bluuebunny/crossref_metadata_2025_split huggingface dataset:
Characterization of viability, mitochondrial activity, acrosomal integrity and capacitation status in boar sperm during in vitro storage at different ambient temperatures
Authors: Huo Li-Jun , Yue Kui-Zhong , Yang Zeng-Ming 
Published: March 2003

Extended storage of unfrozen boar semen becomes an alternative because the use of frozen–thawed boar sperm results in low fertility. Sperm viability, mitochondrial activity, capacitation and acrosome integrity of freshly ejaculated boar semen stored in vitro for up to 48 h at 4°C, 15°C, 20°C and 39°C was characterized during the study. The viability of boar sperm was assessed by both Hoechst 33258 and SYBR-14/PI staining. Mitochondrial function was assessed by JC-1 staining. Capacitation status was determined by chlortetracycline (CTC)/Hoechst 33258 staining. The acrosome integrity was analysed with Coomassie blue staining. These data were derived from three ejaculates each from three crossbred boars. The viabilities assessed with SYBR-14/PI, Hoechst 33258 and JC-1 staining correlated highly (r > 0.980). In freshly ejaculated boar semen, 96 ± 1% of the sperm did not take up the Hoechst 33258, whereas 95 ± 2% were stained by SYBR-14 and 96 ± 2% of the sperm had mitochondria exhibiting positive JC-1 staining. Staining with CTC/Hoechst 33258 suggested that a high percentage of sperm became capacitated after 24 h storage at 15°C and 20°C. There were 62 ± 2% (15°C) and 89�±�2% (20°C) capacitated sperm by 48 h. Moreover, most of the capacitated sperm were acrosome intact. These results suggest that SYBR-14/PI, Hoechst 33258 or JC-1 staining can be used to effectively evaluate the quality of boar sperm during in vitro storage.
A field study on persistence and mobility of metsulfuron-methyl in three tropical agricultural soils
Authors: Ismail B. S. , Tet-Vun Chong 
Published: March 2003

Studies on the persistence and mobility of metsulfuron-methyl in 3 soil types (Lating, Sungai Buluh, and Serdang Series) were conducted under field conditions. Two weeks after treatment (WAT), metsulfuron-methyl applied at 30 g/ha leached downward to the lowest depth of the soil profile of 30–40 cm in the Sungai Buluh and Serdang soils, but not in the Lating soil. At the recommended treatment rate (15 g/ha), no metsulfuron-methyl residue was detected in the lowest depth for the Lating series soil studied. At 1 WAT, a high percentage of the metsulfuron-methyl residue had accumulated in the top layer of the Lating soil compared with the other 2 soil types studied. The residue in the upper layer decreased as the period from application increased. The dissipation rate of metsulfuron-methyl was greater at the recommended rate than at double the recommended rate. The half-life of metsulfuron-methyl, at the application rate of 30 g/ha, was shorter in the Lating (approximately 8.2 days) than in the Sungai Buluh and Serdang soils (13.4 and 13.4�days, respectively) for the whole profile. However, the half-life of metsulfuron-methyl when applied at the recommended rate was shorter than when applied at double the recommended rate.
Microbial Populations Stimulated for Hexavalent Uranium Reduction in Uranium Mine Sediment
Authors: Suzuki Yohey , Kelly Shelly D. , Kemner Kenneth M. , Banfield Jillian F. 
Published: March 2003

ABSTRACTUranium-contaminated sediment and water collected from an inactive uranium mine were incubated anaerobically with organic substrates. Stimulated microbial populations removed U almost entirely from solution within 1 month. X-ray absorption near-edge structure analysis showed that U(VI) was reduced to U(IV) during the incubation. Observations by transmission electron microscopy, selected area diffraction pattern analysis, and energy-dispersive X-ray spectroscopic analysis showed two distinct types of prokaryotic cells that precipitated only a U(IV) mineral uraninite (UO2) or both uraninite and metal sulfides. Prokaryotic cells associated with uraninite and metal sulfides were inferred to be sulfate-reducing bacteria. Phylogenetic analysis of 16S ribosomal DNA obtained from the original and incubated sediments revealed that microbial populations were changed from microaerophilicProteobacteriato anaerobic low-G+C gram-positive sporeforming bacteria by the incubation. Forty-two out of 94 clones from the incubated sediment were related to sulfate-reducingDesulfosporosinusspp., and 23 were related to fermentativeClostridiumspp. The results suggest that, if in situ bioremediation were attempted in the uranium mine ponds,Desulfosporosinusspp. would be a major contributor to U(VI) and sulfate reduction andClostridiumspp. to U(VI) reduction.
Biotransformation of Hexahydro-1,3,5-Trinitro-1,3,5-Triazine (RDX) by a Rabbit Liver Cytochrome P450: Insight into the Mechanism of RDX Biodegradation by
Rhodococcus
sp. Strain DN22
Authors: Bhushan Bharat , Trott Sandra , Spain Jim C. , Halasz Annamaria , Paquet Louise , Hawari Jalal 
Published: March 2003

ABSTRACT

A unique metabolite with a molecular mass of 119 Da (C
2
H
5
N
3
O
3
) accumulated during biotransformation of hexahydro-1,3,5-trinitro-1,3,5-triazine (RDX) by
Rhodococcus
sp. strain DN22 (D. Fournier, A. Halasz, J. C. Spain, P. Fiurasek, and J. Hawari, Appl. Environ. Microbiol. 68:166-172, 2002). The structure of the molecule and the reactions that led to its synthesis were not known. In the present study, we produced and purified the unknown metabolite by biotransformation of RDX with
Rhodococcus
sp. strain DN22 and identified the molecule as 4-nitro-2,4-diazabutanal using nuclear magnetic resonance and elemental analyses. Furthermore, we tested the hypothesis that a cytochrome P450 enzyme was responsible for RDX biotransformation by strain DN22. A cytochrome P450 2B4 from rabbit liver catalyzed a very similar biotransformation of RDX to 4-nitro-2,4-diazabutanal. Both the cytochrome P450 2B4 and intact cells of
Rhodococcus
sp. strain DN22 catalyzed the release of two nitrite ions from each reacted RDX molecule. A comparative study of cytochrome P450 2B4 and
Rhodococcus
sp. strain DN22 revealed substantial similarities in the product distribution and inhibition by cytochrome P450 inhibitors. The experimental evidence led us to propose that cytochrome P450 2B4 can catalyze two single electron transfers to RDX, thereby causing double denitration, which leads to spontaneous hydrolytic ring cleavage and decomposition to produce 4-nitro-2,4-diazabutanal. Our results provide strong evidence that a cytochrome P450 enzyme is the key enzyme responsible for RDX biotransformation by
Rhodococcus
sp. strain DN22.
Purification and Characterization of Brochocin A and Brochocin B(10-43), a Functional Fragment Generated by Heterologous Expression in
Carnobacterium piscicola
Authors: Garneau Sylvie , Ference Claire A. , van Belkum Marco J. , Stiles Michael E. , Vederas John C. 
Published: March 2003

ABSTRACT

Brochothrix campestris
ATCC 43754 produces a heat-stable, two-component, nonlantibiotic, class IIb bacteriocin, brochocin C (BrcC), that is active against a broad range of gram-positive bacteria, including spores of
Clostridium botulinum
. An improved purification method was developed for BrcC, in which
n
-butanol and chloroform extraction are used. Mass spectral characterization of the two components, brochocin A (BrcA) and brochocin B (BrcB), showed that both components are excreted into the medium by
B. campestris
as mature peptides consisting of 59 and 43 amino acids, respectively. Separate expression clones of BrcA and BrcB were constructed previously in
Carnobacterium piscicola
LV17C, but the products were not chemically characterized. Purification by the new protocol showed that BrcA is expressed as the mature 59-amino-acid peptide but that BrcB is produced by
C. piscicola
as a fragment, BrcB(10-43), which is cleaved at an internal Gly-Gly site. This fragment is not antimicrobial by itself, but in combination with BrcA it displays the full activity of the BrcC complex. Circular dichroism measurements revealed a high β-sheet content in the secondary structure of both BrcA and BrcB(10-43), as well as in a 1:1 BrcA-BrcB(10-43) mixture. Separate expression clones of
brcA
and
brcB
were also constructed in
Escherichia coli
, but these clones only produced multiple fragments of the desired peptides with little or no activity.
Community Structure of Ammonia-Oxidizing Bacteria within Anoxic Marine Sediments
Authors: Freitag Thomas E. , Prosser James I. 
Published: March 2003

ABSTRACT

The potential for oxidation of ammonia in anoxic marine sediments exists through anaerobic oxidation by
Nitrosomonas
-like organisms, utilizing nitrogen dioxide, coupling of nitrification, manganese reduction, and anaerobic oxidation of ammonium by planctomycetes (the Anammox process). Here we describe the presence of microbial communities with the potential to carry out these processes in a natural marine sediment system (Loch Duich, Scotland). Natural microbial communities of
Planctomycetales-Verrucomicrobia
and β- and γ-proteobacterial ammonia-oxidizing bacteria were characterized by analysis of 16S rRNA genes amplified using group-specific primers by PCR- and reverse transcription-PCR amplification of 16S rDNA and RNA, respectively. Amplification products were analyzed by sequencing of clones and by denaturant gradient gel electrophoresis (DGGE). Amplification of primers specific for
Planctomycetales-Verrucomicrobia
and β-proteobacterial ammonia-oxidizing bacteria generated products at all sampling sites and depths, but no product was generated using primers specific for γ-proteobacterial ammonia-oxidizing bacteria. 16S rDNA DGGE banding patterns indicated complex communities of β-proteobacterial ammonia-oxidizing bacteria in anoxic marine sediments. Phylogenetic analysis of sequences from clones and those excised from DGGE gels suggests dominance of
Nitrosospira
cluster 1-like organisms and of strains belonging to a novel cluster represented in dominant bands in 16S rRNA DGGE banding patterns. Their presence indicates a group of organisms closely related to recognized β-proteobacterial ammonia-oxidizing bacteria that may be selected in anoxic environments and may be capable of anoxic ammonia oxidation. Sequence analysis of planctomycete clone libraries and sequences excised from DGGE gels also demonstrated a diverse microbial community and suggested the presence of new subdivisions, but no sequence related to recognized Anammox organisms was detected.
Expression of Chlorocatechol 1,2-Dioxygenase and Chlorocatechol 2,3-Dioxygenase Genes in Chlorobenzene-Contaminated Subsurface Samples
Authors: Alfreider Albin , Vogt Carsten , Babel Wolfgang 
Published: March 2003

ABSTRACT

In order to evaluate the in situ degradative capabilities of microorganisms in an underground reactor facility housing two flowthrough columns filled with aquifer soil, we examined the distribution and phylogeny of gene transcripts encoding enzymes capable of catalyzing the cleavage of the chlorinated aromatic ring during transformation of the main pollutant, chlorobenzene. Initial biostimulation of the autochthonous bacteria in the originally anaerobic reactor columns was achieved by injecting nitrate and oxygen in the form of H
2
O
2
. Two broad-range primer pairs were used for reverse transcriptase PCR (RT-PCR) of partial subunit genes of chlorocatechol 1,2-dioxygenase and catechol 2,3-dioxygenase from RNA directly extracted from different groundwater and aquifer samples. Samples retrieved from the lowermost sections of the reactor columns, which were operated in upflow mode, were positive for the presence of chlorocatechol 1,2-dioxygenase and catechol 2,3-dioxygenase mRNA. On the other hand, chlorocatechol 1,2-dioxygenase RT-PCR products were detected in a larger part of each reactor column, up to a zone 5.5 m above the bottom. Phylogenetic analyses of these chlorocatechol 1,2-dioxygenase sequences clearly separated them into two main clusters, one of which was closely affiliated with the broad-spectrum chlorocatechol 1,2-dioxygenase from
Pseudomonas chlororaphis
RW71. Analysis of sequences obtained from RT-PCR products amplified with catechol 2,3-dioxygenase primers revealed that their closest relative was the chlorocatechol 2,3-dioxygenase gene
cbzE
from
Pseudomonas putida
GJ31 (A. E. Mars, J. Kingma, S. R. Kaschabek, W. Reineke, and D. B. Janssen, J. Bacteriol. 181:1309-1318, 1999), with sequence similarities between 97.8 and 99.0%.
New Thermosensitive Delivery Vector and Its Use To Enable Nisin-Controlled Gene Expression in
Lactobacillus gasseri
Authors: Neu T. , Henrich B. 
Published: March 2003

ABSTRACT

Derivatives of a cryptic plasmid from
Lactobacillus curvatus
showed temperature-sensitive replication in thermophilic lactobacilli. The thermosensitive replicon was used to construct the new delivery vector pTN1, which allows site-specific replacement of chromosomal DNA sequences. pTN1 carries an erythromycin resistance marker suitable for selection of single-copy integrants and replicates readily at 35°C, whereas replication is efficiently shut down at 42°C. To demonstrate the functionality of pTN1, the signal transduction genes (
nisRK
) of the nisin-controlled expression system were integrated downstream of the
pepN
gene into the chromosome of
Lactobacillus gasseri
. In the resulting strain, UKLbg1, expression of
nisRK
was likely driven by cotranscription with
pepN
and enabled nisin-dependent induction of a fusion of a reporter gene (
pepI
) to the
nisA
promoter. The induction rates were correlated with the amount of nisin used, and maximum
pepI
expression was achieved with nisin concentrations (above 25 ng/ml) at which growth of the bacteria was already inhibited.
A Real-Time PCR Assay for the Detection of
Campylobacter jejuni
in Foods after Enrichment Culture
Authors: Sails Andrew D. , Fox Andrew J. , Bolton Frederick J. , Wareing David R. A. , Greenway David L. A. 
Published: March 2003

ABSTRACT

A real-time PCR assay was developed for the quantitative detection of
Campylobacter jejuni
in foods after enrichment culture. The specificity of the assay for
C. jejuni
was demonstrated with a diverse range of
Campylobacter
species, related organisms, and unrelated genera. The assay had a linear range of quantification over six orders of magnitude, and the limit of detection was approximately 12 genome equivalents. The assay was used to detect
C. jejuni
in both naturally and artificially contaminated food samples. Ninety-seven foods, including raw poultry meat, offal, raw shellfish, and milk samples, were enriched in blood-free
Campylobacter
enrichment broth at 37°C for 24 h, followed by 42°C for 24 h. Enrichment cultures were subcultured to
Campylobacter
charcoal-cefoperazone-deoxycholate blood-free selective agar, and presumptive
Campylobacter
isolates were identified with phenotypic methods. DNA was extracted from enrichment cultures with a rapid lysis method and used as the template in the real-time PCR assay. A total of 66 samples were positive for
C. jejuni
by either method, with 57 samples positive for
C. jejuni
by subculture to selective agar medium and 63 samples positive in the real-time PCR assay. The results of both methods were concordant for 84 of the samples. The total time taken for detection from enrichment broth samples was approximately 3 h for the real-time PCR assay, with the results being available immediately at the end of PCR cycling, compared to 48 h for subculture to selective agar. This assay significantly reduces the total time taken for the detection of
C. jejuni
in foods and is an important model for other food-borne pathogens.
Detection and Typing of
Campylobacter jejuni
and
Campylobacter coli
and Analysis of Indicator Organisms in Three Waterborne Outbreaks in Finland
Authors: Hänninen Marja-Liisa , Haajanen H. , Pummi T. , Wermundsen K. , Katila M.-L. , Sarkkinen H. , Miettinen I. , Rautelin H. 
Published: March 2003

ABSTRACT

Waterborne outbreaks associated with contamination of drinking water by
Campylobacter jejuni
are rather common in the Nordic countries Sweden, Norway, and Finland, where in sparsely populated districts groundwater is commonly used without disinfection. Campylobacters,
Escherichia coli
, or other coliforms have rarely been detected in potential sources. We studied three waterborne outbreaks in Finland caused by
C. jejuni
and used sample volumes of 4,000 to 20,000 ml for analysis of campylobacters and sample volumes of 1 to 5,000 ml for analysis of coliforms and
E. coli
, depending on the sampling site. Multiple samples obtained from possible sources (water distribution systems and environmental water sources) and the use of large sample volumes (several liters) increased the chance of detecting the pathogen
C. jejuni
in water. Filtration of a large volume (1,000 to 2,000 ml) also increased the rate of detection of coliforms and
E. coli
. To confirm the association between drinking water contamination and illness, a combination of Penner serotyping and pulsed-field gel electrophoresis (digestion with
Sma
I and
Kpn
I) was found to be useful. This combination reliably verified similarity or dissimilarity of
C. jejuni
isolates from patient samples, from drinking water, and from other environmental sources, thus confirming the likely reservoir of an outbreak.
Construction and Screening of Metagenomic Libraries Derived from Enrichment Cultures: Generation of a Gene Bank for Genes Conferring Alcohol Oxidoreductase Activity on
Escherichia coli
Authors: Knietsch Anja , Waschkowitz Tanja , Bowien Susanne , Henne Anke , Daniel Rolf 
Published: March 2003

ABSTRACT

Enrichment of microorganisms with special traits and the construction of metagenomic libraries by direct cloning of environmental DNA have great potential for identifying genes and gene products for biotechnological purposes. We have combined these techniques to isolate novel genes conferring oxidation of short-chain (C
2
to C
4
) polyols or reduction of the corresponding carbonyls. In order to favor the growth of microorganisms containing the targeted genes, samples collected from four different environments were incubated in the presence of glycerol and 1,2-propanediol. Subsequently, the DNA was extracted from the four samples and used to construct complex plasmid libraries. Approximately 100,000
Escherichia coli
strains of each library per test substrate were screened for the production of carbonyls from polyols on indicator agar. Twenty-four positive
E. coli
clones were obtained during the initial screen. Sixteen of them contained a plasmid (pAK101 to pAK116) which conferred a stable carbonyl-forming phenotype. Eight of the positive clones exhibited NAD(H)-dependent alcohol oxidoreductase activity with polyols or carbonyls as the substrates in crude extracts. Sequencing revealed that the inserts of pAK101 to pAK116 encoded 36 complete and 17 incomplete presumptive protein-encoding genes. Fifty of these genes showed similarity to sequenced genes from a broad collection of different microorganisms. The genes responsible for the carbonyl formation of
E. coli
were identified for nine of the plasmids (pAK101, pAK102, pAK105, pAK107 to pAK110, pAK115, and pAK116). Analyses of the amino acid sequences deduced from these genes revealed that three (
orf12
,
orf14
, and
orf22
) encoded novel alcohol dehydrogenases of different types, four (
orf5
,
sucB
,
fdhD
, and
yabF
) encoded novel putative oxidoreductases belonging to groups distinct from alcohol dehydrogenases, one (
glpK
) encoded a putative glycerol kinase, and one (
orf1
) encoded a protein which showed no similarity to any other known gene product.
Isolation and Characterization of Thermophilic Bacilli Degrading Cinnamic, 4-Coumaric, and Ferulic Acids
Authors: Peng Xue , Misawa Norihiko , Harayama Shigeaki 
Published: March 2003

ABSTRACT

Thirty-four thermophilic
Bacillus
sp. strains were isolated from decayed wood bark and a hot spring water sample based on their ability to degrade vanillic acid under thermophilic conditions. It was found that these bacteria were able to degrade a wide range of aromatic acids such as cinnamic, 4-coumaric, 3-phenylpropionic, 3-(
p
-hydroxyphenyl)propionic, ferulic, benzoic, and 4-hydroxybenzoic acids. The metabolic pathways for the degradation of these aromatic acids at 60°C were examined by using one of the isolates, strain B1. Benzoic and 4-hydroxybenzoic acids were detected as breakdown products from cinnamic and 4-coumaric acids, respectively. The β-oxidative mechanism was proposed to be responsible for these conversions. The degradation of benzoic and 4-hydroxybenzoic acids was determined to proceed through catechol and gentisic acid, respectively, for their ring fission. It is likely that a non-β-oxidative mechanism is the case in the ferulic acid catabolism, which involved 4-hydroxy-3-methoxyphenyl-β-hydroxypropionic acid, vanillin, and vanillic acid as the intermediates. Other strains examined, which are V0, D1, E1, G2, ZI3, and H4, were found to have the same pathways as those of strain B1, except that strains V0, D1, and H4 had the ability to transform 3-hydroxybenzoic acid to gentisic acid, which strain B1 could not do.
Population Dynamics of Male-Killing and Non-Male-Killing Spiroplasmas in
Drosophila melanogaster
Authors: Anbutsu Hisashi , Fukatsu Takema 
Published: March 2003

ABSTRACT

The endosymbiotic bacteria
Spiroplasma
spp. are vertically transmitted through female hosts and are known to cause selective death of male offspring in insects. One strain of spiroplasma, NSRO, causes male killing in
Drosophila
species, and a non-male-killing variant of NSRO, designated NSRO-A, has been isolated. It is not known why NSRO-A does not kill males. In an attempt to understand the mechanism of male killing, we investigated the population dynamics of NSRO and NSRO-A throughout the developmental course of the laboratory host
Drosophila melanogaster
by using a quantitative PCR technique. In the early development of the host insect, the titers of NSRO were significantly higher than those of NSRO-A at the first- and second-instar stages, whereas at the egg, third-instar, and pupal stages, the titers of the two spiroplasmas were almost the same. Upon adult emergence, the titers of the two spiroplasmas were similar, around 2 × 10
8
dnaA
copy equivalents. However, throughout host aging, the two spiroplasmas showed strikingly different population growth patterns. The titers of NSRO increased exponentially for 3 weeks, attained a peak value of around 4 × 10
9
dnaA
copy equivalents per insect, and then decreased. In contrast, the titers of NSRO-A were almost constant throughout the adult portion of the life cycle. In adult females, consequently, the titer of NSRO was significantly higher than the titer of NSRO-A except for a short period just after emergence. Although infection of adult females with NSRO resulted in almost 100% male killing, production of some male offspring was observed within 4 days after emergence when the titers of NSRO were as low as those of NSRO-A. Based on these results, we proposed a threshold density hypothesis for the expression of male killing caused by the spiroplasma. The extents of the bottleneck in the vertical transmission through host generations were estimated to be 5 × 10
−5
for NSRO and 3 × 10
−4
for NSRO-A.