bluuebunny/crossref_metadata_2025_split huggingface dataset:
Isolation of Endohyphal Bacteria from Foliar Ascomycota and
In Vitro
Establishment of Their Symbiotic Associations
Authors: Arendt Kayla R. , Hockett Kevin L. , Araldi-Brondolo Sarah J. , Baltrus David A. , Arnold A. Elizabeth 
Published: March 2016

ABSTRACT

Endohyphal bacteria (EHB) can influence fungal phenotypes and shape the outcomes of plant-fungal interactions. Previous work has suggested that EHB form facultative associations with many foliar fungi in the Ascomycota. These bacteria can be isolated in culture, and fungi can be cured of EHB using antibiotics. Here, we present methods for successfully introducing EHB into axenic mycelia of strains representing two classes of Ascomycota. We first establish
in vitro
conditions favoring reintroduction of two strains of EHB (
Luteibacter
sp.) into axenic cultures of their original fungal hosts, focusing on fungi isolated from healthy plant tissue as endophytes:
Microdiplodia
sp. (Dothideomycetes) and
Pestalotiopsis
sp. (Sordariomycetes). We then demonstrate that these EHB can be introduced into a novel fungal host under the same conditions, successfully transferring EHB between fungi representing different classes. Finally, we manipulate conditions to optimize reintroduction in a focal EHB-fungal association. We show that EHB infections were initiated and maintained more often under low-nutrient culture conditions and when EHB and fungal hyphae were washed with MgCl
2
prior to reassociation. Our study provides new methods for experimental assessment of the effects of EHB on fungal phenotypes and shows how the identity of the fungal host and growth conditions can define the establishment of these widespread and important symbioses.
The
apt
/6-Methylpurine Counterselection System and Its Applications in Genetic Studies of the Hyperthermophilic Archaeon Sulfolobus islandicus
Authors: Zhang Changyi , She Qunxin , Bi Hongkai , Whitaker Rachel J. 
Published: March 2016

ABSTRACT

Sulfolobus islandicus
serves as a model for studying archaeal biology as well as linking novel biology to evolutionary ecology using functional population genomics. In the present study, we developed a new counterselectable genetic marker in
S. islandicus
to expand the genetic toolbox for this species. We show that resistance to the purine analog 6-methylpurine (6-MP) in
S. islandicus
M.16.4 is due to the inactivation of a putative adenine phosphoribosyltransferase encoded by
M164_0158
(
apt
). The application of the
apt
gene as a novel counterselectable marker was first illustrated by constructing an unmarked α-amylase deletion mutant. Furthermore, the 6-MP counterselection feature was employed in a forward (loss-of-function) mutation assay to reveal the profile of spontaneous mutations in
S. islandicus
M.16.4 at the
apt
locus. Moreover, the general conservation of
apt
genes in the crenarchaea suggests that the same strategy can be broadly applied to other crenarchaeal model organisms. These results demonstrate that the
apt
locus represents a new tool for genetic manipulation and sequence analysis of the hyperthermophilic crenarchaeon
S. islandicus
.

IMPORTANCE
Currently, the
pyrEF
/5-fluoroorotic acid (5-FOA) counterselection system remains the sole counterselection marker in crenarchaeal genetics. Since most
Sulfolobus
mutants constructed by the research community were derived from genetic hosts lacking the
pyrEF
genes, the
pyrEF
/5-FOA system is no longer available for use in forward mutation assays. Demonstration of the
apt
/6-MP counterselection system for the
Sulfolobus
model renders it possible to again study the mutation profiles in mutants that have already been constructed by the use of strains with a
pyrEF
-deficient background. Furthermore, additional counterselectable markers will allow us to conduct more sophisticated genetic studies, i.e., investigate mechanisms of chromosomal DNA transfer and quantify recombination frequencies among
S. islandicus
strains.
Comparative Single-Cell Genomics of Chloroflexi from the Okinawa Trough Deep-Subsurface Biosphere
Authors: Fullerton Heather , Moyer Craig L. 
Published: March 2016

ABSTRACT

Chloroflexi
small-subunit (SSU) rRNA gene sequences are frequently recovered from subseafloor environments, but the metabolic potential of the phylum is poorly understood. The phylum
Chloroflexi
is represented by isolates with diverse metabolic strategies, including anoxic phototrophy, fermentation, and reductive dehalogenation; therefore, function cannot be attributed to these organisms based solely on phylogeny. Single-cell genomics can provide metabolic insights into uncultured organisms, like the deep-subsurface
Chloroflexi
. Nine SSU rRNA gene sequences were identified from single-cell sorts of whole-round core material collected from the Okinawa Trough at Iheya North hydrothermal field as part of Integrated Ocean Drilling Program (IODP) expedition 331 (Deep Hot Biosphere). Previous studies of subsurface
Chloroflexi
single amplified genomes (SAGs) suggested heterotrophic or lithotrophic metabolisms and provided no evidence for growth by reductive dehalogenation. Our nine
Chloroflexi
SAGs (seven of which are from the order
Anaerolineales
) indicate that, in addition to genes for the Wood-Ljungdahl pathway, exogenous carbon sources can be actively transported into cells. At least one subunit for pyruvate ferredoxin oxidoreductase was found in four of the
Chloroflexi
SAGs. This protein can provide a link between the Wood-Ljungdahl pathway and other carbon anabolic pathways. Finally, one of the seven
Anaerolineales
SAGs contains a distinct reductive dehalogenase homologous (
rdhA
) gene.

IMPORTANCE
Through the use of single amplified genomes (SAGs), we have extended the metabolic potential of an understudied group of subsurface microbes, the
Chloroflexi
. These microbes are frequently detected in the subsurface biosphere, though their metabolic capabilities have remained elusive. In contrast to previously examined
Chloroflexi
SAGs, our genomes (several are from the order
Anaerolineales
) were recovered from a hydrothermally driven system and therefore provide a unique window into the metabolic potential of this type of habitat. In addition, a reductive dehalogenase gene (
rdhA
) has been directly linked to marine subsurface
Chloroflexi
, suggesting that reductive dehalogenation is not limited to the class
Dehalococcoidia
. This discovery expands the nutrient-cycling and metabolic potential present within the deep subsurface and provides functional gene information relating to this enigmatic group.
Development of a Real-Time PCR for a Sensitive One-Step Coprodiagnosis Allowing both the Identification of Carnivore Feces and the Detection of Toxocara spp. and Echinococcus multilocularis
Authors: Knapp Jenny , Umhang Gérald , Poulle Marie-Lazarine , Millon Laurence 
Published: March 2016

ABSTRACT

Studying the environmental occurrence of parasites of concern for humans and animals based on coprosamples is an expanding field of work in epidemiology and the ecology of health. Detecting and quantifying
Toxocara
spp. and
Echinococcus multilocularis
, two predominant zoonotic helminths circulating in European carnivores, in feces may help to better target measures for prevention. A rapid, sensitive, and one-step quantitative PCR (qPCR) allowing detection of
E. multilocularis
and
Toxocara
spp. was developed in the present study, combined with a host fecal test based on the identification of three carnivores (red fox, dog, and cat) involved in the life cycles of these parasites. A total of 68 coprosamples were collected from identified specimens from
Vulpes vulpes
,
Canis lupus familiaris
,
Canis lupus
,
Felis silvestris catus
,
Meles meles
,
Martes foina
, and
Martes martes
. With DNA coprosamples, real-time PCR was performed in duplex with a qPCR inhibitor control specifically designed for this study. All the coprosample host identifications were confirmed by qPCR combined with sequencing, and parasites were detected and confirmed (
E. multilocularis
in red foxes and
Toxocara cati
in cats; 16% of samples presented inhibition). By combining parasite detection and quantification, the host fecal test, and a new qPCR inhibitor control, we created a technique with a high sensitivity that may considerably improve environmental studies of pathogens.
Cellar-Associated Saccharomyces cerevisiae Population Structure Revealed High-Level Diversity and Perennial Persistence at Sauternes Wine Estates
Authors: Börlin Marine , Venet Pauline , Claisse Olivier , Salin Franck , Legras Jean-Luc , Masneuf-Pomarede Isabelle 
Published: March 2016

ABSTRACTThree wine estates (designated A, B, and C) were sampled in Sauternes, a typical appellation of the Bordeaux wine area producing sweet white wine. From those wine estates, 551 yeast strains were collected between 2012 and 2014, added to 102 older strains from 1992 to 2011 from wine estate C. All the strains were analyzed through 15 microsatellite markers, resulting in 503 uniqueSaccharomyces cerevisiaegenotypes, revealing high genetic diversity and a low presence of commercial yeast starters. Population analysis performed usingFstgenetic distance or ancestry profiles revealed that the two closest wine estates, B and C, which have juxtaposed vineyard plots and common seasonal staff, share more related isolates with each other than with wine estate A, indicating exchange between estates. The characterization of isolates collected 23 years ago at wine estate C in relation to recent isolates obtained at wine estate B revealed the long-term persistence of isolates. Last, during the 2014 harvest period, a temporal succession of ancestral subpopulations related to the different batches associated with the selective picking of noble rotted grapes was highlighted.IMPORTANCEHigh genetic diversity ofS. cerevisiaeisolates from spontaneous fermentation on wine estates in the Sauternes appellation of Bordeaux was revealed. Only 7% of all Sauternes strains were considered genetically related to specific commercial strains. The long-term persistence (over 20 years) ofS. cerevisiaeprofiles on a given wine estate is highlighted.
Biological Low-pH Mn(II) Oxidation in a Manganese Deposit Influenced by Metal-Rich Groundwater
Authors: Bohu Tsing , Akob Denise M. , Abratis Michael , Lazar Cassandre S. , Küsel Kirsten 
Published: March 2016

ABSTRACT

The mechanisms, key organisms, and geochemical significance of biological low-pH Mn(II) oxidation are largely unexplored. Here, we investigated the structure of indigenous Mn(II)-oxidizing microbial communities in a secondary subsurface Mn oxide deposit influenced by acidic (pH 4.8) metal-rich groundwater in a former uranium mining area. Microbial diversity was highest in the Mn deposit compared to the adjacent soil layers and included the majority of known Mn(II)-oxidizing bacteria (MOB) and two genera of known Mn(II)-oxidizing fungi (MOF). Electron X-ray microanalysis showed that romanechite [(Ba,H
2
O)
2
(Mn
4+
,Mn
3+
)
5
O
10
] was conspicuously enriched in the deposit. Canonical correspondence analysis revealed that certain fungal, bacterial, and archaeal groups were firmly associated with the autochthonous Mn oxides. Eight MOB within the
Proteobacteria
,
Actinobacteria
, and
Bacteroidetes
and one MOF strain belonging to
Ascomycota
were isolated at pH 5.5 or 7.2 from the acidic Mn deposit. Soil-groundwater microcosms demonstrated 2.5-fold-faster Mn(II) depletion in the Mn deposit than adjacent soil layers. No depletion was observed in the abiotic controls, suggesting that biological contribution is the main driver for Mn(II) oxidation at low pH. The composition and species specificity of the native low-pH Mn(II) oxidizers were highly adapted to
in situ
conditions, and these organisms may play a central role in the fundamental biogeochemical processes (e.g., metal natural attenuation) occurring in the acidic, oligotrophic, and metalliferous subsoil ecosystems.

IMPORTANCE
This study provides multiple lines of evidence to show that microbes are the main drivers of Mn(II) oxidation even at acidic pH, offering new insights into Mn biogeochemical cycling. A distinct, highly adapted microbial community inhabits acidic, oligotrophic Mn deposits and mediates biological Mn oxidation. These data highlight the importance of biological processes for Mn biogeochemical cycling and show the potential for new bioremediation strategies aimed at enhancing biological Mn oxidation in low-pH environments for contaminant mitigation.
Overproduction of Magnetosomes by Genomic Amplification of Biosynthesis-Related Gene Clusters in a Magnetotactic Bacterium
Authors: Lohße Anna , Kolinko Isabel , Raschdorf Oliver , Uebe René , Borg Sarah , Brachmann Andreas , Plitzko Jürgen M. , Müller Rolf , Zhang Youming , Schüler Dirk 
Published: March 2016

ABSTRACT

Magnetotactic bacteria biosynthesize specific organelles, the magnetosomes, which are membrane-enclosed crystals of a magnetic iron mineral that are aligned in a linear chain. The number and size of magnetosome particles have to be critically controlled to build a sensor sufficiently strong to ensure the efficient alignment of cells within Earth's weak magnetic field while at the same time minimizing the metabolic costs imposed by excessive magnetosome biosynthesis. Apart from their biological function, bacterial magnetosomes have gained considerable interest since they provide a highly useful model for prokaryotic organelle formation and represent biogenic magnetic nanoparticles with exceptional properties. However, potential applications have been hampered by the difficult cultivation of these fastidious bacteria and their poor yields of magnetosomes. In this study, we found that the size and number of magnetosomes within the cell are controlled by many different Mam and Mms proteins. We present a strategy for the overexpression of magnetosome biosynthesis genes in the alphaproteobacterium
Magnetospirillum gryphiswaldense
by chromosomal multiplication of individual and multiple magnetosome gene clusters via transposition. While stepwise amplification of the
mms6
operon resulted in the formation of increasingly larger crystals (increase of ∼35%), the duplication of all major magnetosome operons (
mamGFDC
,
mamAB
,
mms6
, and
mamXY
, comprising 29 genes in total) yielded an overproducing strain in which magnetosome numbers were 2.2-fold increased. We demonstrate that the tuned expression of the
mam
and
mms
clusters provides a powerful strategy for the control of magnetosome size and number, thereby setting the stage for high-yield production of tailored magnetic nanoparticles by synthetic biology approaches.

IMPORTANCE
Before our study, it had remained unknown how the upper sizes and numbers of magnetosomes are genetically regulated, and overproduction of magnetosome biosynthesis had not been achieved, owing to the difficulties of large-scale genome engineering in the recalcitrant magnetotactic bacteria. In this study, we established and systematically explored a strategy for the overexpression of magnetosome biosynthesis genes by genomic amplification of single and multiple magnetosome gene clusters via sequential chromosomal insertion by transposition. Our findings also indicate that the expression levels of magnetosome proteins together limit the upper size and number of magnetosomes within the cell. We demonstrate that tuned overexpression of magnetosome gene clusters provides a powerful strategy for the precise control of magnetosome size and number.
Control of Legionella Contamination and Risk of Corrosion in Hospital Water Networks following Various Disinfection Procedures
Authors: Marchesi Isabella , Ferranti Greta , Mansi Antonella , Marcelloni Anna M. , Proietto Anna R. , Saini Navneet , Borella Paola , Bargellini Annalisa 
Published: March 2016

ABSTRACT

Physical and chemical disinfection methods have been proposed with the aim of controlling
Legionella
water contamination. To date, the most effective procedures for reducing bacterial contamination have not yet been defined. The aim of this study was to assess the long-term effectiveness of various disinfection procedures in order to reduce both culturable and nonculturable (NC) legionellae in different hospital water networks treated with heat, chlorine dioxide, monochloramine, and hydrogen peroxide. The temperature levels and biocide concentrations that proved to give reliable results were analyzed. In order to study the possible effects on the water pipes, we verified the extent of corrosion on experimental coupons after applying each method for 6 months. The percentage of positive points was at its lowest after treatment with monochloramine, followed by chlorine dioxide, hydrogen peroxide, and hyperthermia. Different selections of
Legionella
spp. were observed, as networks treated with chlorine-based disinfectants were contaminated mainly by
Legionella pneumophila
serogroup 1, hyperthermia was associated with serogroups 2 to 14, and hydrogen peroxide treatment was associated mainly with non-
pneumophila
species. NC cells were detected only in heat-treated waters, and also when the temperature was approximately 60°C. The corrosion rates of the coupons were within a satisfactory limit for water networks, but the morphologies differed. We confirm here that chemical disinfection controls
Legionella
colonization more effectively than hyperthermia does. Monochloramine was the most effective treatment, while hydrogen peroxide may be a promising alternative to chlorine-based disinfectants due to its ability to select for other, less virulent or nonpathogenic species.
Recognition of Histo-Blood Group Antigen-Like Carbohydrates in Lettuce by Human GII.4 Norovirus
Authors: Gao Xiang , Esseili Malak A. , Lu Zhongyan , Saif Linda J. , Wang Qiuhong 
Published: March 2016

ABSTRACT

Human norovirus (HuNoV) genogroup II genotype 4 (GII.4) strains account for about 80% of the gastroenteritis outbreaks in the United States. Contaminated food is a major transmission vehicle for this virus. In humans, pigs, and oysters, histo-blood group antigens (HBGAs) act as attachment factors for HuNoVs. In lettuce, although the virus-like particles (VLPs) of a GII.4 HuNoV were found to bind to cell wall carbohydrates, the exact binding site has not been investigated. Here, we show the presence of HBGA-like carbohydrates in the cell wall of lettuce. The digestion of lettuce leaves with cell wall-degrading enzymes exposed more binding sites and significantly increased the level of binding of GII.4 HuNoV VLPs. Competition assays showed that both the HBGA monoclonal antibody, recognizing the H type, and plant lectins, recognizing α-
l
-fucose in the H type, effectively inhibited VLP binding to lettuce tissues. Lettuce cell wall components were isolated and their NoV VLP binding characteristics were tested by enzyme-linked immunosorbent assays. The binding was inhibited by pretreatment of the lettuce cell wall materials with α-1,2-fucosidase. Collectively, our results indicate that H-type HBGA-like carbohydrates exist in lettuce tissues and that GII.4 HuNoV VLPs can bind the exposed fucose moiety, possibly in the hemicellulose component of the cell wall.

IMPORTANCE
Salad crops and fruits are increasingly recognized as vehicles for human norovirus (HuNoV) transmission. A recent study showed that HuNoVs specifically bind to the carbohydrates of the lettuce cell wall. Histo-blood group antigens (HBGAs) are carbohydrates and are known as the attachment factors for HuNoV infection in humans. In this study, we show the presence of HBGA-like carbohydrates in lettuce, to which HuNoVs specifically bind. These results suggest that specifically bound HuNoVs cannot be removed by simple washing, which may allow viral transmission to consumers. Our findings provide new information needed for developing potential inhibitors to block binding and prevent contamination.
Application of a Euryarchaeota-Specific Helicase from Thermococcus kodakarensis for Noise Reduction in PCR
Authors: Fujiwara Ayako , Kawato Katsuhiro , Kato Saori , Yasukawa Kiyoshi , Hidese Ryota , Fujiwara Shinsuke 
Published: March 2016

ABSTRACT

DNA/RNA helicases, which are enzymes for eliminating hydrogen bonds between bases of DNA/DNA, DNA/RNA, and RNA/RNA using the energy of ATP hydrolysis, contribute to various biological activities. In the present study, the
Euryarchaeota
-specific helicase EshA (TK0566) from the hyperthermophilic archaeon
Thermococcus kodakarensis
(
Tk
-EshA) was obtained as a recombinant form, and its enzymatic properties were examined.
Tk
-EshA exhibited maximal ATPase activity in the presence of RNA at 80°C. Unwinding activity was evaluated with various double-stranded DNAs (forked, 5′ overhung, 3′ overhung, and blunt end) at 50°C.
Tk
-EshA unwound forked and 3′ overhung DNAs. These activities were expected to unwind the structured template and to peel off misannealed primers when
Tk
-EshA was added to a PCR mixture. To examine the effect of
Tk
-EshA on PCR, various target DNAs were selected, and DNA synthesis was investigated. When 16S rRNA genes were used as a template, several misamplified products (noise DNAs) were detected in the absence of
Tk
-EshA. In contrast, noise DNAs were eliminated in the presence of
Tk
-EshA. Noise reduction by
Tk
-EshA was also confirmed when
Taq
DNA polymerase (a family A DNA polymerase, PolI type) and KOD DNA polymerase (a family B DNA polymerase, α type) were used for PCR. Misamplified bands were also eliminated during
toxA
gene amplification from
Pseudomonas aeruginosa
DNA, which possesses a high GC content (69%).
Tk
-EshA addition was more effective than increasing the annealing temperature to reduce misamplified DNAs during
toxA
amplification.
Tk
-EshA is a useful tool to reduce noise DNAs for accurate PCR.

IMPORTANCE
PCR is a technique that is useful for genetic diagnosis, genetic engineering, and detection of pathogenic microorganisms. However, troubles with nonspecific DNA amplification often occur from primer misannealing. In order to achieve a specific DNA amplification by eliminating noise DNAs derived from primer misannealing, a thermostable
Euryarchaeota
-specific helicase (
Tk
-EshA) was included in the PCR mixture. The addition of
Tk
-EshA has reduced noise DNAs in PCR.
Night-to-night variation of pulse oximetry in children with sleep-disordered breathing
Authors: Burke Rachael M , Maxwell Barbara , Hunter Carolyn , Graham David , O'Donoghue Dara , Shields Michael D 
Published: March 2016

BackgroundSleep-disordered breathing is a common and serious feature of many paediatric conditions and is particularly a problem in children with Down syndrome. Overnight pulse oximetry is recommended as an initial screening test, but it is unclear how overnight oximetry results should be interpreted and how many nights should be recorded.MethodsThis retrospective observational study evaluated night-to-night variation using statistical measures of repeatability for 214 children referred to a paediatric respiratory clinic, who required overnight oximetry measurements. This included 30 children with Down syndrome. We measured length of adequate trace, basal SpO2, number of desaturations (>4% SpO2 drop for >10 s) per hour (‘adjusted index’) and time with SpO2<90%. We classified oximetry traces into normal or abnormal based on physiology.Results132 out of 214 (62%) children had three technically adequate nights’ oximetry, including 13 out of 30 (43%) children with Down syndrome. Intraclass correlation coefficient for adjusted index was 0.54 (95% CI 0.20 to 0.81) among children with Down syndrome and 0.88 (95% CI 0.84 to 0.91) for children with other diagnoses. Negative predictor value of a negative first night predicting two subsequent negative nights was 0.2 in children with Down syndrome and 0.55 in children with other diagnoses.ConclusionsThere is substantial night-to-night variation in overnight oximetry readings among children in all clinical groups undergoing overnight oximetry. This is a more pronounced problem in children with Down syndrome. Increasing the number of attempted nights’ recording from one to three provides useful additional clinical information.