bluuebunny/crossref_metadata_2025_split huggingface dataset:
Enhanced Trehalose Production Improves Growth of
Escherichia coli
under Osmotic Stress
Authors: Purvis J. E. , Yomano L. P. , Ingram L. O. 
Published: July 2005

ABSTRACT

The biosynthesis of trehalose has been previously shown to serve as an important osmoprotectant and stress protectant in
Escherichia coli
. Our results indicate that overproduction of trehalose (integrated
lacI
-P

tac

-
otsBA
) above the level produced by the native regulatory system can be used to increase the growth of
E. coli
in M9-2% glucose medium at 37°C to 41°C and to increase growth at 37°C in the presence of a variety of osmotic-stress agents (hexose sugars, inorganic salts, and pyruvate). Smaller improvements were noted with xylose and some fermentation products (ethanol and pyruvate). Based on these results, overproduction of trehalose may be a useful trait to include in biocatalysts engineered for commodity chemicals.
Environmental Temperature Controls
Cryptosporidium
Oocyst Metabolic Rate and Associated Retention of Infectivity
Authors: King Brendon J. , Keegan Alexandra R. , Monis Paul T. , Saint Christopher P. 
Published: July 2005

ABSTRACT

Cryptosporidium
is a significant cause of water-borne enteric disease throughout the world and represents a challenge to the water industry and a threat to public health. In this study we report the use of a cell culture-TaqMan PCR assay to measure oocyst inactivation rates in reagent-grade and environmental waters over a range of temperatures. While oocysts incubated at 4°C and 15°C remained infective over the 12-week holding period, we observed a 4 log
10
reduction in infectivity for both 20 and 25°C incubation treatments at 12 and 8 weeks, respectively, for all water types examined, a faster rate of inactivation for oocysts than previously reported. This temperature-dependent inactivation was further investigated using a simple and rapid ATP assay described herein. Time course experiments performed in reagent-grade water at incubation temperatures of 4, 15, 20, 25, 30, and 37°C identified a close relationship between oocyst infectivity and oocyst ATP content, demonstrating that temperature inactivation at higher temperatures is a function of increased oocyst metabolic activity. While water quality did not affect oocyst inactivation, biological antagonism appears to be a key factor affecting oocyst removal from environmental waters. Both the cell culture-TaqMan PCR assay and the ATP assay provide a sensitive and quantitative method for the determination of environmental oocyst inactivation, providing an alternative to the more costly and time-consuming mouse infection assay. The findings presented here relating temperature to oocyst inactivation provide valuable information for determining the relative risks associated with
Cryptosporidium
oocysts in water.
An Internal Reference Technique for Accurately Quantifying Specific mRNAs by Real-Time PCR with Application to the
tceA
Reductive Dehalogenase Gene
Authors: Johnson David R. , Lee Patrick K. H. , Holmes Victor F. , Alvarez-Cohen Lisa 
Published: July 2005

ABSTRACT

The accuracy of mRNA quantification by reverse transcription (RT) in conjunction with real-time PCR (qPCR) is limited by mRNA losses during sample preparation (cell lysis, RNA isolation, and DNA removal) and by inefficiencies in reverse transcription. To control for these losses and inefficiencies, a technique was developed that utilizes an exogenous internal reference mRNA (
ref
mRNA) along with mRNA absolute standard curves. The technique was applied to quantify mRNA of the trichloroethene (TCE) reductive dehalogenase-encoding
tceA
gene in an anaerobic TCE-to-ethene dechlorinating microbial enrichment. Compared to RT-qPCR protocols that utilize DNA absolute standard curves, application of the new technique increased measured quantities of
tceA
mRNA by threefold, demonstrating a substantial improvement in quantification. The technique was also effective for quantifying the loss of mRNA during specific steps of the sample processing protocol. Analysis revealed that the efficiency of the RNA isolation (56%) step was significantly less than that of the cell lysis (84%), DNA removal (93%), and RT (88%) steps. The technique was applied to compare the effects of cellular exposure to different chlorinated ethenes on
tceA
expression. Results show that exposure to TCE or
cis
-1,2-dichloroethene resulted in 25-fold-higher quantities of
tceA
mRNA than exposure to vinyl chloride or chlorinated ethene starvation.
Effects of Subtherapeutic Administration of Antimicrobial Agents to Beef Cattle on the Prevalence of Antimicrobial Resistance in
Campylobacter jejuni
and
Campylobacter hyointestinalis
Authors: Inglis G. D. , McAllister T. A. , Busz H. W. , Yanke L. J. , Morck D. W. , Olson M. E. , Read R. R. 
Published: July 2005

ABSTRACT

The influence of antimicrobial agents on the development of antimicrobial resistance (AMR) in
Campylobacter
isolates recovered from 300 beef cattle maintained in an experimental feedlot was monitored over a 315-day period (11 sample times). Groups of calves were assigned to one of the following antimicrobial treatments: chlortetracycline and sulfamethazine (CS), chlortetracycline alone (Ct), virginiamycin, monensin, tylosin phosphate, and no antimicrobial agent (i.e., control treatment). In total, 3,283 fecal samples were processed for campylobacters over the course of the experiment. Of the 2,052 bacterial isolates recovered, 92% were
Campylobacter
(1,518 were
Campylobacter hyointestinalis
and 380 were
C. jejuni
). None of the antimicrobial treatments decreased the isolation frequency of
C. jejuni
relative to the control treatment. In contrast,
C. hyointestinalis
was isolated less frequently from animals treated with CS and to a lesser extent from animals treated with Ct. The majority (≥94%) of
C. jejuni
isolates were sensitive to ampicillin, erythromycin, and ciprofloxacin, but more isolates with resistance to tetracycline were recovered from animals fed Ct. All of the 1,500 isolates of
C. hyointestinalis
examined were sensitive to ciprofloxacin. In contrast, 11%, 10%, and 1% of these isolates were resistant to tetracycline, erythromycin, and ampicillin, respectively. The number of animals from which
C. hyointestinalis
isolates with resistance to erythromycin and tetracycline were recovered differed among the antimicrobial treatments. Only Ct administration increased the carriage rates of erythromycin-resistant isolates of
C. hyointestinalis
, and the inclusion of CS in the diet increased the number of animals from which tetracycline-resistant isolates were recovered. The majority of
C. hyointestinalis
isolates with resistance to tetracycline were obtained from cohorts within a single pen, and most of these isolates were recovered from cattle during feeding of a forage-based diet as opposed to a grain-based diet. The findings of this study show that the subtherapeutic administration of tetracycline, alone and in combination with sulfamethazine, to feedlot cattle can select for the carriage of resistant strains of
Campylobacter
species. Considering the widespread use of in-feed antimicrobial agents and the high frequency of beef cattle that shed campylobacters, the development of AMR should be monitored as part of an on-going surveillance program.
Quantitative Detection of
Clostridium perfringens
in the Broiler Fowl Gastrointestinal Tract by Real-Time PCR
Authors: Wise Mark G. , Siragusa Gregory R. 
Published: July 2005

ABSTRACT

Strains of
Clostridium perfringens
are a frequent cause of food-borne disease and gas gangrene and are also associated with necrotic enteritis in chickens. To detect and quantify the levels of
C. perfringens
in the chicken gastrointestinal tract, a quantitative real-time PCR assay utilizing a fluorogenic, hydrolysis-type probe was developed and utilized to assay material retrieved from the broiler chicken cecum and ileum. Primers and probe were selected following an alignment of 16S rDNA sequences from members of cluster I of the genus
Clostridium
, and proved to be specific for
C. perfringens
. The assay could detect approximately 50 fg of
C. perfringens
genomic DNA and approximately 20 cells in pure culture. Measurements of the analytical sensitivity determined with spiked intestinal contents indicated that the consistent limit of detection with ileal samples was approximately 10
2
CFU/g of ileal material, but only about 10
4
CFU/g of cecal samples. The decreased sensitivity with the cecal samples was due to the presence of an unidentified chemical PCR inhibitor(s) in the cecal DNA purifications. The assay was utilized to rapidly detect and quantify
C. perfringens
levels in the gut tract of broiler chickens reared without supplementary growth-promoting antibiotics that manifested symptoms of necrotic enteritis. The results illustrated that quantitative real-time PCR correlates well with quantification via standard plate counts in samples taken from the ileal region of the gastrointestinal tract.
Highly Hydrolytic Reuteransucrase from Probiotic
Lactobacillus reuteri
Strain ATCC 55730
Authors: Kralj S. , Stripling E. , Sanders P. , van Geel-Schutten G. H. , Dijkhuizen L. 
Published: July 2005

ABSTRACT

Lactobacillus reuteri
strain ATCC 55730 (LB BIO) was isolated as a pure culture from a Reuteri tablet purchased from the BioGaia company. This probiotic strain produces a soluble glucan (reuteran), in which the majority of the linkages are of the α-(1→4) glucosidic type (∼70%). This reuteran also contains α-(1→6)- linked glucosyl units and 4,6-disubstituted α-glucosyl units at the branching points. The LB BIO glucansucrase gene (
gtfO
) was cloned and expressed in
Escherichia coli
, and the GTFO enzyme was purified. The recombinant GTFO enzyme and the LB BIO culture supernatants synthesized identical glucan polymers with respect to linkage type and size distribution. GTFO thus is a reuteransucrase, responsible for synthesis of this reuteran polymer in LB BIO. The preference of GTFO for synthesizing α-(1→4) linkages is also evident from the oligosaccharides produced from sucrose with different acceptor substrates, e.g., isopanose from isomaltose. GTFO has a relatively high hydrolysis/transferase activity ratio. Complete conversion of 100 mM sucrose by GTFO nevertheless yielded large amounts of reuteran, although more than 50% of sucrose was converted into glucose. This is only the second example of the isolation and characterization of a reuteransucrase and its reuteran product, both found in different
L. reuteri
strains. GTFO synthesizes a reuteran with the highest amount of α-(1→4) linkages reported to date.
Rickettsia
Symbiont in the Pea Aphid
Acyrthosiphon pisum
: Novel Cellular Tropism, Effect on Host Fitness, and Interaction with the Essential Symbiont
Buchnera
Authors: Sakurai Makiko , Koga Ryuichi , Tsuchida Tsutomu , Meng Xian-Ying , Fukatsu Takema 
Published: July 2005

ABSTRACT

In natural populations of the pea aphid
Acyrthosiphon pisum
, a facultative bacterial symbiont of the genus
Rickettsia
has been detected at considerable infection frequencies worldwide. We investigated the effects of the
Rickettsia
symbiont on the host aphid and also on the coexisting essential symbiont
Buchnera
. In situ hybridization revealed that the
Rickettsia
symbiont was specifically localized in two types of host cells specialized for endosymbiosis: secondary mycetocytes and sheath cells. Electron microscopy identified bacterial rods, about 2 μm long and 0.5 μm thick, in sheath cells of
Rickettsia
-infected aphids. Virus-like particles were sometimes observed in association with the bacterial cells. By an antibiotic treatment, we generated
Rickettsia
-infected and
Rickettsia
-eliminated aphid strains with an identical genetic background. Comparison of these strains revealed that
Rickettsia
infection negatively affected some components of the host fitness. Quantitative PCR analysis of the bacterial population dynamics identified a remarkable interaction between the coexisting symbionts:
Buchnera
population was significantly suppressed in the presence of
Rickettsia
, particularly at the young adult stage, when the aphid most actively reproduces. On the basis of these results, we discussed the possible mechanisms that enable the prevalence of
Rickettsia
infection in natural host populations in spite of the negative fitness effects observed in the laboratory.
Rapid Method for Enumeration of Viable
Legionella pneumophila
and Other
Legionella
spp. in Water
Authors: Delgado-Viscogliosi Pilar , Simonart Tristan , Parent Virginie , Marchand Grégory , Dobbelaere Marie , Pierlot Eric , Pierzo Véronique , Menard-Szczebara Florence , Gaudard-Ferveur Elisabeth , Delabre Karine , Delattre Jean Marie 
Published: July 2005

ABSTRACT

A sensitive and specific method has been developed to enumerate viable
L. pneumophila
and other
Legionella
spp. in water by epifluorescence microscopy in a short period of time (a few hours). This method allows the quantification of
L. pneumophila
or other
Legionella
spp. as well as the discrimination between viable and nonviable
Legionella
. It simultaneously combines the specific detection of
Legionella
cells using antibodies and a bacterial viability marker (ChemChrome V6), the enumeration being achieved by epifluorescence microscopy. The performance of this immunological double-staining (IDS) method was investigated in 38 natural filterable water samples from different aquatic sources, and the viable
Legionella
counts were compared with those obtained by the standard culture method. The recovery rate of the IDS method is similar to, or higher than, that of the conventional culture method. Under our experimental conditions, the limit of detection of the IDS method was <176
Legionella
cells per liter. The examination of several samples in duplicates for the presence of
L. pneumophila
and other
Legionella
spp. indicated that the IDS method exhibits an excellent intralaboratory reproducibility, better than that of the standard culture method. This immunological approach allows rapid measurements in emergency situations, such as monitoring the efficacy of disinfection shock treatments. Although its field of application is as yet limited to filterable waters, the double-staining method may be an interesting alternative (not equivalent) to the conventional standard culture methods for enumerating viable
Legionella
when rapid detection is required.
High-Level Expression and Secretion of Methyl Parathion Hydrolase in
Bacillus subtilis
WB800
Authors: Zhang Xiao-Zhou , Cui Zhong-Li , Hong Qing , Li Shun-Peng 
Published: July 2005

ABSTRACT

The methyl parathion hydrolase (MPH)-encoding gene
mpd
was placed under the control of the P43 promoter and
Bacillus subtilis nprB
signal peptide-encoding sequence. High-level expression and secretion of mature, authentic, and stable MPH were achieved using the protease-deficient strain
B. subtilis
WB800 as the host.
Analysis of Microbial Gene Transcripts in Environmental Samples
Authors: Poretsky Rachel S. , Bano Nasreen , Buchan Alison , LeCleir Gary , Kleikemper Jutta , Pickering Maria , Pate Whitney M. , Moran Mary Ann , Hollibaugh James T. 
Published: July 2005

ABSTRACT

We analyzed gene expression in marine and freshwater bacterioplankton communities by the direct retrieval and analysis of microbial transcripts. Environmental mRNA, obtained from total RNA by subtractive hybridization of rRNA, was reverse transcribed, amplified with random primers, and cloned. Approximately 400 clones were analyzed, of which ∼80% were unambiguously mRNA derived. mRNAs appeared to be from diverse taxonomic groups, including both
Bacteria
(mainly α- and γ-
Proteobacteria
) and
Archaea
(mainly
Euryarchaeota
). Many transcripts could be linked to environmentally important processes such as sulfur oxidation (
soxA
), assimilation of C1 compounds (
fdh1B
), and acquisition of nitrogen via polyamine degradation (
aphA
). Environmental transcriptomics is a means of exploring functional gene expression within natural microbial communities without bias toward known sequences, and provides a new approach for obtaining community-specific variants of key functional genes.
Elucidating the Key Member of a Linuron-Mineralizing Bacterial Community by PCR and Reverse Transcription-PCR Denaturing Gradient Gel Electrophoresis 16S rRNA Gene Fingerprinting and Cultivation
Authors: Sørensen Sebastian R. , Rasmussen Jim , Jacobsen Carsten S. , Jacobsen Ole S. , Juhler René K. , Aamand Jens 
Published: July 2005

ABSTRACT

A bacterial community from Danish agricultural soil was enriched with linuron [
N
-(3,4-dichlorophenyl)-
N
′-methoxy-
N
′-methylurea] as the sole carbon and nitrogen source. The community mineralized [
ring
-U-
14
C]linuron completely to
14
CO
2
and
14
C-biomass. Denaturing gradient gel electrophoresis analysis and cultivation revealed that a
Variovorax
sp. was responsible for the mineralization activity.
Use of Peptide Library Screening To Detect a Previously Unknown Linear Diagnostic Epitope: Proof of Principle by Use of Lyme Disease Sera
Authors: Hamby Carl V. , Llibre Marta , Utpat Sandeepa , Wormser Gary P. 
Published: July 2005

ABSTRACTDiagnostic peptides previously isolated from phage-displayed libraries by affinity selection with serum antibodies from patients with Lyme disease were found to give reproducible serum reactivity patterns when tested in two different enzyme-linked immunosorbent assay formats. In addition, the hypothetical possibility that peptides selected by this type of “epitope discovery” technique might identify the original antigens eliciting antibody responses was tested by searching for sequence similarities in bacterial protein databases. In support of this hypothesis, our search uncovered similarities between peptides representing two different sequence motifs and sequences in the VlsE and BBA61 antigens ofBorrelia burgdorferi. Utilizing synthetic peptides, we verified that the sequence KAASKETPPALNK, located at the C terminus of the VlsE antigen, had the same reactivity pattern to sera from patients with extracutaneous Lyme disease as the diagnostic peptideSKEKPPSLNWPA, with which it shared a 7-amino-acid-residue match (consensus residues are underlined). A peptide with conservative mutations of five of the consensus residues was nonreactive, strongly suggesting that the VlsE sequence represents the epitope that originally elicited antibody responses in these patients. The diagnostic sensitivity of this new VlsE epitope was relatively low (30%) compared to that (100%) of the well-documented C6diagnostic peptide of VlsE when tested in our small cohort of 10 patients with Lyme disease. Nonetheless, the identification of this previously unknown epitope serves as a proof of the principle of the hypothetical ability of “epitope discovery” techniques to detect specific microbial antigens with diagnostic relevance in infectious diseases.
Immunoglobulin G Subclass Profile of Antimeasles Response in Vaccinated Children and in Adults with Measles History
Authors: Toptygina Anna P. , Pukhalsky Alexander L. , Alioshkin Vladimir A. 
Published: July 2005

ABSTRACT
The aim of this study was to investigate measles-specific immunoglobulin G (IgG) subclass profile in vaccinated children and in adults with natural infection. Serum samples were collected before and 30 days after vaccination. The sera from 51 late convalescent adults and seven adults with natural measles infection at the 12th day after onset of rash have been also investigated. Measles IgG antibodies and specific IgG subclasses were tested by enzyme-linked immunosorbent techniques. In children younger than 3 years, the predominant subclass was IgG3, which contributed, on average, 63.3% of the total IgG response. The contributions of specific IgG1 and IgG4 to the total IgG antimeasles response were lower (19.9% and 16.8%, respectively), whereas IgG2 was not found. In contrast, in the group of children older than 4 years, just IgG2 was a predominant subclass; it contributed 42.6% of the total IgG response. Other subclasses were also present but the contribution was much lower. In adult volunteers with measles history, IgG2 was a predominant subclass of total IgG. Thus, in early convalescence IgG2 contributed 62% of the total IgG response, whereas in late convalescence the contribution was lower (41.4%). There were no visible differences in IgG subclass composition between subjects with natural infection and vaccinated children except those below 3 years of age. The humoral immune response of such subjects is immature and the IgG2 subclass of virus-specific antibodies has not been revealed in the sera.
Enzyme-Linked Immunosorbent Assay Based on a Chimeric Antigen Bearing Antigenic Regions of Structural Proteins E
rns
and E2 for Serodiagnosis of Classical Swine Fever Virus Infection
Authors: Lin Min , Trottier Erin , Mallory Maria 
Published: July 2005

ABSTRACT

The antigenic region (residues 109 to 160) of classical swine fever virus (CSFV) protein E
rns
and the N-terminal antigenic region (residues 1 to 136) of protein E2 were constructed in the form of a fused, chimeric protein, C21E
rns
E2, for use as an enzyme-linked immunosorbent assay (ELISA) antigen for the serodiagnosis of CSFV infection. Tested with 238 negative-field (CSFV-free) sera from Canadian sources, the specificity of the ELISA was determined to be 93.7%. All 20 sera from experimentally infected pigs representing a variety of animals, virus strains, and days postinfection (dpi; range, 7 to 210) were detected as positive (100%). In contrast, an ELISA based on an E
rns
fragment (E
rns
aa 109-160
) or an E2 fragment (E2
aa 1-221
) identified only 18 (90%) of 20 sera from infected pigs as positive, missing two targets collected at 7 dpi. These data suggest that use of the chimeric antigen C21E
rns
E2 would improve serodiagnostic sensitivity and allow for the detection of CSFV infection as early as 7 dpi.
Unique Aspects of Gene Expression during
Candida albicans
Mating and Possible G
1
Dependency
Authors: Zhao Rui , Daniels Karla J. , Lockhart Shawn R. , Yeater Kathleen M. , Hoyer Lois L. , Soll David R. 
Published: July 2005

ABSTRACT

Taking advantage of the high frequency of conjugation tube formation in mating mixtures and α-pheromone-treated
a
/
a
cells derived from saturation phase cultures of opaque cells of
Candida albicans
, 56 up-regulated and 30 down-regulated genes were identified employing microarray and Northern analyses. Combining these results with previous profiling studies of pheromone-induced cells, a more comprehensive transcript profile was developed for comparison with
Saccharomyces cerevisiae
. This comparison revealed the following: (i) that while a majority of mating-associated genes are regulated similarly between the two species, a significant minority are regulated dissimilarly; (ii) that filamentation genes are uniquely up-regulated and opaque-specific genes uniquely down-regulated during
C. albicans
mating; and (iii) that a newly identified class of genes is selectively down-regulated in opaque, but not white, cells that have entered saturation phase in a growth culture and then are up-regulated by pheromone. The observations that opaque cells are uniquely mating competent, that saturation phase facilitates mating, and that a newly identified group of genes is down-regulated only in opaque cells that have entered saturation phase led us to hypothesize that entering saturation phase may be requisite for mating. A test of this hypothesis revealed, however, that cells, whether in the exponential or saturation phase, may simply have to be in G
1
of the cell cycle to respond to pheromone and that the response includes G
1
arrest. These results add to the lists of similarities and dissimilarities between the mating processes of
C. albicans
and
S. cerevisiae
and underscore the unique regulation of filamentation and switching genes in the
C. albicans
mating process.