bluuebunny/crossref_metadata_2025_split huggingface dataset:
Fluorescence In Situ Hybridization Using 16S rRNA-Targeted Oligonucleotides Reveals Localization of Methanogens and Selected Uncultured Bacteria in Mesophilic and Thermophilic Sludge Granules
Authors: Sekiguchi Yuji , Kamagata Yoichi , Nakamura Kazunori , Ohashi Akiyoshi , Harada Hideki 
Published: December 2019

ABSTRACT

16S rRNA-targeted in situ hybridization combined with confocal laser scanning microscopy was used to elucidate the spatial distribution of microbes within two types of methanogenic granular sludge, mesophilic (35°C) and thermophilic (55°C), in upflow anaerobic sludge blanket reactors fed with sucrose-, acetate-, and propionate-based artificial wastewater. The spatial organization of the microbes was visualized in thin sections of the granules by using fluorescent oligonucleotide probes specific to several phylogenetic groups of microbes. In situ hybridization with archaeal- and bacterial-domain probes within granule sections clearly showed that both mesophilic and thermophilic granules had layered structures and that the outer layer harbored mainly bacterial cells while the inner layer consisted mainly of archaeal cells.
Methanosaeta
-,
Methanobacterium
-,
Methanospirillum
-, and
Methanosarcina
-like cells were detected with oligonucleotide probes specific for the different groups of methanogens, and they were found to be localized inside the granules, in both types of which dominant methanogens were members of the genus
Methanosaeta
. For specific detection of bacteria which were previously detected by whole-microbial-community 16S ribosomal DNA (rDNA)-cloning analysis (Y. Sekiguchi, Y. Kamagata, K. Syutsubo, A. Ohashi, H. Harada, and K. Nakamura, Microbiology 144:2655–2665, 1998) we designed probes specific for clonal 16S rDNAs related to unidentified green nonsulfur bacteria and clones related to
Syntrophobacter
species. The probe designed for the cluster closely related to
Syntrophobacter
species hybridized with coccoid cells in the inner layer of the mesophilic granule sections. The probe for the unidentified bacteria which were clustered with the green nonsulfur bacteria detected filamentous cells in the outermost layer of the thermophilic sludge granule sections. These results revealed the spatial organizations of methanogens and uncultivated bacteria and their in situ morphologies and metabolic functions in both mesophilic and thermophilic granular sludges.
Inhibition of Plant-Pathogenic Fungi by a Corn Trypsin Inhibitor Overexpressed in
Escherichia coli
Authors: Chen Zhi-Yuan , Brown Robert L. , Lax Alan R. , Cleveland Thomas E. , Russin John S. 
Published: December 2019

ABSTRACT

The cDNA of a 14-kDa trypsin inhibitor (TI) from corn was subcloned into an
Escherichia coli
overexpression vector. The overexpressed TI was purified based on its insolubility in urea and then refolded into the active form in vitro. This recombinant TI inhibited both conidium germination and hyphal growth of all nine plant pathogenic fungi studied, including
Aspergillus flavus
,
Aspergillus parasiticus
, and
Fusarium moniliforme
. The calculated 50% inhibitory concentration of TI for conidium germination ranged from 70 to more than 300 μg/ml, and that for fungal growth ranged from 33 to 124 μg/ml depending on the fungal species. It also inhibited
A. flavus
and
F. moniliforme
simultaneously when they were tested together. The results suggest that the corn 14-kDa TI may function in host resistance against a variety of fungal pathogens of crops.
Natural Occurrence of the C Series of Fumonisins in Moldy Corn
Authors: Seo Jeong-Ah , Lee Yin-Won 
Published: December 2019

ABSTRACT

We analyzed 44 moldy corn samples for the B and C series of fumonisins by high-performance liquid chromatography. Of the 44 samples, 32 (73%) were contaminated with both the B and C series of fumonisins and 6 were contaminated with only the B series of fumonisins. The incidence of fumonisin C
1
in moldy corn was 71%; the incidence was 11% for fumonisin C
3
and 43% for fumonisin C
4
. Their mean levels ranged from 500 to 1,900 ng/g. This is the first report on the natural occurrence of the C series of fumonisins and fumonisin B
4
in moldy corn.
The Specific Genes for Lantibiotic Mutacin II Biosynthesis in
Streptococcus mutans
T8 Are Clustered and Can Be Transferred En Bloc
Authors: Chen Ping , Qi FengXia , Novak Jan , Caufield Page W. 
Published: December 2019

ABSTRACT

Mutacin II is a ribosomally synthesized peptide lantibiotic produced by group II
Streptococcus mutans
. DNA sequencing has revealed that the mutacin II biosynthetic gene cluster consists of seven specific open reading frames: a regulator (
mutR
), the prepromutacin structural gene (
mutA
), a modifying protein (
mutM
), an ABC transporter (
mutT
), and an immunity cluster (
mutFEG
). Transformations of a non-mutacin-producing strain,
S. mutans
UA159, and a mutacin I-producing strain,
S. mutans
UA140, with chromosomal DNA from
S. mutans
T8 with an
aphIII
marker inserted upstream of the mutacin II structural gene yielded transformants producing mutacin II and mutacins I and II, respectively.
Temperature and pH Conditions That Prevail during Fermentation of Sausages Are Optimal for Production of the Antilisterial Bacteriocin Sakacin K
Authors: Leroy Frédéric , de Vuyst Luc 
Published: December 2019

ABSTRACT

Sakacin K is an antilisterial bacteriocin produced by
Lactobacillus sake
CTC 494, a strain isolated from Spanish dry fermented sausages. The biokinetics of cell growth and bacteriocin production of
L. sake
CTC 494 in vitro during laboratory fermentations were investigated by making use of MRS broth. The data obtained from the fermentations was used to set up a predictive model to describe the influence of the physical factors temperature and pH on microbial behavior. The model was validated successfully for all components. However, the specific bacteriocin production rate seemed to have an upper limit. Both cell growth and bacteriocin activity were very much influenced by changes in temperature and pH. The production of biomass was closely related to bacteriocin activity, indicating primary metabolite kinetics, but was not the only factor of importance. Acidity dramatically influenced both the production and the inactivation of sakacin K; the optimal pH for cell growth did not correspond to the pH for maximal sakacin K activity. Furthermore, cells grew well at 35°C but no bacteriocin production could be detected at this temperature.
L. sake
CTC 494 shows special promise for implementation as a novel bacteriocin-producing sausage starter culture with antilisterial properties, considering the fact that the temperature and acidity conditions that prevail during the fermentation process of dry fermented sausages are optimal for the production of sakacin K.
Formation of Hydride-Meisenheimer Complexes of Picric Acid (2,4,6-Trinitrophenol) and 2,4-Dinitrophenol during Mineralization of Picric Acid by
Nocardioides
sp. Strain CB 22-2
Authors: Behrend Christian , Heesche-Wagner Kerstin 
Published: December 2019

ABSTRACT

There are only a few examples of microbial conversion of picric acid (2,4,6-trinitrophenol). None of the organisms that have been described previously is able to use this compound as a sole source of carbon, nitrogen, and energy at high rates. In this study we isolated and characterized a strain, strain CB 22-2, that was able to use picric acid as a sole source of carbon and energy at concentrations up to 40 mM and at rates of 1.6 mmol · h
−1
· g (dry weight) of cells
−1
in continuous cultures and 920 μmol · h
−1
· g (dry weight) of cells
−1
in flasks. In addition, this strain was able to use picric acid as a sole source of nitrogen at comparable rates in a nitrogen-free medium. Biochemical characterization and 16S ribosomal DNA analysis revealed that strain CB 22-2 is a
Nocardioides
sp. strain. High-pressure liquid chromatography and UV-visible light data, the low residual chemical oxygen demand, and the stoichiometric release of 2.9 ± 0.1 mol of nitrite per mol of picric acid provided strong evidence that complete mineralization of picric acid occurred. During transformation, the metabolites detected in the culture supernatant were the [H
−
]-Meisenheimer complexes of picric acid and 2,4-dinitrophenol (H
−
-DNP), as well as 2,4-dinitrophenol. Experiments performed with crude extracts revealed that H
−
-DNP formation indeed is a physiologically relevant step in picric acid metabolism.
Development and Characterization of a Fluorescent-Bacteriophage Assay for Detection ofEscherichia coliO157:H7
Authors: Goodridge Lawrence , Chen Jinru , Griffiths Mansel 
Published: December 2019

ABSTRACTIn this paper we describe evaluation and characterization of a novel assay that combines immunomagnetic separation and a fluorescently stained bacteriophage for detection ofEscherichia coliO157:H7 in broth. When it was combined with flow cytometry, the fluorescent-bacteriophage assay (FBA) was capable of detecting 104cells/ml. A modified direct epifluorescent-filter technique (DEFT) was employed in an attempt to estimate bacterial concentrations. Using regression analysis, we calculated that the lower detection limit was between 102and 103cells/ml; however, the modified DEFT was found to be an unreliable method for determining bacterial concentrations. The results of this study show that the FBA, when combined with flow cytometry, is a sensitive technique for presumptive detection ofE. coliO157:H7 in broth cultures.
Biphasic Extracellular Proteolytic Enzyme Activity in Benthic Water and Sediment in the Northwestern Mediterranean Sea
Authors: Tholosan Olivier , Lamy François , Garcin Jean , Polychronaki Thalia , Bianchi Armand 
Published: December 2019

ABSTRACT

In this study, we used the fact that bacteria are able to cleave a fluorogenic substrate analog (
l
-leucine-7-amido-4-methylcoumarin) to determine the maximal ectoproteolytic activities (

V
m

) and affinities (

K
m

) of natural benthic microbial communities by the multiconcentration kinetic method. This investigation was performed during the winter and summer of 1997 with a set of 36 samples of near-bottom water and sediment collected from a coastal area and an offshore area in the western part of the Gulf of Lions. The existence of biphasic microbial ectoproteolysis was statistically confirmed for both the near-bottom water and the sediment, regardless of the spatial and seasonal conditions. Globally, 72.2% of the entire set of bacterial consortia collected at the water-sediment boundary layer showed biphasic microbial kinetics. A specific estimator of the biphasicity indicated that deep benthic bacterial consortia responded better with episodic nutrient supplies than shallower benthic bacterial consortia responded.
Levels of Bacterial Community Diversity in Four Arid Soils Compared by Cultivation and 16S rRNA Gene Cloning
Authors: Dunbar John , Takala Shannon , Barns Susan M. , Davis Jody A. , Kuske Cheryl R. 
Published: December 2019

ABSTRACT

Techniques based on amplification of 16S rRNA genes for comparing bacterial communities are now widely used in microbial ecology, but calibration of these techniques with traditional tools, such as cultivation, has been conspicuously absent. In this study, we compared levels of bacterial community diversity in two pinyon rhizosphere soil samples and two between-tree (interspace) soil samples by analyzing 179 cultivated bacterial isolates and 801 16S rRNA genes amplified from extracted soil DNA. Phylotypes were defined by performing a restriction fragment length polymorphism analysis of 16S rRNA gene sequences with the enzymes
Rsa
I and
Bst
UI. The average level of 16S rRNA gene sequence similarity of members of a phylotype was 86.6% based on an analysis of partial sequences. A total of 498 phylotypes were identified among the 16S ribosomal DNA (rDNA) clones, while 34 phylotypes occurred among the cultivated isolates. Analysis of sequences from a subset of the phylotypes showed that at least seven bacterial divisions were represented in the clone libraries, whereas the isolates represented only three. The phylotype richness, frequency distribution (evenness), and composition of the four culture collections and the four clone libraries were investigated by using a variety of diversity indices. Although cultivation and 16S rRNA cloning analyses gave contradictory descriptions of the relative phylotype richness for one of the four environments, the two methods identified qualitatively consistent relationships when levels of evenness were compared. The levels of phylotype similarity between communities were uniformly low (15 to 31%). Both methods consistently indicated that one environment was distinct from the other three. Our data illustrate that while 16S rDNA cloning and cultivation generally describe similar relationships between soil microbial communities, significant discrepancies can occur.
Homologous Expression of Recombinant Lignin Peroxidase in
Phanerochaete chrysosporium
Authors: Sollewijn Gelpke Maarten D. , Mayfield-Gambill Mary , Lin Cereghino Geoffrey P. , Gold Michael H. 
Published: December 2019

ABSTRACT

The glyceraldehyde-3-phosphate dehydrogenase (
gpd
) promoter was used to drive expression of
lip2
, the gene encoding lignin peroxidase (LiP) isozyme H8, in primary metabolic cultures of
Phanerochaete chrysosporium
. The expression vector, pUGL, also contained the
Schizophyllum commune ura1
gene as a selectable marker. pUGL was used to transform a
P. chrysosporium
Ura11 auxotroph to prototrophy. Ura
+
transformants were screened for peroxidase activity in liquid cultures containing high-carbon and high-nitrogen medium. Recombinant LiP (rLiP) was secreted in active form by the transformants after 4 days of growth, whereas endogenous
lip
genes were not expressed under these conditions. Approximately 2 mg of homogeneous rLiP/liter was obtained after purification. The molecular mass, pI, and optical absorption spectrum of rLiPH8 were essentially identical to those of the wild-type LiPh8 (wt LiPH8), indicating that heme insertion, folding, and secretion functioned normally in the transformant. Steady-state and transient-state kinetic properties for the oxidation of veratryl alcohol between wtLiPH8 and rLiPH8 were also identical.
Relationship between Succinate Transport and Production of Extracellular Poly(3-Hydroxybutyrate) Depolymerase in
Pseudomonas lemoignei
Authors: Terpe Kay , Kerkhoff Kirsten , Pluta Elena , Jendrossek Dieter 
Published: December 2019

ABSTRACT

The relationship between extracellular poly(3-hydroxybutyrate) (PHB) depolymerase synthesis and the unusual properties of a succinate uptake system was investigated in
Pseudomonas lemoignei
. Growth on and uptake of succinate were highly pH dependent, with optima at pH 5.6. Above pH 7, growth on and uptake of succinate were strongly reduced with concomitant derepression of PHB depolymerase synthesis. The specific succinate uptake rates were saturable by high concentrations of succinate, and maximal transport rates of 110 nmol/mg of cell protein per min were determined between pH 5.6 and 6.8. The apparent
K
S0.5
values increased with increasing pH from 0.2 mM succinate at pH 5.6 to more than 10 mM succinate at pH 7.6. The uptake of [
14
C]succinate was strongly inhibited by several monocarboxylates. Dicarboxylates also inhibited the uptake of succinate but only at pH values near the dissociation constant of the second carboxylate function (pK
a
2). We conclude that the succinate carrier is specific for the monocarboxylate forms of various carboxylic acids and is not able to utilize the dicarboxylic forms. The inability to take up succinate
2−
accounts for the carbon starvation of
P. lemoignei
observed during growth on succinate at pH values above 7. As a consequence the bacteria produce high levels of extracellular PHB depolymerase activity in an effort to escape carbon starvation by utilization of PHB hydrolysis products.
Quantitative Selective PCR of 16S Ribosomal DNA Correlates Well with Selective Agar Plating in Describing Population Dynamics of Indigenous
Pseudomonas
spp. in Soil Hot Spots
Authors: Johnsen Kaare , Enger Øivind , Jacobsen Carsten S. , Thirup Laila , Torsvik Vigdis 
Published: December 2019

ABSTRACT

We used a quantitative PCR method targeting 16S ribosomal DNA using competitive PCR for specific detection of indigenous
Pseudomonas
DNA in soil hot spots. The amount of
Pseudomonas
DNA corresponded to the number of culturable
Pseudomonas
bacteria on Gould’s S1 agar. This represents the first use of PCR for quantification of indigenous bacteria in more than one sample of soil.
Subspecies-Dependent Regulation of
Bacillus thuringiensis
Protoxin Genes
Authors: Cheng Ping , Wu Lan , Ziniu Yu , Aronson Arthur 
Published: December 2019

ABSTRACT

Bacillus thuringiensis
accumulates, primarily during sporulation, large quantities of insecticidal protoxins which are deposited as crystalline, intracellular inclusions. Most subspecies contain several plasmid-encoded
cry
genes, each of which has a unique specificity. The overall toxicity profile of a subspecies depends not only on the array of
cry
genes present but also on the relative expression of the genes. In general, transcription depends on sporulation-specific sigma factors, but little is known about regulation of expression of the individual genes. In order to determine whether expression of a particular
cry
gene varies in different subspecies,
lacZ
fusions to the
cry
promoters of two protoxin genes (
cry1
class) were constructed. Protoxin accumulation and mRNA contents were also measured by performing immunoblotting and Northern analyses, respectively. The expression of a
cry1Ab-lacZ
fusion, but not the expression of a
cry1C-lacZ
fusion, was three to four times lower in
B. thuringiensis
subsp.
aizawai
strains than in
B. thuringiensis
subsp.
kurstaki
or
B. thuringiensis
subsp.
tolworthi
. Also, the Cry1Ab antigen and steady-state mRNA contents of
B. thuringiensis
subsp.
aizawai
were lower. The regulation of the genes must involve regions upstream of the promoters which are unique to each
cry
gene since (i) mutations in the upstream region of the
cry1Ab
gene resulted in enhanced expression in
B. thuringiensis
subsp.
aizawai
and (ii) no differences were found when the
lacZ
fusions contained the
cry1Ab
promoters but no upstream sequences. The capacity to regulate each of the protoxin genes must be a factor in the overall protoxin composition of a subspecies and thus its toxicity profile.
Combinations of Intervention Treatments Resulting in 5-Log
10
-Unit Reductions in Numbers of
Escherichia coli
O157:H7 and
Salmonella typhimurium
DT104 Organisms in Apple Cider
Authors: Uljas Heidi E. , Ingham Steven C. 
Published: December 2019

ABSTRACT

The U.S. Food and Drug Administration (FDA) recently mandated a warning statement on packaged fruit juices not treated to reduce target pathogen populations by 5 log
10
units. This study describes combinations of intervention treatments that reduced concentrations of mixtures of
Escherichia coli
O157:H7 (strains ATCC 43895, C7927, and USDA-FSIS-380-94) or
Salmonella typhimurium
DT104 (DT104b, U302, and DT104) by 5 log
10
units in apple cider with a pH of 3.3, 3.7, and 4.1. Treatments used were short-term storage at 4, 25, or 35°C and/or freeze-thawing (48 h at −20°C; 4 h at 4°C) of cider with or without added organic acids (0.1% lactic acid, sorbic acid [SA], or propionic acid). Treatments more severe than those for
S. typhimurium
DT104 were always required to destroy
E. coli
O157:H7. In pH 3.3 apple cider, a 5-log
10
-unit reduction in
E. coli
O157:H7 cell numbers was achieved by freeze-thawing or 6-h 35°C treatments. In pH 3.7 cider the 5-log
10
-unit reduction followed freeze-thawing combined with either 6 h at 4°C, 2 h at 25°C, or 1 h at 35°C or 6 h at 35°C alone. A 5-log
10
-unit reduction occurred in pH 4.1 cider after the following treatments: 6 h at 35°C plus freeze-thawing, SA plus 12 h at 25°C plus freeze-thawing, SA plus 6 h at 35°C, and SA plus 4 h at 35°C plus freeze-thawing. Yeast and mold counts did not increase significantly (
P
< 0.05) during the 6-h storage at 35°C. Cider with no added organic acids treated with either 6 h at 35°C, freeze-thawing or their combination was always preferred by consumers over pasteurized cider (
P
< 0.05). The simple, inexpensive intervention treatments described in the present work could produce safe apple cider without pasteurization and would not require the FDA-mandated warning statement.
Cocolonization of the Rhizosphere by Pathogenic
Agrobacterium
Strains and Nonpathogenic Strains K84 and K1026, Used for Crown Gall Biocontrol
Authors: Penyalver Ramón , López María M. 
Published: December 2019

ABSTRACT

The crown gall biocontrol agent strain K84 and three mutants derived from it, K1026 (Tra
−
deletion mutant of pAgK84), K84 Agr
−
(lacking pAgK84), and K1143 (lacking pAgK84 and pNoc), significantly reduced gall formation caused by two pathogenic strains resistant to agrocin 84 in peach × almond seedlings planted in infested soil. Cocolonization of roots by pathogenic and nonpathogenic strains was observed in these biocontrol experiments under field conditions. In spite of the efficient biocontrol observed, average populations consisting of 10
2
and 10
6
pathogenic agrobacteria per g of root were found 8 months after planting. The total numbers of pathogenic bacteria on roots were similar for plants treated with the biocontrol strains and for the untreated plants. Strain K84 and the genetically engineered organism K1026 survived at a level of 10
6
agrocin 84-producing bacteria per g of root. The population size of genetically engineered strain K1026 was not significantly different than the population size of wild-type strain K84 8 months after root inoculation. Strains K84 and K1026 controlled two pathogens resistant to agrocin 84 without reducing the total number of pathogenic bacteria in the root system. In addition, this study shows that some biological control activity of strain K84 against agrocin 84-resistant pathogens is independent of plasmids pAgK84 and pNoc.