bluuebunny/crossref_metadata_2025_split huggingface dataset:
Role of oxidative stress‐induced endothelin‐converting enzyme activity in the alteration of carotid body function by chronic intermittent hypoxia
Authors: Peng Ying‐Jie , Nanduri Jayasri , Raghuraman Gayatri , Wang Ning , Kumar Ganesh K. , Prabhakar Nanduri R. 
Published: August 2013

New findings
What is the central question of this study? What mechanisms mediate chronic intermittent hypoxia‐induced increases in endothelin‐1 in the carotid body?
What is the main finding and its importance? Upregulation of endothelin‐1 by chronic intermittent hypoxia results from reactive oxygen species‐dependent activation of endothelin‐converting enzyme and is not the result of augmented endothelin‐1 gene transcriptional activity. The resultant increased endothelin‐1 acts via endothelin‐1A receptors to induce hypoxic hypersensitivity of the carotid body but does not contribute to the sensory long‐term facilitation observed in this condition. These findings provide mechanistic insight into how chronic intermittent hypoxia alters carotid body function.
Chronic intermittent hypoxia (CIH) leads to remodelling of the carotid body function, manifested by an augmented sensory response to hypoxia and induction of sensory long‐term facilitation (LTF). It was proposed that endothelin‐1 (ET‐1) contributes to CIH‐induced hypoxic hypersensitivity of the carotid body. The objectives of the present study were as follows: (i) to delineate the mechanisms by which CIH upregulates ET‐1 expression in the carotid body; and (ii) to assess whether ET‐1 also contributes to sensory LTF. Experiments were performed on adult, male rats exposed to alternating cycles of 5% O2 (15 s) and room air (5 min), nine episodes per hour and 8 h per day for 10 days. Chronic intermittent hypoxia increased ET‐1 levels in glomus cells without significantly altering prepro‐endothelin‐1 mRNA levels. The activity of endothelin‐converting enzyme increased with concomitant elevation of ET‐1 levels in CIH‐exposed carotid bodies, and MnTMPyP, a membrane‐permeable antioxidant, prevented these effects. Hypoxia facilitated ET‐1 release from CIH‐treated carotid bodies, which is a prerequisite for activation of ET receptors; however, hypoxia had no effect on ET‐1 release from control carotid bodies. In CIH‐exposed carotid bodies, mRNAs encoding ETA receptor were upregulated, and an ETA receptor‐specific antagonist abolished CIH‐induced hypersensitivity of the hypoxic response, whereas it had no effect on the sensory LTF. These results suggest that ECE‐dependent increased production of ET‐1 coupled with hypoxia‐evoked ET‐1 release and the ensuing ETA receptor activation mediate the CIH‐induced carotid body hypersensitivity to hypoxia, but the ETA signalling pathway is not associated with sensory LTF elicited by CIH.
Coherence among Different Microbial Source Tracking Markers in a Small Agricultural Stream with or without Livestock Exclusion Practices
Authors: Wilkes Graham , Brassard Julie , Edge Thomas A. , Gannon Victor , Jokinen Cassandra C. , Jones Tineke H. , Marti Romain , Neumann Norman F. , Ruecker Norma J. , Sunohara Mark , Topp Edward , Lapen David R. 
Published: August 2013

ABSTRACT

Over 1,400 water samples were collected biweekly over 6 years from an intermittent stream protected and unprotected from pasturing cattle. The samples were monitored for host-specific
Bacteroidales
markers,
Cryptosporidium
species/genotypes, viruses and coliphages associated with humans or animals, and bacterial zoonotic pathogens. Ruminant
Bacteroidales
markers did not increase within the restricted cattle access reach of the stream, whereas the ruminant
Bacteroidales
marker increased significantly in the unrestricted cattle access reach. Human
Bacteroidales
markers significantly increased downstream of homes where septic issues were documented. Wildlife
Bacteroidales
markers were detected downstream of the cattle exclusion practice where stream and riparian habitat was protected, but detections decreased after the unrestricted pasture, where the stream and riparian zone was unprotected from livestock. Detection of a large number of human viruses was shown to increase downstream of homes, and similar trends were observed for the human
Bacteroidales
marker. There was considerable interplay among biomarkers with stream flow, season, and the cattle exclusion practices. There were no to very weak associations with
Bacteroidales
markers and bacterial, viral, and parasitic pathogens. Overall, discrete sample-by-sample coherence among the different microbial source tracking markers that expressed a similar microbial source was minimal, but spatial trends were physically meaningful in terms of land use (e.g., beneficial management practice) effects on sources of fecal pollution.
Metagenome Survey of a Multispecies and Alga-Associated Biofilm Revealed Key Elements of Bacterial-Algal Interactions in Photobioreactors
Authors: Krohn-Molt Ines , Wemheuer Bernd , Alawi Malik , Poehlein Anja , Güllert Simon , Schmeisser Christel , Pommerening-Röser Andreas , Grundhoff Adam , Daniel Rolf , Hanelt Dieter , Streit Wolfgang R. 
Published: August 2013

ABSTRACT

Photobioreactors (PBRs) are very attractive for sunlight-driven production of biofuels and capturing of anthropogenic CO
2
. One major problem associated with PBRs however, is that the bacteria usually associated with microalgae in nonaxenic cultures can lead to biofouling and thereby affect algal productivity. Here, we report on a phylogenetic, metagenome, and functional analysis of a mixed-species bacterial biofilm associated with the microalgae
Chlorella vulgaris
and
Scenedesmus obliquus
in a PBR. The biofilm diversity and population dynamics were examined through 16S rRNA phylogeny. Overall, the diversity was rather limited, with approximately 30 bacterial species associated with the algae. The majority of the observed microorganisms were affiliated with
Alphaproteobacteria
,
Betaproteobacteria
, and
Bacteroidetes
. A combined approach of sequencing via GS FLX Titanium from Roche and HiSeq 2000 from Illumina resulted in the overall production of 350 Mbp of sequenced DNA, 165 Mbp of which was assembled in larger contigs with a maximum size of 0.2 Mbp. A KEGG pathway analysis suggested high metabolic diversity with respect to the use of polymers and aromatic and nonaromatic compounds. Genes associated with the biosynthesis of essential B vitamins were highly redundant and functional. Moreover, a relatively high number of predicted and functional lipase and esterase genes indicated that the alga-associated bacteria are possibly a major sink for lipids and fatty acids produced by the microalgae. This is the first metagenome study of microalga- and PBR-associated biofilm bacteria, and it gives new clues for improved biofuel production in PBRs.
ClpB1 Overproduction in Synechocystis sp. Strain PCC 6803 Increases Tolerance to Rapid Heat Shock
Authors: Gonzalez-Esquer C. Raul , Vermaas Wim F. J. 
Published: August 2013

ABSTRACT

ClpB1 is a heat shock protein known to disaggregate large protein complexes. Constitutive, 16-fold ClpB1 overproduction in the cyanobacterium
Synechocystis
sp. strain PCC 6803 increased cell survival by 20-fold when cultures were heated quickly (1°C/s) to 50°C and delayed cell death by an average of 3 min during incubation at high temperatures (>46°C). Cooverexpression of ClpB1 and another heat shock protein, DnaK2, further increased cell survival. According to immunocytochemistry results, ClpB1 is dispersed throughout the cytoplasm but is concentrated in specific areas and is more prevalent near thylakoid membranes. However, ClpB1 overproduction does not lead to a change in the morphology, chlorophyll content, or photosystem ratio. Whereas electron microscopy demonstrated that apparent protein aggregation occurred after heat treatment in the control strain, protein aggregate size was maintained in the ClpB1 overexpresser. Constitutive ClpB1 overproduction allows an earlier response to heat shock and protects from rapid heating of cultures.
Proteomic Analysis of the Quorum-Sensing Regulon in Pantoea stewartii and Identification of Direct Targets of EsaR
Authors: Ramachandran Revathy , Stevens Ann M. 
Published: August 2013

ABSTRACT

The proteobacterium
Pantoea stewartii
subsp.
stewartii
causes Stewart's wilt disease in maize when it colonizes the xylem and secretes large amounts of stewartan, an exopolysaccharide. The success of disease pathogenesis lies in the timing of bacterial virulence factor expression through the different stages of infection. Regulation is achieved through a quorum-sensing (QS) system consisting of the acyl-homoserine lactone (AHL) synthase, EsaI, and the transcription regulator EsaR. At low cell densities, EsaR represses transcription of itself and of
rcsA
, an activator of the stewartan biosynthesis operon; it also activates
esaS
, which encodes a small RNA (sRNA). Repression or activation ceases at high cell densities when EsaI synthesizes sufficient levels of the AHL ligand
N
-3-oxo-hexanoyl-
l
-homoserine lactone to bind and inactivate EsaR. This study aims to identify other genes activated or repressed by EsaR during the QS response. Proteomic analysis identified a QS regulon of more than 30 proteins. Electrophoretic mobility shift assays of promoters of genes encoding differentially expressed proteins distinguished direct targets of EsaR from indirect targets. Additional quantitative reverse transcription-PCR (qRT-PCR) and DNA footprinting analysis established that EsaR directly regulates the promoters of
dkgA
,
glpF
, and
lrhA
. The proteins encoded by
dkgA
,
glpF
, and
lrhA
are a 2,5-diketogluconate reductase, glycerol facilitator, and transcriptional regulator of chemotaxis and motility, respectively, indicating a more global QS response in
P. stewartii
than previously recognized.
Exploring the Biosynthesis of Unsaturated Fatty Acids in Bacillus cereus ATCC 14579 and Functional Characterization of Novel Acyl-Lipid Desaturases
Authors: Chazarreta Cifré Lorena , Alemany Mariana , de Mendoza Diego , Altabe Silvia 
Published: August 2013

ABSTRACT

At low temperatures,
Bacillus cereus
synthesizes large amounts of unsaturated fatty acids (UFAs) with double bonds in positions Δ5 and Δ10, as well as Δ5,10 diunsaturated fatty acids. Through sequence homology searches, we identified two open reading frames (ORFs) encoding a putative Δ5 desaturase and a fatty acid acyl-lipid desaturase in the
B. cereus
ATCC 14579 genome, and these were named BC2983 and BC0400, respectively. Functional characterization of ORFs BC2983 and BC0400 by means of heterologous expression in
Bacillus subtilis
confirmed that they both encode acyl-lipid desaturases that use phospholipids as the substrates and have Δ5 and Δ10 desaturase activities. Thus, these ORFs were correspondingly named
desA
(Δ5 desaturase) and
desB
(Δ10 desaturase). We established that DesA utilizes ferredoxin and flavodoxins (Flds) as electron donors for the desaturation reaction, while DesB preferably employs Flds. In addition, increased amounts of UFAs were found when
B. subtilis
expressing
B. cereus
desaturases was subjected to a cold shock treatment, indicating that the activity or the expression of these enzymes is upregulated in response to a decrease in growth temperature. This represents the first work reporting the functional characterization of fatty acid desaturases from
B. cereus
.
Directed Evolution and Structural Analysis of NADPH-Dependent Acetoacetyl Coenzyme A (Acetoacetyl-CoA) Reductase from Ralstonia eutropha Reveals Two Mutations Responsible for Enhanced Kinetics
Authors: Matsumoto Ken'ichiro , Tanaka Yoshikazu , Watanabe Tsuyoshi , Motohashi Ren , Ikeda Koji , Tobitani Kota , Yao Min , Tanaka Isao , Taguchi Seiichi 
Published: August 2013

ABSTRACT

NADPH-dependent acetoacetyl-coenzyme A (acetoacetyl-CoA) reductase (PhaB) is a key enzyme in the synthesis of poly(3-hydroxybutyrate) [P(3HB)], along with β-ketothiolase (PhaA) and polyhydroxyalkanoate synthase (PhaC). In this study, PhaB from
Ralstonia eutropha
was engineered by means of directed evolution consisting of an error-prone PCR-mediated mutagenesis and a P(3HB) accumulation-based
in vivo
screening system using
Escherichia coli
. From approximately 20,000 mutants, we obtained two mutant candidates bearing Gln47Leu (Q47L) and Thr173Ser (T173S) substitutions. The mutants exhibited
k
cat
values that were 2.4-fold and 3.5-fold higher than that of the wild-type enzyme, respectively. In fact, the PhaB mutants did exhibit enhanced activity and P(3HB) accumulation when expressed in recombinant
Corynebacterium glutamicum
. Comparative three-dimensional structural analysis of wild-type PhaB and highly active PhaB mutants revealed that the beneficial mutations affected the flexibility around the active site, which in turn played an important role in substrate recognition. Furthermore, both the kinetic analysis and crystal structure data supported the conclusion that PhaB forms a ternary complex with NADPH and acetoacetyl-CoA. These results suggest that the mutations affected the interaction with substrates, resulting in the acquirement of enhanced activity.
Metabolism of Four α-Glycosidic Linkage-Containing Oligosaccharides by Bifidobacterium breve UCC2003
Authors: O'Connell Kerry Joan , O'Connell Motherway Mary , O'Callaghan John , Fitzgerald Gerald F. , Ross R. Paul , Ventura Marco , Stanton Catherine , van Sinderen Douwe 
Published: August 2013

ABSTRACT

Members of the genus
Bifidobacterium
are common inhabitants of the gastrointestinal tracts of humans and other mammals, where they ferment many diet-derived carbohydrates that cannot be digested by their hosts. To extend our understanding of bifidobacterial carbohydrate utilization, we investigated the molecular mechanisms by which 11 strains of
Bifidobacterium breve
metabolize four distinct α-glucose- and/or α-galactose-containing oligosaccharides, namely, raffinose, stachyose, melibiose, and melezitose. Here we demonstrate that all
B. breve
strains examined possess the ability to utilize raffinose, stachyose, and melibiose. However, the ability to metabolize melezitose was not common to all
B. breve
strains tested. Transcriptomic and functional genomic approaches identified a gene cluster dedicated to the metabolism of α-galactose-containing carbohydrates, while an adjacent gene cluster, dedicated to the metabolism of α-glucose-containing melezitose, was identified in strains that are able to use this carbohydrate.
Raman Spectroscopy and Chemometrics for Identification and Strain Discrimination of the Wine Spoilage Yeasts Saccharomyces cerevisiae, Zygosaccharomyces bailii, and Brettanomyces bruxellensis
Authors: Rodriguez Susan B. , Thornton Mark A. , Thornton Roy J. 
Published: August 2013

ABSTRACTThe yeastsZygosaccharomyces bailii,Dekkera bruxellensis(anamorph,Brettanomyces bruxellensis), andSaccharomyces cerevisiaeare the major spoilage agents of finished wine. A novel method using Raman spectroscopy in combination with a chemometric classification tool has been developed for the identification of these yeast species and for strain discrimination of these yeasts. Raman spectra were collected for six strains of each of the yeastsZ. bailii,B. bruxellensis, andS. cerevisiae. The yeasts were classified with high sensitivity at the species level: 93.8% forZ. bailii, 92.3% forB. bruxellensis, and 98.6% forS. cerevisiae. Furthermore, we have demonstrated that it is possible to discriminate between strains of these species. These yeasts were classified at the strain level with an overall accuracy of 81.8%.
Oxidative Stress at High Temperatures in Lactococcus lactis Due to an Insufficient Supply of Riboflavin
Authors: Chen Jun , Shen Jing , Solem Christian , Jensen Peter Ruhdal 
Published: August 2013

ABSTRACT

Lactococcus lactis
MG1363 was found to be unable to grow at temperatures above 37°C in a defined medium without riboflavin, and the cause was identified to be dissolved oxygen introduced during preparation of the medium. At 30°C, growth was unaffected by dissolved oxygen and oxygen was consumed quickly. Raising the temperature to 37°C resulted in severe growth inhibition and only slow removal of dissolved oxygen. Under these conditions, an abnormally low intracellular ratio of [ATP] to [ADP] (1.4) was found (normally around 5), which indicates that the cells are energy limited. By adding riboflavin to the medium, it was possible to improve growth and oxygen consumption at 37°C, and this also normalized the [ATP]-to-[ADP] ratio. A codon-optimized redox-sensitive green fluorescent protein (GFP) was introduced into
L. lactis
and revealed a more oxidized cytoplasm at 37°C than at 30°C. These results indicate that
L. lactis
suffers from heat-induced oxidative stress at increased temperatures. A decrease in intracellular flavin adenine dinucleotide (FAD), which is derived from riboflavin, was observed with increasing growth temperature, but the presence of riboflavin made the decrease smaller. The drop was accompanied by a decrease in NADH oxidase and pyruvate dehydrogenase activities, both of which depend on FAD as a cofactor. By overexpressing the riboflavin transporter, it was possible to improve FAD biosynthesis, which resulted in increased NADH oxidase and pyruvate dehydrogenase activities and improved fitness at high temperatures in the presence of oxygen.
The Virulence of Salmonella enterica Serovar Typhimurium in the Insect Model Galleria mellonella Is Impaired by Mutations in RNase E and RNase III
Authors: Viegas Sandra C. , Mil-Homens Dalila , Fialho Arsénio M. , Arraiano Cecília M. 
Published: August 2013

ABSTRACT

Salmonella enterica
serovar Typhimurium is a Gram-negative bacterium able to invade and replicate inside eukaryotic cells. To cope with the host defense mechanisms, the bacterium has to rapidly remodel its transcriptional status. Regulatory RNAs and ribonucleases are the factors that ultimately control the fate of mRNAs and final protein levels in the cell. There is growing evidence of the direct involvement of these factors in bacterial pathogenicity. In this report, we validate the use of a
Galleria mellonela
model in
S
. Typhimurium pathogenicity studies through the parallel analysis of a mutant with a mutation in
hfq
, a well-established
Salmonella
virulence gene. The results obtained with this mutant are similar to the ones reported in a mouse model. Through the use of this insect model, we demonstrate a role for the main endoribonucleases RNase E and RNase III in
Salmonella
virulence. These ribonuclease mutants show an attenuated virulence phenotype, impairment in motility, and reduced proliferation inside the host. Interestingly, the two mutants trigger a distinct immune response in the host, and the two mutations seem to have an impact on distinct bacterial functions.
Carbonylation as a Key Reaction in Anaerobic Acetone Activation by Desulfococcus biacutus
Authors: Gutiérrez Acosta Olga B. , Hardt Norman , Schink Bernhard 
Published: August 2013

ABSTRACT

Acetone is activated by aerobic and nitrate-reducing bacteria via an ATP-dependent carboxylation reaction to form acetoacetate as the first reaction product. In the activation of acetone by sulfate-reducing bacteria, acetoacetate has not been found to be an intermediate. Here, we present evidence of a carbonylation reaction as the initial step in the activation of acetone by the strictly anaerobic sulfate reducer
Desulfococcus biacutus
. In cell suspension experiments, CO was found to be a far better cosubstrate for acetone activation than CO
2
. The hypothetical reaction product, acetoacetaldehyde, is extremely reactive and could not be identified as a free intermediate. However, acetoacetaldehyde dinitrophenylhydrazone was detected by mass spectrometry in cell extract experiments as a reaction product of acetone, CO, and dinitrophenylhydrazine. In a similar assay, 2-amino-4-methylpyrimidine was formed as the product of a reaction between acetoacetaldehyde and guanidine. The reaction depended on ATP as a cosubstrate. Moreover, the specific activity of aldehyde dehydrogenase (coenzyme A [CoA] acylating) tested with the putative physiological substrate was found to be 153 ± 36 mU mg
−1
protein, and its activity was specifically induced in extracts of acetone-grown cells. Moreover, acetoacetyl-CoA was detected (by mass spectrometry) after the carbonylation reaction as the subsequent intermediate after acetoacetaldehyde was formed. These results together provide evidence that acetoacetaldehyde is an intermediate in the activation of acetone by sulfate-reducing bacteria.
Candida albicans
VMA3
Is Necessary for V-ATPase Assembly and Function and Contributes to Secretion and Filamentation
Authors: Rane Hallie S. , Bernardo Stella M. , Raines Summer M. , Binder Jessica L. , Parra Karlett J. , Lee Samuel A. 
Published: August 2013

ABSTRACT

The vacuolar membrane ATPase (V-ATPase) is a protein complex that utilizes ATP hydrolysis to drive protons from the cytosol into the vacuolar lumen, acidifying the vacuole and modulating several key cellular response systems in
Saccharomyces cerevisiae
. To study the contribution of V-ATPase to the biology and virulence attributes of the opportunistic fungal pathogen
Candida albicans
, we created a conditional mutant in which
VMA3
was placed under the control of a tetracycline-regulated promoter (tetR-
VMA3
strain). Repression of
VMA3
in the tetR-
VMA3
strain prevents V-ATPase assembly at the vacuolar membrane and reduces concanamycin A-sensitive ATPase-specific activity and proton transport by more than 90%. Loss of
C. albicans
V-ATPase activity alkalinizes the vacuolar lumen and has pleiotropic effects, including pH-dependent growth, calcium sensitivity, and cold sensitivity. The tetR-
VMA3
strain also displays abnormal vacuolar morphology, indicative of defective vacuolar membrane fission. The tetR-
VMA3
strain has impaired aspartyl protease and lipase secretion, as well as attenuated virulence in an
in vitro
macrophage killing model. Repression of
VMA3
suppresses filamentation, and V-ATPase-dependent filamentation defects are not rescued by overexpression of
RIM8
,
MDS3
,
EFG1
,
CST20
, or
UME6
, which encode positive regulators of filamentation. Specific chemical inhibition of Vma3p function also results in defective filamentation. These findings suggest either that V-ATPase functions downstream of these transcriptional regulators or that V-ATPase function during filamentation involves independent mechanisms and alternative signaling pathways. Taken together, these data indicate that V-ATPase activity is a fundamental requirement for several key virulence-associated traits in
C. albicans
.