bluuebunny/crossref_metadata_2025_split huggingface dataset:
Genetic Variation in
Fusarium
Section
Liseola
from No-Till Maize in Argentina
Authors: Chulze S. N. , Ramirez M. L. , Torres A. , Leslie J. F. 
Published: July 2002

ABSTRACT

Strains of
Fusarium
species belonging to section
Liseola
cause stalk and ear rot of maize and produce important mycotoxins, such as fumonisins. We isolated two species,
Fusarium verticillioides
(
Gibberella fujikuroi
mating population A) and
Fusarium proliferatum
(
G. fujikuroi
mating population D) from maize cultivated under no-till conditions at five locations in the Córdoba province of Argentina. We determined the effective population number for mating population A (

N
e

) and found that the

N
e

for mating type was 89% of the count (total population) and that the

N
e

for male or hermaphrodite status was 36%. Thus, the number of strains that can function as the female parent limits

N
e

, and sexual reproduction needs to occur only once every 54 to 220 asexual generations to maintain this level of sexual fertility. Our results indicate that the fungal populations isolated from no-till maize are similar to those recovered from maize managed with conventional tillage. We placed 36 strains from mating population A into 28 vegetative compatibility groups (VCGs). Of the 13 strains belonging to five multimember VCGs, only 2 isolates belonging to one VCG were clones based on amplified fragment length polymorphism (AFLP) fingerprints. Members of the other four multimember VCGs had an average similarity index of 0.89, and members of one VCG were no more closely related to other members of the same VCG than they were to other members of the population as a whole. This finding suggests that the common assumption that strains in the same VCG are either clonal or very closely related needs to be examined in more detail. The variability observed with AFLPs and VCGs suggests that sexual reproduction may occur more frequently than estimated by

N
e

.
Purification and Molecular Characterization of a Tripeptidase (PepT) from
Lactobacillus helveticus
Authors: Savijoki Kirsi , Palva Airi 
Published: July 2002

ABSTRACT

A tripeptidase (PepT) from a thermophilic dairy starter strain of
Lactobacillus helveticus
was purified by four chromatographic steps. PepT appeared to be a trimeric metallopeptidase with a molecular mass of 150 kDa. PepT exhibited maximum activity against hydrophobic tripeptides, with the highest activity for Met-Gly-Gly (

K
m

, 2.6 mM;
V
max
, 80.2 μmol · min
−1
· μg
−1
). Some of the hydrophobic dipeptides were slowly hydrolyzed, distinguishing the
Lactobacillus
PepT from its counterpart in mesophilic
Lactococcus lactis
. No activity against tetrapeptides or amino acid
p
-nitroanilide derivatives was observed. The
pepT
gene and its flanking regions were isolated by PCR and sequenced by cyclic sequencing. The sequence analyses revealed open reading frames (ORFs) 816 bp (ORF1) and 1,239 bp (ORF2) long. ORF2 encoded a 47-kDa PepT protein which exhibited 53% identity with the PepT from
L. lactis
. The mRNA analyses indicated that
pepT
conforms a novel operon structure with an ORF1 located upstream. Several putative −35/−10 regions preceded the operon, but only one transcription start site located downstream of the first putative −10 region was identified. An inverted repeat structure with Δ
G
of −64.8 kJ/mol was found downstream of the PepT-encoding region.
Detection of DNA Damage in Prokaryotes by Terminal Deoxyribonucleotide Transferase-Mediated dUTP Nick End Labeling
Authors: Rohwer Forest , Azam Farooq 
Published: July 2002

ABSTRACT

Numerous agents can damage the DNA of prokaryotes in the environment (e.g., reactive oxygen species, irradiation, and secondary metabolites such as antibiotics, enzymes, starvation, etc.). The large number of potential DNA-damaging agents, as well as their diverse modes of action, precludes a simple test of DNA damage based on detection of nucleic acid breakdown products. In this study, free 3′-OH DNA ends, produced by either direct damage or excision DNA repair, were used to assess DNA damage. Terminal deoxyribonucleotide transferase (TdT)-mediated dUTP nick end labeling (TUNEL) is a procedure in which 3′-OH DNA ends are enzymatically labeled with dUTP-fluorescein isothiocyanate using TdT. Cells labeled by this method can be detected using fluorescence microscopy or flow cytometry. TUNEL was used to measure hydrogen peroxide-induced DNA damage in the archaeon
Haloferax volcanii
and the bacterium
Escherichia coli
. DNA repair systems were implicated in the hydrogen peroxide-dependent generation of 3′-OH DNA ends by the finding that the protein synthesis inhibitors chloramphenicol and diphtheria toxin blocked TUNEL labeling of
E. coli
and
H. volcanii
, respectively. DNA damage induced by UV light and bacteriophage infection was also measured using TUNEL. This methodology should be useful in applications where DNA damage and repair are of interest, including mutant screening and monitoring of DNA damage in the environment.
Gene Cloning and Nucleotide Sequencing and Properties of a Cocaine Esterase fromRhodococcussp. Strain MB1
Authors: Bresler Matthew M. , Rosser Susan J. , Basran Amrik , Bruce Neil C. 
Published: July 2002

ABSTRACTA strain ofRhodococcusdesignated MB1, which was capable of utilizing cocaine as a sole source of carbon and nitrogen for growth, was isolated from rhizosphere soil of the tropane alkaloid-producing plantErythroxylum coca. A cocaine esterase was found to initiate degradation of cocaine, which was hydrolyzed to ecgonine methyl ester and benzoate; both of these esterolytic products were further metabolized byRhodococcussp. strain MB1. The structural gene encoding a cocaine esterase, designatedcocE, was cloned fromRhodococcussp. strain MB1 genomic libraries by screening recombinant strains ofRhodococcus erythropolisCW25 for growth on cocaine. The nucleotide sequence ofcocEcorresponded to an open reading frame of 1,724 bp that codes for a protein of 574 amino acids. The amino acid sequence of cocaine esterase has a region of similarity with the active serine consensus of X-prolyl dipeptidyl aminopeptidases, suggesting that the cocaine esterase is a serine esterase. ThecocEcoding sequence was subcloned into the pCFX1 expression plasmid and expressed inEscherichia coli. The recombinant cocaine esterase was purified to apparent homogeneity and was found to be monomeric, with anMrof approximately 65,000. The apparentKmof the enzyme (mean ± standard deviation) for cocaine was measured as 1.33 ± 0.085 mM. These findings are of potential use in the development of a linked assay for the detection of illicit cocaine.
Relationships between Microbial Community Structure and Hydrochemistry in a Landfill Leachate-Polluted Aquifer
Authors: Röling Wilfred F. M. , van Breukelen Boris M. , Braster Martin , Lin Bin , van Verseveld Henk W. 
Published: July 2002

ABSTRACT

Knowledge about the relationship between microbial community structure and hydrogeochemistry (e.g., pollution, redox and degradation processes) in landfill leachate-polluted aquifers is required to develop tools for predicting and monitoring natural attenuation. In this study analyses of pollutant and redox chemistry were conducted in parallel with culture-independent profiling of microbial communities present in a well-defined aquifer (Banisveld, The Netherlands). Degradation of organic contaminants occurred under iron-reducing conditions in the plume of pollution, while upstream of the landfill and above the plume denitrification was the dominant redox process. Beneath the plume iron reduction occurred. Numerical comparison of 16S ribosomal DNA (rDNA)-based denaturing gradient gel electrophoresis (DGGE) profiles of
Bacteria
and
Archaea
in 29 groundwater samples revealed a clear difference between the microbial community structures inside and outside the contaminant plume. A similar relationship was not evident in sediment samples. DGGE data were supported by sequencing cloned 16S rDNA. Upstream of the landfill members of the β subclass of the class
Proteobacteria
(β-proteobacteria) dominated. This group was not encountered beneath the landfill, where gram-positive bacteria dominated. Further downstream the contribution of gram-positive bacteria to the clone library decreased, while the contribution of δ-proteobacteria strongly increased and β-proteobacteria reappeared. The β-proteobacteria (
Acidovorax
,
Rhodoferax
) differed considerably from those found upstream (
Gallionella
,
Azoarcus
). Direct comparisons of cloned 16S rDNA with bands in DGGE profiles revealed that the data from each analysis were comparable. A relationship was observed between the dominant redox processes and the bacteria identified. In the iron-reducing plume members of the family
Geobacteraceae
made a strong contribution to the microbial communities. Because the only known aromatic hydrocarbon-degrading, iron-reducing bacteria are
Geobacter
spp., their occurrence in landfill leachate-contaminated aquifers deserves more detailed consideration.
Identification by Subtractive Hybridization of Sequences Specific for
Salmonella enterica
Serovar Enteritidis
Authors: Agron Peter G. , Walker Richard L. , Kinde Hailu , Sawyer Sherilyn J. , Hayes Dawn C. , Wollard Jessica , Andersen Gary L. 
Published: July 2002

ABSTRACT

Salmonella enterica
serovar Enteritidis, a major cause of food poisoning, can be transmitted to humans through intact chicken eggs when the contents have not been thoroughly cooked. Infection in chickens is asymptomatic; therefore, simple, sensitive, and specific detection methods are crucial for efforts to limit human exposure. Suppression subtractive hybridization was used to isolate DNA restriction fragments present in
Salmonella
serovar Enteritidis but absent in other bacteria found in poultry environments. Oligonucleotide primers to candidate regions were used in polymerase chain reactions to test 73 non-Enteritidis
S. enterica
isolates comprising 34 different serovars, including Dublin and Pullorum, two very close relatives of Enteritidis. A primer pair to one
Salmonella
difference fragment (termed Sdf I) clearly distinguished serovar Enteritidis from all other serovars tested, while two other primer pairs only identified a few non-Enteritidis strains. These primer pairs were also useful for the detection of a diverse collection of clinical and environmental
Salmonella
serovar Enteritidis isolates. In addition, five bacterial genera commonly found with
Salmonella
serovar Enteritidis were not detected. By treating total DNA with an exonuclease that degrades sheared chromosomal DNA but not intact circular plasmid DNA, it was shown that Sdf I is located on the chromosome. The Sdf I primers were used to screen a
Salmonella
serovar Enteritidis genomic library and a unique 4,060-bp region was defined. These results provide a basis for developing a rapid, sensitive, and highly specific detection system for
Salmonella
serovar Enteritidis and provide sequence information that may be relevant to the unique characteristics of this serovar.
Identification of Bacteriocin-Like Inhibitors from Rumen
Streptococcus
spp. and Isolation and Characterization of Bovicin 255
Authors: Whitford M. F. , McPherson M. A. , Forster R. J. , Teather R. M. 
Published: July 2002

ABSTRACT

Streptococci obtained from rumen sources were tested for the production of antibacterial compounds using a deferred-antagonism plating assay. Of 35 isolates tested, 7 were identified that inhibited the growth of other streptococci. None of the inhibitory activity was due to bacteriophage. Three isolates, LRC0253, LRC0255, and LRC0476, were selected for further characterization. Analysis of 16S ribosomal DNA indicated that LRC0476 was a strain of
Streptococcus bovis
, while isolates LRC0253 and LRC0255 are likely strains of
Streptococcus gallolyticus
. The antibacterial compounds produced by these bacteria were protease sensitive, remained active in a pH range from 1 to 12, and did not lose activity after heating at 100°C for 15 min. The inhibitory peptide from strain LRC0255 was purified using pH-dependent adsorption and desorption to bacterial cells, followed by ammonium sulfate precipitation and reversed-phase chromatography and gel filtration. The peptide was 6 kDa, as determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. An oligonucleotide probe based on the N-terminal sequence of the purified peptide was used to identify the gene encoding the inhibitory peptide. The antibacterial peptide has characteristics that are very similar to those described for class II bacteriocins of gram-positive bacteria.
Clostridium beijerinckii
and
Clostridium difficile
Detoxify Methylglyoxal by a Novel Mechanism Involving Glycerol Dehydrogenase
Authors: Liyanage Hemachandra , Kashket Shelby , Young Michael , Kashket Eva R. 
Published: July 2002

ABSTRACT

In contrast to gram-negative bacteria, little is known about the mechanisms by which gram-positive bacteria degrade the toxic metabolic intermediate methylglyoxal (MG).
Clostridium beijerinckii
BR54, a Tn
1545
insertion mutant of the NCIMB 8052 strain, formed cultures that contained significantly more (free) MG than wild-type cultures. Moreover, BR54 was more sensitive to growth inhibition by added MG than the wild type, suggesting that it has a reduced ability to degrade MG. The single copy of Tn
1545
in this strain lies just downstream from
gldA
, encoding glycerol dehydrogenase. As a result of antisense RNA production, cell extracts of BR54 possess significantly less glycerol dehydrogenase activity than wild-type cell extracts (H. Liyanage, M. Young, and E. R. Kashket, J. Mol. Microbiol. Biotechnol. 2:87–93, 2000). Inactivation of
gldA
in both
C. beijerinckii
and
Clostridium difficile
gave rise to pinpoint colonies that could not be subcultured, indicating that glycerol dehydrogenase performs an essential function in both organisms. We propose that this role is detoxification of MG. To our knowledge, this is the first report of targeted gene disruption in the
C. difficile
chromosome.
Detection of Acute Bee Paralysis Virus and Black Queen Cell Virus from Honeybees by Reverse Transcriptase PCR
Authors: Benjeddou Mongi , Leat Neil , Allsopp Mike , Davison Sean 
Published: July 2002

ABSTRACT
A reverse transcriptase PCR (RT-PCR) assay was developed for the detection of acute bee paralysis virus (ABPV) and black queen cell virus (BQCV), two honeybee viruses. Complete genome sequences were used to design unique PCR primers within a 1-kb region from the 3′ end of both genomes to amplify a fragment of 900 bp from ABPV and 700 bp from BQCV. The combined guanidinium thiocyanate and silica membrane method was used to extract total RNA from samples of healthy and laboratory-infected bee pupae. In a blind test, RT-PCR successfully identified the samples containing ABPV and BQCV. Sensitivities were approximately 1,600 genome equivalents of purified ABPV and 130 genome equivalents of BQCV.
Genetic Diversity of
Nostoc
Symbionts Endophytically Associated with Two Bryophyte Species
Authors: Costa José-Luis , Paulsrud Per , Rikkinen Jouko , Lindblad Peter 
Published: July 2002

ABSTRACT

The diversity of the endophytic
Nostoc
symbionts of two thalloid bryophytes, the hornwort
Anthoceros fusiformis
and the liverwort
Blasia pusilla
, was examined using the tRNA
Leu
(UAA) intron sequence as a marker. The results confirmed that many different
Nostoc
strains are involved in both associations under natural conditions in the field. The level of
Nostoc
diversity within individual bryophyte thalli varied, but single DNA fragments were consistently amplified from individual symbiotic colonies. Some
Nostoc
strains were widespread and were detected from thalli collected from different field sites and different years. These findings indicate a moderate level of spatial and temporal continuity in bryophyte-
Nostoc
symbioses.
Surface Modifications Created by Using Engineered Hydrophobins
Authors: Scholtmeijer Karin , Janssen Meike I. , Gerssen Bertus , de Vocht Marcel L. , van Leeuwen Babs M. , van Kooten Theo G. , Wösten Han A. B. , Wessels Joseph G. H. 
Published: July 2002

ABSTRACT

Hydrophobins are small (ca. 100 amino acids) secreted fungal proteins that are characterized by the presence of eight conserved cysteine residues and by a typical hydropathy pattern. Class I hydrophobins self-assemble at hydrophilic-hydrophobic interfaces into highly insoluble amphipathic membranes, thereby changing the nature of surfaces. Hydrophobic surfaces become hydrophilic, while hydrophilic surfaces become hydrophobic. To see whether surface properties of assembled hydrophobins can be changed, 25
N
-terminal residues of the mature SC3 hydrophobin were deleted (TrSC3). In addition, the cell-binding domain of fibronectin (RGD) was fused to the N terminus of mature SC3 (RGD-SC3) and TrSC3 (RGD-TrSC3). Self-assembly and surface activity were not affected by these modifications. However, physiochemical properties at the hydrophilic side of the assembled hydrophobin did change. This was demonstrated by a change in wettability and by enhanced growth of fibroblasts on Teflon-coated with RGD-SC3, TrSC3, or RGD-TrSC3 compared to bare Teflon or Teflon coated with SC3. Thus, engineered hydrophobins can be used to functionalize surfaces.
Engineering of Phytase for Improved Activity at Low pH
Authors: Tomschy Andrea , Brugger Roland , Lehmann Martin , Svendsen Allan , Vogel Kurt , Kostrewa Dirk , Lassen Søren F. , Burger Dominique , Kronenberger Alexandra , van Loon Adolphus P. G. M. , Pasamontes Luis , Wyss Markus 
Published: July 2002

ABSTRACTFor industrial applications in animal feed, a phytase of interest must be optimally active in the pH range prevalent in the digestive tract. Therefore, the present investigation describes approaches to rationally engineer the pH activity profiles ofAspergillus fumigatusand consensus phytases. Decreasing the negative surface charge of theA. fumigatusQ27L phytase mutant by glycinamidylation of the surface carboxy groups (of Asp and Glu residues) lowered the pH optimum by ca. 0.5 unit but also resulted in 70 to 75% inactivation of the enzyme. Alternatively, detailed inspection of amino acid sequence alignments and of experimentally determined or homology modeled three-dimensional structures led to the identification of active-site amino acids that were considered to correlate with the activity maxima at low pH ofA. nigerNRRL 3135 phytase,A. nigerpH 2.5 acid phosphatase, andPeniophora lyciiphytase. Site-directed mutagenesis confirmed that, inA. fumigatuswild-type phytase, replacement of Gly-277 and Tyr-282 with the corresponding residues ofA. nigerphytase (Lys and His, respectively) gives rise to a second pH optimum at 2.8 to 3.4. In addition, the K68A single mutation (in bothA. fumigatusand consensus phytase backbones), as well as the S140Y D141G double mutation (inA. fumigatusphytase backbones), decreased the pH optima with phytic acid as substrate by 0.5 to 1.0 unit, with either no change or even a slight increase in maximum specific activity. These findings significantly extend our tools for rationally designing an optimal phytase for a given purpose.
Ethyleneglycol-Bis-(β-Aminoethylether)Tetraacetate as a Blood Anticoagulant: Preservation of Antigen-Presenting Cell Function and Antigen-Specific Proliferative Response of Peripheral Blood Mononuclear Cells from Stored Blood
Authors: Kumar Priti , Satchidanandam Vijaya 
Published: July 2002

ABSTRACTWe examined the progressive and irreversible loss of antigen-specific lymphoproliferative responses in peripheral blood mononuclear cells (PBMC) obtained from blood exposed for prolonged periods to EDTA as an anticoagulant. The responses of these lymphocytes to interleukin-2 or to concanavalin A were, however, unaffected. The observed loss was not due to depletion of metal ions by EDTA, since the addition of several divalent cations to whole blood during storage in EDTA or to lymphocytes from EDTA-stored blood during antigen stimulation in vitro did not alleviate the defect. Reconstitution of antigen-specific T-cell lines or Percoll-purified T cells with adherent antigen-presenting cells in antigen stimulation assays revealed that the presenting cells and not the effector T-cells were the targets of EDTA-mediated damage. The anticoagulant heparin helped to circumvent this problem. Surprisingly, EGTA, another metal ion chelator, could successfully replace EDTA, with a marginal loss in antigen-specific responses. Lymphoproliferative responses to antigens of Japanese encephalitis virus (JEV) andMycobacterium tuberculosiswere both significantly preserved in EGTA. JEV antigen-specific responses of PBMC obtained from the blood of convalescent JEV patients and stored in EGTA for as long as 24 h (n= 20) were comparable to those of fresh PBMC (n= 10), while PBMC from blood stored in EDTA (n= 17) for 16 h or longer failed to respond. We recommend that EGTA be used as the anticoagulant of choice for applications that require the lymphocyte proliferation assay, especially when on-site testing facilities are not available.
Competitive Enzyme-Linked Immunosorbent Assay for Detection of
Leptospira interrogans
Serovar pomona Antibodies in Bovine Sera
Authors: Surujballi Om , Mallory Maria 
Published: July 2002

ABSTRACT

A competitive enzyme-linked immunosorbent assay (ELISA) using a specific monoclonal antibody (M898) was developed for detection of bovine antibodies to
Leptospira interrogans
serovar pomona. This assay was evaluated using field sera (
n
= 190) with serovar pomona microscopic agglutination test (MAT) titers of ≥100 as the positive population (group A); field sera (
n
= 1,445) which were negative in the MAT (1:100 dilution) for serovar pomona (group B); and sera (from a specific-pathogen-free cattle herd [
n
= 210]) which were negative in the MAT (1:100 dilution) for serovars canicola, copenhageni, grippotyphosa, hardjo, pomona, and sejroe (group C). At the cutoff point recommended by receiver operating characteristic (ROC) curve analysis of the combined ELISA results of serum groups A, B, and C, the sensitivity and specificity values were 93.7 and 96.3%, respectively. The value for the area under this ROC curve was 0.977, indicating a high level of accuracy for the ELISA. Similar results were obtained from the analysis of the combined results of serum groups A and B and from the analysis of the combined results of serum groups A and C.
Association of
Lactobacillus
spp. with Peyer's Patches in Mice
Authors: Plant Laura , Conway Patricia 
Published: July 2002

ABSTRACT

Sixteen strains of
Lactobacillus
isolated from humans, mice, and food products were screened for their capacity to associate with Peyer's patches in mice. In preliminary experiments, in vitro binding to tissue pieces was assessed by scanning electron microscopy, and it was demonstrated qualitatively that 5 of the 16 strains showed some affinity for the Peyer's patches, irrespective of their association with the nonlymphoid intestinal tissue.
Lactobacillus fermentum
KLD was selected for further study, since, in addition to its intrinsically high adhesion rate, this organism was found to exhibit a preferential binding to the follicle-associated epithelium of the Peyer's patches compared with its level of binding to the mucus-secreting regions of the small intestine. Quantitative assessment of scanning electron micrographs of tissue sections which had been incubated with
L. fermentum
KLD or a nonbinding control strain,
Lactobacillus delbruckii
subsp.
bulgaricus
, supported these observations, since a marked difference in adhesion was noted (
P
< 0.05). This preferential association of strain KLD with the Peyer's patches was also confirmed with radiolabeled lactobacilli incubated with intestinal tissue in the in vitro adhesion assay. Direct recovery of
L. fermentum
KLD from washed tissue following oral dosing of mice revealed a distinct association (
P
< 0.05) between this organism and the Peyer's patch tissue. In contrast,
L. delbruckii
subsp.
bulgaricus
showed negligible binding to both tissue types in both in vitro and in vivo adhesion assays. It was concluded that
L. fermentum
KLD bound preferentially to Peyer's patches of BALB/c mice.