bluuebunny/crossref_metadata_2025_split huggingface dataset:
A PCR Detection Method for Rapid Identification of
Paenibacillus larvae
Authors: Govan V. A. , Allsopp M. H. , Davison S. 
Published: December 2019

ABSTRACT

American foulbrood is a disease of larval honeybees (
Apis mellifera
) caused by the bacterium
Paenibacillus larvae
. Over the years attempts have been made to develop a selective medium for the detection of
P. larvae
spores from honey samples. The most successful of these is a semiselective medium containing nalidixic acid and pipermedic acid. Although this medium allows the growth of
P. larvae
and prevents the growth of most other bacterial species, the false-positive colonies that grow on it prevent the rapid confirmation of the presence of
P. larvae
. Here we describe a PCR detection method which can be used on the colonies that grow on this semiselective medium and thereby allows the rapid confirmation of the presence of
P. larvae
. The PCR primers were designed on the basis of the 16S rRNA gene of
P. larvae
and selectively amplify a 973-bp amplicon. The PCR amplicon was confirmed as originating from
P. larvae
by sequencing in both directions. Detection was specific for
P. larvae
, and the primers did not hybridize with DNA from closely related bacterial species.
Microbial Biomass and Activity in Lead-Contaminated Soil
Authors: Konopka A. , Zakharova T. , Bischoff M. , Oliver L. , Nakatsu C. , Turco R. F. 
Published: December 2019

ABSTRACT

Microbial community diversity, potential microbial activity, and metal resistance were determined in three soils whose lead contents ranged from 0.00039 to 48 mmol of Pb kg of soil
−1
. Biomass levels were directly related to lead content. A molecular analysis of 16S rRNAs suggested that each soil contained a complex, diverse microbial community. A statistical analysis of the phospholipid fatty acids indicated that the community in the soil having the highest lead content was not related to the communities in the other soils. All of the soils contained active microbial populations that mineralized [
14
C]glucose. In all samples, 10 to 15% of the total culturable bacteria were Pb resistant and had MIC of Pb for growth of 100 to 150 μM.
Divergence of West African and North American Communities of
Aspergillus
Section
Flavi
Authors: Cotty Peter J. , Cardwell Kitty F. 
Published: December 2019

ABSTRACT

West African
Aspergillus flavus
S isolates differed from North American isolates. Both produced aflatoxin B
1
. However, 40 and 100% of West African isolates also produced aflatoxin G
1
in NH
4
medium and urea medium, respectively. No North American S strain isolate produced aflatoxin G
1
. This geographical and physiological divergence may influence aflatoxin management.
Transcriptional Control of
ADH
Genes in the Xylose-Fermenting Yeast
Pichia stipitis
Authors: Cho Jae-Yong , Jeffries Thomas W. 
Published: December 2019

ABSTRACT

We studied the expression of the genes encoding group I alcohol dehydrogenases (
PsADH1
and
PsADH2
) in the xylose-fermenting yeast
Pichia stipitis
CBS 6054. The cells expressed
PsADH1
approximately 10 times higher under oxygen-limited conditions than under fully aerobic conditions when cultivated on xylose. Transcripts of
PsADH2
were not detectable under either aeration condition. We used a
PsADH1
::
lacZ
fusion to monitor
PsADH1
expression and found that expression increased as oxygen decreased. The level of
PsADH1
transcript was repressed about 10-fold in cells grown in the presence of heme under oxygen-limited conditions. Concomitantly with the induction of
PsADH1
,
PsCYC1
expression was repressed. These results indicate that oxygen availability regulates
PsADH1
expression and that regulation may be mediated by heme. The regulation of
PsADH2
expression was also examined in other genetic backgrounds. Disruption of
PsADH1
dramatically increased
PsADH2
expression on nonfermentable carbon sources under fully aerobic conditions, indicating that the expression of
PsADH2
is subject to feedback regulation under these conditions.
Earthworm Egg Capsules as Vectors for the Environmental Introduction of Biodegradative Bacteria
Authors: Daane L. L. , Häggblom M. M. 
Published: December 2019

ABSTRACTEarthworm egg capsules (cocoons) may acquire bacteria from the environment in which they are produced. We found thatRalstonia eutropha(pJP4) can be recovered fromEisenia fetidacocoons formed in soil inoculated with this bacterium. Plasmid pJP4 contains the genes necessary for 2,4-dichlorophenoxyacetic acid (2,4-D) and 2,4-dichlorophenol (2,4-DCP) degradation. In this study we determined that the presence ofR. eutropha(pJP4) within the developing earthworm cocoon can influence the degradation and toxicity of 2,4-D and 2,4-DCP, respectively. The addition of cocoons containingR. eutropha(pJP4) at either low or high densities (102or 105CFU per cocoon, respectively) initiated degradation of 2,4-D in nonsterile soil microcosms. Loss of 2,4-D was observed within the first week of incubation, and respiking the soil with 2,4-D showed depletion within 24 h. Microbial analysis of the soil revealed the presence of approximately 104CFUR. eutropha(pJP4) g−1of soil. The toxicity of 2,4-DCP to developing earthworms was tested by using cocoons with or withoutR. eutropha(pJP4). Results showed that cocoons containingR. eutropha(pJP4) were able to tolerate higher levels of 2,4-DCP. Our results indicate that the biodegradation of 2,4-DCP byR. eutropha(pJP4) within the cocoons may be the mechanism contributing to toxicity reduction. These results suggest that the microbiota may influence the survival of developing earthworms exposed to toxic chemicals. In addition, cocoons can be used as inoculants for the introduction into the environment of beneficial bacteria, such as strains with biodegradative capabilities.
Flow Cytometric Analysis of 5-Cyano-2,3-Ditolyl Tetrazolium Chloride Activity of Marine Bacterioplankton in Dilution Cultures
Authors: Sieracki Michael E. , Cucci Terry L. , Nicinski Justyna 
Published: December 2019

ABSTRACT
The respiratory activity of marine bacteria is an important indication of the ecological functioning of these organisms in marine ecosystems. The redox dye 5-cyano-2,3-ditolyl tetrazolium chloride (CTC) is reduced intracellularly in respiring cells to an insoluble, fluorescent precipitate. This product is detectable and quantifiable by flow cytometry in individual cells. We describe here an evaluation of flow cytometry for measuring CTC activity in natural assemblages of marine bacteria growing in dilution cultures. We found that more CTC-positive cells are detected by flow cytometry than by visual epifluorescence microscopy. Samples can be stored refrigerated or frozen in liquid nitrogen for at least 4 weeks without a significant loss of total cells, CTC-positive cells, or CTC fluorescence. Cytometry still may not detect all active cells, however, since the dimmest fluorescing cells are not clearly separated from background noise. Reduction of CTC is very fast in most active cells, and the number of active cells reaches 80% of the maximum number within 2 to 10 min. The proportion of active cells is correlated with the growth rate, while the amount of fluorescence per cell varies inversely with the growth rate. The CTC reduction kinetics in assemblages bubbled with nitrogen and in assemblages bubbled with air to vary the oxygen availability were the same, suggesting that CTC can effectively compete with oxygen for reducing power. A nonbubbled control, however, contained more CTC-positive cells, and the amount of fluorescence per cell was greater. Activity may have been reduced by bubble-induced turbulence. Addition of an artificial reducing agent, sodium dithionite, after CTC incubation and fixation resulted in a greater number of positive cells but did not “activate” a majority of the cells. This indicated that some of the negative cells actually transported CTC across their cell membranes but did not reduce it to a detectable level. Automated analysis by flow cytometry allows workers to study single-cell variability in marine bacterioplankton activity and changes in activity on a small temporal or spatial scale.
Environmental Factors Modulating Antibiotic and Siderophore Biosynthesis by
Pseudomonas fluorescens
Biocontrol Strains
Authors: Duffy Brion K. , Défago Geneviève 
Published: December 2019

ABSTRACT

Understanding the environmental factors that regulate the biosynthesis of antimicrobial compounds by disease-suppressive strains of
Pseudomonas fluorescens
is an essential step toward improving the level and reliability of their biocontrol activity. We used liquid culture assays to identify several minerals and carbon sources which had a differential influence on the production of the antibiotics 2,4-diacetylphloroglucinol (PHL), pyoluteorin (PLT), and pyrrolnitrin and the siderophores salicylic acid and pyochelin by the model strain CHA0, which was isolated from a natural disease-suppressive soil in Switzerland. Production of PHL was stimulated by Zn
2+
, NH
4
Mo
2+
, and glucose; the precursor compound mono-acetylphloroglucinol was stimulated by the same factors as PHL. Production of PLT was stimulated by Zn
2+
, Co
2+
, and glycerol but was repressed by glucose. Pyrrolnitrin production was increased by fructose, mannitol, and a mixture of Zn
2+
and NH
4
Mo
2+
. Pyochelin production was increased by Co
2+
, fructose, mannitol, and glucose. Interestingly, production of its precursor salicylic acid was increased by different factors, i.e., NH
4
Mo
2+
, glycerol, and glucose. The mixture of Zn
2+
and NH
4
Mo
2+
with fructose, mannitol, or glycerol further enhanced the production of PHL and PLT compared with either the minerals or the carbon sources used alone, but it did not improve siderophore production. Extending fermentation time from 2 to 5 days increased the accumulation of PLT, pyrrolnitrin, and pyochelin but not of PHL. When findings with CHA0 were extended to an ecologically and genetically diverse collection of 41
P. fluorescens
biocontrol strains, the effect of certain factors was strain dependent, while others had a general effect. Stimulation of PHL by Zn
2+
and glucose was strain dependent, whereas PLT production by all strains that can produce this compound was stimulated by Zn
2+
and transiently repressed by glucose. Inorganic phosphate reduced PHL production by CHA0 and seven other strains tested but to various degrees. Production of PLT but not pyrrolnitrin by CHA0 was also reduced by 100 mM phosphate. The use of 1/10-strength nutrient broth-yeast extract, compared with standard nutrient broth-yeast extract, amended with glucose and/or glycerol resulted in dramatically increased accumulations of PHL (but not PLT), pyochelin, and salicylic acid, indicating that the ratio of carbon source to nutrient concentration played a key role in the metabolic flow. The results of this study (i) provide insight into the biosynthetic regulation of antimicrobial compounds, (ii) limit the number of factors for intensive study in situ, and (iii) indicate factors that can be manipulated to improve bacterial inoculants.
Enhancement of Expression and Apparent Secretion of
Erwinia chrysanthemi
Endoglucanase (Encoded by
celZ
) in
Escherichia coli
B
Authors: Zhou Shengde , Yomano Lorraine P. , Saleh Alif Z. , Davis Francis C. , Aldrich Henry C. , Ingram Lonnie O. 
Published: December 2019

ABSTRACT

Escherichia coli
B has been engineered as a biocatalyst for the conversion of lignocellulose into ethanol. Previous research has demonstrated that derivatives of
E. coli
B can produce high levels of
Erwinia chrysanthemi
endoglucanase (encoded by
celZ
) as a periplasmic product and that this enzyme can function with commercial fungal cellulase to increase ethanol production. In this study, we have demonstrated two methods that improve
celZ
expression in
E. coli
B. Initially, with a low-copy-number vector, two
E. coli
glycolytic gene promoters (
gap
and
eno
) were tested and found to be less effective than the original
celZ
promoter. By screening 18,000 random fragments of
Zymomonas mobilis
DNA, a surrogate promoter was identified which increased
celZ
expression up to sixfold. With this promoter, large polar inclusion bodies were clearly evident in the periplasmic space. Sequencing revealed that the most active surrogate promoter is derived from five
Sau
3A1 fragments, one of which was previously sequenced in
Z. mobilis
. Visual inspection indicated that this DNA fragment contains at least five putative promoter regions, two of which were confirmed by primer extension analysis. Addition of the
out
genes from
E. chrysanthemi
EC16 caused a further increase in the production of active enzyme and facilitated secretion or release of over half of the activity into the extracellular environment. With the most active construct, of a total of 13,000 IU of active enzyme per liter of culture, 7,800 IU was in the supernatant. The total active endoglucanase was estimated to represent 4 to 6% of cellular protein.
Differential Expression of Three α-Galactosidase Genes and a Single β-Galactosidase Gene from
Aspergillus niger
Authors: de Vries Ronald P. , van den Broeck Hetty C. , Dekkers Ester , Manzanares Paloma , de Graaff Leo H. , Visser Jaap 
Published: December 2019

ABSTRACT

A gene encoding a third α-galactosidase (AglB) from
Aspergillus niger
has been cloned and sequenced. The gene consists of an open reading frame of 1,750 bp containing six introns. The gene encodes a protein of 443 amino acids which contains a eukaryotic signal sequence of 16 amino acids and seven putative N-glycosylation sites. The mature protein has a calculated molecular mass of 48,835 Da and a predicted pI of 4.6. An alignment of the AglB amino acid sequence with those of other α-galactosidases revealed that it belongs to a subfamily of α-galactosidases that also includes
A. niger
AglA.
A. niger
AglC belongs to a different subfamily that consists mainly of prokaryotic α-galactosidases. The expression of
aglA
,
aglB
,
aglC
, and
lacA
, the latter of which encodes an
A. niger
β-galactosidase, has been studied by using a number of monomeric, oligomeric, and polymeric compounds as growth substrates. Expression of
aglA
is only detected on galactose and galactose-containing oligomers and polymers. The
aglB
gene is expressed on all of the carbon sources tested, including glucose. Elevated expression was observed on xylan, which could be assigned to regulation via XlnR, the xylanolytic transcriptional activator. Expression of
aglC
was only observed on glucose, fructose, and combinations of glucose with xylose and galactose. High expression of
lacA
was detected on arabinose, xylose, xylan, and pectin. Similar to
aglB
, the expression on xylose and xylan can be assigned to regulation via XlnR. All four genes have distinct expression patterns which seem to mirror the natural substrates of the encoded proteins.
Monooxygenase-Mediated 1,2-Dichloroethane Degradation by
Pseudomonas
sp. Strain DCA1
Authors: Hage Jacobus C. , Hartmans Sybe 
Published: December 2019

ABSTRACT

A bacterial strain, designated
Pseudomonas
sp. strain DCA1, was isolated from a 1,2-dichloroethane (DCA)-degrading biofilm. Strain DCA1 utilizes DCA as the sole carbon and energy source and does not require additional organic nutrients, such as vitamins, for optimal growth. The affinity of strain DCA1 for DCA is very high, with a

K
m

value below the detection limit of 0.5 μM. Instead of a hydrolytic dehalogenation, as in other DCA utilizers, the first step in DCA degradation in strain DCA1 is an oxidation reaction. Oxygen and NAD(P)H are required for this initial step. Propene was converted to 1,2-epoxypropane by DCA-grown cells and competitively inhibited DCA degradation. We concluded that a monooxygenase is responsible for the first step in DCA degradation in strain DCA1. Oxidation of DCA probably results in the formation of the unstable intermediate 1,2-dichloroethanol, which spontaneously releases chloride, yielding chloroacetaldehyde. The DCA degradation pathway in strain DCA1 proceeds from chloroacetaldehyde via chloroacetic acid and presumably glycolic acid, which is similar to degradation routes observed in other DCA-utilizing bacteria.
Maintenance Energy Demand and Starvation Recovery Dynamics ofNitrosomonas europaeaandNitrobacter winogradskyiCultivated in a Retentostat with Complete Biomass Retention
Authors: Tappe W. , Laverman A. , Bohland M. , Braster M. , Rittershaus S. , Groeneweg J. , van Verseveld H. W. 
Published: December 2019

ABSTRACTNitrosomonas europaeaandNitrobacter winogradskyi(strain “Engel”) were grown in ammonia-limited and nitrite-limited conditions, respectively, in a retentostat with complete biomass retention at 25°C and pH 8. Fitting the retentostat biomass and oxygen consumption data ofN. europaeaandN. winogradskyito the linear equation for substrate utilization resulted in up to eight-times-lower maintenance requirements compared to the maintenance energy demand (m) calculated from chemostat experiments. Independent of the growth rate at different stages of such a retention culture, the maximum specific oxygen consumption rate measured by mass spectrometric analysis of inlet and outlet gas oxygen content always amounted to approximately 45 μmol of O2mg−1of biomass-C · h−1for bothN. europaeaandN. winogradskyi. When bacteria were starved for different time periods (up to 3 months), the spontaneous respiratory activity after an ammonia or nitrite pulse decreased with increasing duration of the previous starvation time period, but the observed decrease was many times faster forN. winogradskyithan forN. europaea. Likewise, the velocity of resuscitation decreased with extended time periods of starvation. The increase in oxygen consumption rates during resuscitation referred to the reviving population only, since in parallel no significant increase in the cell concentrations was detectable.N. europaeamore readily recovers from starvation thanN. winogradskyi, explaining the occasionally observed nitrite accumulation in the environment after ammonia becomes available. From chloramphenicol (100 μg · ml−1) inhibition experiments withN. winogradskyi, it has been concluded that energy-starved cells must have a lower protein turnover rate than nonstarved cells. As pointed out by Stein and Arp (L. Y. Stein and D. J. Arp, Appl. Environ. Microbiol. 64:1514–1521, 1998), nitrifying bacteria in soil have to cope with extremely low nutrient concentrations. Therefore, a chemostat is probably not a suitable tool for studying their physiological properties during a long-lasting nutrient shortage. In comparison with chemostats, retentostats offer a more realistic approach with respect to substrate provision and availability.
Microbial System for Polysaccharide Depolymerization: Enzymatic Route for Xanthan Depolymerization by
Bacillus
sp. Strain GL1
Authors: Nankai Hirokazu , Hashimoto Wataru , Miki Hikaru , Kawai Shigeyuki , Murata Kousaku 
Published: December 2019

ABSTRACT

An enzymatic route for the depolymerization of a heteropolysaccharide (xanthan) in
Bacillus
sp. strain GL1, which was closely related to
Brevibacillus thermoruber
, was determined by analyzing the structures of xanthan depolymerization products. The bacterium produces extracellular xanthan lyase catalyzing the cleavage of the glycosidic bond between pyruvylated mannosyl and glucuronyl residues in xanthan side chains (W. Hashimoto et al., Appl. Environ. Microbiol. 64:3765–3768, 1998). The modified xanthan after the lyase reaction was then depolymerized by extracellular β-
d
-glucanase to a tetrasaccharide, without the terminal mannosyl residue of the side chain in a pentasaccharide, a repeating unit of xanthan. The tetrasaccharide was taken into cells and converted to a trisaccharide (unsaturated glucuronyl-acetylated mannosyl-glucose) by β-
d
-glucosidase. The trisaccharide was then converted to the unsaturated glucuronic acid and a disaccharide (mannosyl-glucose) by unsaturated glucuronyl hydrolase. Finally, the disaccharide was hydrolyzed to mannose and glucose by α-
d
-mannosidase. This is the first complete report on xanthan depolymerization by bacteria. Novel β-
d
-glucanase, one of the five enzymes involved in the depolymerization route, was purified from the culture fluid. This enzyme was a homodimer with a subunit molecular mass of 173 kDa and was most active at pH 6.0 and 45°C. The enzyme specifically acted on xanthan after treatment with xanthan lyase and released the tetrasaccharide.
Cellobiose Transport by Mixed Ruminal Bacteria from a Cow
Authors: Kajikawa Hiroshi , Masaki Shigehiko 
Published: December 2019

ABSTRACT

The transport of cellobiose in mixed ruminal bacteria harvested from a holstein cow fed an Italian ryegrass hay was determined in the presence of nojirimycin-1-sulfate, which almost inhibited cellobiase activity. The kinetic parameters of cellobiose uptake were 14 μM for the

K
m

and 10 nmol/min/mg of protein for the
V
max
. Extracellular and cell-associated cellobiases were detected in the rumen, with both showing higher
V
max
values and lower affinities than those determined for cellobiose transport. The proportion of cellobiose that was directly transported before it was extracellularly degraded into glucose increased as the cellobiose concentration decreased, reaching more than 20% at the actually observed levels of cellobiose in the rumen, which were less than 0.02 mM. The inhibitor experiment showed that cellobiose was incorporated into the cells mainly by the phosphoenolpyruvate phosphotransferase system and partially by an ATP-dependent and proton-motive-force-independent active transport system. This finding was also supported by determinations of phosphoenolpyruvate phosphotransferase-dependent NADH oxidation with cellobiose and the effects of artificial potentials on cellobiose transport. Cellobiose uptake was sensitive to a decrease in pH (especially below 6.0), and it was weakly but significantly inhibited in the presence of glucose.
Temperature Dependence of Inorganic Nitrogen Uptake: Reduced Affinity for Nitrate at Suboptimal Temperatures in Both Algae and Bacteria
Authors: Reay David S. , Nedwell David B. , Priddle Julian , Ellis-Evans J. Cynan 
Published: December 2019

ABSTRACTNitrate utilization and ammonium utilization were studied by using three algal isolates, six bacterial isolates, and a range of temperatures in chemostat and batch cultures. We quantified affinities for both substrates by determining specific affinities (specific affinity = maximum growth rate/half-saturation constant) based on estimates of kinetic parameters obtained from chemostat experiments. At suboptimal temperatures, the residual concentrations of nitrate in batch cultures and the steady-state concentrations of nitrate in chemostat cultures both increased. The specific affinity for nitrate was strongly dependent on temperature (Q10 ≈ 3, where Q10is the proportional change with a 10°C temperature increase) and consistently decreased at temperatures below the optimum temperature. In contrast, the steady-state concentrations of ammonium remained relatively constant over the same temperature range, and the specific affinity for ammonium exhibited no clear temperature dependence. This is the first time that a consistent effect of low temperature on affinity for nitrate has been identified for psychrophilic, mesophilic, and thermophilic bacteria and algae. The different responses of nitrate uptake and ammonium uptake to temperature imply that there is increasing dependence on ammonium as an inorganic nitrogen source at low temperatures.