bluuebunny/crossref_metadata_2025_split huggingface dataset:
Transport and Utilization of Hexoses and Pentoses in the Halotolerant Yeast
Debaryomyces hansenii
Authors: Nobre Alexandra , Lucas Cândida , Leão Cecília 
Published: December 2019

ABSTRACT

Debaryomyces hansenii
is a yeast species that is known for its halotolerance. This organism has seldom been mentioned as a pentose consumer. In the present work, a strain of this species was investigated with respect to the utilization of pentoses and hexoses in mixtures and as single carbon sources. Growth parameters were calculated for batch aerobic cultures containing pentoses, hexoses, and mixtures of both types of sugars. Growth on pentoses was slower than growth on hexoses, but the values obtained for biomass yields were very similar with the two types of sugars. Furthermore, when mixtures of two sugars were used, a preference for one carbon source did not inhibit consumption of the other. Glucose and xylose were transported by cells grown on glucose via a specific low-affinity facilitated diffusion system. Cells derepressed by growth on xylose had two distinct high-affinity transport systems for glucose and xylose. The sensitivity of labeled glucose and xylose transport to dissipation of the transmembrane proton gradient by the protonophore carbonyl cyanide
m
-chlorophenylhydrazone allowed us to consider these transport systems as proton symports, although the cells displayed sugar-associated proton uptake exclusively in the presence of NaCl or KCl. When the
V
max
values of transport systems for glucose and xylose were compared with glucose- and xylose-specific consumption rates during growth on either sugar, it appeared that transport did not limit the growth rate.
Colonization Pattern of the Biocontrol Strain
Pseudomonas chlororaphis
MA 342 on Barley Seeds Visualized by Using Green Fluorescent Protein
Authors: Tombolini Riccardo , van der Gaag Dirk Jan , Gerhardson Berndt , Jansson Janet K. 
Published: December 2019

ABSTRACT

Pseudomonas chlororaphis
MA 342 is a potent biocontrol agent that can be used against several seed-borne diseases of cereal crops, including net blotch of barley caused by the fungus
Drechslera teres
. In this study, strain MA 342 was tagged with the
gfp
gene (encoding the green fluorescent protein) in order to study the fate of cells after seed inoculation. The
gfp
-tagged strain, MA 342G2, had the same biocontrol efficacy as the wild type when it was applied at high cell concentrations to seeds but was less effective at lower cell concentrations. By comparing cell counts determined by microscopy to the number of CFU, we found that the number of culturable cells was significantly lower than the total number of bacteria on seeds which were inoculated and dried for 20 h. Confocal microscopy and epifluorescence stereomicroscopy were used to determine the pattern of MA 342G2 colonization and cell aggregation on barley seeds. Immediately after inoculation of seeds, bacteria were found mainly under the seed glume, and there was no particular aggregation pattern. However, after the seeds were sown, irregularly distributed areas of bacterial aggregation were found, which reflected epiphytic colonization of glume cells. There was a trend towards bacterial aggregation near the embryo but never within the embryo. Bacterial aggregates were regularly found in the groove of each seed formed by the base of the coleoptile and the scutellum. Based on these results, we suggest that MA 342 colocalizes with the pathogen
D. teres
, which facilitates the action of the fungistatic compound(s) produced by this strain.
The Branched-Chain Dodecylbenzene Sulfonate Degradation Pathway of
Pseudomonas aeruginosa
W51D Involves a Novel Route for Degradation of the Surfactant Lateral Alkyl Chain
Authors: Campos-García Jesús , Esteve Abraham , Vázquez-Duhalt Rafael , Ramos Juán Luis , Soberón-Chávez Gloria 
Published: December 2019

ABSTRACT

Pseudomonas aeruginosa
W51D is able to grow by using branched-chain dodecylbenzene sulfonates (B-DBS) or the terpenic alcohol citronellol as a sole source of carbon. A mutant derived from this strain (W51M1) is unable to degrade citronellol but still grows on B-DBS, showing that the citronellol degradation route is not the main pathway involved in the degradation of the surfactant alkyl moiety. The structures of the main B-DBS isomers and of some intermediates were identified by gas chromatography-mass spectrometric analysis, and a possible catabolic route is proposed.
Improved Method for Purification of Bacterial DNA from Bovine Milk for Detection of
Brucella
spp. by PCR
Authors: Romero C. , Lopez-Goñi I. 
Published: December 2019

ABSTRACT

Different methods of extraction of bacterial DNA from bovine milk to improve the direct detection of
Brucella
by PCR were evaluated. We found that the use of a lysis buffer with high concentrations of Tris, EDTA, and NaCl, high concentrations of sodium dodecyl sulfate and proteinase K, and high temperatures of incubation was necessary for the efficient extraction of
Brucella
DNA. The limit of detection by PCR was 5 to 50
Brucella
CFU/ml of milk.
Comparison and Recovery of
Escherichia coli
and Thermotolerant Coliforms in Water with a Chromogenic Medium Incubated at 41 and 44.5°C
Authors: Alonso Jose L. , Soriano Adela , Carbajo Oscar , Amoros Inmaculada , Garelick Hemda 
Published: December 2019

ABSTRACT

This study compared the performance of a commercial chromogenic medium, CHROMagarECC (CECC), and CECC supplemented with sodium pyruvate (CECCP) with the membrane filtration lauryl sulfate-based medium (mLSA) for enumeration of
Escherichia coli
and non-
E. coli
thermotolerant coliforms (KEC). To establish that we could recover the maximum KEC and
E. coli
population, we compared two incubation temperature regimens, 41 and 44.5°C. Statistical analysis by the Fisher test of data did not demonstrate any statistically significant differences (
P
= 0.05) in the enumeration of
E. coli
for the different media (CECC and CECCP) and incubation temperatures. Variance analysis of data performed on KEC counts showed significant differences (
P
= 0.01) between KEC counts at 41 and 44.5°C on both CECC and CECCP. Analysis of variance demonstrated statistically significant differences (
P
= 0.05) in the enumeration of total thermotolerant coliforms (TTCs) on CECC and CECCP compared with mLSA. Target colonies were confirmed to be
E. coli
at a rate of 91.5% and KEC of likely fecal origin at a rate of 77.4% when using CECCP incubated at 41°C. The results of this study showed that CECCP agar incubated at 41°C is efficient for the simultaneous enumeration of
E. coli
and KEC from river and marine waters.
Production of a New
d
-Amino Acid Oxidase from the Fungus
Fusarium oxysporum
Authors: Gabler Matthias , Fischer Lutz 
Published: December 2019

ABSTRACT

The fungus
Fusarium oxysporum
produced a
d
-amino acid oxidase (EC
1.4.3.3
) in a medium containing glucose as the carbon and energy source and ammonium sulfate as the nitrogen source. The specific
d
-amino acid oxidase activity was increased up to 12.5-fold with various
d
-amino acids or their corresponding derivatives as inducers. The best inducers were
d
-alanine (2.7 μkat/g of dry biomass) and
d
-3-aminobutyric acid (2.6 μkat/g of dry biomass). The addition of zinc ions was necessary to permit the induction of peroxisomal
d
-amino acid oxidase. Bioreactor cultivations were performed on a 50-liter scale, yielding a volumetric
d
-amino acid oxidase activity of 17 μkat liter
−1
with
d
-alanine as an inducer. Under oxygen limitation, the volumetric activity was increased threefold to 54 μkat liter
−1
(3,240 U liter
−1
).
Development of an In Vitro Bioassay for
Clostridium botulinum
Type B Neurotoxin in Foods That Is More Sensitive than the Mouse Bioassay
Authors: Wictome Matthew , Newton Kirsti , Jameson Karen , Hallis Bassam , Dunnigan Paul , Mackay Eric , Clarke Sally , Taylor Richard , Gaze Joy , Foster Keith , Shone Clifford 
Published: December 2019

ABSTRACT

A novel, in vitro bioassay for detection of the botulinum type B neurotoxin in a range of media was developed. The assay is amplified by the enzymic activity of the neurotoxin’s light chain and includes the following three stages: first, a small, monoclonal antibody-based immunoaffinity column captures the toxin; second, a peptide substrate is cleaved by using the endopeptidase activity of the type B neurotoxin; and finally, a modified enzyme-linked immunoassay system detects the peptide cleavage products. The assay is highly specific for type B neurotoxin and is capable of detecting type B toxin at a concentration of 5 pg ml
−1
(0.5 mouse 50% lethal dose ml
−1
) in approximately 5 h. The format of the test was found to be suitable for detecting botulinum type B toxin in a range of foodstuffs with a sensitivity that exceeds the sensitivity of the mouse assay. Using highly specific monoclonal antibodies as the capture phase, we found that the endopeptidase assay was capable of differentiating between the type B neurotoxins produced by proteolytic and nonproteolytic strains of
Clostridium botulinum
type B.
Distribution and Diversity of Sulfur-Oxidizing
Thiomicrospira
spp. at a Shallow-Water Hydrothermal Vent in the Aegean Sea (Milos, Greece)
Authors: Brinkhoff Thorsten , Sievert Stefan M. , Kuever Jan , Muyzer Gerard 
Published: December 2019

ABSTRACT

A shallow-water hydrothermal vent system in the Aegean Sea close to the island of Milos (Greece) was chosen to study the diversity and distribution of the chemolithoautotrophic sulfur-oxidizing bacterium
Thiomicrospira
. Cell numbers in samples from different regions around a solitary vent decreased toward the center of the vent (horizontal distribution), as well as with depth (vertical distribution), corresponding to an increase in temperature (from ca. 25 to 60°C) and a decrease in pH (from ca. pH 7 to 5).
Thiomicrospira
was one of the most abundant culturable sulfur oxidizers and was even dominant in one region. Phylogenetic analysis of
Thiomicrospira
spp. present in the highest most-probable-number (MPN) dilutions revealed that most of the obtained sequences grouped in two new closely related clusters within the
Thiomicrospira
branch. Two different new isolates, i.e., Milos-T1 and Milos-T2, were obtained from high-dilution (10
−5
) enrichments. Phylogenetic analysis indicated that isolate Milos-T1 is related to the recently described
Thiomicrospira kuenenii
and
Hydrogenovibrio marinus
, whereas isolate Milos-T2 grouped with the MPN sequences of cluster 2. The predominance of strain Milos-T2 was indicated by its identification in several environmental samples by hybridization analysis of denaturing gradient gel electrophoresis (DGGE) patterns and by sequencing of one of the corresponding bands, i.e., ML-1, from the DGGE gel. The results shown in this paper support earlier indications that
Thiomicrospira
species are important members of hydrothermal vent communities.
Enzymatic Formation of G-Group Aflatoxins and Biosynthetic Relationship between G- and B-Group Aflatoxins
Authors: Yabe Kimiko , Nakamura Miki , Hamasaki Takashi 
Published: December 2019

ABSTRACT

We detected biosynthetic activity for aflatoxins G
1
and G
2
in cell extracts of
Aspergillus parasiticus
NIAH-26. We found that in the presence of NADPH, aflatoxins G
1
and G
2
were produced from
O
-methylsterigmatocystin and dihydro-
O
-methylsterigmatocystin, respectively. No G-group aflatoxins were produced from aflatoxin B
1
, aflatoxin B
2
, 5-methoxysterigmatocystin, dimethoxysterigmatocystin, or sterigmatin, confirming that B-group aflatoxins are not the precursors of G-group aflatoxins and that G- and B-group aflatoxins are independently produced from the same substrates (
O
-methylsterigmatocystin and dihydro-
O
-methylsterigmatocystin). In competition experiments in which the cell-free system was used, formation of aflatoxin G
2
from dihydro-
O
-methylsterigmatocystin was suppressed when
O
-methylsterigmatocystin was added to the reaction mixture, whereas aflatoxin G
1
was newly formed. This result indicates that the same enzymes can catalyze the formation of aflatoxins G
1
and G
2
. Inhibition of G-group aflatoxin formation by methyrapone, SKF-525A, or imidazole indicated that a cytochrome P-450 monooxygenase may be involved in the formation of G-group aflatoxins. Both the microsome fraction and a cytosol protein with a native mass of 220 kDa were necessary for the formation of G-group aflatoxins. Due to instability of the microsome fraction, G-group aflatoxin formation was less stable than B-group aflatoxin formation. The
ordA
gene product, which may catalyze the formation of B-group aflatoxins, also may be required for G-group aflatoxin biosynthesis. We concluded that at least three reactions, catalyzed by the
ordA
gene product, an unstable microsome enzyme, and a 220-kDa cytosol protein, are involved in the enzymatic formation of G-group aflatoxins from either
O
-methylsterigmatocystin or dihydro-
O
-methylsterigmatocystin.
Occurrence of
Shewanella algae
in Danish Coastal Water and Effects of Water Temperature and Culture Conditions on Its Survival
Authors: Gram Lone , Bundvad Anemone , Melchiorsen Jette , Johansen Charlotte , Fonnesbech Vogel Birte 
Published: December 2019

ABSTRACT

The marine bacterium
Shewanella algae
, which was identified as the cause of human cases of bacteremia and ear infections in Denmark in the summers of 1994 and 1995, was detected in seawater only during the months (July, August, September, and October) when the water temperature was above 13°C. The bacterium is a typical mesophilic organism, and model experiments were conducted to elucidate the fate of the organism under cold and nutrient-limited conditions. The culturable count of
S. algae
decreased rapidly from 10
7
CFU/ml to 10
1
CFU/ml in approximately 1 month when cells grown at 20 to 37°C were exposed to cold (2°C) seawater. In contrast, the culturable count of cells exposed to warmer seawater (10 to 25°C) remained constant. Allowing the bacterium a transition period in seawater at 20°C before exposure to the 2°C seawater resulted in 100% survival over a period of 1 to 2 months. The cold protection offered by this transition (starvation) probably explains the ability of the organism to persist in Danish seawater despite very low (0 to 1°C) winter water temperatures. The culturable counts of samples kept at 2°C increased to 10
5
to 10
7
CFU/ml at room temperature. Most probable number analysis showed this result to be due to regrowth rather than resuscitation. It was hypothesized that
S. algae
would survive cold exposure better if in the biofilm state; however, culturable counts from
S. algae
biofilms decreased as rapidly as did counts of planktonic cells.
A Most-Probable-Number Assay for Enumeration of Infectious
Cryptosporidium parvum
Oocysts
Authors: Slifko Theresa R. , Huffman Debra E. , Rose Joan B. 
Published: December 2019

ABSTRACT

Cryptosporidium
is globally established as a contaminant of drinking and recreational waters. A previously described cell culture infectivity assay capable of detecting infectious oocysts was adapted to quantify viable oocysts through sporozoite invasion and clustering of foci. Eight experiments were performed by using oocysts less than 4 months of age to inoculate host HCT-8 cell monolayers. Oocysts were diluted in a standard 5- or 10-fold multiple dilution format, levels of infection and clustering were determined, and the most probable number (MPN) of infectious oocysts in the stock suspension was calculated. The MPN was compared to the initial oocyst inoculum to determine the level of correlation. For oocysts less than 30 days of age, the correlation coefficient (
r
) was 0.9726 (0.9306 to 0.9893;
n
= 20). A two-tailed
P
value (alpha = 0.05) indicated that
P
was less than 0.0001. This strong correlation suggests that the MPN can be used to effectively enumerate infectious oocysts in a cell culture system. Age affected the degree of oocyst infectivity. Oocyst infectivity was tested by the focus detection method (FDM)-MPN assay and in BALB/c mice before and after treatment with pulsed white light (PureBrite). The FDM-MPN assay and animal infectivity assays both demonstrated more than a 4 log
10
inactivation. Municipal water systems and a host of other water testing organizations could utilize the FDM-MPN assay for routine survival and disinfection studies.
Long-Chain Polyphosphate Causes Cell Lysis and Inhibits
Bacillus cereus
Septum Formation, Which Is Dependent on Divalent Cations
Authors: Maier Simon K. , Scherer Siegfried , Loessner Martin J. 
Published: December 2019

ABSTRACT

We investigated the cellular mechanisms that led to growth inhibition, morphological changes, and lysis of
Bacillus cereus
WSBC 10030 when it was challenged with a long-chain polyphosphate (polyP). At a concentration of 0.1% or higher, polyP had a bacteriocidal effect on log-phase cells, in which it induced rapid lysis and reductions in viable cell counts of up to 3 log units. The cellular debris consisted of empty cell wall cylinders and polar caps, suggesting that polyP-induced lysis was spatially specific. This activity was strictly dependent on active growth and cell division, since polyP failed to induce lysis in cells treated with chloramphenicol and in stationary-phase cells, which were, however, bacteriostatically inhibited by polyP. Similar observations were made with
B. cereus
spores; 0.1% polyP inhibited spore germination and outgrowth, and a higher concentration (1.0%) was even sporocidal. Supplemental divalent metal ions (Mg
2+
and Ca
2+
) could almost completely block and reverse the antimicrobial activity of polyP; i.e., they could immediately stop lysis and reinitiate rapid cell division and multiplication. Interestingly, a sublethal polyP concentration (0.05%) led to the formation of elongated cells (average length, 70 μm) after 4 h of incubation. While DNA replication and chromosome segregation were undisturbed, electron microscopy revealed a complete lack of septum formation within the filaments. Exposure to divalent cations resulted in instantaneous formation and growth of ring-shaped edges of invaginating septal walls. After approximately 30 min, septation was complete, and cell division resumed. We frequently observed a minicell-like phenotype and other septation defects, which were probably due to hyperdivision activity after cation supplementation. We propose that polyP may have an effect on the ubiquitous bacterial cell division protein FtsZ, whose GTPase activity is known to be strictly dependent on divalent metal ions. It is tempting to speculate that polyP, because of its metal ion-chelating nature, indirectly blocks the dynamic formation (polymerization) of the Z ring, which would explain the aseptate phenotype.
Purification and Characterization of an Extracellular Protease from the Fish Pathogen
Yersinia ruckeri
and Effect of Culture Conditions on Production
Authors: Secades P. , Guijarro J. A. 
Published: December 2019

ABSTRACT

A novel protease, hydrolyzing azocasein, was identified, purified, and characterized from the culture supernatant of the fish pathogen
Yersinia ruckeri
. Exoprotease production was detected at the end of the exponential growth phase and was temperature dependent. Activity was detected in peptone but not in Casamino Acid medium. Its synthesis appeared to be under catabolite repression and ammonium control. The protease was purified in a simple two-step procedure involving ammonium sulfate precipitation and ion-exchange chromatography. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) analysis of the purified protein indicated an estimated molecular mass of 47 kDa. The protease had characteristics of a cold-adapted protein, i.e., it was more active in the range of 25 to 42°C and had an optimum activity at 37°C. The activation energy for the hydrolysis of azocasein was determined to be 15.53 kcal/mol, and the enzyme showed a rapid decrease in activity at 42°C. The enzyme had an optimum pH of around 8. Characterization of the protease showed that it required certain cations such as Mg
2+
or Ca
2+
for maximal activity and was inhibited by EDTA, 1,10-phenanthroline, and EGTA but not by phenylmethylsulfonyl fluoride. Two
N
-methyl-
N
-nitro-
N
-nitrosoguanidine mutants were isolated and analyzed; one did not show caseinolytic activity and lacked the 47-kDa protein, while the other was hyperproteolytic and produced increased amounts of the 47-kDa protein. Azocasein activity, SDS-PAGE, immunoblotting by using polyclonal anti-47-kDa-protease serum, and zymogram analyses showed that protease activity was present in 8 of 14 strains tested and that two
Y. ruckeri
groups could be established based on the presence or absence of the 47-kDa protease.
Molecular Standardization of Mating Type Terminology in the
Gibberella fujikuroi
Species Complex
Authors: Kerényi Z. , Zeller K. , Hornok L. , Leslie J. F. 
Published: December 2019

ABSTRACT

Mating type in the
Gibberella fujikuroi
species complex is controlled by a single locus with two alleles and is usually identified following sexual crosses with standard, female-fertile tester isolates. The mating type alleles have been arbitrarily designated “+” and “−” within each biological species, and the nomenclature is tied to the standard tester strains. We developed a pair of PCR primers that can be used to amplify a unique fragment of one of the mating type alleles (
MAT-2
) from at least seven of the biological species in this species complex. Based on the amplification pattern, we propose a replacement for the existing, arbitrary +/− terminology that is presently in use. The new terminology is based on DNA sequence similarities between the mating type allele fragments from the biological species of the
G. fujikuroi
species complex and the corresponding fragments from other filamentous ascomycetes.
Use of Molecular and Isotopic Techniques To Monitor the Response of Autotrophic Ammonia-Oxidizing Populations of the β Subdivision of the Class
Proteobacteria
in Arable Soils to Nitrogen Fertilizer
Authors: Mendum T. A. , Sockett R. E. , Hirsch P. R. 
Published: December 2019

ABSTRACT

This study examined the effects of NH
4
NO
3
fertilizer on the size and activity of nitrifying, autotrophic, ammonia-oxidizing populations of the β subdivision of the class
Proteobacteria
in arable soils. Plots under different long-term fertilizer regimes were sampled before and after NH
4
NO
3
additions, and the rates of nitrification were determined by
15
N isotopic pool dilution assays. Ammonia-oxidizing populations in the plots were quantified by competitive PCR assays based on the
amoA
and ribosomal 16S genes. Prior to fertilizer addition, ammonium concentrations and nitrification rates in the plots were comparatively low; ammonia-oxidizing populations were present at 10
4
to 10
5
gene copies g of soil
−1
. Three days after the application of fertilizer, nitrification rates had risen considerably but the size of the ammonia-oxidizing population was unchanged. Six weeks after fertilizer treatment, ammonium concentrations and nitrification rates had fallen while the ammonia-oxidizing populations in plots receiving fertilizer had increased. The rapidity of the rise in nitrification rates observed after 3 days suggests that it results from phenotypic changes in the ammonia-oxidizing bacterial population. Associated increases in population sizes were only observed after 6 weeks and did not correlate directly with nitrifying activity. Phylogenetic analyses of PCR products from one of the plots revealed a population dominated by
Nitrosospira
-type organisms, similar to those prevalent in other soils.