bluuebunny/crossref_metadata_2025 huggingface dataset:
A Sporadic Third Layer in the Ionosphere of Mars
Authors: Pätzold M. , Tellmann S. , Häusler B. , Hinson D. , Schaa R. , Tyler G. L.
Published: November 2005

The daytime martian ionosphere has been observed as a two-layer structure with electron densities that peak at altitudes between about 110 and 130 kilometers. The Mars Express Orbiter Radio Science Experiment on the European Mars Express spacecraft observed, in 10 out of 120 electron density profiles, a third ionospheric layer at altitude ranges of 65 to 110 kilometers, where electron densities, on average, peaked at 0.8 × 10
10
per cubic meter. Such a layer has been predicted to be permanent and continuous. Its origin has been attributed to ablation of meteors and charge exchange of magnesium and iron. Our observations imply that this layer is present sporadically and locally.
Synthesis and Structure of Sila-Adamantane
Authors: Fischer Jelena , Baumgartner Judith , Marschner Christoph
Published: November 2005

An organosilicon cage compound containing the sila-adamantane structural unit, which is the building block of crystalline silicon, was synthesized by the Lewis acid‐catalyzed rearrangement reaction of a structural isomer.
Regulated Pole-to-Pole Oscillations of a Bacterial Gliding Motility Protein
Authors: Mignot Tâm , Merlie John P. , Zusman David R.
Published: November 2005

Little is known about directed motility of bacteria that move by type IV pilus–mediated (twitching) motility. Here, we found that during periodic cell reversals of
Myxoccocus xanthus
, type IV pili were disassembled at one pole and reassembled at the other pole. Accompanying these reversals, FrzS, a protein required for directed motility, moved in an oscillatory pattern between the cell poles. The frequency of the oscillations was controlled by the Frz chemosensory system, which is essential for directed motility. Pole-to-pole migration of FrzS appeared to involve movement along a filament running the length of the cell. FrzS dynamics may thus regulate cell polarity during directed motility.
Impressions of Our Solar System
Authors: Yeomans Donald K.
Published: November 2005

The Planets
.
By Dava Sobel
. Viking, New York, 2005. 284 pp. $24.95, C$35. ISBN 0-670-03446-0. Fourth Estate, London. £15. ISBN 1-85702-850-3.

In this impressionistic tour of the solar system, the best-selling author emphasizes cultural and historical perspectives on the planets rather than science.
An Explosion in the Desert
Authors: Herken Gregg
Published: November 2005

Dr. Atomic
.
John Adams, composer. Libretto by Peter Sellars
. San Francisco Opera, co-production with Lyric Opera of Chicago and De Nederlandse Opera, Amsterdam. San Francisco, 1 to 22 October 2005.
www.doctor-atomic.com

This thought-provoking opera is centered around Oppenheimer and the detonation of the world's first atomic bomb in the New Mexico desert.
Novel 4-Chlorophenol Degradation Gene Cluster and Degradation Route via Hydroxyquinol in
Arthrobacter chlorophenolicus
A6
Authors: Nordin Karolina , Unell Maria , Jansson Janet K.
Published: November 2005

Arthrobacter chlorophenolicus
A6, a previously described 4-chlorophenol-degrading strain, was found to degrade 4-chlorophenol via hydroxyquinol, which is a novel route for aerobic microbial degradation of this compound. In addition, 10 open reading frames exhibiting sequence similarity to genes encoding enzymes involved in chlorophenol degradation were cloned and designated part of a chlorophenol degradation gene cluster (
cph
genes). Several of the open reading frames appeared to encode enzymes with similar functions; these open reading frames included two genes,
cphA-I
and
cphA-II
, which were shown to encode functional hydroxyquinol 1,2-dioxygenases. Disruption of the
cphA-I
gene yielded a mutant that exhibited negligible growth on 4-chlorophenol, thereby linking the
cph
gene cluster to functional catabolism of 4-chlorophenol in
A. chlorophenolicus
A6. The presence of a resolvase pseudogene in the
cph
gene cluster together with analyses of the G+C content and codon bias of flanking genes suggested that horizontal gene transfer was involved in assembly of the gene cluster during evolution of the ability of the strain to grow on 4-chlorophenol.
Preparation, Characterization, and Optimization of an In Vitro C30Carotenoid Pathway
Authors: Ku Bosung , Jeong Jae-Cheol , Mijts Benjamin N. , Schmidt-Dannert Claudia , Dordick Jonathan S.
Published: November 2005

TheispAgene encoding farnesyl pyrophosphate (FPP) synthase fromEscherichia coliand thecrtMgene encoding 4,4′-diapophytoene (DAP) synthase fromStaphylococcus aureuswere overexpressed and purified for use in vitro. Steady-state kinetics for FPP synthase and DAP synthase, individually and in sequence, were determined under optimized reaction conditions. For the two-step reaction, the DAP product was unstable in aqueous buffer; however, in situ extraction using an aqueous-organic two-phase system resulted in a 100% conversion of isopentenyl pyrophosphate and dimethylallyl pyrophosphate into DAP. This aqueous-organic two-phase system is the first demonstration of an in vitro carotenoid synthesis pathway performed with in situ extraction, which enables quantitative conversions. This approach, if extended to a wide range of isoprenoid-based pathways, could lead to the synthesis of novel carotenoids and their derivatives.
Genetic and Phylogeographic Structures of the Symbiotic Fungus
Tuber magnatum
Authors: Rubini Andrea , Paolocci Francesco , Riccioni Claudia , Vendramin Giovanni G. , Arcioni Sergio
Published: November 2005

The quality and market price of truffles vary with the species and, traditionally, the place of origin. The premium species
Tuber magnatum
produces white truffles and has a patchy distribution restricted to Italy and some Balkan areas. We used polymorphic microsatellites to evaluate 316 specimens grouped into 26 populations sampled across the species' geographic range to determine if natural populations of
T. magnatum
are genetically differentiated. We found that the southernmost and the northwesternmost populations were significantly differentiated from the rest of the populations. The simple sequence repeat data also could be used to make inferences about the postglacial
T. magnatum
expansion pattern. This study is the first to identify a genetic and phylogeographic structure in
T. magnatum.
The presence of a genetic structure can be of practical interest in tracing truffle populations according to their geographic origin for marketing strategies. Evidence for extensive outcrossing in field populations of
T. magnatum
also is provided for the first time.
Intra- and Interspecific Comparisons of Bacterial Diversity and Community Structure Support Coevolution of Gut Microbiota and Termite Host
Authors: Hongoh Yuichi , Deevong Pinsurang , Inoue Tetsushi , Moriya Shigeharu , Trakulnaleamsai Savitr , Ohkuma Moriya , Vongkaluang Charunee , Noparatnaraporn Napavarn , Kudo Toshiaki
Published: November 2005

We investigated the bacterial gut microbiota from 32 colonies of wood-feeding termites, comprising four
Microcerotermes
species (Termitidae) and four
Reticulitermes
species (Rhinotermitidae), using terminal restriction fragment length polymorphism analysis and clonal analysis of 16S rRNA. The obtained molecular community profiles were compared statistically between individuals, colonies, locations, and species of termites. Both analyses revealed that the bacterial community structure was remarkably similar within each termite genus, with small but significant differences between sampling sites and/or termite species. In contrast, considerable differences were found between the two termite genera. Only one bacterial phylotype (defined with 97% sequence identity) was shared between the two termite genera, while 18% and 50% of the phylotypes were shared between two congeneric species in the genera
Microcerotermes
and
Reticulitermes
, respectively. Nevertheless, a phylogenetic analysis of 228 phylotypes from
Microcerotermes
spp. and 367 phylotypes from
Reticulitermes
spp. with other termite gut clones available in public databases demonstrated the monophyly of many phylotypes from distantly related termites. The monophyletic “termite clusters” comprised of phylotypes from more than one termite species were distributed among 15 bacterial phyla, including the novel candidate phyla TG2 and TG3. These termite clusters accounted for 95% of the 960 clones analyzed in this study. Moreover, the clusters in 12 phyla comprised phylotypes from more than one termite (sub)family, accounting for 75% of the analyzed clones. Our results suggest that the majority of gut bacteria are not allochthonous but are specific symbionts that have coevolved with termites and that their community structure is basically consistent within a genus of termites.
On-Site DNA Extraction and Real-Time PCR for Detection of
Phytophthora ramorum
in the Field
Authors: Tomlinson J. A. , Boonham N. , Hughes K. J. D. , Griffin R. L. , Barker I.
Published: November 2005

Phytophthora ramorum
is a recently described pathogen causing oak mortality (sudden oak death) in forests in coastal areas of California and southern Oregon and dieback and leaf blight in a range of tree, shrub, and herbaceous species in the United States and Europe. Due to the threat posed by this organism, stringent quarantine regulations are in place, which restrict the movement of a number of hosts. Fast and accurate diagnostic tests are required in order to characterize the distribution of
P. ramorum
, prevent its introduction into pathogen-free areas, and minimize its spread within affected areas. However, sending samples to a laboratory for testing can cause a substantial delay between sampling and diagnosis. A rapid and simple DNA extraction method was developed for use at the point of sampling and used to extract DNAs from symptomatic foliage and stems in the field. A sensitive and specific single-round real-time PCR (TaqMan) assay for
P. ramorum
was performed using a portable real-time PCR platform (Cepheid SmartCycler II), and a cost-effective method for stabilizing PCR reagents was developed to allow their storage and transportation at room temperature. To our knowledge, this is the first description of a method for DNA extraction and molecular testing for a plant pathogen carried out entirely in the field, independent of any laboratory facilities.
Degradation of Azo Dyes by
Trametes villosa
Laccase over Long Periods of Oxidative Conditions
Authors: Zille Andrea , Górnacka Barbara , Rehorek Astrid , Cavaco-Paulo Artur
Published: November 2005

Trametes villosa
laccase was used for direct azo dye degradation, and the reaction products that accumulated after 72 h of incubation were analyzed. Liquid chromatography-mass spectrometry (LC-MS) analysis showed the formation of phenolic compounds during the dye oxidation process as well as a large amount of polymerized products that retain azo group integrity. The amino-phenol reactions were also investigated by
13
C-nuclear magnetic resonance and LC-MS analysis, and the polymerization character of laccase was shown. This study highlights the fact that laccases polymerize the reaction products obtained during long-term batch decolorization processes with azo dyes. These polymerized products provide unacceptable color levels in effluents, limiting the application of laccases as bioremediation agents.
Detection of
Salmonella enterica
in Naturally Contaminated Liquid Eggs by Loop-Mediated Isothermal Amplification, and Characterization of
Salmonella
Isolates
Authors: Ohtsuka Kayoko , Yanagawa Keiko , Takatori Kosuke , Hara-Kudo Yukiko
Published: November 2005

Loop-mediated isothermal amplification (LAMP) assay was effective in detecting
Salmonella enterica
in naturally contaminated liquid egg samples.
Salmonella
was detected in 110 samples taken from four egg-breaking plants. The egg samples were pre-enriched in buffered peptone water (BPW) at 37°C for 20 h. The selective enrichment was done in Rappaport-Vassiliadis or tetrathionate broth and plated onto xylose lysine deoxycholate agar and brilliant green agar, modified. In addition, the PCR assay was used to detect
Salmonella
after pre-enrichment in BPW at 37°C for 20 h. The culture method and PCR assay were compared to the LAMP assay, which was also performed after pre-enrichment in BPW. PCR failed to detect
Salmonella
in 10% of 110 samples, whereas the culture method and LAMP assay successfully identified
Salmonella
in all samples. However, the LAMP assay was found to be much more rapid than the culture method and as sensitive in detecting
Salmonella
from liquid eggs. In all of the egg-breaking plants studied,
Salmonella
was isolated on most tested days. The positive samples showed that more than 75% of the
Salmonella
strains had identical genetic patterns when analyzed by pulsed-field gel electrophoresis. This suggests that the same
Salmonella
strains having survived long periods of time in the plants were contaminating the production line. The LAMP assay is rapid, specific, and sensitive for
Salmonella
detection in liquid eggs and is able to monitor
Salmonella
contamination in egg-handling plants more reliably.
Effect of Different Carbon Sources on Community Composition of Bacterial Enrichments from Soil
Authors: Wawrik Boris , Kerkhof Lee , Kukor Jerome , Zylstra Gerben
Published: November 2005

Soil is a highly heterogeneous matrix, which can contain thousands of different bacterial species per gram. Only a small component of this diversity (maybe <1%) is commonly captured using standard isolation techniques, although indications are that a larger proportion of the soil community is in fact culturable. Better isolation techniques yielding greater bacterial diversity would be of benefit for understanding the metabolic activity and capability of many soil microorganisms. We studied the response of soil bacterial communities to carbon source enrichment in small matrices by means of terminal restriction fragment length polymorphism (TRFLP) analysis. The community composition of replicate enrichments from soil displayed high variability, likely attributable to soil heterogeneity. An analysis of TRFLP data indicated that enrichment on structurally similar carbon sources selected for similar bacterial communities. The same analysis indicated that communities first enriched on glucose or benzoate and subsequently transferred into medium containing an alternate carbon source retained a distinct community signature induced by the carbon source used in the primary enrichment. Enrichment on leucine presented a selective challenge that was able to override the imprint left by primary enrichment on acetate. In a time series experiment community change was most rapid 18 hours after inoculation, corresponding to exponential growth. Community composition did not stabilize even 4 days after secondary enrichment. Four different soil types were enriched on four different carbon sources. TRFLP analysis indicated that in three out of four cases communities enriched on the same carbon source were more similar regardless of which soil type was used. Conversely, the garden soil samples yielded similar enrichment communities regardless of the enrichment carbon source. Our results indicate that in order to maximize the diversity of bacteria recovered from the environment, multiple enrichments should be performed using a chemically diverse set of carbon sources.
Potential for Quantifying Expression of the
Geobacteraceae
Citrate Synthase Gene To Assess the Activity of
Geobacteraceae
in the Subsurface and on Current-Harvesting Electrodes
Authors: Holmes Dawn E. , Nevin Kelly P. , O'Neil Regina A. , Ward Joy E. , Adams Lorrie A. , Woodard Trevor L. , Vrionis Helen A. , Lovley Derek R.
Published: November 2005

The
Geobacteraceae
citrate synthase is phylogenetically distinct from those of other prokaryotes and is a key enzyme in the central metabolism of
Geobacteraceae
. Therefore, the potential for using levels of citrate synthase mRNA to estimate rates of
Geobacter
metabolism was evaluated in pure culture studies and in four different
Geobacteraceae
-dominated environments. Quantitative reverse transcription-PCR studies with mRNA extracted from cultures of
Geobacter sulfurreducens
grown in chemostats with Fe(III) as the electron acceptor or in batch with electrodes as the electron acceptor indicated that transcript levels of the citrate synthase gene,
gltA
, increased with increased rates of growth/Fe(III) reduction or current production, whereas the expression of the constitutively expressed housekeeping genes
recA
,
rpoD
, and
proC
remained relatively constant. Analysis of mRNA extracted from groundwater collected from a U(VI)-contaminated site undergoing in situ uranium bioremediation revealed a remarkable correspondence between acetate levels in the groundwater and levels of transcripts of
gltA
. The expression of
gltA
was also significantly greater in RNA extracted from groundwater beneath a highway runoff recharge pool that was exposed to calcium magnesium acetate in June, when acetate concentrations were high, than in October, when the levels had significantly decreased. It was also possible to detect
gltA
transcripts on current-harvesting anodes deployed in freshwater sediments. These results suggest that it is possible to monitor the in situ metabolic rate of
Geobacteraceae
by tracking the expression of the citrate synthase gene.
Iron Uptake Mechanisms in the Fish Pathogen
Tenacibaculum maritimum
Authors: Avendaño-Herrera Ruben , Toranzo Alicia E. , Romalde Jesús L. , Lemos Manuel L. , Magariños Beatriz
Published: November 2005

We present here the first evidence of the presence of iron uptake mechanisms in the bacterial fish pathogen
Tenacibaculum maritimum
. Representative strains of this species, with different serotypes and origins, were examined. All of them were able to grow in the presence of the chelating agent ethylenediamine-di- (
o-
hydroxyphenyl acetic acid) (EDDHA) and also produced siderophores. Cross-feeding assays suggest that the siderophores produced are closely related. In addition, all
T. maritimum
strains utilized transferrin, hemin, hemoglobin, and ferric ammonic citrate as iron sources when added to iron-deficient media. Whole cells of all
T. maritimum
strains, grown under iron-supplemented or iron-restricted conditions, were able to bind hemin, indicating the existence of constitutive binding components located at the
T. maritimum
cell surface. This was confirmed by the observation that isolated total and outer membrane proteins from all of the strains, regardless of the iron levels of the media, were able to bind hemin, with the outer membranes showing the strongest binding. proteinase K treatment of whole cells did not affect the hemin binding, indicating that, in addition to proteins, some protease-resistant components could also bind hemin. At least three outer membrane proteins were induced in iron-limiting conditions, and all strains, regardless of their serotype, showed a similar pattern of induced proteins. The results of the present study suggest that
T. maritimum
possesses at least two different systems of iron acquisition: one involving the synthesis of siderophores and another that allows the utilization of heme groups as iron sources by direct binding.
Biocatalytic Conversion of Avermectin to 4"-Oxo-Avermectin: Characterization of Biocatalytically Active Bacterial Strains and of Cytochrome P450 Monooxygenase Enzymes and Their Genes
Authors: Jungmann Volker , Molnár István , Hammer Philip E. , Hill D. Steven , Zirkle Ross , Buckel Thomas G. , Buckel Dagmar , Ligon James M. , Pachlatko J. Paul
Published: November 2005

4"-Oxo-avermectin is a key intermediate in the manufacture of the agriculturally important insecticide emamectin benzoate from the natural product avermectin. Seventeen biocatalytically active
Streptomyces
strains with the ability to oxidize avermectin to 4"-oxo-avermectin in a regioselective manner have been discovered in a screen of 3,334 microorganisms. The enzymes responsible for this oxidation reaction in these biocatalytically active strains were found to be cytochrome P450 monooxygenases (CYPs) and were termed Ema1 to Ema17. The genes for Ema1 to Ema17 have been cloned, sequenced, and compared to reveal a new subfamily of CYPs. Ema1 to Ema16 have been overexpressed in
Escherichia coli
and purified as His-tagged recombinant proteins, and their basic enzyme kinetic parameters have been determined.
Culture-Dependent and Culture-Independent Diversity within the Obligate Marine Actinomycete Genus
Salinispora
Authors: Mincer Tracy J. , Fenical William , Jensen Paul R.
Published: November 2005

Salinispora
is the first obligate marine genus within the order
Actinomycetales
and a productive source of biologically active secondary metabolites. Despite a worldwide, tropical or subtropical distribution in marine sediments, only two
Salinispora
species have thus far been cultivated, suggesting limited species-level diversity. To further explore
Salinispora
diversity and distributions, the phylogenetic diversity of more than 350 strains isolated from sediments collected around the Bahamas was examined, including strains cultured using new enrichment methods. A culture-independent method, using a
Salinispora
-specific seminested PCR technique, was used to detect
Salinispora
from environmental DNA and estimate diversity. Overall, the 16S rRNA gene sequence diversity of cultured strains agreed well with that detected in the environmental clone libraries. Despite extensive effort, no new species level diversity was detected, and 97% of the 105 strains examined by restriction fragment length polymorphism belonged to one phylotype (
S. arenicola
). New intraspecific diversity was detected in the libraries, including an abundant new phylotype that has yet to be cultured, and a new depth record of 1,100 m was established for the genus. PCR-introduced error, primarily from
Taq
polymerase, significantly increased clone library sequence diversity and, if not masked from the analyses, would have led to an overestimation of total diversity. An environmental DNA extraction method specific for vegetative cells provided evidence for active actinomycete growth in marine sediments while indicating that a majority of sediment samples contained predominantly
Salinispora
spores at concentrations that could not be detected in environmental clone libraries. Challenges involved with the direct sequence-based detection of spore-forming microorganisms in environmental samples are discussed.
Development of a
cob
-18S rRNA Gene Real-Time PCR Assay for Quantifying
Pfiesteria shumwayae
in the Natural Environment
Authors: Zhang Huan , Lin Senjie
Published: November 2005

Despite the fact that the heterotrophic dinoflagellate
Pfiesteria shumwayae
is an organism of high interest due to alleged toxicity, its abundance in natural environments is poorly understood. To address this inadequacy, a real-time quantitative PCR assay based on mitochondrial cytochrome
b
(
cob
) and18S rRNA gene was developed and
P. shumwayae
abundance was investigated in several geographic locations. First,
cob
and its 5′-end region were isolated from a
P. shumwayae
culture, revealing three different copies, each consisting of an identical
cob
coding region and an unidentified region (X) of variable length and sequence. The unique sequences in
cob
and the X region were then used to develop a
P. shumwayae
-specific primer set. This primer set was used with reported
P. shumwayae
-specific 18S primers in parallel real-time PCRs to investigate
P. shumwayae
abundance from Maine to North Carolina along the U.S. east coast and along coasts in Chile, Hawaii, and China. Both genes generally gave similar results, indicating that this species was present, but at low abundance (mostly <10 cells · ml
−1
), in all the American coast locations investigated (with the exception of Long Island Sound, where which both genes gave negative results). Genetic variation was detected by use of both genes in most of the locations, and while
cob
consistently detected
P. shumwayae
or close genetic variants, some of the 18S PCR products were unrelated to
P. shumwayae
. We conclude that (i) the real-time PCR assay developed is useful for specific quantification of
P. shumwayae
, and (ii)
P. shumwayae
is distributed widely at the American coasts, but normally only as a minor component of plankton even in high-risk estuaries (Neuse River and the Chesapeake Bay).
Survival of Genetically Modified and Self-Cloned Strains of Commercial Baker's Yeast in Simulated Natural Environments: Environmental Risk Assessment
Authors: Ando Akira , Suzuki Chise , Shima Jun
Published: November 2005

Although genetic engineering techniques for baker's yeast might improve the yeast's fermentation characteristics, the lack of scientific data on the survival of such strains in natural environments as well as the effects on human health prevent their commercial use. Disruption of acid trehalase gene (
ATH1
) improves freeze tolerance, which is a crucial characteristic in frozen-dough baking. In this study,
ATH1
disruptants constructed by genetic modification (GM) and self-cloning (SC) techniques were used as models to study such effects because these strains have higher freeze tolerance and are expected to be used commercially. Behavior of the strains in simulated natural environments, namely, in soil and water, was studied by measuring the change in the number of viable cells and in the concentration of DNA that contains
ATH1
loci. Measurements were made using a real-time PCR method during 40 days of cultivation. Results showed that the number of viable cells of GM and SC strains decreased in a time-dependent manner and that the decrease rate was nearly equal to or higher than that for wild-type (WT) yeast. For all three strains (SC, GM, and WT) in the two simulated natural environments (water and soil), the DNA remained longer than did viable cells but the decrease patterns of either the DNA or the viable cells of SC and GM strains had tendencies similar to those of the WT strain. In conclusion, disruption of
ATH1
by genetic engineering apparently does not promote the survival of viable cells and DNA in natural environments.
Real-Time Nucleic Acid Sequence-Based Amplification Assay for Detection of Hepatitis A Virus
Authors: Abd El Galil Khaled H. , El Sokkary M. A. , Kheira S. M. , Salazar Andre M. , Yates Marylynn V. , Chen Wilfred , Mulchandani Ashok
Published: November 2005

A nucleic acid sequence-based amplification (NASBA) assay in combination with a molecular beacon was developed for the real-time detection and quantification of hepatitis A virus (HAV). A 202-bp, highly conserved 5′ noncoding region of HAV was targeted. The sensitivity of the real-time NASBA assay was tested with 10-fold dilutions of viral RNA, and a detection limit of 1 PFU was obtained. The specificity of the assay was demonstrated by testing with other environmental pathogens and indicator microorganisms, with only HAV positively identified. When combined with immunomagnetic separation, the NASBA assay successfully detected as few as 10 PFU from seeded lake water samples. Due to its isothermal nature, its speed, and its similar sensitivity compared to the real-time RT-PCR assay, this newly reported real-time NASBA method will have broad applications for the rapid detection of HAV in contaminated food or water.
Prevalence of Pathogenic
Yersinia enterocolitica
Strains in Pigs in the United States
Authors: Bhaduri Saumya , Wesley Irene V. , Bush Eric J.
Published: November 2005

Yersinia enterocolitica
is considered an important food-borne pathogen
impacting the pork production and processing industry in the United
States. Since this bacterium is a commensal of swine, the primary goal
of this study was to determine the prevalence of pathogenic
Y
.
enterocolitica
in pigs in the United Sates using feces as the
sample source. A total of 2,793 fecal samples were tested for its
presence in swine. Fecal samples were collected from late finisher pigs
from 77 production sites in the 15 eastern and midwestern
pork-producing states over a period of 27 weeks (6 September 2000 to 20
March 2001). The prevalence of
ail
-positive
Y
.
enterocolitica
was determined in samples using both a
fluorogenic 5′ nuclease PCR assay and a culture method. The
mean prevalence was 13.10% (366 of 2,793 fecal samples tested) when
both PCR- and culture-positive results were combined. Forty-one of 77
premises (53.25%) contained at least one fecal sample positive for the
ail
sequence. The PCR assay indicated a contamination rate of
12.35% (345/2,793) compared to 4.08% (114/2,793) by the culture method.
Of the 345 PCR-positive samples, 252 were culture negative, while of
the 114 culture-positive samples, 21 were PCR negative. Among 77
premises, the PCR assay revealed a significantly (
P
<
0.05) higher percentage (46.75%,
n
= 36 sites) of
samples positive for the pathogen (
ail
sequence) than the
culture method (22.08%,
n
= 17 sites). Thus, higher
sensitivity, with respect to number of samples and sites identified as
positive for the PCR method compared with the culture method for
detecting pathogenic
Y. enterocolitica
, was
demonstrated in this study. The results support the hypothesis that
swine are a reservoir for
Y. enterocolitica
strains
potentially pathogenic for
humans.
Engineering of
Bacillus subtilis
for Enhanced Total Synthesis of Folic Acid
Authors: Zhu T. , Pan Z. , Domagalski N. , Koepsel R. , Ataai M. M. , Domach M. M.
Published: November 2005

We investigated whether the yield of the B vitamin folic acid could be elevated in
Bacillus subtilis
. Strategies for increasing the folic acid yield were investigated by employing computer-aided flux analysis and mutation. Controlling the activity of the enzyme pyruvate kinase by placing it under inducible control was one strategy devised to elevate yield while insuring that a rapid growth rate results. Other single mutation strategies included amplifying the expression of the genes in the folate operon and overexpressing the
Escherichia coli aroH
gene, which encodes 2-dehydro-3-deoxyphosphoheptonate aldolase. The latter could conceivably elevate the abundance of the folic acid precursor,
para
-aminobenzoic acid. Strains that combined two or more mutations were also constructed. Overall, a strain possessing inducible pyruvate kinase, overexpressed
aroH
, and increased transcription and translation of genes from the folic operon exhibited the best yield. The yield was eightfold higher than that displayed by the parent
B. subtilis
168 strain.
Novel DNA Sequences from Natural Strains of the Nitrogen-Fixing Symbiotic Bacterium
Sinorhizobium meliloti
Authors: Guo Hong , Sun Sheng , Finan Turlough M. , Xu Jianping
Published: November 2005

Variation in genome size and content is common among bacterial strains. Identifying these naturally occurring differences can accelerate our understanding of bacterial attributes, such as ecological specialization and genome evolution. In this study, we used representational difference analysis to identify potentially novel sequences not present in the sequenced laboratory strain Rm1021 of the nitrogen-fixing bacterium
Sinorhizobium meliloti
. Using strain Rm1021 as the driver and the type strain of
S. meliloti
ATCC 9930, which has a genome size ∼370 kilobases bigger than that of strain Rm1021, as the tester, we identified several groups of sequences in the ATCC 9930 genome not present in strain Rm1021. Among the 85 novel DNA fragments examined, 55 showed no obvious homologs anywhere in the public databases. Of the remaining 30 sequences, 24 contained homologs to the Rm1021 genome as well as unique segments not found in Rm1021, 3 contained sequences homologous to those published for another
S. meliloti
strain but absent in Rm1021, 2 contained sequences homologous to other symbiotic nitrogen-fixing bacteria (
Rhizobium etli
and
Bradyrhizobium japonicum
), and 1 contained a sequence homologous to a gene in a non-nitrogen-fixing species,
Pseudomonas
sp. NK87. Using PCR, we assayed the distribution of 12 of the above 85 novel sequences in a collection of 59 natural
S. meliloti
strains. The distribution varied widely among the 12 novel DNA fragments, from 1.7% to 72.9%. No apparent correlation was found between the distribution of these novel DNA sequences and their genotypes obtained using multilocus enzyme electrophoresis. Our results suggest potentially high rates of gene gain and loss in
S. meliloti
genomes.
Proteomic Signature of
Lactococcus lactis
NCDO763 Cultivated in Milk
Authors: Gitton Christophe , Meyrand Mickael , Wang Juhui , Caron Christophe , Trubuil Alain , Guillot Alain , Mistou Michel-Yves
Published: November 2005

We have compared the proteomic profiles of
L. lactis
subsp.
cremoris
NCDO763 growing in the synthetic medium M17Lac, skim milk microfiltrate (SMM), and skim milk. SMM was used as a simple model medium to reproduce the initial phase of growth of
L. lactis
in milk. To widen the analysis of the cytoplasmic proteome, we used two different gel systems (pH ranges of 4 to 7 and 4.5 to 5.5), and the proteins associated with the cell envelopes were also studied by two-dimensional electrophoresis. In the course of the study, we analyzed about 800 spots and identified 330 proteins by mass spectrometry. We observed that the levels of more than 50 and 30 proteins were significantly increased upon growth in SMM and milk, respectively. The large redeployment of protein synthesis was essentially associated with an activation of pathways involved in the metabolism of nitrogenous compounds: peptidolytic and peptide transport systems, amino acid biosynthesis and interconversion, and de novo biosynthesis of purines. We also showed that enzymes involved in reactions feeding the purine biosynthetic pathway in one-carbon units and amino acids have an increased level in SMM and milk. The analysis of the proteomic data suggested that the glutamine synthetase (GS) would play a pivotal role in the adaptation to SMM and milk. The analysis of
glnA
expression during growth in milk and the construction of a
glnA
-defective mutant confirmed that GS is an essential enzyme for the development of
L. lactis
in dairy media. This analysis thus provides a proteomic signature of
L. lactis
, a model lactic acid bacterium, growing in its technological environment.
Alteration of Leucine Aminopeptidase from
Streptomyces septatus
TH-2 to Phenylalanine Aminopeptidase by Site-Directed Mutagenesis
Authors: Arima Jiro , Uesugi Yoshiko , Iwabuchi Masaki , Hatanaka Tadashi
Published: November 2005

To tailor leucine aminopeptidase from
Streptomyces septatus
TH-2 (SSAP) to become a convenient biocatalyst, we are interested in Phe221 of SSAP, which is thought to interact with the side chain of the N-terminal residue of the substrate. By using saturation mutagenesis, the feasibility of altering the performance of SSAP was evaluated. The hydrolytic activities of 19 mutants were investigated using aminoacyl
p
-nitroanilide (
p
NA) derivatives as substrates. Replacement of Phe221 resulted in changes in the activities of all the mutants. Three of these mutants, F221G, F221A, and F221S, specifically hydrolyzed
l
-Phe-
p
NA, and F221I SSAP exhibited hydrolytic activity with
l
-Leu-
p
NA exceeding that of the wild type. Although the hydrolytic activities with peptide substrates decreased, the hydrolytic activities with amide and methyl ester substrates were proportional to the changes in the hydrolytic activities with
p
NA derivatives. Furthermore, based on a comparative kinetic study, the mechanism underlying the alteration in the preference of SSAP from leucine to phenylalanine is discussed.
First Genome Data from Uncultured Upland Soil Cluster Alpha Methanotrophs Provide Further Evidence for a Close Phylogenetic Relationship toMethylocapsa acidiphilaB2 and for High-Affinity Methanotrophy Involving Particulate Methane Monooxygenase
Authors: Ricke Peter , Kube Michael , Nakagawa Satoshi , Erkel Christoph , Reinhardt Richard , Liesack Werner
Published: November 2005

Members of upland soil cluster alpha (USCα) are assumed to be methanotrophic bacteria (MB) adapted to the trace level of atmospheric methane. So far, these MB have eluded all cultivation attempts. While the 16S rRNA phylogeny of USCα members is still not known, phylogenies constructed for the active-site polypeptide (encoded bypmoA) of particulate methane monooxygenase (pMMO) placed USCα next to the alphaproteobacterialMethylocapsa acidiphilaB2. To assess whether thepmoAtree reflects the evolutionary identity of USCα, a 42-kb genomic contig of a USCα representative was obtained from acidic forest soil by screening a metagenomic fosmid library of 250,000 clones usingpmoA-targeted PCR. For comparison, a 101-kb genomic contig fromM. acidiphilawas analyzed, including thepmooperon. The following three lines of evidence confirmed a close phylogenetic relationship between USCα andM. acidiphila: (i) tetranucleotide frequency patterns of 5-kb genomic subfragments, (ii) annotation and comparative analysis of the genomic fragments against all completely sequenced genomes available in public domain databases, and (iii) three single gene phylogenies constructed using the deduced amino acid sequences of a putative prephenate dehydratase, a staphylococcal-like nuclease, and a putative zinc metalloprotease. A comparative analysis of thepmooperons of USCα andM. acidiphilacorroborated previous reports that both thepmooperon structure and the predicted secondary structure of deduced pMMO are highly conserved among all MB.
Isolation, Sequence Analysis, and Comparison of Two Plasmids (28 and 29 Kilobases) from the Biomining Bacterium
Leptospirillum ferrooxidans
ATCC 49879
Authors: Coram Nicolette J. , van Zyl Leonardo J. , Rawlings Douglas E.
Published: November 2005

Two plasmids, of 28,878 bp and 28,012 bp, were isolated from
Leptospirillum ferrooxidans
ATCC 49879. Altogether, a total of 67 open reading frames (ORFs) were identified on both plasmids, of which 32 had predicted products with high homology to proteins of known function, while 11 ORFs had predicted products with homology to previously identified proteins of unknown function. Twenty-four ORFs had products with no homologues in the GenBank/NCBI database. An analysis of the ORFs and other features of the two plasmids, the first to be isolated from a bacterium of the genus
Leptospirillum
, is presented.
Symbiotic Hydrogenase Activity in
Bradyrhizobium
sp. (
Vigna
) Increases Nitrogen Content in
Vigna unguiculata
Plants
Authors: Baginsky Cecilia , Brito Belén , Imperial Juan , Ruiz-Argüeso Tomás , Palacios Jose M.
Published: November 2005

Bradyrhizobium
sp. (
Lupinus
) and
Bradyrhizobium
sp. (
Vigna
) mutants in which hydrogenase (
hup
) activity was affected were constructed and analyzed.
Vigna unguiculata
plants inoculated with the
Bradyrhizobium
sp. (
Vigna
)
hup
mutant showed reduced nitrogenase activity and also a significant decrease in nitrogen content, suggesting a relevant contribution of hydrogenase activity to plant yield.
Comparison of the Levels of Heat Resistance of Wild-Type,
cpe
Knockout, and
cpe
Plasmid-Cured
Clostridium perfringens
Type A Strains
Authors: Raju Deepa , Sarker Mahfuzur R.
Published: November 2005

An enterotoxin (
cpe
) plasmid was cured from a
Clostridium perfringens
non-food-borne gastrointestinal disease (NFBGID) isolate, and the heat resistance levels of wild-type,
cpe
knockout, and
cpe
plasmid-cured strains were compared. Our results demonstrated that (i) wild-type
cpe
has no influence in mediating high-level heat resistance in
C. perfringens
and (ii) the
cpe
plasmid does not confer heat sensitivity on NFBGID isolates.
Keep people informed or leave them alone? A suggested tool for identifying research participants who rightly want only limited information
Authors: Eriksson S , Helgesson G
Published: November 2005

People taking part in research vary in the extent to which they understand information concerning their participation. Since they may choose to limit the time and effort spent on such information, lack of understanding is not necessarily an ethical problem. Researchers who notice a lack of understanding are in the quandary of not knowing whether this is due to flaws in the information process or to participants’ deliberate choices. We argue that the two explanations call for different responses.

A tool for identifying those research participants who want limited information is presented. This consists of a restricted number of questions about trust in and appraisal of research, priority of time and privacy, and perception of a duty to participate. It is argued that an important group of participants who purposely lack understanding of the study can be identified with this tool. Some limitations to this approach are also discussed.
The tension between self governance and absolute inner worth in Kant’s moral philosophy
Authors: Häyry M
Published: November 2005

The concepts of autonomy as the self governance of individuals and dignity as the inner worth of human beings play an important role in contemporary bioethics. Since both notions are crucial to Immanuel Kant’s moral theory, it would be tempting to think that Kantian ethics could ease the friction between the two concepts. It is argued in this paper, however, that this line of thought cannot be supported by Kant’s original ideas. While he did make a conscious effort to bring autonomy and dignity together, his emphasis on the absolute inner worth of our collective humanity made it impossible for him to embrace fully the personal self determination of individuals, as it is usually understood in today’s liberal thinking.
“Hello, hello—it’s English I speak!”: a qualitative exploration of patients’ understanding of the science of clinical trials
Authors: Stead M , Eadie D , Gordon D , Angus K
Published: November 2005

Informed consent may be seriously compromised if patients fail to understand the experimental nature of the trial in which they are participating. Using focus groups, the authors explored how prospective trial participants interpret and understand the science of clinical trials by using patient information sheets relative to their medical condition. An opportunity was provided to hear in the patients’ own words how they interpret the information and why there is variable understanding. Respondents struggled to comprehend the meaning and purpose of concepts such as randomisation and double blinding, and found them threatening to their ideas of medical care. Suggestions are made about how to improve the national guidelines on written information for trial participants and pretesting of the information sheets is advocated.
Reproductive cloning combined with genetic modification
Authors: Strong C
Published: November 2005

Although there is widespread opposition to reproductive cloning, some have argued that its use by infertile couples to have genetically related children would be ethically justifiable. Others have suggested that lesbian or gay couples might wish to use cloning to have genetically related children. Most of the main objections to human reproductive cloning are based on the child’s lack of unique nuclear DNA. In the future, it may be possible safely to create children using cloning combined with genetic modifications, so that they have unique nuclear DNA. The genetic modifications could be aimed at giving such children genetic characteristics of both members of the couple concerned. Thus, cloning combined with genetic modification could be appealing to infertile, lesbian, or gay couples who seek genetically related children who have genetic characteristics of both members. In such scenarios, the various objections to human reproductive cloning that are based on the lack of genetic uniqueness would no longer be applicable. The author argues that it would be ethically justifiable for such couples to create children in this manner, assuming these techniques could be used safely.
Ethical considerations in presymptomatic testing for variant CJD
Authors: Duncan R E , Delatycki M B , Collins S J , Boyd A , Masters C L , Savulescu J
Published: November 2005

Variant Creutzfeldt–Jakob disease (vCJD) is a fatal, transmissible, neurodegenerative disorder for which there is currently no effective treatment. vCJD arose from the zoonotic spread of bovine spongiform encephalopathy. There is now compelling evidence for human to human transmission through blood transfusions from presymptomatic carriers and experts are warning that the real epidemic may be yet to come. Imperatives exist for the development of reliable, non-invasive presymptomatic diagnostic tests. Research into such tests is well advanced. In this article the ethical implications of the availability of these tests are elaborated and comparisons drawn with predictive genetic testing for Huntington’s disease and screening for HIV. Paramount to considerations is the issue of whom to test, weighing up respect for personal autonomy against obligations to benefit and protect society. A paradigm is proposed similar to that used for HIV screening but with unique features: compulsory testing of all blood/organ donors and individuals undergoing surgery or invasive procedures who have a significant risk of disease transmission.
Mouse GPR40 heterologously expressed in Xenopus oocytes is activated by short-, medium-, and long-chain fatty acids
Authors: Stewart Gavin , Hira Tohru , Higgins Andrew , Smith Craig P. , McLaughlin John T.
Published: November 2005

Several orphan G protein-coupled receptors, including GPR40, have recently been shown to be responsive to fatty acids. Although previous reports have suggested GPR40 detects medium- and long-chain fatty acids, it has been reported to be unresponsive to short chain fatty acids. In this study, we have heterologously expressed mouse GPR40 in Xenopus laevis oocytes and measured fatty acid-induced increases in intracellular Ca2+, via two electrode voltage clamp recordings of the endogenous Ca2+-activated chloride conductance. Exposure to 500 μM linoleic acid (C18:2), a long-chain fatty acid, stimulated significant currents in mGPR40-injected oocytes ( P < 0.01, ANOVA), but not in water-injected control oocytes (not significant, ANOVA). These currents were confirmed as Ca2+-activated chloride conductances because they were biphasic, sensitive to changes in external pH, and inhibited by DIDS. Similar currents were observed with medium-chain fatty acids, such as lauric acid (C12:0) ( P < 0.01, ANOVA), and more importantly, with short-chain fatty acids, such as butyric acid (C4:0) ( P < 0.01, ANOVA). In contrast, no responses were observed in mGPR40-injected oocytes exposed to either acetic acid (C2:0) or propionic acid (C3:0). Therefore, GPR40 has the capacity to respond to fatty acids with chain lengths of four or greater. This finding has important implications for understanding the structure:function relationship of fatty acid sensors, and potentially for short-chain fatty acid sensing in the gastrointestinal tract.