bluuebunny/crossref_metadata_2025_split huggingface dataset:
Export of Virulence Genes and Shiga Toxin by Membrane Vesicles of
Escherichia coli
O157:H7
Authors: Kolling Glynis L. , Matthews Karl R. 
Published: December 2019

ABSTRACT

Membrane vesicles released by
Escherichia coli
O157:H7 into culture medium were purified and analyzed for protein and DNA content. Electron micrographs revealed vesicles that are spherical, range in size from 20 to 100 nm, and have a complete bilayer. Analysis of vesicle protein by sodium dodecyl sulfate-polyacrylamide gel electrophoresis demonstrates vesicles that contain many proteins with molecular sizes similar to outer membrane proteins and a number of cellular proteins. Immunoblot (Western) analysis of vesicles suggests the presence of cell antigens. Treatment of vesicles with exogenous DNase hydrolyzed surface-associated DNA; PCR demonstrated that vesicles contain DNA encoding the virulence genes
eae
,
stx1
and
stx2
, and
uidA
, which encodes for β-galactosidase. Immunoblot analysis of intact and lysed, proteinase K-treated vesicles demonstrate that Shiga toxins 1 and 2 are contained within vesicles. These results suggest that vesicles contain toxic material and transfer experiments demonstrate that vesicles can deliver genetic material to other gram-negative organisms.
A Colorimetric Technique for Detecting Trichothecenes and Assessing Relative Potencies
Authors: Engler Kathryn H. , Coker Raymond D. , Evans Ivor H. 
Published: December 2019

ABSTRACT

We tested a novel colorimetric toxicity test, based on inhibition of β-galactosidase activity in the yeast
Kluyveromyces marxianus
, for sensitivity to a range of mycotoxins. A variety of trichothecene mycotoxins could be detected. The order of toxicity established with this bioassay was verrucarin A > roridin A > T-2 toxin > diacetoxyscirpenol > HT-2 toxin > acetyl T-2 toxin > neosolaniol > fusarenon X > T-2 triol > scirpentriol > nivalenol > deoxynivalenol > T-2 tetraol. The sensitivity of detection was high, with the most potent trichothecene tested, verrucarin A, having a 50% effective concentration (concentration of toxin causing 50% inhibition) of 2 ng/ml. Other mycotoxins (cyclopiazonic acid, fumonisin B
1
, ochratoxin A, patulin, sterigmatocystin, tenuazonic acid, and zearalenone) could not be detected at up to 10 μg/ml, nor could aflatoxins B
1
and M
1
be detected at concentrations up to 25 μg/ml. This test should be useful for trichothecene detection and for studies of relevant interactions—both between trichothecenes themselves and between trichothecenes and other food constituents.
Differentiation of
Vibrio alginolyticus
Strains Isolated from Sardinian Waters by Ribotyping and a New Rapid PCR Fingerprinting Method
Authors: Zanetti Stefania , Deriu Antonella , Duprè Ilaria , Sanguinetti Maurizio , Fadda Giovanni , Sechi Leonardo A. 
Published: December 2019

ABSTRACT

We investigated the usefulness of a novel PCR fingerprinting technique, based on the specific amplification of genomic regions, to differentiate 30
Vibrio alginolyticus
strains isolated in Sardinian waters. The different profiles obtained were scanned and analyzed by a computer program in order to determine genetic relationships. The results were then compared with the patterns obtained by ribotyping with
Hin
dIII,
Kpn
I, and
Xba
I restriction enzymes. PCR fingerprinting could differentiate the strains analyzed into 12 different patterns, whereas ribotyping with
Xba
I, which produced the highest number of patterns, generated only 7 different profiles. This study revealed the superior discriminative power of the proposed technique for the differentiation of related
V. alginolyticus
strains and the potential use of PCR fingerprinting in epidemiological studies.
Viral Lysis and Bacterivory during a Phytoplankton Bloom in a Coastal Water Microcosm
Authors: Guixa-Boixereu Núria , Lysnes Kristine , Pedrós-Alió Carlos 
Published: December 2019

ABSTRACT

The relative importance of viral lysis and bacterivory as causes of bacterial mortality were estimated. A laboratory experiment was carried out to check the kind of control that viruses could exert over the bacterial assemblage in a non-steady-state situation. Virus-like particles (VLP) were determined by using three methods of counting (DAPI [4′,6-diamidino-2-phenylindole] staining, YOPRO staining, and transmission electron microscopy). Virus counts increased from the beginning until the end of the experiment. However, different methods produced significantly different results. DAPI-stained VLP yielded the lowest numbers, while YOPRO-stained VLP yielded the highest numbers. Bacteria reached the maximal abundance at 122 h (3 × 10
7
bacteria ml
−1
), after the peak of chlorophyll
a
(80 μg liter
−1
). Phototrophic nanoflagellates followed the same pattern as for chlorophyll
a
. Heterotrophic nanoflagellates showed oscillations in abundance throughout the experiment. The specific bacterial growth rate increased until 168 h (2.6 day
−1
). The bacterivory rate reached the maximal value at 96 hours (0.9 day
−1
). Bacterial mortality due to viral infection was measured by using two approaches: measuring the percentage of visibly infected bacteria (%VIB) and measuring the viral decay rates (VDR), which were estimated with cyanide. The %VIB was always lower than 1% during the experiment. VDR were used to estimate viral production. Viral production increased 1 order of magnitude during the experiment (from 10
6
to 10
7
VLP ml
−1
h
−1
). The percentage of heterotrophic bacterial production consumed by bacterivores was higher than 60% during the first 4 days of the experiment; afterwards, this percentage was lower than 10%. The percentage of heterotrophic bacterial production lysed by viruses as assessed by the VDR reached the highest values at the beginning (100%) and at the end (50%) of the experiment. Comparing both sources of mortality at each stage of the bloom, bacterivory was found to be higher than viral lysis at days 2 and 4, and viral lysis was higher than bacterivory at days 7 and 9. A balance between bacterial losses and bacterial production was calculated for each sampling interval. At intervals of 0 to 2 and 2 to 4 days, viral lysis and bacterivory accounted for all the bacterial losses. At intervals of 4 to 7 and 7 to 9 days, bacterial losses were not balanced by the sources of mortality measured. At these time points, bacterial abundance was about 20 times higher than the expected value if viral lysis and bacterivory had been the only factors causing bacterial mortality. In conclusion, mortality caused by viruses can be more important than bacterivory under non-steady-state conditions.
Purification and Characterization of an Extremely Thermostable Cyclomaltodextrin Glucanotransferase from a Newly Isolated Hyperthermophilic Archaeon, a
Thermococcus
sp
Authors: Tachibana Yoshihisa , Kuramura Akiko , Shirasaka Naoki , Suzuki Yuji , Yamamoto Tomoko , Fujiwara Shinsuke , Takagi Masahiro , Imanaka Tadayuki 
Published: December 2019

ABSTRACT

The extremely thermophilic anaerobic archaeon strain B1001 was isolated from a hot-spring environment in Japan. The cells were irregular cocci, 0.5 to 1.0 μm in diameter. The new isolate grew at temperatures between 60 and 95°C (optimum, 85°C), from pH 5.0 to 9.0 (optimum, pH 7.0), and from 1.0 to 6.0% NaCl (optimum, 2.0%). The G+C content of the genomic DNA was 43.0 mol%. The 16S rRNA gene sequencing of strain B1001 indicated that it belongs to the genus
Thermococcus
. During growth on starch, the strain produced a thermostable cyclomaltodextrin glucanotransferase (CGTase). The enzyme was purified 1,750-fold, and the molecular mass was determined to be 83 kDa by sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis. Incubation at 120°C with SDS and 2-mercaptoethanol was required for complete unfolding. The optimum temperatures for starch-degrading activity and cyclodextrin synthesis activity were 110 and 90 to 100°C, respectively. The optimum pH for enzyme activity was pH 5.0 to 5.5. At pH 5.0, the half-life of the enzyme was 40 min at 110°C. The enzyme formed mainly α-cyclodextrin with small amounts of β- and γ-cyclodextrins from starch. This is the first report on the presence of the extremely thermostable CGTase from hyperthermophilic archaea.
Structure and Dynamics of Experimentally Introduced and Naturally Occurring
Laccaria
sp. Discrete Genotypes in a Douglas Fir Plantation
Authors: Selosse Marc-André , Martin Francis , Bouchard Daniel , le Tacon François 
Published: December 2019

ABSTRACT

Ectomycorrhizal fungi have been introduced in forest nurseries to improve seedling growth. Outplanting of inoculated seedlings to forest plantations raises the questions about inoculant persistence and its effects on indigenous fungal populations. We previously showed (M.-A. Selosse et al. Mol. Ecol. 7:561–573, 1998) that the American strain
Laccaria bicolor
S238N persisted 10 years after outplanting in a French Douglas fir plantation, without introgression or selfing and without fruiting on uninoculated adjacent plots. In the present study, the relevance of those results to sympatric strains was assessed for another part of the plantation, planted in 1985 with seedlings inoculated with the French strain
L. bicolor
81306 or left uninoculated. About 720
Laccaria
sp. sporophores, collected from 1994 to 1997, were typed by using randomly amplified polymorphic DNA markers and PCR amplification of the mitochondrial and nuclear ribosomal DNAs. All plots were colonized by small spontaneous discrete genotypes (genets). The inoculant strain 81306 abundantly fruited beneath inoculated trees, with possible introgression in indigenous
Laccaria
populations but without selfing. In contrast to our previous survey of
L. bicolor
S238N,
L. bicolor
81306 colonized a plot of uninoculated trees. Meiotic segregation analysis verified that the invading genet was strain 81306 (
P
< 0.00058), implying a vegetative growth of 1.1 m · year
−1
. This plot was also invaded in 1998 by strain S238N used to inoculate other trees of the plantation. Five other uninoculated plots were free of these inoculant strains. The fate of inoculant strains thus depends less on their geographic origin than on unknown local factors.
Enhanced Nitrogen Fixation in a
Rhizobium etli ntrC
Mutant That Overproduces the
Bradyrhizobium japonicum
Symbiotic Terminal Oxidase
cbb
3
Authors: Soberón Mario , López Oswaldo , Morera Claudia , Girard Maria de Lourdes , Tabche Maria Luisa , Miranda Juan 
Published: December 2019

ABSTRACT

The
ntrC
gene codes for a transcriptional activator protein that modulates gene expression in response to nitrogen. The cytochrome production pattern of a
Rhizobium etli ntrC
mutant (CFN2012) was studied. CO difference spectral analysis of membranes showed that CFN2012 produced a terminal oxidase similar to the symbiotic terminal oxidase of bacteroids in free-living cells under aerobic conditions, with a characteristic trough at 553 nm. CFN2012 produced two
c
-type cytochromes with molecular masses of 27 and 32 kDa, in contrast with the wild-type strain, which produced only a 32-kDa
c
-type cytochrome. The expression levels of the
R. etli fixNOQP
operon, which codes for terminal oxidase
cbb
3
, were not affected by the
ntrC
mutation. However, the production levels of the two
c
-type cytochromes (27 and 32 kDa) were enhanced at least eightfold when the
Bradyrhizobium japonicum fixNOQP
operon was expressed in CFN2012 from the
nptII
promoter (pMSfix
c
), suggesting that these proteins are subunits FixO (27 kDa) and FixP (32 kDa) of
cbb
3
and that CFN2012/pMSfix
c
overproduced this terminal oxidase. CFN2012/pMSfix
c
showed a significant increase in its symbiotic performance as judged by the determination of nitrogenase activities of plants inoculated with this strain, suggesting that the overproduction of
cbb
3
terminal oxidase correlates with an enhancement in symbiotic nitrogen fixation.
Cloning and Characterization of Polyphosphate Kinase and Exopolyphosphatase Genes from
Pseudomonas aeruginosa
8830
Authors: Zago Anna , Chugani Sudha , Chakrabarty A. M. 
Published: December 2019

ABSTRACT

Pseudomonas aeruginosa
accumulates polyphosphates in response to nutrient limitations. To elucidate the function of polyphosphate in this microorganism, we have investigated polyphosphate metabolism by isolating from
P. aeruginosa
8830 the genes encoding polyphosphate kinase (PPK) and exopolyphosphatase (PPX), which are involved in polyphosphate synthesis and degradation, respectively. The 690- and 506-amino-acid polypeptides encoded by the two genes have been expressed in
Escherichia coli
and purified, and their activities have been tested in vitro. Gene replacement was used to construct a PPK-negative strain of
P. aeruginosa
8830. Low residual PPK activity in the
ppk
mutant suggests a possible alternative pathway of polyphosphate synthesis in this microorganism. Primer extension analysis indicated that
ppk
is transcribed from a ς
E
-dependent promoter, which could be responsive to environmental stresses. However, no coregulation between
ppk
and
ppx
promoters has been demonstrated in response to osmotic shock or oxidative stress.
Lignin-Modifying Enzymes of
Flavodon flavus
, a Basidiomycete Isolated from a Coastal Marine Environment
Authors: Raghukumar C. , D’Souza T. M. , Thorn R. G. , Reddy C. A. 
Published: December 2019

ABSTRACT

A basidiomycetous fungus
Flavodon flavus
(Klotzsch) Ryvarden (strain 312), isolated from decaying sea grass from a coral lagoon off the west coast of India, mineralized nearly 24% of
14
C-labeled synthetic lignin to
14
CO
2
in 24 days. When grown in low-nitrogen medium (2.4 mM N) this fungus produced three major classes of extracellular lignin-modifying enzymes (LMEs): manganese-dependent peroxidase (MNP), lignin peroxidase (LIP), and laccase. Low MNP and laccase activities were seen in high-nitrogen medium (24 mM N), but no LIP activity was seen. In media containing lignocellulosic substrates such as pine, poplar, or sugarcane bagasse as the sole source of carbon and nitrogen, relatively high MNP and moderate levels of laccases were seen, but LIP production either was not seen or was minimal. LME production was also seen in media prepared with artificial seawater. Fast protein liquid chromatography and isoelectric focusing resolved LMEs into four isozymes each of MNP and LIP, while laccase isozymes were resolved into two groups, one group containing seven isozymes (pIs 4 to 6) and the other group containing at least three isozymes (pIs < 3). The molecular masses of the different isozymes were 43 to 99 kDa for MNP, 40 and 41.5 kDa for LIP, and 43 and 99 kDa for laccase.
F. flavus
showed effective degradation of various dye pollutants in media prepared with or without artificial seawater. This is the first report on the production of all three major classes of LMEs by
F. flavus
and points to the bioremediation potential of this organism in terrestrial as well as marine environments.
Detection of Viable
Listeria monocytogenes
with a 5′ Nuclease PCR Assay
Authors: Norton Dawn-Marie , Batt Carl A. 
Published: December 2019

ABSTRACT

A 5′ nuclease assay has been developed to detect viable
Listeria monocytogenes
. The assay targets the hemolysin A (
hlyA
) transcript, which is found only in
L. monocytogenes
. The single-tube, reverse transcriptase (RT), fluorogenic probe-based assay was formatted by using
Tth
DNA polymerase whose activity was modulated by using the manganese-chelating morpholinepropanesulfonic acid (MOPS) buffer. This assay was quantitative over a 3-log-unit range of template concentrations when tested with an in vitro-transcribed
hlyA
mRNA. The viability of
L. monocytogenes
was reduced by heating at various temperatures and times up to a maximum of a 9-D inactivation. The location of the primer had a pronounced effect on the utility of the assay, and primers located in the most distal regions of the
hlyA
transcript appeared to correlate with the number of CFU while primers located more internal on the amplicon overestimated the cell viability. The assay with primers that included the 3′ end of the transcript was an accurate indicator of viability as measured by CFU determination or staining with 5-sulfofluorescein diacetate.
Growth from Spores of NonproteolyticClostridium botulinumin Heat-Treated Vegetable Juice
Authors: Stringer Sandra C. , Haque Nuzrul , Peck Michael W. 
Published: December 2019

ABSTRACTUnheated spores of nonproteolyticClostridium botulinumwere able to lead to growth in sterile deoxygenated turnip, spring green, helda bean, broccoli, or potato juice, although the probability of growth was low and the time to growth was longer than the time to growth in culture media. With all five vegetable juices tested, the probability of growth increased when spores were inoculated into the juice and then heated for 2 min in a water bath at 80°C. The probability of growth was greater in bean or broccoli juice than in culture media following 10 min of heat treatment in these media. Growth was prevented by heat treatment of spores in vegetable juices or culture media at 80°C for 100 min. We show for the first time that adding heat-treated vegetable juice to culture media can increase the number of heat-damaged spores ofC. botulinumthat can lead to colony formation.
Cloning, Expression, and Nucleotide Sequence of the
Pseudomonas aeruginosa
142
ohb
Genes Coding for Oxygenolytic
ortho
Dehalogenation of Halobenzoates
Authors: Tsoi Tamara V. , Plotnikova Elena G. , Cole James R. , Guerin William F. , Bagdasarian Michael , Tiedje James M. 
Published: December 2019

ABSTRACT

We have cloned and characterized novel oxygenolytic
ortho
-dehalogenation (
ohb
) genes from 2-chlorobenzoate (2-CBA)- and 2,4-dichlorobenzoate (2,4-dCBA)-degrading
Pseudomonas aeruginosa
142. Among 3,700
Escherichia coli
recombinants, two clones, DH5αF′(pOD22) and DH5αF′(pOD33), converted 2-CBA to catechol and 2,4-dCBA and 2,5-dCBA to 4-chlorocatechol. A subclone of pOD33, plasmid pE43, containing the 3,687-bp minimized
ohb
DNA region conferred to
P. putida
PB2440 the ability to grow on 2-CBA as a sole carbon source. Strain PB2440(pE43) also oxidized but did not grow on 2,4-dCBA, 2,5-dCBA, or 2,6-dCBA. Terminal oxidoreductase ISP
OHB
structural genes
ohbA
and
ohbB
, which encode polypeptides with molecular masses of 20,253 Da (β-ISP) and 48,243 Da (α-ISP), respectively, were identified; these proteins are in accord with the 22- and 48-kDa (as determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis) polypeptides synthesized in
E. coli
and
P. aeruginosa
parental strain 142. The
ortho
-halobenzoate 1,2-dioxygenase activity was manifested in the absence of ferredoxin and reductase genes, suggesting that the ISP
OHB
utilized electron transfer components provided by the heterologous hosts. ISP
OHB
formed a new phylogenetic cluster that includes aromatic oxygenases featuring atypical structural-functional organization and is distant from the other members of the family of primary aromatic oxygenases. A putative IclR-type regulatory gene (
ohbR
) was located upstream of the
ohbAB
genes. An open reading frame (
ohbC
) of unknown function that overlaps lengthwise with
ohbB
but is transcribed in the opposite direction was found. The
ohbC
gene codes for a 48,969-Da polypeptide, in accord with the 49-kDa protein detected in
E. coli
. The
ohb
genes are flanked by an IS
1396
-like sequence containing a putative gene for a 39,715-Da transposase A (
tnpA
) at positions 4731 to 5747 and a putative gene for a 45,247-Da DNA topoisomerase I/III (
top
) at positions 346 to 1563. The
ohb
DNA region is bordered by 14-bp imperfect inverted repeats at positions 56 to 69 and 5984 to 5997.
Molecular Phylogenetic and Biogeochemical Studies of Sulfate-Reducing Bacteria in the Rhizosphere of
Spartina alterniflora
Authors: Hines Mark E. , Evans Robert S. , Sharak Genthner Barbara R. , Willis Stephanie G. , Friedman Stephanie , Rooney-Varga Juliette N. , Devereux Richard 
Published: December 2019

ABSTRACT

The population composition and biogeochemistry of sulfate-reducing bacteria (SRB) in the rhizosphere of the marsh grass
Spartina alterniflora
was investigated over two growing seasons by molecular probing, enumerations of culturable SRB, and measurements of SO
4
2−
reduction rates and geochemical parameters. SO
4
2−
reduction was rapid in marsh sediments with rates up to 3.5 μmol ml
−1
day
−1
. Rates increased greatly when plant growth began in April and decreased again when plants flowered in late July. Results with nucleic acid probes revealed that SRB rRNA accounted for up to 43% of the rRNA from members of the domain
Bacteria
in marsh sediments, with the highest percentages occurring in bacteria physically associated with root surfaces. The relative abundance (RA) of SRB rRNA in whole-sediment samples compared to that of
Bacteria
rRNA did not vary greatly throughout the year, despite large temporal changes in SO
4
2−
reduction activity. However, the RA of root-associated SRB did increase from <10 to >30% when plants were actively growing. rRNA from members of the family
Desulfobacteriaceae
comprised the majority of the SRB rRNA at 3 to 34% of
Bacteria
rRNA, with
Desulfobulbus
spp. accounting for 1 to 16%. The RA of
Desulfovibrio
rRNA generally comprised from <1 to 3% of the
Bacteria
rRNA. The highest
Desulfobacteriaceae
RA in whole sediments was 26% and was found in the deepest sediment samples (6 to 8 cm). Culturable SRB abundance, determined by most-probable-number analyses, was high at >10
7
ml
−1
. Ethanol utilizers were most abundant, followed by acetate utilizers. The high numbers of culturable SRB and the high RA of SRB rRNA compared to that of
Bacteria
rRNA may be due to the release of SRB substrates in plant root exudates, creating a microbial food web that circumvents fermentation.
Removal of Dibenzofuran, Dibenzo-
p
-Dioxin, and 2-Chlorodibenzo-
p
-Dioxin from Soils Inoculated with
Sphingomonas
sp. Strain RW1
Authors: Halden Rolf U. , Halden Barbara G. , Dwyer Daryl F. 
Published: December 2019

ABSTRACT

Removal of dibenzofuran, dibenzo-
p
-dioxin, and 2-chlorodibenzo-
p
-dioxin (2-CDD) (10 ppm each) from soil microcosms to final concentrations in the parts-per-billion range was affected by the addition of
Sphingomonas
sp. strain RW1. Rates and extents of removal were influenced by the density of RW1 organisms. For 2-CDD, the rate of removal was dependent on the content of soil organic matter (SOM), with half-life values ranging from 5.8 h (0% SOM) to 26.3 h (5.5% SOM).
Rapid Microbial Production of Filamentous Sulfur Mats at Hydrothermal Vents
Authors: Taylor Craig D. , Wirsen Carl O. , Gaill Françoise 
Published: December 2019

ABSTRACT

During recent oceanographic cruises to Pacific hydrothermal vent sites (9°N and the Guaymas Basin), the rapid microbial formation of filamentous sulfur mats by a new chemoautotrophic, hydrogen sulfide-oxidizing bacterium was documented in both in situ and shipboard experiments. Observations suggest that formation of these sulfur mats may be a factor in the initial colonization of hydrothermal surfaces by macrofaunal
Alvinella
worms. This novel metabolic capability, previously shown to be carried out by a coastal strain in H
2
S continuous-flow reactors, may be an important, heretofore unconsidered, source of microbial organic matter production at deep-sea hydrothermal vents.
Elevated Growth of
Saccharomyces cerevisiae ATH1
Null Mutants on Glucose Is an Artifact of Nonmatching Auxotrophies of Mutant and Reference Strains
Authors: Chopra Rohini , Sharma Vishva Mitra , Ganesan K. 
Published: December 2019

ABSTRACT

Yeast strains disrupted for
ATH1
, which encodes vacuolar acid trehalase, have been reported to grow to higher cell densities than reference strains. We showed that the increase in cell density is due to the
URA3
gene introduced as a part of the disruption and concluded that the misinterpretation is a result of not using a control strain with matching auxotrophic markers.