bluuebunny/crossref_metadata_2025_split huggingface dataset:
Cell Culture-Taqman PCR Assay for Evaluation of
Cryptosporidium parvum
Disinfection
Authors: Keegan Alexandra R. , Fanok Stella , Monis Paul T. , Saint Christopher P. 
Published: May 2003

ABSTRACT

Cryptosporidium parvum
represents a challenge to the water industry and a threat to public health. In this study, we developed a cell culture-quantitative PCR assay to evaluate the inactivation of
C. parvum
with disinfectants. The assay was validated by using a range of disinfectants in common use in the water industry, including low-pressure UV light (LP-UV), ozone, mixed oxidants (MIOX), and chlorine. The assay was demonstrated to be reliable and sensitive, with a lower detection limit of a single infectious oocyst. Effective oocyst inactivation was achieved (>2 log
10
units) with LP-UV (20 mJ/cm
2
) or 2 mg of ozone/liter (for 10 min). MIOX and chlorine treatments of oocysts resulted in minimal effective disinfection, with <0.1 log
10
unit being inactivated. These results demonstrate the inability of MIOX to inactivate
Cryptosporidium
. The assay is a valuable tool for the evaluation of disinfection systems for drinking water and recycled water.
TheftsHGene of the Wine BacteriumOenococcus oeniIs Involved in Protection against Environmental Stress
Authors: Bourdineaud Jean-Paul , Nehmé Benjamin , Tesse Sonia , Lonvaud-Funel Aline 
Published: May 2003

ABSTRACTThe wine bacteriumOenococcus oenihas to cope with harsh environmental conditions, including an acidic pH, a high alcoholic content, nonoptimal growth temperatures, and growth-inhibitory compounds such as fatty acids, phenolic acids, and tannins. We describe the characterization and cloning of theO. oeni ftsHgene, encoding a protease belonging to the ATP binding cassette protein superfamily. TheO. oeniFtsH protein is closest in sequence similarity to the FtsH homologue ofLactococcus lactis. TheO. oeni ftsHgene proved to be stress-responsive, since its expression increased at high temperatures or under osmotic shock.O. oeniFtsH protein function was tested in anEscherichia coli ftsHmutant strain, and consistent with theO. oeni ftsHgene expression pattern, theO. oeniFtsH protein provided protection for theE. coli ftsHmutant against heat shock.O. oeniandBradyrhizobium japonicumFtsH proteins also triggeredE. coliresistance to wine toxicity. Genes homologous toO. oeni ftsHwere detected in many other lactic acid bacteria found in wine, suggesting that this type of gene constitutes a well-conserved stress-protective molecular device.
Global Expression Profiling and Physiological Characterization ofCorynebacterium glutamicumGrown in the Presence ofl-Valine
Authors: Lange C. , Rittmann D. , Wendisch V. F. , Bott M. , Sahm H. 
Published: May 2003

ABSTRACTAddition ofl-valine (50 to 200 mM) to glucose minimal medium had no effect on the growth of wild-typeCorynebacterium glutamicumATCC 13032 but inhibited the growth of the derived valine production strain VAL1 [13032 ΔilvAΔpanBC(pJC1ilvBNCD)] in a concentration-dependent manner. In order to explore this strain-specific valine effect, genomewide expression profiling was performed using DNA microarrays, which showed that valine caused an increasedilvBNmRNA level in VAL1 but not in the wild type. This unexpected result was confirmed by an increased cellular level of theilvBprotein product, i.e., the large subunit of acetohydroxyacid synthase (AHAS), and by an increased AHAS activity of valine-treated VAL1 cells. The conclusion that valine caused the limitation of another branched-chain amino acid was confirmed by showing that high concentrations ofl-isoleucine could relieve the valine effect on VAL1 whereasl-leucine had the same effect as valine. The valine-caused isoleucine limitation was supported by the finding that the inhibitory valine effect was linked to theilvAdeletion that results in isoleucine auxotrophy. Taken together, these results implied that the valine effect is caused by competition for uptake of isoleucine by the carrier BrnQ, which transports all branched-chained amino acids. Indeed, valine inhibition could also be relieved by supplementing VAL1 with the dipeptide isoleucyl-isoleucine, which is taken up by a dipeptide transport system rather than by BrnQ. Interestingly, addition of external valine stimulated valine production by VAL1. This effect is most probably due to a reduced carbon usage for biomass production and to the increased expression ofilvBN, indicating that AHAS activity may still be a limiting factor for valine production in the VAL1 strain.
Transposon-Like Organization of the Plasmid-Borne Organophosphate Degradation (
opd
) Gene Cluster Found in
Flavobacterium
sp
Authors: Siddavattam Dayananda , Khajamohiddin Syed , Manavathi Bramanandam , Pakala Suresh B. , Merrick Mike 
Published: May 2003

ABSTRACT

Several bacterial strains that can use organophosphate pesticides as a source of carbon have been isolated from soil samples collected from diverse geographical regions. All these organisms synthesize an enzyme called parathion hydrolase, and in each case the enzyme is encoded by a gene (
opd
) located on a large indigenous plasmid. These plasmids show considerable genetic diversity, but the region containing the
opd
gene is highly conserved. Two
opd
plasmids, pPDL2 from
Flavobacterium
sp. and pCMS1 from
Pseudomonas diminuta
, are well characterized, and in each of them a region of about 5.1 kb containing the
opd
gene shows an identical restriction pattern. We now report the complete sequence of the conserved region of plasmid pPDL2. The
opd
gene is flanked upstream by an insertion sequence, ISFlsp
1
, that is a member of the IS
21
family, and downstream by a Tn
3
-like element encoding a transposase and a resolvase. Adjacent to
opd
but transcribed in the opposite direction is an open reading frame (
orf243
) with the potential to encode an aromatic hydrolase somewhat similar to
Pseudomonas putida
TodF. We have shown that
orf243
encodes a polypeptide of 27 kDa, which plays a role in the degradation of
p
-nitrophenol and is likely to act in concert with
opd
in the degradation of parathion. The linkage of
opd
and
orf243
, the organization of the genes flanking
opd
, and the wide geographical distribution of these genes suggest that this DNA sequence may constitute a complex catabolic transposon.
Factors Influencing Survival of
Legionella pneumophila
Serotype 1 in Hot Spring Water and Tap Water
Authors: Ohno Akira , Kato Naoyuki , Yamada Koji , Yamaguchi Keizo 
Published: May 2003

ABSTRACT

The factors involved in the survival of
Legionella pneumophila
in the microcosms of both hot spring water and tap water were studied by examining cultivability and metabolic activity.
L. pneumophila
could survive by maintaining metabolic activity but was noncultivable in all microcosms at 42°C, except for one microcosm with a pH of <2.0. Lower temperatures supported survival without loss of cultivability. The cultivability declined with increasing temperature, although metabolic activity was observed at temperatures of up to 45°C. The optimal range of pH for survival was between 6.0 and 8. The metabolic activity could be maintained for long periods even in microcosms with high concentrations of salt. The cultivability of organisms in the post-exponential phase in a tap water microcosm with a low inoculum size was more rapidly reduced than that of organisms in the exponential phase. In contrast, the loss of cultivability in microcosms of a high inoculum size was significant in the exponential phase. Random(ly) amplified polymorphic DNA analysis of microcosms where cultivability was lost but metabolic activity was retained showed no change compared to cells grown freshly, although an effect on the amplified DNA band pattern by production of stress proteins was expected. Resuscitation by the addition of
Acanthamoeba castellanii
to the microcosm in which cultivability was completely lost but metabolic activity was maintained was observed only in part of the cell population. Our results suggest that
L. pneumophila
cell populations can potentially survive as free organisms for long periods by maintaining metabolic activity but temporarily losing cultivability under strict environments and requiring resuscitation by ingestion by amoebas.
Determining Diversity of Freshwater Fungi on Decaying Leaves: Comparison of Traditional and Molecular Approaches
Authors: Nikolcheva Liliya G. , Cockshutt Amanda M. , Bärlocher Felix 
Published: May 2003

ABSTRACT

Traditional microscope-based estimates of species richness of aquatic hyphomycetes depend upon the ability of the species in the community to sporulate. Molecular techniques which detect DNA from all stages of the life cycle could potentially circumvent the problems associated with traditional methods. Leaf disks from red maple, alder, linden, beech, and oak as well as birch wood sticks were submerged in a stream in southeastern Canada for 7, 14, and 28 days. Fungal biomass, estimated by the amount of ergosterol present, increased with time on all substrates. Alder, linden, and maple leaves were colonized earlier and accumulated the highest fungal biomass. Counts and identifications of released conidia suggested that fungal species richness increased, while community evenness decreased, with time (up to 11 species on day 28). Conidia of
Articulospora tetracladia
dominated. Modifications of two molecular methods—denaturing gradient gel electrophoresis (DGGE) and terminal restriction fragment length polymorphism (T-RFLP) analysis—suggested that both species richness and community evenness decreased with time. The dominant ribotype matched that of
A. tetracladia
. Species richness estimates based on DGGE were consistently higher than those based on T-RFLP analysis and exceeded those based on spore identification on days 7 and 14. Since traditional and molecular techniques assess different aspects of the fungal organism, both are essential for a balanced view of fungal succession on leaves decaying in streams.
Formation of Pseudo-Terminal Restriction Fragments, a PCR-Related Bias Affecting Terminal Restriction Fragment Length Polymorphism Analysis of Microbial Community Structure
Authors: Egert Markus , Friedrich Michael W. 
Published: May 2003

ABSTRACT

Terminal restriction fragment length polymorphism (T-RFLP) analysis of PCR-amplified genes is a widely used fingerprinting technique in molecular microbial ecology. In this study, we show that besides expected terminal restriction fragments (T-RFs), additional secondary T-RFs occur in T-RFLP analysis of amplicons from cloned 16S rRNA genes at high frequency. A total of 50% of 109 bacterial and 78% of 68 archaeal clones from the guts of cetoniid beetle larvae, using
Msp
I and
Alu
I as restriction enzymes, respectively, were affected by the presence of these additional T-RFs. These peaks were called “pseudo-T-RFs” since they can be detected as terminal fluorescently labeled fragments in T-RFLP analysis but do not represent the primary terminal restriction site as indicated by sequence data analysis. Pseudo-T-RFs were also identified in T-RFLP profiles of pure culture and environmental DNA extracts. Digestion of amplicons with the single-strand-specific mung bean nuclease prior to T-RFLP analysis completely eliminated pseudo-T-RFs. This clearly indicates that single-stranded amplicons are the reason for the formation of pseudo-T-RFs, most probably because single-stranded restriction sites cannot be cleaved by restriction enzymes. The strong dependence of pseudo-T-RF formation on the number of cycles used in PCR indicates that (partly) single-stranded amplicons can be formed during amplification of 16S rRNA genes. In a model, we explain how transiently formed secondary structures of single-stranded amplicons may render single-stranded amplicons accessible to restriction enzymes. The occurrence of pseudo-T-RFs has consequences for the interpretation of T-RFLP profiles from environmental samples, since pseudo-T-RFs may lead to an overestimation of microbial diversity. Therefore, it is advisable to establish 16S rRNA gene sequence clone libraries in parallel with T-RFLP analysis from the same sample and to check clones for their in vitro digestion T-RF pattern to facilitate the detection of pseudo-T-RFs.
Enhanced Killing of
Acanthamoeba
Cysts with a Plant Peroxidase-Hydrogen Peroxide-Halide Antimicrobial System
Authors: Hughes Reanne , Andrew Peter W. , Kilvington Simon 
Published: May 2003

ABSTRACT

The activity of H
2
O
2
against the resistant cyst stage of the pathogenic free-living amoeba
Acanthamoeba
was enhanced by the addition of KI and either horseradish peroxidase or soybean peroxidase or, to a lesser degree, lactoperoxidase. This resulted in an increase in the cysticidal activity of 3% (wt/vol) H
2
O
2
, and there was >3-log killing in 2 h, compared with the 6 h required for comparable results with the peroxide solution alone (
P
< 0.05). With 2% H
2
O
2
, enhancement was observed at all time points (
P
< 0.05), and total killing of the cyst inoculum occurred at 4 h, compared with 6 h for the peroxide alone. The activity of sublethal 1% H
2
O
2
was enhanced to give 3-log killing after 8 h of exposure (
P
< 0.05). No enhancement was obtained when KCl or catalase was used as a substitute in the reaction mixtures. The H
2
O
2
was not neutralized in the enhanced system during the experiments. However, in the presence of a platinum disk used to neutralize H
2
O
2
in contact lens care systems, the enhanced 2% H
2
O
2
system gave 2.8-log killing after 6 h or total cyst killing by 8 h, and total neutralization of the H
2
O
2
occurred by 4 h. In contrast, 2% H
2
O
2
alone resulted in <0.8-log killing of cysts in the presence of the platinum disk due to rapid (<1 h) neutralization of the peroxide. Our observations could result in significant improvement in the efficacy of H
2
O
2
contact lens disinfection systems against
Acanthamoeba
cysts and prevention of acanthamoeba keratitis.
Cloning and Sequencing of the Histidine Decarboxylase Genes of Gram-Negative, Histamine-Producing Bacteria and Their Application in Detection and Identification of These Organisms in Fish
Authors: Takahashi Hajime , Kimura Bon , Yoshikawa Miwako , Fujii Tateo 
Published: May 2003

ABSTRACT

The use of molecular tools for early and rapid detection of gram-negative histamine-producing bacteria is important for preventing the accumulation of histamine in fish products. To date, no molecular detection or identification system for gram-negative histamine-producing bacteria has been developed. A molecular method that allows the rapid detection of gram-negative histamine producers by PCR and simultaneous differentiation by single-strand conformation polymorphism (SSCP) analysis using the amplification product of the histidine decarboxylase genes (
hdc
) was developed. A collection of 37 strains of histamine-producing bacteria (8 reference strains from culture collections and 29 isolates from fish) and 470 strains of non-histamine-producing bacteria isolated from fish were tested. Histamine production of bacteria was determined by paper chromatography and confirmed by high-performance liquid chromatography. Among 37 strains of histamine-producing bacteria, all histidine-decarboxylating gram-negative bacteria produced a PCR product, except for a strain of
Citrobacter braakii
. In contrast, none of the non-histamine-producing strains (470 strains) produced an amplification product. Specificity of the amplification was further confirmed by sequencing the 0.7-kbp amplification product. A phylogenetic tree of the isolates constructed using newly determined sequences of partial
hdc
was similar to the phylogenetic tree generated from 16S ribosomal DNA sequences. Histamine accumulation occurred when PCR amplification of
hdc
was positive in all of fish samples tested and the presence of powerful histamine producers was confirmed by subsequent SSCP identification. The potential application of the PCR-SSCP method as a rapid monitoring tool is discussed.
Growth Characteristics and Intraspecies Host Specificity of a Large Virus Infecting the Dinoflagellate
Heterocapsa circularisquama
Authors: Nagasaki Keizo , Tomaru Yuji , Tarutani Kenji , Katanozaka Noriaki , Yamanaka Satoshi , Tanabe Hiroshi , Yamaguchi Mineo 
Published: May 2003

ABSTRACT

The growth characteristics and intraspecies host specificity of
Heterocapsa circularisquama
virus (HcV), a large icosahedral virus specifically infecting the bivalve-killing dinoflagellate
H. circularisquama
, were examined. Exponentially growing host cells were more sensitive to HcV than those in the stationary phase, and host cells were more susceptible to HcV infection in the culture when a higher percent of the culture was replaced with fresh medium each day, suggesting an intimate relationship between virus sensitivity and the physiological condition of the host cells. HcV was infective over a wide range of temperatures, 15 to 30°C, and the latent period and burst size were estimated at 40 to 56 h and 1,800 to 2,440 infective particles, respectively. Transmission electron microscopy revealed that capsid formation began within 16 h postinfection, and mature virus particles appeared within 24 h postinfection at 20°C. Compared to
Heterosigma akashiwo
virus, HcV was more widely infectious to
H. circularisquama
strains that had been independently isolated in the western part of Japan, and only 5.3% of the host-virus combinations (53 host and 10 viral strains) showed resistance to viral infection. The present results are helpful in understanding the ecology of algal host-virus systems in nature.
Genetic Characterization of
Escherichia coli
Populations from Host Sources of Fecal Pollution by Using DNA Fingerprinting
Authors: McLellan Sandra L. , Daniels Annette D. , Salmore Alissa K. 
Published: May 2003

ABSTRACT

Escherichia coli
isolates were obtained from common host sources of fecal pollution and characterized by using repetitive extragenic palindromic (REP) PCR fingerprinting. The genetic relationship of strains within each host group was assessed as was the relationship of strains among different host groups. Multiple isolates from a single host animal (gull, human, or dog) were found to be identical; however, in some of the animals, additional strains occurred at a lower frequency. REP PCR fingerprint patterns of isolates from sewage (
n
= 180), gulls (
n
= 133), and dairy cattle (
n
= 121) were diverse; within a host group, pairwise comparison similarity indices ranged from 98% to as low as 15%. A composite dendrogram of
E. coli
fingerprint patterns did not cluster the isolates into distinct host groups but rather produced numerous subclusters (approximately >80% similarity scores calculated with the cosine coefficient) that were nearly exclusive for a host group. Approximately 65% of the isolates analyzed were arranged into host-specific groups. Comparable results were obtained by using enterobacterial repetitive intergenic consensus PCR and pulsed-field gel electrophoresis (PFGE), where PFGE gave a higher differentiation of closely related strains than both PCR techniques. These results demonstrate that environmental studies with genetic comparisons to detect sources of
E. coli
contamination will require extensive isolation of strains to encompass
E. coli
strain diversity found in host sources of contamination. These findings will assist in the development of approaches to determine sources of fecal pollution, an effort important for protecting water resources and public health.
Insertional Mutagenesis of a Fungal Biocontrol Agent Led to Discovery of a Rare Cellobiose Lipid with Antifungal Activity
Authors: Cheng Yali , McNally David J. , Labbé Caroline , Voyer Normand , Belzile François , Bélanger Richard R. 
Published: May 2003

ABSTRACT

Insertional mutagenesis was applied for the first time to a fungal biocontrol agent,
Pseudozyma flocculosa
, in an attempt to obtain mutants with altered antagonistic properties. Transformants were obtained via DNA-mediated transformation. Molecular analyses of the transformants revealed that multiple copies of the plasmid were integrated in tandem at one to many chromosomal loci. The transformants were screened for their biocontrol properties using standard bioassays, and the 160 tested transformants were classified into four groups: group I mutants (22 transformants) showed a stronger antagonistic effect than the wild type (WT) while those of group II (107 transformants) had a comparable antagonistic effect; group III mutants (17 transformants) had a decreased antagonistic effect relative to WT and group IV mutants (14 transformants) had lost their biocontrol properties. Culture extracts of the mutants (group IV) and WT were analyzed and compared for the presence of active metabolites which were then separated by solid-phase extraction and purified using conventional methods. Nuclear magnetic resonance experiments and analytical studies on a metabolite specifically produced by the WT revealed the presence of 2-(2′,4′-diacetoxy-5′-carboxy-pentanoyl) octadecyl cellobioside (flocculosin), a novel glycolipid with strong antifungal properties; the production of this compound would account for the biocontrol activity of
P. flocculosa
.
A Novel NAD-Dependent Dehydrogenase, Highly Specific for 1,5-Anhydro-
d
-Glucitol, from
Trichoderma longibrachiatum
Strain 11-3
Authors: Yoshida Nobuyuki , Uchida Etsuko , Katsuragi Tohoru , Tani Yoshiki 
Published: May 2003

ABSTRACT

A novel NAD-dependent dehydrogenase highly specific for 1,5-anhydro-
d
-glucitol (1,5-AG) was found in the cell extract of an imperfect fungus,
Trichoderma longibrachiatum
strain 11-3. This fungus used 1,5-AG as a sole carbon source for growth and transformed 1,5-AG into glucose. 1,5-AG dehydrogenase (AGH) was purified to homogeneity, as determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). The molecular mass of the purified enzyme was estimated to be 36 and 141 kDa by SDS-PAGE and by gel filtration, respectively, suggesting that the enzyme was homotetrameric. The enzyme was highly specific for 1,5-AG and did not exhibit activity with any sugar or sugar alcohol tested in this study other than 1,5-AG. A linear relationship between the initial rate of the enzyme reaction and the concentration of 1,5-AG at the physiological level was observed. The presence of glucose in abundance did not interfere with the relationship. The optimum temperature for the enzyme reaction was 50°C, and the enzyme was stable at temperatures up to 70°C. These results suggested that AGH is a novel enzyme and is useful for specifically diagnosing diabetes mellitus.