bluuebunny/crossref_metadata_2025_split huggingface dataset:
Mannan-Degrading Enzymes from
Cellulomonas fimi
Authors: Stoll Dominik , Stålbrand Henrik , Warren R. Antony J. 
Published: December 2019

ABSTRACT

The genes
man26a
and
man2A
from
Cellulomonas fimi
encode mannanase 26A (Man26A) and β-mannosidase 2A (Man2A), respectively. Mature Man26A is a secreted, modular protein of 951 amino acids, comprising a catalytic module in family 26 of glycosyl hydrolases, an S-layer homology module, and two modules of unknown function. Exposure of Man26A produced by
Escherichia coli
to
C. fimi
protease generates active fragments of the enzyme that correspond to polypeptides with mannanase activity produced by
C. fimi
during growth on mannans, indicating that it may be the only mannanase produced by the organism. A significant fraction of the Man26A produced by
C. fimi
remains cell associated. Man2A is an intracellular enzyme comprising a catalytic module in a subfamily of family 2 of the glycosyl hydrolases that at present contains only mammalian β-mannosidases.
PCR Comparison of
Mycobacterium avium
Isolates Obtained from Patients and Foods
Authors: Yoder S. , Argueta C. , Holtzman A. , Aronson T. , Berlin O. G. W. , Tomasek P. , Glover N. , Froman S. , Stelma G. 
Published: December 2019

ABSTRACT

Mycobacterium avium
is a cause of disseminated disease in AIDS patients. A need for a better understanding of possible sources and routes of transmission of this organism has arisen. This study utilized a PCR typing method designed to amplify DNA segments located between the insertion sequences IS
1245
and IS
1311
to compare levels of relatedness of
M. avium
isolates found in patients and foods. Twenty-five of 121 food samples yielded 29 mycobacterial isolates, of which 12 were
M. avium
. Twelve food and 103 clinical
M. avium
isolates were tested. A clinical isolate was found to be identical to a food isolate, and close relationships were found between two patient isolates and two food isolates. Relatedness between food isolates and patient isolates suggests the possibility that food is a potential source of
M. avium
infection. This study demonstrates a rapid, inexpensive method for typing
M. avium
, possibly replacing pulsed-field gel electrophoresis.
BACTOX, a Rapid Bioassay That Uses Protozoa To Assess the Toxicity of Bacteria
Authors: Schlimme Wolfram , Marchiani Marcello , Hanselmann Kurt , Jenni Bernard 
Published: December 2019

ABSTRACT

A new type of toxicity test based on the protozoan
Tetrahymena pyriformis
has been developed to assess the overall toxicity of bacterial strains given as prey. This simple and rapid test is able to detect toxicant-producing bacteria, which may present a biohazard. It can also be used for the risk assessment of microbes designed for deliberate release.
Conjugal Transfer but Not Quorum-Dependent
tra
Gene Induction of pTiC58 Requires a Solid Surface
Authors: Piper Kevin R. , Farrand Stephen K. 
Published: December 2019

ABSTRACT

Donors of
Agrobacterium tumefaciens
harboring a transfer-constitutive derivative of the nopaline-type Ti plasmid pTiC58 transferred this element at frequencies 3 to 4 orders of magnitude higher in matings conducted on solid surfaces than in those conducted in liquid medium. However, as measured with a
lacZ
reporter fusion, the
tra
genes of the wild-type Ti plasmid were inducible by opines to indistinguishable levels on solid and in liquid medium. Donors induced in liquid transferred the Ti plasmid at high frequency when mated with recipients on solid medium. We conclude that while formation of stable mating pairs and subsequent transfer of the Ti plasmid is dependent on a solid stratum, the regulatory system can activate
tra
gene expression to equivalent levels in liquid and on solid surfaces.
Method for Detection and Enumeration ofCryptosporidium parvumOocysts in Feces, Manures, and Soils
Authors: Kuczynska Ewa , Shelton Daniel R. 
Published: December 2019

ABSTRACTEight concentration and purification methods were evaluated to determine percentages of recovery ofCryptosporidium parvumoocysts from calf feces. The NaCl flotation method generally resulted in the highest percentages of recovery. Based on the percentages of recovery, the amounts of fecal debris in the final oocyst preparations, the relatively short processing time (<3 h), and the low expense, the NaCl flotation method was chosen for further evaluation. Extraction efficiency was evaluated by using oocyst concentrations of 25, 50, 102, 103, 104, and 105oocysts g of bovine feces−1. The percentages of recovery ranged from 10.8% (25 oocysts g−1) to 17.0% (104oocysts g−1) (r2= 0.996). A conservative estimate of the detection limit for bovine feces is ca. 30 oocysts g of feces−1. Percentages of recovery were determined for six different types of animal feces (cow, horse, pig, sheep, deer, and chicken feces) at a single oocyst concentration (104oocysts g−1). The percentages of recovery were highest for bovine feces (17.0%) and lowest for chicken feces (3.2%). Percentages of recovery were determined for bovine manure after 3 to 7 days of storage. The percentages of recovery ranged from 1.9 to 3.5% depending on the oocyst concentration, the time of storage, and the dispersing solution. The percentages of oocyst recovery from soils were evaluated by using different flotation solutions (NaCl, cold sucrose, ZnSO4), different dispersing solutions (Triton X-100, Tween 80, Tris plus Tween 80), different dispersion techniques (magnetic stirring, sonication, blending), and different dispersion times (5, 15, and 30 min). Twenty-five-gram soil samples were used to reduce the spatial variability. The highest percentages of recovery were obtained when we used 50 mM Tris–0.5% Tween 80 as the dispersing solution, dispersion for 15 min by stirring, and saturated NaCl as the flotation solution. The percentages of oocyst recovery from freshly spiked sandy loam, silty clay loam, and clay loam soils were ca. 12 to 18, 8, and 6%, respectively. The theoretical detection limits were ca. 1 to 2 oocysts g of soil−1depending on the soil type. The percentages of recovery without dispersant (distilled H2O or phosphate-buffered saline) were less than 0.1%, which indicated that oocysts adhere to soil particles. The percentages of recovery decreased with storage time, although the addition of dispersant (Tris-Tween 80) before storage appeared to partially prevent adhesion. These data indicate that the NaCl flotation method is suitable for routine detection and enumeration of oocysts from feces, manures, soils, or soil-manure mixtures.
Influence of Kernel Age on Fumonisin B
1
Production in Maize by
Fusarium moniliforme
Authors: Warfield Colleen Y. , Gilchrist David G. 
Published: December 2019

ABSTRACT

Production of fumonisins by
Fusarium moniliforme
on naturally infected maize ears is an important food safety concern due to the toxic nature of this class of mycotoxins. Assessing the potential risk of fumonisin production in developing maize ears prior to harvest requires an understanding of the regulation of toxin biosynthesis during kernel maturation. We investigated the developmental-stage-dependent relationship between maize kernels and fumonisin B
1
production by using kernels collected at the blister (R2), milk (R3), dough (R4), and dent (R5) stages following inoculation in culture at their respective field moisture contents with
F. moniliforme
. Highly significant differences (
P
≤ 0.001) in fumonisin B
1
production were found among kernels at the different developmental stages. The highest levels of fumonisin B
1
were produced on the dent stage kernels, and the lowest levels were produced on the blister stage kernels. The differences in fumonisin B
1
production among kernels at the different developmental stages remained significant (
P
≤ 0.001) when the moisture contents of the kernels were adjusted to the same level prior to inoculation. We concluded that toxin production is affected by substrate composition as well as by moisture content. Our study also demonstrated that fumonisin B
1
biosynthesis on maize kernels is influenced by factors which vary with the developmental age of the tissue. The risk of fumonisin contamination may begin early in maize ear development and increases as the kernels reach physiological maturity.
Purification and Characterization of an α-Glucosidase from
Rhizobium
sp. (
Robinia pseudoacacia
L.) Strain USDA 4280
Authors: Berthelot Karine , Delmotte Francis M. 
Published: December 2019

ABSTRACT

A novel α-glucosidase with an apparent subunit mass of 59 ± 0.5 kDa was purified from protein extracts of
Rhizobium
sp. strain USDA 4280, a nodulating strain of black locust (
Robinia pseudoacacia
L), and characterized. After purification to homogeneity (475-fold; yield, 18%) by ammonium sulfate precipitation, cation-exchange chromatography, hydrophobic chromatography, dye chromatography, and gel filtration, this enzyme had a pI of 4.75 ± 0.05. The enzyme activity was optimal at pH 6.0 to 6.5 and 35°C. The activity increased in the presence of NH
4
+
and K
+
ions but was inhibited by Cu
2+
, Ag
+
, Hg
+
, and Fe
2+
ions and by various phenyl, phenol, and flavonoid derivatives. Native enzyme activity was revealed by native gel electrophoresis and isoelectrofocusing-polyacrylamide gel electrophoresis with fluorescence detection in which 4-methylumbelliferyl α-glucoside was the fluorogenic substrate. The enzyme was more active with α-glucosides substituted with aromatic aglycones than with oligosaccharides. This α-glucosidase exhibited Michaelis-Menten kinetics with 4-methylumbelliferyl α-
d
-glucopyranoside (

K
m

, 0.141 μM;
V
max
, 6.79 μmol min
−1
mg
−1
) and with
p
-nitrophenyl α-
d
-glucopyranoside (

K
m

, 0.037 μM;
V
max
, 2.92 μmol min
−1
mg
−1
). Maltose, trehalose, and sucrose were also hydrolyzed by this enzyme.
Factors Influencing In Vitro Killing of Bacteria by Hemocytes of the Eastern Oyster (Crassostrea virginica)
Authors: Genthner Fred J. , Volety Aswani K. , Oliver Leah M. , Fisher William S. 
Published: December 2019

ABSTRACTA tetrazolium dye reduction assay was used to study factors governing the killing of bacteria by oyster hemocytes. In vitro tests were performed on bacterial strains by using hemocytes from oysters collected from the same location in winter and summer.Vibrio parahaemolyticusstrains, altered in motility or colonial morphology (opaque and translucent), andListeria monocytogenesmutants lacking catalase, superoxide dismutase, hemolysin, and phospholipase activities were examined in winter and summer.Vibrio vulnificusstrains, opaque and translucent (with and without capsules), were examined only in summer. AmongV. parahaemolyticusandL. monocytogenes, significantly (P< 0.05) higher levels of killing by hemocytes were observed in summer than in winter.L. monocytogeneswas more resistant thanV. parahaemolyticusorV. vulnificusto the bactericidal activity of hemocytes. In winter, both translucent strains ofV. parahaemolyticusshowed significantly (P< 0.05) higher susceptibility to killing by hemocytes than did the wild-type opaque strain. In summer, only one of theV. parahaemolyticustranslucent strains showed significantly (P< 0.05) higher susceptibility to killing by hemocytes than did the wild-type opaque strain. No significant differences (P> 0.05) in killing by hemocytes were observed between opaque (encapsulated) and translucent (nonencapsulated) pairs ofV. vulnificus. Activities of 19 hydrolytic enzymes were measured in oyster hemolymph collected in winter and summer. Only one enzyme, esterase (C4), showed a seasonal difference in activity (higher in winter than in summer). These results suggest that differences existed between bacterial genera in their ability to evade killing by oyster hemocytes, that a trait(s) associated with the opaque phenotype may have enabledV. parahaemolyticusto evade killing by the oyster’s cellular defense, and that bactericidal activity of hemocytes was greater in summer than in winter.
A Sensitive Nested Reverse Transcriptase PCR Assay To Detect Viable Cells of the Fish Pathogen
Renibacterium salmoninarum
in Atlantic Salmon (
Salmo salar
L.)
Authors: Cook Marcia , Lynch William H. 
Published: December 2019

ABSTRACT

A nested reverse transcriptase (RT) PCR assay detected mRNA of the salmonid pathogen
Renibacterium salmoninarum
in samples of RNA extracts of between 1 and 10 cells. Total RNA was extracted from cultured bacteria, Atlantic salmon (
Salmo salar
L.) kidney tissue and ovarian fluid seeded with the pathogen, and kidney tissue from both experimentally challenged and commercially raised fish. Following DNase treatment, extracted RNA was amplified by both RT PCR and PCR by using primers specific for the gene encoding the major protein antigen of
R. salmoninarum
. A 349-bp amplicon was detected by polyacrylamide gel electrophoresis and silver stain. Inactivation of cultured bacteria by rifampin or erythromycin produced a loss of nested RT PCR mRNA detection corresponding to a loss of bacterial cell viability determined from plate counts but no loss of DNA detection by PCR. In subclinically diseased fish, nested RT PCR identified similar levels of infected fish as determined by viable pathogen culture. Higher percentages of fish testing positive were generated by PCR, particularly in samples from fish previously subjected to antibiotic chemotherapy where 93% were PCR positive, but only 7% were nested RT PCR and culture positive. PCR can generate false-positive data from amplification of target DNA from nonviable pathogen cells. Therefore, nested RT PCR may prove useful for monitoring cultured Atlantic salmon for the presence of viable
R. salmoninarum
within a useful time frame, particularly samples from broodstock where antibiotic chemotherapy is used prior to spawning to reduce vertical pathogen transmission.
Purification and Characterization of a Secreted Laccase of
Gaeumannomyces graminis
var.
tritici
Authors: Edens William A. , Goins Tresa Q. , Dooley David , Henson Joan M. 
Published: December 2019

ABSTRACT

We purified a secreted fungal laccase from filtrates of
Gaeumannomyces graminis
var.
tritici
cultures induced with copper and xylidine. The active protein had an apparent molecular mass of 190 kDa and yielded subunits with molecular masses of 60 kDa when denatured and deglycosylated. This laccase had a pI of 5.6 and an optimal pH of 4.5 with 2,6-dimethoxyphenol as its substrate. Like other, previously purified laccases, this one contained several copper atoms in each subunit, as determined by inductively coupled plasma spectroscopy. The active enzyme catalyzed the oxidation of 2,6-dimethoxyphenol (

K
m

= 2.6 × 10
−5
± 7 × 10
−6
M), catechol (

K
m

= 2.5 × 10
−4
± 1 × 10
−5
M), pyrogallol (

K
m

= 3.1 × 10
−4
± 4 × 10
−5
M), and guaiacol (

K
m

= 5.1 × 10
−4
± 2 × 10
−5
M). In addition, the laccase catalyzed the polymerization of 1,8-dihydroxynaphthalene, a natural fungal melanin precursor, into a high-molecular-weight melanin and catalyzed the oxidation, or decolorization, of the dye poly B-411, a lignin-like polymer. These findings indicate that this laccase may be involved in melanin polymerization in this phytopathogen’s hyphae and/or in lignin depolymerization in its infected plant host.
Effect of Growth Temperature on Hydrolytic and Esterifying Activities from
Pseudomonas fragi
CRDA 037 Grown on Whey
Authors: Fonchy E. , Morin A. , Rodrigue N. , Müller B. , Chalier P. 
Published: December 2019

ABSTRACT

The production of hydrolytic and esterifying activities of
Pseudomonas fragi
CRDA 037 grown on a whey-based medium was investigated at different temperatures over time. The optimal temperature was found to be critical and different for the production of both activities. The highest hydrolytic activity was detected with bacteria cultivated at between 24°C (149.2 U/liter) and 27°C (133.8 U/liter), while the highest production of ethyl valerate (esterifying activity) was observed by using biomass grown at 15°C (0.75 U/liter). When the fermentation temperature was increased, the incubation time necessary to reach the maximal concentration of both activities was reduced. Studies of the thermostability of both activities showed that the hydrolytic activity was more stable than the esterifying activity at 15 and 24°C. Statistical analysis allowed the determination of the equations that predicted the production of hydrolytic and esterifying activities as a function of time and growth temperature. The optimal assay temperatures for the hydrolytic and esterifying activities were 37°C and 12 to 15°C, respectively.
Identification of a New Gene Family Expressed during the Onset of Sexual Reproduction in the Centric Diatom
Thalassiosira weissflogii
Authors: Armbrust E. Virginia 
Published: December 2019

ABSTRACT

An intriguing feature of the diatom life cycle is that sexual reproduction and the generation of genetic diversity are coupled to the control of cell size. A PCR-based cDNA subtraction technique was used to identify genes that are expressed as small cells of the centric diatom
Thalassiosira weissflogii
initiate gametogenesis. Ten genes that are up-regulated during the early stages of sexual reproduction have been identified thus far. Three of the sexually induced genes,
Sig1
,
Sig2
, and
Sig3
, were sequenced to completion and are members of a novel gene family. The three polypeptides encoded by these genes possess different molecular masses and charges but display many features in common: they share five highly conserved domains; they each contain three or more cysteine-rich epithelial growth factor (EGF)-like repeats; and they each display homology to the EGF-like region of the vertebrate extracellular matrix glycoprotein tenascin X. Interestingly, the five conserved domains appear in the same order in each polypeptide but are separated by variable numbers of nonconserved amino acids. SIG1 and SIG2 display putative regulatory domains within the nonconserved regions. A calcium-binding, EF-hand motif is found in SIG1, and an ATP/GTP binding motif is present in SIG2. The striking similarity between the SIG polypeptides and extracellular matrix components commonly involved in cell-cell interactions suggests that the SIG polypeptides may play a role in sperm-egg recognition. The SIG polypeptides are thus important molecular targets for determining when and where sexual reproduction occurs in the field.
Discriminant Analysis of Ribotype Profiles of
Escherichia coli
for Differentiating Human and Nonhuman Sources of Fecal Pollution
Authors: Parveen Salina , Portier Kenneth M. , Robinson Kevin , Edmiston Lee , Tamplin Mark L. 
Published: December 2019

ABSTRACT

Estuarine waters receive fecal pollution from a variety of sources, including humans and wildlife.
Escherichia coli
is a ubiquitous bacterium in the intestines of warm-blooded animals and is used as an indicator of fecal pollution. However, its presence does not specifically differentiate sources of pollution. A total of 238
E. coli
isolates from human sources (HS) and nonhuman sources (NHS) were collected from the Apalachicola National Estuarine Research Reserve, from associated sewage treatment plants, and directly from animals and tested for ribotype (RT) profile. HS and NHS isolates showed 41 and 61 RT profiles, respectively. At a similarity index of ca. 50%, HS and NHS isolates demonstrated four clusters, with the majority of HS and NHS isolates located in clusters C and D; isolates obtained directly from human and animal feces also could be grouped within these clusters. Discriminant analysis (DA) of RT profiles showed that 97% of the NHS isolates and 100% of the animal fecal isolates were correctly classified. The average rate of correct classification for HS and NHS isolates was 82%. We conclude that DA of RT profiles may be a useful method for identifying HS and NHS fecal pollution and may potentially facilitate management practices.
Use of Conserved Randomly Amplified Polymorphic DNA (RAPD) Fragments and RAPD Pattern for Characterization of
Lactobacillus fermentum
in Ghanaian Fermented Maize Dough
Authors: Hayford Alice E. , Petersen Anne , Vogensen Finn K. , Jakobsen Mogens 
Published: December 2019

ABSTRACT

The present work describes the use of randomly amplified polymorphic DNA (RAPD) for the characterization of 172 dominant
Lactobacillus
isolates from present and previous studies of Ghanaian maize fermentation. Heterofermentative lactobacilli dominate the fermentation flora, since approximately 85% of the isolates belong to this group. Cluster analysis of the RAPD profiles obtained showed the presence of two main clusters. Cluster 1 included
Lactobacillus fermentum
, whereas cluster 2 comprised the remaining
Lactobacillus
spp. The two distinct clusters emerged at the similarity level of <50%. All isolates in cluster 1 showed similarity in their RAPD profile to the reference strains of
L. fermentum
included in the study. These isolates, yielding two distinct bands of approximately 695 and 773 bp with the primers used, were divided into four subclusters, indicating that several strains are involved in the fermentation and remain dominant throughout the process. The two distinct RAPD fragments were cloned, sequenced, and used as probes in Southern hybridization experiments. With one exception,
Lactobacillus reuteri
LMG 13045, the probes hybridized only to fragments of different sizes in
Eco
RI-digested chromosomal DNA of
L. fermentum
strains, thus indicating the specificity of the probes and variation within the
L. fermentum
isolates.
Engineering of Stable Recombinant Bacteria for Production of Chiral Medium-Chain-Length Poly-3-Hydroxyalkanoates
Authors: Prieto Maria A. , Kellerhals Michele B. , Bozzato Gian B. , Radnovic Dragan , Witholt Bernard , Kessler Birgit 
Published: December 2019

ABSTRACT

In order to scale up medium-chain-length polyhydroxyalkanoate (mcl-PHA) production in recombinant microorganisms, we generated and investigated different recombinant bacteria containing a stable regulated expression system for
phaC1
, which encodes one of the mcl-PHA polymerases of
Pseudomonas oleovorans
. We used the mini-Tn
5
system as a tool to construct
Escherichia coli
193MC1 and
P. oleovorans
POMC1, which had stable antibiotic resistance and PHA production phenotypes when they were cultured in a bioreactor in the absence of antibiotic selection. The molecular weight and the polydispersity index of the polymer varied, depending on the inducer level.
E. coli
193MC1 produced considerably shorter polyesters than
P. oleovorans
produced; the weight average molecular weight ranged from 67,000 to 70,000, and the polydispersity index was 2.7. Lower amounts of inducer added to the media shifted the molecular weight to a higher value and resulted in a broader molecular mass distribution. In addition, we found that
E. coli
193MC1 incorporated exclusively the
R
configuration of the 3-hydroxyoctanoate monomer into the polymer, which corroborated the enantioselectivity of the PhaC1 polymerase enzyme.