bluuebunny/crossref_metadata_2025_split huggingface dataset:
Biotransformation of 2,4,6-Trinitrotoluene with
Phanerochaete chrysosporium
in Agitated Cultures at pH 4.5
Authors: Hawari Jalal , Halasz Annamaria , Beaudet Sylvie , Paquet Louise , Ampleman Guy , Thiboutot Sonia 
Published: December 2019

ABSTRACT

The biotransformation of 2,4,6-trinitrotoluene (TNT) (175 μM) by
Phanerochaete chrysosporium
with molasses and citric acid at pH 4.5 was studied. In less than 2 weeks, TNT disappeared completely, but mineralization (liberated
14
CO
2
) did not exceed 1%. A time study revealed the presence of several intermediates, marked by the initial formation of two monohydroxylaminodinitrotoluenes (2- and 4-HADNT) followed by their successive transformation to several other products, including monoaminodinitrotoluenes (ADNT). A group of nine acylated intermediates were also detected. They included 2-
N
-acetylamido-4,6-dinitrotoluene and its
p
isomer, 2-formylamido-4,6-dinitrotoluene and its
p
isomer (as acylated ADNT), 4-
N
-acetylamino-2-amino-6-nitrotoluene and 4-
N
-formylamido-2-amino-6-nitrotoluene (as acetylated DANT), 4-
N
-acetylhydroxy-2,6-dinitrotoluene and 4-
N
-acetoxy-2,6-dinitrotoluene (as acetylated HADNT), and finally 4-
N
-acetylamido-2-hydroxylamino-6-nitrotoluene. Furthermore, a fraction of HADNTs were found to rearrange to their corresponding phenolamines (Bamberger rearrangement), while another group dimerized to azoxytoluenes which in turn transformed to azo compounds and eventually to the corresponding hydrazo derivatives. After 30 days, all of these metabolites, except traces of 4-ADNT and the hydrazo derivatives, disappeared, but mineralization did not exceed 10% even after the incubation period was increased to 120 days. The biotransformation of TNT was accompanied by the appearance of manganese peroxidase (MnP) and lignin-dependent peroxidase (LiP) activities. MnP activity was observed almost immediately after TNT disappearance, which was the period marked by the appearance of the initial metabolites (HADNT and ADNT), whereas the LiP activity was observed after 8 days of incubation, corresponding to the appearance of the acyl derivatives. Both MnP and LiP activities reached their maximum levels (100 and 10 U/liter, respectively) within 10 to 15 days after inoculation.
Purification, Characterization, Gene Cloning, Sequencing, and Overexpression of Aminopeptidase N fromStreptococcus thermophilusA
Authors: Chavagnat Frederic , Casey Michael G. , Meyer Jacques 
Published: December 2019

ABSTRACTThe general aminopeptidase PepN fromStreptococcus thermophilusA was purified to protein homogeneity by hydroxyapatite, anion-exchange, and gel filtration chromatographies. The PepN enzyme was estimated to be a monomer of 95 kDa, with maximal activity onN-Lys–7-amino-4-methylcoumarin at pH 7 and 37°C. It was strongly inhibited by metal chelating agents, suggesting that it is a metallopeptidase. The activity was greatly restored by the bivalent cations Co2+, Zn2+, and Mn2+. Except for proline, glycine, and acidic amino acid residues, PepN has a broad specificity on theN-terminal amino acid of small peptides, but no significant endopeptidase activity has been detected. TheN-terminal and short internal amino acid sequences of purified PepN were determined. By using synthetic primers and a battery of PCR techniques, thepepNgene was amplified, subcloned, and further sequenced, revealing an open reading frame of 2,541 nucleotides encoding a protein of 847 amino acids with a molecular weight of 96,252. Amino acid sequence analysis of thepepNgene translation product shows high homology with other PepN enzymes from lactic acid bacteria and exhibits the signature sequence of the zinc metallopeptidase family. ThepepNgene was cloned in a T7 promoter-based expression plasmid and the 452-fold overproduced PepN enzyme was purified to homogeneity from the periplasmic extract of the hostEscherichia colistrain. The overproduced enzyme showed the same catalytic characteristics as the wild-type enzyme.
High-Level Production of Human Leptin by Fed-Batch Cultivation of Recombinant
Escherichia coli
and Its Purification
Authors: Jeong Ki Jun , Lee Sang Yup 
Published: December 2019

ABSTRACT

Human leptin is a 16-kDa (146-amino-acid) protein that is secreted from adipocytes and influences body weight homeostasis. In order to obtain high-level production of leptin, the human
obese
gene coding for leptin was expressed in
Escherichia coli
BL21(DE3) under the strong inducible T7 promoter. The recombinant leptin was produced as inclusion bodies in
E. coli
, and the recombinant leptin content was as high as 54% of the total protein content. For production of recombinant human leptin in large amounts, pH-stat fed-batch cultures were grown. Expression of leptin was induced at three different cell optical densities at 600 nm (OD
600
), 30, 90, and 140. When cells were induced at an OD
600
of 90, the amount of leptin produced was 9.7 g/liter (37% of the total protein). After simple purification steps consisting of inclusion body isolation, denaturation and refolding, and anion-exchange chromatography, 144.9 mg of leptin that was more than 90% pure was obtained from a 50-ml culture, and the recovery yield was 41.1%.
The
groESL
Chaperone Operon of
Lactobacillus johnsonii
Authors: Walker D. Carey , Girgis Hany S. , Klaenhammer Todd R. 
Published: December 2019

ABSTRACT

The
Lactobacillus johnsonii
VPI 11088
groESL
operon was localized on the chromosome near the insertion element IS
1223
. The operon was initially cloned as a series of three overlapping PCR fragments, which were sequenced and used to design primers to amplify the entire operon. The amplified fragment was used as a probe to recover the chromosomal copy of the
groESL
operon from a partial library of
L. johnsonii
VPI 11088 (NCK88) DNA, cloned in the shuttle vector pTRKH2. The 2,253-bp
groESL
fragment contained three putative open reading frames, two of which encoded the ubiquitous GroES and GroEL chaperone proteins. Analysis of the
groESL
promoter region revealed three transcription initiation sites, as well as three sets of inverted repeats (IR) positioned between the transcription and translation start sites. Two of the three IR sets bore significant homology to the CIRCE elements, implicated in negative regulation of the heat shock response in many bacteria. Northern analysis and primer extension revealed that multiple temperature-sensitive promoters preceded the
groESL
chaperone operon, suggesting that stress protein production in
L. johnsonii
is strongly regulated. Maximum
groESL
transcription activity was observed following a shift to 55°C, and a 15 to 30-min exposure of log-phase cells to this temperature increased the recovery of freeze-thawed
L. johnsonii
VPI 11088. These results suggest that a brief, preconditioning heat shock can be used to trigger increased chaperone production and provide significant cross-protection from the stresses imposed during the production of frozen culture concentrates.
Microbial Communities Associated with Anaerobic Benzene Degradation in a Petroleum-Contaminated Aquifer
Authors: Rooney-Varga Juliette N. , Anderson Robert T. , Fraga Jocelyn L. , Ringelberg David , Lovley Derek R. 
Published: December 2019

ABSTRACT

Microbial community composition associated with benzene oxidation under in situ Fe(III)-reducing conditions in a petroleum-contaminated aquifer located in Bemidji, Minn., was investigated. Community structure associated with benzene degradation was compared to sediment communities that did not anaerobically oxidize benzene which were obtained from two adjacent Fe(III)-reducing sites and from methanogenic and uncontaminated zones. Denaturing gradient gel electrophoresis of 16S rDNA sequences amplified with bacterial or
Geobacteraceae
-specific primers indicated significant differences in the composition of the microbial communities at the different sites. Most notable was a selective enrichment of microorganisms in the
Geobacter
cluster seen in the benzene-degrading sediments. This finding was in accordance with phospholipid fatty acid analysis and most-probable-number–PCR enumeration, which indicated that members of the family
Geobacteraceae
were more numerous in these sediments. A benzene-oxidizing Fe(III)-reducing enrichment culture was established from benzene-degrading sediments and contained an organism closely related to the uncultivated
Geobacter
spp. This genus contains the only known organisms that can oxidize aromatic compounds with the reduction of Fe(III). Sequences closely related to the Fe(III) reducer
Geothrix fermentans
and the aerobe
Variovorax paradoxus
were also amplified from the benzene-degrading enrichment and were present in the benzene-degrading sediments. However, neither
G. fermentans
nor
V. paradoxus
is known to oxidize aromatic compounds with the reduction of Fe(III), and there was no apparent enrichment of these organisms in the benzene-degrading sediments. These results suggest that
Geobacter
spp. play an important role in the anaerobic oxidation of benzene in the Bemidji aquifer and that molecular community analysis may be a powerful tool for predicting a site’s capacity for anaerobic benzene degradation.
Expression of the
Escherichia coli
Catabolic Threonine Dehydratase in
Corynebacterium glutamicum
and Its Effect on Isoleucine Production
Authors: Guillouet S. , Rodal A. A. , An G.-H. , Lessard P. A. , Sinskey A. J. 
Published: December 2019

ABSTRACT

The catabolic or biodegradative threonine dehydratase (E.C. 4.2.1.16) of
Escherichia coli
is an isoleucine feedback-resistant enzyme that catalyzes the degradation of threonine to α-ketobutyrate, the first reaction of the isoleucine pathway. We cloned and expressed this enzyme in
Corynebacterium glutamicum
. We found that while the native threonine dehydratase of
C. glutamicum
was totally inhibited by 15 mM isoleucine, the heterologous catabolic threonine dehydratase expressed in the same strain was much less sensitive to isoleucine; i.e., it retained 60% of its original activity even in the presence of 200 mM isoleucine. To determine whether expressing the catabolic threonine dehydratase (encoded by the
tdcB
gene) provided any benefit for isoleucine production compared to the native enzyme (encoded by the
ilvA
gene), fermentations were performed with the wild-type strain, an
ilvA
-overexpressing strain, and a
tdcB
-expressing strain. By expressing the heterologous catabolic threonine dehydratase in
C. glutamicum
, we were able to increase the production of isoleucine 50-fold, whereas overexpression of the native threonine dehydratase resulted in only a fourfold increase in isoleucine production. Carbon balance data showed that when just one enzyme, the catabolic threonine dehydratase, was overexpressed, 70% of the carbon available for the lysine pathway was redirected into the isoleucine pathway.
Specific Detection of the Gene for the Extracellular Neutral Protease of
Bacillus cereus
by PCR and Blot Hybridization
Authors: Bach H.-J. , Errampalli D. , Leung K. T. , Lee H. , Hartmann A. , Trevors J. T. , Munch J. C. 
Published: December 2019

ABSTRACT

A pair of primers and a gene probe for the amplification and detection of the
Bacillus cereus
neutral protease gene (NPRC) were developed. Specificity for the
npr
genes of the
B. cereus
group members
B. cereus
,
B. mycoides
, and
B. thuringiensis
was shown. Restriction polymorphism patterns of the PCR products confirmed the presence of the NPRC gene in all three species.
Viability and Virulence of Experimentally Stressed Nonculturable
Salmonella typhimurium
Authors: Caro Audrey , Got Patrice , Lesne Jean , Binard Sylvie , Baleux Bernard 
Published: December 2019

ABSTRACT

Maintenance of pathogenicity of viable but nonculturable
Salmonella typhimurium
cells experimentally stressed with UV-C and seawater, was investigated relative to the viability level of the cellular population. Pathogenicity, tested in a mouse model, was lost concomitantly with culturability, whereas cell viability remained undamaged, as determined by respiratory activity and cytoplasmic membrane and genomic integrities.
Effect of Cattle Diet on
Escherichia coli
O157:H7 Acid Resistance
Authors: Hovde Carolyn J. , Austin Paula R. , Cloud Karen A. , Williams Christopher J. , Hunt Carl W. 
Published: December 2019

ABSTRACT

The duration of shedding of
Escherichia coli
O157 isolates by hay-fed and grain-fed steers experimentally inoculated with
E. coli
O157:H7 was compared, as well as the acid resistance of the bacteria. The hay-fed animals shed
E. coli
O157 longer than the grain-fed animals, and irrespective of diet, these bacteria were equally acid resistant. Feeding cattle hay may increase human infections with
E. coli
O157:H7.
Influence of 1-[(
E
)-2-(2-Methyl-4-Nitrophenyl)Diaz-1-enyl]Pyrrolidine-2-Carboxylic Acid and Diphenyliodonium Chloride on Ruminal Protein Metabolism and Ruminal Microorganisms
Authors: Floret Fabienne , Chaudhary Lal C. , Ellis William C. , El Hassan Suliman , McKain Nest , Newbold C. James , Wallace R. John 
Published: December 2019

ABSTRACT

The effects of 1-[(
E
)-2-(2-methyl-4-nitrophenyl)diaz-1-enyl]pyrrolidine-2-carboxylic acid (LY29) and diphenyliodonium chloride (DIC) on the degradation of protein to ammonia were determined in a mixed rumen microbial population taken from sheep on a grass hay-concentrate diet. Both compounds decreased NH
3
production by inhibiting deamination of amino acids. LY29, but not DIC, inhibited growth of the high-activity ammonia-producing species,
Clostridium aminophilum
and
Clostridium sticklandii
.
The Spheroplast Lysis Assay for Yeast in Microtiter Plate Format
Authors: Ovalle Rafael , Spencer Moyah , Thiwanont Monthiwa , Lipke Peter N. 
Published: December 2019

ABSTRACT
A yeast lysis assay in the microtiter plate format improved precision and throughput and led to an improved algorithm for estimating lag time. The assay reproducibly revealed differences of 10% or greater in the maximal lysis rate and 50% or greater in the lag time. Clonal differences were determined to be the major source of variation. Microtiter-based assays should be useful for screening for drug susceptibility and for analyzing mutant phenotypes.
Determination of Abundance and Biovolume of Bacteria in Sediments by Dual Staining with 4′,6-Diamidino-2-Phenylindole and Acridine Orange: Relationship to Dispersion Treatment and Sediment Characteristics
Authors: Kuwae Tomohiro , Hosokawa Yasushi 
Published: December 2019

ABSTRACT

We measured the abundance and biovolume of bacteria in intertidal sediments from Tokyo Bay, Japan, by using a dual-staining technique (4′,6-diamidino-2-phenylindole and acridine orange) and several dispersion techniques (ultrasonic cleaner, ultrasonic sonicator, and tissue homogenizer). Dual staining reduced serious background fluorescence, particularly when used for silt-, clay-, and detritus-rich sediments, and allowed us to distinguish bacteria from other objects during both counting and sizing. Within the studied samples, the number of bacterial cells ranged from 0.20 × 10
9
to 3.54 × 10
9
g of wet sediment
−1
. With the cleaner and sonicator treatments, the bacterial numbers for all of the sites initially increased with dispersion time and then became constant. For the homogenizer treatments, the highest bacterial numbers were observed with the shortest (0.5- to 2-min) treatments, and the counts then declined steeply as the homogenization time increased, indicating that cell destruction occurred. The cleaner treatment had the possibility of insufficient dispersion of bacteria for fine-grain sediments. Within the studied samples, the bacterial biovolume ranged from 0.07 to 0.22 μm
3
. With the cleaner and sonicator treatments, the biovolume peaked during the shorter dispersion time. This pattern was caused not by cell destruction but by the incremental portion of dispersed small cells. We concluded that with the cleaner and sonicator treatments, the longer dispersion time reflected the real size spectrum and was preferable for accurate estimation of mean bacterial biovolumes.
Molecular Karyotype of the White Rot Fungus
Pleurotus ostreatus
Authors: Larraya Luis M. , Pérez Gumer , Peñas María M. , Baars Johan J. P. , Mikosch Thomas S. P. , Pisabarro Antonio G. , Ramírez Lucía 
Published: December 2019

ABSTRACT

The white rot fungus
Pleurotus ostreatus
is an edible basidiomycete with increasing agricultural and biotechnological importance. Genetic manipulation and breeding of this organism are restricted because of the lack of knowledge about its genomic structure. In this study, we analyzed the genomic constitution of
P. ostreatus
by using pulsed-field gel electrophoresis optimized for the separation of its chromosomes. We have determined that it contains 11 pairs of chromosomes with sizes ranging from 1.4 to 4.7 Mbp. In addition to chromosome separation, the use of single-copy DNA probes allowed us to resolve the ambiguities caused by chromosome comigration. When the two nuclei present in the dikaryon were separated by protoplasting, analysis of their karyotypes revealed length polymorphisms affecting various chromosomes. This is, to our knowledge, the clearest chromosome separation available for this species.
Culturable Populations of
Sporomusa
spp. and
Desulfovibrio
spp. in the Anoxic Bulk Soil of Flooded Rice Microcosms
Authors: Rosencrantz Dirk , Rainey Frederick A. , Janssen Peter H. 
Published: December 2019

ABSTRACT

Most-probable-number (MPN) counts were made of homoacetogenic and other bacteria present in the anoxic flooded bulk soil of laboratory microcosms containing 90- to 95-day-old rice plants. MPN counts with substrates known to be useful for the selective enrichment or the cultivation of homoacetogenic bacteria (betaine, ethylene glycol, 2,3-butanediol, and 3,4,5-trimethoxybenzoate) gave counts of 2.3 × 10
3
to 2.8 × 10
5
cells per g of dry soil. Homoacetogens isolated from the terminal positive steps of these dilution cultures belonged to the genus
Sporomusa
. Counts with succinate, ethanol, and lactate gave much higher MPNs of 5.9 × 10
5
to 3.4 × 10
7
cells per g of dry soil and led to the isolation of
Desulfovibrio
spp. Counting experiments on lactate and ethanol which included
Methanospirillum hungatei
in the medium gave MPNs of 2.3 × 10
6
to 7.5 × 10
8
cells per g of dry soil and led to the isolation of
Sporomusa
spp. The latter strains could grow with betaine, ethylene glycol, 2,3-butanediol, and/or 3,4,5-trimethoxybenzoate, but apparently most cells of
Sporomusa
spp. did not initiate growth in counting experiments with those substrates. Spores apparently accounted for 2.2% or less of the culturable bacteria. It appears that culturable
Desulfovibrio
spp. and
Sporomusa
spp. were present in approximately equal numbers in the bulk soil. Multiple, phylogenetically-distinct, phenotypically-different, strains of each genus were found in the same soil system.
Induction of Protease Activity in
Vibrio anguillarum
by Gastrointestinal Mucus
Authors: Denkin Steven M. , Nelson David R. 
Published: December 2019

ABSTRACT

The effect of gastrointestinal mucus on protease activity in
Vibrio anguillarum
was investigated. Protease activity was measured by using an azocasein hydrolysis assay. Cells grown to stationary phase in mucus (200 μg of mucus protein/ml) exhibited ninefold-greater protease activity than cells grown in Luria-Bertani broth plus 2% NaCl (LB20). Protease induction was examined with cells grown in LB20 and resuspended in mucus, LB20, nine-salts solution (NSS [a carbon-, nitrogen-, and phosphorus-free salt solution]), or marine minimal medium (3M) (∼10
9
CFU/ml). Induction of protease activity occurred 60 to 90 min after addition of mucus and was ≥70-fold greater than protease activity measured in cells incubated in either LB20 or 3M. Mucus was fractionated into aqueous and chloroform-methanol-soluble fractions. The aqueous fraction supported growth of
V. anguillarum
cells, but did not induce protease activity. The chloroform-methanol-soluble fraction did not support growth, nor did it induce protease activity. When the two fractions were mixed, protease activity was induced. The chloroform-methanol-soluble fraction did not induce protease activity in cells growing in LB20. EDTA (50 mM) inhibited the protease induced by mucus. Upon addition of divalent cations, Mg
2+
(100 mM) was more effective than equimolar amounts of either Ca
2+
or Zn
2+
in restoring activity, suggesting that the mucus-inducible protease was a magnesium-dependent metalloprotease. An
empA
mutant strain of
V. anguillarum
did not exhibit protease activity after exposure to mucus, but did grow in mucus. Southern analysis and PCR amplification confirmed that
V. anguillarum
M93 contained
empA
. These data demonstrate that the
empA
metalloprotease of
V. anguillarum
is specifically induced by gastrointestinal mucus.
Plasticizers Increase Adhesion of the Deteriogenic Fungus
Aureobasidium pullulans
to Polyvinyl Chloride
Authors: Webb Jeremy S. , Van der Mei Henny C. , Nixon Marianne , Eastwood Ian M. , Greenhalgh Malcolm , Read Simon J. , Robson Geoffrey D. , Handley Pauline S. 
Published: December 2019

ABSTRACT

Initial adhesion of fungi to plasticized polyvinyl chloride (pPVC) may determine subsequent colonization and biodeterioration processes. The deteriogenic fungus
Aureobasidium pullulans
was used to investigate the physicochemical nature of adhesion to both unplasticized PVC (uPVC) and pPVC containing the plasticizers dioctyl phthalate (DOP) and dioctyl adipate (DOA). A quantitative adhesion assay using image analysis identified fundamental differences in the mechanism of adhesion of
A. pullulans
blastospores to these substrata. Adhesion to pPVC was greater than that to uPVC by a maximum of 280% after a 4-h incubation with 10
8
blastospores ml
−1
. That plasticizers enhance adhesion to PVC was confirmed by incorporating a dispersion of both DOA and DOP into the blastospore suspension. Adhesion to uPVC was increased by up to 308% in the presence of the dispersed plasticizers. Hydrophobic interactions were found to dominate adhesion to uPVC because (i) a strong positive correlation was observed between substratum hydrophobicity (measured by using a dynamic contact angle analyzer) and adhesion to a range of unplasticized polymers including uPVC, and (ii) neither the pH nor the electrolyte concentration of the suspension buffer, both of which influence electrostatic interactions, affected adhesion to uPVC. In contrast, adhesion to pPVC is principally controlled by electrostatic interactions. Enhanced adhesion to pPVC occurred despite a relative reduction of 13° in the water contact angle of pPVC compared to that of uPVC. Furthermore, adhesion to pPVC was strongly dependent on both the pH and electrolyte concentration of the suspension medium, reaching maximum levels at pH 8 and with an electrolyte concentration of 10 mM NaCl. Plasticization with DOP and DOA therefore increases adhesion of
A. pullulans
blastospores to pPVC through an interaction mediated by electrostatic forces.