bluuebunny/crossref_metadata_2025_split huggingface dataset:
Generation of Food-Grade Recombinant Lactic Acid Bacterium Strains by Site-Specific Recombination
Authors: Martín M. Cruz , Alonso Juan C. , Suárez Juan E. , Alvarez Miguel A. 
Published: July 2002

ABSTRACT

The construction of a delivery and clearing system for the generation of food-grade recombinant lactic acid bacterium strains, based on the use of an integrase (Int) and a resolvo-invertase (β-recombinase) and their respective target sites (
attP-attB
and
six
, respectively) is reported. The delivery system contains a heterologous replication origin and antibiotic resistance markers surrounded by two directly oriented
six
sites, a multiple cloning site where passenger DNA could be inserted (e.g., the
c
I gene of bacteriophage A2), the
int
gene, and the
attP
site of phage A2. The clearing system provides a plasmid-borne gene encoding β-recombinase. The nonreplicative vector-borne delivery system was transformed into
Lactobacillus casei
ATCC 393 and, by site-specific recombination, integrated as a single copy in an orientation- and Int-dependent manner into the
attB
site present in the genome of the host strain. The transfer of the clearing system into this strain, with the subsequent expression of the β-recombinase, led to site-specific DNA resolution of the non-food-grade DNA. These methods were validated by the construction of a stable food-grade
L. casei
ATCC 393-derived strain completely immune to phage A2 infection during milk fermentation.
Contamination of Foods by Food Handlers: Experiments on Hepatitis A Virus Transfer to Food and Its Interruption
Authors: Bidawid S. , Farber J. M. , Sattar S. A. 
Published: July 2002

ABSTRACT

Hepatitis A virus (HAV) is an important pathogen which has been responsible for many food-borne outbreaks. HAV-excreting food handlers, especially those with poor hygienic practices, can contaminate the foods which they handle. Consumption of such foods without further processing has been known to result in cases of infectious hepatitis. Since quantitative data on virus transfer during contact of hands with foods is not available, we investigated the transfer of HAV from artificially contaminated fingerpads of adult volunteers to pieces of fresh lettuce. Touching the lettuce with artificially contaminated fingerpads for 10 s at a pressure of 0.2 to 0.4 kg/cm
2
resulted in transfer of 9.2% ± 0.9% of the infectious virus. The pretreatments tested to interrupt virus transfer from contaminated fingerpads included (i) hard-water rinsing and towel drying, (ii) application of a domestic or commercial topical agent followed by water rinsing and towel drying, and (iii) exposure to a hand gel containing 62% ethanol or 75% liquid ethanol without water rinsing or towel drying. When the fingerpads were treated with the topical agents or alcohol before the lettuce was touched, the amount of infectious virus transferred to lettuce was reduced from 9.2% to between 0.3 and 0.6% (depending on the topical agent used), which was a reduction in virus transfer of up to 30-fold. Surprisingly, no virus transfer to lettuce was detected when the fingerpads were rinsed with water alone before the lettuce was touched. However, additional experiments with water rinsing in which smaller volumes of water were used (1 ml instead of 15 ml) showed that the rate of virus transfer to lettuce was 0.3% ± 0.1%. The variability in virus transfer rates following water rinsing may indicate that the volume of water at least in part influences virus removal from the fingerpads differently, a possibility which should be investigated further. This study provided novel information concerning the rate of virus transfer to foods and a model for investigating the transfer of viral and other food-borne pathogens from contaminated hands to foods, as well as techniques for interrupting such transfer to improve food safety.
Terminal Restriction Fragment Length Polymorphism Analysis Program, a Web-Based Research Tool for Microbial Community Analysis
Authors: Marsh Terence L. , Saxman Paul , Cole James , Tiedje James 
Published: July 2002

ABSTRACT

Rapid analysis of microbial communities has proven to be a difficult task. This is due, in part, to both the tremendous diversity of the microbial world and the high complexity of many microbial communities. Several techniques for community analysis have emerged over the past decade, and most take advantage of the molecular phylogeny derived from 16S rRNA comparative sequence analysis. We describe a web-based research tool located at the Ribosomal Database Project web site (
http://www.cme.msu.edu/RDP/html/analyses.html
) that facilitates microbial community analysis using terminal restriction fragment length polymorphism of 16S ribosomal DNA. The analysis function (designated TAP T-RFLP) permits the user to perform in silico restriction digestions of the entire 16S sequence database and derive terminal restriction fragment sizes, measured in base pairs, from the 5′ terminus of the user-specified primer to the 3′ terminus of the restriction endonuclease target site. The output can be sorted and viewed either phylogenetically or by size. It is anticipated that the site will guide experimental design as well as provide insight into interpreting results of community analysis with terminal restriction fragment length polymorphisms.
Anaerobic Cometabolic Conversion of Benzothiophene by a Sulfate-Reducing Enrichment Culture and in a Tar-Oil-Contaminated Aquifer
Authors: Annweiler Eva , Michaelis Walter , Meckenstock Rainer U. 
Published: July 2002

ABSTRACT

Anaerobic cometabolic conversion of benzothiophene was studied with a sulfate-reducing enrichment culture growing with naphthalene as the sole source of carbon and energy. The sulfate-reducing bacteria were not able to grow with benzothiophene as the primary substrate. Metabolite analysis was performed with culture supernatants obtained by cometabolization experiments and revealed the formation of three isomeric carboxybenzothiophenes. Two isomers were identified as 2-carboxybenzothiophene and 5-carboxybenzothiophene. In some experiments, further reduced dihydrocarboxybenzothiophene was identified. No other products of benzothiophene degradation could be determined. In isotope-labeling experiments with a [
13
C]bicarbonate-buffered culture medium, carboxybenzothiophenes which were significantly enriched in the
13
C content of the carboxyl group were formed, indicating the addition of a C
1
unit from bicarbonate to benzothiophene as the initial activation reaction. This finding was consistent with the results of earlier studies on anaerobic naphthalene degradation with the same culture, and we therefore propose that benzothiophene was cometabolically converted by the same enzyme system. Groundwater analyses of the tar-oil-contaminated aquifer from which the naphthalene-degrading enrichment culture was isolated exhibited the same carboxybenzothiophene isomers as the culture supernatants. In addition, the benzothiophene degradation products, in particular, dihydrocarboxybenzothiophene, were significantly enriched in the contaminated groundwater to concentrations almost the same as those of the parent compound, benzothiophene. The identification of identical metabolites of benzothiophene conversion in the sulfate-reducing enrichment culture and in the contaminated aquifer indicated that the same enzymatic reactions were responsible for the conversion of benzothiophene in situ.
Successional Changes in an Evolving Anaerobic Chlorophenol-Degrading Community Used To Infer Relationships between Population Structure and System-Level Processes
Authors: Becker Jennifer G. , Berardesco Gina , Rittmann Bruce E. , Stahl David A. 
Published: July 2002

ABSTRACT

The response of a complex methanogenic sediment community to 2-chlorophenol (2-CP) was evaluated by monitoring the concentrations of this model contaminant and important metabolic intermediates and products and by using rRNA-targeted probes to track several microbial populations. Key relationships between the evolving population structure, formation of metabolic intermediates, and contaminant mineralization were identified. The nature of these relationships was intrinsically linked to the metabolism of benzoate, an intermediate that transiently accumulated during the mineralization of 2-CP. Before the onset of benzoate fermentation, reductive dehalogenation of 2-CP competed with methanogenesis for endogenous reducing equivalents. This suppressed H
2
levels, methane production, and archaeal small-subunit (SSU)-rRNA concentrations in the sediment community. The concentrations of bacterial SSU rRNA, including SSU rRNA derived from “
Desulfovibrionaceae
” populations, tracked with 2-CP levels, presumably reflecting changes in the activity of dehalogenating organisms. After the onset of benzoate fermentation, the abundance of
Syntrophus
-like SSU rRNA increased, presumably because these syntrophic organisms fermented benzoate to methanogenic substrates. Consequently, although the parent substrate 2-CP served as an electron acceptor, cleavage of its aromatic nucleus also influenced the sediment community by releasing the electron donors H
2
and acetate. Increased methane production and archaeal SSU-rRNA levels, which tracked with the
Syntrophus
-like SSU-rRNA concentrations, revealed that methanogenic populations in particular benefited from the input of reducing equivalents derived from 2-CP.
Fungal Metabolism of Toluene: Monitoring of Fluorinated Analogs by
19
F Nuclear Magnetic Resonance Spectroscopy
Authors: Prenafeta-Boldú Francesc X. , Luykx Dion M. A. M. , Vervoort Jacques , de Bont Jan A. M. 
Published: July 2002

ABSTRACT

We used isomeric fluorotoluenes as model substrates to study the catabolism of toluene by five deuteromycete fungi and one ascomycete fungus capable of growth on toluene as the sole carbon and energy source, as well as by two fungi (
Cunninghamella echinulata
and
Aspergillus niger
) that cometabolize toluene. Whole cells were incubated with 2-, 3-, and 4-fluorotoluene, and metabolites were characterized by
19
F nuclear magnetic resonance. Oxidation of fluorotoluene by
C. echinulata
was initiated either at the aromatic ring, resulting in fluorinated
o-
cresol, or at the methyl group to form fluorobenzoate. The initial conversion of the fluorotoluenes by toluene-grown fungi occurred only at the side chain and resulted in fluorinated benzoates. The latter compounds were the substrate for the ring hydroxylation and, depending on the fluorine position, were further metabolized up to catecholic intermediates. From the
19
F nuclear magnetic resonance metabolic profiles, we propose that diverse fungi that grow on toluene assimilate toluene by an initial oxidation of the methyl group.
Sensitivities of Germinating Spores and Carvacrol-Adapted Vegetative Cells and Spores of
Bacillus cereus
to Nisin and Pulsed-Electric-Field Treatment
Authors: Pol Irene E. , van Arendonk Willy G. C. , Mastwijk Hennie C. , Krommer Judit , Smid Eddy J. , Moezelaar Roy 
Published: July 2002

ABSTRACT

Treatment of
Bacillus cereus
spores with nisin and/or pulsed-electric-field (PEF) treatment did not lead to direct inactivation of the spores or increased heat sensitivity as a result of sublethal damage. In contrast, germinating spores were found to be sensitive to PEF treatment. Nisin treatment was more efficient than PEF treatment for inactivating germinating spores. PEF resistance was lost after 50 min of germination, and not all germinated spores could be inactivated. Nisin, however, was able to inactivate the germinating spores to the same extent as heat treatment. Resistance to nisin was lost immediately when the germination process started. A decrease in the membrane fluidity of vegetative cells caused by incubation in the presence of carvacrol resulted in a dramatic increase in the sensitivity to nisin. On the other hand, inactivation by PEF treatment or by a combination of nisin and PEF treatments did not change after adaptation to carvacrol. Spores grown in the presence of carvacrol were not susceptible to nisin and/or PEF treatment in any way.
Purification and Characterization of Cellobiose Dehydrogenase from the Plant PathogenSclerotium(Athelia)rolfsii
Authors: Baminger Ursula , Subramaniam Sai S. , Renganathan V. , Haltrich Dietmar 
Published: July 2002

ABSTRACTCellobiose dehydrogenase (CDH) is an extracellular hemoflavoenzyme produced by several wood-degrading fungi. In the presence of a suitable electron acceptor, e.g., 2,6-dichloro-indophenol (DCIP), cytochromec, or metal ions, CDH oxidizes cellobiose to cellobionolactone. The phytopathogenic fungusSclerotium rolfsii(teleomorph:Athelia rolfsii) strain CBS 191.62 produces remarkably high levels of CDH activity when grown on a cellulose-containing medium. Of the 7,500 U of extracellular enzyme activity formed per liter, less than 10% can be attributed to the proteolytic product cellobiose:quinone oxidoreductase. As with CDH from wood-rotting fungi, the intact, monomeric enzyme fromS. rolfsiicontains one hemeband one flavin adenine dinucleotide cofactor per molecule. It has a molecular size of 101 kDa, of which 15% is glycosylation, and a pI value of 4.2. The preferred substrates are cellobiose and cellooligosaccharides; additionally, β-lactose, thiocellobiose, and xylobiose are efficiently oxidized. Cytochromec(equine) and the azino-di-(3-ethyl-benzthiazolin-6-sulfonic acid) cation radical were the best electron acceptors, while DCIP, 1,4-benzoquinone, phenothiazine dyes such as methylene blue, phenoxazine dyes such as Meldola's blue, and ferricyanide were also excellent acceptors. In addition, electrons can be transferred to oxygen. Limited in vitro proteolysis with papain resulted in the formation of several protein fragments that are active with DCIP but not with cytochromec. Such a flavin-containing fragment, with a mass of 75 kDa and a pI of 5.1 and lacking the heme domain, was isolated and partially characterized.
Characterization of Non-Starter Lactic Acid Bacteria from Italian Ewe Cheeses Based on Phenotypic, Genotypic, and Cell Wall Protein Analyses
Authors: De Angelis M. , Corsetti A. , Tosti N. , Rossi J. , Corbo M. R. , Gobbetti M. 
Published: July 2002

ABSTRACT

Non-starter lactic acid bacteria (NSLAB) were isolated from 12 Italian ewe cheeses representing six different types of cheese, which in several cases were produced by different manufacturers. A total of 400 presumptive
Lactobacillus
isolates were obtained, and 123 isolates and 10 type strains were subjected to phenotypic, genetic, and cell wall protein characterization analyses. Phenotypically, the cheese isolates included 32%
Lactobacillus plantarum
isolates, 15%
L. brevis
isolates, 12%
L. paracasei
subsp.
paracasei
isolates, 9%
L. curvatus
isolates, 6%
L. fermentum
isolates, 6%
L. casei
subsp.
casei
isolates, 5%
L. pentosus
isolates, 3%
L. casei
subsp.
pseudoplantarum
isolates, and 1%
L. rhamnosus
isolates. Eleven percent of the isolates were not phenotypically identified. Although a randomly amplified polymorphic DNA (RAPD) analysis based on three primers and clustering by the unweighted pair group method with arithmetic average (UPGMA) was useful for partially differentiating the 10 type strains, it did not provide a species-specific DNA band or a combination of bands which permitted complete separation of all the species considered. In contrast, sodium dodecyl sulfate-polyacrylamide gel electrophoresis cell wall protein profiles clustered by UPGMA were species specific and resolved the NSLAB. The only exceptions were isolates phenotypically identified as
L. plantarum
and
L. pentosus
or as
L. casei
subsp.
casei
and
L. paracasei
subsp.
paracasei
, which were grouped together. Based on protein profiles, Italian ewe cheeses frequently contained four different species and 3 to 16 strains. In general, the cheeses produced from raw ewe milk contained a larger number of more diverse strains than the cheeses produced from pasteurized milk. The same cheese produced in different factories contained different species, as well as strains that belonged to the same species but grouped in different RAPD clusters.
Degradation of Substituted Phenylurea Herbicides by
Arthrobacter globiformis
Strain D47 and Characterization of a Plasmid-Associated Hydrolase Gene,
puhA
Authors: Turnbull Gillian A. , Ousley Margaret , Walker Allan , Shaw Eve , Morgan J. Alun W. 
Published: July 2002

ABSTRACT

Arthrobacter globiformis
D47 was shown to degrade a range of substituted phenylurea herbicides in soil. This strain contained two plasmids of approximately 47 kb (pHRIM620) and 34 kb (pHRIM621). Plasmid-curing experiments produced plasmid-free strains as well as strains containing either the 47- or the 34-kb plasmid. The strains were tested for their ability to degrade diuron, which demonstrated that the degradative genes were located on the 47-kb plasmid. Studies on the growth of these strains indicated that the ability to degrade diuron did not offer a selective advantage to
A. globiformis
D47 on minimal medium designed to contain the herbicide as a sole carbon source. The location of the genes on a plasmid and a lack of selection would explain why the degradative phenotype, as with many other pesticide-degrading bacteria, can be lost on subculture. A 22-kb
Eco
RI fragment of plasmid pHRIM620 was expressed in
Escherichia coli
and enabled cells to degrade diuron. Transposon mutagenesis of this fragment identified one open reading frame that was essential for enzyme activity. A smaller subclone of this gene (2.5 kb) expressed in
E. coli
coded for the protein that degraded diuron. This gene and its predicted protein sequence showed only a low level of protein identity (25% over ca. 440 amino acids) to other database sequences and was named after the enzyme it encoded, phenylurea hydrolase (
puhA
gene).
Microbial Populations Associated with Treatment of an Industrial Dye Effluent in an Anaerobic Baffled Reactor
Authors: Plumb Jason J. , Bell Joanne , Stuckey David C. 
Published: July 2002

ABSTRACT

Fluorescent in situ hybridization (FISH) using 16S and 23S rRNA-targeted probes together with construction of an archaeal 16S ribosomal DNA (rDNA) clone library was used to characterize the microbial populations of an anaerobic baffled reactor successfully treating industrial dye waste. Wastewater produced during the manufacture of food dyes containing several different azo and other dye compounds was decolorized and degraded under sulfidogenic and methanogenic conditions. Use of molecular methods to describe microbial populations showed that a diverse group of
Bacteria
and
Archaea
was involved in this treatment process. FISH enumeration showed that members of the gamma subclass of the class
Proteobacteria
and bacteria in the
Cytophaga-Flexibacter-Bacteroides
phylum, together with sulfate-reducing bacteria, were prominent members of a mixed bacterial population. A combination of FISH probing and analysis of 98 archaeal 16S rDNA clone inserts revealed that together with the bacterial population, a methanogenic population dominated by
Methanosaeta
species and containing species of
Methanobacterium
and
Methanospirillum
and a relatively unstudied methanogen,
Methanomethylovorans hollandica
, contributed to successful anaerobic treatment of the industrial waste. We suggest that sulfate reducers, or more accurately sulfidogenic bacteria, together with
M. hollandica
contribute considerably to the treatment process through metabolism of dye-associated sulfonate groups and subsequent conversion of sulfur compounds to carbon dioxide and methane.
Bacterial Species Determination from DNA-DNA Hybridization by Using Genome Fragments and DNA Microarrays
Authors: Cho Jae-Chang , Tiedje James M. 
Published: July 2002

ABSTRACT

Whole genomic DNA-DNA hybridization has been a cornerstone of bacterial species determination but is not widely used because it is not easily implemented. We have developed a method based on random genome fragments and DNA microarray technology that overcomes the disadvantages of whole-genome DNA-DNA hybridization. Reference genomes of four fluorescent
Pseudomonas
species were fragmented, and 60 to 96 genome fragments of approximately 1 kb from each strain were spotted on microarrays. Genomes from 12 well-characterized fluorescent
Pseudomonas
strains were labeled with Cy dyes and hybridized to the arrays. Cluster analysis of the hybridization profiles revealed taxonomic relationships between bacterial strains tested at species to strain level resolution, suggesting that this approach is useful for the identification of bacteria as well as determining the genetic distance among bacteria. Since arrays can contain thousands of DNA spots, a single array has the potential for broad identification capacity. In addition, the method does not require laborious cross-hybridizations and can provide an open database of hybridization profiles, avoiding the limitations of traditional DNA-DNA hybridization.
Isolation and Regulation of
Sinorhizobium meliloti
1021 Loci Induced by Oxygen Limitation
Authors: Trzebiatowski Jodi R. , Ragatz Daniel M. , de Bruijn Frans J. 
Published: July 2002

ABSTRACT

Eleven
Sinorhizobium meliloti
1021 loci whose expression was induced under low oxygen concentrations were identified in a collection of 5,000 strains carrying Tn
5
-
1063
(
luxAB
) transcriptional reporter gene fusions. The 11 Tn
5
-
1063
-tagged loci were cloned and characterized. The dependence of the expression of the tagged loci on the FixL/FixJ oxygen-sensing two-component regulatory system was examined. Three of the loci were found to be dependent upon
fixL
and
fixJ
for their expression, while one locus showed a partial dependence. The remaining seven loci showed
fixL
- and
fixJ
-independent induction of expression in response to oxygen limitation. This suggests that in
S. meliloti
, additional regulatory system(s) exist that respond either directly or indirectly to oxygen limitation conditions.
Induction of Phase Variation Events in the Life Cycle of the Marine Coccolithophorid
Emiliania huxleyi
Authors: Laguna Richard , Romo Jesus , Read Betsy A. , Wahlund Thomas M. 
Published: July 2002

ABSTRACT

Emiliania huxleyi
is a unicellular marine alga that is considered to be the world's major producer of calcite. The life cycle of this alga is complex and is distinguished by its ability to synthesize exquisitely sculptured calcium carbonate cell coverings known as coccoliths. These structures have been targeted by materials scientists for applications relating to the chemistry of biomedical materials, robust membranes for high-temperature separation technology, lightweight ceramics, and semiconductor design. To date, however, the molecular and biochemical events controlling coccolith production have not been determined. In addition, little is known about the life cycle of
E. huxleyi
and the environmental and physiological signals triggering phase switching between the diploid and haploid life cycle stages. We have developed laboratory methods for inducing phase variation between the haploid (S-cell) and diploid (C-cell) life cycle stages of
E. huxleyi
. Plating
E. huxleyi
C cells on solid media was shown to induce phase switching from the C-cell to the S-cell life cycle stage, the latter of which has been maintained for over 2 years under these conditions. Pure cultures of S cells were obtained for the first time. Laboratory conditions for inducing phase switching from the haploid stage to the diploid stage were also established. Regeneration of the C-cell stage from pure cultures of S cells followed a predictable pattern involving formation of large aggregations of S cells and the subsequent production of cultures consisting predominantly of diploid C cells. These results demonstrate the ability to manipulate the life cycle of
E. huxleyi
under controlled laboratory conditions, providing us with powerful tools for the development of genetic techniques for analysis of coccolithogenesis and for investigating the complex life cycle of this important marine alga.