bluuebunny/crossref_metadata_2025_split huggingface dataset:
Cystine dimethylester loading promotes oxidative stress and a reduction in ATP independent of lysosomal cystine accumulation in a human proximal tubular epithelial cell line
Authors: Sumayao Rodolfo , McEvoy Bernadette , Martin‐Martin Natalia , McMorrow Tara , Newsholme Philip 
Published: June 2013

New findings
What is the central question of this study?Are the molecular and cellular consequences of cystine dimethylester loading different from an alternative and more specific model of the cystinotic kidney proximal tubule epithelial cell, obtained by CTNS gene silencing.
What is the main finding and its importance?Using a human‐derived kidney proximal tubular cell line, HK‐2, we demonstrated that cystine dimethylester loading induces detrimental effects independent of lysosomal cystine accumulation. CTNS gene silencing, which resulted in comparable levels of cystine accumulation, evidence of oxidative stress and reduced ATP concentration but unaltered ATP generation capacity, represents a more useful model for investigating biochemical alterations in cystinosis.
Using the cystine dimethylester (CDME) loading technique to achieve elevated lysosomal cystine levels, ATP depletion has previously been postulated to be responsible for the renal dysfunction in cystinosis, a genetic disorder characterized by an excessive accumulation of cystine in the lysosomes. However, this is unlikely to be the sole factor responsible for the complexity of cell stress associated with cystinosis. Moreover, CDME has been shown to induce a direct toxic effect on mitochondrial ATP generation. Using a human‐derived proximal tubular epithelial cell line, we compared the effects of CDME loading with small interfering RNA‐mediated cystinosin, lysosomal cystine transporter (CTNS) gene silencing on glutathione redox status, reactive oxygen species levels, oxidative stress index, antioxidant enzyme activities and ATP generating capacity. The CDME‐loaded cells displayed increased total glutathione content, extensive superoxide depletion, augmented oxidative stress index, decreased catalase activity, normal superoxide dismutase activity and compromised ATP generation. In contrast, cells subjected to CTNS gene inhibition demonstrated decreased total glutathione content, increased superoxide levels, unaltered oxidative stress index, unaltered catalase activity, induction of superoxide dismutase activity and normal ATP generation. Our data indicate that many CDME‐induced effects are independent of lysosomal cystine accumulation, which further underscores the limited value of CDME loading for studying the pathogenesis of cystinosis. CTNS gene inhibition, which results in intracellular cystine accumulation, is a more realistic approach for investigating biochemical alterations in cystinosis.
Combined Genomics and Experimental Analyses of Respiratory Characteristics of Shewanella putrefaciens W3-18-1
Authors: Qiu Dongru , Wei Hehong , Tu Qichao , Yang Yunfeng , Xie Ming , Chen Jingrong , Pinkerton Mark H. , Liang Yili , He Zhili , Zhou Jizhong 
Published: June 2013

ABSTRACTIt has previously been shown that theShewanella putrefaciensW3-18-1 strain produces remarkably high current in microbial fuel cells (MFCs) and can form magnetite at 0°C. To explore the underlying mechanisms, we developed a genetic manipulation method by deleting the restriction-modification system genes of the SGI1 (Salmonellagenome island 1)-like prophage and analyzed the key genes involved in bacterial respiration. W3-18-1 has less respiratory flexibility than the well-characterizedS. oneidensisMR-1 strain, as it possesses fewer cytochromecgenes and lacks the ability to oxidize sulfite or reduce dimethyl sulfoxide (DMSO) and timethylamine oxide (TMAO). W3-18-1 lacks the hydrogen-producing Fe-only hydrogenase, and the hydrogen-oxidizing Ni-Fe hydrogenase genes were split into two separate clusters. Two periplasmic nitrate reductases (NapDAGHB and NapDABC) were functionally redundant in anaerobic growth of W3-18-1 with nitrate as the electron acceptor, thoughnapDABCwas not regulated by Crp. Moreover, nitrate respiration started earlier in W3-18-1 than in MR-1 (with NapDAGHB only) under microoxic conditions. These results indicate thatShewanella putrefaciensW3-18-1 is well adapted to habitats with higher oxygen levels. Taken together, the results of this study provide valuable insights into bacterial genome evolution.
Assessment of Anaerobic Toluene Biodegradation Activity by
bssA
Transcript/Gene Ratios
Authors: Brow Christina N. , O'Brien Johnson Reid , Johnson Richard L. , Simon Holly M. 
Published: June 2013

ABSTRACT

Benzylsuccinate synthase (
bssA
) genes associated with toluene degradation were profiled across a groundwater contaminant plume under nitrate-reducing conditions and were detected in significant numbers throughout the plume. However, differences between groundwater and core sediment samples suggested that microbial transport, rather than local activity, was the underlying cause of the high copy numbers within the downgradient plume. Both gene transcript and reactant concentrations were consistent with this hypothesis. Expression of
bssA
genes from denitrifying toluene degraders was induced by toluene but only in the presence of nitrate, and transcript abundance dropped rapidly following the removal of either toluene or nitrate. The drop in
bssA
transcripts following the removal of toluene could be described by an exponential decay function with a half-life on the order of 1 h. Interestingly,
bssA
transcripts never disappeared completely but were always detected at some level if either inducer was present. Therefore, the detection of transcripts alone may not be sufficient evidence for contaminant degradation. To avoid mistakenly associating basal-level gene expression with actively degrading microbial populations, an integrated approach using the ratio of functional gene transcripts to gene copies is recommended. This approach minimizes the impact of microbial transport on activity assessment and allows reliable assessments of microbial activity to be obtained from water samples.
Fermentation Temperature Modulates Phosphatidylethanolamine and Phosphatidylinositol Levels in the Cell Membrane of Saccharomyces cerevisiae
Authors: Henderson Clark M. , Zeno Wade F. , Lerno Larry A. , Longo Marjorie L. , Block David E. 
Published: June 2013

ABSTRACTDuring alcoholic fermentation,Saccharomyces cerevisiaeis exposed to a host of environmental and physiological stresses. Extremes of fermentation temperature have previously been demonstrated to induce fermentation arrest under growth conditions that would otherwise result in complete sugar utilization at “normal” temperatures and nutrient levels. Fermentations were carried out at 15°C, 25°C, and 35°C in a defined high-sugar medium using threeSaccharomyces cerevisiaestrains with diverse fermentation characteristics. The lipid composition of these strains was analyzed at two fermentation stages, when ethanol levels were low early in stationary phase and in late stationary phase at high ethanol concentrations. Several lipids exhibited dramatic differences in membrane concentration in a temperature-dependent manner. Principal component analysis (PCA) was used as a tool to elucidate correlations between specific lipid species and fermentation temperature for each yeast strain. Fermentations carried out at 35°C exhibited very high concentrations of several phosphatidylinositol species, whereas at 15°C these yeast strains exhibited higher levels of phosphatidylethanolamine and phosphatidylcholine species with medium-chain fatty acids. Furthermore, membrane concentrations of ergosterol were highest in the yeast strain that experienced stuck fermentations at all three temperatures. Fluorescence anisotropy measurements of yeast cell membrane fluidity during fermentation were carried out using the lipophilic fluorophore diphenylhexatriene. These measurements demonstrate that the changes in the lipid composition of these yeast strains across the range of fermentation temperatures used in this study did not significantly affect cell membrane fluidity. However, the results from this study indicate that fermentingS. cerevisiaemodulates its membrane lipid composition in a temperature-dependent manner.
Measurement of Predation and Biofilm Formation under Different Ambient Oxygen Conditions Using a Simple Gasbag-Based System
Authors: Kadouri Daniel E. , Tran Aimy 
Published: June 2013

ABSTRACT

Bdellovibrio bacteriovorus
and
Micavibrio aeruginosavorus
are Gram-negative bacteria characterized by predatory behavior. The aim of this study was to evaluate the ability of the predators to prey in different oxygen environments. When placed on an orbital shaker, a positive association between the rate of aeration and predation was observed. To further examine the effects of elevated ambient oxygen levels on predation, a simple gasbag system was developed. Using the system, we were able to conduct experiments at ambient oxygen levels of 3% to 86%. When placed in gasbags and inflated with air, 50% O
2
, and 100% O
2
, positive predation was seen on both planktonic and biofilm-grown prey cells. However, in low-oxygen environments, predatory bacteria were able to attack only prey cells grown as biofilms. To further evaluate the gasbag system, biofilm development of Gram-positive and Gram-negative microorganisms was also measured. Although the gasbag system was found to be suitable for culturing bacteria that require a low-oxygen environment, it was not capable of supporting, with its current configuration, the growth of obligate anaerobes in liquid or agar medium.
Establishment of a Simple Lactobacillus plantarum Cell Consortium for Cellulase-Xylanase Synergistic Interactions
Authors: Moraïs Sarah , Shterzer Naama , Grinberg Inna Rozman , Mathiesen Geir , Eijsink Vincent G. H. , Axelsson Lars , Lamed Raphael , Bayer Edward A. , Mizrahi Itzhak 
Published: June 2013

ABSTRACT

Lactobacillus plantarum
is an attractive candidate for bioprocessing of lignocellulosic biomass due to its high metabolic variability, including its ability to ferment both pentoses and hexoses, as well as its high acid tolerance, a quality often utilized in industrial processes. This bacterium grows naturally on biomass; however, it lacks the inherent ability to deconstruct lignocellulosic substrates. As a first step toward engineering lignocellulose-converting lactobacilli, we have introduced genes coding for a GH6 cellulase and a GH11 xylanase from a highly active cellulolytic bacterium into
L. plantarum
. For this purpose, we employed the recently developed pSIP vectors for efficient secretion of heterologous proteins. Both enzymes were secreted by
L. plantarum
at levels estimated at 0.33 nM and 3.3 nM, for the cellulase and xylanase, respectively, in culture at an optical density at 600 nm (OD
600
) of 1. Transformed cells demonstrated the ability to degrade individually either cellulose or xylan and wheat straw. When mixed together to form a two-strain cell-based consortium secreting both cellulase and xylanase, they exhibited synergistic activity in the overall release of soluble sugar from wheat straw. This result paves the way toward metabolic harnessing of
L. plantarum
for novel biorefining applications, such as production of ethanol and polylactic acid directly from plant biomass.
Frequent Occurrence of Mixed Enterocytozoon bieneusi Infections in Humans
Authors: Widmer Giovanni , Dilo Julia , Tumwine James K. , Tzipori Saul , Akiyoshi Donna E. 
Published: June 2013

ABSTRACT

Enterocytozoon bieneusi
(phylum
Microsporidia
) is a human pathogen with a broad host range. Following the sequencing of 3.8 Mb of the estimated 6-Mb
E. bieneusi
genome, simple sequence repeats (micro- and minisatellites) were identified. Sequencing of four such repeats from various human and animal
E. bieneusi
isolates identified extensive sequence polymorphism and enabled the development of a multilocus genotyping method to study the epidemiology of this pathogen. We genotyped
E. bieneusi
DNA extracted from 197 fecal samples originating from children with diarrhea who were residing in Kampala, Uganda. Three newly identified microsatellite markers and the internal transcribed spacer were PCR amplified, and multiple cloned amplicons for each marker were sequenced from each individual. Most microsatellite sequences were unique to the Ugandan population. Significantly, polymorphism not only was present among isolates but was also found within isolates. This observation suggests that infections with heterogeneous
E. bieneusi
populations are common in this region. However, the data do not exclude that some of the polymorphism originates from divergent paralogs within the genome. The frequent occurrence of multiple sequences within an isolate precluded the identification of multilocus genotypes. This observation raises the possibility that in a region in which the prevalence of
E. bieneusi
is high, sequencing of uncloned PCR products may not be adequate for multilocus genotyping.
pA506, a Conjugative Plasmid of the Plant Epiphyte Pseudomonas fluorescens A506
Authors: Stockwell Virginia O. , Davis Edward W. , Carey Alyssa , Shaffer Brenda T. , Mavrodi Dmitri V. , Hassan Karl A. , Hockett Kevin , Thomashow Linda S. , Paulsen Ian T. , Loper Joyce E. 
Published: June 2013

ABSTRACT

Conjugative plasmids are known to facilitate the acquisition and dispersal of genes contributing to the fitness of
Pseudomonas
spp. Here, we report the characterization of pA506, the 57-kb conjugative plasmid of
Pseudomonas fluorescens
A506, a plant epiphyte used in the United States for the biological control of fire blight disease of pear and apple. Twenty-nine of the 67 open reading frames (ORFs) of pA506 have putative functions in conjugation, including a type IV secretion system related to that of MOB
P6
family plasmids and a gene cluster for type IV pili. We demonstrate that pA506 is self-transmissible via conjugation between A506 and strains of
Pseudomonas
spp. or the
Enterobacteriaceae
. The origin of vegetative replication (
oriV
) of pA506 is typical of those in pPT23A family plasmids, which are present in many pathovars of
Pseudomonas syringae
, but pA506 lacks
repA
, a defining locus for pPT23A plasmids, and has a novel partitioning region. We selected a plasmid-cured derivative of A506 and compared it to the wild type to identify plasmid-encoded phenotypes. pA506 conferred UV resistance, presumably due to the plasmid-borne
rulAB
genes, but did not influence epiphytic fitness of A506 on pear or apple blossoms in the field. pA506 does not appear to confer resistance to antibiotics or other toxic elements. Based on the conjugative nature of pA506 and the large number of its genes that are shared with plasmids from diverse groups of environmental bacteria, the plasmid is likely to serve as a vehicle for genetic exchange between A506 and its coinhabitants on plant surfaces.
Chitin Amendment Increases Soil Suppressiveness toward Plant Pathogens and Modulates the Actinobacterial and Oxalobacteraceal Communities in an Experimental Agricultural Field
Authors: Cretoiu Mariana Silvia , Korthals Gerard W. , Visser Johnny H. M. , van Elsas Jan Dirk 
Published: June 2013

ABSTRACT

A long-term experiment on the effect of chitin addition to soil on the suppression of soilborne pathogens was set up and monitored for 8 years in an experimental field, Vredepeel, The Netherlands. Chitinous matter obtained from shrimps was added to soil top layers on two different occasions, and the suppressiveness of soil toward
Verticillium dahliae
, as well as plant-pathogenic nematodes, was assessed, in addition to analyses of the abundances and community structures of members of the soil microbiota. The data revealed that chitin amendment had raised the suppressiveness of soil, in particular toward
Verticillium dahliae
, 9 months after the (second) treatment, extending to 2 years following treatment. Moreover, major effects of the added chitin on the soil microbial communities were detected. First, shifts in both the abundances and structures of the chitin-treated soil microbial communities, both of total soil bacteria and fungi, were found. In addition, the abundances and structures of soil actinobacteria and the
Oxalobacteraceae
were affected by chitin. At the functional gene level, the abundance of specific (family-18 glycoside hydrolase) chitinase genes carried by the soil bacteria also revealed upshifts as a result of the added chitin. The effects of chitin noted for the
Oxalobacteraceae
were specifically related to significant upshifts in the abundances of the species
Duganella violaceinigra
and
Massilia plicata
. These effects of chitin persisted over the time of the experiment.
Quantification of Endospore-Forming Firmicutes by Quantitative PCR with the Functional Genespo0A
Authors: Bueche Matthieu , Wunderlin Tina , Roussel-Delif Ludovic , Junier Thomas , Sauvain Loic , Jeanneret Nicole , Junier Pilar 
Published: June 2013

ABSTRACTBacterial endospores are highly specialized cellular forms that allow endospore-formingFirmicutes(EFF) to tolerate harsh environmental conditions. EFF are considered ubiquitous in natural environments, in particular, those subjected to stress conditions. In addition to natural habitats, EFF are often the cause of contamination problems in anthropogenic environments, such as industrial production plants or hospitals. It is therefore desirable to assess their prevalence in environmental and industrial fields. To this end, a high-sensitivity detection method is still needed. The aim of this study was to develop and evaluate an approach based on quantitative PCR (qPCR). For this, the suitability of functional genes specific for and common to all EFF were evaluated. Seven genes were considered, but onlyspo0Awas retained to identify conserved regions for qPCR primer design. An approach based on multivariate analysis was developed for primer design. Two primer sets were obtained and evaluated with 16 pure cultures, including representatives of the generaBacillus,Paenibacillus,Brevibacillus,Geobacillus,Alicyclobacillus,Sulfobacillus,Clostridium, andDesulfotomaculum, as well as with environmental samples. The primer sets developed gave a reliable quantification when tested on laboratory strains, with the exception ofSulfobacillusandDesulfotomaculum. A test using sediment samples with a diverse EFF community also gave a reliable quantification compared to 16S rRNA gene pyrosequencing. A detection limit of about 104cells (or spores) per gram of initial material was calculated, indicating this method has a promising potential for the detection of EFF over a wide range of applications.
Functional Characterization of Key Enzymes involved in
l
-Glutamate Synthesis and Degradation in the Thermotolerant and Methylotrophic Bacterium Bacillus methanolicus
Authors: Krog Anne , Heggeset Tonje Marita Bjerkan , Ellingsen Trond Erling , Brautaset Trygve 
Published: June 2013

ABSTRACT

Bacillus methanolicus
wild-type strain MGA3 secretes 59 g/liter
−1
of
l
-glutamate in fed-batch methanol cultivations at 50°C. We recently sequenced the MGA3 genome, and we here characterize key enzymes involved in
l
-glutamate synthesis and degradation. One glutamate dehydrogenase (GDH) that is encoded by
yweB
and two glutamate synthases (GOGATs) that are encoded by the
gltAB
operon and by
gltA2
were found, in contrast to
Bacillus subtilis
, which has two different GDHs and only one GOGAT.
B. methanolicus
has a glutamine synthetase (GS) that is encoded by
glnA
and a 2-oxoglutarate dehydrogenase (OGDH) that is encoded by the
odhAB
operon. The
yweB
,
gltA
,
gltB
, and
gltA2
gene products were purified and characterized biochemically
in vitro
. YweB has a low

K
m

value for ammonium (10 mM) and a high

K
m

value for
l
-glutamate (250 mM), and the
V
max
value is 7-fold higher for
l
-glutamate synthesis than for the degradation reaction. GltA and GltA2 displayed similar

K
m

values (1 to 1.4 mM) and
V
max
values (4 U/mg) for both
l
-glutamate and 2-oxoglutarate as the substrates, and GltB had no effect on the catalytic activities of these enzymes
in vitro
. Complementation assays indicated that GltA and not GltA2 is dependent on GltB for GOGAT activity
in vivo
. To our knowledge, this is the first report describing the presence of two active GOGATs in a bacterium.
In vivo
experiments indicated that OGDH activity and, to some degree, GOGAT activity play important roles in regulating
l
-glutamate production in this organism.
Psychrophilic and Psychrotolerant Fungi on Bats and the Presence of Geomyces spp. on Bat Wings Prior to the Arrival of White Nose Syndrome
Authors: Johnson Lynnaun J. A. N. , Miller Andrew N. , McCleery Robert A. , McClanahan Rod , Kath Joseph A. , Lueschow Shiloh , Porras-Alfaro Andrea 
Published: June 2013

ABSTRACT

Since 2006,
Geomyces destructans
, the causative agent of white nose syndrome (WNS), has killed over 5.7 million bats in North America. The current hypothesis suggests that this novel fungus is an invasive species from Europe, but little is known about the diversity within the genus
Geomyces
and its distribution on bats in the United States. We documented the psychrophilic and psychrotolerant fungal flora of hibernating bats prior to the arrival of WNS using culture-based techniques. A total of 149 cultures, which were obtained from 30 bats in five bat hibernacula located in four caves and one mine, were sequenced for the entire internal transcribed spacer (ITS) nuclear ribosomal DNA (nrDNA) region. Approximately 53 operational taxonomic units (OTUs) at 97% similarity were recovered from bat wings, with the community dominated by fungi within the genera
Cladosporium
,
Fusarium
,
Geomyces
,
Mortierella
,
Penicillium
, and
Trichosporon
. Eleven
Geomyces
isolates were obtained and placed in at least seven distinct
Geomyces
clades based on maximum-likelihood phylogenetic analyses. Temperature experiments revealed that all
Geomyces
strains isolated are psychrotolerant, unlike
G. destructans
, which is a true psychrophile. Our results confirm that a large diversity of fungi, including several
Geomyces
isolates, occurs on bats prior to the arrival of WNS. Most of these isolates were obtained from damaged wings. Additional studies need to be conducted to determine potential ecological roles of these abundant
Geomyces
strains isolated from bats.
Rapid Detection of Ceratocystis platani Inoculum by Quantitative Real-Time PCR Assay
Authors: Luchi Nicola , Ghelardini Luisa , Belbahri Lassaâd , Quartier Marion , Santini Alberto 
Published: June 2013

ABSTRACT

Ceratocystis platani
is the causal agent of canker stain of plane trees, a lethal disease able to kill mature trees in one or two successive growing seasons. The pathogen is a quarantine organism and has a negative impact on anthropogenic and natural populations of plane trees. Contaminated sawdust produced during pruning and sanitation fellings can contribute to disease spread. The goal of this study was to design a rapid, real-time quantitative PCR assay to detect a
C. platani
airborne inoculum. Airborne inoculum traps (AITs) were placed in an urban setting in the city of Florence, Italy, where the disease was present. Primers and TaqMan minor groove binder (MGB) probes were designed to target cerato-platanin (CP) and internal transcribed spacer 2 (ITS2) genes. The detection limits of the assay were 0.05 pg/μl and 2 fg/μl of fungal DNA for CP and ITS, respectively. Pathogen detection directly from AITs demonstrated specificity and high sensitivity for
C. platani
, detecting DNA concentrations as low as 1.2 × 10
−2
to 1.4 × 10
−2
pg/μl, corresponding to ∼10 conidia per ml. Airborne inoculum traps were able to detect the
C. platani
inoculum within 200 m of the closest symptomatic infected plane tree. The combination of airborne trapping and real-time quantitative PCR assay provides a rapid and sensitive method for the specific detection of a
C. platani
inoculum. This technique may be used to identify the period of highest risk of pathogen spread in a site, thus helping disease management.