bluuebunny/crossref_metadata_2025_split huggingface dataset:
Impaired Secretion of a Hydrophobic Cutinase by
Saccharomyces cerevisiae
Correlates with an Increased Association with Immunoglobulin Heavy-Chain Binding Protein (BiP)
Authors: Sagt C. M. J. , Müller W. H. , Boonstra J. , Verkleij A. J. , Verrips C. T. 
Published: December 2019

ABSTRACT

This study focuses on the different efficiencies of secretion of two fungal cutinases by
Saccharomyces cerevisiae
, a wild-type cutinase (CY000) and a hydrophobic mutant cutinase (CY028). Both cutinases are placed under control of the
GAL7
promoter, by which the expression levels can be regulated. Wild-type cutinase was secreted at up to 25 mg per g (dry weight), while CY028 was secreted at a level of 2 mg per g (dry weight); this difference is nearly independent of the expression level. Pulse-chase experiments revealed that whereas CY000 cutinase is secreted, CY028 is irreversibly retained in the cell. Immunogold labelling followed by electron microscopy revealed colocalization of CY028 with immunoglobulin heavy-chain binding protein (BiP) in the endoplasmic reticulum (ER). The increase of wild-type cutinase expression did not result in higher levels of the molecular chaperone BiP, but BiP levels are raised by increased induction of the hydrophobic mutant cutinase. Immunoprecipitation studies showed that in contrast to the wild-type cutinase, the hydrophobic mutant cutinase interacts with BiP. These results indicate that the introduction of two exposed hydrophobic patches in cutinase results in a higher affinity for BiP which might cause the retention of this mutant cutinase in the ER.
Electron and Fluorescence Microscopy of Extracellular Glucan and Aryl-Alcohol Oxidase during Wheat-Straw Degradation by
Pleurotus eryngii
Authors: Barrasa J. M. , Gutiérrez A. , Escaso V. , Guillén F. , Martínez M. J. , Martínez A. T. 
Published: December 2019

ABSTRACT

The ligninolytic fungus
Pleurotus eryngii
grown in liquid medium secreted extracellular polysaccharide (87% glucose) and the H
2
O
2
-producing enzyme aryl-alcohol oxidase (AAO). The production of both was stimulated by wheat-straw. Polyclonal antibodies against purified AAO were obtained, and a complex of glucanase and colloidal gold was prepared. With these tools, the localization of AAO and extracellular glucan in mycelium from liquid medium and straw degraded under solid-state fermentation conditions was investigated by transmission electron microscopy (TEM) and fluorescence microscopy. These studies revealed that
P. eryngii
produces a hyphal sheath consisting of a thin glucan layer. This sheath appeared to be involved in both mycelial adhesion to the straw cell wall during degradation and AAO immobilization on hyphal surfaces, with the latter evidenced by double labeling. AAO distribution during differential degradation of straw tissues was observed by immunofluorescence microscopy. Finally, TEM immunogold studies confirmed that AAO penetrates the plant cell wall during
P. eryngii
degradation of wheat straw.
PCR Amplification and Species Determination of Microsporidia in Formalin-Fixed Feces after Immunomagnetic Separation
Authors: Dowd Scot E. , Gerba Charles P. , Enriquez F. Javier , Pepper Ian L. 
Published: December 2019

ABSTRACT

The term microsporidia is used to describe several species of opportunistic protozoan parasites.
Encephalitozoon intestinalis
and
Enterocytozoon bieneusi
have been found in stools of more than 40% of AIDS patients with diarrhea. Diagnosis of infection with these small protozoans has been difficult, and until recently their occurrence has not been well documented. Formalin is widely used to preserve clinical specimens, but due to the nature of the fixation process, subsequent analysis, especially analysis by the PCR, is difficult. This study evaluated methods used to prepare formalin-fixed fecal specimens for PCR amplification of microsporidial DNA. Two methods were devised to allow PCR detection and subsequent identification of microsporidia in formalin-fixed fecal specimens to the species level. One method involved immunomagnetic separation to concentrate microsporidial spores from fecal specimens. In the second method Chelex resin (Bio-Rad, Hercules, Calif.) was used to remove inhibitory substances, followed by a DNA concentration step. Both methods resulted in reproducible, confirmed detection of microsporidia in formalinized fecal specimens and subsequent species determination by PCR sequencing. The detection sensitivity was two in vitro culture-derived spores (
Encephalitozoon intestinalis
) for the direct PCR. The reproducible detection sensitivity for DNA amplification from formalin-fixed fecal samples was 200 spores for either the Chelex method or the immunomagnetic bead separation method. Thus, we developed two methods for rapid, inexpensive detection of microsporidial spores in formalin-fixed fecal specimens.
Occurrence of
Vibrio vulnificus
Biotypes in Danish Marine Environments
Authors: Høi L. , Larsen J. L. , Dalsgaard I. , Dalsgaard A. 
Published: December 2019

ABSTRACT

During the unusually warm summer in Denmark in 1994, 11 clinical cases of
Vibrio vulnificus
infection were reported. These reports initiated an investigation of the occurrence of
V. vulnificus
biotypes in Danish marine environments. Samples of coastal water, sediment, shellfish, and wild fish were analyzed by preenrichment in alkaline peptone water amended with polymyxin B (2.0 × 10
4
U/liter) followed by streaking onto modified cellobiose-polymyxin B-colistin agar.
V. vulnificus
-like colonies were tested with a
V. vulnificus
-specific DNA probe. Low densities of
V. vulnificus
were detected in water (0.8 to 19 CFU/liter) from June until mid-September and in sediment (0.04 to >11 CFU/g) from July until mid-November. The presence of
V. vulnificus
was strongly correlated with water temperature. However, we isolated
V. vulnificus
from water from a mussel farm at a lower temperature than previously reported (7°C). In 1 of the 13 locations studied,
V. vulnificus
was found in mussels in 7 of 17 samples analyzed; this is the first report of
V. vulnificus
in European shellfish.
V. vulnificus
was also isolated from gills, intestinal contents, and mucus from wild fish. Although biotyping of 706
V. vulnificus
strains isolated during our investigations revealed that the majority of the strains (99.6%) belonged to biotype 1, biotype 2 was detected in seawater at a low frequency (0.4%). Our findings provide further evidence that seawater can serve as a reservoir and might facilitate spread of
V. vulnificus
biotype 2 to eels, with subsequent spread to persons handling eels. In conclusion, our data demonstrate that
V. vulnificus
is ubiquitous in a temperate marine environment and that
V. vulnificus
biotype 2 is not strictly confined to eels.
Molecular Analysis of Hypervirulent Somatic Hybrids of the Entomopathogenic Fungi
Beauveria bassiana
and
Beauveria sulfurescens
Authors: Viaud Muriel , Couteaudier Yvonne , Riba Guy 
Published: December 2019

ABSTRACT

Protoplast fusion of diauxotrophic mutants of a
Beauveria bassiana
entomopathogenic strain (Bb28) and a
Beauveria sulfurescens
toxinogenic strain (Bs2) produced hybrids which were significantly different from the parents in pathogenicity. Some of the hybrids were hypervirulent and killed insects more quickly than the Bb28 strain, probably because these hybrids had acquired the toxic activity of the Bs2 strain. By using six nuclear genes and a telomeric fingerprint probe, the molecular structures of the hybrids were studied. The results demonstrated the occurrence of parasexual events. Hybrids appeared to be diploid or aneuploid, with portions of the genome being heterozygous. A mitochondrial molecular marker indicated homoplasmy of the hybrids and inheritance of mitochondria from strain Bs2 or Bb28. The pathogenicities and the ploidies of the hybrids remained stable after passage through the host insect, showing that somatic hybridization provides an attractive method for the genetic improvement of biocontrol efficiency in the genus
Beauveria.
Leaching of Zinc Sulfide by
Thiobacillus ferrooxidans
: Experiments with a Controlled Redox Potential Indicate No Direct Bacterial Mechanism
Authors: Fowler T. A. , Crundwell F. K. 
Published: December 2019

ABSTRACT

The role of
Thiobacillus ferrooxidans
in bacterial leaching of mineral sulfides is controversial. Much of the controversy is due to the fact that the solution conditions, especially the concentrations of ferric and ferrous ions, change during experiments. The role of the bacteria would be more easily discernible if the concentrations of ferric and ferrous ions were maintained at set values throughout the experimental period. In this paper we report results obtained by using the constant redox potential apparatus described previously (P. I. Harvey and F. K. Crundwell, Appl. Environ. Microbiol. 63:2586–2592, 1997). This apparatus is designed to control the redox potential in the leaching compartment of an electrolytic cell by reduction or oxidation of dissolved iron. By controlling the redox potential the apparatus maintains the concentrations of ferrous and ferric ions at their initial values. Experiments were conducted in the presence of
T. ferrooxidans
and under sterile conditions. Analysis of the conversion of zinc sulfide in the absence of the bacteria and analysis of the conversion of zinc sulfate in the presence of the bacteria produced the same results. This indicates that the only role of the bacteria under the conditions used is regeneration of ferric ions in solution. In this work we found no evidence that there is a direct mechanism for bacterial leaching.
Purification, Characterization, and Substrate Specificity of a Novel Highly Glucose-Tolerant β-Glucosidase fromAspergillus oryzae
Authors: Riou Christine , Salmon Jean-Michel , Vallier Marie-Jose , Günata Ziya , Barre Pierre 
Published: December 2019

ABSTRACTAspergillus oryzaewas found to secrete two distinct β-glucosidases when it was grown in liquid culture on various substrates. The major form had a molecular mass of 130 kDa and was highly inhibited by glucose. The minor form, which was induced most effectively on quercetin (3,3′,4′,5,7-pentahydroxyflavone)-rich medium, represented no more than 18% of total β-glucosidase activity but exhibited a high tolerance to glucose inhibition. This highly glucose-tolerant β-glucosidase (designated HGT-BG) was purified to homogeneity by ammonium sulfate precipitation, gel filtration, and anion-exchange chromatography. HGT-BG is a monomeric protein with an apparent molecular mass of 43 kDa and a pI of 4.2 as determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and isoelectric focusing polyacrylamide gel electrophoresis, respectively. Usingp-nitrophenyl-β-d-glucoside as the substrate, we found that the enzyme was optimally active at 50°C and pH 5.0 and had a specific activity of 1,066 μmol min−1mg of protein−1and aKmof 0.55 mM under these conditions. The enzyme is particularly resistant to inhibition by glucose (Ki, 1.36 M) or glucono-δ-lactone (Ki, 12.5 mM), another powerful β-glucosidase inhibitor present in wine. A comparison of the enzyme activities on various glycosidic substrates indicated that HGT-BG is a broad-specificity type of fungal β-glucosidase. It exhibits exoglucanase activity and hydrolyzes (1→3)- and (1→6)-β-glucosidic linkages most effectively. This enzyme was able to release flavor compounds, such as geraniol, nerol, and linalol, from the corresponding monoterpenyl-β-d-glucosides in a grape must (pH 2.9, 90 g of glucose liter−1). Other flavor precursors (benzyl- and 2-phenylethyl-β-d-glucosides) and prunin (4′,5,7-trihydroxyflavanone-7-glucoside), which contribute to the bitterness of citrus juices, are also substrates of the enzyme. Thus, this novel β-glucosidase is of great potential interest in wine and fruit juice processing because it releases aromatic compounds from flavorless glucosidic precursors.
Characterization of an Extremely Thermostable Restriction Enzyme,
Psp
GI, from a
Pyrococcus
Strain and Cloning of the
Psp
GI Restriction-Modification System in
Escherichia coli
Authors: Morgan Richard , Xiao Jian-ping , Xu Shuang-yong 
Published: December 2019

ABSTRACT

An extremely thermostable restriction endonuclease,
Psp
GI, was purified from
Pyrococcus
sp. strain GI-H.
Psp
GI is an isoschizomer of
Eco
RII and cleaves DNA before the first C in the sequence 5′ ^CCWGG 3′ (W is A or T).
Psp
GI digestion can be carried out at 65 to 85°C. To express
Psp
GI at high levels, the
Psp
GI restriction-modification genes (
pspGIR
and
pspGIM
) were cloned in
Escherichia coli
. M.
Psp
GI contains the conserved sequence motifs of α-aminomethyltransferases; therefore, it must be an N4-cytosine methylase. M.
Psp
GI shows 53% similarity to (44% identity with) its isoschizomer, M.
Mva
I from
Micrococcus variabilis
. In a segment of 87 amino acid residues,
Psp
GI shows significant sequence similarity to
Eco
RII and to regions of
Sso
II and
Sty
D4I which have a closely related recognition sequence (5′ ^CCNGG 3′).
Psp
GI was expressed in
E. coli
via a T7 expression system. Recombinant
Psp
GI was purified to near homogeneity and had a half-life of 2 h at 95°C.
Psp
GI remained active following 30 cycles of thermocycling; thus, it can be used in DNA-based diagnostic applications.
Regulation ofaflRand Its Product, AflR, Associated with Aflatoxin Biosynthesis
Authors: Liu Biing-Hui , Chu Fun Sun 
Published: December 2019

ABSTRACTWe studied the role of the regulatory geneaflRand its product, AflR, in the biosynthesis of aflatoxin inAspergillus. Western blot and enzyme-linked immunosorbent assay analyses revealed that aflatoxin B1accumulation was directly related to AflR expression and was regulated by various environmental and nutritional conditions, including temperature, air supply, carbon source, nitrogen source, and zinc availability. Expression of an aflatoxin biosynthetic pathway structural gene,omtA, was regulated by the presence of AflR. Induction patterns foraflRmRNA and AflR were correlated with that foromtAmRNA in an aflatoxin-producing strain ofAspergillus parasiticus. Analysis of non-aflatoxin-producing strains ofA. flavus,A. sojae, andA. oryzaegrown in medium suitable for aflatoxin B1production showed that bothaflRmRNA and AflR production were present; however,omtAmRNA production was not detected in any of these examined strains. AflR in theA. oryzaestrain was regulated by carbon source and temperature in a manner similar to that seen withA. parasiticus.
Development of PCR Primer Systems for Amplification of Nitrite Reductase Genes (nirKandnirS) To Detect Denitrifying Bacteria in Environmental Samples
Authors: Braker Gesche , Fesefeldt Andreas , Witzel Karl-Paul 
Published: December 2019

ABSTRACTA system was developed for the detection of denitrifying bacteria by the amplification of specific nitrite reductase gene fragments with PCR. Primer sequences were found for the amplification of fragments from both nitrite reductase genes (nirKandnirS) after comparative sequence analysis. Whenever amplification was tried with these primers, the knownnirtype of denitrifying laboratory cultures could be confirmed. Likewise, the method allowed a determination of thenirtype of five laboratory strains. ThenirKgene could be amplified fromBlastobacter denitrificans,Alcaligenes xylosoxidans, andAlcaligenessp. (DSM 30128); thenirSgene was amplified fromAlcaligenes eutrophusDSM 530 and from the denitrifying isolate IFAM 3698. For each of the two genes, at least one primer combination amplified successfully for all of the test strains. Specific amplification products were not obtained with nondenitrifying bacteria or with strains of the othernirtype. The specificity of the amplified products was confirmed by subsequent sequencing. These results suggest the suitability of the method for the qualitative detection of denitrifying bacteria in environmental samples. This was shown by applying one generally amplifying primer combination for eachnirgene developed in this study to total DNA preparations from aquatic habitats.
Temperature Gradient Gel Electrophoresis Analysis of 16S rRNA from Human Fecal Samples Reveals Stable and Host-Specific Communities of Active Bacteria
Authors: Zoetendal Erwin G. , Akkermans Antoon D. L. , De Vos Willem M. 
Published: December 2019

ABSTRACT

The diversity of the predominant bacteria in the human gastrointestinal tract was studied by using 16S rRNA-based approaches. PCR amplicons of the V6 to V8 regions of fecal 16S rRNA and ribosomal DNA (rDNA) were analyzed by temperature gradient gel electrophoresis (TGGE). TGGE of fecal 16S rDNA amplicons from 16 individuals showed different profiles, with some bands in common. Fecal samples from two individuals were monitored over time and showed remarkably stable profiles over a period of at least 6 months. TGGE profiles derived from 16S rRNA and rDNA amplicons showed similar banding patterns. However, the intensities of bands with similar mobilities differed in some cases, indicating a different contribution to the total active fraction of the prominent fecal bacteria. Most 16S rRNA amplicons in the TGGE pattern of one subject were identified by cloning and sequence analysis. Forty-five of the 78 clones matched 15 bands, and 33 clones did not match any visible band in the TGGE pattern. Nested PCR of amplified 16S rDNA indicated preferential amplification of a sequence corresponding to 12 of the 33 nonmatching clones with similar mobilities in TGGE. The sequences matching 15 bands in the TGGE pattern showed 91.5 to 98.7% homology to sequences derived from different
Clostridium
clusters. Most of these were related to strains derived from the human intestine. The results indicate that the combination of cloning and TGGE analysis of 16S rDNA amplicons is a reliable approach to monitoring different microbial communities in feces.
The Introduction into
Bacillus sphaericus
of the
Bacillus thuringiensis
subsp.
medellin cyt1Ab1
Gene Results in Higher Susceptibility of Resistant Mosquito Larva Populations to
B. sphaericus
Authors: Thiéry I. , Hamon S. , Delécluse A. , Orduz S. 
Published: December 2019

ABSTRACT

The fragment containing the gene encoding the cytolytic Cyt1Ab1 protein from
Bacillus thuringiensis
subsp.
medellin
and its flanking sequences (I. Thiery, A. Delécluse, M. C. Tamayo, and S. Orduz, Appl. Environ. Microbiol. 63:468–473, 1997) was introduced into
Bacillus sphaericus
toxic strains 2362, 2297, and Iab872 by electroporation with the shuttle vector pMK3. Only small amounts of the protein were produced in recombinant strains 2362 and Iab872. The protein was detected in these strains only by Western blotting and immunodetection with antibody raised against Cyt1Ab1 protein. Large amounts of Cyt1Ab1 protein were produced in
B. sphaericus
recombinant strain 2297, and there was an additional crystal, other than that of the binary toxin, within the exosporium. The production of the Cyt1Ab1 protein in addition to the binary toxin did not increase the larvicidal activity of the
B. sphaericus
recombinant strain against susceptible mosquito populations of
Culex pipiens
or
Aedes aegypti
. However, it partially restored (10 to 20 times) susceptibility of the resistant mosquito populations of
C. pipiens
(SPHAE) and
Culex quinquefasciatus
(GeoR) to the binary toxin. The Cyt1Ab1 protein produced in recombinant
B. thuringiensis
SPL407(p
cyt1Ab1
) was synthesized in two types of crystal—one round and with various dense areas, surrounded by an envelope, and the other a regular cuboid crystal, very similar to that found in the
B. sphaericus
recombinant strain.
Construction of a Range of Derivatives of the Biological Control Strain
Agrobacterium rhizogenes
K84: a Study of Factors Involved in Biological Control of Crown Gall Disease
Authors: McClure Nicholas C. , Ahmadi Ali-Reza , Clare Bruce G. 
Published: December 2019

ABSTRACT

The biological control strain
Agrobacterium rhizogenes
K84 is an effective agent in the control of
Agrobacterium
pathogens, the causative agents of crown gall disease. A number of factors are thought to play a role in the control process, including production of the specific agrocins 84 and 434, which differ in the spectra of pathogenic strains that they inhibit in vitro. A range of derivatives of strain K84 has been developed with every combination of the three resident plasmids, pAgK84, pAgK434, and pAtK84b, including a plasmid-free strain. These derivatives produced either both, one, or neither of the characterized agrocins 84 and 434 and were isolated by plasmid curing, conjugation, and Tn
5
transposon mutagenesis. The ability of the derivative strains to inhibit gall formation on almond roots was compared to that of the wild-type K84 parent. Treatment with the plasmid-free derivative did not result in a significant level of control of an
A. rhizogenes
pathogen based on numbers or dry weight of galls formed on injured almond roots. The presence of plasmid pAgK84, pAgK434, or pAtK84b significantly enhanced the biological control efficacy of K84 derivatives, and the highest level of control was observed with strains harboring two or more plasmids. The results observed with strains deficient in agrocin 434 production suggest that this product may play an important role in the biological control of
A. rhizogenes
pathogens. The involvement of plasmid pAgK84b in biological control has not previously been reported. This study supports the conclusion that multiple factors are involved in the success of strain K84 as a biological control agent.
A New Operation for Producing Disease-Suppressive Compost from Grass Clippings
Authors: Nakasaki Kiyohiko , Hiraoka Sachiko , Nagata Hiroyuki 
Published: December 2019

ABSTRACT

This study evaluated the use of grass clippings discharged from golf courses as the raw material for production of a suppressive compost to control
Rhizoctonia
large-patch disease in mascarene grass.
Bacillus subtilis
N4, a mesophilic bacterium with suppressive effects on the pathogenic fungus
Rhizoctonia solani
AG2-2, was used as an inoculum in a procedure developed with the aim of controlling composting temperatures and inoculation timing. The population density of mesophilic bacteria in the raw material was reduced to around 5 log
10
CFU/g (dry weight) of composting material in the self-heating reaction at the initial stage of composting by maintaining a temperature of 80°C for 1 day. The inoculum was applied immediately, and the composting material was maintained at 40°C for 3 days. This served both to highly concentrate the suppressive bacterium and to achieve sporulation. The temperature was then raised to 60°C and maintained, enabling hygienic, high-speed composting while maintaining the population density of the suppressive bacterium as high as 8 log
10
CFU/g (dry weight) in the compost. The suppressiveness of compost made in this way was confirmed in a turf grass disease prevention assay.