bluuebunny/crossref_metadata_2025_split huggingface dataset:
Effects of Transgenic Hybrid Aspen Overexpressing Polyphenol Oxidase on Rhizosphere Diversity
Authors: Oliver Kathryn L. , Hamelin Richard C. , Hintz William E. 
Published: June 2008

ABSTRACT

This study assessed the potential effects of transgenic aspen overexpressing a polyphenol oxidase gene on diversity in rhizosphere communities. Cultivation-independent methods were used to better delineate bacterial and fungal populations associated with transgenic and nontransgenic trees. Gene libraries for the bacterial component of the rhizosphere were established using 16S rRNA and chaperonin-60 (CPN-60) gene sequences, while the fungal community was characterized using 18S rRNA gene sequences. The 16S rRNA gene libraries were dominated by alphaproteobacterial sequences, while the CPN-60 gene libraries were dominated by members of the
Bacteroidetes
/
Chlorobi
group. In both the CPN-60 and 16S rRNA libraries, there were differences in only minor components of the bacterial community between transgenic and unmodified trees, and no significant differences in species diversity were observed. Compared to the bacterial gene libraries, greater coverage of the underlying population was achieved with the fungal 18S rRNA libraries. Members of the
Zygomycota, Chytridiomycota, Ascomycota
, and
Basidiomycota
were recovered from both libraries. The dominant groups of fungi associated with each tree type were very similar, although there were some qualitative differences in the recovery of less-abundant fungi, likely as a result of the underlying heterogeneity of the fungal population. The methods employed revealed only minor differences between the bacterial and fungal communities associated with transgenic and unmodified trees.
High-Throughput, Single-Cell Analysis of Macrophage Interactions with Fluorescently LabeledBacillus anthracisSpores
Authors: Stojkovic Bojana , Torres Eric M. , Prouty Angela M. , Patel Hetal K. , Zhuang Lefan , Koehler Theresa M. , Ballard Jimmy D. , Blanke Steven R. 
Published: June 2008

ABSTRACTThe engulfment ofBacillus anthracisspores by macrophages is an important step in the pathogenesis of inhalational anthrax. However, from a quantitative standpoint, the magnitude to which macrophages interact with and engulf spores remains poorly understood, in part due to inherent limitations associated with commonly used assays. To analyze phagocytosis of spores by RAW264.7 macrophage-like cells in a high-throughput, nonsubjective manner, we labeledB. anthracisSterne 7702 spores prior to infection with an Alexa Fluor 488 amine-reactive dye in a manner that did not alter their germination, growth kinetics, and heat resistance. Using flow cytometry, large numbers of cells exposed to labeled spores were screened to concurrently discriminate infected from uninfected cells and surface-associated from internalized spores. These experiments revealed that spore uptake was not uniform, but instead, highly heterogeneous and characterized by subpopulations of infected and uninfected cells, as well as considerable variation in the number of spores associated with individual cells. Flow cytometry analysis of infections demonstrated that spore uptake was independent of the presence or absence of fetal bovine serum, a germinant that, while routinely used in vitro, complicates the interpretation of the outcome of infections. Two commonly used macrophage cell lines, RAW264.7 and J774A.1 cells, were compared, revealing significant disparity between these two models in the rates of phagocytosis of labeled spores. These studies provide the experimental framework for investigating mechanisms of spore phagocytosis, as well as quantitatively evaluating strategies for interfering with macrophage binding and uptake of spores.
Molecular Identification of a SNAP-25-Like SNARE Protein inParamecium
Authors: Schilde Christina , Lutter Kaya , Kissmehl Roland , Plattner Helmut 
Published: June 2008

ABSTRACTUsing database searches of the completedParamecium tetraureliamacronuclear genome with the metazoan SNAP-25 homologues, we identified a single 21-kDa Qb/c-SNARE in this ciliated protozoan, namedP. tetraureliaSNAP (PtSNAP), containing the characteristic dual heptad repeat SNARE motifs of SNAP-25. The presence of only a single Qb/c class SNARE inP. tetraureliais surprising in view of the multiple genome duplications and the high number of SNAREs found in other classes of this organism. As inferred from the subcellular localization of a green fluorescent protein (GFP) fusion construct, the protein is localized on a variety of intracellular membranes, and there is a large soluble pool of PtSNAP. Similarly, the PtSNAP that is detected with a specific antibody in fixed cells is associated with a number of intracellular membrane structures, including food vacuoles, the contractile vacuole system, and the sites of constitutive endo- and exocytosis. Surprisingly, using gene silencing, we could not assign a role to PtSNAP in the stimulated exocytosis of dense core vesicles (trichocysts), but we found an increased number of food vacuoles in PtSNAP-silenced cells. In conclusion, we identify PtSNAP as aParameciumhomologue of metazoan SNAP-25 that shows several divergent features, like resistance to cleavage by botulinum neurotoxins.
Saccharomyces cerevisiae
Afr1 Protein Is a Protein Phosphatase 1/Glc7-Targeting Subunit That Regulates the Septin Cytoskeleton during Mating
Authors: Bharucha Jennifer P. , Larson Jennifer R. , Konopka James B. , Tatchell Kelly 
Published: June 2008

ABSTRACT

Glc7, the type1 serine/threonine phosphatase in the yeast
Saccharomyces cerevisiae
, is targeted by auxiliary subunits to numerous locations in the cell, where it regulates a range of physiological pathways. We show here that the accumulation of Glc7 at mating projections requires Afr1, a protein required for the formation of normal projections.
AFR1
-null mutants fail to target Glc7 to projections, and an Afr1 variant specifically defective in binding to Glc7 [Afr1(V546A F548A)] forms aberrant projections. The septin filaments in mating projections of
AFR1
mutants initiate normally but then rearrange asymmetrically as the projection develops, suggesting that the Afr1-Glc7 holoenzyme may regulate the maintenance of septin complexes during mating. These results demonstrate a previously unknown role for Afr1 in targeting Glc7 to mating projections and in regulating the septin architecture during mating.
Dolichyl-Phosphate-Glucose Is Used To Make O-Glycans on Glycoproteins of
Trichomonas vaginalis
Authors: Grabińska Kariona A. , Ghosh Sudip K. , Guan Ziqiang , Cui Jike , Raetz Christian R. H. , Robbins Phillips W. , Samuelson John 
Published: June 2008

ABSTRACT

Trichomonas vaginalis
, the protist that causes vaginal itching, has a huge genome with numerous gene duplications. Recently we found that
Trichomonas
has numerous genes encoding putative dolichyl-phosphate-glucose (Dol-P-Glc) synthases (encoded by
ALG5
genes) despite the fact that
Trichomonas
lacks the glycosyltransferases (encoded by
ALG6
,
ALG8
, and
ALG10
genes) that use Dol-P-Glc to glucosylate dolichyl-PP-linked glycans. In addition,
Trichomonas
does not have a canonical
DPM1
gene, encoding a dolichyl-P-mannose (Dol-P-Man) synthase. Here we show
Trichomonas
membranes have roughly 300 times the Dol-P-Glc synthase activity of
Saccharomyces cerevisiae
membranes and about one-fifth the Dol-P-Man synthase activity of
Saccharomyces
membranes. Endogenous Dol-P-hexoses of
Trichomonas
are relatively abundant and contain 16 isoprene units. Five paralogous
Trichomonas ALG5
gene products have Dol-P-Glc synthase activity when expressed as recombinant proteins, and these
Trichomonas
Alg5s correct a carboxypeptidase N glycosylation defect in a
Saccharomyces alg5
mutant in vivo. A recombinant
Trichomonas
Dpm1, which is deeply divergent in its sequence, has Dol-P-Man synthase activity. When radiolabeled Dol-P-Glc is incubated with
Trichomonas
membranes, Glc is incorporated into reducing and nonreducing sugars of O-glycans of endogenous glycoproteins. To our knowledge, this is the first demonstration of Dol-P-Glc as a sugar donor for O-glycans on glycoproteins.
Involvement of
Saccharomyces cerevisiae
Avo3p/Tsc11p in Maintaining TOR Complex 2 Integrity and Coupling to Downstream Signaling
Authors: Ho Hsiang-Ling , Lee Hsin-Yi , Liao Hsien-Ching , Chen Mei-Yu 
Published: June 2008

ABSTRACT

Target-of-rapamycin proteins (TORs) are Ser/Thr kinases serving a central role in cell growth control. TORs function in two conserved multiprotein complexes, TOR complex 1 (TORC1) and TORC2; the mechanisms underlying their actions and regulation are not fully elucidated.
Saccharomyces
TORC2, containing Tor2p, Avo1p, Avo2p, Avo3p/Tsc11p, Bit61p, and Lst8p, regulates cell integrity and actin organization. Two classes of
avo3
temperature-sensitive (
avo3
ts
) mutants that we previously identified display cell integrity and actin defects, yet one is suppressed by
AVO1
while the other is suppressed by
AVO2
or
SLM1
, defining two TORC2 downstream signaling mechanisms, one mediated by Avo1p and the other by Avo2p/Slm1p. Employing these mutants, we explored Avo3p functions in TORC2 structure and signaling. By observing binary protein interactions using coimmunoprecipitation, we discovered that the composition of TORC2 and its recruitment of the downstream effectors Slm1p and Slm2p were differentially affected in different
avo3
ts
mutants. These molecular defects can be corrected only by expressing
AVO3
, not by expressing suppressors, highlighting the role of Avo3p as a structural and signaling scaffold for TORC2. Phenotypic modifications of
avo3
ts
mutants by deletion of individual Rho1p-GTPase-activating proteins indicate that two TORC2 downstream signaling branches converge on Rho1p activation. Our results also suggest that Avo2p/Slm1p-mediated signaling, but not Avo1p-mediated signaling, links to Rho1p activation specifically through the Rho1p-guanine nucleotide exchange factor Tus1p.
Expression Quantitative Trait Locus Mapping of
Toxoplasma
Genes Reveals Multiple Mechanisms for Strain-Specific Differences in Gene Expression
Authors: Boyle Jon P. , Saeij Jeroen P. J. , Harada Scott Y. , Ajioka Jim W. , Boothroyd John C. 
Published: June 2008

ABSTRACT

Toxoplasma gondii
is an intracellular parasite with a significant impact on human health, especially in cases where individuals are immunocompromised (e.g., due to human immunodeficiency virus/AIDS). In Europe and North America, only a few clonal genotypes appear to be responsible for the vast majority of
Toxoplasma
infections, and these clonotypes have been intensely studied to identify strain-specific phenotypes that may play a role in the manifestation of more-severe disease. To identify and genetically map strain-specific differences in gene expression, we have carried out expression quantitative trait locus analysis on
Toxoplasma
gene expression phenotypes by using spotted cDNA microarrays. This led to the identification of 16
Toxoplasma
genes that had significant and mappable strain-specific variation in hybridization intensity. While the analysis should identify both
cis
- and
trans
-mapping hybridization profiles, we identified only loci with strain-specific hybridization differences that are most likely due to differences in the locus itself (i.e.,
cis
mapping). Interestingly, a larger number of these
cis
-mapping genes than would be expected by chance encode either confirmed or predicted secreted proteins, many of which are known to localize to the specialized secretory organelles characteristic of members of the phylum Apicomplexa. For six of the
cis
-mapping loci, we determined if the strain-specific hybridization differences were due to true transcriptional differences or rather to strain-specific differences in hybridization efficiency because of extreme polymorphism and/or deletion, and we found examples of both scenarios.
Isolates of
Cryptococcus neoformans
from Infected Animals Reveal Genetic Exchange in Unisexual, α Mating Type Populations
Authors: Bui Tien , Lin Xiaorong , Malik Richard , Heitman Joseph , Carter Dee 
Published: June 2008

ABSTRACT

Sexual reproduction and genetic exchange are important for the evolution of fungal pathogens and for producing potentially infective spores. Studies to determine whether sex occurs in the pathogenic yeast
Cryptococcus neoformans
var
. grubii
have produced enigmatic results, however: basidiospores are the most likely infective propagules, and clinical isolates are fertile and genetically diverse, consistent with a sexual species, but almost all populations examined consist of a single mating type and have little evidence for genetic recombination. The choice of population is critical when looking for recombination, particularly when significant asexual propagation is likely and when latency may complicate assessing the origin of an isolate. We therefore selected isolates from infected animals living in the region of Sydney, Australia, with the assumption that the relatively short life spans and limited travels of the animal hosts would provide a very defined population. All isolates were mating type α and were of molecular genotype VNI or VNII. A lack of linkage disequilibrium among loci suggested that genetic exchange occurred within both genotype groups. Four diploid VNII isolates that produced filaments and basidium-like structures when cultured in proximity to an a mating type strain were found. Recent studies suggest that compatible α-α unions can occur in
C. neoformans
var.
neoformans
populations and in populations of the sibling species
Cryptococcus gattii
. As a mating type strains of
C. neoformans
var.
grubii
have never been found in Australia, or in the VNII molecular type globally, the potential for α-α unions is evidence that α-α unisexual mating maintains sexual recombination and diversity in this pathogen and may produce infectious propagules.
Contribution of Galactofuranose to the Virulence of the Opportunistic Pathogen
Aspergillus fumigatus
Authors: Schmalhorst Philipp S. , Krappmann Sven , Vervecken Wouter , Rohde Manfred , Müller Meike , Braus Gerhard H. , Contreras Roland , Braun Armin , Bakker Hans , Routier Françoise H. 
Published: June 2008

ABSTRACT

The filamentous fungus
Aspergillus fumigatus
is responsible for a lethal disease called invasive aspergillosis that affects immunocompromised patients. This disease, like other human fungal diseases, is generally treated by compounds targeting the primary fungal cell membrane sterol. Recently, glucan synthesis inhibitors were added to the limited antifungal arsenal and encouraged the search for novel targets in cell wall biosynthesis. Although galactomannan is a major component of the
A. fumigatus
cell wall and extracellular matrix, the biosynthesis and role of galactomannan are currently unknown. By a targeted gene deletion approach, we demonstrate that UDP-galactopyranose mutase, a key enzyme of galactofuranose metabolism, controls the biosynthesis of galactomannan and galactofuranose containing glycoconjugates. The
glfA
deletion mutant generated in this study is devoid of galactofuranose and displays attenuated virulence in a low-dose mouse model of invasive aspergillosis that likely reflects the impaired growth of the mutant at mammalian body temperature. Furthermore, the absence of galactofuranose results in a thinner cell wall that correlates with an increased susceptibility to several antifungal agents. The UDP-galactopyranose mutase thus appears to be an appealing adjunct therapeutic target in combination with other drugs against
A. fumigatus
. Its absence from mammalian cells indeed offers a considerable advantage to achieve therapeutic selectivity.
Characterization of Kl
GRR1
and
SMS1
Genes, Two New Elements of the Glucose Signaling Pathway of
Kluyveromyces lactis
Authors: Hnatova Martina , Wésolowski-Louvel Micheline , Dieppois Guenaëlle , Deffaud Julien , Lemaire Marc 
Published: June 2008

ABSTRACT

The expression of the major glucose transporter gene,
RAG1
, is induced by glucose in
Kluyveromyces lactis
. This regulation involves several pathways, including one that is similar to Snf3/Rgt2-ScRgt1 in
Saccharomyces cerevisiae
. We have identified missing key components of the
K. lactis
glucose signaling pathway by comparison to the same pathway of
S. cerevisiae
. We characterized a new mutation,
rag19
, which impairs
RAG1
regulation. The Rag19 protein is 43% identical to the F-box protein ScGrr1 of
S. cerevisiae
and is able to complement an Sc
grr1
mutation. In the
K. lactis
genome, we identified a single gene,
SMS1
(for
s
imilar to
M
th1 and
S
td1), that encodes a protein showing an average of 50% identity with Mth1 and Std1, regulators of the ScRgt1 repressor. The suppression of the
rag4
(glucose sensor),
rag8
(casein kinase I), and
rag19
mutations by the Δ
sms1
deletion, together with the restoration of
RAG1
transcription in the double mutants, demonstrates that Sms1 is a negative regulator of
RAG1
expression and is acting downstream of Rag4, Rag8, and Rag19 in the cascade. We report that Sms1 regulates KlRgt1 repressor activity by preventing its phosphorylation in the absence of glucose, and that
SMS1
is regulated by glucose, both at the transcriptional and the posttranslational level. Two-hybrid interactions of Sms1 with the glucose sensor and KlRgt1 repressor suggest that Sms1 mediates the glucose signal from the plasma membrane to the nucleus. All of these data demonstrated that Sms1 was the
K. lactis
homolog of
MTH1
and
STD1
of
S. cerevisiae
. Interestingly,
MTH1
and
STD1
were unable to complement a Δ
sms1
mutation.
Transfer of the First Arabinofuranose Residue to Galactan Is Essential for
Mycobacterium smegmatis
Viability
Authors: Shi Libin , Zhou Roukun , Liu Zhentong , Lowary Todd L. , Seeberger Peter H. , Stocker Bridget L. , Crick Dean C. , Khoo Kay-Hooi , Chatterjee Delphi 
Published: June 2008

ABSTRACT

The mycobacterial arabinan is an elaborate component of the cell wall with multiple glycosyl linkages and no repeating units. In
Mycobacterium
spp., the Emb proteins (EmbA, EmbB, and EmbC) have been identified as putative mycobacterial arabinosyltransferases implicated in the biogenesis of the cell wall arabinan. Furthermore, it is now evident that the EmbA and EmbB proteins are involved in the assembly of the nonreducing terminal motif of arabinogalactan and EmbC is involved in transferring arabinose, perhaps in the early stage of arabinan synthesis in lipoarabinomannan. It has also been shown that the Emb proteins are a target of the antimycobacterial drug ethambutol (EMB). In the search for additional mycobacterial arabinosyltransferases in addition to the Emb proteins, we disrupted MSMEG_6386 (an orthologue of Rv3792 and a gene upstream of
embC
) in
Mycobacterium smegmatis
. Allelic exchange at the chromosomal MSMEG_6386 locus of
M. smegmatis
could only be achieved in the presence of a rescue plasmid carrying a functional copy of MSMEG_6386 or Rv3792, strongly suggesting that MSMEG_6386 is essential. An in vitro arabinosyltransferase assay using a membrane preparation from
M. smegmatis
expressing Rv3792 and synthetic β-
d
-Gal
f
-(1→5)-β-
d
-Gal
f
-(1→6)-β-
d
-Gal
f
-octyl and β-
d
-Gal
f
-(1→6)-β-
d
-Gal
f
-(1→5)-β-
d
-Gal
f
-octyl showed that Rv3792 gene product can transfer an arabinose residue to the C-5 position of the internal 6-linked galactose. The reactions were insensitive to EMB, and when α-
d
-Man
p
-(1→6)-α-
d
-Man
p
-(1→6)-α-
d
-Man
p
-octylthiomethyl was used as an acceptor, no product was formed. These observations indicate that transfer of the first arabinofuranose residue to galactan is essential for
M. smegmatis
viability.
Replacement of Adenylate Cyclase Toxin in a Lineage of
Bordetella bronchiseptica
Authors: Buboltz Anne M. , Nicholson Tracy L. , Parette Mylisa R. , Hester Sara E. , Parkhill Julian , Harvill Eric T. 
Published: June 2008

ABSTRACT

Bordetella bronchiseptica
is a gram-negative respiratory pathogen that infects a wide range of hosts and causes a diverse spectrum of disease. This diversity is likely affected by multiple factors, such as host immune status, polymicrobial infection, and strain diversity. In a murine model of infection, we found that the virulence of
B. bronchiseptica
strains, as measured by the mean lethal dose, varied widely. Strain 253 was less virulent than the typically studied strain, RB50. Transcriptome analysis showed that
cyaA
, the gene encoding adenylate cyclase toxin (CyaA), was the most downregulated transcript identified in strain 253 compared to that in strain RB50. Comparative genomic hybridization and genome sequencing of strain 253 revealed that the
cya
locus, which encodes, activates, and secretes CyaA, was replaced by an operon (
ptp
) predicted to encode peptide transport proteins. Other
B. bronchiseptica
strains from the same phylogenetic lineage as that of strain 253 also lacked the
cya
locus, contained the
ptp
genes, and were less virulent than strain RB50. Although the loss of CyaA would be expected to be counterselected since it is conserved among the classical bordetellae and believed to be important to their success, our data indicate that the loss of this toxin and the gain of the
ptp
genes occurred in an ancestral strain that then expanded into a lineage. This suggests that there may be ecological niches in which CyaA is not critical for the success of
B. bronchiseptica
.
Characterization of Lipooligosaccharide-Biosynthetic Loci ofCampylobacter jejuniReveals New Lipooligosaccharide Classes: Evidence of Mosaic Organizations
Authors: Parker Craig T. , Gilbert Michel , Yuki Nobuhiro , Endtz Hubert P. , Mandrell Robert E. 
Published: June 2008

ABSTRACTThe lipooligosaccharide (LOS) biosynthesis region is one of the more variable genomic regions between strains ofCampylobacter jejuni. Indeed, eight classes of LOS biosynthesis loci have been established previously based on gene content and organization. In this study, we characterize additional classes of LOS biosynthesis loci and analyze various mechanisms that result in changes to LOS structures. To gain further insights into the genomic diversity ofC. jejuniLOS biosynthesis region, we sequenced the LOS biosynthesis loci of 15 strains that possessed gene content that was distinct from the eight classes. This analysis identified 11 new classes of LOS loci that exhibited examples of deletions and insertions of genes and cassettes of genes found in other LOS classes or capsular biosynthesis loci leading to mosaic LOS loci. The sequence analysis also revealed both missense mutations leading to “allelic” glycosyltransferases and phase-variable and non-phase-variable gene inactivation by the deletion or insertion of bases. Specifically, we demonstrated that gene inactivation is an important mechanism for altering the LOS structures of strains possessing the same class of LOS biosynthesis locus. Together, these observations suggest that LOS biosynthesis region is a hotspot for genetic exchange and variability, often leading to changes in the LOS produced.