bluuebunny/crossref_metadata_2025_split huggingface dataset:
A Simple Methodological Approach for Counting and Identifying Culturable Viruses Adsorbed to Cellulose Nitrate Membrane Filters
Authors: Papageorgiou Georgios T. , Mocé-Llivina Laura , Christodoulou Christina G. , Lucena Francisco , Akkelidou Dina , Ioannou Eleni , Jofre Juan 
Published: April 2009

ABSTRACT
We identified conditions under which Buffalo green monkey cells grew on the surfaces of cellulose nitrate membrane filters in such a way that they covered the entire surface of each filter and penetrated through the pores. When such conditions were used, poliovirus that had previously been adsorbed on the membranes infected the cells and replicated. A plaque assay method and a quantal method (most probable number of cytopathic units) were used to detect and count the viruses adsorbed on the membrane filters. Polioviruses in aqueous suspensions were then concentrated by adsorption to cellulose membrane filters and were subsequently counted without elution, a step which is necessary when the commonly used methods are employed. The pore size of the membrane filter, the sample contents, and the sample volume were optimized for tap water, seawater, and a 0.25 M glycine buffer solution. The numbers of viruses recovered under the optimized conditions were more than 50% greater than the numbers counted by the standard plaque assay. When ceftazidime was added to the assay medium in addition to the antibiotics which are typically used, the method could be used to study natural samples with low and intermediate levels of microbial pollution without decontamination of the samples. This methodological approach also allowed plaque hybridization either directly on cellulose nitrate membranes or on Hybond N+ membranes after the preparations were transferred.
Development of Methods To Detect “Norwalk-Like Viruses” (NLVs) and Hepatitis A Virus in Delicatessen Foods: Application to a Food-Borne NLV Outbreak
Authors: Schwab Kellogg J. , Neill Frederick H. , Fankhauser Rebecca L. , Daniels Nicholas A. , Monroe Stephan S. , Bergmire-Sweat David A. , Estes Mary K. , Atmar Robert L. 
Published: April 2009

ABSTRACT

“Norwalk-like viruses” (NLVs) and hepatitis A virus (HAV) are the most common causes of virus-mediated food-borne illness. Epidemiological investigations of outbreaks associated with these viruses have been hindered by the lack of available methods for the detection of NLVs and HAV in foodstuffs. Although reverse transcription (RT)-PCR methods have been useful in detecting NLVs and HAV in bivalve mollusks implicated in outbreaks, to date such methods have not been available for other foods. To address this need, we developed a method to detect NLVs and HAV recovered from food samples. The method involves washing of food samples with a guanidinium-phenol-based reagent, extraction with chloroform, and precipitation in isopropanol. Recovered viral RNA is amplified with HAV- or NLV-specific primers in RT-PCRs, using a viral RNA internal standard control to identify potential sample inhibition. By this method, 10 to 100 PCR units (estimated to be equivalent to 10
2
to 10
3
viral genome copies) of HAV and Norwalk virus seeded onto ham, turkey, and roast beef were detected. The method was applied to food samples implicated in an NLV-associated outbreak at a university cafeteria. Sliced deli ham was positive for a genogroup II NLV as determined by using both polymerase- and capsid-specific primers and probes. Sequence analysis of the PCR-amplified capsid region of the genome indicated that the sequence was identical to the sequence from virus detected in the stools of ill students. The developed method is rapid, simple, and efficient.
Safety Evaluation of Sous Vide-Processed Products with Respect to Nonproteolytic
Clostridium botulinum
by Use of Challenge Studies and Predictive Microbiological Models
Authors: Hyytiä-Trees Eija , Skyttä Eija , Mokkila Mirja , Kinnunen Arvo , Lindström Miia , Lähteenmäki Liisa , Ahvenainen Raija , Korkeala Hannu 
Published: April 2009

ABSTRACT

Sixteen different types of sous vide-processed products were evaluated for safety with respect to nonproteolytic group II
Clostridium botulinum
by using challenge tests with low (2.0-log-CFU/kg) and high (5.3-log-CFU/kg) inocula and two currently available predictive microbiological models, Food MicroModel (FMM) and Pathogen Modeling Program (PMP). After thermal processing, the products were stored at 4 and 8°C and examined for the presence of botulinal spores and neurotoxin on the sell-by date and 7 days after the sell-by date. Most of the thermal processes were found to be inadequate for eliminating spores, even in low-inoculum samples. Only 2 of the 16 products were found to be negative for botulinal spores and neurotoxin at both sampling times. Two products at the high inoculum level showed toxigenesis during storage at 8°C, one of them at the sell-by date. The predictions generated by both the FMM thermal death model and the FMM and PMP growth models were found to be inconsistent with the observed results in a majority of the challenges. The inaccurate predictions were caused by the limited number and range of the controlling factors in the models. Based on this study, it was concluded that the safety of sous vide products needs to be carefully evaluated product by product. Time-temperature combinations used in thermal treatments should be reevaluated to increase the efficiency of processing, and the use of additional antibotulinal hurdles, such as biopreservatives, should be assessed.
Elucidation of Enzymes in Fermentation Pathways Used by
Clostridium thermosuccinogenes
Growing on Inulin
Authors: Sridhar Jayanth , Eiteman Mark A. , Wiegel Juergen W. 
Published: April 2009

ABSTRACT

Based on the presence and absence of enzyme activities, the biochemical pathways for the fermentation of inulin by
Clostridium thermosuccinogenes
DSM 5809 are proposed. Activities of nine enzymes (lactate dehydrogenase, phosphoenolpyruvate carboxylase, malate dehydrogenase, fumarase, fumarate reductase, phosphotransacetylase, acetate kinase, pyruvate kinase, and alcohol dehydrogenase) were measured at four temperatures (37, 47, 58, and 70°C). Each of the enzymes increased 1.5 to 2.0-fold in activity between 37 and 58°C, but only lactate dehydrogenase, fumarate reductase, malate dehydrogenase, and fumarase increased at a similar rate between 58 and 70°C. No acetate kinase activity was observed at 70°C. Arrhenius energies were calculated for each of these nine enzymes and were in the range of 9.8 to 25.6 kcal/mol. To determine if a relationship existed between product formation and enzyme activity, serum bottle fermentations were completed at the four temperatures. Maximum yields (in moles per mole hexose unit) for succinate (0.23) and acetate (0.79) and for biomass (29.5 g/mol hexose unit) occurred at 58°C, whereas the maximum yields for lactate (0.19) and hydrogen (0.25) and the lowest yields for acetate (0.03) and biomass (19.2 g/mol hexose unit) were observed at 70°C. The ratio of oxidized products to reduced products changed significantly, from 0.52 to 0.65, with an increase in temperature from 58 to 70°C, and there was an unexplained detection of increased reduced products (ethanol, lactate, and hydrogen) with a concomitant decrease in oxidized-product formation at the higher temperature.
Increased Production of Hydrogen Peroxide by
Lactobacillus delbrueckii
subsp.
bulgaricus
upon Aeration: Involvement of an NADH Oxidase in Oxidative Stress
Authors: Marty-Teysset C. , de la Torre F. , Garel J.-R. 
Published: April 2009

ABSTRACT

The growth of
Lactobacillus delbrueckii
subsp.
bulgaricus
(
L. delbrueckii
subsp.
bulgaricus
) on lactose was altered upon aerating the cultures by agitation. Aeration caused the bacteria to enter early into stationary phase, thus reducing markedly the biomass production but without modifying the maximum growth rate. The early entry into stationary phase of aerated cultures was probably related to the accumulation of hydrogen peroxide in the medium. Indeed, the concentration of hydrogen peroxide in aerated cultures was two to three times higher than in unaerated ones. Also, a similar shift from exponential to stationary phase could be induced in unaerated cultures by adding increasing concentrations of hydrogen peroxide. A significant fraction of the hydrogen peroxide produced by
L. delbrueckii
subsp.
bulgaricus
originated from the reduction of molecular oxygen by NADH catalyzed by an NADH:H
2
O
2
oxidase. The specific activity of this NADH oxidase was the same in aerated and unaerated cultures, suggesting that the amount of this enzyme was not directly regulated by oxygen. Aeration did not change the homolactic character of lactose fermentation by
L. delbrueckii
subsp.
bulgaricus
and most of the NADH was reoxidized by lactate dehydrogenase with pyruvate. This indicated that NADH oxidase had no (or a very small) energetic role and could be involved in eliminating oxygen.
Detection and Identification of Gastrointestinal
Lactobacillus
Species by Using Denaturing Gradient Gel Electrophoresis and Species-Specific PCR Primers
Authors: Walter J. , Tannock G. W. , Tilsala-Timisjarvi A. , Rodtong S. , Loach D. M. , Munro K. , Alatossava T. 
Published: April 2009

ABSTRACT

Denaturing gradient gel electrophoresis (DGGE) of DNA fragments obtained by PCR amplification of the V2-V3 region of the 16S rRNA gene was used to detect the presence of
Lactobacillus
species in the stomach contents of mice.
Lactobacillus
isolates cultured from human and porcine gastrointestinal samples were identified to the species level by using a combination of DGGE and species-specific PCR primers that targeted 16S-23S rRNA intergenic spacer region or 16S rRNA gene sequences. The identifications obtained by this approach were confirmed by sequencing the V2-V3 region of the 16S rRNA gene and by a BLAST search of the GenBank database.
Efficient Production of Artificially Designed Gelatins with a
Bacillus brevis
System
Authors: Kajino Tsutomu , Takahashi Haruo , Hirai Masana , Yamada Yukio 
Published: April 2009

ABSTRACT

Artificially designed gelatins comprising tandemly repeated 30-amino-acid peptide units derived from human αI collagen were successfully produced with a
Bacillus brevis
system. The DNA encoding the peptide unit was synthesized by taking into consideration the codon usage of the host cells, but no clones having a tandemly repeated gene were obtained through the above-mentioned strategy. Minirepeat genes could be selected in vivo from a mixture of every possible sequence encoding an artificial gelatin by randomly ligating the mixed sequence unit and transforming it into
Escherichia coli
. Larger repeat genes constructed by connecting minirepeat genes obtained by in vivo selection were also stable in the expression host cells. Gelatins derived from the eight-unit and six-unit repeat genes were extracellularly produced at the level of 0.5 g/liter and easily purified by ammonium sulfate fractionation and anion-exchange chromatography. The purified artificial gelatins had the predicted N-terminal sequences and amino acid compositions and a solgel property similar to that of the native gelatin. These results suggest that the selection of a repeat unit sequence stable in an expression host is a shortcut for the efficient production of repetitive proteins and that it can conveniently be achieved by the in vivo selection method. This study revealed the possible industrial application of artificially designed repetitive proteins.
Molecular Characterization of the Toxic Cyanobacterium
Cylindrospermopsis raciborskii
and Design of a Species-Specific PCR
Authors: Wilson Kim M. , Schembri Mark A. , Baker Peter D. , Saint Christopher P. 
Published: April 2009

ABSTRACT

Cylindrospermopsis raciborskii
is a toxic-bloom-forming cyanobacterium that is commonly found in tropical to subtropical climatic regions worldwide, but it is also recognized as a common component of cyanobacterial communities in temperate climates. Genetic profiles of
C. raciborskii
were examined in 19 cultured isolates originating from geographically diverse regions of Australia and represented by two distinct morphotypes. A 609-bp region of
rpoC1
, a DNA-dependent RNA polymerase gene, was amplified by PCR from these isolates with cyanobacterium-specific primers. Sequence analysis revealed that all isolates belonged to the same species, including morphotypes with straight or coiled trichomes. Additional
rpoC1
gene sequences obtained for a range of cyanobacteria highlighted clustering of
C. raciborskii
with other heterocyst-producing cyanobacteria (orders
Nostocales
and
Stigonematales
). In contrast, randomly amplified polymorphic DNA and short tandemly repeated repetitive sequence profiles revealed a greater level of genetic heterogeneity among
C. raciborskii
isolates than did
rpoC1
gene analysis, and unique band profiles were also found among each of the cyanobacterial genera examined. A PCR test targeting a region of the
rpoC1
gene unique to
C. raciborskii
was developed for the specific identification of
C. raciborskii
from both purified genomic DNA and environmental samples. The PCR was evaluated with a number of cyanobacterial isolates, but a PCR-positive result was only achieved with
C. raciborskii
. This method provides an accurate alternative to traditional morphological identification of
C. raciborskii
.
A Theoretical Study on the Metabolic Requirements Resulting from α-Ketoglutarate-Dependent Cleavage of Phenoxyalkanoates
Authors: Müller R. H. , Babel W. 
Published: April 2009

ABSTRACT
The etherolytic cleavage of phenoxyalkanoic acids in various bacteria is catalyzed by an α-ketoglutarate-dependent dioxygenase. In this reaction, the electron acceptor is oxidatively decarboxylated to succinate, whereas the proper substrate is cleaved by forming the oxidized alkanoic acid and the phenolic intermediate. The necessity of regenerating α-ketoglutarate and the consequences for the overall metabolism were investigated in a theoretical study. It was found that the dioxygenase mechanism is accompanied by a significant loss of carbon amounting to up to 62.5% in the assimilatory branch, thus defining the upper limit of carbon conversion efficiency. This loss in carbon is almost compensated for in comparison to a monooxygenase-catalyzed initial step when the dissimilatory efforts of the entire metabolism are included: the yield coefficients become similar. The α-ketoglutarate-dependent dioxygenase mechanism has more drastic consequences for microorganisms which are restricted in their metabolism to the first step of phenoxyalkanoate degradation by excreting the phenolic intermediate as a dead-end product. In the case of phenoxyacetate derivatives, the cleavage reaction would quickly cease due to the exhaustion of α-ketoglutarate and no growth would be possible. With the cleavage products of phenoxypropionate and phenoxybutyrate herbicides, i.e., pyruvate and succinate(semialdehyde), respectively, as the possible products, the regeneration of α-ketoglutarate will be guaranteed for stoichiometric reasons. However, the maintenance of the cleavage reaction ought to be restricted due to physiological factors owing to the involvement of other metabolic reactions in the pool of metabolites. These effects are discussed in terms of a putative recalcitrance of these compounds.
Requirement of Monooxygenase-Mediated Steps for Sterigmatocystin Biosynthesis by
Aspergillus nidulans
Authors: Keller Nancy P. , Watanabe Coran M. H. , Kelkar Hemant S. , Adams Thomas H. , Townsend Craig A. 
Published: April 2009

ABSTRACT

Sterigmatocystin (ST) and aflatoxin B
1
(AFB
1
) are two polyketide-derived
Aspergillus
mycotoxins synthesized by functionally identical sets of enzymes. ST, the compound produced by
Aspergillus nidulans
, is a late intermediate in the AFB
1
pathway of
A. parasiticus
and
A. flavus
. Previous biochemical studies predicted that five oxygenase steps are required for the formation of ST. A 60-kb ST gene cluster in
A. nidulans
contains five genes,
stcB
,
stcF
,
stcL
,
stcS
, and
stcW
, encoding putative monooxygenase activities. Prior research showed that
stcL
and
stcS
mutants accumulated versicolorins B and A, respectively. We now show that strains disrupted at
stcF
, encoding a P-450 monooxygenase similar to
A. parasiticus avnA
, accumulate averantin. Disruption of either StcB (a putative P-450 monooxygenase) or StcW (a putative flavin-requiring monooxygenase) led to the accumulation of averufin as determined by radiolabeled feeding and extraction studies.
Characterization of the Kexin-Like Maturase of
Aspergillus niger
Authors: Jalving Ruud , van de Vondervoort Peter J. I. , Visser Jaap , Schaap Peter J. 
Published: April 2009

ABSTRACT

Secreted yields of foreign proteins may be enhanced in filamentous fungi through the use of translational fusions in which the target protein is fused to an endogenous secreted carrier protein. The fused proteins are usually separated in vivo by cleavage of an engineered Kex2 endoprotease recognition site at the fusion junction. We have cloned the kexin-encoding gene of
Aspergillus niger
(
kexB
). We constructed strains that either overexpressed KexB or lacked a functional
kexB
gene. Kexin-specific activity doubled in membrane-protein fractions of the strain overexpressing KexB. In contrast, no kexin-specific activity was detected in the similar protein fractions of the
kexB
disruptant. Expression in this loss-of-function strain of a glucoamylase human interleukin-6 fusion protein with an engineered Kex2 dibasic cleavage site at the fusion junction resulted in secretion of unprocessed fusion protein. The results show that KexB is the endoproteolytic proprotein processing enzyme responsible for the processing of (engineered) dibasic cleavage sites in target proteins that are transported through the secretion pathway of
A. niger
.
Role of Leaf Surface Sugars in Colonization of Plants by Bacterial Epiphytes
Authors: Mercier Julien , Lindow S. E. 
Published: April 2009

ABSTRACT

The relationship between nutrients leached onto the leaf surface and the colonization of plants by bacteria was studied by measuring both the abundance of simple sugars and the growth of
Pseudomonas fluorescens
on individual bean leaves. Data obtained in this study indicate that the population size of epiphytic bacteria on plants under environmentally favorable conditions is limited by the abundance of carbon sources on the leaf surface. Sugars were depleted during the course of bacterial colonization of the leaf surface. However, about 20% of readily utilizable sugar, such as glucose, present initially remained on fully colonized leaves. The amounts of sugars on a population of apparently identical individual bean leaves before and after microbial colonization exhibited a similar right-hand-skewed distribution and varied by about 25-fold from leaf to leaf. Total bacterial population sizes on inoculated leaves under conditions favorable for bacterial growth also varied by about 29-fold and exhibited a right-hand-skewed distribution. The amounts of sugars on leaves of different plant species were directly correlated with the maximum bacterial population sizes that could be attained on those species. The capacity of bacteria to deplete leaf surface sugars varied greatly among plant species. Plants capable of supporting high bacterial population sizes were proportionally more depleted of leaf surface nutrients than plants with low epiphytic populations. Even in species with a high epiphytic bacterial population, a substantial amount of sugar remained after bacterial colonization. It is hypothesized that residual sugars on colonized leaves may not be physically accessible to the bacteria due to limitations in wettability and/or diffusion of nutrients across the leaf surface.
Quantification of Different
Eubacterium
spp. in Human Fecal Samples with Species-Specific 16S rRNA-Targeted Oligonucleotide Probes
Authors: Schwiertz Andreas , Le Blay Gwenaelle , Blaut Michael 
Published: April 2009

ABSTRACT

Species-specific 16S rRNA-targeted, Cy3 (indocarbocyanine)-labeled oligonucleotide probes were designed and validated to quantify different
Eubacterium
species in human fecal samples. Probes were directed at
Eubacterium barkeri
,
E. biforme
,
E. contortum
,
E. cylindroides
(two probes),
E. dolichum
,
E. hadrum
,
E. lentum
,
E. limosum
,
E. moniliforme
, and
E. ventriosum
. The specificity of the probes was tested with the type strains and a range of common intestinal bacteria. With one exception, none of the probes showed cross-hybridization under stringent conditions. The species-specific probes were applied to fecal samples obtained from 12 healthy volunteers.
E. biforme
,
E. cylindroides
,
E. hadrum
,
E. lentum
, and
E. ventriosum
could be determined. All other
Eubacterium
species for which probes had been designed were under the detection limit of 10
7
cells g (dry weight) of feces
−1
. The cell counts obtained are essentially in accordance with the literature data, which are based on colony counts. This shows that whole-cell in situ hybridization with species-specific probes is a valuable tool for the enumeration of
Eubacterium
species in feces.
Dual Labeling with Green Fluorescent Proteins for Confocal Microscopy
Authors: Cowan Stacie E. , Gilbert Eric , Khlebnikov Artem , Keasling J. D. 
Published: April 2009

ABSTRACT
We report a dual labeling technique involving two green fluorescent protein (GFP) variants that is compatible with confocal microscopy. Two lasers were used to obtain images of (i) mixed cultures of cells, where one species contained GFPuv and another species contained GFPmut2 or GFPmut3, and (ii) a single species containing both GFPuv and GFPmut2 in the same cell. This method shows promise for monitoring gene expression and as a nondestructive and in situ technique for confocal microscopy of multispecies biofilms.
Molecular and Functional Analyses of the
metC
Gene of
Lactococcus lactis
, Encoding Cystathionine β-Lyase
Authors: Fernández María , van Doesburg Wim , Rutten Ger A. M. , Marugg Joey D. , Alting Arno C. , van Kranenburg Richard , Kuipers Oscar P. 
Published: April 2009

ABSTRACT

The enzymatic degradation of amino acids in cheese is believed to generate aroma compounds and therefore to be essential for flavor development. Cystathionine β-lyase (CBL) can convert cystathionine to homocysteine but is also able to catalyze an α,γ elimination. With methionine as a substrate, it produces volatile sulfur compounds which are important for flavor formation in Gouda cheese. The
metC
gene, which encodes CBL, was cloned from the
Lactococcus lactis
model strain MG1363 and from strain B78, isolated from a cheese starter culture and known to have a high capacity to produce volatile compounds. The
metC
gene was found to be cotranscribed with a downstream
cysK
gene, which encodes a putative cysteine synthase. The MetC proteins of both strains were overproduced in strain MG1363 with the NICE (nisin-controlled expression) system, resulting in a >25-fold increase in cystathionine lyase activity. A disruption of the
metC
gene was achieved in strain MG1363. Determination of enzymatic activities in the overproducing and knockout strains revealed that MetC is essential for the degradation of cystathionine but that at least one lyase other than CBL contributes to methionine degradation via α,γ elimination to form volatile aroma compounds.
Phoma glomerataas a Mycoparasite of Powdery Mildew
Authors: Sullivan Raymond F. , White James F. 
Published: April 2009

ABSTRACTAmpelomycesandPhomaspecies are frequently confused with each other. Isolates previously attributed to the genusAmpelomyceswere shown to bePhomaisolates through studies of their morphology and life cycle and ribosomal DNA internal transcribed spacer region 1 sequence analysis.Phoma glomeratacan colonize and suppress development of powdery mildew on oak and may have utility as a mycoparasitic agent.