bluuebunny/crossref_metadata_2025 huggingface dataset:
Binaural hearing is impaired in children with hearing loss who use bilateral hearing aids
Authors: Gorodensky Jonah H. , Alemu Robel Z. , Gill Simrat S. , Sandor Mark T. , Papsin Blake C. , Cushing Sharon L. , Gordon Karen A.
Published: December 2019

This paper asked whether children fitted with bilateral hearing aids (BHA) develop normal perception of binaural cues which are the basis of spatial hearing. Data from children with BHA (n = 26, age = 12.6 ± 2.84 years) were compared to data from a control group (n = 12, age = 12.36 ± 2.83 years). Stimuli were 250 Hz click-trains of 36 ms and a 40 ms consonant-vowel /da/ at 1 Hz presented through ER3A insert-earphones unilaterally or bilaterally. Bilateral stimuli were presented at different interaural level difference (ILD) and interaural timing difference (ITD) conditions. Participants indicated whether the sound came from the left or right side (lateralization) or whether one sound or two could be heard (binaural fusion). BHA children lateralized ILDs similarly to the control group but had impaired lateralization of ITDs. Longer response times relative to controls suggest that lateralization of ITDs was challenging for children with BHA. Most, but not all, of the BHA group were able to fuse click and speech sounds similarly to controls. Those unable to fuse showed particularly poor ITD lateralization. Results suggest that ITD perception is abnormal in children using BHAs, suggesting persistent effects of hearing loss that are not remediated by present clinical rehabilitation protocols.
Evolutionary transitions toward pair living in nonhuman primates as stepping stones toward more complex societies
Authors: Kappeler Peter M. , Pozzi Luca
Published: December 2019

A new scenario for primate social evolution: Pair living evolved repeatedly from solitary ancestors.
An autonomous untethered fast soft robotic insect driven by low-voltage dielectric elastomer actuators
Authors: Ji Xiaobin , Liu Xinchang , Cacucciolo Vito , Imboden Matthias , Civet Yoan , El Haitami Alae , Cantin Sophie , Perriard Yves , Shea Herbert
Published: December 2019

Low-voltage dielectric elastomer actuators propel an autonomous untethered subgram robust soft legged robot along complex paths.
A new hope for neuroblastoma treatment?
Authors: Montero Joan
Published: December 2019

Combined ALK and PIM1 inhibition may treat high-risk neuroblastomas.
Repression of Phenol Catabolism by Organic Acids in
Ralstonia eutropha
Authors: Ampe Frédéric , Léonard David , Lindley Nicholas D.
Published: December 2019

During batch growth of
Ralstonia eutropha
(previously named
Alcaligenes eutrophus
) on phenol in the presence of acetate, acetate was found to be the preferred substrate; this organic acid was rapidly metabolized, and the specific rate of phenol consumption was considerably decreased, although phenol consumption was not abolished. This decrease corresponded to a drop in phenol hydroxylase and catechol-2,3-dioxygenase specific activities, and the synthesis of the latter was repressed at the transcriptional level. Studies with a mutant not able to consume acetate indicated that the organic acid itself triggers the repression. Other organic acids were also found to repress phenol degradation. One of these, benzoate, was found to completely block the catabolism of phenol (diauxic growth). A mutant unable to metabolize benzoate was also unable to develop on benzoate-phenol mixtures, indicating that the organic acid rather than a metabolite involved in benzoate degradation was responsible for the repression observed.
In Vivo Characterization of Dimethylsulfoniopropionate Lyase in the Fungus
Fusarium lateritium
Authors: Bacic Melissa K. , Yoch Duane C.
Published: December 2019

A fungus,
Fusarium lateritium
, with dimethylsulfoniopropionate (DMSP) lyase activity was isolated from both seawater and a salt marsh due to its ability to grow on DMSP (with the evolution of dimethyl sulfide) as the sole source of carbon. This is the first reported case of DMSP lyase activity in a fungus. Several other common fungal genera tested did not have DMSP lyase activity. DMSP was taken up more rapidly by
F. lateritium
than it was utilized, leading to its intracellular accumulation. Inhibitor studies with nystatin and cyanide indicated that DMSP uptake was an energy-dependent process. The lyase was inducible by its substrate, DMSP (

K
m

, 1.2 mM), and by the substrate analogs choline and glycine betaine. During induction, DMSP lyase activity increased with time and then dropped rapidly. This loss of activity could be prevented by spiking the culture with fresh DMSP or choline. The
V
max
for DMSP lyase was 34.7 mU · mg of protein
−1
. The inhibitory effects of nystatin, and
p
-chloromercuriphenylsulfonate on DMSP lyase activity suggested that the enzyme is cytosolic. Because plants like
Spartina
(a marsh grass) and marine algae contain high concentrations of DMSP, we speculate that DMSP-utilizing fungi may be involved in their decay.
Morpholine Degradation Pathway of
Mycobacterium aurum
MO1: Direct Evidence of Intermediates by In Situ
1
H Nuclear Magnetic Resonance
Authors: Combourieu B. , Besse P. , Sancelme M. , Veschambre H. , Delort A. M. , Poupin P. , Truffaut N.
Published: December 2019

Resting
Mycobacterium aurum
MO1 cells were incubated with morpholine, a waste from the chemical industry. The kinetics of biodegradation was monitored by using in situ nuclear magnetic resonance (NMR). The incubation medium was directly analyzed by
1
H NMR. This technique allowed the unambiguous identification of two intermediates of the metabolic pathway involved in the biodegradation process, glycolate and 2-(2-aminoethoxy)acetate. The latter compound, which was not commercially available, was synthesized, in three steps, from 2-(2-aminoethoxy)ethanol. Quantitative analysis of the kinetics of degradation of morpholine was performed by integrating the signals of the different metabolites in
1
H-NMR spectra. Morpholine was degraded within 10 h. The intermediates increased during the first 10 h and finally disappeared after 20 h incubation. Assays of degradation were also carried out with glycolate and ethanolamine, hypothetical intermediates of the morpholine degradation pathway. They were degraded within 4 and 8 h, respectively. Until now, no tool for direct detection of intermediates or even morpholine has been available, consequently, only hypothetical pathways have been proposed. The approach described here gives both qualitative and quantitative information about the metabolic routes used in morpholine degradation by
M. aurum
MO1. It could be used to investigate many biodegradative processes.
Effect of Metal-Rich Sludge Amendments on the Soil Microbial Community
Authors: Bååth Erland , Díaz-Raviña Montserrat , Frostegård Åsa , Campbell Colin D.
Published: December 2019

The effects of heavy-metal-containing sewage sludge on the soil microbial community were studied in two agricultural soils of different textures, which had been contaminated separately with three predominantly single metals (Cu, Zn, and Ni) at two different levels more than 20 years ago. We compared three community-based microbiological measurements, namely, phospholipid fatty acid (PLFA) analysis to reveal changes in species composition, the Biolog system to indicate metabolic fingerprints of microbial communities, and the thymidine incorporation technique to measure bacterial community tolerance. In the Luddington soil, bacterial community tolerance increased in all metal treatments compared to an unpolluted-sludge-treated control soil. Community tolerance to specific metals increased the most when the same metal was added to the soil; for example, tolerance to Cu increased most in Cu-polluted treatments. A dose-response effect was also evident. There were also indications of cotolerance to metals whose concentration had not been elevated by the sludge treatment. The PLFA pattern changed in all metal treatments, but the interpretation was complicated by the soil moisture content, which also affected the results. The Biolog measurements indicated similar effects of metals and moisture to the PLFA measurements, but due to high variation between replicates, no significant differences compared to the uncontaminated control were found. In the Lee Valley soil, significant increases in community tolerance were found for the high levels of Cu and Zn, while the PLFA pattern was significantly altered for the soils with high levels of Cu, Ni, and Zn. No effects on the Biolog measurements were found in this soil.
Effects of UV-A (320 to 399 Nanometers) on Grazing Pressure of a Marine Heterotrophic Nanoflagellate on Strains of the Unicellular Cyanobacteria
Synechococcus
spp
Authors: Ochs Clifford A. , Eddy Laura P.
Published: December 2019

In the open ocean, where turbidity is very low, UV radiation may be an important factor regulating interactions among planktonic microorganisms. The effect of exposure to UV radiation on grazing by a commonly isolated marine heterotrophic nanoflagellate,
Paraphysomonas bandaiensis
, on two strains of the cyanobacteria
Synechococcus
spp. was investigated. Laboratory cultures were exposed to a range of irradiances of artificially produced UV-B (290 to 319 nm) and UV-A (320 to 399 nm) for up to 10 h. At a UV-B irradiance of 0.19 W m
−2
, but not 0.12 W m
−2
, grazing mortality of
Synechococcus
spp. and nanoflagellate-specific grazing rates were reduced compared to mortality and grazing rates with UV-A treatment. Within 6 h of exposure, UV-A alone suppressed grazing mortality at irradiances as low as 3.02 W m
−2
. The extent to which grazing mortality and nanoflagellate-specific grazing rates were suppressed by UV-A increased with both irradiance and duration of exposure. Over a 6-h exposure period, differences in grazing mortality were largely attributable to differential survival of nanoflagellates. Over a longer period of exposure, there was impairment by UV-A alone of nanoflagellate-specific grazing rates. Rates of primary productivity of
Synechococcus
spp. were also reduced by UV-A. The extent to which
Synechococcus
productivity was reduced, compared to the reduction in
Synechococcus
grazing mortality, depended on the duration of UV-A exposure. These results support the hypothesis that UV-A alone influences the composition and biomass of marine microbial communities by affecting predator-prey interactions and primary production.
Measurement of Antimicrobial-Resistant
Escherichia coli
in Pig Feces with a Hydrophobic Grid Membrane Filter Interpreter System
Authors: Dunlop R. Hugo , McEwen Scott A. , Meek Alan H. , Clarke Robert C. , Friendship Robert M. , Black William D. , Sharpe Anthony N.
Published: December 2019

Hydrophobic grid membrane filter technology was used to measure resistance among
Escherichia coli
in pig fecal samples to ampicillin, sulfisoxazole, and tetracycline. The method accurately measured resistance, with sensitivities ranging from 96.5 to 99.5% and specificities ranging from 87.0 to 98.3%, and it identified
E. coli
with 96% confidence.
Optimization of Differential Display of Prokaryotic mRNA: Application to Pure Culture and Soil Microcosms
Authors: Fleming James T. , Yao Wen-Hsiang , Sayler Gary S.
Published: December 2019

The differential display (DD) technique, which is widely used almost exclusively for eukaryotic gene discovery, was optimized to detect differential mRNA transcription from both pure-culture and soil-derived bacterial RNA. A model system which included toluene induction oftodC1inPseudomonas putidaF1 was used to optimize the procedure. At 24-htodinduction was determined to be approximately 8 × 107transcripts/μg or 0.08% of the total mRNA. The primer concentration, primer length, annealing temperature, and template, deoxynucleoside triphosphate, and MgCl2concentrations were varied to optimize amplification of atodC1fragment. The limit of detection oftodC1by DD was found to be 0.015 ng of total RNA template or approximately 103transcripts. Once optimized, atodC1C2gene fragment fromP. putidaF1 RNA was detected by using an arbitrary primer for the reverse transcriptase step in conjunction with the same arbitrary primer and a Shine-Dalgarno primer in the PCR. To verify the results, an arbitrary primer was used to detect recovery of a new salicylate-inducible naphthalene dioxygenase inBurkholderia cepaciaJS150. The method was then used to detect mRNA induction in both inoculated and uninoculated toluene-induced soil microcosms. Several putative differentially expressed partial gene sequences obtained from the uninoculated microcosms were examined, and one novel fragment was found to be differentially expressed.
Structural and Functional Dynamics of Sulfate-Reducing Populations in Bacterial Biofilms
Authors: Santegoeds Cecilia M. , Ferdelman Timothy G. , Muyzer Gerard , de Beer Dirk
Published: December 2019

We describe the combined application of microsensors and molecular techniques to investigate the development of sulfate reduction and of sulfate-reducing bacterial populations in an aerobic bacterial biofilm. Microsensor measurements for oxygen showed that anaerobic zones developed in the biofilm within 1 week and that oxygen was depleted in the top 200 to 400 μm during all stages of biofilm development. Sulfate reduction was first detected after 6 weeks of growth, although favorable conditions for growth of sulfate-reducing bacteria (SRB) were present from the first week. In situ hybridization with a 16S rRNA probe for SRB revealed that sulfate reducers were present in high numbers (approximately 10
8
SRB/ml) in all stages of development, both in the oxic and anoxic zones of the biofilm. Denaturing gradient gel electrophoresis (DGGE) showed that the genetic diversity of the microbial community increased during the development of the biofilm. Hybridization analysis of the DGGE profiles with taxon-specific oligonucleotide probes showed that
Desulfobulbus
and
Desulfovibrio
were the main sulfate-reducing bacteria in all biofilm samples as well as in the bulk activated sludge. However, different
Desulfobulbus
and
Desulfovibrio
species were found in the 6th and 8th weeks of incubation, respectively, coinciding with the development of sulfate reduction. Our data indicate that not all SRB detected by molecular analysis were sulfidogenically active in the biofilm.
Colonization of Wheat Roots by an Exopolysaccharide-Producing
Pantoea agglomerans
Strain and Its Effect on Rhizosphere Soil Aggregation
Authors: Amellal N. , Burtin G. , Bartoli F. , Heulin T.
Published: December 2019

The effect of bacterial secretion of an exopolysaccharide (EPS) on rhizosphere soil physical properties was investigated by inoculating strain NAS206, which was isolated from the rhizosphere of wheat (
Triticum durum
L.) growing in a Moroccan vertisol and was identified as
Pantoea aglomerans
. Phenotypic identification of this strain with the Biotype-100 system was confirmed by amplified ribosomal DNA restriction analysis. After inoculation of wheat seedlings with strain NAS206, colonization increased at the rhizoplane and in root-adhering soil (RAS) but not in bulk soil. Colonization further increased under relatively dry conditions (20% soil water content; matric potential, −0.55 MPa). By means of genetic fingerprinting using enterobacterial repetitive intergenic consensus PCR, we were able to verify that colonies counted as strain NAS206 on agar plates descended from inoculated strain NAS206. The intense colonization of the wheat rhizosphere by these EPS-producing bacteria was associated with significant soil aggregation, as shown by increased ratios of RAS dry mass to root tissue (RT) dry mass (RAS/RT) and the improved water stability of adhering soil aggregates. The maximum effect of strain NAS206 on both the RAS/RT ratio and aggregate stability was measured at 24% average soil water content (matric potential, −0.20 MPa). Inoculated strain NAS206 improved RAS macroporosity (pore diameter, 10 to 30 μm) compared to the noninoculated control, particularly when the soil was nearly water saturated (matric potential, −0.05 MPa). Our results suggest that
P. agglomerans
NAS206 can play an important role in the regulation of the water content (excess or deficit) of the rhizosphere of wheat by improving soil aggregation.
Phenotypic and Phylogenetic Characterization of Ruminal Tannin-Tolerant Bacteria
Authors: Nelson Karen E. , Thonney Michael L. , Woolston Tina K. , Zinder Stephen H. , Pell Alice N.
Published: December 2019

The 16S rRNA sequences and selected phenotypic characteristics were determined for six recently isolated bacteria that can tolerate high levels of hydrolyzable and condensed tannins. Bacteria were isolated from the ruminal contents of animals in different geographic locations, including Sardinian sheep (
Ovis aries
), Honduran and Colombian goats (
Capra hircus
), white-tail deer (
Odocoileus virginianus
) from upstate New York, and Rocky Mountain elk (
Cervus elaphus nelsoni
) from Oregon. Nearly complete sequences of the small-subunit rRNA genes, which were obtained by PCR amplification, cloning, and sequencing, were used for phylogenetic characterization. Comparisons of the 16S rRNA of the six isolates showed that four of the isolates were members of the genus
Streptococcus
and were most closely related to ruminal strains of
Streptococcus bovis
and the recently described organism
Streptococcus gallolyticus
. One of the other isolates, a gram-positive rod, clustered with the clostridia in the low-G+C-content group of gram-positive bacteria. The sixth isolate, a gram-negative rod, was a member of the family
Enterobacteriaceae
in the gamma subdivision of the class
Proteobacteria
. None of the 16S rRNA sequences of the tannin-tolerant bacteria examined was identical to the sequence of any previously described microorganism or to the sequence of any of the other organisms examined in this study. Three phylogenetically distinct groups of ruminal bacteria were isolated from four species of ruminants in Europe, North America, and South America. The presence of tannin-tolerant bacteria is not restricted by climate, geography, or host animal, although attempts to isolate tannin-tolerant bacteria from cows on low-tannin diets failed.
Comparison ofPaenibacillus azotofixansStrains Isolated from Rhizoplane, Rhizosphere, and Non-Root-Associated Soil from Maize Planted in Two Different Brazilian Soils
Authors: Seldin Lucy , Rosado Alexandre Soares , da Cruz Davi William , Nobrega Alberto , van Elsas Jan Dirk , Paiva Edilson
Published: December 2019

Paenibacillus azotofixansis a nitrogen-fixing bacterium often found in soil and in the rhizospheres of different grasses. In this study, two Brazilian clay soils were planted with cross-hybrid maize (BR-201) and four stages of plant growth were analyzed to characterize theP. azotofixanspopulations present in the rhizoplanes, rhizospheres, and non-root-associated soils (herein called nonrhizospheres). A total of 106 strains were isolated and identified asP. azotofixanswith an API 50CH kit, by classical biochemical tests, and via the use of specific primers based on the 16S rRNA gene in PCRs. To compare the isolated strains, phenotypic characteristics were determined and three different probes were used in hybridization experiments: twonifprobes and one probe comprising a 0.58-kb fragment cloned from theP. azotofixansC3L4 genome. These results were used to construct a dendrogram, in which two main clusters could be observed. One cluster contained exclusively strains from Várzea soil, and the other contained the majority of strains from Cerrado soil. The 60 strains from Várzea soil and the 46 strains from Cerrado soil were further analyzed with REP and BOX primers, respectively. Based on the patterns obtained, it was possible to identify 21 different groups among strains from Várzea soil and 4 different groups among strains from Cerrado soil. These different patterns were tested by multivariate analysis of variance, and differences in the populations ofP. azotofixansduring the four stages of plant growth were demonstrated. Moreover, strains isolated from the rhizoplanes, rhizospheres, and nonrhizospheres of maize planted in Cerrado and Várzea soils were shown to be statistically different; the diversity ofP. azotofixansstrains was affected by the soil type.
Occurrence of Fusaproliferin and Beauvericin in
Fusarium
-Contaminated Livestock Feed in Iowa
Authors: Munkvold Gary , Stahr H. M. , Logrieco Antonio , Moretti Antonio , Ritieni Alberto
Published: December 2019

Fusarium
fungal contaminants and related mycotoxins were investigated in eight maize feed samples submitted to the Iowa State University Veterinary Diagnostic Laboratory.
Fusarium moniliforme
,
F. proliferatum
, and
F. subglutinans
were isolated from seven, eight, and five samples, respectively. These strains belonged to mating populations A, D, and E of the teleomorph
Gibberella fujikuroi
. Fusaproliferin was detected at concentrations of 0.1 to 30 μg/g in four samples, and beauvericin was detected (0.1 to 3.0 μg/g) in five samples. Fumonisins were detected in all eight samples (1.1 to 14 μg/g). Ten of 11 strains of
F. proliferatum
and all 12 strains of
F. subglutinans
isolated from the samples produced fusaproliferin in culture on whole maize kernels (4 to 350 and 100 to 1,000 μg/g, respectively). Nine
F. proliferatum
strains also produced beauvericin in culture (85 to 350 μg/g), but none of the
F. subglutinans
strains produced beauvericin. Fumonisin B
1
was produced by all nine
F. moniliforme
strains (50 to 2,000 μg/g) and by 10 of the
F. proliferatum
strains (1,000 to 2,000 μg/g). This is the first report of the natural occurrence of fusaproliferin outside Italy and of the natural occurrence of beauvericin in North America.
Purification and Characterization of an NAD-Malic Enzyme from
Bradyrhizobium japonicum
A1017
Authors: Chen Fan , Okabe Youichi , Osano Kaoru , Tajima Shigeyuki
Published: December 2019

An NAD-malic enzyme was purified to homogeneity from
Bradyrhizobium japonicum
A1017, and its molecular characteristics were surveyed. The enzyme exhibited native and subunit molecular masses of 388 and 85 kDa, respectively, suggesting that it exists as a homotetramer, and was activated by metabolic intermediates in glycolysis. The role of the enzyme in bacteroids’ carbon metabolism is discussed.
Balance of Activities of Alcohol Acetyltransferase and Esterase in
Saccharomyces cerevisiae
Is Important for Production of Isoamyl Acetate
Authors: Fukuda Kiyoshi , Yamamoto Nagi , Kiyokawa Yoshifumi , Yanagiuchi Toshiyasu , Wakai Yoshinori , Kitamoto Katsuhiko , Inoue Yoshiharu , Kimura Akira
Published: December 2019

Isoamyl acetate is synthesized from isoamyl alcohol and acetyl coenzyme A by alcohol acetyltransferase (AATFase) in
Saccharomyces cerevisiae
and is hydrolyzed by esterases at the same time. We hypothesized that the balance of both enzyme activities was important for optimum production of isoamyl acetate in sake brewing. To test this hypothesis, we constructed yeast strains with different numbers of copies of the AATFase gene (
ATF1
) and the isoamyl acetate-hydrolyzing esterase gene (
IAH1
) and used these strains in small-scale sake brewing. Fermentation profiles as well as components of the resulting sake were largely alike; however, the amount of isoamyl acetate in the sake increased with an increasing ratio of AATFase/Iah1p esterase activity. Therefore, we conclude that the balance of these two enzyme activities is important for isoamyl acetate accumulation in sake mash.
Persistence of PCR-Detectable
Bacteroides distasonis
from Human Feces in River Water
Authors: Kreader Carol A.
Published: December 2019

To evaluate the persistence of PCR-detectable
Bacteroides distasonis
in surface water, whole human feces were dispersed into water from the Ohio River and incubated in flasks in the laboratory or in diffusion chambers in situ. Duplicate samples were taken daily, and material that pelleted at 16,000 ×
g
was assayed by PCR. Persistence of PCR-detectable DNA from this anaerobe depended upon temperature and predation, two of the factors shown by others to influence the survival of aerobic bacteria detected by culture.
B. distasonis
was detected by PCR for at least 2 weeks at 4°C but for only 4 to 5 days at 14°C, 1 to 2 days at 24°C, and 1 day at 30°C. In filtered water or in the presence of cycloheximide, a eukaryotic inhibitor, persistence at 24°C was extended by at least a week.
Survival of
Enterococcus faecalis
in an Oligotrophic Microcosm: Changes in Morphology, Development of General Stress Resistance, and Analysis of Protein Synthesis
Authors: Hartke Axel , Giard Jean-Christophe , Laplace Jean-Marie , Auffray Yanick
Published: December 2019

The ability of
Enterococcus faecalis
to metabolically adapt to an oligotrophic environment has been analyzed.
E. faecalis
is able to survive for prolonged periods under conditions of complete starvation established by incubation in tap water. During incubation in this microcosm, cells developed a rippled cell surface with irregular shapes. Exponentially growing cells survived to the same extent as cells starved for glucose prior to exposure to the multiple nutrient deficient stress. Chloramphenicol treatment during incubation in tap water led to a rapid decline in plate counts for exponentially growing cells but showed progressively reduced influence on stationary-phase cells harvested after different times of glucose starvation. During incubation in the oligotrophic environment, cells from the exponential-growth phase and early-stationary phase became progressively more resistant to other environmental stresses (heat [62°C], acid [pH 3.3], UV
254 nm
light [180 J/m
2
], and sodium hypochlorite [0.05%]) until they reached a maximum of survival characteristic for each treatment. In contrast, cells starved of glucose for 24 h did not become more resistant to the different treatments during incubation in tap water. Our combined data suggest that energy starvation induces a response similar to that triggered by oligotrophy. Analysis of protein synthesis by two-dimensional gel electrophoresis revealed the enhanced synthesis of 51 proteins which were induced in the oligotrophic environment. A comparison of these oligotrophy-inducible proteins with the 42 glucose starvation-induced polypeptides (J. C. Giard, A. Hartke, S. Flahaut, P. Boutibonnes, and Y. Auffray, Res. Microbiol. 148:27–35, 1997) showed that 16 are common between the two different starvation conditions. These proteins and the corresponding genes seem to play a key role in the observed phenomena of long-term survival and development of general stress resistance of starved cultures of
E. faecalis
.
Threonine Aldolase Overexpression plus Threonine Supplementation Enhanced Riboflavin Production in
Ashbya gossypii
Authors: Monschau Nicole , Sahm Hermann , Stahmann K.-Peter
Published: December 2019

Riboflavin production in the filamentous fungus
Ashbya gossypii
is limited by glycine, an early precursor required for purine synthesis. We report an improvement of riboflavin production in this fungus by overexpression of the glycine biosynthetic enzyme threonine aldolase. The
GLY1
gene encoding the threonine aldolase of
A. gossypii
was isolated by heterologous complementation of the glycine-auxotrophic
Saccharomyces cerevisiae
strain YM13 with a genomic library from
A. gossypii
. The deduced amino acid sequence of
GLY1
showed 88% similarity to threonine aldolase from
S. cerevisiae
. In the presence of the
GLY1
gene, 25 mU of threonine aldolase specific activity mg
−1
was detectable in crude extracts of
S. cerevisiae
YM13. Disruption of
GLY1
led to a complete loss of threonine aldolase activity in
A. gossypii
crude extracts, but growth of and riboflavin production by the knockout mutant were not affected. This indicated a minor role of the enzyme in glycine biosynthesis of
A. gossypii
. However, overexpression of
GLY1
under the control of the constitutive
TEF
promoter and terminator led to a 10-fold increase of threonine aldolase specific activity in crude extracts along with a 9-fold increase of riboflavin production when the medium was supplemented with threonine. This strong enhancement, which could not be achieved by supplementation with glycine alone, was attributed to an almost quantitative uptake of threonine and its intracellular conversion into glycine. This became evident by a subsequent partial efflux of the glycine formed.
Low-Concentration Kinetics of Atmospheric CH
4
Oxidation in Soil and Mechanism of NH
4
+
Inhibition
Authors: Gulledge Jay , Schimel Joshua P.
Published: December 2019

NH
4
+
inhibition kinetics for CH
4
oxidation were examined at near-atmospheric CH
4
concentrations in three upland forest soils. Whether NH
4
+
-independent salt effects could be neutralized by adding nonammoniacal salts to control samples in lieu of deionized water was also investigated. Because the levels of exchangeable endogenous NH
4
+
were very low in the three soils, desorption of endogenous NH
4
+
was not a significant factor in this study. The

K
m(app)

values for water-treated controls were 9.8, 22, and 57 nM for temperate pine, temperate hardwood, and birch taiga soils, respectively. At CH
4
concentrations of ≤15 μl liter
−1
, oxidation followed first-order kinetics in the fine-textured taiga soil, whereas the coarse-textured temperate soils exhibited Michaelis-Menten kinetics. Compared to water controls, the

K
m(app)

values in the temperate soils increased in the presence of NH
4
+
salts, whereas the
V

max(
app
)

values decreased substantially, indicating that there was a mixture of competitive and noncompetitive inhibition mechanisms for whole NH
4
+
salts. Compared to the corresponding K
+
salt controls, the

K
m(app)

values for NH
4
+
salts increased substantially, whereas the
V

max
(app)

values remained virtually unchanged, indicating that NH
4
+
acted by competitive inhibition. Nonammoniacal salts caused inhibition to increase with increasing CH
4
concentrations in all three soils. In the birch taiga soil, this trend occurred with both NH
4
+
and K
+
salts, and the slope of the increase was not affected by the addition of NH
4
+
. Hence, the increase in inhibition resulted from an NH
4
+
-independent mechanism. These results show that NH
4
+
inhibition of atmospheric CH
4
oxidation resulted from enzymatic substrate competition and that additional inhibition that was not competitive resulted from a general salt effect that was independent of NH
4
+
.
Rice Hull Ash and Silicic Acid as Adsorbents for Concentration of Bacteriocins
Authors: Janes M. E. , Nannapaneni R. , Proctor A. , Johnson M. G.
Published: December 2019

A model procedure has been developed for the rapid extraction of five bacteriocins (nisin, pediocin RS2, leucocin BC2, lactocin GI3, and enterocin CS1) from concentrated freeze-dried crude culture supernatants by adsorption onto acid or alkaline rice hull ash (RHA) or silicic acid (SA). Bacteriocins were adsorbed onto RHA or SA by a pH-dependent method and desorbed by decreasing the pH to 2.5 or 3.0 and heating at 90°C for 5 min. The maximum adsorption and optimal pH range for different bacteriocins were as follows: nisin, 97% at pH 7.0; lactocin GI3, 94% at pH 6.0; pediocin RS2, 97% at pH 8.0 to 9.0; leucocin BC2, 88% at pH 9.0; and enterocin CS1, 94% at pH 5.0. The desorption level of lactocin GI3 or enterocin CS1 from the surfaces of both RHA and SA was 94%, while the desorption level of pediocin RS2 and leucocin BC2 was 50% or less. Nisin was desorbed readily from SA (91%) but not from RHA (50% or less). The adsorption of bacteriocins onto RHA and SA increased with the increasing concentration of bacteriocins. Analysis of the desorbed bacteriocins after dialysis and sodium dodecyl sulfate–16% polyacrylamide gel electrophoresis showed a single band that gave a single inhibition zone when overlaid with
Lactobacillus plantarum
for detection of lactocin GI3, enterocin CS1, and nisin. RHA appears useful for extraction, concentration, and partial purification of the five bacteriocins.
Accumulation of α-Keto Acids as Essential Components in Cyanide Assimilation by
Pseudomonas fluorescens
NCIMB 11764
Authors: Kunz Daniel A. , Chen Jui-Lin , Pan Guangliang
Published: December 2019

Pyruvate (Pyr) and α-ketoglutarate (αKg) accumulated when cells of
Pseudomonas fluorescens
NCIMB 11764 were cultivated on growth-limiting amounts of ammonia or cyanide and were shown to be responsible for the nonenzymatic removal of cyanide from culture fluids as previously reported (J.-L. Chen and D. A. Kunz, FEMS Microbiol. Lett. 156:61–67, 1997). The accumulation of keto acids in the medium paralleled the increase in cyanide-removing activity, with maximal activity (760 μmol of cyanide removed min
−1
ml of culture fluid
−1
) being recovered after 72 h of cultivation, at which time the keto acid concentration was 23 mM. The reaction products that formed between the biologically formed keto acids and cyanide were unambiguously identified as the corresponding cyanohydrins by
13
C nuclear magnetic resonance spectroscopy. Both the Pyr and α-Kg cyanohydrins were further metabolized by cell extracts and served also as nitrogenous growth substrates. Radiotracer experiments showed that CO
2
(and NH
3
) were formed as enzymatic conversion products, with the keto acid being regenerated as a coproduct. Evidence that the enzyme responsible for cyanohydrin conversion is cyanide oxygenase, which was shown previously to be required for cyanide utilization, is based on results showing that (i) conversion occurred only when extracts were induced for the enzyme, (ii) conversion was oxygen and reduced-pyridine nucleotide dependent, and (iii) a mutant strain defective in the enzyme was unable to grow when it was provided with the cyanohydrins as a growth substrate. Pyr and αKg were further shown to protect cells from cyanide poisoning, and excretion of the two was directly linked to utilization of cyanide as a growth substrate. The results provide the basis for a new mechanism of cyanide detoxification and assimilation in which keto acids play an essential role.
A Small, Dilute-Cytoplasm, High-Affinity, Novel Bacterium Isolated by Extinction Culture and Having Kinetic Constants Compatible with Growth at Ambient Concentrations of Dissolved Nutrients in Seawater
Authors: Button D. K. , Robertson Betsy R. , Lepp Paul W. , Schmidt Thomas M.
Published: December 2019

Dilutions of raw seawater produced a bacterial isolate capable of extended growth in unamended seawater. Its 2.9-Mb genome size and 40-fg dry mass were similar to values for many naturally occurring aquatic organotrophs, but water and DNA comprised a large portion of this small chemoheterotroph, as compared to
Escherichia coli
. The isolate used only a few aromatic hydrocarbons and acetate, and glucose and amino acid incorporation were entirely absent, although many membrane and cytoplasmic proteins were inducible; it was named
Cycloclasticus oligotrophus
. A general rate equation that incorporates saturation phenomena into specific affinity theory is derived. It is used to relate the kinetic constants for substrate uptake by the isolate to its cellular proteins. The affinity constant

K
A

for toluene was low at 1.3 μg/liter under optimal conditions, similar to those measured in seawater, and the low value was ascribed to an unknown slow step such as limitation by a cytoplasmic enzyme;

K
A

increased with increasing specific affinities. Specific affinities,

a°
s

, were protocol sensitive, but under optimal conditions were 47.4 liters/mg of cells/h, the highest reported in the literature and a value sufficient for growth in seawater at concentrations sometimes found. Few rRNA operons, few cytoplasmic proteins, a small genome size, and a small cell size, coupled with a high

a°
s

and a low solids content and the ability to grow without intentionally added substrate, are consistent with the isolation of a marine bacterium with properties typical of the bulk of those present.
Sequence Polymorphism in the β-Tubulin Gene Reveals Heterogeneous and Variable Population Structures in
Cryptosporidium parvum
Authors: Widmer Giovanni , Tchack Laurie , Chappell Cynthia L. , Tzipori Saul
Published: December 2019

Restriction fragment length polymorphism (RFLP) analysis of isolates of
Cryptosporidium parvum
has revealed two subgroups, termed H and C. The limited resolution of the RFLP method precludes an in-depth study of the genetic structure of
C. parvum
populations. Published
C. parvum
restriction polymorphisms lie within protein-coding regions known to be more homogeneous than noncoding sequences. To better assess the degrees of heterogeneity between and within
C. parvum
isolates, sequence polymorphism in the β-tubulin intron, the only
C. parvum
intron described to date, was investigated. In contrast to the two genotypes distinguished by multilocus RFLP, several alleles were detected by sequence and RFLP analysis of the β-tubulin intron and adjacent exon 2. Isolates carrying different β-tubulin alleles were found. Significantly, one of the β-tubulin alleles present in two geographically unrelated isolates combined features of C- and H-type isolates, suggesting that it might have arisen from a recombination event. A comparison of multiple samples of a calf-propagated laboratory isolate showed that the ratio of different β-tubulin alleles fluctuated during serial passage.
In Situ Detection of an Uncultured Predominant Bacillus in Dutch Grassland Soils
Authors: Felske Andreas , Akkermans Antoon D. L. , De Vos Willem M.
Published: December 2019

Uncultured predominant
Bacillus
ribotype DA001 in Dutch Drentse A grassland soils, as revealed by its 16S rRNA sequence, was detected in soil by fluorescent whole-cell in situ hybridization. A prominent rod-shaped cell type was identified in bacterial suspensions prepared from soil by a multiple 16S rRNA probing approach.
The Mitochondrial Toxin Produced by
Streptomyces griseus
Strains Isolated from an Indoor Environment Is Valinomycin
Authors: Andersson M. A. , Mikkola R. , Kroppenstedt R. M. , Rainey F. A. , Peltola J. , Helin J. , Sivonen K. , Salkinoja-Salonen M. S.
Published: December 2019

Actinomycete isolates from indoor air and dust in water-damaged schools and children’s day care centers were tested for toxicity by using boar spermatozoa as an indicator. Toxicity was detected in extracts of four strains which caused a loss of sperm motility, and the 50% effective concentrations (EC
50
) were 10 to 63 ng (dry weight) ml of extended boar semen
−1
. The four strains were identified as
Streptomyces griseus
strains by 16S ribosomal DNA and chemotaxonomic methods. The four
S. griseus
strains had similar effects on sperm cells, including loss of motility and swelling of mitochondria, but we observed no loss of plasma membrane integrity or depletion of cellular ATP. None of the effects was observed with sperm cells exposed to extracts of other indoor actinomycete isolates at concentrations of ≥5,000 to 72,000 ng ml
−1
. The toxin was purified from all four strains and was identified as a dodecadepsipeptide, and the fragmentation pattern obtained by tandem mass spectrometry was identical to that of valinomycin. Commercial valinomycin had effects in sperm cells that were identical to the effects of the four indoor isolates of
S. griseus
. The EC
50
of purified toxin from the
S. griseus
strains were 1 to 3 ng ml of extended boar semen
−1
, and the EC
50
of commercial valinomycin was 2 ng ml of extended boar semen
−1
. To our knowledge, this is the first report of the presence of ionophoric toxin producers in an indoor environment and the first report of valinomycin-producing strains identified as
S. griseus.
A Technique To Quantify the Population Size and Composition of the Biofilm Component in Communities of Bacteria in the Phyllosphere
Authors: Morris Cindy E. , Monier Jean-Michel , Jacques Marie-Agnès
Published: December 2019

The presence of microbial biofilms in the phyllosphere of terrestrial plants has recently been demonstrated, but few techniques to study biofilms associated with living plant tissues are available. Here we report a technique to estimate the proportion of the bacterial population on leaves that is assembled in biofilms and to quantitatively isolate bacteria from the biofilm and nonbiofilm (solitary) components of phyllosphere microbial communities. This technique is based on removal of bacteria from leaves by gentle washing, separation of biofilm and solitary bacteria by filtration, and disintegration of biofilms by ultrasonication. The filters used for this technique were evaluated for their nonspecific retention rates of solitary bacteria and for the efficiency of filtration for different concentrations of solitary bacteria in the presence of biofilms and other particles. The lethality and efficiency of disintegration of the sonication conditions used here were also evaluated. Isolation and quantification of bacteria by this technique is based on use of culture media. However, oligonucleotide probes, sera, or epifluorescent stains could also be used for direct characterization of the biofilm and solitary bacteria in the suspensions generated by this technique. Preliminary results from estimates of biofilm abundance in phyllosphere communities show that bacteria in biofilms constitute between about 10 and 40% of the total bacterial population on broad-leaf endive and parsley leaves.
Psychrotolerant Bacteria Isolated from Arctic Soil That Degrade Polychlorinated Biphenyls at Low Temperatures
Authors: Master Emma R. , Mohn William W.
Published: December 2019

Psychrotolerant polychlorinated biphenyl (PCB)-degrading bacteria were isolated at 7°C from PCB-contaminated Arctic soil by using biphenyl as the sole organic carbon source. These isolates were distinguished from each other by differences in substrates that supported growth and substrates that were oxidized. 16S ribosomal DNA sequences suggest that these isolates are most closely related to the genus
Pseudomonas
. Total removal of Aroclor 1242, and rates of removal of selected PCB congeners, by cell suspensions of Arctic soil isolates and the mesophile
Burkholderia cepacia
LB400 were determined at 7, 37, and 50°C. Total removal values of Aroclor 1242 at 7°C by LB400 and most Arctic soil isolates were similar (between 2 and 3.5 μg of PCBs per mg of cell protein). However the rates of removal of some individual PCB congeners by Arctic isolates were up to 10 times higher than corresponding rates of removal by LB400. Total removal of Aroclor 1242 and the rates of removal of individual congeners by the Arctic soil bacteria were higher at 37°C than at 7°C but as much as 90% lower at 50°C than at 37°C. In contrast, rates of PCB removal by LB400 were higher at 50°C than at 37°C. In all cases, temperature did not affect the congener specificity of the bacteria. These observations suggest that the PCB-degrading enzyme systems of the bacteria isolated from Arctic soil are cold adapted.
Cloning of theAlcaligenes latusPolyhydroxyalkanoate Biosynthesis Genes and Use of These Genes for Enhanced Production of Poly(3-hydroxybutyrate) inEscherichia coli
Authors: Choi Jong-il , Lee Sang Yup , Han Kyuboem
Published: December 2019

Polyhydroxyalkanoates (PHAs) are microbial polyesters that can be used as completely biodegradable polymers, but the high production cost prevents their use in a wide range of applications. RecombinantEscherichia colistrains harboring theRalstonia eutrophaPHA biosynthesis genes have been reported to have several advantages as PHA producers compared with wild-type PHA-producing bacteria. However, the PHA productivity (amount of PHA produced per unit volume per unit time) obtained with these recombinantE. colistrains has been lower than that obtained with the wild-type bacteriumAlcaligenes latus. To endow the potentially superior PHA biosynthetic machinery toE. coli, we cloned the PHA biosynthesis genes fromA. latus. The three PHA biosynthesis genes formed an operon with the order PHA synthase, β-ketothiolase, and reductase genes and were constitutively expressed from the natural promoter inE. coli. RecombinantE. colistrains harboring theA. latusPHA biosynthesis genes accumulated poly(3-hydroxybutyrate) (PHB), a model PHA product, more efficiently than those harboring theR. eutrophagenes. With a pH-stat fed-batch culture of recombinantE. coliharboring a stable plasmid containing theA. latusPHA biosynthesis genes, final cell and PHB concentrations of 194.1 and 141.6 g/liter, respectively, were obtained, resulting in a high productivity of 4.63 g of PHB/liter/h. This improvement should allow recombinantE. colito be used for the production of PHB with a high level of economic competitiveness.
NADP-Isocitrate Dehydrogenase from
Pseudomonas nautica
: Kinetic Constant Determination and Carbon Limitation Effects on the Pool of Intracellular Substrates
Authors: Roy Sylvie O. , Packard Ted T.
Published: December 2019

Variations of intracellular concentrations of isocitrate and NADP
+
were measured throughout all growth phases of the marine bacterium
Pseudomonas nautica
. The intracellular isocitrate concentration tracked the intracellular protein concentration throughout all phases of growth. It rapidly increased in early exponential phase to a maximum and fell to nearly zero in parallel with pyruvate exhaustion in the culture medium. The intracellular NADP
+
and protein concentrations increased in parallel during the exponential phase but were poorly correlated. Even after carbon exhaustion, the intracellular NADP
+
concentration stayed high, as did protein levels. The results demonstrated that the intracellular isocitrate concentration, but not the intracellular NADP
+
concentration, was affected by the carbon availability in the culture. They also suggest that, because of its variability, isocitrate, but not NADP
+
, plays the larger role in the control of the respiratory CO
2
production rate (
R

CO
2

). From initial rate studies, bisubstrate Michaelis constants and the dissociation constant were determined for NADP
+
-specific isocitrate dehydrogenase (IDH) from
P. nautica
. These studies support the hypothesis that the mechanism of IDH’s activity involves the ordered addition of the substrates,
d
-isocitrate and NADP
+
. Furthermore, the results support the use of a bisubstrate enzyme kinetic equation to model
R

CO
2

in
P. nautica.
Novel Euryarchaeotal Lineages Detected on Rice Roots and in the Anoxic Bulk Soil of Flooded Rice Microcosms
Authors: Großkopf Regine , Stubner Stephan , Liesack Werner
Published: December 2019

Because excised, washed roots of rice (Oryza sativa) immediately produce CH4when they are incubated under anoxic conditions (P. Frenzel and U. Bosse, FEMS Microbiol. Ecol. 21:25–36, 1996), we employed a culture-independent molecular approach to identify the methanogenic microbial community present on roots of rice plants. Archaeal small-subunit rRNA-encoding genes were amplified directly from total root DNA by PCR and then cloned. Thirty-two archaeal rice root (ARR) gene clones were randomly selected, and the amplified primary structures of ca. 750 nucleotide sequence positions were compared. Only 10 of the environmental sequences were affiliated with known methanogens; 5 were affiliated withMethanosarcinaspp., and 5 were affiliated withMethanobacteriumspp. The remaining 22 ARR gene clones formed four distinct lineages (rice clusters I through IV) which were not closely related to any known cultured member of theArchaea. Rice clusters I and II formed distinct clades within the phylogenetic radiation of the orders “Methanosarcinales” andMethanomicrobiales. Rice cluster I was novel, and rice cluster II was closely affiliated with environmental sequences obtained from bog peat in northern England. Rice cluster III occurred on the same branch asThermoplasma acidophilumand marine group II but was only distantly related to these taxa. Rice cluster IV was a deep-branching crenarchaeotal assemblage that was closely related to clone pGrfC26, an environmental sequence recovered from a temperate marsh environment. The use of a domain-specific oligonucleotide probe in a fluorescent in situ hybridization analysis revealed that viable members of theArchaeawere present on the surfaces of rice roots. In addition, we describe a novel euryarchaeotal main line of descent, designated rice cluster V, which was detected in anoxic rice paddy soil. These results indicate that there is an astonishing richness of archaeal diversity present on rice roots and in the surrounding paddy soil.