaeon Pyrococcus furiosus, which encodes a thermostable beta-glucosidase with a high level of beta-galactosidase activity. The celB gene is highly suitable as a marker for studying plant-bacterium interaction because endogenous background beta-glucosidase and beta-galactosidase enzyme activity can readily be inactivated by heat and because inexpensive substrates for detection are commercially available. Two celB-expressing transposons were constructed for use in ecological studies of a variety of gram-negative bacteria. The combined use of the gusA marker gene and celB allowed the simultaneous detection of several Rhizobium strains on a plant, and multiple-strain occupancy of individual modules also could be easily detected.
Degradation of the fluoroquinolone enrofloxacin by wood-rotting fungi
Authors: Martens R , Wetzstein H G , Zadrazil F , Capelari M , Hoffmann P , Schmeer N
Published: January 2020

The veterinary fluoroquinolone enrofloxacin was degraded in vitro by four species of wood-rotting fungi growing on wetted wheat straw containing carbonyl-14C-labeled drug. A maximum 14CO2 production of 17% per week was observed with the brown rot fungus Gloeophyllum striatum, resulting in up to 53% after 8 weeks. However, rates reached at most 0.2 and 0.9% per week, if enrofloxacin was preadsorbed to native or gamma ray-sterilized soil, respectively.
Hemolytic activity reevaluation of putative nonpathogenic Listeria monocytogenes strains
Authors: Lachica R V
Published: January 2020

Identification of 12 strains originally characterized as nonpathogenic Listeria monocytogenes was reassured following the evaluation of their hemolytic capability with a newly developed horse blood agar plate. Seven of the strains were observed consistently to be hemolytic and confirmed as L. monocytogenes with the use of two commercial systems: the Gene-Trak L. monocytogenes-specific colorimetric DNA hybridization assay and the API Listeria system. Except for one strain that formed typical smooth colonies, these hemolytic strains formed rough colonies on a selective medium, lithium chloride-ceftazidime agar. The rest of the strains were nonhemolytic and did not hybridize with the DNA probe; they were identified as Listeria innocua on the basis of their API Listeria system biochemical profile. All but one of these nonhemolytic strains formed smooth colonies on lithium chloride-ceftazidime agar.
Growth Physiology of the Hyperthermophilic Archaeon Thermococcus litoralis: Development of a Sulfur-Free Defined Medium, Characterization of an Exopolysaccharide, and Evidence of Biofilm Formation
Authors: Rinker K D , Kelly R M
Published: January 2020

Nutritional characteristics of the hyperthermophilic archaeon Thermococcus litoralis have been investigated with emphasis on the development of a sulfur-free, defined growth medium, analysis of an exocellular polysaccharide, and formation of a biofilm. An artificial-seawater-based medium, containing 16 amino acids, adenine, uracil, vitamins, and trace elements, allowed T. litoralis to attain growth rates and cell densities similar to those found with complex media. Four amino acids (alanine, asparagine, glutamine, and glutamate) were not included due to their lack of effect on growth rates and cell yields. In this medium, cultures reached densities of 10(sup8) cells per ml, with doubling times of 55 min (without maltose) or 43 min (with maltose). Neither the addition of elemental sulfur nor the presence of H(inf2) significantly affected cell growth. A sparingly soluble exopolysaccharide was produced by T. litoralis grown in either defined or complex media. Analysis of the acid-hydrolyzed exopolysaccharide yielded mannose as the only monosaccharidic constituent. This exopolysaccharide is apparently involved in the formation of a biofilm on polycarbonate filters and glass slides, which is inhabited by high levels of T. litoralis. Biofilm formation by hyperthermophilic microorganisms in geothermal environments has not been examined to any extent, but further work in this area may provide information related to the interactions among high-temperature organisms.
Purification of the bacteriocin bavaricin MN and characterization of its mode of action against Listeria monocytogenes Scott A cells and lipid vesicles
Authors: Kaiser A L , Montville T J
Published: January 2020

Bavaricin MN was purified from Lactobacillus sake culture supernatant 135-fold with a final yield of 11%. Sequence analysis revealed bavaricin MN to be a 42-amino-acid peptide having a molecular weight of 4,769 and a calculated pI of 10.0. Computer analysis indicated that the C-terminal region may form an alpha-helical structure with an amphipathic nature deemed important in the interaction of bacteriocins with biological membranes. Bavaricin MN rapidly depleted the membrane potential (delta p) of energized Listeria monocytogenes cells in a concentration-dependent fashion. At a bavaricin MN concentration of 9.0 micrograms/ml, the delta p decreased by 85%. Both the electrical potential (delta psi) and Z delta pH components of the delta p were depleted, and this depletion was not dependent on a threshold level of proton motive force. In addition to studying the effect of bavaricin MN on the delta p of vegetative cells, bavaricin MN-induced carboxyfluorescein (CF) efflux from L. monocytogenes-derived lipid vesicles was also characterized. Bavaricin MN-induced CF leakage was also concentration dependent with an optimum of pH 6.0. The rate of CF efflux was 63% greater in lipid vesicles in which a delta psi was generated compared with that in lipid vesicles in the absence of a delta psi.
Isolation of expressed sequence tags of Agaricus bisporus and their assignment to chromosomes
Authors: Sonnenberg A S , de Groot P W , Schaap P J , Baars J J , Visser J , Van Griensven L J
Published: January 2020

The genome of the cultivated basidiomycete Agaricus bisporus Horst U1 and of its homokaryotic parents has been characterized by using an optimized method of pulsed-field gel electrophoresis. Expressed sequence tags obtained as expressed cDNAs from a primordial tissue-derived cDNA library and a number of previously isolated genes were used to identify the individual chromosomes of the parental lines of Horst U1. The genome consists of 13 chromosomes, and its total size is 31 Mb. For those chromosomes that could not be resolved by contour-clamped homogeneous electric field electrophoresis, the segregation of marker genes was studied in a set of 86 homokaryotic offspring of Horst U1. At least two markers were assigned to each individual chromosome. In this way all individual chromosomes were unequivocally identified. The large size difference observed between the homologous chromosomes IX, harboring the rDNA repeat, was shown to be largely due to a higher copy number of rDNA in parental strain H97 than in parental strain H39.
Application of a Mycoplasma group-specific PCR for monitoring decontamination of Mycoplasma-infected Chlamydia sp. strains
Authors: Ossewaarde J M , de Vries A , Bestebroer T , Angulo A F
Published: January 2020

Mycoplasma contamination of biological materials remains a major problem. Most contaminations are caused by the use of Mycoplasma-contaminated cell lines. We adapted a Mycoplasma group-specific PCR to detect Mycoplasma contamination in cell lines and demonstrate its use in monitoring decontamination procedures with Mycoplasma-contaminated suspensions of Chlamydia spp. Three different methods were investigated: the use of Mycoplasma-specific antiserum in cell culture, physical separation by the combined use of enzymatic treatment and differential centrifugation, and the use of detergents. With these methods only incubation with Triton X-100 resulted in decontamination of Mycoplasma-contaminated suspensions of several laboratory strains of Chlamydia pneumoniae, C. pecorum, and C. trachomatis. Only one C. pneumoniae strain, UZG-1, was sensitive to Triton X-100 treatment. Since 39 of 40 throat swabs from patients with symptoms of an upper respiratory tract infection had positive reactions in the Mycoplasma group-specific PCR, this procedure could also have clinical significance in attempts to propagate C. pneumoniae strains from clinical specimens.
Phylogeny of not-yet-cultured spirochetes from termite guts
Authors: Paster B J , Dewhirst F E , Cooke S M , Fussing V , Poulsen L K , Breznak J A
Published: January 2020

Comparisons of 16S rDNA sequences were used to determine the phylogeny of not-yet-cultured spirochetes from hindguts of the African higher termite, Nasutitermes lujae (Wasmann). The 16S rRNA genes were amplified directly from spirochete-rich hindguts by using universal primers, and the amplified products were cloned into Escherichia coli. Clones were screened with a spirochete-specific DNA probe. Analysis of 1,410 base positions of the 16S rDNA insert from one spirochete clone, designated NL1, supported its assignment to the genus Treponema, with average interspecies similarities of ca. 85%. The sequence of NL1 was most closely related (ca. 87 to 88% similarity) to sequences of Spirochaeta stenostrepta and Spirochaeta caldaria and to a previously published sequence (ca. 87% similarity) of spirochetal clone MDS1 from the Australian lower termite, Mastotermes darwiniensis (Froggatt). On the basis of 16S rRNA sequence comparisons and individual base signatures, clones NL1 and MDS1 clearly represent two novel species of Treponema, although specific epithets have not yet been proposed. The gross morphology of NL1 was determined from in situ hybridization experiments with an NL1-specific, fluorescently labeled oligonucleotide probe. Cells were approximately 0.3 to 0.4 by 30 microns in size, with a wavelength and amplitude of about 10 microns and 0.8 to 1.6 micron, respectively. Moreover, electron microscopy of various undulate cells present in gut contents confirmed that they possessed ultrastructural features typical of spirochetes, i.e., a wavy protoplasmic cylinder, periplasmic flagella, and an outer sheath. The sequence data suggest that termite gut spirochetes may represent a separate line of descent from other treponemes and that they constitute a significant reservoir of previously unrecognized spirochetal biodiversity.
Degradation of Chlorinated Dibenzofurans and Dibenzo-p-Dioxins by Sphingomonas sp. Strain RW1
Authors: Wilkes H , Wittich R , Timmis K N , Fortnagel P , Francke W
Published: January 2020

The ability of the dibenzofuran- and dibenzo-p-dioxin-mineralizing bacterium Sphingomonas sp. strain RW1 (R.-M. Wittich, H. Wilkes, V. Sinnwell, W. Francke, and P. Fortnagel, Appl. Environ. Microbiol. 58:1005-1010, 1992) to oxidize chlorinated derivatives of dibenzofuran and dibenzo-p-dioxin was analyzed. Strain RW1 degraded several mono- and dichlorinated dibenzofurans and dibenzo-p-dioxins, but it did not degrade more highly chlorinated congeners. Most mono- and dichlorinated dibenzofurans and dibenzo-p-dioxins investigated in this study were degraded to the corresponding mono- and dichlorinated salicylates and catechols, respectively, together with salicylate and catechol. This indicates an initial dioxygenolytic attack on the substituted as well as on the nonsubstituted aromatic nucleus of most of the target compounds. Strain RW1 could not grow at the expense of monochlorinated dibenzo-p-dioxins and dibenzofurans as carbon sources, with the exception of 4-chlorodibenzofuran, which was stoichiometrically converted to 3-chlorosalicylate.
Metabolic pathway for poly(3-hydroxybutyrate-co-3-hydroxyvalerate) formation in Nocardia corallina: inactivation of mutB by chromosomal integration of a kanamycin resistance gene
Authors: Valentin H F , Dennis D
Published: January 2020

The gene encoding the large subunit of the methylmalonyl-coenzyme A (CoA) mutase in Nocardia corallina (mutBNc) was cloned. A 4.3-kbp BamHI fragment containing almost the entire mutBNc was identified by Southern hybridization experiments employing a digoxigenin-labeled probe deduced from mutB of Streptomyces cinnamonensis, mutBNc was interrupted by insertion of a kanamycin resistance gene block (mutB::kan or mutB::neo) and introduced into N. corallina to obtain mutB-negative strains by homologous recombination. Four of sixteen kanamycin-resistant clones occurred via double-crossover events and harbored only the interrupted mutBNc. These exhibited no growth on odd-chain fatty acids in the presence of kanamycin but exhibited wild-type growth on even-chain fatty acids, glucose, and succinate. Whereas the wild type of N. corallina accumulates a copolyester of 3-hydroxybutyrate (3HB) and 3-hydroxyvalerate (3HV) containing more than 60 mol% 3HV from most carbon sources, mutB-negative strains accumulated poly(3HB-co-3HV) containing only 2 to 6 mol% 3HV. Methylmalonyl-CoA mutase activity was not found in these clones. Therefore, this study provides strong evidence that the majority of 3HV units in poly(3HB-co-3HV) accumulated by N. corallina are synthesized via the methylmalonyl-CoA pathway.
Phylogenetic diversity of the intestinal bacterial community in the termite Reticulitermes speratus
Authors: Ohkuma M , Kudo T
Published: January 2020

The phylogenetic diversity of the intestinal microflora of a lower termite, Reticulitermes speratus, was examined by a strategy which does not rely on cultivation of the resident microorganisms. Small-subunit rRNA genes (16S rDNAs) were directly amplified from the mixed-population DNA of the termite gut by the PCR and were clonally isolated. Analysis of partial 16S rDNA sequences showed the existence of well-characterized genera as well as the presence of bacterial species for which no 16S rDNA sequence data are available. Of 55 clones sequenced, 45 were phylogenetically affiliated with four of the major groups of the domain Bacteria: the Proteobacteria, the spirochete group, the Bacteroides group, and the low-G+C-content gram-positive bacteria. Within the Proteobacteria, the 16S rDNA clones showed a close relationship to those of cultivated species of enteric bacteria and sulfate-reducing bacteria, while the 16S rDNA clones in the remaining three groups showed only distant relationships to those of known organisms in these groups. Of the remaining 10 clones, among which 8 clones formed a cluster, there was only very low sequence similarity to known 16S rRNA sequences. None of these clones were affiliated with any of the major groups within the domain Bacteria. The 16S rDNA gene sequence data show that the majority of the intestinal microflora of R. speratus consists of new, uncultured species previously unknown to microbiologists.
Assessment of competitiveness of rhizobia infecting Galega orientalis on the basis of plant yield, nodulation, and strain identification by antibiotic resistance and PCR
Authors: Tas E , Leinonen P , Saano A , Räsänen L A , Kaijalainen S , Piippola S , Hakola S , Lindström K
Published: January 2020

Competition between effective and ineffective Rhizobium galegae strains nodulating Galega orientalis was examined on the basis of plant growth, nodulation, antibiotic resistance, and PCR results. In a preliminary experiment in Leonard's jars, ineffective R. galegae strains HAMBI 1207 and HAMBI 1209 competed in similar manners with the effective strain R. galegae HAMBI 1174. In a pot experiment, soil was inoculated with 0 to 10(5) HAMBI 1207 cells per g before G. orientalis was sown. Seeds of G. orientalis were surface inoculated with 2 x 10(4) and 2 x 10(5) cells of HAMBI 1174 per seed (which represent half and fivefold the commercially recommended amount of inoculant, respectively). Plant yield and nodulation by the effective strain were significantly reduced, with as few as 10(2) ineffective rhizobia per g of soil, and the inoculation response was not improved by the 10-fold greater dose of the inoculant. Bacteria occupying the nodules were identified by antibiotic resistance and PCR with primers specific for R. galegae HAMBI 1174, R. galegae, and genes coding for bacterial 16S rRNA (bacterial 16S rDNA). Sixty-two large nodules examined were occupied by the effective strain HAMBI 1174, as proven by antibiotic resistance and amplification of the strain-specific fragment. From 20 small nodules, only the species-specific fragment could be amplified, and isolated bacteria had the same antibiotic resistance and 16S PCR restriction pattern as strain HAMBI 1207. PCR with our strain-specific and species-specific primers provides a powerful tool for strain identification of R. galegae directly from nodules without genetic modification of the bacteria.
The peptide synthetase gene phsA from Streptomyces viridochromogenes is not juxtaposed with other genes involved in nonribosomal biosynthesis of peptides
Authors: Schwartz D , Alijah R , Nussbaumer B , Pelzer S , Wohlleben W
Published: January 2020

By complementation of a previously described non-phosphinothricin tripeptide (PTT)-producing mutant, NTG1, which is blocked in nonribosomal synthesis of the peptide, a DNA fragment including the putative peptide synthetase gene phsA was isolated (W. Wohlleben, R. Alijah, J. Dorendorf, D. Hillemann, B. Nussbaumer, and S. Pelzer, Gene 115:127-132, 1992). Sequence analysis of phsA revealed that it encodes a protein of 622 amino acids with regions which are highly similar to core motifs characteristic for peptide synthetases. PhsA represents one functional domain of a peptide synthetase which is necessary for activation and condensation of one amino acid, probably N-acetyl-demethyl-phosphinothricin. With regard to the arrangement of the flanking genes, phsA is the first peptide synthetase gene which is not in the direct neighborhood of additional peptide synthetase genes involved in the formation of peptide antibiotics. Gene disruption mutants with internal fragments of phsA subcloned in temperature-sensitive pGM vectors were generated. Integration occurred either into the chromosomal copy of phsA or into a gene outside the known phsA locus, resulting in two classes of non-PTT-producing mutants. In cofeeding experiments the former phsA mutants showed the same phenotype as did NTG1, which confirmed participation of phsA in nonribosomal synthesis of PTT. A truncated phsA gene was overexpressed in Escherichia coli, and the resulting protein of 593 amino acids was purified for raising antibodies. By performing immunoblotting experiments, the expression of phsA could be detected in Streptomyces viridochromogenes Tü494 in the stationary-growth phase after 4 days of incubation.
Glucose Transport in Stationary-Phase Cultures of an Asporogenous Strain of Bacillus licheniformis
Authors: Tangney M , Tate J E , Priest F G , Mitchell W J
Published: January 2020

The sporulation-deficient industrial organism Bacillus licheniformis HWL10 possesses two distinct glucose transport systems in log-phase cells, a glucose phosphotransferase system (PTS) and a non-PTS mechanism. The strain continues to take up glucose at a significant though reduced rate during prolonged stationary-phase incubation, but only the PTS is active.
Utilization of individual cellodextrins by three predominant ruminal cellulolytic bacteria
Authors: Shi Y , Weimer P J
Published: January 2020

Growth of the ruminal bacteria Fibrobacter succinogenes S85, Ruminococcus flavefaciens FD-1, and R. albus 7 followed Monod kinetics with respect to concentrations of individual pure cellodextrins (cellobiose, cellotriose, cellotetraose, cellopentaose, and cellohexaose). Under the conditions tested, R. flavefaciens FD-1 possesses the greatest capacity to compete for low concentrations of these cellodextrins.
Purification and partial characterization of an alkaline lipase from Pseudomonas pseudoalcaligenes F-111
Authors: Lin S F , Chiou C M , Yeh C M , Tsai Y C
Published: January 2020

An extracellular alkaline lipase of alkalophilic Pseudomonas pseudoalcaligenes F-111 was purified to homogeneity. The apparent molecular weight determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis was 32,000, and the isoelectric point was 7.3. With p-nitrophenyl esters as its substrates, the enzyme shows preference for C12 acyl and C14 acyl groups. It was stable in the pH range of 6 to 10, which coincides with the optimum pH range.
Transformation of phenol into phenylalanine by a methanogenic consortium
Authors: Lepine F , Bisaillon J , Milot S , Khalid T H , Beaudet R , Villemur R
Published: January 2020

The conversion by a methanogenic consortium of phenol into phenylalanine, with benzoic and phenylpropionic acid as intermediates, was investigated. When (sup14)C-labelled phenol was fed to the consortium, the radioactivity was mostly transferred into methane and CO(inf2), but 4% of the radioactivity was found in the water fraction after extraction of the culture medium with an organic solvent. Utilization of labelled compounds and analysis by gas chromatography coupled with mass spectrometry revealed that a fraction of the benzoic acid produced was transformed into 3-phenylpropionic acid. When fully (sup13)C-labelled acetic acid was fed to the consortium, the labels were incorporated at the 1 and 2 positions of 3-phenylpropionic acid. When deuterium-labelled 3-phenylpropionic acid was fed to the consortium, part of the phenylalanine of the biomass was labelled. These metabolic transformations are reversible, since deuterium-labelled phenylalanine generated labelled 3-phenylpropionic acid. Cinnamic acid was also transformed into 3-phenylpropionic acid.
Comparison of factors influencing trichloroethylene degradation by toluene-oxidizing bacteria
Authors: Leahy J G , Byrne A M , Olsen R H
Published: January 2020

The degradation of trichloroethylene (TCE) by toluene-oxidizing bacteria has been extensively studied, and yet the influence of environmental conditions and physiological characteristics of individual strains has received little attention. To consider these effects, the levels of TCE degradation by strains distinguishable on the basis of toluene and nitrate metabolism were compared under aerobic or hypoxic conditions in the presence and absence of nitrate and an exogenous electron donor, lactate. Under aerobic conditions with toluene-induced cells, strains expressing toluene dioxygenases (Pseudomonas putida F1, Pseudomonas sp. strain JS150, Pseudomonas fluorescens CFS215, and Pseudomonas sp. strain W31) degraded TCE at low rates, with less than 12% of the TCE removed in 18 h. In contrast, strains expressing toluene monooxygenases (Burkholderia cepacia G4, Burkholderia pickettii PKO1, and Pseudomonas mendocina KR1) degraded 36 to 67% of the TCE over the same period. Under hypoxic conditions (1.7 mg of dissolved oxygen per liter) or when lactate was added as an electron donor, the extent of TCE degradation by toluene-induced cells was generally lower. In the presence of lactate, degradation of TCE by denitrifying strain PKO1 was enhanced by nitrate under conditions in which dissimilatory nitrate reduction was observed. The results of experiments performed with strains F1, G4, PKO1, and KR1 suggested that TCE or an oxidation product induces toluene degradation and that TCE induces its own degradation in the monooxygenase strains. The role of TCE as an inducer of toluene oxygenase activity in PKO1 was confirmed by performing a promoter probe analysis, in which we found that TCE activates transcription from the PKO1 3-monooxygenase operon promoter.