bluuebunny/crossref_metadata_2025 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

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

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

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

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

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

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

In 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

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

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

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

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

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

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

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

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.
Use of a Sentinel System for Field Measurements ofCryptosporidium parvumOocyst Inactivation in Soil and Animal Waste
Authors: Jenkins M. B. , Walker M. J. , Bowman D. D. , Anthony L. C. , Ghiorse W. C.
Published: December 2019

A small-volume sentinel chamber was developed to assess the effects of environmental stresses on survival of sucrose-Percoll-purifiedCryptosporidium parvumoocysts in soil and animal wastes. Chambers were tested for their ability to equilibrate with external chemical and moisture conditions. Sentinel oocysts were then exposed to stresses of the external environment that affected their viability (potential infectivity), as indicated by results of a dye permeability assay. Preliminary laboratory experiments indicated that temperatures between 35 and 50°C and decreases in soil water potential (−0.003 to −3.20 MPa) increased oocyst inactivation rates. The effects of two common animal waste management practices on oocyst survival were investigated on three dairy farms in Delaware County, N.Y., within the New York City watershed: (i) piling wastes from dairy youngstock (including neonatal calves) and (ii) spreading wastes as a soil amendment on an agricultural field. Sentinel containers filled with air-dried and sieved (2-mm mesh) youngstock waste or field soil were wetted and inoculated with 2 million oocysts in an aqueous suspension and then placed in waste piles on two different farms and in soil within a cropped field on one farm. Controls consisted of purified oocysts in either phosphate-buffered saline or distilled water contained in sealed microcentrifuge tubes. Two microdata loggers recorded the ambient temperature at each field site. Sentinel experiments were conducted during the fall and winter (1996 to 1997) and winter (1998). Sentinel containers and controls were removed at 2- to 4-week intervals, and oocysts were extracted and tested by the dye permeability assay. The proportions of potentially infective oocysts exposed to the soil and waste pile material decreased more rapidly than their counterpart controls exposed to buffer or water, indicating that factors other than temperature affected oocyst inactivation in the waste piles and soil. The effect of soil freeze-thaw cycles was evident in the large proportion of empty sentinel oocysts. The potentially infective sentinel oocysts were reduced to <1% while the proportions in controls did not decrease below 50% potentially infective during the first field experiment. Microscopic observations of empty oocyst fragments indicated that abrasive effects of soil particles were a factor in oocyst inactivation. A similar pattern was observed in a second field experiment at the same site.
Division of
Listeria monocytogenes
Serovar 1/2a Strains into Two Groups by PCR and Restriction Enzyme Analysis
Authors: Unnerstad Helle , Nilsson Inger , Ericsson Henrik , Danielsson-Tham Marie-Louise , Bille Jacques , Bannerman Elizabeth , Tham Wilhelm
Published: December 2019

Altogether, 100 strains of
Listeria monocytogenes
serovar 1/2a isolated from humans, animals, food, and the environment were typed by a combination of PCR and restriction enzyme analysis (REA). A PCR product of 2,916 bp, containing the downstream end of the gene
inlA
(955 bp), the space between
inlA
and
inlB
(85 bp), and 1,876 bp of the gene
inlB
, was cleaved with the enzyme
Alu
I, and the fragments generated were separated by gel electrophoresis. By this method two different cleavage patterns were obtained. Seventy of the 100 strains shared one restriction profile, and the remaining 30 strains shared the second one. No relation was found between the types differentiated by PCR-REA and the origins of the strains.
Localized, Positive Charge Mediates Adhesion of
Rhodosporidium toruloides
to Barley Leaves and Polystyrene
Authors: Buck James W. , Andrews John H.
Published: December 2019

The physicochemical forces that mediate attachment of yeasts to the phylloplane are unknown. Cell surface charge and hydrophobicity and adhesion to polystyrene, glass, and barley were assessed for wild-type
Rhodosporidium toruloides
and attachment-minus (Att
−
) mutants. Cells were grown under conditions promoting (excess carbon) or not promoting (excess nitrogen) capsule production. Hydrophobicity was measured by adhesion to xylenes, and surface charge characteristics were assessed by attachment to either DEAE (positive)- or carboxymethyl (CM) (negative)-Sephadex ion-exchange beads. Hydrophobicity and adhesiveness of nonencapsulated, wild-type
R. toruloides
decreased from mid-log to late stationary phase. Encapsulated wild-type
R. toruloides
cells were more hydrophobic and more adhesive than nonencapsulated cells. However, two encapsulated Att
−
mutants were more hydrophobic than the wild type and levels of adhesion of
R. toruloides
were similar on polystyrene and less hydrophobic glass surfaces. Adhesion of wild-type yeast to barley and polystyrene was correlated with attachment to CM-Sephadex beads, indicating a positive cell surface charge. Sixteen Att
−
mutants did not exhibit a positive cell surface charge, and wild-type yeast cells that did not attach to CM-Sephadex did not adhere to either polystyrene or barley. Wild-type
R. toruloides
attached to CM-Sephadex beads by the poles of the cells, indicating a localization of positive charge which was also visualized with India ink. We conclude that localized, positive charge, and not hydrophobic interactions, mediates attachment of
R. toruloides
to barley leaves.
Excision of the Conjugative Transposon Tn
916
in
Lactococcus lactis
Authors: Marra Diana , Smith Jennifer G. , Scott June R.
Published: December 2019

In
Lactococcus lactis
excision of Tn
916
is limited by the concentration of integrase and is increased by providing more excisionase. However, even with increased excision of Tn
916
in
L. lactis
, no conjugative transfer is detectable. This suggests that
L. lactis
is deficient in a host factor(s) required for conjugative transposition.
Methods for Detection of
Anticarsia gemmatalis
Nucleopolyhedrovirus DNA in Soil
Authors: de Moraes R. R. , Maruniak J. E. , Funderburk J. E.
Published: December 2019

Two methods, phenol-ether and magnetic capture-hybridization (MCH), were developed and compared with regard to their sensitivities and abilities to extract the DNA of the insect baculovirus
Anticarsia gemmatalis
nucleopolyhedrovirus (Ag
M
NPV) from soil and to produce DNA amplifiable by PCR. Laboratory experiments were performed with 0.25 g of autoclaved soil inoculated with different viral concentrations to optimize both methods of baculovirus DNA extraction and to determine their sensitivities. Both procedures produced amplifiable DNA; however, the MCH method was 100-fold more sensitive than the phenol-ether procedure. The removal of PCR inhibitors from the soil appeared to be complete when MCH was used as the viral DNA isolation method, because undiluted aliquots of the DNA preparations could be amplified by PCR. The phenol-ether procedure probably did not completely remove PCR inhibitors from the soil, since PCR products were observed only when the Ag
M
NPV DNA preparations were diluted 10- or 100-fold. Ag
M
NPV DNA was detected in field-collected soil samples from 15 to 180 days after virus application when the MCH procedure to isolate DNA was coupled with PCR amplification of the polyhedrin region.
Quantitative Immunofluorescence of Regulated
eps
Gene Expression in Single Cells of
Ralstonia solanacearum
Authors: Kang Yaowei , Saile Elke , Schell Mark A. , Denny Timothy P.
Published: December 2019

Ralstonia solanacearum
, a phytopathogenic bacterium, uses an environmentally sensitive and complex regulatory network to control expression of multiple virulence genes. Part of this network is an unusual autoregulatory system that produces and senses 3-hydroxypalmitic acid methyl ester. In culture, this autoregulatory system ensures that expression of virulence genes, such as those of the
eps
operon encoding biosynthesis of the acidic extracellular polysaccharide, occurs only at high cell density (>10
7
cells/ml). To determine if regulation follows a similar pattern within tomato plants, we first developed a quantitative immunofluorescence (QIF) method that measures the relative amount of a target protein within individual bacterial cells. For
R. solanacearum
, QIF was used to determine the amount of β-galactosidase protein within wild-type cells containing a stable
eps-lacZ
reporter allele. When cultured cells were examined to test the method, QIF accurately detected both low and high levels of
eps
gene expression. QIF analysis of
R. solanacearum
cells recovered from stems of infected tomato plants showed that expression of
eps
during pathogenesis was similar to that in culture. These results suggest that there are no special signals or conditions within plants that override or short-circuit the regulatory processes observed in
R. solanacearum
in culture. Because QIF is a robust, relatively simple procedure that uses generally accessible equipment, it should be useful in many situations where gene expression in single bacterial cells must be determined.
Deletions in the Gibberellin Biosynthesis Gene Cluster of
Gibberella fujikuroi
by Restriction Enzyme-Mediated Integration and Conventional Transformation-Mediated Mutagenesis
Authors: Linnemannstöns Pia , Voß Thorsten , Hedden Peter , Gaskin Paul , Tudzynski Bettina
Published: December 2019

We induced mutants of
Gibberella fujikuroi
deficient in gibberellin (GA) biosynthesis by transformation-mediated mutagenesis with the vector pAN7-1. We recovered 24 GA-defective mutants in one of nine transformation experiments performed without the addition of a restriction enzyme. Each mutant had a similar Southern blot pattern, suggesting the integration of the vector into the same site. The addition of a restriction enzyme by restriction enzyme-mediated integration (REMI) significantly increased the transformation rate and the rate of single-copy integration events. Of 1,600 REMI transformants, two produced no GAs. Both mutants had multiple copies of the vector pAN7-1 and one had a Southern blot pattern similar to those of the 24 conventionally transformed GA-deficient mutants. Biochemical analysis of the two REMI mutants confirmed that they cannot produce
ent
-kaurene, the first specific intermediate of the GA pathway. Feeding the radioactively labelled precursors
ent
-kaurene and GA
12
-aldehyde followed by high-performance liquid chromatography and gas chromatography-mass spectrometry analysis showed that neither of these intermediates was converted to GAs in the mutants. Southern blot analysis and pulsed-field gel electrophoresis of the transformants using the bifunctional
ent
-copalyl diphosphate/
ent
-kaurene synthase gene (
cps/ks
) and the flanking regions as probes revealed a large deletion in the GA-deficient REMI transformants and in the GA-deficient transformants obtained by conventional insertional transformation. We conclude that transformation procedures with and without the addition of restriction enzymes can lead to insertion-mediated mutations and to deletions and chromosome translocations.
Purification and Characterization of a Keratinolytic Serine Proteinase from
Streptomyces albidoflavus
Authors: Bressollier Philippe , Letourneau François , Urdaci Maria , Verneuil Bernard
Published: December 2019

Streptomyces
strain K
1-02
, which was identified as a strain of
Streptomyces albidoflavus
, secreted at least six extracellular proteases when it was cultured on feather meal-based medium. The major keratinolytic serine proteinase was purified to homogeneity by a two-step procedure. This enzyme had a molecular weight of 18,000 and was optimally active at pH values ranging from 6 to 9.5 and at temperatures ranging from 40 to 70°C. Its sensitivity to protease inhibitors, its specificity on synthetic substrates, and its remarkably high level of NH
2
-terminal sequence homology with
Streptomyces griseus
protease B (SGPB) showed that the new enzyme, designated SAKase, was homologous to SGPB. We tested the activity of SAKase with soluble and fibrous substrates (elastin, keratin, and type I collagen) and found that it was very specific for keratinous substrates compared to SGPB and proteinase K.
Nitrous Oxide Production and Methane Oxidation by Different Ammonia-Oxidizing Bacteria
Authors: Jiang Qing-Qiao , Bakken Lars R.
Published: December 2019

Ammonia-oxidizing bacteria (AOB) are thought to contribute significantly to N
2
O production and methane oxidation in soils. Most of our knowledge derives from experiments with
Nitrosomonas europaea
, which appears to be of minor importance in most soils compared to
Nitrosospira
spp. We have conducted a comparative study of levels of aerobic N
2
O production in six phylogenetically different
Nitrosospira
strains newly isolated from soils and in two
N. europaea
and
Nitrosospira multiformis
type strains. The fraction of oxidized ammonium released as N
2
O during aerobic growth was remarkably constant (0.07 to 0.1%) for all the
Nitrosospira
strains, irrespective of the substrate supply (urea versus ammonium), the pH, or substrate limitation.
N. europaea
and
Nitrosospira multiformis
released similar fractions of N
2
O when they were supplied with ample amounts of substrates, but the fractions rose sharply (to 1 to 5%) when they were restricted by a low pH or substrate limitation. Phosphate buffer (versus HEPES) doubled the N
2
O release for all types of AOB. No detectable oxidation of atmospheric methane was detected. Calculations based on detection limits as well as data in the literature on CH
4
oxidation by AOB bacteria prove that none of the tested strains contribute significantly to the oxidation of atmospheric CH
4
in soils.
Piriformospora indica
, a Cultivable Plant-Growth-Promoting Root Endophyte
Authors: Varma Ajit , Savita Verma  , Sudha  , Sahay Nirmal , Bütehorn Britta , Franken Philipp
Published: December 2019

Piriformospora indica
(Hymenomycetes, Basidiomycota) is a newly described cultivable endophyte that colonizes roots. Inoculation with the fungus and application of fungal culture filtrate promotes plant growth and biomass production. Due to its ease of culture, this fungus provides a model organism for the study of beneficial plant-microbe interactions and a new tool for improving plant production systems.
Influence of Temperature and Growth Phase on Expression of a 104-Kilodalton
Listeria
Adhesion Protein in
Listeria monocytogenes
Authors: Santiago Nivia I. , Zipf Allan , Bhunia Arun K.
Published: December 2019

Interaction of
Listeria monocytogenes
with mammalian intestinal cells is believed to be an important first step in
Listeria
pathogenesis. Transposon (Tn
916
) mutagenesis provided strong evidence that a 104-kDa surface protein, designated the
Listeria
adhesion protein (LAP), was involved in adherence of
L. monocytogenes
to a human enterocyte-like Caco-2 cell line (V. Pandiripally, D. Westbrook, G. Sunki, and A. Bhunia, J. Med. Microbiol. 48:117–124, 1999). In this study, expression of LAP in
L. monocytogenes
at various growth temperatures (25, 37, and 42°C) and in various growth phases was determined by performing an enzyme-linked immunoassay (ELISA) and Western blotting with a specific monoclonal antibody (monoclonal antibody H7). The ELISA and Western blot results indicated that there was a significant increase in LAP expression over time only at 37 and 42°C and that the level of LAP expression was low during the exponential phase and high during the stationary phase. In contrast, there were not significant differences in LAP expression between the exponential and stationary phases at 25°C. Examination of the adhesion of
L. monocytogenes
cells from exponential-phase (12-h) or stationary-phase (24-h) cultures grown at 37°C to Caco-2 cells revealed that there were not significant differences in adhesion. Although expression of
L. monocytogenes
LAP was different at different growth temperatures and in different growth phases, enhanced expression did not result in increased adhesion, possibly because only a few LAP molecules were sufficient to initiate binding to Caco-2 cells.
Multiple Antibiotic Resistance Patterns of
Escherichia coli
Isolates from Swine Farms
Authors: Mathew A. G. , Saxton A. M. , Upchurch W. G. , Chattin S. E.
Published: December 2019

Antibiotic resistance of
Escherichia coli
from sows and pigs was determined to compare patterns between pigs of various ages and degrees of antibiotic use. Resistance patterns differed between farm types and pigs of differing ages, indicating that pig age and degree of antibiotic use affect resistance of fecal
E. coli
.
Response of Pathogenic
Vibrio
Species to High Hydrostatic Pressure
Authors: Berlin Daniel L. , Herson Diane S. , Hicks Doris T. , Hoover Dallas G.
Published: December 2019

Vibrio parahaemolyticus
ATCC 17802,
Vibrio vulnificus
ATCC 27562,
Vibrio cholerae
O:1 ATCC 14035,
Vibrio cholerae
non-O:1 ATCC 14547,
Vibrio hollisae
ATCC 33564, and
Vibrio mimicus
ATCC 33653 were treated with 200 to 300 MPa for 5 to 15 min at 25°C. High hydrostatic pressure inactivated all strains of pathogenic
Vibrio
without triggering a viable but nonculturable (VBNC) state; however, cells already existing in a VBNC state appeared to possess greater pressure resistance.
A New Biocatalyst for Production of Optically Pure Aryl Epoxides by Styrene Monooxygenase from
Pseudomonas fluorescens
ST
Authors: Di Gennaro Patrizia , Colmegna Andrea , Galli Enrica , Sello Guido , Pelizzoni Francesca , Bestetti Giuseppina
Published: December 2019

We developed a biocatalyst by cloning the styrene monooxygenase genes (
styA
and
styB
) from
Pseudomonas fluorescens
ST responsible for the oxidation of styrene to its corresponding epoxide. Recombinant
Escherichia coli
was able to oxidize different aryl vinyl and aryl ethenyl compounds to their corresponding optically pure epoxides. The results of bioconversions indicate the broad substrate preference of styrene monooxygenase and its potential for the production of several fine chemicals.
Production of Monoclonal Antibodies to
Listeria monocytogenes
and Their Application To Determine the Virulence of Isolates from Channel Catfish
Authors: Erdenlig Sevil , Ainsworth A. Jerald , Austin Frank W.
Published: December 2019

We produced monoclonal antibodies (MAbs) to the extracellular proteins of
Listeria monocytogenes
EGD grown in Chelex-treated improved minimal medium. Ten of the positive hybridomas generated were chosen for further characterization. Seven of the MAbs reacted with a protein having a molecular mass of 60 kDa. These MAbs inhibited listeriolysin (LLO)-mediated hemolysis, and two of them were specific for LLO and none of the other thiol-activated toxins tested. In an enzyme-linked immunosorbent assay and Western blot analysis, five of the anti-LLO MAbs reacted with ivanolysin from
Listeria ivanovii
. Three of the 10 MAbs reacted with a 29-kDa protein on Western blots and neutralized the phosphatidylcholine-specific phospholipase C (PC-PLC) activity of
L. monocytogenes
. These three anti-PC-PLC MAbs did not react with phospholipases from five different gram-positive bacteria. However, the anti-PC-PLC MAbs recognized a 27-kDa extracellular protein from
L. ivanovii
and neutralized sphingomyelinase activity in a hemolysis test that demonstrates the antigenic relatedness of listerial phospholipases. These data indicate that listerial thiol-activated toxins possess species-specific epitopes and share group-specific epitopes. This is the first description of MAbs that neutralize listerial PC-PLC, and the data suggest that there is antigenic similarity between
L. monocytogenes
PC-PLC and
L. ivanovii
sphingomyelinase. The reactions of the MAbs with catfish isolates of
L. monocytogenes
suggested that some of the isolates examined lack the LLO and/or PC-PLC required for pathogenicity. The MAbs described here differentiated some catfish isolates from previously described type strain-pathogenic isolates and could be useful for detecting and determining the virulence of
L. monocytogenes
in food and clinical samples and for detecting
L. ivanovii
in veterinary clinical samples.
Stress Tolerance in Doughs of
Saccharomyces cerevisiae
Trehalase Mutants Derived from Commercial Baker’s Yeast
Authors: Shima Jun , Hino Akihiro , Yamada-Iyo Chie , Suzuki Yasuo , Nakajima Ryouichi , Watanabe Hajime , Mori Katsumi , Takano Hiroyuki
Published: December 2019

Accumulation of trehalose is widely believed to be a critical determinant in improving the stress tolerance of the yeast
Saccharomyces cerevisiae
, which is commonly used in commercial bread dough. To retain the accumulation of trehalose in yeast cells, we constructed, for the first time, diploid homozygous neutral trehalase mutants (Δ
nth1
), acid trehalase mutants (Δ
ath1
), and double mutants (Δ
nth1 ath1
) by using commercial baker’s yeast strains as the parent strains and the gene disruption method. During fermentation in a liquid fermentation medium, degradation of intracellular trehalose was inhibited with all of the trehalase mutants. The gassing power of frozen doughs made with these mutants was greater than the gassing power of doughs made with the parent strains. The Δ
nth1
and Δ
ath1
strains also exhibited higher levels of tolerance of dry conditions than the parent strains exhibited; however, the Δ
nth1 ath1
strain exhibited lower tolerance of dry conditions than the parent strain exhibited. The improved freeze tolerance exhibited by all of the trehalase mutants may make these strains useful in frozen dough.
Interaction between Functional Domains of
Bacillus thuringiensis
Insecticidal Crystal Proteins
Authors: Rang Cécile , Vachon Vincent , de Maagd Ruud A. , Villalon Mario , Schwartz Jean-Louis , Bosch Dirk , Frutos Roger , Laprade Raynald
Published: December 2019

Interactions among the three structural domains of
Bacillus thuringiensis
Cry1 toxins were investigated by functional analysis of chimeric proteins. Hybrid genes were prepared by exchanging the regions coding for either domain I or domain III among Cry1Ab, Cry1Ac, Cry1C, and Cry1E. The activity of the purified trypsin-activated chimeric toxins was evaluated by testing their effects on the viability and plasma membrane permeability of Sf9 cells. Among the parental toxins, only Cry1C was active against these cells and only chimeras possessing domain II from Cry1C were functional. Combination of domain I from Cry1E with domains II and III from Cry1C, however, resulted in an inactive toxin, indicating that domain II from an active toxin is necessary, but not sufficient, for activity. Pores formed by chimeric toxins in which domain I was from Cry1Ab or Cry1Ac were slightly smaller than those formed by toxins in which domain I was from Cry1C. The properties of the pores formed by the chimeras are therefore likely to result from an interaction between domain I and domain II or III. Domain III appears to modulate the activity of the chimeric toxins: combination of domain III from Cry1Ab with domains I and II of Cry1C gave a protein which was more strongly active than Cry1C.
Biotransformation of 2,4,6-Trinitrotoluene with
Phanerochaete chrysosporium
in Agitated Cultures at pH 4.5
Authors: Hawari Jalal , Halasz Annamaria , Beaudet Sylvie , Paquet Louise , Ampleman Guy , Thiboutot Sonia
Published: December 2019

The biotransformation of 2,4,6-trinitrotoluene (TNT) (175 μM) by
Phanerochaete chrysosporium
with molasses and citric acid at pH 4.5 was studied. In less than 2 weeks, TNT disappeared completely, but mineralization (liberated
14
CO
2
) did not exceed 1%. A time study revealed the presence of several intermediates, marked by the initial formation of two monohydroxylaminodinitrotoluenes (2- and 4-HADNT) followed by their successive transformation to several other products, including monoaminodinitrotoluenes (ADNT). A group of nine acylated intermediates were also detected. They included 2-
N
-acetylamido-4,6-dinitrotoluene and its
p
isomer, 2-formylamido-4,6-dinitrotoluene and its
p
isomer (as acylated ADNT), 4-
N
-acetylamino-2-amino-6-nitrotoluene and 4-
N
-formylamido-2-amino-6-nitrotoluene (as acetylated DANT), 4-
N
-acetylhydroxy-2,6-dinitrotoluene and 4-
N
-acetoxy-2,6-dinitrotoluene (as acetylated HADNT), and finally 4-
N
-acetylamido-2-hydroxylamino-6-nitrotoluene. Furthermore, a fraction of HADNTs were found to rearrange to their corresponding phenolamines (Bamberger rearrangement), while another group dimerized to azoxytoluenes which in turn transformed to azo compounds and eventually to the corresponding hydrazo derivatives. After 30 days, all of these metabolites, except traces of 4-ADNT and the hydrazo derivatives, disappeared, but mineralization did not exceed 10% even after the incubation period was increased to 120 days. The biotransformation of TNT was accompanied by the appearance of manganese peroxidase (MnP) and lignin-dependent peroxidase (LiP) activities. MnP activity was observed almost immediately after TNT disappearance, which was the period marked by the appearance of the initial metabolites (HADNT and ADNT), whereas the LiP activity was observed after 8 days of incubation, corresponding to the appearance of the acyl derivatives. Both MnP and LiP activities reached their maximum levels (100 and 10 U/liter, respectively) within 10 to 15 days after inoculation.