bluuebunny/crossref_metadata_2025_split huggingface dataset:
Degradation of Polycyclic Aromatic Hydrocarbons at Low Temperature under Aerobic and Nitrate-Reducing Conditions in Enrichment Cultures from Northern Soils
Authors: Eriksson Mikael , Sodersten Erik , Yu Zhongtang , Dalhammar Gunnel , Mohn William W. 
Published: January 2003

ABSTRACT

Thepotential for biodegradation of polycyclic aromatic hydrocarbons (PAHs)at low temperature and under anaerobic conditions is not wellunderstood, but such biodegradation would be very useful forremediation of polluted sites. Biodegradation of a mixture of 11different PAHs with two to five aromatic rings, each at a concentrationof 10 μg/ml, was studied in enrichment cultures inoculated withsamples of four northern soils. Under aerobic conditions, lowtemperature severely limited PAH biodegradation. After 90 days, aerobiccultures at 20°C removed 52 to 88% of the PAHs. The mostextensive PAH degradation under aerobic conditions at 7°C,53% removal, occurred in a culture from creosote-contaminatedsoil. Low temperature did not substantially limit PAH biodegradationunder nitrate-reducing conditions. Under nitrate-reducing conditions,naphthalene, 2-methylnaphthalene, fluorene, and phenanthrene weredegraded. The most extensive PAH degradation under nitrate-reducingconditions at 7°C, 39% removal, occurred in a culturefrom fuel-contaminated Arctic soil. In separate transfer cultures fromthe above Arctic soil, incubated anaerobically at 7°C, removalof 2-methylnaphthalene and fluorene was stoichiometrically coupled tonitrate removal. Ribosomal intergenic spacer analysis suggested thatenrichment resulted in a few predominant bacterial populations,including members of the genera
Acidovorax
,
Bordetella
,
Pseudomonas
,
Sphingomonas
, and
Variovorax
. Predominant populations from different soils oftenincluded phylotypes with nearly identical partial 16S rRNA genesequences (i.e., same genus) but never included phylotypes withidentical ribosomal intergenic spacers (i.e., different species orsubspecies). The composition of the enriched communities appeared to bemore affected by presence of oxygen, than by temperature or source oftheinoculum.
Elevated Abundance of Bacteriophage Infecting Bacteria in Soil
Authors: Ashelford Kevin E. , Day Martin J. , Fry John C. 
Published: January 2003

ABSTRACTHere we report the first direct counts of soil bacteriophage and show that substantial populations of these viruses exist in soil (grand mean = 1.5 × 107g−1), at least 350-fold more than the highest numbers estimated from traditional viable plaque counts. Adding pure cultures of aSerratiaphage to soil showed that the direct counting methods with electron microscopy developed here underestimated the added phage populations by at least eightfold. So, assuming natural phages were similarly underestimated, virus numbers in soil averaged 1.5 × 108g−1, which is equivalent to 4% of the total population of bacteria. This high abundance was to some extent confirmed by hybridizing colonies grown onSerratiaandPseudomonasselective media with cocktails of phage infecting these bacteria. This showed that 8.9 and 3.9%, respectively, hybridized with colonies from the two media and confirmed the presence of phage DNA sequences in the cultivable fraction of the natural population. Thus, soil phage, like their aquatic counterparts, are likely to be important in controlling bacterial populations and mediating gene transfer in soil.
Multicenter Validation of the Analytical Accuracy ofSalmonellaPCR: towards an International Standard
Authors: Malorny Burkhard , Hoorfar Jeffrey , Bunge Cornelia , Helmuth Reiner 
Published: January 2003

ABSTRACTAs part of a major international project for the validation and standardization of PCR for detection of five major food-borne pathogens, four primer sets specific forSalmonellaspecies were evaluated in-house for their analytical accuracy (selectivity and detection limit) in identifying 43Salmonellaspp. and 47 non-Salmonellastrains. The most selective primer set was found to be 139-141 (K. Rahn, S. A. De Grandis, R. C. Clarke, S. A. McEwen, J. E. Galán, C. Ginocchio, R. Curtiss III, and C. L. Gyles, Mol. Cell. Probes 6:271-279, 1992), which targets theinvAgene. An extended determination of selectivity by using 364 strains showed that the inclusivity was 99.6% and the exclusivity was 100% for theinvAprimer set. To indicate possible PCR inhibitors derived from the sample DNA, an internal amplification control (IAC), which was coamplified with theinvAtarget gene, was constructed. In the presence of 300 DNA copies of the IAC, the detection probability for primer set 139-141 was found to be 100% when a cell suspension containing 104CFU/ml was used as the template in the PCR (50 CFU per reaction). The primer set was further validated in an international collaborative study that included 16 participating laboratories. Analysis with 28 coded (“blind”) DNA samples revealed an analytical accuracy of 98%. Thus, a simple PCR assay that is specific forSalmonellaspp. and amplifies a chromosomal DNA fragment detected by gel electrophoresis was established through extensive validation and is proposed as an international standard. This study addresses the increasing demand of quality assurance laboratories for standard diagnostic methods and presents findings that can facilitate the international comparison and exchange of epidemiological data.
Fluorigenic Substrates for the Protease Activities of Botulinum Neurotoxins, Serotypes A, B, and F
Authors: Schmidt James J. , Stafford Robert G. 
Published: January 2003

ABSTRACT

The seven botulinum neurotoxins (BoNTs) are zinc metalloproteases that cleave neuronal proteins involved in neurotransmitter release and are among the most toxic natural products known. High-throughput BoNT assays are needed for use in antibotulinum drug discovery and to characterize BoNT protease activities. Compared to other proteases, BoNTs exhibit unusually stringent substrate requirements with respect to amino acid sequences and polypeptide lengths. Nonetheless, we have devised a strategy for development of fluorigenic BoNT protease assays, based on earlier structure-function studies, that has proven successful for three of the seven serotypes: A, B, and F. In synthetic peptide substrates, the P
1
and P
3
′ residues were substituted with 2,4-dinitrophenyl-lysine and
S
-(
N
-[4-methyl-7-dimethylamino-coumarin-3-yl]-carboxamidomethyl)-cysteine, respectively. By monitoring the BoNT-catalyzed increase in fluorescence over time, initial hydrolysis rates could be obtained in 1 to 2 min when BoNT concentrations were 60 ng/ml (about 1 nM) or higher. Each BoNT cleaved its fluorigenic substrate at the same location as in the neuronal target protein, and kinetic constants indicated that the substrates were selective and efficient. The fluorigenic assay for BoNT B was used to characterize a new competitive inhibitor of BoNT B protease activity with a

K
i

value of 4 μM. In addition to real-time activity measurements, toxin concentration determinations, and kinetic studies, the BoNT substrates described herein may be directly incorporated into automated high-throughput assay systems to screen large numbers of compounds for potential antibotulinum drugs.
Plasmid-Encoded Diacetyl (Acetoin) Reductase in
Leuconostoc pseudomesenteroides
Authors: Rattray Fergal P. , Myling-Petersen Dorte , Larsen Dianna , Nilsson Dan 
Published: January 2003

ABSTRACT

A plasmid-borne diacetyl (acetoin) reductase (
butA
) from
Leuconostoc pseudomesenteroides
CHCC2114 was sequenced and cloned. Nucleotide sequence analysis revealed an open reading frame encoding a protein of 257 amino acids which had high identity at the amino acid level to diacetyl (acetoin) reductases reported previously. Downstream of the
butA
gene of
L. pseudomesenteroides
, but coding in the opposite orientation, a putative DNA recombinase was identified. A two-step PCR approach was used to construct FPR02, a
butA
mutant of the wild-type strain, CHCC2114. FPR02 had significantly reduced diacetyl (acetoin) reductase activity with NADH as coenzyme, but not with NADPH as coenzyme, suggesting the presence of another diacetyl (acetoin)-reducing activity in
L. pseudomesenteroides
. Plasmid-curing experiments demonstrated that the
butA
gene is carried on a 20-kb plasmid in
L. pseudomesenteroides
.
Effect of Raw-Milk Cheese Consumption on the Enterococcal Flora of Human Feces
Authors: Gelsomino Roberto , Vancanneyt Marc , Cogan Timothy M. , Swings Jean 
Published: January 2003

ABSTRACT

Enterococci are one of the major facultative anaerobic bacterial groups that reside in the human gastrointestinal tract. In the present study, the composition of the enterococcal fecal flora in three healthy humans was analyzed before, during, and after the daily consumption of ∼125 g of a raw-milk Cheddar-type cheese containing 3.2 × 10
4
enterococci/g of cheese. Enterococcal counts ranged between 1.4 × 10
2
and 2.5 × 10
8
CFU/g of feces and differed from subject to subject and from week to week. The cheese contained mainly
Enterococcus casseliflavus
and a small population of
Enterococcus faecalis
. Clonal relationships were determined by pulsed-field gel electrophoresis. Before and after consumption of the cheese, samples from humans contained mainly
Enterococcus faecium
, with some of the clones being resident. During consumption of the cheese, one particular transient clone of
E. faecalis
, clone Fs2, which was present in small numbers in the cheese, largely dominated the feces. Two clones of
E. casseliflavus
from the cheese were also found in the feces of one of the subjects during cheese consumption. These results suggest that a clone need not be present in a food in high numbers to establish itself in the intestine.
Evaluation of PCR Amplification Bias by Terminal Restriction Fragment Length Polymorphism Analysis of Small-Subunit rRNA and
mcrA
Genes by Using Defined Template Mixtures of Methanogenic Pure Cultures and Soil DNA Extracts
Authors: Lueders Tillmann , Friedrich Michael W. 
Published: January 2003

ABSTRACT

Terminal restriction fragment length polymorphism (T-RFLP) analysis is a widely used method for profiling microbial community structure in different habitats by targeting small-subunit (SSU) rRNA and also functional marker genes. It is not known, however, whether relative gene frequencies of individual community members are adequately represented in post-PCR amplicon frequencies as shown by T-RFLP. In this study, precisely defined artificial template mixtures containing genomic DNA of four different methanogens in various ratios were prepared for subsequent T-RFLP analysis. PCR amplicons were generated from defined mixtures targeting not only the SSU rRNA but also the methyl-coenzyme M reductase (
mcrA/mrtA
) genes of methanogens. Relative amplicon frequencies of microorganisms were quantified by comparing fluorescence intensities of characteristic terminal restriction fragments. SSU ribosomal DNA (rDNA) template ratios in defined template mixtures of the four-membered community were recovered absolutely by PCR-T-RFLP analysis, which demonstrates that the T-RFLP analysis evaluated can give a quantitative view of the template pool. SSU rDNA-targeted T-RFLP analysis of a natural community was found to be highly reproducible, independent of PCR annealing temperature, and unaffected by increasing PCR cycle numbers. Ratios of
mcrA
-targeted T-RFLP analysis were biased, most likely by PCR selection due to the degeneracy of the primers used. Consequently, for microbial community analyses, each primer system used should be evaluated carefully for possible PCR bias. In fact, such bias can be detected by using T-RFLP analysis as a tool for the precise quantification of the PCR product pool.
Molecular Identification of Ectomycorrhizal Mycelium in Soil Horizons
Authors: Landeweert Renske , Leeflang Paula , Kuyper Thom W. , Hoffland Ellis , Rosling Anna , Wernars Karel , Smit Eric 
Published: January 2003

ABSTRACT
Molecular identification techniques based on total DNA extraction provide a unique tool for identification of mycelium in soil. Using molecular identification techniques, the ectomycorrhizal (EM) fungal community under coniferous vegetation was analyzed. Soil samples were taken at different depths from four horizons of a podzol profile. A basidiomycete-specific primer pair (ITS1F-ITS4B) was used to amplify fungal internal transcribed spacer (ITS) sequences from total DNA extracts of the soil horizons. Amplified basidiomycete DNA was cloned and sequenced, and a selection of the obtained clones was analyzed phylogenetically. Based on sequence similarity, the fungal clone sequences were sorted into 25 different fungal groups, or operational taxonomic units (OTUs). Out of 25 basidiomycete OTUs, 7 OTUs showed high nucleotide homology (≥99%) with known EM fungal sequences and 16 were found exclusively in the mineral soil. The taxonomic positions of six OTUs remained unclear. OTU sequences were compared to sequences from morphotyped EM root tips collected from the same sites. Of the 25 OTUs, 10 OTUs had ≥98% sequence similarity with these EM root tip sequences. The present study demonstrates the use of molecular techniques to identify EM hyphae in various soil types. This approach differs from the conventional method of EM root tip identification and provides a novel approach to examine EM fungal communities in soil.
Bacillus anthracis
Diverges from Related Clades of the
Bacillus cereus
Group in 16S-23S Ribosomal DNA Intergenic Transcribed Spacers Containing tRNA Genes
Authors: Cherif Ameur , Borin Sara , Rizzi Aurora , Ouzari Hadda , Boudabous Abdellatif , Daffonchio Daniele 
Published: January 2003

ABSTRACT

Mung bean nuclease treatment of 16S-23S ribosomal DNA intergenic transcribed spacers (ITS) amplified from several strains of the six species of the
Bacillus cereus
group showed that
B. anthracis
Davis TE702 and
B. mycoides
G2 have other intermediate fragments in addition to the 220- and 550-bp homoduplex fragments typical of the
B. cereus
group. Long and intermediate homoduplex ITS fragments from strains Davis TE702 and G2 and from another 19 strains of the six species were sequenced. Two main types of ITS were found, either with two tRNA genes (tRNA
Ile
and tRNA
Ala
) or without any at all. Strain Davis TE702 harbors an additional ITS with a single tRNA gene, a hybrid between the tRNA
Ile
and tRNA
Ala
genes, suggesting that a recombination event rather than a deletion generated the single tDNA-containing ITS. Strain G2 showed an additional ITS of intermediate length with no tDNA and no similarity to other known sequences. Neighbor-joining analysis of tDNA-containing long ITS indicated that
B. cereus
and
B. thuringiensis
represent a single clade. Three signature sequences discriminated
B. anthracis
from
B. cereus
and
B. thuringiensis
, indicating that the anthrax agent started evolving separately from the related clades of the
B. cereus
group.
B. mycoides
and
B. weienstephanensis
were very closely related, while
B. pseudomycoides
appeared the most distant species.
mRNA Differential Display in a Microbial Enrichment Culture: Simultaneous Identification of Three Cyclohexanone Monooxygenases from Three Species
Authors: Brzostowicz Patricia C. , Walters Dana M. , Thomas Stuart M. , Nagarajan Vasantha , Rouvière Pierre E. 
Published: January 2003

ABSTRACT

mRNA differential display has been used to identify cyclohexanone oxidation genes in a mixed microbial community derived from a wastewater bioreactor. Thirteen DNA fragments randomly amplified from the total RNA of an enrichment subculture exposed to cyclohexanone corresponded to genes predicted to be involved in the degradation of cyclohexanone. Nine of these DNA fragments are part of genes encoding three distinct Baeyer-Villiger cyclohexanone monooxygenases from three different bacterial species present in the enrichment culture. In
Arthrobacter
sp. strain BP2 and
Rhodococcus
sp. strain Phi2, the monooxygenase is part of a gene cluster that includes all the genes required for the degradation of cyclohexanone, while in
Rhodococcus
sp. strain Phi1 the genes surrounding the monooxygenase are not predicted to be involved in this degradation pathway but rather seem to belong to a biosynthetic pathway. Furthermore, in the case of
Arthrobacter
strain BP2, three other genes flanking the monooxygenase were identified by differential display, demonstrating that the repeated sampling of bacterial operons shown earlier for a pure culture (D. M. Walters, R. Russ, H. Knackmuss, and P. E. Rouvière, Gene
273:
305-315, 2001) is also possible for microbial communities. The activity of the three cyclohexanone monooxygenases was confirmed and characterized following their expression in
Escherichia coli
.
Mitogen-Activated Protein Kinase in
Pfiesteria piscicida
and Its Growth Rate-Related Expression
Authors: Lin Senjie , Zhang Huan 
Published: January 2003

ABSTRACT

A full-length cDNA (1,434 bp) of mitogen-activated protein kinase (MAPK), a key molecule of a signal transduction cascade, was isolated from the estuarine heterotrophic dinoflagellate
Pfiesteria piscicida
. This cDNA (
Ppmapk1
) encoded a protein (PpMAPK1) of 428 amino acid residues that shared about 30 to 40% amino acid similarity with MAPKs in other organisms. Phylogenetic analysis indicated that PpMAPK1 was tightly clustered with MAPK3 in protozoans. Using reverse transcription-PCR, expression of this gene was evaluated for
P. piscicida
cultures grown under different conditions. While salinity shock, heat shock, starvation, and a subsequent encounter with prey did not appear to affect expression of this gene,
Ppmapk1
expression level was correlated with growth rate, suggesting involvement of this gene in the regulation of cell proliferation in the organism.
Capsid Functions of Inactivated Human Picornaviruses and Feline Calicivirus
Authors: Nuanualsuwan Suphachai , Cliver Dean O. 
Published: January 2003

ABSTRACT
The exceptional stability of enteric viruses probably resides in their capsids. The capsid functions of inactivated human picornaviruses and feline calicivirus (FCV) were determined. Viruses were inactivated by UV, hypochlorite, high temperature (72°C), and physiological temperature (37°C), all of which are pertinent to transmission via food and water. Poliovirus (PV) and hepatitis A virus (HAV) are transmissible via water and food, and FCV is the best available surrogate for the Norwalk-like viruses, which are leading causes of food-borne and waterborne disease in the United States. The capsids of all 37°C-inactivated viruses still protected the viral RNA against RNase, even in the presence of proteinase K, which contrasted with findings with viruses inactivated at 72°C. The loss of ability of the virus to attach to homologous cell receptors was universal, regardless of virus type and inactivation method, except for UV-inactivated HAV, and so virus inactivation was almost always accompanied by the loss of virus attachment. Inactivated HAV and FCV were captured by homologous antibodies. However, inactivated PV type 1 (PV-1) was not captured by homologous antibody and 37°C-inactivated PV-1 was only partially captured. The epitopes on the capsids of HAV and FCV are evidently discrete from the receptor attachment sites, unlike those of PV-1. These findings indicate that the primary target of UV, hypochlorite, and 72°C inactivation is the capsid and that the target of thermal inactivation (37°C versus 72°C) is temperature dependent.
Secretion of Active-Form
Streptoverticillium mobaraense
Transglutaminase by
Corynebacterium glutamicum
: Processing of the Pro-Transglutaminase by a Cosecreted Subtilisin-Like Protease from
Streptomyces albogriseolus
Authors: Kikuchi Yoshimi , Date Masayo , Yokoyama Kei-ichi , Umezawa Yukiko , Matsui Hiroshi 
Published: January 2003

ABSTRACT

The transglutaminase secreted by
Streptoverticillium mobaraense
is a useful enzyme in the food industry. A fragment of transglutaminase was secreted by
Corynebacterium glutamicum
when it was coupled on a plasmid to the promoter and signal peptide of a cell surface protein from
C. glutamicum
. We analyzed the signal peptide and the pro-domain of the transglutaminase gene and found that the signal peptide consists of 31 amino acid residues and the pro-domain consists of 45 residues. When the pro-domain of the transglutaminase was used, the pro-transglutaminase was secreted efficiently by
C. glutamicum
but had no enzymatic activity. However, when the plasmid carrying the
S. mobaraense
transglutaminase also encoded SAM-P45, a subtilisin-like serine protease derived from
Streptomyces albogriseolus
, the peptide bond to the C side of 41-Ser of the pro-transglutaminase was hydrolyzed, and the pro-transglutaminase was converted to an active form. Our findings suggest that
C. glutamicum
has potential as a host for industrial-scale protein production.
A Labeling Study To Elucidate the Biosynthesis of 4-(4-Hydroxyphenyl)-Butan-2-one (Raspberry Ketone) by
Nidula niveo-tomentosa
Authors: Zorn H. , Fischer-Zorn M. , Berger R. G. 
Published: January 2003

ABSTRACT

Submerged cells of the basidiomycete
Nidula niveo-tomentosa
, a microbial producer of 4-(4-hydroxyphenyl)-butan-2-one, were supplemented with
13
C-labeled
l
-phenylalanines and with [1-
13
C]glucose. Labeled transformation products were detected by a novel method of analyzing stable isotope-labeled metabolites, gas chromatography (GC) coupled to an atomic emission detector, and by GC-mass spectrometry. A benzoate moiety was side chain elongated according to the poly-β-keto scheme. The presence of an acetyl coenzyme A-carboxylase inhibitor shifted the spectrum of products to benzyl compounds. Hence, the fungal pathway differs from the one established for plant tissues.
Enhanced Heterologous Expression of Two
Streptomyces griseolus
Cytochrome P450s and
Streptomyces coelicolor
Ferredoxin Reductase as Potentially Efficient Hydroxylation Catalysts
Authors: Hussain Haitham A. , Ward John M. 
Published: January 2003

ABSTRACT

The herbicide-inducible, soluble cytochrome P450s CYP105A1 and CYP105B1 and their adjacent ferredoxins, Fd1 and Fd2, of
Streptomyces griseolus
were expressed in
Escherichia coli
to high levels. Conditions for high-level expression of active enzyme able to catalyze hydroxylation have been developed. Analysis of the expression levels of the P450 proteins in several different
E. coli
expression hosts identified
E. coli
BL21 Star(DE3)pLysS as the optimal host cell to express CYP105B1 as judged by CO difference spectra. Examination of the codons used in the CYP1051A1 sequence indicated that it contains a number of codons corresponding to rare
E. coli
tRNA species. The level of its expression was improved in the modified forms of
E. coli
BL21(DE3), which contain extra copies of rare codon
E. coli
tRNA genes. The activity of correctly folded cytochrome P450s was further enhanced by cloning a ferredoxin reductase from
Streptomyces coelicolor
downstream of CYP105A1 and CYP105B1 and their adjacent ferredoxins. Expression of CYP105A1 and CYP105B1 was also achieved in
Streptomyces lividans
1326 by cloning the P450 genes and their ferredoxins into the expression vector pBW160.
S. lividans
1326 cells containing CYP105A1 or CYP105B1 were able efficiently to dealkylate 7-ethoxycoumarin.