bluuebunny/crossref_metadata_2025_split huggingface dataset:
Quantitative Detection of
Listeria monocytogenes
and
Listeria innocua
by Real-Time PCR: Assessment of
hly
,
iap
, and
lin02483
Targets and AmpliFluor Technology
Authors: Rodríguez-Lázaro David , Hernández Marta , Scortti Mariela , Esteve Teresa , Vázquez-Boland José A. , Pla Maria 
Published: March 2004

ABSTRACT

We developed and assessed real-time PCR (RTi-PCR) assays for the detection and quantification of the food-borne pathogen
Listeria monocytogenes
and the closely related nonpathogenic species
L. innocua
. The target genes were
hly
and
iap
for
L. monocytogenes
and
lin02483
for
L. innocua
. The assays were 100% specific, as determined with 100
Listeria
strains and 45 non-
Listeria
strains, and highly sensitive, with detection limits of one target molecule in 11 to 56% of the reactions with purified DNA and 3 CFU in 56 to 89% of the reactions with bacterial suspensions. Quantification was possible over a 5-log dynamic range, with a limit of 15 target molecules and
R
2
values of >0.996. There was an excellent correspondence between the predicted and the actual numbers of CFU in the samples (deviations of <23%). The
hly
-based assay accurately quantified
L. monocytogenes
in all of the samples tested. The
iap
-based assay, in contrast, was unsuitable for quantification purposes, underestimating the bacterial counts by 3 to 4 log units in a significant proportion of the samples due to serovar-related target sequence variability. The combination of the two assays enabled us to classify
L. monocytogenes
isolates into one of the two major phylogenetic divisions of the species, I and II. We also assessed the new AmpliFluor technology for the quantitative detection of
L. monocytogenes
by RTi-PCR. The performance of this system was similar to that of the TaqMan system, although the former system was slightly less sensitive (detection limit of 15 molecules in 45% of the reactions) and had a higher quantification limit (60 molecules).
Interaction ofBacillus thuringiensisToxins with Larval Midgut Binding Sites ofHelicoverpa armigera(Lepidoptera: Noctuidae)
Authors: Estela Anna , Escriche Baltasar , Ferré Juan 
Published: March 2004

ABSTRACTIn 1996, Bt-cotton (cotton expressing aBacillus thuringiensistoxin gene) expressing the Cry1Ac protein was commercially introduced to control cotton pests. A threat to this first generation of transgenic cotton is the evolution of resistance by the insects. Second-generation Bt-cotton has been developed with either newB. thuringiensisgenes or with a combination ofcrygenes. However, one requirement for the “stacked” gene strategy to work is that the stacked toxins bind to different binding sites. In the present study, the binding of125I-labeled Cry1Ab protein (125I-Cry1Ab) and125I-Cry1Ac to brush border membrane vesicles (BBMV) ofHelicoverpa armigerawas analyzed in competition experiments with 11 nonlabeled Cry proteins. The results indicate that Cry1Aa, Cry1Ab, and Cry1Ac competed for common binding sites. No other Cry proteins tested competed for either125I-Cry1Ab or125I-Cry1Ac binding, except Cry1Ja, which competed only at the highest concentrations used. Furthermore, BBMV from fourH. armigerapopulations were also tested with125I-Cry1Ac and Cry1Ab to check the influence of the insect population on the binding results. Finally, the inhibitory effect of selected sugars and lectins was also determined.125I-Cry1Ac binding was strongly inhibited byN-acetylgalactosamine, sialic acid, and concanavalin A and moderately inhibited by soybean agglutinin. In contrast,125I-Cry1Ab binding was only significantly inhibited by concanavalin A. These results show that Cry1Ac and Cry1Ab use different epitopes for binding to BBMV.
Aerobic and Anaerobic Toluene Degradation by a Newly Isolated Denitrifying Bacterium,
Thauera
sp. Strain DNT-1
Authors: Shinoda Yoshifumi , Sakai Yasuyoshi , Uenishi Hiroshi , Uchihashi Yasumitsu , Hiraishi Akira , Yukawa Hideaki , Yurimoto Hiroya , Kato Nobuo 
Published: March 2004

ABSTRACT

A newly isolated denitrifying bacterium,
Thauera
sp. strain DNT-1, grew on toluene as the sole carbon and energy source under both aerobic and anaerobic conditions. When this strain was cultivated under oxygen-limiting conditions with nitrate, first toluene was degraded as oxygen was consumed, while later toluene was degraded as nitrate was reduced. Biochemical observations indicated that initial degradation of toluene occurred through a dioxygenase-mediated pathway and the benzylsuccinate pathway under aerobic and denitrifying conditions, respectively. Homologous genes for toluene dioxygenase (
tod
) and benzylsuccinate synthase (
bss
), which are the key enzymes in aerobic and anaerobic toluene degradation, respectively, were cloned from genomic DNA of strain DNT-1. The results of Northern blot analyses and real-time quantitative reverse transcriptase PCR suggested that transcription of both sets of genes was induced by toluene. In addition, the
tod
genes were induced under aerobic conditions, whereas the
bss
genes were induced under both aerobic and anaerobic conditions. On the basis of these results, it is concluded that strain DNT-1 modulates the expression of two different initial pathways of toluene degradation according to the availability of oxygen in the environment.
Comparison of PCR, Electrochemical Enzyme-Linked Immunosorbent Assays, and the Standard Culture Method for Detecting
Salmonella
in Meat Products
Authors: Croci Luciana , Delibato Elisabetta , Volpe Giulia , De Medici Dario , Palleschi Giuseppe 
Published: March 2004

ABSTRACT
An electrochemical enzyme-linked immunosorbent assay (ELISA) coupled with flow injection analysis (ELISA-FIA) and a PCR-based method using ST11 and ST15 primers for detecting salmonellae in meat were evaluated in comparison with the International Organization for Standardization (ISO) culture method. The methods were applied to experimentally contaminated and naturally contaminated meat samples. The results showed that both ELISA-FIA and PCR allowed detection of salmonella in a product contaminated with a low number of the microorganisms (1 to 10 salmonellae/25 g) after only 5 h of incubation of preenrichment broth, and they were just as effective as the ISO method.
Characterization of a Thermostable
l
-Arabinose (
d
-Galactose) Isomerase from the Hyperthermophilic Eubacterium
Thermotoga maritima
Authors: Lee Dong-Woo , Jang Hyeung-Jin , Choe Eun-Ah , Kim Byoung-Chan , Lee Sang-Jae , Kim Seong-Bo , Hong Young-Ho , Pyun Yu-Ryang 
Published: March 2004

ABSTRACT

The
araA
gene encoding
l
-arabinose isomerase (AI) from the hyperthermophilic bacterium
Thermotoga maritima
was cloned and overexpressed in
Escherichia coli
as a fusion protein containing a C-terminal hexahistidine sequence. This gene encodes a 497-amino-acid protein with a calculated molecular weight of 56,658. The recombinant enzyme was purified to homogeneity by heat precipitation followed by Ni
2+
affinity chromatography. The native enzyme was estimated by gel filtration chromatography to be a homotetramer with a molecular mass of 232 kDa. The purified recombinant enzyme had an isoelectric point of 5.7 and exhibited maximal activity at 90°C and pH 7.5 under the assay conditions used. Its apparent

K
m

values for
l
-arabinose and
d
-galactose were 31 and 60 mM, respectively; the apparent
V
max
values (at 90°C) were 41.3 U/mg (
l
-arabinose) and 8.9 U/mg (
d
-galactose), and the catalytic efficiencies (
k
cat
/

K
m

) of the enzyme were 74.8 mM
−1
· min
−1
(
l
-arabinose) and 8.5 mM
−1
· min
−1
(
d
-galactose). Although the
T. maritima
AI exhibited high levels of amino acid sequence similarity (>70%) to other heat-labile mesophilic AIs, it had greater thermostability and higher catalytic efficiency than its mesophilic counterparts at elevated temperatures. In addition, it was more thermostable in the presence of Mn
2+
and/or Co
2+
than in the absence of these ions. The enzyme carried out the isomerization of
d
-galactose to
d
-tagatose with a conversion yield of 56% for 6 h at 80°C.
Vanadium(V) Reduction by
Shewanella oneidensis
MR-1 Requires Menaquinone and Cytochromes from the Cytoplasmic and Outer Membranes
Authors: Myers Judith M. , Antholine William E. , Myers Charles R. 
Published: March 2004

ABSTRACT

The metal-reducing bacterium
Shewanella oneidensis
MR-1 displays remarkable anaerobic respiratory plasticity, which is reflected in the extensive number of electron transport components encoded in its genome. In these studies, several cell components required for the reduction of vanadium(V) were determined. V(V) reduction is mediated by an electron transport chain which includes cytoplasmic membrane components (menaquinone and the tetraheme cytochrome CymA) and the outer membrane (OM) cytochrome OmcB. A partial role for the OM cytochrome OmcA was evident. Electron spin resonance spectroscopy demonstrated that V(V) was reduced to V(IV). V(V) reduction did not support anaerobic growth. This is the first report delineating specific electron transport components that are required for V(V) reduction and of a role for OM cytochromes in the reduction of a soluble metal species.
PCR-Denaturing Gradient Gel Electrophoresis Profiling of Inter- and Intraspecies 18S rRNA Gene Sequence Heterogeneity Is an Accurate and Sensitive Method To Assess Species Diversity of Arbuscular Mycorrhizal Fungi of the GenusGigaspora
Authors: de Souza Francisco A. , Kowalchuk George A. , Leeflang Paula , van Veen Johannes A. , Smit Eric 
Published: March 2004

ABSTRACTDespite the importance of arbuscular mycorrhizal fungi in the majority of terrestrial ecosystems, their ecology, genetics, and evolution are poorly understood, partly due to difficulties associated with detecting and identifying species. We explored the inter- and intraspecies variations of the 18S rRNA genes of the genusGigasporato assess the use of this marker for the discrimination ofGigasporaisolates and ofGigasporaceaepopulations from environmental samples. Screening of 48Gigasporaisolates by PCR-denaturing gradient gel electrophoresis (DGGE) revealed that the V3-V4 region of the 18S rRNA gene contained insufficient variation to discriminate between differentGigasporaspecies. In contrast, the patterns of 18S ribosomal DNA (rDNA) heterogeneity within the V9 region of this marker could be used for reliable identification of all recognized species within this genus. PCR-DGGE patterns provided insight into some putative misidentifications and could be used to differentiate geographic isolates ofG. albida,G. gigantea, andG. margaritabut notG. rosea. Two major clusters were apparent based upon PCR-DGGE ribotype patterns, one containingG. albida,G. candida,G. ramisporophora, andG. roseaand the other containingG. decipiensandG. margarita. Dissection of the DGGE patterns by cloning, DGGE screening, and sequencing confirmed these groupings and revealed that some ribotypes were shared across species boundaries. Of the 48 isolates examined, only two displayed any spore-to-spore variation, and these exceptions may be indicative of coisolation of more than one species or subspecies within these cultures. Two Brazilian agricultural soils were also analyzed with aGigasporaceae-specific nested PCR approach, revealing a dominance ofG. margaritawithin this family.
Development of a Markerless Genetic Exchange Method for
Methanosarcina acetivorans
C2A and Its Use in Construction of New Genetic Tools for Methanogenic Archaea
Authors: Pritchett Matthew A. , Zhang Jun Kai , Metcalf William W. 
Published: March 2004

ABSTRACT

A new genetic technique for constructing mutants of
Methanosarcina acetivorans
C2A by using
hpt
as a counterselectable marker was developed. Mutants with lesions in the
hpt
gene, encoding hypoxanthine phosphoribosyltransferase, were shown to be >35-fold more resistant to the toxic base analog 8-aza-2,6-diaminopurine (8ADP) than was the wild type. Reintroduction of the
hpt
gene into a Δ
hpt
host restored 8ADP sensitivity and provided the basis for a two-step strategy involving plasmid integration and excision for recombination of mutant alleles onto the
M. acetivorans
chromosome. We have designated this method markerless exchange because, although selectable markers are used during the process, they are removed in the final mutants. Thus, the method can be repeated many times in the same cell line. The method was validated by construction of Δ
proC
Δ
hpt
mutants, which were recovered at a frequency of 22%. Additionally, a
Methanosarcina-Escherichia
shuttle vector, encoding the
Escherichia coli proC
gene as a new selectable marker, was constructed for use in
proC
hosts. Finally, the markerless exchange method was used to recombine a series of
uidA
reporter gene fusions into the
M. acetivorans proC
locus. In vitro assay of β-glucuronidase activity in extracts of these recombinants demonstrated, for the first time, the utility of
uidA
as a reporter gene in
Methanosarcina.
A >5,000-fold range of promoter activities could be measured by using
uidA
: the methyl-coenzyme M reductase operon fusion displayed ∼300-fold-higher activity than did the
serC
gene fusion, which in turn had 16-fold-higher activity than did a fusion to the unknown
orf2
gene.
Symbiont-Induced Changes in Host Actin during the Onset of a Beneficial Animal-Bacterial Association
Authors: Kimbell Jennifer R. , McFall-Ngai Margaret J. 
Published: March 2004

ABSTRACT

The influence of bacteria on the cytoskeleton of animal cells has been studied extensively only in pathogenic associations. We characterized changes in host cytoskeletal actin induced by the bacterial partner during the onset of a cooperative animal-bacteria association using the squid-vibrio model. Two-dimensional sodium dodecyl sulfate-polyacrylamide gel electrophoresis and Western blot analysis revealed that
Vibrio fischeri
induced a dramatic increase in actin protein abundance in the bacteria-associated host tissues during the onset of the symbiosis. Immunocytochemistry revealed that this change in actin abundance correlated with a two- to threefold increase in actin in the apical cell surface of the epithelium-lined ducts, the route of entry of symbionts into host tissues. Real-time reverse transcriptase PCR and in situ hybridization did not detect corresponding changes in actin mRNA. Temporally correlated with the bacteria-induced changes in actin levels was a two- to threefold decrease in duct circumference, a 20% loss in the average number of cells interfacing with the duct lumina, and dramatic changes in duct cell shape. When considered with previous studies of the biomechanical and biochemical characteristics of the duct, these findings suggest that the bacterial symbionts, upon colonizing the host organ, induce modifications that physically and chemically limit the opportunity for subsequent colonizers to pass through the ducts. Continued study of the squid-vibrio system will allow further comparisons of the mechanisms by which pathogenic and cooperative bacteria influence cytoskeleton dynamics in host cells.
Comparison of Prevalence and Antimicrobial Susceptibilities ofCampylobacterspp. Isolates from Organic and Conventional Dairy Herds in Wisconsin
Authors: Sato K. , Bartlett P. C. , Kaneene J. B. , Downes F. P. 
Published: March 2004

ABSTRACTThe prevalence and antimicrobial susceptibilities ofCampylobacterspp.isolates from bovine feces were compared between organic and conventional dairy herds. Thirty organic dairy herds, where antimicrobials are rarely used for calves and never used for cows, were compared with 30 neighboring conventional dairy farms, where antimicrobials were routinely used for animals for all ages. Fecal specimens from 10 cows and 10 calves on 120 farm visits yielded 332Campylobacterisolates. The prevalence ofCampylobacterspp. in organic and conventional farms was 26.7 and 29.1%, and the prevalence was not statistically different between the two types of farms.Campylobacterprevalence was significantly higher in March than in September, higher in calves than in cows, and higher in smaller farms than in large farms. The rates of retained placenta, pneumonia, mastitis, and abortion were associated with the proportion ofCampylobacterisolation from fecal samples. The gradient disk diffusion MIC method (Etest) was used for testing susceptibility to four antimicrobial agents: ciprofloxacin, gentamicin, erythromycin, and tetracycline. Two isolates were resistant to ciprofloxacin, and none of isolates was resistant to gentamicin or erythromycin. Resistance to tetracycline was 45% (148 of 332 isolates). Tetracycline resistance was found more frequently in calves than in cows (P= 0.042), but no difference was observed between organic and conventional farms. When we usedCampylobacterspp.as indicator bacteria, we saw no evidence that restriction of antimicrobial use on dairy farms was associated with prevalence of resistance to ciprofloxacin, gentamicin, erythromycin, and tetracycline.
Detection of Bovine and Porcine Adenoviruses for Tracing the Source of Fecal Contamination
Authors: Maluquer de Motes Carlos , Clemente-Casares Pilar , Hundesa Ayalkibet , Martín Margarita , Girones Rosina 
Published: March 2004

ABSTRACT

In this study, a molecular procedure for the detection of adenoviruses of animal origin was developed to evaluate the level of excretion of these viruses by swine and cattle and to design a test to facilitate the tracing of specific sources of environmental viral contamination. Two sets of oligonucleotides were designed, one to detect porcine adenoviruses and the other to detect bovine and ovine adenoviruses. The specificity of the assays was assessed in 31 fecal samples and 12 sewage samples that were collected monthly during a 1-year period. The data also provided information on the environmental prevalence of animal adenoviruses. Porcine adenoviruses were detected in 17 of 24 (70%) pools of swine samples studied, with most isolates being closely related to serotype 3. Bovine adenoviruses were present in 6 of 8 (75%) pools studied, with strains belonging to the genera
Mastadenovirus
and
Atadenovirus
and being similar to bovine adenoviruses of types 2, 4, and 7. These sets of primers produced negative results in nested PCR assays when human adenovirus controls and urban-sewage samples were tested. Likewise, the sets of primers previously designed for detection of human adenovirus also produced negative results with animal adenoviruses. These results indicate the importance of further studies to evaluate the usefulness of these tests to trace the source of fecal contamination in water and food and for environmental studies.
Development and Testing of a DNA Macroarray To Assess Nitrogenase (
nifH
) Gene Diversity
Authors: Steward Grieg F. , Jenkins Bethany D. , Ward Bess B. , Zehr Jonathan P. 
Published: March 2004

ABSTRACT

A DNA macroarray was developed and evaluated for its potential to distinguish variants of the dinitrogenase reductase (
nifH
) gene. Diverse
nifH
gene fragments amplified from a clone library were spotted onto nylon membranes. Amplified, biotinylated
nifH
fragments from individual clones or a natural picoplankton community were hybridized to the array and detected by chemiluminescence. A hybridization test with six individual targets mixed in equal proportions resulted in comparable relative signal intensities for the corresponding probes (standard deviation, 14%). When the targets were mixed in unequal concentrations, there was a predictable, but nonlinear, relationship between target concentration and relative signal intensity. Results implied a detection limit of roughly 13 pg of target ml
−1
, a half-saturation of signal at 0.26 ng ml
−1
, and a dynamic range of about 2 orders of magnitude. The threshold for cross-hybridization varied between 78 and 88% sequence identity. Hybridization patterns were reproducible with significant correlations between signal intensities of duplicate probes (
r
= 0.98,
P
< 0.0001,
n
= 88). A mixed
nifH
target amplified from a natural Chesapeake Bay water sample hybridized strongly to 6 of 88 total probes and weakly to 17 additional probes. The natural community results were well simulated (
r
= 0.941,
P
< 0.0001,
n
= 88) by hybridizing a defined mixture of six individual targets corresponding to the strongly hybridizing probes. Our results indicate that macroarray hybridization can be a highly reproducible, semiquantitative method for assessing the diversity of functional genes represented in mixed pools of PCR products amplified from the environment.
Engineering
Lactococcus lactis
for Production of Mannitol: High Yields from Food-Grade Strains Deficient in Lactate Dehydrogenase and the Mannitol Transport System
Authors: Gaspar Paula , Neves Ana Rute , Ramos Ana , Gasson Michael J. , Shearman Claire A. , Santos Helena 
Published: March 2004

ABSTRACT

Mannitol is a sugar polyol claimed to have health-promoting properties. A mannitol-producing strain of
Lactococcus lactis
was obtained by disruption of two genes of the phosphoenolpyruvate (PEP)-mannitol phosphotransferase system (PTS
Mtl
). Genes
mtlA
and
mtlF
were independently deleted by double-crossover recombination in strain
L. lactis
FI9630 (a food-grade lactate dehydrogenase-deficient strain derived from MG1363), yielding two mutant (Δ
ldh
Δ
mtlA
and Δ
ldh
Δ
mtlF
) strains. The new strains, FI10091 and FI10089, respectively, do not possess any selection marker and are suitable for use in the food industry. The metabolism of glucose in nongrowing cell suspensions of the mutant strains was characterized by in vivo
13
C-nuclear magnetic resonance. The intermediate metabolite, mannitol-1-phosphate, accumulated intracellularly to high levels (up to 76 mM). Mannitol was a major end product, one-third of glucose being converted to this hexitol. The double mutants, in contrast to the parent strain, were unable to utilize mannitol even after glucose depletion, showing that mannitol was taken up exclusively by PEP-PTS
Mtl
. Disruption of this system completely blocked mannitol transport in
L. lactis
, as intended. In addition to mannitol, approximately equimolar amounts of ethanol, 2,3-butanediol, and lactate were produced. A mixed-acid fermentation (formate, ethanol, and acetate) was also observed during growth under controlled conditions of pH and temperature, but mannitol production was low. The reasons for the alteration in the pattern of end products under nongrowing and growing conditions are discussed, and strategies to improve mannitol production during growth are proposed.