bluuebunny/crossref_metadata_2025 huggingface dataset:
Purification, Characterization, Gene Cloning, Sequencing, and Overexpression of Aminopeptidase N fromStreptococcus thermophilusA
Authors: Chavagnat Frederic , Casey Michael G. , Meyer Jacques
Published: December 2019

The 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

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

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

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

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

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

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

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

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

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

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

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

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

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

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.
A Novel Mineral Flotation Process Using
Thiobacillus ferrooxidans
Authors: Nagaoka Toru , Ohmura Naoya , Saiki Hiroshi
Published: December 2019

Oxidative leaching of metals by
Thiobacillus ferrooxidans
has proven useful in mineral processing. Here, we report on a new use for
T. ferrooxidans
, in which bacterial adhesion is used to remove pyrite from mixtures of sulfide minerals during flotation. Under control conditions, the floatabilities of five sulfide minerals tested (pyrite, chalcocite, molybdenite, millerite, and galena) ranged from 90 to 99%. Upon addition of
T. ferrooxidans
, the floatability of pyrite was significantly suppressed to less than 20%. In contrast, addition of the bacterium had little effect on the floatabilities of the other minerals, even when they were present in relatively large quantities: their floatabilities remained in the range of 81 to 98%.
T. ferrooxidans
thus appears to selectively suppress pyrite floatability. As a consequence, 77 to 95% of pyrite was removed from mineral mixtures while 72 to 100% of nonpyrite sulfide minerals was recovered. The suppression of pyrite floatability was caused by bacterial adhesion to pyrite surfaces. When normalized to the mineral surface area, the number of cells adhering to pyrite was significantly larger than the number adhering to other minerals. These results suggest that flotation with
T. ferrooxidans
may provide a novel approach to mineral processing in which the biological functions involved in cell adhesion play a key role in the separation of minerals.
Sunlight Inactivation of Fecal Bacteriophages and Bacteria in Sewage-Polluted Seawater
Authors: Sinton Lester W. , Finlay Rochelle K. , Lynch Philippa A.
Published: December 2019

Sunlight inactivation rates of somatic coliphages, F-specific RNA bacteriophages (F-RNA phages), and fecal coliforms were compared in seven summer and three winter survival experiments. Experiments were conducted outdoors, using 300-liter 2% (vol/vol) sewage-seawater mixtures held in open-top chambers. Dark inactivation rates (

k
D

s), measured from exponential survival curves in enclosed (control) chambers, were higher in summer (temperature range: 14 to 20°C) than in winter (temperature range: 8 to 10°C). Winter

k
D

s were highest for fecal coliforms and lowest for F-RNA phages but were the same or similar for all three indicators in summer. Sunlight inactivation rates (

k
S

), as a function of cumulative global solar radiation (insolation), were all higher than the

k
D

s with a consistent

k
S

ranking (from greatest to least) as follows: fecal coliforms, F-RNA phages, and somatic coliphages. Phage inactivation was exponential, but bacterial curves typically exhibited a shoulder. Phages from raw sewage exhibited

k
S

s similar to those from waste stabilization pond effluent, but raw sewage fecal coliforms were inactivated faster than pond effluent fecal coliforms. In an experiment which included F-DNA phages and
Bacteroides fragilis
phages, the

k
S

ranking (from greatest to least) was as follows: fecal coliforms, F-RNA phages,
B. fragilis
phages, F-DNA phages, and somatic coliphages. In a 2-day experiment which included enterococci, the initial concentration ranking (from greatest to least: fecal coliforms, enterococci, F-RNA phages, and somatic coliphages) was reversed during sunlight exposure, with only the phages remaining detectable by the end of day 2. Inactivation rates under different optical filters decreased with the increase in spectral cutoff wavelength (50% light transmission) and indicated that F-RNA phages and fecal coliforms are more susceptible than somatic coliphages to longer solar wavelengths, which predominate in seawater. The consistently superior survival of somatic coliphages in our experiments suggests that they warrant further consideration as fecal, and possibly viral, indicators in marine waters.
Seasonal Variations in Microbial Populations and Environmental Conditions in an Extreme Acid Mine Drainage Environment
Authors: Edwards Katrina J. , Gihring Thomas M. , Banfield Jillian F.
Published: December 2019

Microbial populations, their distributions, and their aquatic environments were studied over a year (1997) at an acid mine drainage (AMD) site at Iron Mountain, Calif. Populations were quantified by fluorescence in situ hybridizations with group-specific probes. Probes were used for the domains
Eucarya
,
Bacteria
, and
Archaea
and the two species most widely studied and implicated for their role in AMD production,
Thiobacillus ferrooxidans
and
Leptospirillum ferrooxidans
. Results show that microbial populations, in relative proportions and absolute numbers, vary spatially and seasonally and correlate with geochemical and physical conditions (pH, temperature, conductivity, and rainfall). Bacterial populations were in the highest proportion (>95%) in January. Conversely, archaeal populations were in the highest proportion in July and September (∼50%) and were virtually absent in the winter. Bacterial and archaeal populations correlated with conductivity and rainfall. High concentrations of dissolved solids, as reflected by high conductivity values (up to 125 mS/cm), occurred in the summer and correlated with high archaeal populations and proportionally lower bacterial populations. Eukaryotes were not detected in January, when total microbial cell numbers were lowest (<10
5
cells/ml), but eukaryotes increased at low-pH sites (∼0.5) during the remainder of the year. This correlated with decreasing water temperatures (50 to 30°C; January to November) and increasing numbers of prokaryotes (10
8
to 10
9
cells/ml).
T. ferrooxidans
was in highest abundance (>30%) at moderate pHs and temperatures (∼2.5 and 20°C) in sites that were peripheral to primary acid-generating sites and lowest (0 to 5%) at low-pH sites (pH ∼0.5) that were in contact with the ore body.
L. ferrooxidans
was more widely distributed with respect to geochemical conditions (pH = 0 to 3; 20 to 50°C) but was more abundant at higher temperatures and lower pHs (∼40°C; pH ∼0.5) than
T. ferrooxidans
.
Microbial Reduction of Fe(III) in Acidic Sediments: Isolation of
Acidiphilium cryptum
JF-5 Capable of Coupling the Reduction of Fe(III) to the Oxidation of Glucose
Authors: Küsel Kirsten , Dorsch Tanja , Acker Georg , Stackebrandt Erko
Published: December 2019

To evaluate the microbial populations involved in the reduction of Fe(III) in an acidic, iron-rich sediment, the anaerobic flow of supplemental carbon and reductant was evaluated in sediment microcosms at the in situ temperature of 12°C. Supplemental glucose and cellobiose stimulated the formation of Fe(II); 42 and 21% of the reducing equivalents that were theoretically obtained from glucose and cellobiose, respectively, were recovered in Fe(II). Likewise, supplemental H
2
was consumed by acidic sediments and yielded additional amounts of Fe(II) in a ratio of approximately 1:2. In contrast, supplemental lactate did not stimulate the formation of Fe(II). Supplemental acetate was not consumed and inhibited the formation of Fe(II). Most-probable-number estimates demonstrated that glucose-utilizing acidophilic Fe(III)-reducing bacteria approximated to 1% of the total direct counts of 4′,6-diamidino-2-phenylindole-stained bacteria. From the highest growth-positive dilution of the most-probable-number series at pH 2.3 supplemented with glucose, an isolate, JF-5, that could dissimilate Fe(III) was obtained. JF-5 was an acidophilic, gram-negative, facultative anaerobe that completely oxidized the following substrates via the dissimilation of Fe(III): glucose, fructose, xylose, ethanol, glycerol, malate, glutamate, fumarate, citrate, succinate, and H
2
. Growth and the reduction of Fe(III) did not occur in the presence of acetate. Cells of JF-5 grown under Fe(III)-reducing conditions formed blebs, i.e., protrusions that were still in contact with the cytoplasmic membrane. Analysis of the 16S rRNA gene sequence of JF-5 demonstrated that it was closely related to an Australian isolate of
Acidiphilium cryptum
(99.6% sequence similarity), an organism not previously shown to couple the complete oxidation of sugars to the reduction of Fe(III). These collective results indicate that the in situ reduction of Fe(III) in acidic sediments can be mediated by heterotrophic
Acidiphilium
species that are capable of coupling the reduction of Fe(III) to the complete oxidation of a large variety of substrates including glucose and H
2
.
Multiplex PCR Screening To Detectcry9Genes inBacillus thuringiensisStrains
Authors: Ben-Dov Eitan , Wang Qingfeng , Zaritsky Arieh , Manasherob Robert , Barak Ze’ev , Schneider Bert , Khamraev Aloviddin , Baizhanov Mukhtar , Glupov Victor , Margalith Yoel
Published: December 2019

An extended PCR method was established to rapidly identify and classifyBacillus thuringiensisstrains containingcry(crystal protein) genes toxic to lepidopteran, coleopteran, and dipteran pests (Ben-Dov et al., Appl. Environ. Microbiol. 63:4883–4890, 1997). To optimize identification of all reportedcrygenes, this methodology needs a complete PCR set of primers. In the study reported here, a set of universal (Un9) and specific primers for multiplex rapid screening for all four known genes from thecry9group was designed. PCR analyses were performed forcry9genes on 16 standard strains and 215 field isolates ofB. thuringiensis. Among the standard strains, onlyB. thuringiensissubsp.aizawaiHD-133, which harborscry1andcry2genes, was positive with Un9 but negative to all four specific primers forcry9genes. DNA of 22 field-collected isolates was also found to be positive with Un9. These isolates were classified into threecry9profiles using specific primers; all of them harborcry1andcry2. This newly designed set of primers complements the existing PCR methodology for most currently knowncrygenes.
Biocontrol of
Escherichia coli
O157 with O157-Specific Bacteriophages
Authors: Kudva Indira T. , Jelacic Srdjan , Tarr Phillip I. , Youderian Philip , Hovde Carolyn J.
Published: December 2019

Escherichia coli
O157 antigen-specific bacteriophages were isolated and tested to determine their ability to lyse laboratory cultures of
Escherichia coli
O157:H7. A total of 53 bovine or ovine fecal samples were enriched for phage, and 5 of these samples were found to contain lytic phages that grow on
E. coli
O157:H7. Three bacteriophages, designated KH1, KH4, and KH5, were evaluated. At 37 or 4°C, a mixture of these three O157-specific phages lysed all of the
E. coli
O157 cultures tested and none of the non-O157
E. coli
or non-
E. coli
cultures tested. These results required culture aeration and a high multiplicity of infection. Without aeration, complete lysis of the bacterial cells occurred only after 5 days of incubation and only at 4°C. Phage infection and plaque formation were influenced by the nature of the host cell O157 lipopolysaccharide (LPS). Strains that did not express the O157 antigen or expressed a truncated LPS were not susceptible to plaque formation or lysis by phage. In addition, strains that expressed abundant mid-range-molecular-weight LPS did not support plaque formation but were lysed in liquid culture. Virulent O157 antigen-specific phages could play a role in biocontrol of
E. coli
O157:H7 in animals and fresh foods without compromising the viability of other normal flora or food quality.
An Effective Strategy, Applicable to
Streptococcus salivarius
and Related Bacteria, To Enhance or Confer Electroporation Competence
Authors: Buckley Nicole D. , Vadeboncoeur Christian , LeBlanc Donald J. , Lee Linda N. , Frenette Michel
Published: December 2019

Despite the large number of techniques available for transformation of bacteria, certain species and strains are still resistant to introduction of foreign DNA. Some oral streptococci are among the organisms that can be particularly difficult to transform. We performed a series of experiments that involved manipulation of growth and recovery media and cell wall weakening, in which the electroporation conditions, cell concentration, and type and concentration of the transforming plasmid were varied. The variables were optimized such that a previously untransformable
Streptococcus salivarius
strain, ATCC 25975, could be transformed reproducibly at a level of 10
5
transformants per μg of DNA. The technique was used to introduce a plasmid into other strains of
S. salivarius
, including a fresh isolate. Moreover, the same technique was applied successfully to a wide range of oral streptococci and other gram-positive bacteria.
Marine Bacterial Isolates Display Diverse Responses to UV-B Radiation
Authors: Joux Fabien , Jeffrey Wade H. , Lebaron Philippe , Mitchell David L.
Published: December 2019

The molecular and biological consequences of UV-B radiation were investigated by studying five species of marine bacteria and one enteric bacterium. Laboratory cultures were exposed to an artificial UV-B source and subjected to various post-UV irradiation treatments. Significant differences in survival subsequent to UV-B radiation were observed among the isolates, as measured by culturable counts. UV-B-induced DNA photodamage was investigated by using a highly specific radioimmunoassay to measure cyclobutane pyrimidine dimers (CPDs). The CPDs determined following UV-B exposure were comparable for all of the organisms except
Sphingomonas
sp. strain RB2256, a facultatively oligotrophic ultramicrobacterium. This organism exhibited little DNA damage and a high level of UV-B resistance. Physiological conditioning by growth phase and starvation did not change the UV-B sensitivity of marine bacteria. The rates of photoreactivation following exposure to UV-B were investigated by using different light sources (UV-A and cool white light). The rates of photoreactivation were greatest during UV-A exposure, although diverse responses were observed. The differences in sensitivity to UV-B radiation between strains were reduced after photoreactivation. The survival and CPD data obtained for
Vibrio natriegens
when we used two UV-B exposure periods interrupted by a repair period (photoreactivation plus dark repair) suggested that photoadaptation could occur. Our results revealed that there are wide variations in marine bacteria in their responses to UV radiation and subsequent repair strategies, suggesting that UV-B radiation may affect the microbial community structure in surface water.
Degradation of 4-Fluorobiphenyl by Mycorrhizal Fungi as Determined by
19
F Nuclear Magnetic Resonance Spectroscopy and
14
C Radiolabelling Analysis
Authors: Green N. A. , Meharg A. A. , Till C. , Troke J. , Nicholson J. K.
Published: December 2019

The pathways of biotransformation of 4-fluorobiphenyl (4FBP) by the ectomycorrhizal fungus
Tylospora fibrilosa
and several other mycorrhizal fungi were investigated by using
19
F nuclear magnetic resonance (NMR) spectroscopy in combination with
14
C radioisotope-detected high-performance liquid chromatography (
14
C-HPLC). Under the conditions used in this study
T. fibrillosa
and some other species degraded 4FBP.
14
C-HPLC profiles indicated that there were four major biotransformation products, whereas
19
F NMR showed that there were six major fluorine-containing products. We confirmed that 4-fluorobiphen-4′-ol and 4-fluorobiphen-3′-ol were two of the major products formed, but no other products were conclusively identified. There was no evidence for the expected biotransformation pathway (namely,
meta
cleavage of the less halogenated ring), as none of the expected products of this route were found. To the best of our knowledge, this is the first report describing intermediates formed during mycorrhizal degradation of halogenated biphenyls.
Detection of Viral Pathogens by Reverse Transcriptase PCR and of Microbial Indicators by Standard Methods in the Canals of the Florida Keys
Authors: Griffin Dale W. , Gibson Charles J. , Lipp Erin K. , Riley Kelley , Paul John H. , Rose Joan B.
Published: December 2019

In order to assess the microbial water quality in canal waters throughout the Florida Keys, a survey was conducted to determine the concentration of microbial fecal indicators and the presence of human pathogenic microorganisms. A total of 19 sites, including 17 canal sites and 2 nearshore water sites, were assayed for total coliforms, fecal coliforms,
Escherichia coli
,
Clostridium perfringens
, enterococci, coliphages, F-specific (F
+
) RNA coliphages,
Giardia lamblia
,
Cryptosporidium parvum
, and human enteric viruses (polioviruses, coxsackie A and B viruses, echoviruses, hepatitis A viruses, Norwalk viruses, and small round-structured viruses). Numbers of coliforms ranged from <1 to 1,410,
E. coli
organisms from <1 to 130,
Clostridium
spp. from <1 to 520, and enterococci from <1 to 800 CFU/100 ml of sample. Two sites were positive for coliphages, but no F
+
phages were identified. The sites were ranked according to microbial water quality and compared to various water quality standards and guidelines. Seventy-nine percent of the sites were positive for the presence of enteroviruses by reverse transcriptase PCR (polioviruses, coxsackie A and B viruses, and echoviruses). Sixty-three percent of the sites were positive for the presence of hepatitis A viruses. Ten percent of the sites were positive for the presence of Norwalk viruses. Ninety-five percent of the sites were positive for at least one of the virus groups. These results indicate that the canals and nearshore waters throughout the Florida Keys are being impacted by human fecal material carrying human enteric viruses through current wastewater treatment strategies such as septic tanks. Exposure to canal waters through recreation and work may be contributing to human health risks.
Community Size and Metabolic Rates of Psychrophilic Sulfate-Reducing Bacteria in Arctic Marine Sediments
Authors: Knoblauch Christian , Jørgensen Bo Barker , Harder Jens
Published: December 2019

The numbers of sulfate reducers in two Arctic sediments with in situ temperatures of 2.6 and −1.7°C were determined. Most-probable-number counts were higher at 10°C than at 20°C, indicating the predominance of a psychrophilic community. Mean specific sulfate reduction rates of 19 isolated psychrophiles were compared to corresponding rates of 9 marine, mesophilic sulfate-reducing bacteria. The results indicate that, as a physiological adaptation to the permanently cold Arctic environment, psychrophilic sulfate reducers have considerably higher specific metabolic rates than their mesophilic counterparts at similarly low temperatures.
Maximising and evaluating the uptake, use and impact of golf and health studies
Authors: Murray Andrew , Kelly Paul , Morton Sarah , Glover Danny , Duncan Jennifer , Hawkes Roger , Grant Liz , Mutrie Nanette
Published: December 2019

Introduction

The dissemination of research, and evaluation of its impact is an increasing priority for the scientific community and funders. We take the topic of golf and health and aim to outline processes that may contribute to improved research uptake, use and impact proposing a research impact (RI) tool. We then evaluate our published research using the Research Contributions Framework (RCF).

Methods

Building on existing research and frameworks we i) assessed the need for, ii) carried out and iii) published research, before iv) creating digital resources, v) sharing these resources widely and vi) evaluating our research.

To evaluate uptake, use and impact of our three principal golf and health research outputs, we performed a contributions analysis, using the RCF first proposed by Morton.

Results/Discussion

We developed a specific six-step Research Impact tool. Having implemented this, research uptake and use included over 300 press articles, a dedicated website and social media channels. Golf’s global industry leadership dispersed information across >150 countries, embedded golf and health into curricula for industry professionals and used leading tournaments to promote health. National policy makers hosted dedicated meetings regarding golf and health and began to implement policy change.

Conclusion

To date, strong uptake and use can be demonstrated for these studies, while a final contribution to impact requires further time to determine.

Frameworks we used aiming to maximise impact (Research Impact tool) and evaluate its contribution to uptake, use and impact (Research Contribution Framework) could potentially add value to public health/sports medicine researchers.
Opportunities for antibiotic optimisation and outcome improvement in patients with negative blood cultures: study protocol for a cluster-randomised crossover trial, the NO-BACT study
Authors: Jiménez-Jorge Silvia , Palacios-Baena Zaira R , Rosso-Fernández Clara M , Girón-Ortega José A , Rodriguez-Baño Jesús , Retamar Pilar
Published: December 2019

Introduction

Patients with negative blood cultures (BCx) represent 85%–90% of all patients with BCx taken during hospital admission. This population usually includes a heterogeneous group of patients admitted with infectious diseases or febrile syndromes that require a blood culture. There is very little evidence of the clinical characteristics and antibiotic treatment given to these patients.

Methods and analysis

In a preliminary exploratory prospective cohort study of patients with BCx taken, the clinical/therapeutic characteristics and outcomes/antimicrobial stewardship opportunities of a population of patients with negative BCx will be analysed. In the second phase, using a cluster randomised crossover design, the implementation of an antimicrobial stewardship intervention targeting patients with negative BCx will be evaluated in terms of quality of antimicrobial use (duration and de-escalation), length of hospital stay and mortality.

Ethics and dissemination

This study has been and registered with clinicaltrials.gov. The findings of our study may support the implementation in clinical practice of an antimicrobial stewardship intervention to optimise the use of antibiotics in patients with negative BCx. The results of this study will be published in peer-reviewed journals and disseminated at national and international conferences.

Trial registration number

NCT03535324.
Beyond forest plots: clinical gestalt and its influence on COPD telemonitoring studies and outcomes review
Authors: Smith Sheree M , Holland Anne E , McDonald Christine F
Published: December 2019

Background

Chronic obstructive pulmonary disease (COPD) is a progressive chronic condition. Improvements in therapies have resulted in better patient outcomes. The use of technology such as telemonitoring as an additional intervention is aimed at enhancing care and reducing unnecessary acute hospital service use. The influence of verbal communication between health staff and patients to inform decision making regarding use of acute hospital services within telemonitoring studies has not been assessed.

Method

A systematic overview of published systematic reviews of COPD and telemonitoring was conducted using ana prioriprotocol to ascertain the impact of verbal communication in telemonitoring studies on health service outcomes such as emergency department attendances, hospitalisation and hospital length of stay. The search of the following electronic databases: Cochrane Library, Medline, Pubmed, CINAHL, Embase, TROVE, Australian Digital Thesis and Proquest International Dissertations and Theses was conducted in 2017 and updated in September 2019.

Results

Six systematic reviews were identified. All reviews involved home monitoring of COPD symptoms and biometric data. Included reviews reported 5–28 studies with sample sizes ranging from 310 to 2891 participants. Many studies reported in the systematic reviews were excluded as they were telephone support, cost effectiveness studies, and/or did not report the outcomes of interest for this overview. Irrespective of group assignment, verbal communication with the health or research team did not alter the emergency attendance or hospitalisation outcome. The length of stay was longer for those who were assigned home telemonitoring in the majority of studies.

Conclusion

This overview of telemonitoring for COPD had small sample sizes and a wide variety of included studies. Communication was not consistent in all included studies. Understanding the context of communication with study participants and the decision-making process for referring patients to various health services needs to be reported in future studies of telemonitoring and COPD.
Relapse in rheumatoid arthritis patients undergoing dose reduction and withdrawal of biologics: are predictable factors more relevant than predictive parameters? An observational prospective real-life study
Authors: Vittecoq Olivier , Desouches Sandra , Kozyreff Marie , Nicolau Julia , Pouplin Sophie , Rottenberg Pascal , Sens Nicolas , Lequerre Thierry , Avenel Gilles
Published: December 2019

Objective

To determine predictive/predictable factors of relapse in rheumatoid arthritis (RA) patients undergoing biologic Disease-Modifying Anti-Rheumatic Drugs (bDMARDs) dose reduction/discontinuation.

Patients and methods

RA patients receiving the same bDMARD for more than 1 year, in Simplified Disease Activity Index (SDAI) remission, were selected in an observational monocentric real-life study. The 18-month follow-up included spacing (6 months) and withdrawal (12 months) periods of bDMARD. Clinical, biological and ultrasonographic (US) parameters were collected regularly. Relapse was defined by SDAI>11.

Results

Fifty-three RA patients (mean age: 58 years; 72% women; median duration: 11 years) were enrolled. Forty-two received anti-cytokinic bDMARD targeting tumour necrosis factor (n=39) or interleukin-6R (n=3) and 11 were treated by abatacept. The number of relapses during the spacing and discontinuation periods were 19 and 20, respectively. After 18 months of follow-up, among the 53 patients, 12 maintained bDMARD-free remission, 39 had relapsed and 2 were lost of follow-up. Median time to relapse was 11.8 months. In multivariate analysis, baseline factors predictive of relapse were corticosteroid intake, female gender, longer disease duration and no methotrexate intake with bDMARD. Concerning the survival analysis, also taking into account the factors of predictability, the main risk factor of relapse after discontinuation was an increase of SDAI >0 during the spacing period (p=0.03). US findings were not contributive.

Conclusion

In the context of RA in remission under bDMARDs, variation of SDAI during the dose-reduction phase is more relevant than baseline parameters to predict success of drug withdrawal.