bluuebunny/crossref_metadata_2025_split huggingface dataset:
The Host as the Driver of the Microbiota in the Gut and External Environment of Drosophila melanogaster
Authors: Wong Adam C.-N. , Luo Yuan , Jing Xiangfeng , Franzenburg Soeren , Bost Alyssa , Douglas Angela E. 
Published: July 2015

ABSTRACT

Most associations between animals and their gut microbiota are dynamic, involving sustained transfer of food-associated microbial cells into the gut and shedding of microorganisms into the external environment with feces, but the interacting effects of host and microbial factors on the composition of the internal and external microbial communities are poorly understood. This study on laboratory cultures of the fruit fly
Drosophila melanogaster
reared in continuous contact with their food revealed time-dependent changes of the microbial communities in the food that were strongly influenced by the presence and abundance of
Drosophila
. When germfree
Drosophila
eggs were aseptically added to nonsterile food, the microbiota in the food and flies converged to a composition dramatically different from that in fly-free food, showing that
Drosophila
has microbiota-independent effects on the food microbiota. The microbiota in both the flies that developed from unmanipulated eggs (bearing microorganisms) and the associated food was dominated by the bacteria most abundant on the eggs, demonstrating effective vertical transmission via surface contamination of eggs. Food coinoculated with a four-species defined bacterial community of
Acetobacter
and
Lactobacillus
species revealed the progressive elimination of
Lactobacillus
from the food bearing few or no
Drosophila
, indicating the presence of antagonistic interactions between
Acetobacter
and
Lactobacillus. Drosophila
at high densities ameliorated the
Acetobacter/Lactobacillus
antagonism, enabling
Lactobacillus
to persist. This study with
Drosophila
demonstrates how animals can have major, coordinated effects on the composition of microbial communities in the gut and immediate environment.
Influence of
prgH
on the Persistence of Ingested Salmonella enterica in the Leafhopper Macrosteles quadrilineatus
Authors: Dundore-Arias José Pablo , Groves Russell L. , Barak Jeri D. 
Published: July 2015

ABSTRACT

Phytophagous insects can encounter
Salmonella enterica
on contaminated plant surfaces and transmit externally adhered and internalized bacteria on and among leaves. Excretion of ingested
S. enterica
by the leafhopper
Macrosteles quadrilineatus
has been previously reported; however, the sites of persistence of ingested bacteria remain undetermined. Fluorescence microscopy revealed the presence and persistence of
S. enterica
in various organs of
M. quadrilineatus
fed an inoculated diet for 12 h and then moved to two consecutive noninoculated diets for a total of 48 h. Ingested
S. enterica
was predominantly observed in the filter chamber, midgut, and Malpighian tubules of
M. quadrilineatus
dissected immediately after acquisition and at 24- and 48-h post-acquisition access periods (post-AAPs). Additionally, we examined the potential roles of the
Salmonella
pathogenicity island 1 (SPI-1) and SPI-2 type III secretion systems (T3SSs) in the persistence and excretion of ingested
S. enterica
. In competition assays, a
prgH
mutant lacking a functional SPI-1 T3SS was recovered at significantly lower levels than the WT in insect homogenates at 24 h post-AAP, and complementation with
prgH
restored
S. enterica
persistence in
M. quadrilineatus
. Moreover, expression of
prgH
inside
M. quadrilineatus
was observed up to 48 post-AAP. No differences were observed between the WT and an
ssaK
mutant lacking a functional SPI-2 T3SS in insect homogenates or between the WT and either mutant in insect excretions. This study provides novel insight into the presence and persistence of
S. enterica
inside
M. quadrilineatus
and demonstrates that the SPI-1 T3SS influences the persistence of the pathogen in the gut of a potential vector.
Novel Strategies for Genomic Manipulation of Trichoderma reesei with the Purpose of Strain Engineering
Authors: Derntl Christian , Kiesenhofer Daniel P. , Mach Robert L. , Mach-Aigner Astrid R. 
Published: July 2015

ABSTRACT

The state-of-the-art procedure for gene insertions into
Trichoderma reesei
is a cotransformation of two plasmids, one bearing the gene of interest and the other a marker gene. This procedure yields up to 80% transformation efficiency, but both the number of integrated copies and the loci of insertion are unpredictable. This can lead to tremendous pleiotropic effects. This study describes the development of a novel transformation system for site-directed gene insertion based on auxotrophic markers. For this purpose, we tested the applicability of the genes
asl1
(encoding an enzyme of the
l
-arginine biosynthesis pathway), the
hah1
(encoding an enzyme of the
l
-lysine biosynthesis pathway), and the
pyr4
(encoding an enzyme of the uridine biosynthesis pathway). The developed transformation system yields strains with an additional gene at a defined locus that are prototrophic and ostensibly isogenic compared to their parental strain. A positive transformation rate of 100% was achieved due to the developed split-marker system. Additionally, a double-auxotrophic strain that allows multiple genomic manipulations was constructed, which facilitates metabolic engineering purposes in
T. reesei
. By employing
goxA
of
Aspergillus niger
as a reporter system, the influence on the expression of an inserted gene caused by the orientation of the insertion and the transformation strategy used could be demonstrated. Both are important aspects to be considered during strain engineering.
Differences in Physical and Biochemical Properties of Thermus scotoductus SA-01 Cultured with Dielectric or Convection Heating
Authors: Cockrell Allison L. , Fitzgerald Lisa A. , Cusick Kathleen D. , Barlow Daniel E. , Tsoi Stanislav D. , Soto Carissa M. , Baldwin Jeffrey W. , Dale Jason R. , Morris Robert E. , Little Brenda J. , Biffinger Justin C. 
Published: July 2015

ABSTRACTA thermophile,Thermus scotoductusSA-01, was cultured within a constant-temperature (65°C) microwave (MW) digester to determine if MW-specific effects influenced the growth and physiology of the organism. As a control,T. scotoductuscells were also cultured using convection heating at the same temperature as the MW studies. Cell growth was analyzed by optical density (OD) measurements, and cell morphologies were characterized using electron microscopy imaging (scanning electron microscopy [SEM] and transmission electron microscopy [TEM]), dynamic light scattering (DLS), and atomic force microscopy (AFM). Biophysical properties (i.e., turgor pressure) were also calculated with AFM, and biochemical compositions (i.e., proteins, nucleic acids, fatty acids) were analyzed by attenuated total reflectance-Fourier transform infrared (ATR-FTIR) spectroscopy. Gas chromatography-mass spectrometry (GC-MS) was used to analyze the fatty acid methyl esters extracted from cell membranes. Here we report successful cultivation of a thermophile with only dielectric heating. Under the MW conditions for growth, cell walls remained intact and there were no indications of membrane damage or cell leakage. Results from these studies also demonstrated thatT. scotoductuscells grown with MW heating exhibited accelerated growth rates in addition to altered cell morphologies and biochemical compositions compared with oven-grown cells.
Novel pH-Stable Glycoside Hydrolase Family 3 β-Xylosidase from Talaromyces amestolkiae: an Enzyme Displaying Regioselective Transxylosylation
Authors: Nieto-Domínguez Manuel , de Eugenio Laura I. , Barriuso Jorge , Prieto Alicia , Fernández de Toro Beatriz , Canales-Mayordomo Ángeles , Martínez María Jesús 
Published: July 2015

ABSTRACT

This paper reports on a novel β-xylosidase from the hemicellulolytic fungus
Talaromyces amestolkiae
. The expression of this enzyme, called BxTW1, could be induced by beechwood xylan and was purified as a glycoprotein from culture supernatants. We characterized the gene encoding this enzyme as an intronless gene belonging to the glycoside hydrolase gene family 3 (GH3). BxTW1 exhibited transxylosylation activity in a regioselective way. This feature would allow the synthesis of oligosaccharides or other compounds not available from natural sources, such as alkyl glycosides displaying antimicrobial or surfactant properties. Regioselective transxylosylation, an uncommon combination, makes the synthesis reproducible, which is desirable for its potential industrial application. BxTW1 showed high pH stability and Cu
2+
tolerance. The enzyme displayed a pI of 7.6, a molecular mass around 200 kDa in its active dimeric form, and

K
m

and
V
max
values of 0.17 mM and 52.0 U/mg, respectively, using commercial
p
-nitrophenyl-β-
d
-xylopyranoside as the substrate. The catalytic efficiencies for the hydrolysis of xylooligosaccharides were remarkably high, making it suitable for different applications in food and bioenergy industries.
NADP
+
-Preferring
d
-Lactate Dehydrogenase from Sporolactobacillus inulinus
Authors: Zhu Lingfeng , Xu Xiaoling , Wang Limin , Dong Hui , Yu Bo , Ma Yanhe 
Published: July 2015

ABSTRACT

Hydroxy acid dehydrogenases, including
l
- and
d
-lactate dehydrogenases (L-LDH and D-LDH), are responsible for the stereospecific conversion of 2-keto acids to 2-hydroxyacids and extensively used in a wide range of biotechnological applications. A common feature of LDHs is their high specificity for NAD
+
as a cofactor. An LDH that could effectively use NADPH as a coenzyme could be an alternative enzymatic system for regeneration of the oxidized, phosphorylated cofactor. In this study, a
d
-lactate dehydrogenase from a
Sporolactobacillus inulinus
strain was found to use both NADH and NADPH with high efficiencies and with a preference for NADPH as its coenzyme, which is different from the coenzyme utilization of all previously reported LDHs. The biochemical properties of the D-LDH enzyme were determined by X-ray crystal structural characterization and
in vivo
and
in vitro
enzymatic activity analyses. The residue Asn
174
was demonstrated to be critical for NADPH utilization. Characterization of the biochemical properties of this enzyme will contribute to understanding of the catalytic mechanism and provide referential information for shifting the coenzyme utilization specificity of 2-hydroxyacid dehydrogenases.
Interplay between Gliotoxin Resistance, Secretion, and the Methyl/Methionine Cycle in Aspergillus fumigatus
Authors: Owens Rebecca A. , O'Keeffe Grainne , Smith Elizabeth B. , Dolan Stephen K. , Hammel Stephen , Sheridan Kevin J. , Fitzpatrick David A. , Keane Thomas M. , Jones Gary W. , Doyle Sean 
Published: July 2015

ABSTRACT

Mechanistic studies on gliotoxin biosynthesis and self-protection in
Aspergillus fumigatus
, both of which require the gliotoxin oxidoreductase GliT, have revealed a rich landscape of highly novel biochemistries, yet key aspects of this complex molecular architecture remain obscure. Here we show that an
A. fumigatus
Δ
gliA
strain is completely deficient in gliotoxin secretion but still retains the ability to efflux bisdethiobis(methylthio)gliotoxin (BmGT). This correlates with a significant increase in sensitivity to exogenous gliotoxin because gliotoxin trapped inside the cell leads to (i) activation of the
gli
cluster, as disabling
gli
cluster activation, via
gliZ
deletion, attenuates the sensitivity of an
A. fumigatus
Δ
gliT
strain to gliotoxin, thus implicating cluster activation as a factor in gliotoxin sensitivity, and (ii) increased methylation activity due to excess substrate (dithiol gliotoxin) for the gliotoxin
bis
-thiomethyltransferase GtmA. Intracellular dithiol gliotoxin is oxidized by GliT and subsequently effluxed by GliA. In the absence of GliA, gliotoxin persists in the cell and is converted to BmGT, with levels significantly higher than those in the wild type. Similarly, in the Δ
gliT
strain, gliotoxin oxidation is impeded, and methylation occurs unchecked, leading to significant
S
-adenosylmethionine (SAM) depletion and
S
-adenosylhomocysteine (SAH) overproduction. This in turn significantly contributes to the observed hypersensitivity of
gliT
-deficient
A. fumigatus
to gliotoxin. Our observations reveal a key role for GliT in preventing dysregulation of the methyl/methionine cycle to control intracellular SAM and SAH homeostasis during gliotoxin biosynthesis and exposure. Moreover, we reveal attenuated GliT abundance in the
A. fumigatus
Δ
gliK
strain, but not the Δ
gliG
strain, following exposure to gliotoxin, correlating with relative sensitivities. Overall, we illuminate new systems interactions that have evolved in gliotoxin-producing, compared to gliotoxin-naive, fungi to facilitate their cellular presence.
Characterization of Virulence-Related Phenotypes in Candida Species of the CUG Clade
Authors: Priest Shelby J. , Lorenz Michael C. 
Published: July 2015

ABSTRACT

Candida
species cause a variety of mucosal and invasive infections and are, collectively, the most important human fungal pathogens in the developed world. The majority of these infections result from a few related species within the “CUG clade,” so named because they use a nonstandard translation for that codon. Some members of the CUG clade, such as
Candida albicans
, present significant clinical problems, whereas others, such as
Candida
(
Meyerozyma
)
guilliermondii
, are uncommon in patients. The differences in incidence rates are imperfectly correlated with virulence in animal models of infection, but comparative analyses that might provide an explanation for why some species are effective pathogens and others are not have been rare or incomplete. To better understand the phenotypic basis for these differences, we characterized eight CUG clade species—
C. albicans
,
C. dubliniensis
,
C. tropicalis
,
C. parapsilosis
,
Clavispora lusitaniae
,
M. guilliermondii
,
Debaryomyces hansenii
, and
Lodderomyces elongisporus
—for host-relevant phenotypes, including nutrient utilization, stress tolerance, morphogenesis, interactions with phagocytes, and biofilm formation. Two species deviated from expectations based on animal studies and human incidence.
C. dubliniensis
was quite robust, grouping in nearly all assays with the most virulent species,
C. albicans
and
C. tropicalis
, whereas
C. parapsilosis
was substantially less fit than might be expected from its clinical importance. These findings confirm the utility of
in vitro
measures of virulence and provide insight into the evolution of virulence in the CUG clade.
Heparan Sulfate Modulates Neutrophil and Endothelial Function in Antibacterial Innate Immunity
Authors: Xu Ding , Olson Joshua , Cole Jason N. , van Wijk Xander M. , Brinkmann Volker , Zychlinsky Arturo , Nizet Victor , Esko Jeffrey D. , Chang Yung-Chi 
Published: July 2015

ABSTRACT

Recently, we showed that endothelial heparan sulfate facilitates entry of a bacterial pathogen into the central nervous system. Here, we show that normal bactericidal activity of neutrophils is influenced by the sulfation pattern of heparan sulfate. Inactivation of heparan sulfate uronyl 2-
O
-sulfotransferase (Hs2st) in neutrophils substantially reduced their bactericidal activity, and Hs2st deficiency rendered mice more susceptible to systemic infection with the pathogenic bacterium group B
Streptococcus
. Specifically, altered sulfation of heparan sulfate in mutant neutrophils affected formation of neutrophil extracellular traps while not influencing phagocytosis, production of reactive oxygen species, or secretion of granular proteases. Heparan sulfate proteoglycan(s) is present in neutrophil extracellular traps, modulates histone affinity, and modulates their microbial activity. Hs2st-deficient brain endothelial cells show enhanced binding to group B
Streptococcus
and are more susceptible to apoptosis, likely contributing to the observed increase in dissemination of group B
Streptococcus
into the brain of Hs2st-deficient mice following intravenous challenge. Taken together, our data provide strong evidence that heparan sulfate from both neutrophils and the endothelium plays important roles in modulating innate immunity.
BB0744 Affects Tissue Tropism and Spatial Distribution of Borrelia burgdorferi
Authors: Wager Beau , Shaw Dana K. , Groshong Ashley M. , Blevins Jon S. , Skare Jon T. 
Published: July 2015

ABSTRACT

Borrelia burgdorferi
, the etiologic agent of Lyme disease, produces a variety of proteins that promote survival and colonization in both the
Ixodes
species vector and various mammalian hosts. We initially identified BB0744 (also known as p83/100) by screening for
B. burgdorferi
strain B31 proteins that bind to α
1
β
1
integrin and hypothesized that, given the presence of a signal peptide, BB0744 may be a surface-exposed protein. In contrast to this expectation, localization studies suggested that BB0744 resides in the periplasm. Despite its subsurface location, we were interested in testing whether BB0744 is required for borrelial pathogenesis. To this end, a
bb0744
deletion was isolated in a
B. burgdorferi
strain B31 infectious background, complemented, and queried for the role of BB0744 following experimental infection. A combination of bioluminescent imaging, cultivation of infected tissues, and quantitative PCR (qPCR) demonstrated that Δ
bb0744
mutant
B. burgdorferi
bacteria were attenuated in the ability to colonize heart tissue, as well as skin locations distal to the site of infection. Furthermore, qPCR indicated a significantly reduced spirochetal load in distal skin and joint tissue infected with Δ
bb0744
mutant
B. burgdorferi
. Complementation with
bb0744
restored infectivity, indicating that the defect seen in Δ
bb0744
mutant
B. burgdorferi
was due to the loss of BB0744. Taken together, these results suggest that BB0744 is necessary for tissue tropism, particularly in heart tissue, alters the ability of
B. burgdorferi
to disseminate efficiently, or both. Additional studies are warranted to address the mechanism employed by BB0744 that alters the pathogenic potential of
B. burgdorferi
.
A Candida albicans Strain Expressing Mammalian Interleukin-17A Results in Early Control of Fungal Growth during Disseminated Infection
Authors: Huppler Anna R. , Whibley Natasha , Woolford Carol A. , Childs Erin E. , He Jie , Biswas Partha S. , McGeachy Mandy J. , Mitchell Aaron P. , Gaffen Sarah L. 
Published: July 2015

ABSTRACT

Candida albicans
is normally a commensal fungus of the human mucosae and skin, but it causes life-threatening systemic infections in hospital settings in the face of predisposing conditions, such as indwelling catheters, abdominal surgery, or antibiotic use. Immunity to
C. albicans
involves various immune parameters, but the cytokine interleukin-17A (IL-17A) (also known as IL-17) has emerged as a centrally important mediator of immune defense against both mucosal and systemic candidiasis. Conversely, IL-17A has been suggested to enhance the virulence of
C. albicans
, indicating that it may exert detrimental effects on pathogenesis. In this study, we hypothesized that a
C. albicans
strain expressing IL-17A would exhibit reduced virulence
in vivo
. To that end, we created a
Candida
-optimized expression cassette encoding murine IL-17A, which was transformed into the DAY286 strain of
C. albicans
.
Candida
-derived IL-17A was indistinguishable from murine IL-17A in terms of biological activity and detection in standard enzyme-linked immunosorbent assays (ELISAs). Expression of IL-17A did not negatively impact the growth of these strains
in vitro
. Moreover, the IL-17A-expressing
C. albicans
strains showed significantly reduced pathogenicity in a systemic model of
Candida
infection, mainly evident during the early stages of disease. Collectively, these findings suggest that IL-17A mitigates the virulence of
C. albicans
.
Phenotypic Suppression of Streptomycin Resistance by Mutations in Multiple Components of the Translation Apparatus
Authors: Carr Jennifer F. , Lee Hannah J. , Jaspers Joshua B. , Dahlberg Albert E. , Jogl Gerwald , Gregory Steven T. 
Published: July 2015

ABSTRACT

The bacterial ribosome and its associated translation factors are frequent targets of antibiotics, and antibiotic resistance mutations have been found in a number of these components. Such mutations can potentially interact with one another in unpredictable ways, including the phenotypic suppression of one mutation by another. These phenotypic interactions can provide evidence of long-range functional interactions throughout the ribosome and its functional complexes and potentially give insights into antibiotic resistance mechanisms. In this study, we used genetics and experimental evolution of the thermophilic bacterium
Thermus thermophilus
to examine the ability of mutations in various components of the protein synthesis apparatus to suppress the streptomycin resistance phenotypes of mutations in ribosomal protein S12, specifically those located distant from the streptomycin binding site. With genetic selections and strain constructions, we identified suppressor mutations in EF-Tu or in ribosomal protein L11. Using experimental evolution, we identified amino acid substitutions in EF-Tu or in ribosomal proteins S4, S5, L14, or L19, some of which were found to also relieve streptomycin resistance. The wide dispersal of these mutations is consistent with long-range functional interactions among components of the translational machinery and indicates that streptomycin resistance can result from the modulation of long-range conformational signals.

IMPORTANCE
The thermophilic bacterium
Thermus thermophilus
has become a model system for high-resolution structural studies of macromolecular complexes, such as the ribosome, while its natural competence for transformation facilitates genetic approaches. Genetic studies of
T. thermophilus
ribosomes can take advantage of existing high-resolution crystallographic information to allow a structural interpretation of phenotypic interactions among mutations. Using a combination of genetic selections, strain constructions, and experimental evolution, we find that certain mutations in the translation apparatus can suppress the phenotype of certain antibiotic resistance mutations. Suppression of resistance can occur by mutations located distant in the ribosome or in a translation factor. These observations suggest the existence of long-range conformational signals in the translating ribosome, particularly during the decoding of mRNA.