bluuebunny/crossref_metadata_2025_split huggingface dataset:
Functional Cell Surface Display and Controlled Secretion of Diverse Agarolytic Enzymes by Escherichia coli with a Novel Ligation-Independent Cloning Vector Based on the Autotransporter YfaL
Authors: Ko Hyeok-Jin , Park Eunhye , Song Joseph , Yang Taek Ho , Lee Hee Jong , Kim Kyoung Heon , Choi In-Geol 
Published: February 2012

ABSTRACT

Autotransporters have been employed as the anchoring scaffold for cell surface display by replacing their passenger domains with heterologous proteins to be displayed. We adopted an autotransporter (YfaL) of
Escherichia coli
for the cell surface display system. The critical regions in YfaL for surface display were identified for the construction of a ligation-independent cloning (LIC)-based display system. The designed system showed no detrimental effect on either the growth of the host cell or overexpressing heterologous proteins on the cell surface. We functionally displayed monomeric red fluorescent protein (mRFP1) as a reporter protein and diverse agarolytic enzymes from
Saccharophagus degradans
2-40, including Aga86C and Aga86E, which previously had failed to be functional expressed. The system could display different sizes of proteins ranging from 25.3 to 143 kDa. We also attempted controlled release of the displayed proteins by incorporating a tobacco etch virus protease cleavage site into the C termini of the displayed proteins. The maximum level of the displayed protein was 6.1 × 10
4
molecules per a single cell, which corresponds to 5.6% of the entire cell surface of actively growing
E. coli
.
Versatile Transformation System That Is Applicable to both Multiple Transgene Expression and Gene Targeting for Thraustochytrids
Authors: Sakaguchi Keishi , Matsuda Takanori , Kobayashi Takumi , Ohara Jun-ichiro , Hamaguchi Rie , Abe Eriko , Nagano Naoki , Hayashi Masahiro , Ueda Mayumi , Honda Daiske , Okita Yuji , Taoka Yousuke , Sugimoto Shinichi , Okino Nozomu , Ito Makoto 
Published: February 2012

ABSTRACT

A versatile transformation system for thraustochytrids, a promising producer for polyunsaturated fatty acids and fatty acid-derived fuels, was established. G418, hygromycin B, blasticidin, and zeocin inhibited the growth of thraustochytrids, indicating that multiple selectable marker genes could be used in the transformation system. A neomycin resistance gene (
neo
r
), driven with an ubiquitin or an EF-1α promoter-terminator from
Thraustochytrium aureum
ATCC 34304, was introduced into representatives of two thraustochytrid genera,
Aurantiochytrium
and
Thraustochytrium
. The
neo
r
marker was integrated into the chromosomal DNA by random recombination and then functionally translated into
neo
r
mRNA. Additionally, we confirmed that another two genera,
Parietichytrium
and
Schizochytrium
, could be transformed by the same method. By this method, the enhanced green fluorescent protein was functionally expressed in thraustochytrids. Meanwhile,
T. aureum
ATCC 34304 could be transformed by two 18S ribosomal DNA-targeting vectors, designed to cause single- or double-crossover homologous recombination. Finally, the fatty acid Δ5 desaturase gene was disrupted by double-crossover homologous recombination in
T. aureum
ATCC 34304, resulting in an increase of dihomo-γ-linolenic acid (C
20:3n-6
) and eicosatetraenoic acid (C
20:4n-3
), substrates for Δ5 desaturase, and a decrease of arachidonic acid (C
20:4n-6
) and eicosapentaenoic acid (C
20:5n-3
), products for the enzyme. These results clearly indicate that a versatile transformation system which could be applicable to both multiple transgene expression and gene targeting was established for thraustochytrids.
Transcriptome Mapping of pAR060302, a
bla
CMY-2
-Positive Broad-Host-Range IncA/C Plasmid
Authors: Lang Kevin S. , Danzeisen Jessica L. , Xu Wayne , Johnson Timothy J. 
Published: February 2012

ABSTRACT

The multidrug resistance-encoding plasmids belonging to the IncA/C incompatibility group have recently emerged among
Escherichia coli
and
Salmonella enterica
strains in the United States. These plasmids have a unique genetic structure compared to other enterobacterial plasmid types, a broad host range, and a propensity to acquire large numbers of antimicrobial resistance genes via their accessory regions. Using
E. coli
strain DH5α harboring the prototype IncA/C plasmid pAR060302, we sought to define the baseline transcriptome of IncA/C plasmids under laboratory growth and in the face of selective pressure. The effects of ampicillin, florfenicol, or streptomycin exposure were compared to those on cells left untreated at logarithmic phase using Illumina platform-based RNA sequencing (RNA-Seq). Under growth in Luria-Bertani broth lacking antibiotics, much of the backbone of pAR060302 was transcriptionally inactive, including its putative transfer regions. A few plasmid backbone genes of interest were highly transcribed, including genes of a putative toxin-antitoxin system and an H-NS-like transcriptional regulator. In contrast, numerous genes within the accessory regions of pAR060302 were highly transcribed, including the resistance genes
floR
,
bla
CMY-2
,
aadA
, and
aacA
. Treatment with ampicillin or streptomycin resulted in no genes being differentially expressed compared to controls lacking antibiotics, suggesting that many of the resistance-associated genes are not differentially expressed due to exposure to these antibiotics. In contrast, florfenicol treatment resulted in the upregulation of
floR
and numerous chromosomal genes. Overall, the transcriptome mapping of pAR060302 suggests that it mitigates the fitness costs of carrying resistance-associated genes through global regulation with its transcriptional regulators.
Antimicrobial Mechanism of Monocaprylate
Authors: Hyldgaard Morten , Sutherland Duncan S. , Sundh Maria , Mygind Tina , Meyer Rikke Louise 
Published: February 2012

ABSTRACT

Monoglyceride esters of fatty acids occur naturally and encompass a broad spectrum of antimicrobial activity. Monocaprylate is generally regarded as safe (GRAS) and can function both as an emulsifier and as a preservative in food. However, knowledge about its mode of action is lacking. The aim of this study was therefore to elucidate the mechanism behind monocaprylate's antimicrobial effect. The cause of cell death in
Escherichia coli
,
Staphylococcus xylosus
, and
Zygosaccharomyces bailii
was investigated by examining monocaprylate's effect on cell structure, membrane integrity, and its interaction with model membranes. Changes in cell structure were visible by atomic force microscopy (AFM), and propidium iodide staining showed membrane disruption, indicating the membrane as a site of action. This indication was confirmed by measuring calcein leakage from membrane vesicles exposed to monocaprylate. AFM imaging of supported lipid bilayers visualized the integration of monocaprylate into the liquid disordered, and not the solid ordered, phase of the membrane. The integration of monocaprylate was confirmed by quartz crystal microbalance measurements, showing an abrupt increase in mass and hydration of the membrane after exposure to monocaprylate above a threshold concentration. We hypothesize that monocaprylate destabilizes membranes by increasing membrane fluidity and the number of phase boundary defects. The sensitivity of cells to monocaprylate will therefore depend on the lipid composition, fluidity, and curvature of the membrane.
Leptospire Genomic Diversity Revealed by Microarray-Based Comparative Genomic Hybridization
Authors: Eribo Broderick , Mingmongkolchai Sirima , Yan Tingfen , Dubbs Padunsri , Nelson Karen E. 
Published: February 2012

ABSTRACT

Comparative genomic hybridization was used to compare genetic diversity of five strains of
Leptospira
(
Leptospira interrogans
serovars Bratislava, Canicola, and Hebdomadis and
Leptospira kirschneri
serovars Cynopteri and Grippotyphosa). The array was designed based on two available sequenced
Leptospira
reference genomes, those of
L. interrogans
serovar Copenhageni and
L. interrogans
serovar Lai. A comparison of genetic contents showed that
L. interrogans
serovar Bratislava was closest to the reference genomes while
L. kirschneri
serovar Grippotyphosa had the least similarity to the reference genomes. Cluster analysis indicated that
L. interrogans
serovars Bratislava and Hebdomadis clustered together first, followed by
L. interrogans
serovar Canicola, before the two
L. kirschneri
strains. Confirmed/potential virulence factors identified in previous research were also detected in the tested strains.
Aceticlastic and NaCl-Requiring Methanogen “Methanosaeta pelagica” sp. nov., Isolated from Marine Tidal Flat Sediment
Authors: Mori Koji , Iino Takao , Suzuki Ken-Ichiro , Yamaguchi Kaoru , Kamagata Yoichi 
Published: February 2012

ABSTRACT

Among methanogens, only 2 genera,
Methanosaeta
and
Methanosarcina
, are known to contribute to methanogenesis from acetate, and
Methanosaeta
is a specialist that uses acetate specifically. However,
Methanosaeta
strains so far have mainly been isolated from anaerobic digesters, despite the fact that it is widespread, not only in anaerobic methanogenic reactors and freshwater environments, but also in marine environments, based upon extensive 16S rRNA gene-cloning analyses. In this study, we isolated an aceticlastic methanogen, designated strain 03d30q
T
, from a tidal flat sediment. Phylogenetic analyses based on 16S rRNA and
mcrA
genes revealed that the isolate belongs to the genus
Methanosaeta
. Unlike the other known
Methanosaeta
species, this isolate grows at Na
+
concentrations of 0.20 to 0.80 M, with an optimum concentration of 0.28 M. Quantitative estimation using real-time PCR detected the 16S rRNA gene of the genus
Methanosaeta
in the marine sediment, and relative abundance ranged from 3.9% to 11.8% of the total archaeal 16S rRNA genes. In addition, the number of
Methanosaeta
organisms increased with increasing depth and was much higher than that of
Methanosarcina
organisms, suggesting that aceticlastic methanogens contribute to acetate metabolism to a greater extent than previously thought in marine environments, where sulfate-reducing acetate oxidation prevails. This is the first report on marine
Methanosaeta
species, and based on phylogenetic and characteristic studies, the name “
Methanosaeta pelagica
” sp. nov. is proposed for this novel species, with type strain 03d30q.
Impact of Xynthia Tempest on Viral Contamination of Shellfish
Authors: Grodzki Marco , Ollivier Joanna , Le Saux Jean-Claude , Piquet Jean-Côme , Noyer Mathilde , Le Guyader Françoise S. 
Published: February 2012

ABSTRACT
Viral contamination in oyster and mussel samples was evaluated after a massive storm with hurricane wind named “Xynthia tempest” destroyed a number of sewage treatment plants in an area harboring many shellfish farms. Although up to 90% of samples were found to be contaminated 2 days after the disaster, detected viral concentrations were low. A 1-month follow-up showed a rapid decrease in the number of positive samples, even for norovirus.
One-Step Triplex High-Resolution Melting Analysis for Rapid Identification and Simultaneous Subtyping of Frequently Isolated Salmonella Serovars
Authors: Zeinzinger Josef , Pietzka Ariane T. , Stöger Anna , Kornschober Christian , Kunert Renate , Allerberger Franz , Mach Robert , Ruppitsch Werner 
Published: February 2012

ABSTRACT

Salmonellosis is one of the most important food-borne diseases worldwide. For outbreak investigation and infection control, accurate and fast subtyping methods are essential. A triplex gene-scanning assay was developed and evaluated for serotype-specific subtyping of
Salmonella enterica
isolates based on specific single-nucleotide polymorphisms in fragments of
fljB
,
gyrB
, and
ycfQ
. Simultaneous gene scanning of
fljB
,
gyrB
, and
ycfQ
by high-resolution melting-curve analysis of 417
Salmonella
isolates comprising 46 different serotypes allowed the unequivocal, simple, and fast identification of 37 serotypes. Identical melting-curve profiles were obtained in some cases from
Salmonella enterica
serotype Enteritidis and
Salmonella enterica
serotype Dublin, in all cases from
Salmonella enterica
serotype Ohio and
Salmonella enterica
serotype Rissen, from
Salmonella enterica
serotype Mbandaka and
Salmonella enterica
serotype Kentucky, and from
Salmonella enterica
serotype Bredeney,
Salmonella enterica
serotype Give, and
Salmonella enterica
serotype Schwarzengrund. To differentiate the most frequent
Salmonella
serotype, Enteritidis, from some
S
. Dublin isolates, an additional single PCR assay was developed for specific identification of
S
. Enteritidis. The closed-tube triplex high-resolution melting-curve assay developed, in combination with an
S
. Enteritidis-specific PCR, represents an improved protocol for accurate, cost-effective, simple, and fast subtyping of 39
Salmonella
serotypes. These 39 serotypes represent more than 94% of all human and more than 85% of all nonhuman
Salmonella
isolates (including isolates from veterinary, food, and environmental samples) obtained in the years 2008 and 2009 in Austria.
Induction of Holomycin Production and Complex Metabolic Changes by the
argR
Mutation in Streptomyces clavuligerus NP1
Authors: Yin Hua , Xiang Sihai , Zheng Jianting , Fan Keqiang , Yu Tingting , Yang Xu , Peng Yanfeng , Wang Haibin , Feng Deqin , Luo Yuanming , Bai Hua , Yang Keqian 
Published: February 2012

ABSTRACT

In bacteria, arginine biosynthesis is tightly regulated by a universally conserved regulator, ArgR, which regulates the expression of arginine biosynthetic genes, as well as other important genes. Disruption of
argR
in
Streptomyces clavuligerus
NP1 resulted in complex phenotypic changes in growth and antibiotic production levels. To understand the metabolic changes underlying the phenotypes, comparative proteomic studies were carried out between NP1 and its
argR
disruption mutant (designated CZR). In CZR, enzymes involved in holomycin biosynthesis were overexpressed; this is consistent with its holomycin overproduction phenotype. The effects on clavulanic acid (CA) biosynthesis are more complex. Several proteins from the CA cluster were moderately overexpressed, whereas several proteins from the 5S clavam biosynthetic cluster and from the paralog cluster of CA and 5S clavam biosynthesis were severely downregulated. Obvious changes were also detected in primary metabolism, which are mainly reflected in the altered expression levels of proteins involved in acetyl-coenzyme A (CoA) and cysteine biosynthesis. Since acetyl-CoA and cysteine are precursors for holomycin synthesis, overexpression of these proteins is consistent with the holomycin overproduction phenotype. The complex interplay between primary and secondary metabolism and between secondary metabolic pathways were revealed by these analyses, and the insights will guide further efforts to improve production levels of CA and holomycin in
S. clavuligerus
.
Increased Competitive Fitness of Bacillus subtilis under Nonsporulating Conditions via Inactivation of Pleiotropic Regulators AlsR, SigD, and SigW
Authors: Nicholson Wayne L. 
Published: February 2012

ABSTRACT

Previous studies implicated loss of motility and mutations of the
alsR
and
sigW
regulatory genes in enhanced fitness of the
Bacillus subtilis
evolved strain WN716 over that of its ancestral strain WN624. The fitness of strains carrying knockout mutations
alsR
::
spc
,
sigD
::
kan
, and/or
sigW
::
erm
was measured and compared to that of the congenic ancestral strain by competition experiments.
Corals Form Characteristic Associations with Symbiotic Nitrogen-Fixing Bacteria
Authors: Lema Kimberley A. , Willis Bette L. , Bourne David G. 
Published: February 2012

ABSTRACT

The complex symbiotic relationship between corals and their dinoflagellate partner
Symbiodinium
is believed to be sustained through close associations with mutualistic bacterial communities, though little is known about coral associations with bacterial groups able to fix nitrogen (diazotrophs). In this study, we investigated the diversity of diazotrophic bacterial communities associated with three common coral species (
Acropora millepora
,
Acropora muricata
, and
Pocillopora damicormis
) from three midshelf locations of the Great Barrier Reef (GBR) by profiling the conserved subunit of the
nifH
gene, which encodes the dinitrogenase iron protein. Comparisons of diazotrophic community diversity among coral tissue and mucus microenvironments and the surrounding seawater revealed that corals harbor diverse
nifH
phylotypes that differ between tissue and mucus microhabitats. Coral mucus
nifH
sequences displayed high heterogeneity, and many bacterial groups overlapped with those found in seawater. Moreover, coral mucus diazotrophs were specific neither to coral species nor to reef location, reflecting the ephemeral nature of coral mucus. In contrast, the dominant diazotrophic bacteria in tissue samples differed among coral species, with differences remaining consistent at all three reefs, indicating that coral-diazotroph associations are species specific. Notably, dominant diazotrophs for all coral species were closely related to the bacterial group rhizobia, which represented 71% of the total sequences retrieved from tissue samples. The species specificity of coral-diazotroph associations further supports the coral holobiont model that bacterial groups associated with corals are conserved. Our results suggest that, as in terrestrial plants, rhizobia have developed a mutualistic relationship with corals and may contribute fixed nitrogen to
Symbiodinium
.
Emission of Methane by Eudrilus eugeniae and Other Earthworms from Brazil
Authors: Depkat-Jakob Peter S. , Hunger Sindy , Schulz Kristin , Brown George G. , Tsai Siu M. , Drake Harold L. 
Published: February 2012

ABSTRACT

Earthworms emit denitrification-derived nitrous oxide and fermentation-derived molecular hydrogen. The present study demonstrated that the earthworm
Eudrilus eugeniae
, obtained in Brazil, emitted methane. Other worms displayed a lesser or no capacity to emit methane. Gene and transcript analyses of
mcrA
(encoding the alpha subunit of methyl-CoM reductase) in gut contents of
E. eugeniae
suggested that
Methanosarcinaceae
,
Methanobacteriaceae
, and
Methanomicrobiaceae
might be associated with this emission.
Molecular Characterization of the Putative Transcription Factor SebA Involved in Virulence in Aspergillus fumigatus
Authors: Dinamarco Taísa Magnani , Almeida Ricardo S. , Alves de Castro Patrícia , Brown Neil Andrew , dos Reis Thaila Fernanda , Zambelli Ramalho Leandra Naira , Savoldi Marcela , Goldman Maria Helena S. , Goldman Gustavo Henrique 
Published: February 2012

ABSTRACT

Aspergillus fumigatus
is a major opportunistic pathogen and allergen of mammals. Nutrient sensing and acquisition mechanisms, as well as the capability to cope with different stressing conditions, are essential for
A. fumigatus
virulence and survival in the mammalian host. This study characterized the
A. fumigatus
SebA transcription factor, which is the putative homologue of the factor encoded by
Trichoderma atroviride
seb1
. The Δ
sebA
mutant demonstrated reduced growth in the presence of paraquat, hydrogen peroxide, CaCl
2
, and poor nutritional conditions, while viability associated with
sebA
was also affected by heat shock exposure. Accordingly, SebA::GFP (SebA::green fluorescent protein) was shown to accumulate in the nucleus upon exposure to oxidative stress and heat shock conditions. In addition, genes involved in either the oxidative stress or heat shock response had reduced transcription in the Δ
sebA
mutant. The
A. fumigatus
Δ
sebA
strain was attenuated in virulence in a murine model of invasive pulmonary aspergillosis. Furthermore, killing of the Δ
sebA
mutant by murine alveolar macrophages was increased compared to killing of the wild-type strain.
A. fumigatus
SebA plays a complex role, contributing to several stress tolerance pathways and growth under poor nutritional conditions, and seems to be integrated into different stress responses.
Deciphering Transcriptional Regulatory Mechanisms Associated with Hemicellulose Degradation in Neurospora crassa
Authors: Sun Jianping , Tian Chaoguang , Diamond Spencer , Glass N. Louise 
Published: February 2012

ABSTRACT

Hemicellulose, the second most abundant plant biomass fraction after cellulose, is widely viewed as a potential substrate for the production of liquid fuels and other value-added materials. Degradation of hemicellulose by filamentous fungi requires production of many different enzymes, which are induced by biopolymers or its derivatives and regulated mainly at the transcriptional level through transcription factors (TFs).
Neurospora crassa
, a model filamentous fungus, expresses and secretes enzymes required for plant cell wall deconstruction. To better understand genes specifically associated with degradation of hemicellulose, we applied secretome and transcriptome analysis to
N. crassa
grown on beechwood xylan. We identified 34 secreted proteins and 353 genes with elevated transcription on xylan. The xylanolytic phenotype of strains with deletions in genes identified from the secretome and transcriptome analysis of the wild type was assessed, revealing functions for known and unknown proteins associated with hemicellulose degradation. By evaluating phenotypes of strains containing deletions of predicted TF genes in
N. crassa
, we identified a TF (XLR-1;
x
y
l
an degradation
r
egulator
1
) essential for hemicellulose degradation that is an ortholog to XlnR/XYR1 in
Aspergillus
and
Trichoderma
species, respectively, a major transcriptional regulator of genes encoding both cellulases and hemicellulases. Deletion of
xlr-1
in
N. crassa
abolished growth on xylan and xylose, but growth on cellulose and cellulolytic activity were only slightly affected. To determine the regulatory mechanisms for hemicellulose degradation, we explored the transcriptional regulon of XLR-1 under xylose, xylanolytic, and cellulolytic conditions. XLR-1 regulated only some predicted hemicellulase genes in
N. crassa
and was required for a full induction of several cellulase genes. Hemicellulase gene expression was induced by a combination of release from carbon catabolite repression (CCR) and induction. This systematic analysis illustrates the similarities and differences in regulation of hemicellulose degradation among filamentous fungi.
Transcriptional Organization and Physiological Contributions of the
relQ
Operon of Streptococcus mutans
Authors: Kim Jeong Nam , Ahn Sang-Joon , Seaton Kinda , Garrett Steven , Burne Robert A. 
Published: February 2012

ABSTRACT

The molecular alarmone (p)ppGpp functions as a global regulator of gene expression in bacteria. In
Streptococcus mutans
, (p)ppGpp synthesis is catalyzed by three gene products: RelA, RelP, and RelQ. RelA is responsible for (p)ppGpp production during a stringent response, and RelP is the primary source of (p)ppGpp during exponential growth, but the role of RelQ has not been thoroughly investigated. In this study, we analyzed the four-gene
relQ
operon to establish how these gene products may affect homeostasis and stress tolerance in the dental caries pathogen
S. mutans
. Northern blotting and reverse transcriptase PCR demonstrated that
relQ
is cotranscribed with the downstream genes
ppnK
(NAD kinase),
rluE
(pseudouridine synthase), and
pta
(phosphotransacetylase). In addition, a promoter located within the
rluE
gene was shown to drive transcription of
pta
. Inactivation of
relQ
,
ppnK
, or
rluE
did not significantly affect growth of or stress tolerance by
S. mutans
, whereas strains lacking
pta
were more sensitive to acid and oxidative stresses. Interestingly, introduction of an
rluE
deletion into the
pta
mutant reversed the deleterious effects of the
pta
mutation on growth and stress tolerance. Accumulation of (p)ppGpp was also decreased in a
pta
mutant strain, whereas inactivation of
relQ
caused enhanced (p)ppGpp synthesis in exponential-phase cells. The results reveal an important role for the
relQ
operon in the expression of traits that are essential for persistence and pathogenesis by
S. mutans
and provide evidence for a molecular connection of acetate and (p)ppGpp metabolism with tolerance of acid and oxidative stresses.