bluuebunny/crossref_metadata_2025 huggingface dataset:
Identification of
Gremmeniella abietina
Races with Random Amplified Polymorphic DNA Markers
Authors: Hamelin Richard C. , Ouellette G. B. , Bernier Louis
Published: January 2020

Seven random amplified polymorphic DNA (RAPD) markers amplified from four oligonucleotides (10-mers) by the polymerase chain reaction were used to distinguish between the North American and European races of
Gremmeniella abietina
, the causal agent of Scleroderris canker of conifers. Forty-three isolates of the pathogen from 11 different host species originating from 11 countries, states, and provinces were tested; race designation was consistent with results from immunogenic and soluble-protein assays. By using RAPD markers, it was possible to identify
G. abietina
races by DNA amplifications directly from fruiting bodies, thus eliminating the need to culture the fungus, as is necessary with immunogenic and soluble-protein assays. Two isolates which had been previously classified as intermediate were clearly identified as belonging to either one of the two races by using RAPD markers. No interracial hybrids were detected in our survey. Patterns of amplification products from the European race in North America were identical to patterns of European isolates, further substantiating that this is an introduced race to the North American continent.
Degradation of 2,4,5-trichlorophenol by the lignin-degrading basidiomycete Phanerochaete chrysosporium
Authors: Joshi D K , Gold M H
Published: January 2020

Under secondary metabolic conditions the white rot basidiomycete Phanerochaete chrysosporium rapidly mineralizes 2,4,5-trichlorophenol. The pathway for degradation of 2,4,5-trichlorophenol was elucidated by the characterization of fungal metabolites and oxidation products generated by purified lignin peroxidase (LiP) and manganese peroxidase (MnP). The multistep pathway involves cycles of peroxidase-catalyzed oxidative dechlorination reactions followed by quinone reduction reactions to yield the key intermediate 1,2,4,5-tetrahydroxybenzene, which is presumably ring cleaved. In the first step of the pathway, 2,4,5-trichlorophenol is oxidized to 2,5-dichloro-1,4-benzoquinone by either MnP or Lip. 2,5-Dichloro-1,4-benzoquinone is then reduced to 2,5-dichloro-1,4-hydroquinone. The 2,5-dichloro-1,4-hydroquinone is oxidized by MnP to generate 5-chloro-4-hydroxy-1,2-benzoquinone. The orthoquinone is in turn reduced to 5-chloro-1,2,4-trihydroxybenzene. Finally, the 5-chlorotrihydroxybenzene undergoes another cycle of oxidative dechlorination and reduction reactions to generate 1,2,4,5-tetrahydroxybenzene. The latter is presumably ring cleaved, with subsequent degradation to CO2. In this pathway, the substrate is oxidatively dechlorinated by LiP or MnP in a reaction which produces a quinone. The quinone intermediate is recycled by a reduction reaction to regenerate an intermediate which is again a substrate for peroxidase-catalyzed oxidative dechlorination. This pathway apparently results in the removal of all three chlorine atoms before ring cleavage occurs.
Two Different
Bacillus thuringiensis
Delta-Endotoxin Receptors in the Midgut Brush Border Membrane of the European Corn Borer,
Ostrinia nubilalis
(Hübner) (Lepidoptera: Pyralidae)
Authors: Denolf P. , Jansens S. , Peferoen M. , Degheele D. , Van Rie J.
Published: January 2020

Binding of three
Bacillus thuringiensis
insecticidal crystal proteins (ICPs) to the midgut epithelium of
Ostrinia nubilalis
larvae was characterized by performing binding experiments with both isolated brush border membrane vesicles and gut tissue sections. Our results demonstrate that two independent ICP receptors are present in the brush border of
O. nubilalis
gut epithelium. From competition binding experiments performed with
125
I-labeled and native ICPs it was concluded that CryIA(b) and CryIA(c) are recognized by the same receptor. An 11-fold-higher binding affinity of CryIA(b) for this receptor correlated with a 10-fold-higher toxicity of this ICP compared with CryIA(c). The CryIB toxin did not compete for the binding site of CryIA(b) and CryIA(c). Immunological detection of ingested
B. thuringiensis
ICPs on gut sections of
O. nubilalis
larvae revealed binding only along the epithelial brush border membrane. CryID and CryIE, two ICPs that are not toxic to
O. nubilalis,
were not bound to the apical microvilli of gut epithelial cells. In vitro binding experiments performed with native and biotinylated ICPs on tissue sections confirmed the correlation between ICP binding and toxicity. Moreover, by performing heterologous competition experiments with biotinylated and native ICPs, it was confirmed that the CryIB receptor is different from the receptor for CryIA(b) and CryIA(c). Retention of activated crystal proteins by the peritrophic membrane was not correlated with toxicity. Furthermore, it was demonstrated that CryIA(b), CryIA(c), and CryIB toxins interact in vitro with the epithelial microvilli of Malpighian tubules. In addition, CryIA(c) toxin also adheres to the basement membrane of the midgut epithelium.
Indigenous Bacteria in Hemolymph and Tissues of Marine Bivalves at Low Temperatures
Authors: Olafsen Jan A. , Mikkelsen Helene V. , Giæver Hanne M. , Høvik Hansen Geir
Published: January 2020

Hemolymph and soft tissues of Pacific oysters (
Crassostrea gigas
) kept in sand-filtered seawater at temperatures between 1 and 8°C were normally found to contain bacteria, with viable counts (CFU) in hemolymph in the range 1.4 × 10
2
to 5.6 × 10
2
bacteria per ml.
Pseudomonas, Alteromonas, Vibrio,
and
Aeromonas
organisms dominated, with a smaller variety of morphologically different unidentified strains. Hemolymph and soft tissues of horse mussels (
Modiolus modiolus
), locally collected from a 6- to 10-m depth in the sea at temperatures between 4 and 6°C, also contained bacteria. The CFU in horse mussel hemolymph was of the same magnitude as that in oysters (mean, 2.6 × 10
4
), and the bacterial flora was dominated by
Pseudomonas
(61.3%),
Vibrio
(27.0%), and
Aeromonas
(11.7%) organisms. In soft tissues of horse mussels, a mean CFU of 2.9 × 10
4
bacteria per g was found, with
Vibrio
(38.5%),
Pseudomonas
(33.0%), and
Aeromonas
(28.5%) constituting the major genera. After the challenge of oysters in seawater at 4°C to the psychrotrophic fish pathogen
Vibrio salmonicida
(strains NCIMB 2245 from Scotland and TEO 84001 from Norway) and a commensal
Aeromonas
sp. isolated from oysters, the viable count in hemolymph increased 1,000-fold to about 10
5
bacteria per ml. In soft tissues, about a 1,000-fold increase in CFU to 6 × 10
7
was observed.
V. salmonicida
NCIMB 2245 invaded hemolymph and soft tissues after 14 days and dominated these compartments after 41 days, whereas strain TEO 84001 did not invade soft tissues to the same extent. Challenge with
V. salmonicida
NCIMB 2245 resulted in 100% mortality, whereas about 50% of the oysters survived challenge with the Norwegian strain, TEO 84001. The commensal
Aeromonas
sp. invaded hemolymph and soft tissues and caused 100% mortality. Oyster hemolymph contained agglutinins for
Vibrio anguillarum
but not for
V. salmonicida,
whereas we did not find agglutinins for either of these bacteria in horse mussels. Agglutinins for horse and human erythrocytes were found in hemolymph from both animals. We found no differences in agglutinin titers in oysters from different Norwegian locations, and long-term challenge with bacteria in seawater did not result in changes of agglutinin activity. These studies demonstrate that bacteria exist in hemolymph and soft tissues of marine bivalves at temperatures below 8°C. Increased bacterial numbers in seawater at 4°C result in augmented invasion of bacteria in hemolymph and soft tissues.
V. salmonicida,
a bacterium pathogenic for fish at low temperatures, invades bivalve hemolymph and soft tissues, and thus bivalves may serve as a reservoir for pathogens of fish at low seawater temperatures.
5-Hydroxyquinoline-2-Carboxylic Acid, a Dead-End Metabolite from the Bacterial Oxidation of 5-Aminonaphthalene-2-Sulfonic Acid
Authors: Nörtemann Bernd , Glässer Andrea , Machinek Reinhard , Remberg Gerd , Knackmuss Hans-Joachim
Published: January 2020

5-Aminonaphthalene-2-sulfonate (5A2NS) is converted by strain BN6 into 5-hydroxyquinoline-2-carboxylate (5H2QC). The authenticity of this new compound is confirmed by nuclear magnetic resonance and mass spectrometry. Its formation is explained by a spontaneous cyclization of the hypothetical metabolite 6′-amino-2′-hydroxybenzalpyruvate. The formation of 5H2QC as a dead-end product of 5A2NS prevents NADH regeneration so that 5A2NS oxidation is limited by the internal NADH pool.
Ethylene Production by Root Nodules and Effect of Ethylene on Nodulation in
Glycine max
Authors: Hunter William J.
Published: January 2020

Nodulated soybean roots produced more ethylene and contained more 1-aminocyclopropane-1-carboxylic acid than uninoculated roots. Nodules produced more ethylene and contained more 1-aminocyclopropane-1-carboxylic acid per gram of material than roots. Almost all of the ethylene produced by the nodules was produced by the plant fractions of the nodules. Ethylene, at physiological concentrations, did not inhibit nodulation in soybeans.
Quantitative Determination of the Spatial Distribution of
Nitrosomonas europaea
and
Nitrobacter agilis
Cells Immobilized in κ-Carrageenan Gel Beads by a Specific Fluorescent-Antibody Labelling Technique
Authors: Hunik Jan H. , van den Hoogen Marijke P. , de Boer Wietse , Smit Marieke , Tramper Johannes
Published: January 2020

A novel technique, combining labelling and stereological methods, for the determination of spatial distribution of two microorganisms in a biofilm is presented. Cells of
Nitrosomonas europaea
(ATCC 19718) and
Nitrobacter agilis
(ATCC 14123) were homogeneously distributed in a κ-carrageenan gel during immobilization and allowed to grow out to colonies. The gel beads were sliced in thin cross sections after fixation and embedding. A two-step labelling method resulted in green fluorescent colonies of either
N. europaea
or
N. agilis
in the respective cross sections. The positions and surface areas of the colonies of each species were determined, and from that a biomass volume distribution for
N. europaea
and
N. agilis
in κ-carrageenan gel beads was estimated. This technique will be useful for the validation of biofilm models, which predict such biomass distributions.
Effect of Temperature on Fatty Acid Composition of a White
Thermus
Strain
Authors: Nordström Katrina M.
Published: January 2020

A white
Thermus
sp. strain, NCIMB 11245, showed high levels of anteiso C
17:0
, anteiso C
17:1
, normal C
16:1
, and iso C
16:0
with low levels of iso C
15:0
+ iso C
17:0
in comparison to yellow-pigmented strains. The fatty acid composition may be associated with precursor metabolism or the absence of carotene pigmentation.
Method for Investigation of Competition between Bacteria as a Function of Three Environmental Factors Varied Simultaneously
Authors: Thomas Linda V. , Wimpenny Julian W. T.
Published: January 2020

Competition between microorganisms as affected by temperature, pH, and the sodium chloride (NaCl) concentration was investigated by selective replication from gradient plates.
Salmonella typhimurium
was inhibited by
Pseudomonas putida
at 20 and 23°C but not 30 and 35°C.
P. putida
no longer grew at the extremes of pH and NaCl concentration, particularly at 30 and 35°C.
Survival and growth of Escherichia coli O157:H7 on salad vegetables
Authors: Abdul-Raouf U M , Beuchat L R , Ammar M S
Published: January 2020

The influence of modified-atmosphere packaging, storage temperature, and time on survival and growth of Escherichia coli O157:H7 inoculated onto shredded lettuce, sliced cucumber, and shredded carrot was determined. Growth of psychotrophic and mesophilic microorganisms and changes in pH and sensory qualities of vegetables, as judged by subjective evaluation, were also monitored. Packaging under an atmosphere containing 3% oxygen and 97% nitrogen had no apparent effect on populations of E. coli O157:H7, psychotrophs, or mesophiles. Populations of viable E. coli O157:H7 declined on vegetables stored at 5 degrees C and increased on vegetables stored at 12 and 21 degrees C for up to 14 days. The most rapid increases in populations of E. coli O157:H7 occurred on lettuce and cucumbers stored at 21 degrees C. These results suggest that an unknown factor(s) associated with carrots may inhibit the growth of E. coli O157:H7. The reduction in pH of vegetables was correlated with initial increases in populations of E. coli O157:H7 and naturally occurring microfloras. Eventual decreases in E. coli O157:H7 in some samples, e.g., those stored at 21 degrees C, are attributed to the toxic effect of accumulated acids. Changes in visual appearance of vegetables were not influenced substantially by growth of E. coli O157:H7. The ability of E. coli O157:H7 to growth on raw salad vegetables subjected to processing and storage conditions simulating those routinely used in commercial practice has been demonstrated.
Kinetics of Bacterial Growth on Chlorinated Aliphatic Compounds
Authors: van den Wijngaard Arjan J. , Wind Richèle D. , Janssen Dick B.
Published: January 2020

With the pure bacterial cultures
Ancylobacter aquaticus
AD20 and AD25,
Xanthobacter autotrophicus
GJ10, and
Pseudomonas
sp. strain AD1, Monod kinetics was observed during growth in chemostat cultures on 1,2-dichloroethane (AD20, AD25, and GJ10), 2-chloroethanol (AD20 and GJ10), and 1,3-dichloro-2-propanol (AD1). Both the Michaelis-Menten constants (
K

m

) of the first catabolic (dehalogenating) enzyme and the Monod half-saturation constants (
K

s

) followed the order 2-chloroethanol, 1,3-dichloro-2-propanol, epichlorohydrin, and 1,2-dichloroethane. The
K

s

values of strains GJ10, AD20, and AD25 for 1,2-dichloroethane were 260, 222, and 24 μM, respectively. The low
K

s

value of strain AD25 was correlated with a higher haloalkane dehalogenase content of this bacterium. The growth rates of strains AD20 and GJ10 in continuous cultures on 1,2-dichloroethane were higher than the rates predicted from the kinetics of the haloalkane dehalogenase and the concentration of the enzyme in the cells. The results indicate that the efficiency of chlorinated compound removal is indeed influenced by the kinetic properties and cellular content of the first catabolic enzyme. The cell envelope did not seem to act as a barrier for permeation of 1,2-dichloroethane.
Enhancement of Population Densities of
Pseudomonas putida
PpG7 in Agricultural Ecosystems by Selective Feeding with the Carbon Source Salicylate
Authors: Colbert Stephen F. , Schroth Milton N. , Weinhold Albert R. , Hendson Mavis
Published: January 2020

Sodium salicylate (1,000 μg/ml) was delivered through a drip irrigation system to agricultural field soils planted to tomato and infested with
Pseudomonas putida
PpG7, the host of the salicylate catabolic plasmid NAH7. In nonfumigated soils infested with approximately 10
3
CFU of PpG7 per g in the top 30 cm, population densities were increased up to 112-fold within 14 days of the initial application of salicylate compared with the densities in the respective nonamended soils. Mean season-long population densities of PpG7 in the top 30 cm of soil were significantly increased (
P
< 0.01) from 216 CFU/g in nonamended soils to 1,370 CFU/g in salicylate-amended soils. In the respective rhizosphere soils, mean population densities of PpG7 were significantly increased (
P
< 0.01) from 92 to 2,066 CFU/cm of root. Soil fumigation interacted (
P
< 0.01) with salicylate amendment and further increased the mean population densities of PpG7 in nonrhizosphere soil by an additional 5,689 CFU/g of soil. This fumigation effect was not detected in rhizosphere soils. The effect of salicylate in increasing population densities of PpG7 in soil also was affected by inoculum level, field site, and soil depth. Proportionate differences were greater in soils infested with approximately 10
3
CFU of PpG7 per g than in comparable soils infested with 10
5
CFU/g. In low-inoculum soils, increases from salicylate amendments were 26- and 29-fold in rhizosphere and nonrhizosphere soils, respectively, and in high-inoculum soils, the respective increases were 5.6- and 5-fold. No increases of fungi able to utilize salicylate were detected in soils amended with salicylate. However, soil fumigation with metham-sodium significantly reduced (
P
< 0.01) population densities of fungal salicylate utilizers in rhizosphere and nonrhizosphere soils.
Enhanced Growth and Activity of a Biocontrol Bacterium Genetically Engineered To Utilize Salicylate
Authors: Colbert Stephen F. , Hendson Mavis , Ferri Mario , Schroth Milton N.
Published: January 2020

Plasmid NAH7 was transferred from
Pseudomonas putida
PpG7 to
P. putida
R20 [R20(NAH7)], an antagonist of
Pythium ultimum
. The plasmid did not affect growth or survival of R20(NAH7) and was stably maintained under nonselective conditions in broth and soil and on sugar beet seeds. Plasmid NAH7 conferred to R20(NAH7) the ability to utilize salicylate in culture, agricultural field soil, and on sugar beet seeds. The metabolic activity of R20(NAH7), but not the wild-type R20, was greatly increased in soil by amendment with salicylate (250 μg/g) as measured by induced respiration. Population densities of R20(NAH7) were also enhanced in salicylate-amended soil, increasing from approximately 1 × 10
5
CFU/g to approximately 3 × 10
8
CFU/g after 35 h of incubation. In contrast, population densities of R20(NAH7) in nonamended soil were approximately 3 × 10
6
CFU/g of soil after 35 h of incubation. The concentration of salicylate in soil affected the rate and extent of population increase by R20(NAH7). At 50 to 250 μg of salicylate per g of soil, population densities of R20(NAH7) increased to approximately 10
8
CFU/g of soil by 48 h of incubation, with the fastest increase at 100 μg/g. A lag phase of approximately 24 h occurred before the population density increased in the presence of salicylate at 500 μg/g; at 1,000 μg/g, population densities of R20(NAH7) declined over the time period of the experiment. Population densities of R20(NAH7) on sugar beet seeds in soils amended with 100 μg of salicylate per g were not increased while ample carbon was present in the spermosphere. However, after carbon from the seed had been utilized, population densities of R20(NAH7) decreased significantly less (
P
= 0.005) on sugar beet seeds in soil amended with salicylate than in nonamended soil.
Sodium chloride, potassium chloride, and virulence in Listeria monocytogenes
Authors: Myers E R , Dallmier A W , Martin S E
Published: January 2020

Virulence, as determined in a mouse model, and the virulence factor activities of catalase, superoxide dismutase, and listeriolysin O were examined in a parental strain (10403S) and in a nonhemolytic mutant strain (DP-L224) of Listeria monocytogenes. The cells were propagated in media containing various concentrations of sodium chloride or potassium chloride. Strains 10403S and DP-L224 exhibited significant increases in catalase activity and listeriolysin O activity when grown in medium containing either salt at 428 mM. The superoxide dismutase activities for both strains increased when they were grown in medium containing either salt. The superoxide dismutase activity was significantly increased only when cells were propagated in medium containing no salt compared with that when they were propagated in medium containing either salt at 1,112 mM. In addition, the listeriolysin O activity was highest for cells propagated in medium containing KCl at 428 mM, while the activity was significantly less for cells propagated in medium containing NaCl at an equal concentration. Virulence was examined in mouse livers and spleens after intravenous infection, and approximate 50% lethal doses were determined after intragastric and intraperitoneal infection. Each method of infection indicated that listeriolysin O is required for virulence, while growth in salt-containing medium or the production of higher levels of catalase, superoxide dismutase, and listeriolysin O do not appear to enhance the virulence of L. monocytogenes.
Transformation of
Kluyveromyces lactis
by Electroporation
Authors: Sánchez Manuel , Iglesias Francisco J. , Santamaría Carlos , Domínguez Angel
Published: January 2020

The physical and biological parameters involved in efficient transformation of
Kluyveromyces lactis
by electroporation have been analyzed. By using an optimum voltage and a constant volume of cell suspension in a cuvette, the efficiency of transformation increased with increases in cell numbers and plasmid concentration. However, the most important parameter was the time of the pulse. Changes of 1 ms decreased the efficiency of transformation more than 70 to 80%. Under our best conditions, between 10
6
and 10
7
transformants per μg of plasmid DNA could be obtained. Under certain conditions, the size of the plasmid also affected electroporation efficiency. In any case, we did not obtain integrative transformation with an autonomously replicating plasmid.
Effects of Condensed Tannins on Endoglucanase Activity and Filter Paper Digestion by
Fibrobacter succinogenes
S85
Authors: Bae Hee Dong , McAllister Tim A. , Yanke Jay , Cheng K.-J. , Muir A. D.
Published: January 2020

The effect of condensed tannins from birdsfoot trefoil (
Lotus corniculatus
L.) on the cellulolytic rumen bacterium
Fibrobacter succinogenes
S85 was examined. Condensed tannins inhibited endoglucanase activity in the extracellular culture fluid, at concentrations as low as 25 μg ml
-1
. In contrast, cell-associated endoglucanase activity increased in concentrations of condensed tannins between 100 and 300 μg ml
-1
. Inhibition of endoglucanase activity in both the extracellular and the cell-associated fractions was virtually complete at 400 μg of condensed tannins ml
-1
. Despite the sharp decline in extracellular endoglucanase activity with increasing concentrations of condensed tannins, filter paper digestion declined only moderately between 0 and 200 μg of condensed tannins ml
-1
. However, at 300 μg ml
-1
, filter paper digestion was dramatically reduced and at 400 μg ml
-1
, almost no filter paper was digested.
F. succinogenes
S85 was seen to form digestive grooves on the surface of cellulose, and at 200 μg ml
-1
, digestive pits were formed which penetrated into the interior of cellulose fibers. Cells grown with condensed tannins (100 to 300 μg ml
-1
) possessed large amounts of surface material, and although this material may have been capsular carbohydrate, its osmiophilic nature suggested that it had arisen from the formation of tannin-protein complexes on the cell surface. The presence of electron-dense extracellular material suggested that similar complexes were formed with extracellular protein.
Enantiomeric Composition of the
trans
-Dihydrodiols Produced from Phenanthrene by Fungi
Authors: Sutherland John B. , Fu Peter P. , Yang Shen K. , Von Tungeln Linda S. , Casillas Robert P. , Crow Sidney A. , Cerniglia Carl E.
Published: January 2020

The
trans
-dihydrodiols produced during the metabolism of phenanthrene by
Cunninghamella elegans, Syncephalastrum racemosum,
and
Phanerochaete chrysosporium
were purified by high-performance liquid chromatography (HPLC). The enantiomeric compositions and optical purities of the
trans
-dihydrodiols were determined to compare interspecific differences in the regio- and stereoselectivity of the fungal enzymes. Circular dichroism spectra of the
trans
-dihydrodiols were obtained, and the enantiomeric composition of each preparation was analyzed by HPLC with a chiral stationary-phase column. The phenanthrene
trans
-1,2-dihydrodiol produced by
C. elegans
was a mixture of the 1
R
,2
R
and 1
S
,2
S
enantiomers in variable proportions. The phenanthrene
trans
-3,4-dihydrodiol produced by
P. chrysosporium
was the optically pure 3
R
,4
R
enantiomer, but that produced by
S. racemosum
was a 68:32 mixture of the 3
R
,4
R
and 3
S
,4
S
enantiomers. The phenanthrene
trans
-9,10-dihydrodiol produced by
P. chrysosporium
was predominantly the 9
S
,10
S
enantiomer, but those produced by
C. elegans
and
S. racemosum
were predominantly the 9
R
,10
R
enantiomer. The results indicate that although different fungi may exhibit similar regioselectivity, there still may be differences in stereoselectivity that depend on the species and the cultural conditions.
Presumptive fecal streptococci in environmental samples characterized by one-dimensional sodium dodecyl sulfate-polyacrylamide gel electrophoresis
Authors: Niemi R M , Niemelä S I , Bamford D H , Hantula J , Hyvärinen T , Forsten T , Raateland A
Published: January 2020

The use of fecal streptococci as fecal indicators requires better knowledge of the ecology of these bacteria. We isolated 371 presumptive fecal streptococci from environmental samples--domestic wastewater, forest industry wastewater, contaminated surface and seawater, well water, cow dung, bird droppings, and pristine waters--and clustered them according to their protein profiles in one-dimensional sodium dodecyl sulfate-polyacrylamide gel electrophoresis analysis. Some clusters could be tentatively identified with the help of reference strains. Samples from each environment had a typical composition of streptococcus types. Enterococcus faecalis was present, but not as a dominating enterococcal species, in samples in which fecal contamination was probable. Enterococcus faecium, Enterococcus durans, Enterococcus hirae, and Enterococcus mundtii had protein profiles that were difficult to distinguish from each other. These bacteria were found in a variety of samples. Enterococcus casseliflavus and Enterococcus gallinarum had identical protein profiles. On the basis of the maximum temperatures for growth and pigment production, isolates of this protein profile group common in forest industry wastewaters were identified as E. casseliflavus. Lactococcus lactis subsp. lactis was also found in this environment. Nearly all strains from pristine waters belonged to protein profile groups which could not be identified with the aid of known Aerococcus, Enterococcus, Lactococcus, or Streptococcus strains. The maximum temperatures for growth and the results of fatty acid analysis were in general agreement within each protein profile group.
Genetic instability and strain degeneration in Streptomyces rimosus
Authors: Gravius B , Bezmalinović T , Hranueli D , Cullum J
Published: January 2020

During a strain selection program to improve oxytetracycline production in Streptomyces rimosus R6, isolates that showed extreme morphological instability appeared. Propagation via spores gave much higher instability than did propagation via mycelial fragments. Five phenotypic traits were affected: sporulation, pigmentation, colony morphology, oxytetracycline production, and oxytetracycline resistance. The variants were classified on the basis of oxytetracycline resistance into three classes. Class I variants (99% of variants) showed parental levels of resistance but were very heterogeneous regarding the other phenotypes. No DNA rearrangements were detected in primary class I variants. Class II variants (1% of variants, oxytetracycline sensitive) were phenotypically uniform, and most variants carried the same large deletion of ca. 455 kb, including the oxytetracycline resistance gene otrB. Class III variants (0.1% of variants, increased oxytetracycline resistance) were phenotypically uniform and overproduced a brown pigment and oxytetracycline. Most of these variants also showed a reproducible large-scale DNA rearrangement, which probably included deletion and a low-level reiteration (three or four copies) of a DNA fragment. "Revertants" of some class I variants show a similar DNA rearrangement to the class III variants, but there is extensive reiteration of sequences of about 200 kb, including the otrB gene. The significance of these results for the problem of strain degeneration and overproduction of antibiotics is discussed.
Microbial transformations of ferulic acid by Saccharomyces cerevisiae and Pseudomonas fluorescens
Authors: Huang Z , Dostal L , Rosazza J P
Published: January 2020

Saccharomyces cerevisiae (dry baker's yeast) and Pseudomonas fluorescens were used to convert trans-ferulic acid into 4-hydroxy-3-methoxystyrene in 96 and 89% yields, respectively. The metabolites were isolated by solid-phase extraction and analyzed by thin-layer chromatography and high-performance liquid chromatography. The identities of the metabolites were determined by 1H- and 13C-nuclear magnetic resonance spectroscopy and by mass spectrometry. The mechanism of the decarboxylation of ferulic acid was investigated by measuring the degree and position of deuterium incorporated into the styrene derivative from D2O by mass spectrometry and by both proton and deuterium nuclear magnetic resonance spectroscopies. Resting cells of baker's yeast reduced ferulic acid to 4-hydroxy-3-methoxyphenylpropionic acid in 54% yield when incubations were under an argon atmosphere.
Reductive Dechlorination of the Nitrogen Heterocyclic Herbicide Picloram
Authors: Ramanand K. , Nagarajan A. , Suflita Joseph M.
Published: January 2020

The anaerobic biodegradation of picloram (3,5,6-trichloro-4-amino-2-pyridinecarboxylic acid) in freshwater sediment was favored under methanogenic conditions but not when sulfate or nitrate was available as a terminal electron acceptor. Under the former conditions, more than 85% of the parent substrate (340 μM) was removed from nonsterile incubations in 30 days, following a 50-day acclimation period. Concomitant with substrate decay, an intermediate transiently accumulated in the sediment slurries. By liquid chromatography-mass spectrometry, the intermediate was identified as an isomer of dichloro-4-amino-2-pyridinecarboxylic acid. Proton nuclear magnetic resonance evidence suggested that a chlorine was reductively removed from the parent substrate at the position
meta
to the nitrogen heteroatom. Upon continued incubation, the dechlorinated product was transformed into an unidentified compound which accumulated and resisted further decay. The addition of sulfate or bromoethanesulfonic acid to sediment slurries inhibited picloram dehalogenation, but molybdate reversed the inhibitory effect of sulfate on pesticide metabolism. These findings help clarify the fate of a halogenated nitrogen heterocyclic herbicide in anaerobic environments.
Estimation of ruminal bacteriophage numbers by pulsed-field gel electrophoresis and laser densitometry
Authors: Klieve A V , Swain R A
Published: January 2020

To investigate phage activity in the rumen, a method for quantifying phage has been developed. By differential centrifugation and ultrafiltration, phage particles were separated and concentrated from ruminal fluid. Linear double-stranded DNA from this fraction containing predominantly tailed phage was isolated and separated by size, using pulsed-field gel electrophoresis (PFGE). Laser densitometry of gel photographs allowed the numbers of phages with DNA in each size region to be calculated and, therefore, the total numbers per milliliter of ruminal fluid to be estimated. Phage numbers were estimated to be between 3 x 10(9) and 1.6 x 10(10) particles ml of ruminal fluid-1. The phage population, as gauged by the appearance of DNA on PFGE gels, had two major components. A broad region of DNA between 30 and 200 kb was always present on PFGE gels. It appears this region comprises DNA from a great many different phages and would include most of the temperate phages. In addition, discrete DNA bands ranging in size from 10 to 850 kb were frequently observed. DNA from one such band, of 12 kb in size, was shown to consist primarily of a single DNA type, suggesting that it originated from a specific phage. It is postulated that the discrete bands are due to epidemics or blooms of phage activity from specific, probably lytic, phages. The method that has been developed will greatly enhance future investigations into the interactions between the ruminal phage population, the ruminal bacterial population, and animal nutrition and growth.(ABSTRACT TRUNCATED AT 250 WORDS)
Effect of treated-sewage contamination upon bacterial energy charge, adenine nucleotides, and DNA content in a sandy aquifer on Cape Cod
Authors: Metge D W , Brooks M H , Smith R L , Harvey R W
Published: January 2020

Changes in adenylate energy charge (ECA) and in total adenine nucleotides (A(T) and DNA content (both normalized to the abundance of free-living, groundwater bacteria) in response to carbon loading were determined for a laboratory-grown culture and for a contaminated aquifer. The latter study involved a 3-km-long transect through a contaminant plume resulting from continued on-land discharge of secondary sewage to a shallow, sandy aquifer on Cape Cod, Mass. With the exception of the most contaminated groundwater immediately downgradient from the contaminant source, DNA and adenylate levels correlated strongly with bacterial abundance and decreased exponentially with increasing distance downgradient. ECAS (0.53 to 0.60) and the ratios of ATP to DNA (0.001 to 0.003) were consistently low, suggesting that the unattached bacteria in this groundwater study are metabolically stressed, despite any eutrophication that might have occurred. Elevated ECAS (up to 0.74) were observed in glucose-amended groundwater, confirming that the metabolic state of this microbial community could be altered. In general, per-bacterium DNA and ATP contents were approximately twofold higher in the plume than in surrounding groundwater, although ECA and per-bacterium levels of A(T) differed little in the plume and the surrounding uncontaminated groundwater. However, per-bacterium levels of DNA and A(T) varied six- and threefold, respectively, during a 6-h period of decreasing growth rate for an unidentified pseudomonad isolated from contaminated groundwater and grown in batch culture.(ABSTRACT TRUNCATED AT 250 WORDS)
Purification and some properties of an alkaline xylanase from alkaliphilic Bacillus sp. strain 41M-1
Authors: Nakamura S , Wakabayashi K , Nakai R , Aono R , Horikoshi K
Published: January 2020

An alkaliphilic Bacillus sp. strain, 41M-1, isolated from soil produced multiple xylanases extracellularly. One of these xylanases was purified to homogeneity by ammonium sulfate fractionation and anion-exchange chromatography. The moleculr mass of this enzyme (xylanase J) was 36 kDa, and the isoelectric point was pH 5.3. Xylanase J was most active at pH 9.0. The optimum temperature for the activity at pH 9.0 was around 50 degrees C. The enzyme was stable up to 55 degrees C at pH 9.0 for 30 min. Xylanase J was completely inhibited by the Hg2+ion and N-bromosuccinimide. The predominant products of xylan hydrolysate were xylobiose, xylotriose, and higher oligosaccharides, indicating that the enzyme was an endoxylanase. The apparent Km and Vmax values on xylan were 3.3 mg/ml and 1,100 micromol-1 mg-1, respectively. Xylanase J showed high sequence homology with the xylanases from Bacillus pumilus and Clostridium acetobutylicum in the N-terminal region. Xylanase J acted on neither crystalline cellulose nor carboxymethyl cellulose, indicating a possible application of the enzyme in biobleaching processes.
Cell Density and Growth Phase as Factors in the Resistance of a Biofilm of
Pseudomonas aeruginosa
(ATCC 27853) to Iodine
Authors: Brown Matthew L. , Gauthier Joseph J.
Published: January 2020

Previous studies have shown that biofilms exhibit enhanced resistance to iodine. Investigations were conducted to determine the relative importance of growth phase versus cell density on biofilm resistance of
Pseudomonas aeruginosa
(ATCC 27853) to iodine. Cell density is a contributing factor to resistance, whereas growth to the stationary phase is not sufficient to achieve resistance.
Is an activator protein-2-like transcription factor involved in regulating gene expression during nitrogen limitation in fungi?
Authors: Dhawale S S
Published: January 2020

The upstream sequences of all published lignin peroxidase and manganese peroxidase genomic clones from Phanerochaete chrysosporium were analyzed. This analysis revealed the presence of putative activator protein-2 (AP-2) recognition sequences in 11 of 15 lignin peroxidase genes. The lignin peroxidase clone GLG6 and the manganese peroxidase gene (mnp-1) have two copies of putative AP-2 sequence in the upstream region. Interestingly, the lignin peroxidase gene VLG4 of another white rot fungus, Trametes versicolor, and the nit-2 gene of Neurospora crassa also contain putative AP-2-binding sequences. Since all of these genes are regulated by nutrient nitrogen, I hypothesize that an AP-2-like transcription factor may be involved in inducing gene expression during nitrogen limitation in fungi.
Colony immunoblot assay of botulinal toxin
Authors: Goodnough M C , Hammer B , Sugiyama H , Johnson E A
Published: January 2020

Botulinal neurotoxin in and around colonies of Clostridium botulinum types A, B, and E and of toxigenic Clostridium butyricum was detected by an enzyme-linked immunoassay procedure whereby the toxin was transferred from the agar medium to a nitrocellulose support and the immobilized toxin was probed with type-specific antibodies. The method identified the toxin types of the colonies grown from a mixed inoculum of C. botulinum serotypes. The specificity of the antitoxins for type A and B toxins was improved by adsorption of the antitoxins with the antigens of heterologous type cultures.
Localization and Characterization of α-Glucosidase Activity in
Brettanomyces lambicus
Authors: Kumara H. M. C. Shantha , De Cort S. , Verachtert H.
Published: January 2020

Brettanomyces lambicus
was isolated and identified from a typical overattenuating Belgian lambic beer and exhibited extracellular and intracellular α-glucosidase activities. Production of the intracellular enzyme was higher than production of the extracellular enzyme, and localization studies showed that the intracellular α-glucosidase is mostly soluble and partially cell wall bound. Both intracellular and extracellular enzymes were purified by ammonium sulfate precipitation, gel filtration (Sephadex G-150, Sephadex G-200, Ultrogel AcA-44), and ion-exchange chromatography (sulfopropyl-Sephadex C-50, (carboxymethyl-Sephadex C-50). The intracellular α-glucosidase exhibited optimum activity at 39°C and pH 6.2. The extracellular enzyme exhibited optimum catalytic activity at 40°C and pH 6.0. The molecular masses of purified intracellular and extracellular α-glucosidases, as determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, were 72,500 and 77,250, respectively. For both enzymes there was a decrease in the rate of hydrolysis with an increase in the degree of polymerization, and both enzymes hydrolyzed dextrins isolated from lambic wort (degrees of polymerization, 3 to 9 and more than 9). The

K
m

values for
p
-nitrophenyl-α-
d
-glucopyranoside, maltose, and maltotriose for the intracellular enzyme were 0.9, 3.4, and 3.7 mM, respectively. The

K
i

values for both enzymes were between 28.5 and 57 μM for acarbose and between 7.45 and 15.7 mM for Tris. These enzymes are probably involved in the overattenuation of spontaneously fermented lambic beer.
Analysis of Tox5 gene expression in Gibberella pulicaris strains with different trichothecene production phenotypes
Authors: Hohn T M , Desjardins A E , McCormick S P
Published: January 2020

The Tox5 gene encodes trichodiene synthase, the first unique enzyme in the trichothecene biosynthetic pathway. In Gibberella pulicaris R-6380, the level of Tox5 mRNA was found to increase 47-fold in early stationary phase. Sequence analysis of the Tox5 promoter regions from geographically distinct strains of G. pulicaris revealed the existence of two Tox5 alleles (Tox5-1 and Tox5-2). All G. pulicaris strains that produce high levels of trichothecenes in liquid culture carry a 42-nucleotide (nt) tandem repeat sequence (Tox5-1) in the Tox5 promoter region, whereas strains that produce low levels of trichothecenes carry a single copy of this sequence (Tox5-2). A genetic cross between high- and low-level trichothecene producers resulted in the cosegregation of higher-level trichothecene production with the Tox5-1 allele. To determine the importance of the 42-nt repeat sequence in the regulation of Tox5 expression, reporter gene constructs carrying either the Tox5-1 or the Tox5-2 promoter region fused to the beta-galactosidase gene of Escherichia coli were introduced into the high-level-trichothecene-producing strain, R-6380. Expression of reporter gene activity in transformants was found to be regulated in a manner similar to Tox5 expression but appeared to be independent of the 42-nt sequence copy number. These results indicate that transcriptional controls play an important role in the regulation of Tox5 expression and that genes involved in trichothecene biosynthesis in G. pulicaris may be linked to Tox5.
Coal Depyritization by the Thermophilic Archaeon
Metallosphaera sedula
Authors: Clark Thomas R. , Baldi Franco , Olson Gregory J.
Published: January 2020

The kinetics of pyrite oxidation by
Metallosphaera sedula
were investigated with mineral pyrite and two coals with moderate (Pittsburgh no. 8) and high (New Brunswick, Canada) pyritic sulfur content.
M. sedula
oxidized mineral pyrite at a greater rate than did another thermophile,
Acidianus brierleyi
, or a mesophile,
Thiobacillus ferrooxidans
. Maximum rates of coal depyritization were also greater with
M. sedula
, although the magnitude of biological stimulation above abiotic rates was notably less than with mineral pyrite. Coal depyritization appears to be limited by the oxidation of pyrite with ferric ions and not by the rate of biotic oxidation of ferrous iron, as evidenced by the maintenance of a high ratio of ferric to ferrous iron in solution by
M. sedula
. Significant precipitation of hydronium jarosite at elevated temperature occurred only with New Brunswick coal.
Measurement of rRNA Variations in Natural Communities of Microorganisms on the Southeastern U.S. Continental Shelf
Authors: Kramer Jonathan G. , Singleton Fred L.
Published: January 2020

The development of a clear understanding of the physiology of marine prokaryotes is complicated by the difficulties inherent in resolving the activity of various components of natural microbial communities. Application of appropriate molecular biological techniques offers a means of overcoming some of these problems. In this regard, we have used direct probing of bulk RNA purified from selective size fractions to examine variations in the rRNA content of heterotrophic communities and
Synechococcus
populations on the southeastern U.S. continental shelf. Heterotrophic communities in natural seawater cultures amended with selected substrates were examined.
Synechococcus
populations were isolated from the water column by differential filtration. The total cellular rRNA content of the target populations was assayed by probing RNA purified from these samples with an oligonucleotide complementing a universally conserved region in the eubacterial 16S rRNA (heterotrophs) or with a 1.5-kbp fragment encoding the
Synechococcus
sp. strain WH 7803 16S rRNA (cyanobacteria). The analyses revealed that heterotrophic bacteria responded to the addition of glucose and trace nutrients after a 6-h lag period. However, no response was detected after amino acids were added. The cellular rRNA content increased 48-fold before dropping to a value 20 times that detected before nutrients were added. Variations in the rRNA content from
Synechococcus
spp. followed a distinct diel pattern imposed by the phasing of cell division within the irradiance cycle. The results indicate that careful application of these appropriate molecular biological techniques can be of great use in discerning basic physiological characteristics of selected natural populations and the mechanisms which regulate growth at the subcellular level.
Structural stability of Bacillus thuringiensis delta-endotoxin homolog-scanning mutants determined by susceptibility to proteases
Authors: Almond B D , Dean D H
Published: January 2020

Forty homolog-scanning (double-reciprocal-crossover) mutant proteins of two Bacillus thuringiensis delta-endotoxin genes (cryIAa and cryIAc) were examined for potential structural alterations by a series of proteolytic assays. Three groups of mutants could be identified. Group 1, consisting of 13 mutants, showed no delta-endotoxin present during overexpression conditions in Escherichia coli (48 h at 37 degrees C, with a ptac promoter). These mutants produced full-sized delta-endotoxin detectable by polyacrylamide gel electrophoresis with Coomassie blue staining or Western immunoanalysis after 24 h of growth but not after 48 h, suggesting sensitivity to intracellular proteases. Group 2 consisted of 13 mutants that produced stable delta-endotoxins that were completely digested by 2% bovine trypsin. In contrast, native delta-endotoxin produces a 65,000-Da trypsin-resistant peptide, which is the active toxin. Group 3 mutants expressed delta-endotoxin and trypsin-stable toxins, similar to the wild type. In this study, 12 group 3 mutant toxins were compared with wild type toxins by thermolysin digestion at a range of temperatures. The two wild-type toxins exhibited significant differences in thermolysin digestion midpoints. Among the group 3 mutants, most possessed significantly different protein stabilities relative to their parental toxins. Two of the group 3 mutants were observed to have exchanged the thermolysin sensitivity properties of the parental toxins.
Methylmercury Resistance in
Desulfovibrio desulfuricans
Strains in Relation to Methylmercury Degradation
Authors: Baldi Franco , Pepi Milva , Filippelli Marco
Published: January 2020

Two strains of
Desulfovibrio desulfuricans
, one known to synthesize monomethylmercury from ionic mercury, were grown to determine methylmercury toxicity and for comparison with an anaerobic strain of
Clostridium pasteurianum
, a H
2
producer, and with the broad-spectrum mercury-resistant
Pseudomonas putida
strain FB-1, capable of degrading 1 μg of methylmercury to methane and elemental mercury in 2 h. The CH
3
HgCl resistance of
D. desulfuricans
strains was 10 times that of
P. putida
FB-1 and 100 times that of
C. pasteurianum
. The methylmercury resistance of
D. desulfuricans
was related to the disappearance of methylmercury from cultures by transformation to dimethylmercury, metacinnabar, methane, and traces of ionic mercury. During a 15-day experiment the kinetics of the two volatile compounds dimethylmercury [(CH
3
)
2
Hg] and methane were monitored in the liquid by a specific new technique with purge-and-trap gas chromatography in line with Fourier transform infrared spectroscopy and in the headspace by gas chromatography with flame ionization detection. Insoluble metacinnabar (cubic HgS) of biological origin was detected by X-ray diffractometry in the gray precipitate from the insoluble residue of the pellet of a 1-liter culture spiked with 100 mg of CH
3
HgCl. This was compared with a 1-liter culture of
D. desulfuricans
LS spiked with 100 mg of HgCl
2
. In a further experiment, it was demonstrated that insoluble, decomposable, white dimethylmercury sulfide [(CH
3
Hg)
2
S] formed instantly in the reaction of methylmercury with hydrogen sulfide. This organomercurial was extracted with chloroform and identified by gas chromatography in line with mass spectrometry. The
D. desulfuricans
strains were resistant to high concentrations of methylmercury because they produced insoluble dimethylmercury sulfide, which slowly decomposed under anaerobic conditions to metacinnabar and volatilized to dimethylmercury and methane between pHs 6.2 and 6.5 for high (4.5-g · liter
-1
) or low (0.09-g · liter
-1
) sulfate contents. Methane was produced from CH
3
HgCl at a lower rate than by the broad-spectrum Hg-resistant
P. putida
strain FB-1.