bluuebunny/crossref_metadata_2025 huggingface dataset:
Spatial Flow-Field Approximation Using Few Thermodynamic Measurements—Part II: Uncertainty Assessments
Authors: Seshadri Pranay , Duncan Andrew , Simpson Duncan , Thorne George , Parks Geoffrey
Published: December 2019

In this second part of our two-part paper, we provide a detailed, frequentist framework for propagating uncertainties within our multivariate linear least squares model. This permits us to quantify the impact of uncertainties in thermodynamic measurements—arising from calibrations and the data acquisition system—and the correlations therein, along with uncertainties in probe positions. We show how the former has a much larger effect (relatively) than uncertainties in probe placement. We use this non-deterministic framework to demonstrate why the well-worn metric for assessing spatial sampling uncertainty falls short of providing an accurate characterization of the effect of a few spatial measurements. In other words, it does not accurately describe the uncertainty associated with sampling a non-uniform pattern with a few circumferentially scattered rakes. To this end, we argue that our data-centric framework can offer a more rigorous characterization of this uncertainty. Our paper proposes two new uncertainty metrics: one for characterizing spatial sampling uncertainty and another for capturing the impact of measurement imprecision in individual probes. These metrics are rigorously derived in our paper and their ease in computation permits them to be widely adopted by the turbomachinery community for carrying out uncertainty assessments.
Infrared absorption cross section of SiNx thin films
Authors: DiGregorio Sara N. , Habermehl Scott D.
Published: December 2019

At the molecular level, resonant coupling of infrared radiation with oscillations of the electric dipole moment determines the absorption cross section, σ. The parameter σ relates the bond density to the total integrated absorption. In this work, σ was measured for the Si–N asymmetric stretch mode in SiNx thin films of varying composition and thickness. Thin films were deposited by low pressure chemical vapor deposition at 850 °C from mixtures of dichlorosilane and ammonia. σ for each film was determined from Fourier transform infrared spectroscopy and ellipsometric measurements. Increasing the silicon content from 0% to 25% volume fraction amorphous silicon led to increased optical absorption and a corresponding systematic increase in σ from 4.77 × 10−20 to 6.95 × 10−20 cm2, which is consistent with literature values. The authors believe that this trend is related to charge transfer induced structural changes in the basal SiNx tetrahedron as the volume fraction of amorphous silicon increases. Experimental σ values were used to calculate the effective dipole oscillating charge, q, for four films of varying composition. The authors find that q increases with increasing amorphous silicon content, indicating that compositional factors contribute to modulation of the Si–N dipole moment. Additionally, in the composition range investigated, the authors found that σ agrees favorably with trends observed in films deposited by plasma enhanced chemical vapor deposition.
A comparison of acoustic and articulatory parameters for the GOOSE vowel across British Isles Englishes
Authors: Lawson Eleanor , Stuart-Smith Jane , Rodger Lydia
Published: December 2019

This study quantifies vocalic variation that cannot be measured from the acoustic signal alone and develops methods of standardisation and measurement of articulatory parameters for vowels. Articulatory-acoustic variation in the GOOSE vowel was measured across 3 regional accents of the British Isles using a total of 18 speakers from the Republic of Ireland, Scotland, and England, recorded with synchronous ultrasound tongue imaging, lip camera, and audio. Single co-temporal measures were taken of tongue-body height and backness, lip protrusion, F1, and F2. After normalisation, mixed-effects modelling identified statistically significant variations per region; tongue-body position was significantly higher and fronter for Irish and English speakers. Region was also significant for lip-protrusion measures with Scottish speakers showing significantly smaller degrees of protrusion than English speakers. However, the region was only significant for acoustic height and not for frontness. Correlational analyses of all measures showed a significant positive correlation between tongue-body height and acoustic height, a negative correlation between lip-protrusion and acoustic frontness, but no correlation between tongue-body frontness and acoustic frontness. Effectively, two distinct regional production strategies were found to result in similar normalised acoustic frontness measures for GOOSE. Scottish tongue-body positions were backer and lips less protruded, while English and Irish speakers had fronter tongue-body positions, but more protruded lips.
Statistical sources for broadband shock-associated noise using the Navier-Stokes equations
Authors: Patel Trushant K. , Miller Steven A. E.
Published: December 2019

A decomposition of the Navier-Stokes equations is used to identify the equivalent source term for broadband shock-associated noise (BBSAN). An analytical closed-form model to predict BBSAN is developed using an acoustic analogy based on the Navier-Stokes equations. The field-variables are decomposed into the base flow, aerodynamic fluctuations, and acoustic fluctuations. The spectral densities of fluctuating acoustic quantities are obtained by convolving the vector Green's function with the source terms involving the two-point cross-correlation of the aerodynamic quantities. The scaling of the source term with the off-design parameter β=(|Mj2−Md2|)1/2 is compared with experimental results. The base flow is obtained using a Reynolds-averaged Navier-Stokes solution, while the fluctuating statistical quantities are obtained using theoretical and experimental results. This paper identifies the equivalent source of BBSAN based on the scaling analysis and the physical mechanism of shock-associated noise. The identified source term resides within the Navier-Stokes equations without further rearrangement and correlates very highly with BBSAN. Predictions for BBSAN are made at multiple observer angles and nozzle pressure ratios using the identified source term, and these predictions compare favorably with the experimental results. Finally, identification of the source locations in the jet exhaust responsible for BBSAN at different Strouhal numbers is performed.
The maximum audible low-pass cutoff frequency for speech
Authors: Monson Brian B. , Caravello Jacob
Published: December 2019

Speech energy beyond 8 kHz is often audible for listeners with normal hearing. Limits to audibility in this frequency range are not well described. This study assessed the maximum audible low-pass cutoff frequency for speech, relative to full-bandwidth speech. The mean audible cutoff frequency was approximately 13 kHz, with a small but significant effect of talker sex. Better pure tone thresholds at extended high frequencies correlated with higher audible cutoff frequency. These findings demonstrate that bandlimiting speech even at 13 kHz results in a detectable loss for the average normal-hearing listener, suggesting there is information regarding the speech signal beyond 13 kHz.
Explainable robotics in science fiction
Authors: Murphy Robin R.
Published: December 2019

Would we trust a robot? Science fiction says no, but explainable robotics may find a way.
Advances in clinical MRI technology
Authors: Harisinghani Mukesh G. , O’Shea Aileen , Weissleder Ralph
Published: December 2019

Advances in MRI technologies have the potential to detect, characterize, and monitor a wide variety of diseases.
Rhizoremediation of Trichloroethylene by a Recombinant, Root-Colonizing
Pseudomonas fluorescens
Strain Expressing Toluene
ortho
-Monooxygenase Constitutively
Authors: Yee Dennis C. , Maynard Jennifer A. , Wood Thomas K.
Published: December 2019

Trichloroethylene (TCE) was removed from soils by using a wheat rhizosphere established by coating seeds with a recombinant, TCE-degrading
Pseudomonas fluorescens
strain that expresses the
tomA
+
(toluene
o
-monooxygenase) genes from
Burkholderia cepacia
PR1
23
(TOM
23C
). A transposon integration vector was used to insert
tomA
+
into the chromosome of
P. fluorescens
2-79, producing a stable strain that expressed constitutively the monooxygenase at a level of 1.1 nmol/min · mg of protein (initial TCE concentration, 10 μM, assuming that all of the TCE was in the liquid) for more than 280 cell generations (36 days). We also constructed a salicylate-inducible
P. fluorescens
strain that degraded TCE at an initial rate of 2.6 nmol/min · mg of protein in the presence of 10 μM TCE [cf.
B. cepacia
G4 PR1
23
(TOM
23C
), which degraded TCE at an initial rate of 2.5 nmol/min · mg of protein]. A constitutive strain,
P. fluorescens
2-79TOM, grew (maximum specific growth rate, 0.78 h
−1
) and colonized wheat (3 × 10
6
CFU/cm of root) as well as wild-type
P. fluorescens
2-79 (maximum specific growth rate, 0.77 h
−1
; level of colonization, 4 × 10
6
CFU/cm of root). Rhizoremediation of TCE was demonstrated by using microcosms containing the constitutive monooxygenase-expressing microorganism, soil, and wheat. These closed microcosms degraded an average of 63% of the initial TCE in 4 days (20.6 nmol of TCE/day · plant), compared to the 9% of the initial TCE removed by negative controls consisting of microcosms containing wild-type
P. fluorescens
2-79-inoculated wheat, uninoculated wheat, or sterile soil.
Protozoan Bacterivory and
Escherichia coli
Survival in Drinking Water Distribution Systems
Authors: Sibille I. , Sime-Ngando T. , Mathieu L. , Block J. C.
Published: December 2019

The development of bacterial communities in drinking water distribution systems leads to a food chain which supports the growth of macroorganisms incompatible with water quality requirements and esthetics. Nevertheless, very few studies have examined the microbial communities in drinking water distribution systems and their trophic relationships. This study was done to quantify the microbial communities (especially bacteria and protozoa) and obtain direct and indirect proof of protozoan feeding on bacteria in two distribution networks, one of GAC water (i.e., water filtered on granular activated carbon) and the other of nanofiltered water. The nanofiltered water-supplied network contained no organisms larger than bacteria, either in the water phase (on average, 5 × 10
7
bacterial cells liter
−1
) or in the biofilm (on average, 7 × 10
6
bacterial cells cm
−2
). No protozoa were detected in the whole nanofiltered water-supplied network (water plus biofilm). In contrast, the GAC water-supplied network contained bacteria (on average, 3 × 10
8
cells liter
−1
in water and 4 × 10
7
cells cm
−2
in biofilm) and protozoa (on average, 10
5
cells liter
−1
in water and 10
3
cells cm
−2
in biofilm). The water contained mostly flagellates (93%), ciliates (1.8%), thecamoebae (1.6%), and naked amoebae (1.1%). The biofilm had only ciliates (52%) and thecamoebae (48%). Only the ciliates at the solid-liquid interface of the GAC water-supplied network had a measurable grazing activity in laboratory test (estimated at 2 bacteria per ciliate per h). Protozoan ingestion of bacteria was indirectly shown by adding
Escherichia coli
to the experimental distribution systems. Unexpectedly,
E. coli
was lost from the GAC water-supplied network more rapidly than from the nanofiltered water-supplied network, perhaps because of the grazing activity of protozoa in GAC water but not in nanofiltered water. Thus, the GAC water-supplied network contained a functional ecosystem with well-established and structured microbial communities, while the nanofiltered water-supplied system did not. The presence of protozoa in drinking water distribution systems must not be neglected because these populations may regulate the autochthonous and allochthonous bacterial populations.
Effect of Trichloroethylene on the Competitive Behavior of Toluene-Degrading Bacteria
Authors: Mars Astrid E. , Prins Gjalt T. , Wietzes Pieter , de Koning Wim , Janssen Dick B.
Published: December 2019

The influence of trichloroethylene (TCE) on a mixed culture of four different toluene-degrading bacterial strains (
Pseudomonas putida
mt-2,
P. putida
F1,
P. putida
GJ31, and
Burkholderia cepacia
G4) was studied with a fed-batch culture. The strains were competing for toluene, which was added at a very low rate (31 nmol mg of cells [dry weight]
−1
h
−1
). All four strains were maintained in the mixed culture at comparable numbers when TCE was absent. After the start of the addition of TCE, the viabilities of
B. cepacia
G4 and
P. putida
F1 and GJ31 decreased 50- to 1,000-fold in 1 month. These bacteria can degrade TCE, although at considerably different rates.
P. putida
mt-2, which did not degrade TCE, became the dominant organism. Kinetic analysis showed that the presence of TCE caused up to a ninefold reduction in the affinity for toluene of the three disappearing strains, indicating that inhibition of toluene degradation by TCE occurred. While
P. putida
mt-2 took over the culture, mutants of this strain which could no longer grow on
p
-xylene arose. Most of them had less or no
meta
-cleavage activity and were able to grow on toluene with a higher growth rate. The results indicate that cometabolic degradation of TCE has a negative effect on the maintenance and competitive behavior of toluene-utilizing organisms that transform TCE.
Isolation of Aerobic Anoxygenic Photosynthetic Bacteria from Black Smoker Plume Waters of the Juan de Fuca Ridge in the Pacific Ocean
Authors: Yurkov Vladimir , Beatty J. Thomas
Published: December 2019

A strain of the aerobic anoxygenic photosynthetic bacteria was isolated from a deep-ocean hydrothermal vent plume environment. The in vivo absorption spectra of cells indicate the presence of bacteriochlorophyll
a
incorporated into light-harvesting complex I and a reaction center. The general morphological and physiological characteristics of this new isolate are described.
Functional Display of a Heterologous Protein on the Surface of
Lactococcus lactis
by Means of the Cell Wall Anchor of
Staphylococcus aureus
Protein A
Authors: Steidler Lothar , Viaene Jasmine , Fiers Walter , Remaut Erik
Published: December 2019

In this study, we showed that the cell wall anchor of protein A from
Staphylococcus aureus
is functional in the food-grade organism
Lactococcus lactis
. A fusion protein composed of the lactococcal Usp45 secretion signal peptide, streptavidin monomer, and the
S. aureus
protein A anchor became covalently attached to the peptidoglycan when expressed in
L. lactis
. The streptavidin moiety of the fusion protein was functionally exposed at the cellular surface.
L. lactis
cells expressing the anchored fusion polypeptide could be specifically immobilized on a biotinylated alkaline phosphatase-coated polystyrene support.
Phages InfectingVibrio vulnificusAre Abundant and Diverse in Oysters (Crassostrea virginica) Collected from the Gulf of Mexico
Authors: DePaola Angelo , Motes Miles L. , Chan Amy M. , Suttle Curtis A.
Published: December 2019

Phages infectingVibrio vulnificuswere abundant (>104phages g of oyster tissue−1) throughout the year in oysters (Crassostrea virginica) collected from estuaries adjacent to the Gulf of Mexico (Apalachicola Bay, Fla.; Mobile Bay, Ala.; and Black Bay, La.). Estimates of abundance ranged from 101to 105phages g of oyster tissue−1and were dependent on the bacterial strain used to assay the sample.V. vulnificuswas near or below detection limits (<0.3 cell g−1) from January through March and was most abundant (103to 104cells g−1) during the summer and fall, when phage abundances also tended to be greatest. The phages isolated were specific to strains ofV. vulnificus, except for one isolate that caused lysis in a few strains ofV. parahaemolyticus. Based on morphological evidence obtained by transmission electron microscopy, the isolates belonged to thePodoviridae,Styloviridae, andMyoviridae, three families of double-stranded DNA phages. One newly described morphotype belonging to thePodoviridaeappears to be ubiquitous in Gulf Coast oysters. Isolates of this morphotype have an elongated capsid (mean, 258 nm; standard deviation, 4 nm;n= 35), with some isolates having a relatively broad host range among strains ofV. vulnificus. Results from this study indicate that a morphologically diverse group of phages which infectV. vulnificusis abundant and widely distributed in oysters from estuaries bordering the northeastern Gulf of Mexico.
Effects of pH and Oxygen and Ammonium Concentrations on the Community Structure of Nitrifying Bacteria from Wastewater
Authors: Prinčič Alenka , Mahne Ivan , Megušar France , Paul Eldor A. , Tiedje James M.
Published: December 2019

Shifts in nitrifying community structure and function in response to different ammonium concentrations (50, 500, 1,000, and 3,000 mg of N liter
−1
), pH values (pH 6.0, 7.0, and 8.2), and oxygen concentrations (1, 7, and 21%) were studied in experimental reactors inoculated with nitrifying bacteria from a wastewater treatment plant. The abilities of the communities selected for these conditions to regain their original structures after conditions were returned to the original conditions were also determined. Changes in nitrifying community structure were determined by performing an amplified ribosomal DNA (rDNA) restriction analysis of PCR products obtained with ammonia oxidizer-specific rDNA primers, by phylogenetic probing, by small-subunit (SSU) rDNA sequencing, and by performing a cellular fatty acid analysis. Digestion of ammonia-oxidizer SSU rDNA with five restriction enzymes showed that a high ammonium level resulted in a great community structure change that was reversible once the ammonium concentration was returned to its original level. The smaller changes in community structure brought about by the two pH extremes, however, were irreversible. Sequence analysis revealed that the highest ammonium environment stimulated growth of a nitrifier strain that exhibited 92.6% similarity in a partial SSU rRNA sequence to its nearest relative,
Nitrosomonas eutropha
C-91, although the PCR product did not hybridize with a general phylogenetic probe for ammonia oxidizers belonging to the β subgroup of the class
Proteobacteria
. A principal-component analysis of fatty acid methyl ester data detected changes from the starter culture in all communities under the new selective conditions, but after the standard conditions were restored, all communities produced the original fatty acid profiles.
Intracellular Changes in Ions and Organic Solutes in Halotolerant
Brevibacterium
sp. Strain JCM 6894 after Exposure to Hyperosmotic Shock
Authors: Nagata Shinichi , Adachi Kyoko , Sano Hiroshi
Published: December 2019

In the present study we aimed to observe the intracellular responses when there was a hyperosmotic shock with a large shift in ionic strength in nutrient-rich and nutrient-poor external environments in order to clarify the availability of substrates. To do this, we used the halotolerant organism
Brevibacterium
sp. strain JCM 6894, which is able to grow in the presence of a wide range of salt concentrations. Hyperosmotic shock was induced by transferring cells in the late exponential phase of growth in a complex medium containing 0.5 M NaCl into either old or fresh culture medium containing 2 M NaCl. Changes in the growth rate, in the pH of the medium, and in the internal cation or organic solute concentrations in the cytosol after an upshock were analyzed as a function of incubation time. The cells exhibited very different responses to upshocks in fresh culture medium and in old culture medium; in fresh culture medium, growth was stimulated and the medium became more acidic, whereas the old culture medium repressed growth and the medium became more alkaline. The intracellular free Na
+
concentrations remained low (80 nmol mg of protein
−1
) after an upshock in fresh culture medium, although they quickly increased twofold in the old culture medium. In contrast, K
+
ions immediately accumulated in the cells in fresh culture medium, whereas K
+
ions were taken up quite slowly in old culture medium. Furthermore, the cells placed in fresh culture medium transiently accumulated alanine and glutamine in response to the upshock, but the cells placed in old culture medium did not. Growth of the
Brevibacterium
strain at higher levels of salinity was supported by ectoine synthesis but was not observed after the shift to high-osmolarity conditions in the old culture. In the fresh culture, however, ectoine was vigorously synthesized in cells for more than 5 h after the upshock; the concentration of ectoine in cells was more than 3,500 nmol mg of protein
−1
at 10 h, which corresponded to a ninefold increase compared to the concentration before the shock. These findings are consistent with the results of an analysis of the extracellular medium composition before and after the upshock.
Vibrio cholerae
O1 Strain TSI-4 Produces the Exopolysaccharide Materials That Determine Colony Morphology, Stress Resistance, and Biofilm Formation
Authors: Wai Sun Nyunt , Mizunoe Yoshimitsu , Takade Akemi , Kawabata Shun-Ichiro , Yoshida Shin-Ichi
Published: December 2019

Vibrio cholerae
O1 strain TSI-4 (El Tor, Ogawa) can shift to a rugose colony morphology from its normal translucent colony morphology in response to nutrient starvation. We have investigated differences between the rugose and translucent forms of
V. cholerae
O1 strain TSI-4. Electron microscopic examination of the rugose form of TSI-4 (TSI-4/R) revealed thick, electron-dense exopolysaccharide materials surrounding polycationic ferritin-stained cells, while the ferritin-stained material was absent around the translucent form of TSI-4 (TSI-4/T). The exopolysaccharide produced by
V. cholerae
TSI-4/R was found to have a composition of
N
-acetyl-
d
-glucosamine,
d
-mannose, 6-deoxy-
d
-galactose, and
d
-galactose (7.4:10.2:2.4:3.0). The expression of an amorphous exopolysaccharide promotes biofilm development under static culture conditions. Biofilm formation by the rugose strain was determined by scanning electron microscopy, and most of the surface of the film was colonized by actively dividing rod cells. The corresponding rugose and translucent strains were compared for stress resistance. By having exopolysaccharide materials, the rugose strains acquired resistance to osmotic and oxidative stress. Our data indicated that an exopolysaccharide material on the surface of the rugose strain promoted biofilm formation and resistance to the effects of two stressing agents.
A Bioluminescence Assay Using
Nitrosomonas europaea
for Rapid and Sensitive Detection of Nitrification Inhibitors
Authors: Iizumi Taro , Mizumoto Masahiro , Nakamura Kanji
Published: December 2019

An expression vector for the
luxAB
genes, derived from
Vibrio harveyi
, was introduced into
Nitrosomonas europaea
. Although the recombinant strain produced bioluminescence due to the expression of the
luxAB
genes under normal growing conditions, the intensity of the light emission decreased immediately, in a time-and dose-dependent manner, with the addition of ammonia monooxygenase inhibitors, such as allylthiourea, phenol, and nitrapyrin. When whole cells were challenged with several nitrification inhibitors and toxic compounds, a close relationship was found between the change in the intensity of the light emission and the level of ammonia-oxidizing activity. The response of bioluminescence to the addition of allylthiourea was considerably faster than the change in the ammonia-oxidizing rate, measured as both the O
2
uptake and NO
2
−
production rates. The bioluminescence of cells inactivated by ammonia monooxygenase inhibitor was recovered rapidly by the addition of certain substrates for hydroxylamine oxidoreductase. These results suggested that the inhibition of bioluminescence was caused by the immediate decrease of reducing power in the cell due to the inactivation of ammonia monooxygenase, as well as by the destruction of other cellular metabolic pathways. We conclude that the assay system using luminous
Nitrosomonas
can be applied as a rapid and sensitive detection test for nitrification inhibitors, and it will be used to monitor the nitrification process in wastewater treatment plants.
Estimation of the Relative Abundance of Different
Bacteroides
and
Prevotella
Ribotypes in Gut Samples by Restriction Enzyme Profiling of PCR-Amplified 16S rRNA Gene Sequences
Authors: Wood Jacqueline , Scott Karen P. , Avguštin Gorazd , Newbold C. James , Flint Harry J.
Published: December 2019

We describe an approach for determining the genetic composition of
Bacteroides
and
Prevotella
populations in gut contents based on selective amplification of 16S rRNA gene sequences (rDNA) followed by cleavage of the amplified material with restriction enzymes. The relative contributions of different ribotypes to total
Bacteroides
and
Prevotella
16S rDNA are estimated after end labelling of one of the PCR primers, and the contribution of
Bacteroides
and
Prevotella
sequences to total eubacterial 16S rDNA is estimated by measuring the binding of oligonucleotide probes to amplified DNA.
Bacteroides
and
Prevotella
16S rDNA accounted for between 12 and 62% of total eubacterial 16S rDNA in samples of ruminal contents from six sheep and a cow. Ribotypes 4, 5, 6, and 7, which include most cultivated rumen
Prevotella
strains, together accounted for between 20 and 86% of the total amplified
Bacteroides
and
Prevotella
rDNA in these samples. The most abundant
Bacteroides
or
Prevotella
ribotype in four animals, however, was ribotype 8, for which there is only one known cultured isolate, while ribotypes 1 and 2, which include many colonic
Bacteroides
spp., were the most abundant in two animals. This indicates that some abundant
Bacteroides
and
Prevotella
groups in the rumen are underrepresented among cultured rumen
Prevotella
isolates. The approach described here provides a rapid, convenient, and widely applicable method for comparing the genotypic composition of bacterial populations in gut samples.
Toxic Effects of Modified Fenton Reactions on
Xanthobacter flavus
FB71
Authors: Büyüksönmez Fatïh , Hess Thomas F. , Crawford Ronald L. , Watts Richard J.
Published: December 2019

The toxic effects of modified Fenton reactions on
Xanthobacter flavus
FB71, measured as microbial survival rates, were determined as part of an investigation of simultaneous abiotic and biotic oxidations of xenobiotic chemicals. A central composite, rotatable experimental design was developed to study the survival rates of
X. flavus
under various concentrations of hydrogen peroxide and iron(II) and at different initial cell populations. A model based on the experimental results, relating microorganism survival to the variables of peroxide, iron, and cellular concentrations was formulated and fit the data reasonably well, with a coefficient of determination of 0.76. The results of this study indicate that the use of simultaneous abiotic and biotic processes for the treatment of xenobiotic compounds may be possible.
Improvement of Nitrogen Assimilation and Fermentation Kinetics under Enological Conditions by Derepression of Alternative Nitrogen-Assimilatory Pathways in an IndustrialSaccharomyces cerevisiaeStrain
Authors: Salmon Jean-Michel , Barre Pierre
Published: December 2019

Metabolism of nitrogen compounds by yeasts affects the efficiency of wine fermentation. Ammonium ions, normally present in grape musts, reduce catabolic enzyme levels and transport activities for nonpreferred nitrogen sources. This nitrogen catabolite repression severely impairs the utilization of proline and arginine, both common nitrogen sources in grape juice that require the proline utilization pathway for their assimilation. We attempted to improve fermentation performance by genetic alteration of the regulation of nitrogen-assimilatory pathways inSaccharomyces cerevisiae. One mutant carrying a recessive allele ofure2was isolated from an industrialS. cerevisiaestrain. This mutation strongly deregulated the proline utilization pathway. Fermentation kinetics of this mutant were studied under enological conditions on simulated standard grape juices with various nitrogen levels. Mutant strains produced more biomass and exhibited a higher maximum CO2production rate than the wild type. These differences were primarily due to the derepression of amino acid utilization pathways. When low amounts of dissolved oxygen were added, the mutants could assimilate proline. Biomass yield and fermentation rate were consequently increased, and the duration of the fermentation was substantially shortened.S. cerevisiaestrains lacking URE2 function could improve alcoholic fermentation of natural media where proline and other poorly assimilated amino acids are the major potential nitrogen source, as is the case for most fruit juices and grape musts.
Acid-Sensitive Enteric Pathogens Are Protected from Killing under Extremely Acidic Conditions of pH 2.5 when They Are Inoculated onto Certain Solid Food Sources
Authors: Waterman Scott R. , Small P. L. C.
Published: December 2019

Gastric acidity is recognized as the first line of defense against food-borne pathogens, and the ability of pathogens to resist this pH corresponds to their oral infective dose (ID). Naturally occurring and genetically engineered acid-sensitive enteric pathogens were examined for their ability to survive under acidic conditions of pH 2.5 for 2 h at 37°C when inoculated onto ground beef. Each of the strains displayed significantly high survival rates under these normally lethal conditions. The acid-sensitive pathogens
Campylobacter jejuni
and
Vibrio cholerae
, which were protected at lower levels from acid-induced killing by ground beef under these conditions, were sensitive to killing in acidified media at pH 5.0 but survived at pH 6.0.
Salmonella
inoculated onto the surface of preacidified ground beef could not survive if the pH on the surface of the beef was 2.61 or lower but was viable if the surface pH was 3.27. This implies that the pH of the microenvironment occupied by the bacteria on the surface of the food source is critical for their survival.
Salmonella
was also shown to be protected from killing when inoculated onto boiled egg white, a food source high in protein and low in fat. These results may explain why
Salmonella
species have a higher oral ID of approximately 10
5
cells when administered under defined conditions but have been observed to cause disease at doses as low as 50 to 100 organisms when consumed as part of a contaminated food source. They may also help explain why some pathogens are associated primarily with food-borne modes of transmission rather than fecal-oral transmission.
Optimization of Cry3A Yields in
Bacillus thuringiensis
by Use of Sporulation-Dependent Promoters in Combination with the STAB-SD mRNA Sequence
Authors: Park Hyun-Woo , Ge Baoxue , Bauer Leah S. , Federici Brian A.
Published: December 2019

The insecticidal activity of
Bacillus thuringiensis
strains toxic to coleopterous insects is due to Cry3 proteins assembled into small rectangular crystals. Toxin synthesis in these strains is dependent primarily upon a promoter that is active in the stationary phase and a STAB-SD sequence that stabilizes the
cry3
transcript-ribosome complex. Here we show that significantly higher yields of Cry3A can be obtained by using dual sporulation-dependent
cyt1Aa
promoters to drive the expression of
cry3Aa
when the STAB-SD sequence is included in the construct. The Cry3A yield per unit of culture medium obtained with this expression system was 12.7-fold greater than that produced by DSM 2803, the wild-type strain of
B. thuringiensis
from which Cry3Aa was originally described, and 1.4-fold greater than that produced by NB176, a mutant of the same strain containing two or three copies of
cry3Aa
, which is the active ingredient of the commercial product Novodor, used for control of beetle pests. The toxicities of Cry3A produced with this construct or the wild-type strain were similar when assayed against larvae of the cottonwood leaf beetle,
Chrysomela scripta
. The volume of Cry3A crystals produced with
cyt1Aa
promoters and the STAB-SD sequence was 1.3-fold that of typical bipyramidal Cry1 crystals toxic to lepidopterous insects. The dual-promoter/STAB-SD system offers an additional method for potentially improving the efficacy of insecticides based on
B. thuringiensis
.
Closely Related Plasmid Replicons Coexisting in the PhytopathogenPseudomonas syringaeShow a Mosaic Organization of the Replication Region and Altered Incompatibility Behavior
Authors: Sesma Ane , Sundin George W. , Murillo Jesús
Published: December 2019

ManyPseudomonas syringaestrains contain native plasmids that are important for host-pathogen interactions, and most of them contain several coexisting plasmids (pPT23A-like plasmids) that cross-hybridize to replication sequences from pPT23A, which also carries a gene cluster coding for the phytotoxin coronatine inP. syringaepv. tomato PT23. In this study, three functional pPT23A-like replicons were cloned fromP. syringaepv. glycinea race 6, suggesting that the compatibility of highly related replicons is a common feature ofP. syringaestrains. Hybridization experiments using three separate incompatibility determinants previously identified from pPT23A and therulAB(UV radiation tolerance) genes showed that the organization of the replication region among pPT23A-like plasmids from severalP. syringaepathovars is poorly conserved. The putativerepAgene from four pPT23A-like replicons fromP. syringaepv. glycinea race 6 was amplified by using specific primers. The restriction profiles of the resulting PCR products for the race 6 plasmids were more similar to each other than they were to that of pPT23A. These data, together with the existence of other cross-hybridizing DNA regions around the replicon among the race 6 pPT23A-like plasmids, suggest that some of these plasmids may have originated from duplication events. Our results also imply that modifications of therepAsequences and the poor conservation of putative maintenance determinants contribute to the suppression of incompatibility among members of the pPT23A-like family, thus enhancing the genomic plasticity ofP. syringae.
A Novel ATP-Binding Cassette Transporter Involved in Multidrug Resistance in the Phytopathogenic Fungus
Penicillium digitatum
Authors: Nakaune Ryoji , Adachi Kiichi , Nawata Osamu , Tomiyama Masamitsu , Akutsu Katsumi , Hibi Tadaaki
Published: December 2019

Demethylation inhibitor (DMI)-resistant strains of the plant pathogenic fungus
Penicillium digitatum
were shown to be simultaneously resistant to cycloheximide, 4-nitroquinoline-
N
-oxide (4NQO), and acriflavine. A
PMR1
(
Penicillium
multidrug resistance) gene encoding an ATP-binding cassette (ABC) transporter (P-glycoprotein) was cloned from a genomic DNA library of a DMI-resistant strain (LC2) of
Penicillium digitatum
by heterologous hybridization with a DNA fragment containing an ABC-encoding region from
Botrytis cinerea
. Sequence analysis revealed significant amino acid homology to the primary structures of PMR1 (protein encoded by the
PMR1
gene) and ABC transporters of
Saccharomyces cerevisiae
(PDR5 and SNQ2),
Schizosaccharomyces pombe
(HBA2),
Candida albicans
(CDR1), and
Aspergillus nidulans
(AtrA and AtrB). Disruption of the
PMR1
gene of
P. digitatum
DMI-resistant strain LC2 demonstrated that PMR1 was an important determinant of resistance to DMIs. The effective concentrations inhibiting radial growth by 50% (EC
50
s) and the MICs of fenarimol and bitertanol for the
PMR1
disruptants (Δ
pmr1
mutants) were equivalent to those for DMI-sensitive strains. Northern blot analysis indicated that severalfold more
PMR1
transcript accumulated in the DMI-resistant strains compared with those in DMI-sensitive strains in the absence of fungicide. In both DMI-resistant and -sensitive strains, transcription of
PMR1
was strongly enhanced within 10 min after treatment with the DMI fungicide triflumizole. These results suggested that the toxicant efflux system comprised of PMR1 participates directly in the DMI resistance of the fungus.
Molecular Diversity of Rhizobia Occurring on Native Shrubby Legumes in Southeastern Australia
Authors: Lafay Bénédicte , Burdon Jeremy J.
Published: December 2019

The structure of rhizobial communities nodulating native shrubby legumes in open eucalypt forest of southeastern Australia was investigated by a molecular approach. Twenty-one genomic species were characterized by small-subunit ribosomal DNA PCR-restriction fragment length polymorphism and phylogenetic analyses, among 745 rhizobial strains isolated from nodules sampled on 32 different legume host species at 12 sites. Among these rhizobial genomic species, 16 belonged to theBradyrhizobiumsubgroup, 2 to theRhizobium leguminosarumsubgroup, and 3 to theMesorhizobiumsubgroup. Only one genomic species corresponded to a known species (Rhizobium tropici). The distribution of the various genomic species was highly unbalanced among the 745 isolates, legume hosts, and sites.Bradyrhizobiumspecies were by far the most abundant, andRhizobium tropicidominated among theRhizobiumandMesorhizobiumisolates in the generally acid soils where nodules were collected. Although a statistically significant association occurred between the eight most common genomic species and the 32 hosts, there was sufficient overlap in distributions that no clear specificity between rhizobial genomic species and legume taxa was observed. However, for three legume species, some preference for particular genomic species was suggested. Similarly, no geographical partitioning was found.
Polymorphisms in
Phytophthora infestans
: Four Mitochondrial Haplotypes Are Detected after PCR Amplification of DNA from Pure Cultures or from Host Lesions
Authors: Griffith Gareth W. , Shaw David S.
Published: December 2019

Four pairs of primers were designed for PCR amplification of known polymorphic regions of the mitochondrial genome of
Phytophthora infestans
. Digestion of the amplified products with restriction enzymes allows identification of previously identified haplotypes. Product P2 cut with
Msp
I uniquely identifies haplotypes Ib and IIa, while types Ia and IIb are differentiated by digestion of product P4 with
Eco
RI. Digestion of products P1 and P3 gave results similar to that with digestion of P4, but amplification of these products was less robust. Thus, all four common haplotypes are identified by amplifying and digesting products P2 and P4. Identification of haplotypes was also possible from DNA extracted directly from small, late-blight lesions on both tomato and potato leaves, making isolation of the fungus unnecessary. A rapid and efficient method of monitoring changes in the pathogen population is facilitated. These PCR primers were also useful for differentiating other
Phytophthora
species.
Spatial Physiological Heterogeneity in
Pseudomonas aeruginosa
Biofilm Is Determined by Oxygen Availability
Authors: Xu Karen D. , Stewart Philip S. , Xia Fuhu , Huang Ching-Tsan , McFeters Gordon A.
Published: December 2019

The role of oxygen availability in determining the local physiological activity of
Pseudomonas aeruginosa
growing in biofilms was investigated. Biofilms grown in an ambient-air environment expressed approximately 1/15th the alkaline phosphatase specific activity of planktonic bacteria subjected to the same phosphate limitation treatment. Biofilms grown in a gaseous environment of pure oxygen exhibited 1.9 times the amount of alkaline phosphatase specific activity of air-grown biofilms, whereas biofilms grown in an environment in which the air was replaced with pure nitrogen prior to the inducing treatment did not develop alkaline phosphatase activity. Frozen cross sections of biofilms stained for alkaline phosphatase activity with a fluorogenic stain demonstrated that alkaline phosphatase activity was concentrated in distinct bands adjacent to the gaseous interfaces. These bands were approximately 30 μm thick with biofilms grown in air, 2 μm thick with biofilms grown in pure nitrogen, and 46 μm thick with biofilms grown in pure oxygen. Overall biofilm thickness ranged from approximately 117 to approximately 151 μm. Measurements with an oxygen microelectrode indicated that oxygen was depleted locally within the biofilm and that the oxygen-replete zone was of a dimension similar to that of the biologically active zone, as indicated by alkaline phosphatase induction. These experiments revealed marked spatial physiological heterogeneity within
P. aeruginosa
biofilms in which active protein synthesis was restricted by oxygen availability to the upper 30 μm of the biofilm. Such physiological heterogeneity has implications for microbial ecology and for understanding the reduced susceptibilities of biofilms to antimicrobial agents.
Specific Cell Wall Proteins Confer Resistance to Nisin upon Yeast Cells
Authors: Dielbandhoesing S. K. , Zhang H. , Caro L. H. P. , van der Vaart J. M. , Klis F. M. , Verrips C. T. , Brul S.
Published: December 2019

The cell wall of a yeast cell forms a barrier for various proteinaceous and nonproteinaceous molecules. Nisin, a small polypeptide and a well-known preservative active against gram-positive bacteria, was tested with wild-type
Saccharomyces cerevisiae
. This peptide had no effect on intact cells. However, removal of the cell wall facilitated access of nisin to the membrane and led to cell rupture. The roles of individual components of the cell wall in protection against nisin were studied by using synchronized cultures. Variation in nisin sensitivity was observed during the cell cycle. In the S phase, which is the phase in the cell cycle in which the permeability of the yeast wall to fluorescein isothiocyanate dextrans is highest, the cells were most sensitive to nisin. In contrast, the cells were most resistant to nisin after a peak in expression of the mRNA of cell wall protein 2 (Cwp2p), which coincided with the G2 phase of the cell cycle. A mutant lacking Cwp2p has been shown to be more sensitive to cell wall-interfering compounds and Zymolyase (J. M. Van der Vaart, L. H. Caro, J. W. Chapman, F. M. Klis, and C. T. Verrips, J. Bacteriol. 177:3104–3110, 1995). Here we show that of the single cell wall protein knockouts, a Cwp2p-deficient mutant is most sensitive to nisin. A mutant with a double knockout of Cwp1p and Cwp2p is hypersensitive to the peptide. Finally, in yeast mutants with impaired cell wall structure, expression of both
CWP1
and
CWP2
was modified. We concluded that Cwp2p plays a prominent role in protection of cells against antimicrobial peptides, such as nisin, and that Cwp1p and Cwp2p play a key role in the formation of a normal cell wall.
Possible Transmission of
Streptococcus iniae
from Wild Fish to Cultured Marine Fish
Authors: Zlotkin Amir , Hershko Hannah , Eldar Avi
Published: December 2019

Streptococcus iniae
was isolated from diseased wild fish collected near a mariculture facility where gilthead sea bream and European sea bass exhibited a similar infection. Species-specific PCR and ribotyping confirmed that wild and cultured fish were infected by a single
S. iniae
clone. Wild fish are therefore potential amplifiers of pathogenic
S. iniae
strains.
A Gliding Bacterium Strain Inhibits Adhesion and Motility of Another Gliding Bacterium Strain in a Marine Biofilm
Authors: Burchard Robert P. , Sorongon Maria L.
Published: December 2019

Two species of gliding bacteria were isolated from a marine biofilm. They were described and identified as members of the genusCytophaga. One of them (RB1057) produced an extracellular inhibitor of colony expansion of the other (RB1058). The inhibitor was characterized as a glycoprotein with an apparent molecular mass of 60 kDa. It inhibited RB1058 adhesion to and gliding on substrata. Motility and adhesion of several other aquatic gliding bacteria were not measurably affected by this agent.
Effective Recovery of Bacterial DNA and Percent-Guanine-Plus-Cytosine-Based Analysis of Community Structure in the Gastrointestinal Tract of Broiler Chickens
Authors: Apajalahti Juha H. A. , Särkilahti Laura K. , Mäki Brita R. E. , Heikkinen J. Pekka , Nurminen Päivi H. , Holben William E.
Published: December 2019

A DNA-based, direct method for initial characterization of the total bacterial community in ileum and cecum of the chicken gastrointestinal (GI) tract was developed. The efficiencies of bacterial extraction and lysis were >95 and >99%, respectively, and therefore the DNA recovered should accurately reflect the bacterial communities of the ileal and cecal digesta. Total bacterial DNA samples were fractionated according to their percent G+C content. The profiles reflecting the composition of the bacterial community were reproducible within each compartment, but different between the compartments of the GI tract. This approach is independent of the culturability of the bacteria in the consortium and can be used to improve our understanding of how diet and other variables modulate the microbial communities of the GI tracts of animals.
PCR-Ribotyping of
Xenorhabdus
and
Photorhabdus
Isolates from the Caribbean Region in Relation to the Taxonomy and Geographic Distribution of Their Nematode Hosts
Authors: Fischer-Le Saux Marion , Mauléon Hervé , Constant Philippe , Brunel Brigitte , Boemare Noël
Published: December 2019

The genetic diversity of symbiotic
Xenorhabdus
and
Photorhabdus
bacteria associated with entomopathogenic nematodes was examined by a restriction fragment length polymorphism analysis of PCR-amplified 16S rRNA genes (rDNAs). A total of 117 strains were studied, most of which were isolated from the Caribbean basin after an exhaustive soil sampling. The collection consisted of 77 isolates recovered from entomopathogenic nematodes in 14 Caribbean islands and of 40 reference strains belonging to
Xenorhabdus
and
Photorhabdus
spp. collected at various localities worldwide. Thirty distinctive 16S rDNA genotypes were identified, and cluster analysis was used to distinguish the genus
Xenorhabdus
from the genus
Photorhabdus
. The genus
Xenorhabdus
appears more diverse than the genus
Photorhabdus
, and for both genera the bacterial genotype diversity is in congruence with the host-nematode taxonomy. The occurrence of symbiotic bacterial genotypes was related to the ecological distribution of host nematodes.
Method To Immobilize the Aphid-Pathogenic FungusErynia neoaphidisin an Alginate Matrix for Biocontrol
Authors: Shah P. A. , Aebi M. , Tuor U.
Published: December 2019

Erynia neoaphidisis an important fungal pathogen of aphid pests worldwide. There have been few reported attempts to formulate this natural agent for use in biocontrol. In the current study, factors involved in the immobilization ofE. neoaphidishyphae in an alginate matrix were investigated. Hyphae of two isolates cultured in liquid medium were 220 to 620 μm in length and 7 to 19 μm in diameter with a 74 to 83% cytoplasmic content. The optimal concentration of low-viscosity sodium alginate for production of conidia from entrapped hyphae was 1.5% (wt/vol), and 0.1 and 0.25 M calcium chloride were equally suitable for use as the gelling solution. Alginate beads were rinsed with 10% sucrose after gelling. However, beads should not be left for longer than 40 min in 0.1 M calcium chloride or 10% sucrose to prevent a 10% loss in conidial production. A 40% (vol/vol) concentration of fungal biomass produced significantly more conidia than either 20% or the standard concentration of 10%. This effect persisted even after beads were dried overnight in a laminar flow hood and stored at 4°C for 4 days. Conidia from freshly produced alginate beads caused 27 to 32% infection in Pea aphids as determined by standardized laboratory bioassays. This finding was not significantly different from infections in aphids inoculated with fresh mycelial mats or plugs from Petri dish cultures. In conclusion, algination appears to be a promising technique for utilizingE. neoaphidisin the biocontrol of aphid pests.