bluuebunny/crossref_metadata_2025_split huggingface dataset:
The Weak Acid Preservative Sorbic Acid Inhibits Conidial Germination and Mycelial Growth of
Aspergillus niger
through Intracellular Acidification
Authors: Plumridge Andrew , Hesse Stephan J. A. , Watson Adrian J. , Lowe Kenneth C. , Stratford Malcolm , Archer David B. 
Published: June 2004

ABSTRACT

The growth of the filamentous fungus
Aspergillus niger
, a common food spoilage organism, is inhibited by the weak acid preservative sorbic acid (
trans-trans
-2,4-hexadienoic acid). Conidia inoculated at 10
5
/ml of medium showed a sorbic acid MIC of 4.5 mM at pH 4.0, whereas the MIC for the amount of mycelia at 24 h developed from the same spore inoculum was threefold lower. The MIC for conidia and, to a lesser extent, mycelia was shown to be dependent on the inoculum size.
A. niger
is capable of degrading sorbic acid, and this ability has consequences for food preservation strategies. The mechanism of action of sorbic acid was investigated using
31
P nuclear magnetic resonance (NMR) spectroscopy. We show that a rapid decline in cytosolic pH (pH
cyt
) by more than 1 pH unit and a depression of vacuolar pH (pH
vac
) in
A. niger
occurs in the presence of sorbic acid. The pH gradient over the vacuole completely collapsed as a result of the decline in pH
cyt
. NMR spectra also revealed that sorbic acid (3.0 mM at pH 4.0) caused intracellular ATP pools and levels of sugar-phosphomonoesters and -phosphodiesters of
A. niger
mycelia to decrease dramatically, and they did not recover. The disruption of pH homeostasis by sorbic acid at concentrations below the MIC could account for the delay in spore germination and retardation of the onset of subsequent mycelial growth.
Prevalence of the
stx
2
Gene in Coliform Populations from Aquatic Environments
Authors: García-Aljaro Cristina , Muniesa Maite , Jofre Juan , Blanch Anicet R. 
Published: June 2004

ABSTRACT

Shiga toxin-producing
Escherichia coli
strains are human pathogens linked to hemorrhagic colitis and hemolytic uremic syndrome. The major virulence factors of these strains are Shiga toxins Stx1 and Stx2. The majority of the genes coding for these toxins are borne by bacteriophages. Free Stx2-encoding bacteriophages have been found in aquatic environments, but there is limited information about the lysogenic strains and bacteria present in the environment that are susceptible to phage infection. The aim of this work was to study the prevalence and the distribution of the
stx
2
gene in coliform bacteria in sewage samples of different origins. The presence of the
stx
2
gene was monitored every 2 weeks over a 1-year period in a municipal sewage treatment plant. A mean value of 10
2
genes/ml was observed without significant variation during the study period. This concentration was of the same order of magnitude in raw municipal sewage of various origins and in animal wastewater from several slaughterhouses. A total of 138 strains carrying the
stx
2
gene were isolated by colony hybridization. This procedure detected approximately 1 gene-carrying colony per 1,000 fecal coliform colonies in municipal sewage and around 1 gene-carrying colony per 100 fecal coliform colonies in animal wastewaters. Most of the isolates belonged to
E. coli
serotypes other than
E. coli
O157, suggesting a low prevalence of strains of this serotype carrying the
stx
2
gene in the wastewater studied.
Biotransformation of Explosives by the Old Yellow Enzyme Family of Flavoproteins
Authors: Williams Richard E. , Rathbone Deborah A. , Scrutton Nigel S. , Bruce Neil C. 
Published: June 2004

ABSTRACT
Several independent studies of bacterial degradation of nitrate ester explosives have demonstrated the involvement of flavin-dependent oxidoreductases related to the old yellow enzyme (OYE) of yeast. Some of these enzymes also transform the nitroaromatic explosive 2,4,6-trinitrotoluene (TNT). In this work, catalytic capabilities of five members of the OYE family were compared, with a view to correlating structure and function. The activity profiles of the five enzymes differed substantially; no one compound proved to be a good substrate for all five enzymes. TNT is reduced, albeit slowly, by all five enzymes. The nature of the transformation products differed, with three of the five enzymes yielding products indicative of reduction of the aromatic ring. Our findings suggest two distinct pathways of TNT transformation, with the initial reduction of TNT being the key point of difference between the enzymes. Characterization of an active site mutant of one of the enzymes suggests a structural basis for this difference.
Medium-Chain Fatty Acids Decrease Colonization and Invasion through
hilA
Suppression Shortly after Infection of Chickens with
Salmonella enterica
Serovar Enteritidis
Authors: Van Immerseel F. , De Buck J. , Boyen F. , Bohez L. , Pasmans F. , Volf J. , Sevcik M. , Rychlik I. , Haesebrouck F. , Ducatelle R. 
Published: June 2004

ABSTRACT

The most common source of
Salmonella
infections in humans is food of poultry origin.
Salmonella enterica
serovar Enteritidis has a particular affinity for the contamination of the egg supply. In this study, the medium-chain fatty acids (MCFA), caproic, caprylic, and capric acid, were evaluated for the control of
Salmonella
serovar Enteritidis in chickens. All MCFA were growth inhibiting at low concentrations in vitro, with caproic acid being the most potent. Contact of
Salmonella
serovar Enteritidis with low concentrations of MCFA decreased invasion in the intestinal epithelial cell line T84. By using transcriptional fusions between the promoter of the regulatory gene of the
Salmonella
pathogenicity island I,
hilA
, and
luxCDABE
genes, it was shown that all MCFA decreased the expression of
hilA
, a key regulator related to the invasive capacity of
Salmonella
. The addition of caproic acid (3 g/kg of feed) to the feed of chicks led to a significant decrease in the level of colonization of ceca and internal organs by
Salmonella
serovar Enteritidis at 3 days after infection of 5-day-old chicks. These results suggest that MCFA have a synergistic ability to suppress the expression of the genes required for invasion and to reduce the numbers of bacteria in vivo. Thus, MCFA are potentially useful products for reducing the level of colonization of chicks and could ultimately aid in the reduction of the number of contaminated eggs in the food supply.
Enrichment Followed by Quantitative PCR both for Rapid Detection and as a Tool for Quantitative Risk Assessment of Food-Borne Thermotolerant Campylobacters
Authors: Josefsen M. H. , Jacobsen N. R. , Hoorfar J. 
Published: June 2004

ABSTRACT

As part of a large international project for standardization of PCR (Food-PCR;
www.pcr.dk
), a multiplex, multiplatform, ready-to-go enrichment followed by a real-time PCR method, including an internal amplification control, was developed for detection of food-borne thermotolerant campylobacters in chickens. Chicken rinse samples were enriched in Bolton broth for 20 h, a simple and rapid (1-h) resin-based DNA extraction was performed, and DNA samples were then tested with two instrument platforms: ABI-PRISM 7700 and RotorGene 3000. The method was validated against an International Standard Organization (ISO)-based culture method by testing low, medium, and high levels of 12 spiked and 66 unspiked, presumably naturally contaminated, chicken rinse samples. In the RotorGene, a positive PCR response was detected in 40 samples of the 66. This was in complete agreement with the enriched ISO culture. The ABI-PRISM 7700 missed one culture-positive sample. Positive samples contained 10
2
to 10
7
CFU/ml after enrichment in Bolton broth. In the enriched samples a detection probability of 95% was obtained at levels of 1 × 10
3
and 2 × 10
3
CFU/ml in the RotorGene and ABI-PRISM, respectively. The amplification efficiency in both platforms was 90%, although the linear range of amplification of purified genomic DNA was 1.5 × 10
1
to 1 × 10
7
(
R
2
= 1.00) for the RotorGene and 10
3
to 10
7
(
R
2
= 0.99) for the ABI-PRISM. In RotorGene and ABI-PRISM the levels of precision of detection as determined by standard deviation (coefficients of variation) of 6-carboxyfluorescein (FAM) threshold cycle (Ct) values were 0.184 to 0.417 (0.65 to 2.57%) and 0.119 to 0.421 (0.59 to 1.82%), respectively. The results showed a correlation (
R
2
) of 0.94 between the target FAM Ct values and CFU per milliliter of enriched naturally contaminated chicken samples, which indicates PCR's additional potential as a tool for quantitative risk assessment. Signal from the internal amplification control was detected in all culture-negative samples (VIC Ct: 23.1 to 28.1). The method will be taken further and validated in an international collaborative trial with regard to standardization.
Establishment of a Real-Time PCR-Based Approach for Accurate Quantification of Bacterial RNA Targets in Water, Using
Salmonella
as a Model Organism
Authors: Fey Axel , Eichler Stefan , Flavier Sébastien , Christen Richard , Höfle Manfred G. , Guzmán Carlos A. 
Published: June 2004

ABSTRACT

Quantitative PCR (Q-PCR) is a fast and efficient tool to quantify target genes. In eukaryotic cells, quantitative reverse transcription-PCR (Q-RT-PCR) is also used to quantify gene expression, with stably expressed housekeeping genes as standards. In bacteria, such stable expression of housekeeping genes does not occur, and the use of DNA standards leads to a broad underestimation. Therefore, an accurate quantification of RNA is feasible only by using appropriate RNA standards. We established and validated a Q-PCR method which enables the quantification of not only the number of copies of target genes (i.e., the number of bacterial cells) but also the number of RNA copies. The genes coding for InvA and the 16S rRNA of
Salmonella enterica
serovar Typhimurium were selected for the evaluation of the method. As DNA standards, amplified fragments of the target genes were used, whereas the same DNA standards were transcribed in vitro for the development of appropriate RNA standards.
Salmonella
cultures and environmental water samples inoculated with bacteria were then employed for the final testing. Both experimental approaches led to a sensitive, accurate, and reproducible quantification of the selected target genes and RNA molecules by Q-PCR and Q-RT-PCR. It is the first time that RNA standards have been successfully used for a precise quantification of the number of RNA molecules in prokaryotes. This demonstrates the potential of this approach for determining the presence and metabolic activity of pathogenic bacteria in environmental samples.
Biotransformation in Double-Phase Systems: Physiological Responses of
Pseudomonas putida
DOT-T1E to a Double Phase Made of Aliphatic Alcohols and Biosynthesis of Substituted Catechols
Authors: Rojas Antonia , Duque Estrella , Schmid Andreas , Hurtado Ana , Ramos Juan-Luis , Segura Ana 
Published: June 2004

ABSTRACT

Pseudomonas putida
strain DOT-T1E is highly tolerant to organic solvents, with a logP
ow
(the logarithm of the partition coefficient of a solvent in a two-phase water-octanol system of ≥2.5. Solvent tolerant microorganisms can be exploited to develop double-phase (organic solvent and water) biotransformation systems in which toxic substrates or products are kept in the organic phase. We tested
P. putida
DOT-T1E tolerance to different aliphatic alcohols with a logP
ow
value between 2 and 4, such as decanol, nonanol, and octanol, which are potentially useful in biotransformations in double-phase systems in which compounds with a logP
ow
around 1.5 are produced.
P. putida
DOT-T1E responds to aliphatic alcohols as the second phase through
cis-
to-
trans
isomerization of unsaturated
cis
fatty acids and through efflux of these aliphatic alcohols via a series of pumps that also extrude aromatic hydrocarbons. These defense mechanisms allow
P. putida
DOT-T1E to survive well in the presence of high concentrations of the aliphatic alcohols, and growth with nonanol or decanol occurred at a high rate, whereas in the presence of an octanol double-phase growth was compromised. Our results support that the logP
ow
of aliphatic alcohols correlates with their toxic effects, as octanol (logP
ow
= 2.9) has more negative effects in
P. putida
cells than 1-nonanol (logP
ow
= 3.4) or 1-decanol (logP
ow
= 4). A
P. putida
DOT-T1E derivative bearing plasmid pWW0-
xylE
::Km transforms
m-
xylene (logP
ow
= 3.2) into 3-methylcatechol (logP
ow
= 1.8). The amount of 3-methylcatechol produced in an aliphatic alcohol/water bioreactor was 10- to 20-fold higher than in an aqueous medium, demonstrating the usefulness of double-phase systems for this particular biotransformation.
Sequence-Specific Cleavage of Small-Subunit (SSU) rRNA with Oligonucleotides and RNase H: a Rapid and Simple Approach to SSU rRNA-Based Quantitative Detection of Microorganisms
Authors: Uyeno Yutaka , Sekiguchi Yuji , Sunaga Akiko , Yoshida Hiroki , Kamagata Yoichi 
Published: June 2004

ABSTRACT
A
rapid and simple approach to the small-subunit (SSU) rRNA-based
quantitative detection of a specific group of microorganisms in complex
ecosystems has been developed. The method employs sequence-specific
cleavage of rRNA molecules with oligonucleotides and RNase H. Defined
mixtures of SSU rRNAs were mixed with an oligonucleotide (referred to
as a “scissor probe”) that was specifically designed to
hybridize with a particular site of targeted rRNA and were subsequently
digested with RNase H to proceed to sequence-dependent rRNA scission at
the hybridization site. Under appropriate reaction conditions, the
targeted rRNAs were correctly cut into two fragments, whereas
nontargeted rRNAs remained intact under the same conditions. The
specificity of the cleavage could be properly adjusted by controlling
the hybridization stringency between the rRNA and the oligonucleotides,
i.e., by controlling either the temperature of the reaction or the
formamide concentration in the hybridization-digestion buffer used for
the reaction. This enabled the reliable discrimination of completely
matched rRNA sequences from single-base mismatched sequences. For the
detection of targeted rRNAs, the resulting RNA fragment patterns were
analyzed by gel electrophoresis with nucleotide-staining fluorescent
dyes in order to separate cleaved and intact rRNA molecules. The
relative abundance of the targeted SSU rRNA fragments in the total SSU
rRNA could easily be calculated without the use of an external standard
by determining the signal intensity of individual SSU rRNA bands in the
electropherogram. This approach provides a fast and easy means of
identification, detection, and quantification of a particular group of
microbes in clinical and environmental specimens based on
rRNA.
Identification and Characterization of the Novel LysM Domain-Containing Surface Protein Sep from
Lactobacillus fermentum
BR11 and Its Use as a Peptide Fusion Partner in
Lactobacillus
and
Lactococcus
Authors: Turner Mark S. , Hafner Louise M. , Walsh Terry , Giffard Philip M. 
Published: June 2004

ABSTRACT

Examination of supernatant fractions from broth cultures of
Lactobacillus fermentum
BR11 revealed the presence of a number of proteins, including a 27-kDa protein termed Sep. The amino-terminal sequence of Sep was determined, and the gene encoding it was cloned and sequenced. Sep is a 205-amino-acid protein and contains a 30-amino-acid secretion signal and has overall homology (between 39 and 92% identity) with similarly sized proteins of
Lactobacillus reuteri
,
Enterococcus faecium
,
Streptococcus pneumoniae
,
Streptococcus agalactiae
, and
Lactobacillus plantarum
. The carboxy-terminal 81 amino acids of Sep also have strong homology (86% identity) to the carboxy termini of the aggregation-promoting factor (APF) surface proteins of
Lactobacillus gasseri
and
Lactobacillus johnsonii
. The mature amino terminus of Sep contains a putative peptidoglycan-binding LysM domain, thereby making it distinct from APF proteins. We have identified a common motif within LysM domains that is shared with carbohydrate binding YG motifs which are found in streptococcal glucan-binding proteins and glucosyltransferases. Sep was investigated as a heterologous peptide expression vector in
L. fermentum
,
Lactobacillus rhamnosus
GG and
Lactococcus lactis
MG1363. Modified Sep containing an amino-terminal six-histidine epitope was found associated with the cells but was largely present in the supernatant in the
L. fermentum
,
L. rhamnosus
, and
L. lactis
hosts. Sep as well as the previously described surface protein BspA were used to express and secrete in
L. fermentum
or
L. rhamnosus
a fragment of human E-cadherin, which contains the receptor region for
Listeria monocytogenes
. This study demonstrates that Sep has potential for heterologous protein expression and export in lactic acid bacteria.
Endogenous Xylose Pathway in
Saccharomyces cerevisiae
Authors: Toivari Mervi H. , Salusjärvi Laura , Ruohonen Laura , Penttilä Merja 
Published: June 2004

ABSTRACT

The baker's yeast
Saccharomyces cerevisiae
is generally classified as a non-xylose-utilizing organism. We found that
S. cerevisiae
can grow on
d
-xylose when only the endogenous genes
GRE3
(
YHR104w
), coding for a nonspecific aldose reductase, and
XYL2
(
YLR070c
,
ScXYL2
), coding for a xylitol dehydrogenase (XDH), are overexpressed under endogenous promoters. In nontransformed
S. cerevisiae
strains, XDH activity was significantly higher in the presence of xylose, but xylose reductase (XR) activity was not affected by the choice of carbon source. The expression of
SOR1
, encoding a sorbitol dehydrogenase, was elevated in the presence of xylose as were the genes encoding transketolase and transaldolase. An
S. cerevisiae
strain carrying the XR and XDH enzymes from the xylose-utilizing yeast
Pichia stipitis
grew more quickly and accumulated less xylitol than did the strain overexpressing the endogenous enzymes. Overexpression of the
GRE3
and
ScXYL2
genes in the
S. cerevisiae
CEN.PK2 strain resulted in a growth rate of 0.01 g of cell dry mass liter
−1
h
−1
and a xylitol yield of 55% when xylose was the main carbon source.
Uptake and Replication ofSalmonella entericainAcanthamoeba rhysodes
Authors: Tezcan-Merdol Dilek , Ljungström Marianne , Winiecka-Krusnell Jadwiga , Linder Ewert , Engstrand Lars , Rhen Mikael 
Published: June 2004

ABSTRACTThe ability of salmonellae to become internalized and to survive and replicate in amoebae was evaluated by using three separate serovars ofSalmonella entericaand five different isolates of axenicAcanthamoebaspp. In gentamicin protection assays,Salmonella entericaserovar Dublin was internalized more efficiently thanSalmonella entericaserovar Enteritidis orSalmonella entericaserovar Typhimurium in all of the amoeba isolates tested. The bacteria appeared to be most efficiently internalized byAcanthamoeba rhysodes. Variations in bacterial growth conditions affected internalization efficiency, but this effect was not altered by inactivation ofhilA, a key regulator in the expression of the invasion-associatedSalmonellapathogenicity island 1. Microscopy of infectedA. rhysodesrevealed thatS. entericaresided within vacuoles. Prolonged incubation resulted in a loss of intracellular bacteria associated with morphological changes and loss of amoebae. In part, these alterations were associated withhilAand theSalmonellavirulence plasmid. The data show thatAcanthamoebaspp. can differentiate between different serovars of salmonellae and that internalization is associated with cytotoxic effects mediated by definedSalmonellavirulence loci.
Cryptosporidium parvum
and
Giardia lamblia
Recovered from Flies on a Cattle Farm and in a Landfill
Authors: Szostakowska Beata , Kruminis-Lozowska Wieslawa , Racewicz Maria , Knight Ronald , Tamang Leena , Myjak Przemyslaw , Graczyk Thaddeus K. 
Published: June 2004

ABSTRACT

Filth flies associated with a cattle barn and a municipal landfill were tested positive by combined immunofluorescent antibody and fluorescent in situ hybridization for
Cryptosporidium parvum
and
Giardia lamblia
on their exoskeletons and in their guts. More pathogens were carried by flies from the cattle barn than from the landfill; 81% of
C. parvum
and 84% of
G. lamblia
pathogens were presumptively viable.
Optimization of Procedures for Isolation of Mycobacteria from Soil and Water Samples Obtained in Northern India
Authors: Parashar Deepti , Chauhan D. S. , Sharma V. D. , Chauhan Aradhana , Chauhan S. V. S. , Katoch V. M. 
Published: June 2004

ABSTRACT
For isolation of environmental mycobacteria, a decontamination procedure has been standardized by which treatment with 3% sodium dodecyl sulfate plus 4% NaOH (15 and 30 min for rapid and slow growers, respectively) is followed by incubation with 2% cetrimide (5 and 15 min for fast- and slow-growing mycobacteria, respectively); this procedure was found to completely eliminate contamination with other organisms and resulted in the isolation of only mycobacteria.
Diverse Tetracycline Resistance Genotypes of
Megasphaera elsdenii
Strains Selectively Cultured from Swine Feces
Authors: Stanton Thaddeus B. , McDowall Jennifer S. , Rasmussen Mark A. 
Published: June 2004

ABSTRACT

A
total of 30
Megasphaera elsdenii
strains, selectively isolated
from the feces of organically raised swine by using Me109 M medium, and
one bovine strain were analyzed for tetracycline resistance genotypic
and phenotypic traits. Tetracycline-resistant strains carried
tet
(O),
tet
(W), or a
tet
gene mosaic of
tet
(O) and
tet
(W).
M. elsdenii
strains
carrying
tet
(OWO) genes exhibited the highest tetracycline
MICs (128 to >256 μg/ml), suggesting that
tet
(O)-
tet
(W) mosaic genes provide the selective
advantage of greater tetracycline resistance for this species. Seven
tet
genotypes are now known for
M. elsdenii
, an
archetype commensal anaerobe and model for
tet
gene evolution
in the mammalian intestinal
tract.
Molecular and Biological Characterization of a
Cryptosporidium molnari
-Like Isolate from a Guppy (
Poecilia reticulata
)
Authors: Ryan Una , O'Hara Amanda , Xiao Lihua 
Published: June 2004

ABSTRACT

Histological, morphological, genetic, and phylogenetic analyses of a
Cryptosporidium molnari
-like isolate from a guppy (
Poecilia reticulata
) identified stages consistent with those of
C. molnari
and revealed that
C. molnari
is genetically very distinct from all other species of
Cryptosporidium
. This study represents the first genetic characterization of
C. molnari
.
Immobilization of
Saccharomyces cerevisiae
Cystathionine γ-Lyase and Application of the Product to Cystathionine Synthesis
Authors: Yamagata Shuzo , Akamatsu Tsuyoshi , Iwama Tomonori 
Published: June 2004

ABSTRACT

Cystathionine γ-lyase of
Saccharomyces cerevisiae
was immobilized to aminohexyl-Sepharose through the cofactor pyridoxal 5′-phosphate and was characterized with respect to its cystathionine γ-synthase activity. The immobilized product was so stable that it repeatedly catalyzed as many as five cycles of the reaction without losing activity.
Novel Epibiotic
Thiothrix
Bacterium on a Marine Amphipod
Authors: Gillan David C. , Dubilier Nicole 
Published: June 2004

ABSTRACT

Comparative analysis of the 16S rRNA gene and fluorescent in situ hybridization (FISH) was used to identify epibiotic filamentous bacteria living on the marine amphipod crustacean
Urothoe poseidonis
. The epibionts belong to the gamma proteobacteria and represent a novel marine phylotype within the genus
Thiothrix
. FISH and denaturing gradient gel electrophoresis revealed that the
Thiothrix
filaments are present on the majority of the amphipods examined.