bluuebunny/crossref_metadata_2025_split huggingface dataset:
Quantification of Cyprinid Herpesvirus 3 in Environmental Water by Using an External Standard Virus
Authors: Honjo Mie N. , Minamoto Toshifumi , Matsui Kazuaki , Uchii Kimiko , Yamanaka Hiroki , Suzuki Alata A. , Kohmatsu Yukihiro , Iida Takaji , Kawabata Zen'ichiro 
Published: November 2009

ABSTRACTCyprinid herpesvirus 3 (CyHV-3), a lethal DNA virus that spreads in natural lakes and rivers, infects common carp and koi. We established a quantification method for CyHV-3 that includes a viral concentration method and quantitative PCR combined with an external standard virus. Viral concentration methods were compared using the cation-coated filter and ultrafiltration methods. The recovery of virus-like particles was similar for the two methods (cation-coated filter method, 44% ± 19%,n= 3; ultrafiltration method, 50% ± 3%,n= 3); however, the former method was faster and more suitable for routine determinations. The recovery of seeded CyHV-3 based on the cation-coated filter method varied by more than 3 orders of magnitude among the water samples. The recovery yield of CyHV-3 was significantly correlated with that of the seeded λ phage, and the average ratio of λ to the CyHV-3 recovery yield was 1.4, indicating that λ is useful as an external standard virus for determining the recovery yield of CyHV-3. Therefore, to quantify CyHV-3 in environmental water, a known amount of λ was added as an external standard virus to each water sample. Using this method, CyHV-3 DNA was detected in 6 of the 10 (60%) types of environmental water tested; the highest concentration of CyHV-3 DNA was 2 × 105copies liter−1. The lowest recovery limit of CyHV-3 DNA was 60 copies liter−1. This method is practical for monitoring CyHV-3 abundance in environmental water.
Tertiary Structure and Characterization of a Glycoside Hydrolase Family 44 Endoglucanase from
Clostridium acetobutylicum
Authors: Warner Christopher D. , Hoy Julie A. , Shilling Taran C. , Linnen Michael J. , Ginder Nathaniel D. , Ford Clark F. , Honzatko Richard B. , Reilly Peter J. 
Published: November 2009

ABSTRACT

A gene encoding a glycoside hydrolase family 44 (GH44) protein from
Clostridium acetobutylicum
ATCC 824 was synthesized and transformed into
Escherichia coli.
The previously uncharacterized protein was expressed with a C-terminal His tag and purified by nickel-nitrilotriacetic acid affinity chromatography. Crystallization and X-ray diffraction to a 2.2-Å resolution revealed a triose phosphate isomerase (TIM) barrel-like structure with additional Greek key and β-sandwich folds, similar to other GH44 crystal structures. The enzyme hydrolyzes cellotetraose and larger cellooligosaccharides, yielding an unbalanced product distribution, including some glucose. It attacks carboxymethylcellulose and xylan at approximately the same rates. Its activity on carboxymethylcellulose is much higher than that of the isolated
C. acetobutylicum
cellulosome. It also extensively converts lichenan to oligosaccharides of intermediate size and attacks Avicel to a limited extent. The enzyme has an optimal temperature in a 10-min assay of 55°C and an optimal pH of 5.0.
Traceability of Marketable Japanese Shoro in New Zealand: Using Multiplex PCR To Exploit Phylogeographic Variation among Taxa in the
Rhizopogon
Subgenus
Roseoli
Authors: Visnovsky Sandra B. , Guerin-Laguette Alexis , Wang Yun , Pitman Andrew R. 
Published: November 2009

ABSTRACT

Rhizopogon roseolus
Corda (synonym
Rhizopogon rubescens
Tul.), an economically important edible mushroom associated with the Pinaceae (mostly
Pinus
sp.), has a global distribution resulting from the introduction of exotic trees into the Southern Hemisphere for plantation forestry. However, the marketability of
R. roseolus
varies with the place of origin.
R. roseolus
strains cultivated in New Zealand from local carpophores for the Japanese market are morphologically and biologically distinct from those produced in Japan and are consequently considered less valuable. In this study, the ITS1-5.8S-ITS2 rRNA (internal transcribed spacer [ITS]) region was used to examine the phylogenetic relationships of
R. roseolus
and other closely related fungi belonging to
Rhizopogon
subgenus
Roseoli
to determine the genetic basis for phenotypic differences among
R. roseolus
isolates from different geographic regions. Phylogenetic comparison revealed phylogeographic variation within
Rhizopogon
subgenus
Roseoli
. Collections from the United States and Europe grouped into four distinct clades.
Rhizopogon roseolus
isolates found in New Zealand were closely related to those from the United States, likely due to introduction of
Pinus radiata
from its native California in the United States. In contrast, Japanese
R. roseolus
isolates clustered closely with European collections. Phylogenetic differences between Japanese and New Zealand
R. roseolus
isolates may explain the morphological and biological properties attributed to these geographical variants. The ITS region was subsequently used to design a multiplex PCR for the simultaneous identification of Japanese and New Zealand
R. roseolus
isolates to track the establishment of ectomycorrhiza on
P. radiata
seedlings inoculated with commercially valuable
R. roseolus
. This diagnostic demonstrated the first fruiting of Japanese shoro cultivated on
P. radiata
in the Southern Hemisphere.
Identification of theGeobacter metallireducensBamVW Two-Component System, Involved in Transcriptional Regulation of Aromatic Degradation
Authors: Juárez Javier F. , Zamarro María Teresa , Barragán María J. L. , Blázquez Blas , Boll Matthias , Kuntze Kevin , García José Luis , Díaz Eduardo , Carmona Manuel 
Published: November 2009

ABSTRACTRegulation of aromatic degradation in obligate anaerobes was studied in the Fe(III)-respiring model organismGeobacter metallireducensGS-15. A two-component system and a σ54-dependent promoter were identified that are both involved in the regulation of the gene coding for benzoate-coenzyme A ligase, catalyzing the initial step of benzoate degradation.
The Malaria Parasite Cyclic GMP-Dependent Protein Kinase Plays a Central Role in Blood-Stage Schizogony
Authors: Taylor Helen M. , McRobert Louisa , Grainger Munira , Sicard Audrey , Dluzewski Anton R. , Hopp Christine S. , Holder Anthony A. , Baker David A. 
Published: November 2009

ABSTRACT

A role for the
Plasmodium falciparum
cyclic GMP (cGMP)-dependent protein kinase (PfPKG) in gametogenesis in the malaria parasite was elucidated previously. In the present study we examined the role of PfPKG in the asexual blood-stage of the parasite life cycle, the stage that causes malaria pathology. A specific PKG inhibitor (compound 1, a trisubstituted pyrrole) prevented the progression of
P. falciparum
schizonts through to ring stages in erythrocyte invasion assays. Addition of compound 1 to ring-stage parasites allowed normal development up to 30 h postinvasion, and segmented schizonts were able to form. However, synchronized schizonts treated with compound 1 for ≥6 h became large and dysmorphic and were unable to rupture or liberate merozoites. To conclusively demonstrate that the effect of compound 1 on schizogony was due to its selective action on PfPKG, we utilized genetically manipulated
P. falciparum
parasites expressing a compound 1-insensitive PfPKG. The mutant parasites were able to complete schizogony in the presence of compound 1 but not in the presence of the broad-spectrum protein kinase inhibitor staurosporine. This shows that PfPKG is the primary target of compound 1 during schizogony and provides direct evidence of a role for PfPKG in this process. Discovery of essential roles for the
P. falciparum
PKG in both asexual and sexual development demonstrates that cGMP signaling is a key regulator of both of these crucial life cycle phases and defines this molecule as an exciting potential drug target for both therapeutic and transmission blocking action against malaria.
Evolutionary Dynamics of Mating-Type Loci of
Mycosphaerella
spp. Occurring on Banana
Authors: Arzanlou Mahdi , Crous Pedro W. , Zwiers Lute-Harm 
Published: November 2009

ABSTRACT

The devastating Sigatoka disease complex of banana is primarily caused by three closely related heterothallic fungi belonging to the genus
Mycosphaerella
:
M. fijiensis
,
M. musicola
, and
M. eumusae
. Previous phylogenetic work showing common ancestry led us to analyze the mating-type loci of these
Mycosphaerella
species occurring on banana. We reasoned that this might provide better insight into the evolutionary history of these species. PCR and chromosome-walking approaches were used to clone the mating-type loci of
M. musicola
and
M. eumusae
. Sequences were compared to the published mating-type loci of
M. fijiensis
and other
Mycosphaerella
spp., and a novel organization of the MAT loci was found. The mating-type loci of the examined
Mycosphaerella
species are expanded, containing two additional
Mycosphaerella
-specific genes in a unique genomic organization. The proteins encoded by these novel genes show a higher interspecies than intraspecies homology. Moreover,
M. fijiensis
,
M. musicola
, and
M. eumusae
contain two additional mating-type-like loci, containing parts of both
MAT
1
-
1
-
1
and
MAT
1
-
2
-
1
. The data indicate that
M. fijiensis
,
M. musicola
, and
M. eumusae
share an ancestor in which a fusion event occurred between
MAT
1
-
1
-
1
and
MAT
1
-
2
-
1
sequences and in which additional genes became incorporated into the idiomorph. The new genes incorporated have since then evolved independently in the MAT1-1 and MAT1-2 loci. Thus, these data are an example of the evolutionary dynamics of fungal MAT loci in general and show the great flexibility of the MAT loci of
Mycosphaerella
species in particular.
RNA Interference Silencing of a Major Lipid Droplet Protein Affects Lipid Droplet Size in
Chlamydomonas reinhardtii
Authors: Moellering Eric R. , Benning Christoph 
Published: November 2009

ABSTRACT

Eukaryotic cells store oils in the chemical form of triacylglycerols in distinct organelles, often called lipid droplets. These dynamic storage compartments have been intensely studied in the context of human health and also in plants as a source of vegetable oils for human consumption and for chemical or biofuel feedstocks. Many microalgae accumulate oils, particularly under conditions limiting to growth, and thus have gained renewed attention as a potentially sustainable feedstock for biofuel production. However, little is currently known at the cellular or molecular levels with regard to oil accumulation in microalgae, and the structural proteins and enzymes involved in the biogenesis, maintenance, and degradation of algal oil storage compartments are not well studied. Focusing on the model green alga
Chlamydomonas reinhardtii
, the accumulation of triacylglycerols and the formation of lipid droplets during nitrogen deprivation were investigated. Mass spectrometry identified 259 proteins in a lipid droplet-enriched fraction, among them a major protein, tentatively designated major lipid droplet protein (MLDP). This protein is specific to the green algal lineage of photosynthetic organisms. Repression of MLDP gene expression using an RNA interference approach led to increased lipid droplet size, but no change in triacylglycerol content or metabolism was observed.
Identification and Partial Characterization of a Dynamin-Like Protein, EhDLP1, from the Protist Parasite
Entamoeba histolytica
Authors: Jain Ruchi , Shrimal Shiteshu , Bhattacharya Sudha , Bhattacharya Alok 
Published: November 2009

ABSTRACT

The dynamin superfamily of proteins includes a large repertoire of evolutionarily conserved GTPases that interact with different subcellular organelle membranes in eukaryotes. Dynamins are thought to participate in a number of cellular processes involving membrane remodeling and scission. Dynamin-like proteins (DLPs) form a subfamily of this vast class and play important roles in cellular processes, such as mitochondrial fission, cytokinesis, and endocytosis. In the present study, a gene encoding a dynamin-like protein (EhDLP1) from the protist parasite
Entamoeba histolytica
was identified and the protein was partially characterized using a combination of
in silico
, biochemical, and imaging methods. The protein was capable of GTP binding and hydrolysis, lipid binding, and oligomerization. Immunofluorescence studies showed the protein to be associated with the nuclear membrane. A mutant of EhDLP1 lacking GTP binding and hydrolyzing activities did not associate with the nuclear membrane. The results suggest a nucleus-associated function for EhDLP1.
abc3
+
Encodes an Iron-Regulated Vacuolar ABC-Type Transporter in
Schizosaccharomyces pombe
Authors: Pouliot Benoît , Jbel Mehdi , Mercier Alexandre , Labbé Simon 
Published: November 2009

ABSTRACT

Studies have shown the fundamental contribution of the yeast vacuole as a site for storage and detoxification of metals. Whereas the transmembrane proteins responsible for iron transport into and out of the vacuole have been identified in
Saccharomyces cerevisiae
, less information is available concerning the mobilization of vacuolar iron stores in
Schizosaccharomyces pombe
. In this study, we report the identification of a gene designated
abc3

+

that encodes a protein which exhibits sequence homology with the ABCC subfamily of ATP-binding cassette transporters. The transcription of
abc3

+

is induced by low concentrations of iron but repressed by high levels of iron. The iron-mediated repression of
abc3

+

required a functional
fep1

+

gene. Chromatin immunoprecipitation assays showed that Fep1 associates with the
abc3

+

promoter in vivo, in an iron-dependent manner. Microscopic analyses revealed that a functional Abc3-green fluorescent protein localizes to the membrane vacuole when iron levels were low. Abc3 was required for growth in low-iron medium in the absence of the transport system mediated by Fio1 and Fip1.
abc3Δ
cells exhibited increased levels of expression of the
frp1

+

-encoded ferric reductase, suggesting a loss of Fep1 repression and, consequently, the activation of Fep1-regulated genes. When
abc3

+

was expressed using the
nmt1

+

promoter system, its induction led to a reduced transcriptional activity of the
frp1

+

gene. Because
S. pombe
does not possess vacuolar membrane-localized orthologs to
S. cerevisiae
Fth1, Fet5, and Smf3, our findings suggested that Abc3 may be responsible for mobilizing stored iron from the vacuole to the cytosol in response to iron deficiency.
Nucleosomes Are Depleted at the
VSG
Expression Site Transcribed by RNA Polymerase I in African Trypanosomes
Authors: Figueiredo Luisa M. , Cross George A. M. 
Published: November 2009

ABSTRACT

In most eukaryotes, RNA polymerase I (Pol I) exclusively transcribes long arrays of identical rRNA genes (ribosomal DNA [rDNA]). African trypanosomes have the unique property of using Pol I to also transcribe the variant surface glycoprotein
VSG
genes. VSGs are important virulence factors because their switching allows trypanosomes to escape the host immune system, a mechanism known as antigenic variation. Only one
VSG
is transcribed at a time from one of 15 bloodstream-form expression sites (BESs). Although it is clear that switching among BESs does not involve DNA rearrangements and that regulation is probably epigenetic, it remains unknown why BESs are transcribed by Pol I and what roles are played by chromatin structure and histone modifications. Using chromatin immunoprecipitation, micrococcal nuclease digestion, and chromatin fractionation, we observed that there are fewer nucleosomes at the active BES and that these are irregularly spaced compared to silent BESs. rDNA coding regions are also depleted of nucleosomes, relative to the rDNA spacer. In contrast, genes transcribed by Pol II are organized in a more compact, regularly spaced, nucleosomal structure. These observations provide new insight on antigenic variation by showing that chromatin remodeling is an intrinsic feature of BES regulation.
A Photostable Green Fluorescent Protein Variant for Analysis of Protein Localization in
Candida albicans
Authors: Zhang Chengda , Konopka James B. 
Published: November 2009

ABSTRACT

Fusions to the green fluorescent protein (GFP) are an effective way to monitor protein localization. However, altered codon usage in
Candida
species has delayed implementation of new variants. Examination of three new GFP variants in
Candida albicans
showed that one has higher signal intensity and increased resistance to photobleaching.
Control of Flagellar Gene Regulation in
Legionella pneumophila
and Its Relation to Growth Phase
Authors: Albert-Weissenberger Christiane , Sahr Tobias , Sismeiro Odile , Hacker Jörg , Heuner Klaus , Buchrieser Carmen 
Published: November 2009

ABSTRACT

The bacterial pathogen
Legionella pneumophila
responds to environmental changes by differentiation. At least two forms are well described: replicative bacteria are avirulent; in contrast, transmissive bacteria express virulence traits and flagella. Phenotypic analysis, Western blotting, and electron microscopy of mutants of the regulatory genes encoding RpoN, FleQ, FleR, and FliA demonstrated that flagellin expression is strongly repressed and that the mutants are nonflagellated in the transmissive phase. Transcriptome analyses elucidated that RpoN, together with FleQ, enhances transcription of 14 out of 31 flagellar class II genes, which code for the basal body, hook, and regulatory proteins. Unexpectedly, FleQ independent of RpoN enhances the transcription of
fliA
encoding sigma 28. Expression analysis of a
fliA
mutant showed that FliA activates three out of the five remaining flagellar class III genes and the flagellar class IV genes. Surprisingly, FleR does not induce but inhibits expression of at least 14 flagellar class III genes on the transcriptional level. Thus, we propose that flagellar class II genes are controlled by FleQ and RpoN, whereas the transcription of the class III gene
fliA
is controlled in a FleQ-dependent but RpoN-independent manner. However, RpoN and FleR might influence flagellin synthesis on a posttranscriptional level. In contrast to the commonly accepted view that enhancer-binding proteins such as FleQ always interact with RpoN to fullfill their regulatory functions, our results strongly indicate that FleQ regulates gene expression that is RpoN dependent and RpoN independent. Finally, FliA induces expression of flagellar class III and IV genes leading to the complete synthesis of the flagellum.
The Zinc-Responsive Regulator Zur Controls Expression of the Coelibactin Gene Cluster inStreptomyces coelicolor
Authors: Kallifidas Dimitris , Pascoe Ben , Owen Gillian A. , Strain-Damerell Claire M. , Hong Hee-Jeon , Paget Mark S. B. 
Published: November 2009

ABSTRACTStreptomyces coelicolormutants lacking the zinc-responsive Zur repressor are conditionally defective in sporulation, presumably due to the overexpression of one or more Zur target genes. Gene disruption analyses revealed that deregulation of previously known Zur targets was not responsible for the sporulation phenotype. We used microarrays to identify further Zur targets and discovered that Zur controls a cluster of genes predicted to direct synthesis of an uncharacterized siderophore-related non-ribosomally encoded peptide designated coelibactin. Disruption of a key coelibactin biosynthetic gene suppressed the Zur sporulation phenotype, suggesting that deregulation of coelibactin synthesis inhibits sporulation.
Structure-Function Analysis of IntDOT
Authors: Kim Seyeun , Swalla Brian M. , Gardner Jeffrey F. 
Published: November 2009

ABSTRACTCTnDOT integrase (IntDOT) is a member of the tyrosine family of site-specific DNA recombinases. IntDOT is unusual in that it catalyzes recombination between nonidentical sequences. Previous mutational analyses centered on mutants with substitutions of conserved residues in the catalytic (CAT) domain or residues predicted by homology modeling to be close to DNA in the core-binding (CB) domain. That work suggested that a conserved active-site residue (Arg I) of the CAT domain is missing and that some residues in the CB domain are involved in catalysis. Here we used a genetic approach and constructed anEscherichia coliindicator strain to screen for random mutations in IntDOT that disrupt integrative recombinationin vivo. Twenty-five IntDOT mutants were isolated and characterized for DNA binding, DNA cleavage, and DNA ligation activities. We found that mutants with substitutions in the amino-terminal (N) domain were catalytically active but defective in forming nucleoprotein complexes, suggesting that they have altered protein-protein interactions or altered interactions with DNA. Replacement of Ala-352 of the CAT domain disrupted DNA cleavage but not DNA ligation, suggesting that Ala-352 may be important for positioning the catalytic tyrosine (Tyr-381) during cleavage. Interestingly, our biochemical data and homology modeling of the CAT domain suggest that Arg-285 is the missing Arg I residue of IntDOT. The predicted position of Arg-285 shows it entering the active site from a position on the polypeptide backbone that is not utilized in other tyrosine recombinases. IntDOT may therefore employ a novel active-site architecture to catalyze recombination.
RecA-Independent DNA Damage Induction of
Mycobacterium tuberculosis ruvC
Despite an Appropriately Located SOS Box
Authors: Dawson Lisa F. , Dillury Joanna , Davis Elaine O. 
Published: November 2009

ABSTRACT

Mycobacterium tuberculosis ruvC
was induced by DNA damage in a Δ
recA
strain despite having an appropriately positioned SOS box to which LexA binds
in vitro
. An inducible transcript start mapped within the SOS box, and transcriptional fusions identified the promoter. Disruption of the SOS box did not prevent induction, indicating that an alternative mechanism plays a significant role in the control of
ruvC
expression.
Triskelion Structure of the Gli521 Protein, Involved in the Gliding Mechanism of
Mycoplasma mobile
Authors: Nonaka Takahiro , Adan-Kubo Jun , Miyata Makoto 
Published: November 2009

ABSTRACT

Mycoplasma mobile
binds to solid surfaces and glides smoothly and continuously by a unique mechanism. A huge protein, Gli521 (521 kDa), is involved in the gliding machinery, and it is localized in the cell neck, the base of the membrane protrusion. This protein is thought to have the role of force transmission. In this study, the Gli521 protein was purified from
M. mobile
cells, and its molecular shape was studied. Gel filtration analysis showed that the isolated Gli521 protein forms mainly a monomer in Tween 80-containing buffer and oligomers in Triton X-100-containing buffer. Rotary shadowing electron microscopy showed that the Gli521 monomer consisted of three parts: an oval, a rod, and a hook. The oval was 15 nm long by 11 nm wide, and the filamentous part composed of the rod and the hook was 106 nm long and 3 nm in diameter. The Gli521 molecules form a trimer, producing a “triskelion” reminiscent of eukaryotic clathrin, through association at the hook end. Image averaging of the central part of the triskelion suggested that there are stable and rigid structures. The binding site of a previously isolated monoclonal antibody on Gli521 images showed that the hook end and oval correspond to the C- and N-terminal regions, respectively. Partial digestion of Gli521 showed that the molecule could be divided into three domains, which we assigned to the oval, rod, and hook of the molecular image. The Gli521 molecule's role in the gliding mechanism is discussed.