bluuebunny/crossref_metadata_2025_split huggingface dataset:
Sample Size, Library Composition, and Genotypic Diversity among Natural Populations of
Escherichia coli
from Different Animals Influence Accuracy of Determining Sources of Fecal Pollution
Authors: Johnson LeeAnn K. , Brown Mary B. , Carruthers Ethan A. , Ferguson John A. , Dombek Priscilla E. , Sadowsky Michael J. 
Published: August 2004

ABSTRACT

A horizontal, fluorophore-enhanced, repetitive extragenic palindromic-PCR (rep-PCR) DNA fingerprinting technique (HFERP) was developed and evaluated as a means to differentiate human from animal sources of
Escherichia coli
. Box A1R primers and PCR were used to generate 2,466 rep-PCR and 1,531 HFERP DNA fingerprints from
E. coli
strains isolated from fecal material from known human and 12 animal sources: dogs, cats, horses, deer, geese, ducks, chickens, turkeys, cows, pigs, goats, and sheep. HFERP DNA fingerprinting reduced within-gel grouping of DNA fingerprints and improved alignment of DNA fingerprints between gels, relative to that achieved using rep-PCR DNA fingerprinting. Jackknife analysis of the complete rep-PCR DNA fingerprint library, done using Pearson's product-moment correlation coefficient, indicated that animal and human isolates were assigned to the correct source groups with an 82.2% average rate of correct classification. However, when only unique isolates were examined, isolates from a single animal having a unique DNA fingerprint, Jackknife analysis showed that isolates were assigned to the correct source groups with a 60.5% average rate of correct classification. The percentages of correctly classified isolates were about 15 and 17% greater for rep-PCR and HFERP, respectively, when analyses were done using the curve-based Pearson's product-moment correlation coefficient, rather than the band-based Jaccard algorithm. Rarefaction analysis indicated that, despite the relatively large size of the known-source database, genetic diversity in
E. coli
was very great and is most likely accounting for our inability to correctly classify many environmental
E. coli
isolates. Our data indicate that removal of duplicate genotypes within DNA fingerprint libraries, increased database size, proper methods of statistical analysis, and correct alignment of band data within and between gels improve the accuracy of microbial source tracking methods.
Differential Protein Expression during Growth of
Acidithiobacillus ferrooxidans
on Ferrous Iron, Sulfur Compounds, or Metal Sulfides
Authors: Ramírez Pablo , Guiliani Nicolas , Valenzuela Lissette , Beard Simon , Jerez Carlos A. 
Published: August 2004

ABSTRACT

A set of proteins that changed their levels of synthesis during growth of
Acidithiobacillus ferrooxidans
ATCC 19859 on metal sulfides, thiosulfate, elemental sulfur, and ferrous iron was characterized by using two-dimensional polyacrylamide gel electrophoresis. N-terminal amino acid sequencing and mass spectrometry analysis of these proteins allowed their identification and the localization of the corresponding genes in the available genomic sequence of
A. ferrooxidans
ATCC 23270. The genomic context around several of these genes suggests their involvement in the energetic metabolism of
A. ferrooxidans
. Two groups of proteins could be distinguished. The first consisted of proteins highly upregulated by growth on sulfur compounds (and downregulated by growth on ferrous iron): a 44-kDa outer membrane protein, an exported 21-kDa putative thiosulfate sulfur transferase protein, a 33-kDa putative thiosulfate/sulfate binding protein, a 45-kDa putative capsule polysaccharide export protein, and a putative 16-kDa protein of unknown function. The second group of proteins comprised those downregulated by growth on sulfur (and upregulated by growth on ferrous iron): rusticyanin, a cytochrome
c
552
, a putative phosphate binding protein (PstS), the small and large subunits of ribulose biphosphate carboxylase, and a 30-kDa putative CbbQ protein, among others. The results suggest in general a separation of the iron and sulfur utilization pathways. Rusticyanin, in addition to being highly expressed on ferrous iron, was also newly synthesized, as determined by metabolic labeling, although at lower levels, during growth on sulfur compounds and iron-free metal sulfides. During growth on metal sulfides containing iron, such as pyrite and chalcopyrite, both proteins upregulated on ferrous iron and those upregulated on sulfur compounds were synthesized, indicating that the two energy-generating pathways are induced simultaneously depending on the kind and concentration of oxidizable substrates available.
Purification and Characterization of Chitosanase from
Bacillus
sp. Strain KCTC 0377BP and Its Application for the Production of Chitosan Oligosaccharides
Authors: Choi Yeon Jin , Kim Eun Jung , Piao Zhe , Yun Young Chul , Shin Yong Chul 
Published: August 2004

ABSTRACT

For the enzymatic production of chitosan oligosaccharides from chitosan, a chitosanase-producing bacterium,
Bacillus
sp. strain KCTC 0377BP, was isolated from soil. The bacterium constitutively produced chitosanase in a culture medium without chitosan as an inducer. The production of chitosanase was increased from 1.2 U/ml in a minimal chitosan medium to 100 U/ml by optimizing the culture conditions. The chitosanase was purified from a culture supernatant by using CM-Toyopearl column chromatography and a Superose 12HR column for fast-performance liquid chromatography and was characterized according to its enzyme properties. The molecular mass of the enzyme was estimated to be 45 kDa by means of sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The enzyme demonstrated bifunctional chitosanase-glucanase activities, although it showed very low glucanase activity, with less than 3% of the chitosanase activity. Activity of the enzyme increased with an increase of the degrees of deacetylation (DDA) of the chitosan substrate. However, the enzyme still retained 72% of its relative activity toward the 39% DDA of chitosan, compared with the activity of the 94% DDA of chitosan. The enzyme produced chitosan oligosaccharides from chitosan, ranging mainly from chitotriose to chitooctaose. By controlling the reaction time and by monitoring the reaction products with gel filtration high-performance liquid chromatography, chitosan oligosaccharides with a desired oligosaccharide content and composition were obtained. In addition, the enzyme was efficiently used for the production of low-molecular-weight chitosan and highly acetylated chitosan oligosaccharides. A gene (
csn
45) encoding chitosanase was cloned, sequenced, and compared with other functionally related genes. The deduced amino acid sequence of
csn
45 was dissimilar to those of the classical chitosanase belonging to glycoside hydrolase family 46 but was similar to glucanases classified with glycoside hydrolase family 8.
Purification, Cloning, and Sequencing of a 3,5-Dichlorophenol Reductive Dehalogenase from
Desulfitobacterium frappieri
PCP-1
Authors: Thibodeau Jacinthe , Gauthier Annie , Duguay Marie , Villemur Richard , Lépine François , Juteau Pierre , Beaudet Réjean 
Published: August 2004

ABSTRACT

A membrane-associated 3,5-dichlorophenol reductive dehalogenase was isolated from
Desulfitobacterium frappieri
PCP-1. The highest dehalogenase activity was observed with the biomass cultured at 22°C, compared to 30 and 37°C, where the cell suspensions were 2.2 and 9.6 times less active, respectively. The reductive dehalogenase was purified 12.7-fold to apparent homogeneity. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis revealed a single band with an apparent molecular mass of 57 kDa. Its dechlorinating activity was not inhibited by sulfate and nitrate but was completely inhibited by 2.5 mM sulfite and 10 mM KCN. A mixture of iodopropane and titanium citrate caused a light-reversible inhibition of the dechlorinating activities, suggesting the involvement of a corrinoid cofactor. Several polychlorophenols were dechlorinated at the
meta
and
para
positions. The apparent

K
m

for 3,5-dicholorophenol was 49.3 ± 3.1 μM at a methyl viologen concentration of 2 mM. Six internal tryptic peptides were sequenced by mass spectrometry. One open reading frame (ORF) was found in the
Desulfitobacterium hafniense
genome containing these peptide sequences. This ORF corresponds to a gene coding for a CprA-type reductive dehalogenase. The corresponding ORF (named
cprA5
) in
D. frappieri
PCP-1 was cloned and sequenced. The
cprA5
gene codes for a 548-amino-acid protein that contains a twin-arginine-type signal for secretion. The gene product has a cobalamin binding site motif and two iron-sulfur binding motifs and shows 66% identity (76 to 77% similarity) with some tetrachloroethene reductive dehalogenases. This is the first CprA-type reductive dehalogenase that can dechlorinate chlorophenols at the
meta
and
para
positions.
Effects of Phosphate and Light on Growth of and Bioactive Peptide Production by the Cyanobacterium
Anabaena
Strain 90 and Its Anabaenopeptilide Mutant
Authors: Repka Sari , Koivula Minna , Harjunpä Vesa , Rouhiainen Leo , Sivonen Kaarina 
Published: August 2004

ABSTRACT

Cyanobacteria synthesize several types of bioactive secondary metabolites.
Anabaena
strain 90 produces three types of bioactive peptides, microcystins (inhibitors of protein phosphatases 1 and 2A), anabaenopeptilides, and anabaenopeptins (serine protease inhibitors). To investigate the role of the anabaenopeptilides in
Anabaena
, wild-type strain 90 (WT) and its anabaenopeptilide deficient mutant (MU) were cultured with various light and phosphate levels to evaluate the effects and coeffects of these growth factors on the concentrations of the three classes of peptides and the growth characteristics. WT and MU grew in comparable ways under the different growth conditions. The total peptide concentration in WT was significantly higher than that in MU (2.5 and 1.4 μg/mg [dry weight], respectively). Interestingly, the average concentration of anabaenopeptins was significantly higher in MU than in WT (0.59 and 0.24 μg/mg [dry weight], respectively). The concentration of microcystins was slightly but not statistically significantly higher in MU than in WT (1.0 and 0.86 μg/mg [dry weight], respectively). In WT, the highest peptide concentrations were usually found after 13 days in cultures grown at medium light intensities (23 μmol m
−2
s
−1
) and with the highest phosphate concentrations (2,600 μg liter
−1
). In MU, the highest peptide concentrations were found in 13-day-old cultures grown at medium light intensities (23 μmol m
−2
s
−1
) and with phosphate concentrations greater than 100 μg liter
−1
. The higher concentrations of anabaenopeptins in MU may compensate for the absence of anabaenopeptilides. These findings clearly indicate that these compounds may have some linked function in the producer organism, the nature of which remains to be discovered.
Treatment ofSalmonella entericaSerovar Enteritidis with a Sublethal Concentration of Trisodium Phosphate or Alkaline pH Induces Thermotolerance
Authors: Sampathkumar Balamurugan , Khachatourians George G. , Korber Darren R. 
Published: August 2004

ABSTRACTThe responses ofSalmonella entericaserovar Enteritidis to a sublethal dose of trisodium phosphate (TSP) and its equivalent alkaline pH made with NaOH were examined. Pretreatment ofS. entericaserovar Enteritidis cells with 1.5% TSP or pH 10.0 solutions resulted in a significant increase in thermotolerance, resistance to 2.5% TSP, resistance to high pH, and sensitivity to acid and H2O2. Protein inhibition studies with chloramphenicol revealed that thermotolerance, unlike resistance to high pH, was dependent on de novo protein synthesis. Two-dimensional polyacrylamide gel electrophoresis (PAGE) of total cellular proteins from untreated control cells resolved as many as 232 proteins, of which 22 and 15% were absent in TSP- or alkaline pH-pretreated cells, respectively. More than 50% of the proteins that were either up- or down-regulated by TSP pretreatment were also up- or down-regulated by alkaline pH pretreatment. Sodium dodecyl sulfate-PAGE analysis of detergent-insoluble outer membrane proteins revealed the up-regulation of at least four proteins. Mass spectrometric analysis showed the up-regulated proteins to include those involved in the transport of small hydrophilic molecules across the cytoplasmic membrane and those that act as chaperones and aid in the export of newly synthesized proteins by keeping them in open conformation. Other up-regulated proteins included common housekeeping proteins like those involved in amino acid biosynthesis, nucleotide metabolism, and aminoacyl-tRNA biosynthesis. In addition to the differential expression of proteins following TSP or alkaline pH treatment, changes in membrane fatty acid composition were also observed. Alkaline pH- or TSP-pretreated cells showed a higher saturated and cyclic to unsaturated fatty acid ratio than did the untreated control cells. These results suggest that the cytoplasmic membrane could play a significant role in the induction of thermotolerance and resistance to other stresses following TSP or alkaline pH treatment.
Use of Microautoradiography Combined with Fluorescence In Situ Hybridization To Determine Dimethylsulfoniopropionate Incorporation by Marine Bacterioplankton Taxa
Authors: Vila Maria , Simó Rafel , Kiene Ronald P. , Pinhassi Jarone , González José M. , Moran Mary Ann , Pedrós-Alió Carlos 
Published: August 2004

ABSTRACT

The fraction of planktonic heterotrophic bacteria capable of incorporating dissolved dimethylsulfoniopropionate (DMSP) and leucine was determined at two coastal sites by microautoradioagraphy (AU). In Gulf of Mexico seawater microcosm experiments, the proportion of prokaryotes that incorporated sulfur from [
35
S]DMSP ranged between 27 and 51% of 4′,6-diamidino-2-phenylindole (DAPI)-positive cells, similar to or slightly lower than the proportion incorporating [
3
H]leucine. In the northwest Mediterranean coast, the proportion of cells incorporating sulfur from [
35
S]DMSP increased from 5 to 42% from January to March, coinciding with the development of a phytoplankton bloom. At the same time, the proportion of cells incorporating [
3
H]leucine increased from 21 to 40%. The combination of AU and fluorescence in situ hybridization (FISH) revealed that the
Roseobacter
clade (α-proteobacteria) accounted for 13 to 43% of the microorganisms incorporating [
35
S]DMSP at both sampling sites. Significant uptake of sulfur from DMSP was also found among members of the γ-proteobacteria and
Cytophaga-Flavobacterium
groups.
Roseobacter
and γ-proteobacteria exhibited the highest percentage of DAPI-positive cells incorporating
35
S from DMSP (around 50%). Altogether, the application of AU with [
35
S]DMSP combined with FISH indicated that utilization of S from DMSP is a widespread feature among active marine bacteria, comparable to leucine utilization. These results point toward DMSP as an important substrate for a broad and diverse fraction of marine bacterioplankton.
Chemotaxis of
Silicibacter
sp. Strain TM1040 toward Dinoflagellate Products
Authors: Miller Todd R. , Hnilicka Kristin , Dziedzic Amanda , Desplats Paula , Belas Robert 
Published: August 2004

ABSTRACT

The α-proteobacteria phylogenetically related to the
Roseobacter
clade are predominantly responsible for the degradation of organosulfur compounds, including the algal osmolyte dimethylsulfoniopropionate (DMSP).
Silicibacter
sp. strain TM1040, isolated from a DMSP-producing
Pfiesteria piscicida
dinoflagellate culture, degrades DMSP, producing 3-methylmercaptopropionate. TM1040 possesses three lophotrichous flagella and is highly motile, leading to a hypothesis that TM1040 interacts with
P. piscicida
through a chemotactic response to compounds produced by its dinoflagellate host. A combination of a rapid chemotaxis screening assay and a quantitative capillary assay were used to measure chemotaxis of TM1040. These bacteria are highly attracted to dinoflagellate homogenates; however, the response decreases when homogenates are preheated to 80°C. To help identify the essential attractant molecules within the homogenates, a series of pure compounds were tested for their ability to serve as attractants. The results show that TM1040 is strongly attracted to amino acids and DMSP metabolites, while being only mildly responsive to sugars and the tricarboxylic acid cycle intermediates. Adding pure DMSP, methionine, or valine to the chemotaxis buffer resulted in a decreased response to the homogenates, indicating that exogenous addition of these chemicals blocks chemotaxis and suggesting that DMSP and amino acids are essential attractant molecules in the dinoflagellate homogenates. The implication of
Silicibacter
sp. strain TM1040 chemotaxis in establishing and maintaining its interaction with
P. piscicida
is discussed.
Identification of an Na+-Dependent Transporter Associated with Saxitoxin-Producing Strains of the CyanobacteriumAnabaena circinalis
Authors: Pomati Francesco , Burns Brendan P. , Neilan Brett A. 
Published: August 2004

ABSTRACTBlooms of the freshwater cyanobacteriumAnabaena circinalisare recognized as an important health risk worldwide due to the production of a range of toxins such as saxitoxin (STX) and its derivatives. In this study we used HIP1 octameric-palindrome repeated-sequence PCR to compare the genomic structure of phylogenetically similar Australian isolates ofA. circinalis. STX-producing and nontoxic cyanobacterial strains showed different HIP1 (highly iterated octameric palindrome 1) DNA patterns, and characteristic interrepeat amplicons for each group were identified. Suppression subtractive hybridization (SSH) was performed using HIP1 PCR-generated libraries to further identify toxic-strain-specific genes. An STX-producing strain and a nontoxic strain ofA. circinaliswere chosen as testers in two distinct experiments. The two categories of SSH putative tester-specific sequences were characterized by different families of encoded proteins that may be representative of the differences in metabolism between STX-producing and nontoxicA. circinalisstrains. DNA-microarray hybridization and genomic screening revealed a toxic-strain-specific HIP1 fragment coding for a putative Na+-dependent transporter. Analysis of this gene demonstrated analogy to themrpFgene ofBacillus subtilis, whose encoded protein is involved in Na+-specific pH homeostasis. The application of this gene as a molecular probe in laboratory and environmental screening for STX-producingA. circinalisstrains was demonstrated. The possible role of this putative Na+-dependent transporter in the toxic cyanobacterial phenotype is also discussed, in light of recent physiological studies of STX-producing cyanobacteria.
Detection and Quantification of the Red Tide Dinoflagellate
Karenia brevis
by Real-Time Nucleic Acid Sequence-Based Amplification
Authors: Casper Erica T. , Paul John H. , Smith Matthew C. , Gray Michael 
Published: August 2004

ABSTRACT

Nucleic acid sequence-based amplification (NASBA) is an isothermal method of RNA amplification that has been previously used in clinical diagnostic testing. A real-time NASBA assay has been developed for the detection of
rbcL
mRNA from the red tide dinoflagellate
Karenia brevis
. This assay is sensitive to one
K. brevis
cell and 1.0 fg of in vitro transcript, with occasional detection of lower concentrations of transcript. The assay did not detect
rbcL
mRNA from a wide range of nontarget organisms and environmental clones, while 10 strains (all tested) of
K. brevis
were detected. By the use of standard curves based on time to positivity, concentrations of
K. brevis
in environmental samples were predicted by NASBA and classified into different levels of blooms per the Florida Fish and Wildlife Conservation Commission (FWC) system. NASBA classification matched FWC classification (based on cell counts) 72% of the time. Those samples that did not match were off by only one class. NASBA is sensitive, rapid, and effective and may be used as an additional or alternative method to detect and quantify
K. brevis
in the marine environment.
veA
Is Required for Toxin and Sclerotial Production in
Aspergillus parasiticus
Authors: Calvo Ana M. , Bok Jinwoo , Brooks Wilhelmina , Keller Nancy P. 
Published: August 2004

ABSTRACT

It was long been noted that secondary metabolism is associated with fungal development. In
Aspergillus nidulans
, conidiation and mycotoxin production are linked by a G protein signaling pathway. Also in
A. nidulans
, cleistothecial development and mycotoxin production are controlled by a gene called
veA
. Here we report the characterization of a
veA
ortholog in the aflatoxin-producing fungus
A. parasiticus
. Cleistothecia are not produced by
Aspergillus parasiticus
; instead, this fungus produces spherical structures called sclerotia that allow for survival under adverse conditions. Deletion of
veA
from
A. parasiticus
resulted in the blockage of sclerotial formation as well as a blockage in the production of aflatoxin intermediates. Our results indicate that
A. parasiticus veA
is required for the expression of
aflR
and
aflJ
, which regulate the activation of the aflatoxin gene cluster. In addition to these findings, we observed that deletion of
veA
reduced conidiation both on the culture medium and on peanut seed. The fact that
veA
is necessary for conidiation, production of resistant structures, and aflatoxin biosynthesis makes
veA
a good candidate gene to control aflatoxin biosynthesis or fungal development and in this way to greatly decrease its devastating impact on health and the economy.
Comparisons of Different Hypervariable Regions ofrrsGenes for Use in Fingerprinting of Microbial Communities by PCR-Denaturing Gradient Gel Electrophoresis
Authors: Yu Zhongtang , Morrison Mark 
Published: August 2004

ABSTRACTDenaturing gradient gel electrophoresis (DGGE) has become a widely used tool to examine microbial diversity and community structure, but no systematic comparison has been made of the DGGE profiles obtained when different hypervariable (V) regions are amplified from the same community DNA samples. We report here a study to make such comparisons and establish a preferred choice of V region(s) to examine by DGGE, when community DNA extracted from samples of digesta is used. When the members of the phylogenetically representative set of 218rrsgenes archived in the RDP II database were compared, the V1 region was found to be the most variable, followed by the V9 and V3 regions. The temperature of the lowest-melting-temperature (Tm(L)) domain for each V region was also calculated for theserrsgenes, and the V1 to V4 region was found to be most heterogeneous with respect toTm(L). The averageTm(L)values and their standard deviations for each V region were then used to devise the denaturing gradients suitable for separating 95% of all the sequences, and the PCR-DGGE profiles produced from the same community DNA samples with these conditions were compared. The resulting DGGE profiles were substantially different in terms of the number, resolution, and relative intensity of the amplification products. The DGGE profiles of the V3 region were best, and the V3 to V5 and V6 to V8 regions produced better DGGE profiles than did other multiple V-region amplicons. Introduction of degenerate bases in the primers used to amplify the V1 or V3 region alone did not improve DGGE banding profiles. Our results show that DGGE analysis of gastrointestinal microbiomes is best accomplished by the amplification of either the V3 or V1 region ofrrsgenes, but if a longer amplification product is desired, then the V3 to V5 or V6 to V8 region should be targeted.