bluuebunny/crossref_metadata_2025_split huggingface dataset:
Identification, Characterization, and In Situ Detection of a Fruit-Body-Specific Hydrophobin of
Pleurotus ostreatus
Authors: Peñas María M. , Ásgeirsdóttir Sigridur A. , Lasa Iñigo , Culiañez-Macià Francisco A. , Pisabarro Antonio G. , Wessels Joseph G. H. , Ramírez Lucía 
Published: December 2019

ABSTRACT

Hydrophobins are small (length, about 100 ± 25 amino acids), cysteine-rich, hydrophobic proteins that are present in large amounts in fungal cell walls, where they form part of the outermost layer (rodlet layer); sometimes, they can also be secreted into the medium. Different hydrophobins are associated with different developmental stages of a fungus, and their biological functions include protection of the hyphae against desiccation and attack by either bacterial or fungal parasites, hyphal adherence, and the lowering of surface tension of the culture medium to permit aerial growth of the hyphae. We identified and isolated a hydrophobin (fruit body hydrophobin 1 [Fbh1]) present in fruit bodies but absent in both monokaryotic and dikaryotic mycelia of the edible mushroom
Pleurotus ostreatus
. In order to study the temporal and spatial expression of the
fbh1
gene, we determined the N-terminal amino acid sequence of Fbh1. We also synthesized and cloned the double-stranded cDNA corresponding to the full-length mRNA of Fbh1 to use it as a probe in both Northern blot and in situ hybridization experiments. Fbh1 mRNA is detectable in specific parts of the fruit body, and it is absent in other developmental stages.
A Method for DNA Extraction from the Desert Cyanobacterium
Chroococcidiopsis
and Its Application to Identification of
ftsZ
Authors: Billi Daniela , Grilli Caiola Maria , Paolozzi Luciano , Ghelardini Patrizia 
Published: December 2019

ABSTRACT

A method was developed for extraction of DNA from
Chroococcidiopsis
that overcomes obstacles posed by bacterial contamination and the presence of a thick envelope surrounding the cyanobacterial cells. The method is based on the resistance of
Chroococcidiopsis
to lysozyme and consists of a lysozyme treatment followed by osmotic shock that reduces the bacterial contamination by 3 orders of magnitude. Then DNase treatment is performed to eliminate DNA from the bacterial lysate. Lysis of
Chroococcidiopsis
cells is achieved by grinding with glass beads in the presence of hot phenol. Extracted DNA is further purified by cesium-chloride density gradient ultracentrifugation. This method permitted the first molecular approach to the study of
Chroococcidiopsis
, and a 570-bp fragment of the gene
ftsZ
was cloned and sequenced.
Variable Cross-Resistance to Cry11B from
Bacillus thuringiensis
subsp.
jegathesan
in
Culex quinquefasciatus
(Diptera: Culicidae) Resistant to Single or Multiple Toxins of
Bacillus thuringienisis
subsp.
israelensis
Authors: Wirth Margaret C. , Delécluse Armelle , Federici Brian A. , Walton William E. 
Published: December 2019

ABSTRACT

A novel mosquitocidal bacterium,
Bacillus thuringiensis
subsp.
jegathesan
, and one of its toxins, Cry11B, in a recombinant
B. thuringiensis
strain were evaluated for cross-resistance with strains of the mosquito
Culex quinquefasciatus
that are resistant to single and multiple toxins of
Bacillus thuringiensis
subsp.
israelensis
. The levels of cross-resistance (resistance ratios [RR]) at concentrations which caused 95% mortality (LC
95
) between
B. thuringiensis
subsp.
jegathesan
and the different
B. thuringiensis
subsp.
israelensis
-resistant mosquito strains were low, ranging from 2.3 to 5.1. However, the levels of cross-resistance to Cry11B were much higher and were directly related to the complexity of the
B. thuringiensis
subsp.
israelensis
Cry toxin mixtures used to select the resistant mosquito strains. The LC
95
RR obtained with the mosquito strains were as follows: 53.1 against
Cq
4D, which was resistant to Cry11A; 80.7 against
Cq
4AB, which was resistant to Cry4A plus Cry4B; and 347 against
Cq
4ABD, which was resistant to Cry4A plus Cry4B plus Cry11A. Combining Cyt1A with Cry11B at a 1:3 ratio had little effect on suppressing Cry11A resistance in
Cq
4D but resulted in synergism factors of 4.8 and 11.2 against strains
Cq
4AB and
Cq
4ABD, respectively; this procedure eliminated cross-resistance in the former mosquito strain and reduced it markedly in the latter strain. The high levels of activity of
B. thuringiensis
subsp.
jegathesan
and
B. thuringiensis
subsp.
israelensis
, both of which contain a complex mixture of Cry and Cyt proteins, against Cry4- and Cry11-resistant mosquitoes suggest that novel bacterial strains with multiple Cry and Cyt proteins may be useful in managing resistance to bacterial insecticides in mosquito populations.
An Automated Fluorescent PCR Method for Detection of Shiga Toxin-Producing
Escherichia coli
in Foods
Authors: Chen Shu , Xu Renlin , Yee Arlene , Wu Kai Yuan , Wang Chang-Ning , Read Susan , De Grandis Stephanie A. 
Published: December 2019

ABSTRACT

An automated fluorescence-based PCR system (a model AG-9600 AmpliSensor analyzer) was investigated to determine whether it could detect Shiga toxin-producing
Escherichia coli
(STEC). The AmpliSensor PCR assay involves amplification-mediated disruption of a fluorogenic DNA signal duplex (AmpliSensor) that is homologous to conserved target sequences in a 323-bp amplified fragment of Shiga toxin genes
stx
1
,
stx
2
, and
stx
e
. Using the Amplisensor assay, we detected 113 strains of STEC belonging to 50 different serotypes, while 18 strains of non-Shiga-toxin-producing
E. coli
and 68 strains of other bacteria were not detected. The detection limits of the assay were less than 1 to 5 CFU per PCR mixture when pure cultures of five reference strains were used and 3 CFU per 25 g of food when spiked ground beef samples that were preenriched overnight were used. The performance of the assay was also evaluated by using 53 naturally contaminated meat samples and 48 raw milk samples. Thirty-two STEC-positive samples that were confirmed to be positive by the culture assay were found to be positive when the AmpliSensor assay was used. Nine samples that were found to be positive when the PCR assay was used were culture negative. The system described here is an automated PCR-based system that can be used for detection of all serotypes of STEC in food or clinical samples.
Effect of Specific Growth Rate on Fermentative Capacity of Baker’s Yeast
Authors: Van Hoek Pim , Van Dijken Johannes P. , Pronk Jack T. 
Published: December 2019

ABSTRACT

The specific growth rate is a key control parameter in the industrial production of baker’s yeast. Nevertheless, quantitative data describing its effect on fermentative capacity are not available from the literature. In this study, the effect of the specific growth rate on the physiology and fermentative capacity of an industrial
Saccharomyces cerevisiae
strain in aerobic, glucose-limited chemostat cultures was investigated. At specific growth rates (dilution rates,
D
) below 0.28 h
−1
, glucose metabolism was fully respiratory. Above this dilution rate, respirofermentative metabolism set in, with ethanol production rates of up to 14 mmol of ethanol · g of biomass
−1
· h
−1
at
D
= 0.40 h
−1
. A substantial fermentative capacity (assayed offline as ethanol production rate under anaerobic conditions) was found in cultures in which no ethanol was detectable (
D
< 0.28 h
−1
). This fermentative capacity increased with increasing dilution rates, from 10.0 mmol of ethanol · g of dry yeast biomass
−1
· h
−1
at
D
= 0.025 h
−1
to 20.5 mmol of ethanol · g of dry yeast biomass
−1
· h
−1
at
D
= 0.28 h
−1
. At even higher dilution rates, the fermentative capacity showed only a small further increase, up to 22.0 mmol of ethanol · g of dry yeast biomass
−1
· h
−1
at
D
= 0.40 h
−1
. The activities of all glycolytic enzymes, pyruvate decarboxylase, and alcohol dehydrogenase were determined in cell extracts. Only the in vitro activities of pyruvate decarboxylase and phosphofructokinase showed a clear positive correlation with fermentative capacity. These enzymes are interesting targets for overexpression in attempts to improve the fermentative capacity of aerobic cultures grown at low specific growth rates.
Seasonal Community and Population Dynamics of Pelagic Bacteria and Archaea in a High Mountain Lake
Authors: Pernthaler Jakob , Glöckner Frank-Oliver , Unterholzner Stefanie , Alfreider Albin , Psenner Roland , Amann Rudolf 
Published: December 2019

ABSTRACT

The seasonal variations in community structure and cell morphology of pelagic procaryotes from a high mountain lake (Gossenköllesee, Austria) were studied by in situ hybridization with rRNA-targeted fluorescently labeled oligonucleotide probes (FISH) and image-analyzed microscopy. Compositional changes and biomass fluctuations within the assemblage were observed both in summer and beneath the winter ice cover and are discussed in the context of physicochemical and biotic parameters. Proteobacteria of the beta subclass (beta-proteobacteria) formed a dominant fraction of the bacterioplankton (annual mean, 24% of the total counts), whereas alpha-proteobacteria were of similar relative importance only during spring (mean, 11%). Bacteria of the
Cytophaga-Flavobacterium
cluster, although less abundant, constituted the largest fraction of the filamentous morphotypes during most of the year, thus contributing significantly to the total microbial biomass. Successive peaks of threadlike and rod-shaped archaea were observed during autumn thermal mixing and the period of ice cover formation, respectively. A set of oligonucleotide probes targeted to single phylotypes was constructed from 16S rRNA-encoding gene clone sequences. Three distinct populations of uncultivated microbes, affiliated with the alpha- and beta-proteobacteria, were subsequently monitored by FISH. About one-quarter of all of the beta-proteobacteria (range, 6 to 53%) could be assigned to only two phylotypes. The bacterial populations studied were annually recurrent, seasonally variable, and vertically stratified, except during the periods of lake overturn. Their variability clearly exceeded the fluctuations of the total microbial assemblage, suggesting that the apparent stability of total bacterioplankton abundances may mask highly dynamic community fluctuations.
Metal Accumulation and Vanadium-Induced Multidrug Resistance by Environmental Isolates of
Escherichia hermannii
and
Enterobacter cloacae
Authors: Hernández Alicia , Mellado Rafael P. , Martínez José L. 
Published: December 2019

ABSTRACT

Contaminated soils from an oil refinery were screened for the presence of microorganisms capable of accumulating either nickel, vanadium, or both metals. Three strains of bacteria that belonged to the family
Enterobacteriaceae
were selected. Two of them were
Escherichia hermannii
strains, and outer membrane profile (OMP) analysis showed that they were similar to a strain of clinical origin; the other one was an
Enterobacter cloacae
strain that differed from clinical isolates. The selected bacteria accumulated both nickel and vanadium. Growth in the presence of vanadium induced multidrug resistance phenotypes in
E. hermannii
and
E. cloacae
. Incubation with this metal changed the OMP profile of
E. hermannii
but did not produce variations in the expression of the major OMPs of
E. cloacae.
Molecular Detection, Isolation, and Physiological Characterization of Functionally Dominant Phenol-Degrading Bacteria in Activated Sludge
Authors: Watanabe Kazuya , Teramoto Maki , Futamata Hiroyuki , Harayama Shigeaki 
Published: December 2019

ABSTRACT

DNA was isolated from phenol-digesting activated sludge, and partial fragments of the 16S ribosomal DNA (rDNA) and the gene encoding the largest subunit of multicomponent phenol hydroxylase (LmPH) were amplified by PCR. An analysis of the amplified fragments by temperature gradient gel electrophoresis (TGGE) demonstrated that two major 16S rDNA bands (bands R2 and R3) and two major LmPH gene bands (bands P2 and P3) appeared after the activated sludge became acclimated to phenol. The nucleotide sequences of these major bands were determined. In parallel, bacteria were isolated from the activated sludge by direct plating or by plating after enrichment either in batch cultures or in a chemostat culture. The bacteria isolated were classified into 27 distinct groups by a repetitive extragenic palindromic sequence PCR analysis. The partial nucleotide sequences of 16S rDNAs and LmPH genes of members of these 27 groups were then determined. A comparison of these nucleotide sequences with the sequences of the major TGGE bands indicated that the major bacterial populations, R2 and R3, possessed major LmPH genes P2 and P3, respectively. The dominant populations could be isolated either by direct plating or by chemostat culture enrichment but not by batch culture enrichment. One of the dominant strains (R3) which contained a novel type of LmPH (P3), was closely related to
Valivorax paradoxus
, and the result of a kinetic analysis of its phenol-oxygenating activity suggested that this strain was the principal phenol digester in the activated sludge.
Analysis of the Influence of Environmental Parameters on
Clostridium botulinum
Time-to-Toxicity by Using Three Modeling Approaches
Authors: Schaffner Donald W. , Ross William H. , Montville Thomas J. 
Published: December 2019

ABSTRACT

This study used the technique of waiting time modeling to analyze the combined effects of temperature, pH, carbohydrate, protein, and lipid on the time-to-toxicity of
Clostridium botulinum
56A. Waiting time models can be used whenever the time to the occurrence of some event is the variable of interest. In the case of the time-to-toxicity data, the variable is the time from the beginning of an experiment until a tube is identified as positive. The statistical analysis used the SAS procedure LIFEREG and included determination of the form of the response surface, identification of the error distribution, and simplification of the response surface. We found that increasing the macromolecule concentration decreased the probability of toxin formation. The probability of toxin formation also decreased at lower temperatures and at pHs further from the optimum. The waiting time modeling approach to developing models for botulinal toxin formation compared favorably with other approaches but had one specific advantage. Waiting time models have the inherent advantage that safety concerns regarding predictions are automatically quantified in the analysis by formally identifying a distribution of times-to-toxicity. The use of this time-to-toxicity distribution permits a customizable margin of safety (e.g., one in a million) not possible with other approaches.
Gene Cloning, DNA Sequencing, and Expression of Thermostable β-Mannanase from
Bacillus stearothermophilus
Authors: Ethier Nathalie , Talbot Guylaine , Sygusch Jurgen 
Published: December 2019

ABSTRACT

A DNA genomic library constructed from
Bacillus stearothermophilus
, a gram-positive, facultative thermophilic aerobe that secretes a thermostable β-mannanase, was screened for mannan hydrolytic activity. Recombinant β-mannanase activity was detected on the basis of the clearing of halos around
Escherichia coli
colonies grown on a dye-labelled substrate, Remazol brilliant blue-locust bean gum. The nucleotide sequence of the mannanase gene,
manF
, corresponded to an open reading frame of 2,085 bp that codes for a 32-amino-acid signal peptide and a mature protein with a molecular mass of 76,089 Da. From sequence analysis, ManF belongs to glycosyl hydrolase family 5 and exhibits higher similarity to eukaryotic than to bacterial mannanases. The
manF
coding sequence was subcloned into the pH6EX3 expression plasmid and expressed in
E. coli
as a recombinant fusion protein containing a hexahistidine N-terminal sequence. The fusion protein has thermostability similar to the native enzyme and was purified by Ni
2+
affinity chromatography. The values for the kinetic parameters
V
max
and

K
m

were 384 U/mg and 2.4 mg/ml, respectively, for the recombinant mannanase and were comparable to those of the native enzyme.
Kinetic Bias in Estimates of Coastal Picoplankton Community Structure Obtained by Measurements of Small-Subunit rRNA Gene PCR Amplicon Length Heterogeneity
Authors: Suzuki Marcelino , Rappé Michael S. , Giovannoni Stephen J. 
Published: December 2019

ABSTRACT
Marine bacterioplankton diversity was examined by quantifying natural length variation in the 5′ domain of small-subunit (SSU) rRNA genes (rDNA) amplified by PCR from a DNA sample from the Oregon coast. This new technique, length heterogeneity analysis by PCR (LH-PCR), determines the relative proportions of amplicons originating from different organisms by measuring the fluorescence emission of a labeled primer used in the amplification reaction. Relationships between the sizes of amplicons and gene phylogeny were predicted by an analysis of 366 SSU rDNA sequences from cultivated marine bacteria and from bacterial genes cloned directly from environmental samples. LH-PCR was used to compare the distribution of bacterioplankton SSU rDNAs from a coastal water sample with that of an SSU rDNA clone library prepared from the same sample and also to examine the distribution of genes in the PCR products from which the clone library was prepared. The analysis revealed that the relative frequencies of genes amplified from natural communities are highly reproducible for replicate sets of PCRs but that a bias possibly caused by the reannealing kinetics of product molecules can skew gene frequencies when PCR product concentrations exceed threshold values.
Spectrum of the Reductive Dehalogenation Activity of
Desulfitobacterium frappieri
PCP-1
Authors: Dennie D. , Gladu I. , Lépine F. , Villemur R. , Bisaillon J.-G. , Beaudet R. 
Published: December 2019

ABSTRACT

Desulfitobacterium frappieri
PCP-1 was induced for
ortho
- and
para
-dechlorinating activities by different chlorophenols. Dehalogenation rates ranging from 25 to 1,158 nmol/min/mg of cell protein were observed according to the chlorophenol tested and the position of the chlorine removed.
D. frappieri
shows a broad substrate specificity; in addition to tetrachloroethylene and pentachloropyridine, strain PCP-1 can dehalogenate at
ortho
,
meta
, and
para
positions a large variety of aromatic molecules with substituted hydroxyl or amino groups. Reactions of O demethylation and reduction of nitro to amino substituents on aromatic molecules were also observed.
Effects of Starvation on Physiological Activity and Chlorine Disinfection Resistance in
Escherichia coli
O157:H7
Authors: Lisle John T. , Broadaway Susan C. , Prescott Annette M. , Pyle Barry H. , Fricker Colin , McFeters Gordon A. 
Published: December 2019

ABSTRACT

Escherichia coli
O157:H7 can persist for days to weeks in microcosms simulating natural conditions. In this study, we used a suite of fluorescent, in situ stains and probes to assess the influence of starvation on physiological activity based on membrane potential (rhodamine 123 assay), membrane integrity (LIVE/DEAD
Bac
Light kit), respiratory activity (5-cyano-2,3-di-4-tolyl-tetrazolium chloride assay), intracellular esterase activity (ScanRDI assay), and 16S rRNA content. Growth-dependent assays were also used to assess substrate responsiveness (direct viable count [DVC] assay), ATP activity (MicroStar assay), and culturability (R2A agar assay). In addition, resistance to chlorine disinfection was assessed. After 14 days of starvation, the DVC values decreased, while the values in all other assays remained relatively constant and equivalent to each other. Chlorine resistance progressively increased through the starvation period. After 29 days of starvation, there was no significant difference in chlorine resistance between control cultures that had not been exposed to the disinfectant and cultures that had been exposed. This study demonstrates that
E. coli
O157:H7 adapts to starvation conditions by developing a chlorine resistance phenotype.
AbiQ, an Abortive Infection Mechanism fromLactococcus lactis
Authors: Emond Eric , Dion Eric , Walker Shirley A. , Vedamuthu Ebenezer R. , Kondo Jeffery K. , Moineau Sylvain 
Published: December 2019

ABSTRACTLactococcus lactisW-37 is highly resistant to phage infection. The cryptic plasmids from this strain were coelectroporated, along with the shuttle vector pSA3, into the plasmid-free hostL. lactisLM0230. In addition to pSA3, erythromycin- and phage-resistant isolates carried pSRQ900, an 11-kb plasmid fromL. lactisW-37. This plasmid made the host bacteria highly resistant (efficiency of plaquing <10−8) to c2- and 936-like phages. pSRQ900 did not confer any resistance to phages of the P335 species. Adsorption, cell survival, and endonucleolytic activity assays showed that pSRQ900 encodes an abortive infection mechanism. The phage resistance mechanism is limited to a 2.2-kbEcoRV/BclI fragment. Sequence analysis of this fragment revealed a complete open reading frame (abiQ), which encodes a putative protein of 183 amino acids. A frameshift mutation withinabiQcompletely abolished the resistant phenotype. The predicted peptide has a high content of positively charged residues (pI = 10.5) and is, in all likelihood, a cytosolic protein. AbiQ has no homology to known or deduced proteins in the databases. DNA replication assays showed that phage c21 (c2-like) and phage p2 (936-like) can still replicate in cells harboring AbiQ. However, phage DNA accumulated in its concatenated form in the infected AbiQ+cells, whereas the AbiQ−cells contained processed (mature) phage DNA in addition to the concatenated form. The production of the major capsid protein of phage c21 was not hindered in the cells harboring AbiQ.
Genetic Characterization and Heterologous Expression of Brochocin-C, an Antibotulinal, Two-Peptide Bacteriocin Produced by
Brochothrix campestris
ATCC 43754
Authors: McCormick John K. , Poon Alison , Sailer Miloslav , Gao Yan , Roy Ken L. , McMullen Lynn M. , Vederas John C. , Stiles Michael E. , Van Belkum Marco J. 
Published: December 2019

ABSTRACT

Brochocin-C, produced by
Brochothrix campestris
ATCC 43754, is active against many strains of the closely related meat spoilage organism
Brochothrix thermosphacta
and a wide range of other gram-positive bacteria, including spores of
Clostridium botulinum
. Purification of the active compound and genetic characterization of brochocin-C revealed that it is a chromosomally encoded, two-peptide nonlantibiotic bacteriocin. Both peptides of brochocin-C are ribosomally synthesized as prepeptides that are typical of class II bacteriocins. They are cleaved following Gly-Gly cleavage sites to yield the mature peptides, BrcA and BrcB, containing 59 and 43 amino acids, respectively. Fusion of the nucleotides encoding the signal peptide of the bacteriocin divergicin A in front of the structural genes for either BrcA or BrcB allowed independent expression of each component by the general protein secretion pathway. This revealed the two-component nature of brochocin-C and the necessity for both peptides for activity. A 53-amino-acid peptide encoded downstream of
brcB
functions as the immunity protein (BrcI) for brochocin-C. In addition, the cloned chromosomal fragment revealed open reading frames downstream of
brcI
, designated
brcT
and
brcD
, that encode proteins with homology to ATP-binding cassette translocator and accessory proteins, respectively, involved in the secretion of Gly-Gly-type bacteriocins.