bluuebunny/crossref_metadata_2025_split huggingface dataset:
Molecular Analysis of the Acinetobacter baumannii Biofilm-Associated Protein
Authors: Goh H. M. Sharon , Beatson Scott A. , Totsika Makrina , Moriel Danilo G. , Phan Minh-Duy , Szubert Jan , Runnegar Naomi , Sidjabat Hanna E. , Paterson David L. , Nimmo Graeme R. , Lipman Jeffrey , Schembri Mark A. 
Published: August 2013

ABSTRACT

Acinetobacter baumannii
is a multidrug-resistant pathogen associated with hospital outbreaks of infection across the globe, particularly in the intensive care unit. The ability of
A. baumannii
to survive in the hospital environment for long periods is linked to antibiotic resistance and its capacity to form biofilms. Here we studied the prevalence, expression, and function of the
A. baumannii
biofilm-associated protein (Bap) in 24 carbapenem-resistant
A. baumannii
ST92 strains isolated from a single institution over a 10-year period. The
bap
gene was highly prevalent, with 22/24 strains being positive for
bap
by PCR. Partial sequencing of
bap
was performed on the index case strain MS1968 and revealed it to be a large and highly repetitive gene approximately 16 kb in size. Phylogenetic analysis employing a 1,948-amino-acid region corresponding to the C terminus of Bap showed that Bap
MS1968
clusters with Bap sequences from clonal complex 2 (CC2) strains ACICU, TCDC-AB0715, and 1656-2 and is distinct from Bap in CC1 strains. By using overlapping PCR, the
bap
MS1968
gene was cloned, and its expression in a recombinant
Escherichia coli
strain resulted in increased biofilm formation. A Bap-specific antibody was generated, and Western blot analysis showed that the majority of
A. baumannii
strains expressed an ∼200-kDa Bap protein. Further analysis of three Bap-positive
A. baumannii
strains demonstrated that Bap is expressed at the cell surface and is associated with biofilm formation. Finally, biofilm formation by these Bap-positive strains could be inhibited by affinity-purified Bap antibodies, demonstrating the direct contribution of Bap to biofilm growth by
A. baumannii
clinical isolates.
Temporal Changes and Altitudinal Distribution of Aerobic Anoxygenic Phototrophs in Mountain Lakes
Authors: Čuperová Zuzana , Holzer Evelyn , Salka Ivette , Sommaruga Ruben , Koblížek Michal 
Published: August 2013

ABSTRACTAerobic anoxygenic phototrophs (AAPs) are bacteriochlorophylla-containing microorganisms that use organic substrates for growth but can supplement their energy requirements with light. They have been reported from various marine and limnic environments; however, their ecology remains largely unknown. Here infrared epifluorescence microscopy was used to monitor temporal changes in AAPs in the alpine lake Gossenköllesee, located in the Tyrolean Alps, Austria. AAP abundance was low (103cells ml−1) until mid-July and reached a maximum of ∼1.3 × 105cells ml−1(29% of all prokaryotes) in mid-September. We compared the studied lake with other mountain lakes located across an altitudinal gradient (913 to 2,799 m above sea level). The concentration of dissolved organic carbon and water transparency seem to be the main factors influencing AAP abundance during the seasonal cycle as well as across the altitudinal gradient. While the AAP populations inhabiting the alpine lakes were composed of intensely pigmented large rods (5 to 12 μm), the lakes below the tree line were inhabited by a variety of smaller morphotypes. Analysis ofpufMdiversity revealed that AAPs in Gossenköllesee were almost exclusivelySphingomonadalesspecies, which indicates that AAP communities inhabiting alpine lakes are relatively homogeneous compared to those in low-altitude lakes.
A Fungal Insecticide Engineered for Fast
Per Os
Killing of Caterpillars Has High Field Efficacy and Safety in Full-Season Control of Cabbage Insect Pests
Authors: Liu Yong-Jie , Liu Jing , Ying Sheng-Hua , Liu Shu-Sheng , Feng Ming-Guang 
Published: August 2013

ABSTRACT

Fungal insecticides developed from filamentous pathogens of insects are notorious for their slow killing action through cuticle penetration, depressing commercial interest and practical application. Genetic engineering may accelerate their killing action but cause ecological risk. Here we show that a
Beauveria bassiana
formulation, HV8 (BbHV8), engineered for fast
per os
killing of caterpillars by an insect midgut-acting toxin (Vip3Aa1) overexpressed in conidia has both high field efficacy and safety in full-season protection of cabbage from the damage of an insect pest complex dominated by
Pieris rapae
larvae, followed by
Plutella xylostella
larvae and aphids. In two fields repeatedly sprayed during summer, BbHV8 resulted in overall mean efficacies of killing of 71% and 75%, which were similar or close to the 70% and 83% efficacies achieved by commercially recommended emamectin benzoate but much higher than the 31% and 48% efficacies achieved by the same formulation of the parental wild-type strain (WT). Both BbHV8 and WT sprays exerted no adverse effect on a nontarget spider community during the trials, and the sprays did not influence saprophytic fungi in soil samples taken from the field plots during 4 months after the last spray. Strikingly, BbHV8 and the WT showed low fitness when they were released into the environment because both were decreasingly recovered from the field lacking native
B. bassiana
strains (undetectable 5 months after the spray), and the recovered isolates became much less tolerant to high temperature and UV-B irradiation. Our results highlight for the first time that a rationally engineered fungal insecticide can compete with a chemical counterpart to combat insect pests at an affordable cost and with low ecological risk.
The MAP Kinase Slt2 Is Involved in Vacuolar Function and Actin Remodeling in Saccharomyces cerevisiae Mutants Affected by Endogenous Oxidative Stress
Authors: Pujol-Carrion Nuria , Petkova Mima I. , Serrano Luis , de la Torre-Ruiz Maria Angeles 
Published: August 2013

ABSTRACT

Oxidative stress causes transient actin cytoskeleton depolarization and also provokes vacuole fragmentation in wild-type cells. Under conditions of oxidative stress induced by hydrogen peroxide, the Slt2 protein is required to repolarize the actin cytoskeleton and to promote vacuole fusion. In this study, we show that
grx3 grx4
and
grx5
mutants are cellular models of endogenous oxidative stress. This stress is the result of alterations in iron homeostasis that lead to impairment of vacuolar function and also to disorganization of the actin cytoskeleton. Slt2 overexpression suppresses defects in vacuolar function and actin cytoskeleton organization in the
grx3 grx4
mutant. Slt2 exerts this effect independently of the intracellular levels of reactive oxygen species (ROS) and of iron homeostasis. The deletion of
SLT2
in the
grx3 grx4
mutant results in synthetic lethality related to vacuolar function with substantial vacuole fragmentation. The observation that both Vps4 and Vps73 (two proteins related to vacuole sorting) suppress vacuole fragmentation and actin depolarization in the
grx3 grx4 slt2
triple mutant strengthens the hypothesis that Slt2 plays a role in vacuole homeostasis related to actin dynamics. Here we show that in
sod1
,
grx5
, and
grx3 grx4 slt2
mutants, all of which are affected by chronic oxidative stress, the overexpression of Slt2 favors vacuole fusion through a mechanism dependent on an active actin cytoskeleton.
Methanosarcinaceae and Acetate-Oxidizing Pathways Dominate in High-Rate Thermophilic Anaerobic Digestion of Waste-Activated Sludge
Authors: Ho Dang P. , Jensen Paul D. , Batstone Damien J. 
Published: August 2013

ABSTRACT

This study investigated the process of high-rate, high-temperature methanogenesis to enable very-high-volume loading during anaerobic digestion of waste-activated sludge. Reducing the hydraulic retention time (HRT) from 15 to 20 days in mesophilic digestion down to 3 days was achievable at a thermophilic temperature (55°C) with stable digester performance and methanogenic activity. A volatile solids (VS) destruction efficiency of 33 to 35% was achieved on waste-activated sludge, comparable to that obtained via mesophilic processes with low organic acid levels (<200 mg/liter chemical oxygen demand [COD]). Methane yield (VS basis) was 150 to 180 liters of CH
4
/kg of VS
added
. According to 16S rRNA pyrotag sequencing and fluorescence in situ hybridization (FISH), the methanogenic community was dominated by members of the
Methanosarcinaceae
, which have a high level of metabolic capability, including acetoclastic and hydrogenotrophic methanogenesis. Loss of function at an HRT of 2 days was accompanied by a loss of the methanogens, according to pyrotag sequencing. The two acetate conversion pathways, namely, acetoclastic methanogenesis and syntrophic acetate oxidation, were quantified by stable carbon isotope ratio mass spectrometry. The results showed that the majority of methane was generated by nonacetoclastic pathways, both in the reactors and in off-line batch tests, confirming that syntrophic acetate oxidation is a key pathway at elevated temperatures. The proportion of methane due to acetate cleavage increased later in the batch, and it is likely that stable oxidation in the continuous reactor was maintained by application of the consistently low retention time.
Fluorescence-Based Bacterial Bioreporter for Specific Detection of Methyl Halide Emissions in the Environment
Authors: Farhan Ul Haque Muhammad , Nadalig Thierry , Bringel Françoise , Schaller Hubert , Vuilleumier Stéphane 
Published: August 2013

ABSTRACT

Methyl halides are volatile one-carbon compounds responsible for substantial depletion of stratospheric ozone. Among them, chloromethane (CH
3
Cl) is the most abundant halogenated hydrocarbon in the atmosphere. Global budgets of methyl halides in the environment are still poorly understood due to uncertainties in their natural sources, mainly from vegetation, and their sinks, which include chloromethane-degrading bacteria. A bacterial bioreporter for the detection of methyl halides was developed on the basis of detailed knowledge of the physiology and genetics of
Methylobacterium extorquens
CM4, an aerobic alphaproteobacterium which utilizes chloromethane as the sole source of carbon and energy. A plasmid construct with the promoter region of the chloromethane dehalogenase gene
cmuA
fused to a promotorless yellow fluorescent protein gene cassette resulted in specific methyl halide-dependent fluorescence when introduced into
M. extorquens
CM4. The bacterial whole-cell bioreporter allowed detection of methyl halides at femtomolar levels and quantification at concentrations above 10 pM (approximately 240 ppt). As shown for the model chloromethane-producing plant
Arabidopsis thaliana
in particular, the bioreporter may provide an attractive alternative to analytical chemical methods to screen for natural sources of methyl halide emissions.
Use of Aliphatic
n
-Alkynes To Discriminate Soil Nitrification Activities of Ammonia-Oxidizing Thaumarchaea and Bacteria
Authors: Taylor Anne E. , Vajrala Neeraja , Giguere Andrew T. , Gitelman Alix I. , Arp Daniel J. , Myrold David D. , Sayavedra-Soto Luis , Bottomley Peter J. 
Published: August 2013

ABSTRACT

Ammonia (NH
3
)-oxidizing bacteria (AOB) and thaumarchaea (AOA) co-occupy most soils, yet no short-term growth-independent method exists to determine their relative contributions to nitrification
in situ
. Microbial monooxygenases differ in their vulnerability to inactivation by aliphatic
n
-alkynes, and we found that NH
3
oxidation by the marine thaumarchaeon
Nitrosopumilus maritimus
was unaffected during a 24-h exposure to ≤20 μM concentrations of 1-alkynes C
8
and C
9
. In contrast, NH
3
oxidation by two AOB (
Nitrosomonas europaea
and
Nitrosospira multiformis
) was quickly and irreversibly inactivated by 1 μM C
8
(octyne). Evidence that nitrification carried out by soilborne AOA was also insensitive to octyne was obtained. In incubations (21 or 28 days) of two different whole soils, both acetylene and octyne effectively prevented NH
4
+
-stimulated increases in AOB population densities, but octyne did not prevent increases in AOA population densities that were prevented by acetylene. Furthermore, octyne-resistant, NH
4
+
-stimulated net nitrification rates of 2 and 7 μg N/g soil/day persisted throughout the incubation of the two soils. Other evidence that octyne-resistant nitrification was due to AOA included (i) a positive correlation of octyne-resistant nitrification in soil slurries of cropped and noncropped soils with allylthiourea-resistant activity (100 μM) and (ii) the finding that the fraction of octyne-resistant nitrification in soil slurries correlated with the fraction of nitrification that recovered from irreversible acetylene inactivation in the presence of bacterial protein synthesis inhibitors and with the octyne-resistant fraction of NH
4
+
-saturated net nitrification measured in whole soils. Octyne can be useful in short-term assays to discriminate AOA and AOB contributions to soil nitrification.
Comparison of PCR versus Culture for Detection of Mycobacterium bovis after Experimental Inoculation of Various Matrices Held under Environmental Conditions for Extended Periods
Authors: Adams Angela P. , Bolin Steven R. , Fine Amanda E. , Bolin Carole A. , Kaneene John B. 
Published: August 2013

ABSTRACT

The purpose of this study was to compare the performance of a molecular detection technique (nested PCR) with that of mycobacterial culture in the detection of
Mycobacterium bovis
DNA in a set of 687 samples of experimentally inoculated environmental substrates (hay, soil, corn, water) exposed to natural weather conditions in Michigan. Four replicates of each substrate were used; half were autoclaved for sterilization, all were inoculated with 50,000 CFU of
M. bovis
isolated from Michigan livestock, and all were placed in outdoor enclosures, with half under shade and the other half exposed to direct sunlight. Samples were tested for the presence of
M. bovis
during one 12-month period, with monthly sample testing and during three 12-week periods (winter, spring, summer) with weekly sample testing. Samples were subjected to mycobacterial culture for isolation of
M. bovis
and a nested PCR with two primer sets targeting IS
6110
to detect
M. bovis
DNA. In 128 samples tested during the 12-month period,
M. bovis
was not detectable by culture after 2 months but
M. bovis
DNA was detectable by PCR for at least 7 months. Of the 559 samples tested during the 12-week periods, PCR detected
M. bovis
DNA for up to 88 days in all of the sample types. There were no significant differences in the detection of
M. bovis
between shade and sun samples or between sterile and unsterilized samples, regardless of the detection method (PCR or culture). For use in epidemiologic investigations, the PCR assay was more rapid than mycobacterial culture, was not hindered by contaminating organisms, and detected
M. bovis
DNA in environment samples much longer after initial contamination than mycobacterial culture did.
Benchmarking Various Green Fluorescent Protein Variants in Bacillus subtilis, Streptococcus pneumoniae, and Lactococcus lactis for Live Cell Imaging
Authors: Overkamp Wout , Beilharz Katrin , Detert Oude Weme Ruud , Solopova Ana , Karsens Harma , Kovács Ákos T. , Kok Jan , Kuipers Oscar P. , Veening Jan-Willem 
Published: August 2013

ABSTRACT

Green fluorescent protein (GFP) offers efficient ways of visualizing promoter activity and protein localization
in vivo
, and many different variants are currently available to study bacterial cell biology. Which of these variants is best suited for a certain bacterial strain, goal, or experimental condition is not clear. Here, we have designed and constructed two “superfolder” GFPs with codon adaptation specifically for
Bacillus subtilis
and
Streptococcus pneumoniae
and have benchmarked them against five other previously available variants of GFP in
B. subtilis
,
S. pneumoniae
, and
Lactococcus lactis
, using promoter-
gfp
fusions. Surprisingly, the best-performing GFP under our experimental conditions in
B. subtilis
was the one codon optimized for
S. pneumoniae
and
vice versa
. The data and tools described in this study will be useful for cell biology studies in low-GC-rich Gram-positive bacteria.
Integrating Terminal Truncation and Oligopeptide Fusion for a Novel Protein Engineering Strategy To Improve Specific Activity and Catalytic Efficiency: Alkaline α-Amylase as a Case Study
Authors: Yang Haiquan , Liu Long , Shin Hyun-dong , Chen Rachel R. , Li Jianghua , Du Guocheng , Chen Jian 
Published: August 2013

ABSTRACT

In this work, we integrated terminal truncation and N-terminal oligopeptide fusion as a novel protein engineering strategy to improve specific activity and catalytic efficiency of alkaline α-amylase (AmyK) from
Alkalimonas amylolytica
. First, the C terminus or N terminus of AmyK was partially truncated, yielding 12 truncated mutants, and then an oligopeptide (AEAEAKAKAEAEAKAK) was fused at the N terminus of the truncated AmyK, yielding another 12 truncation-fusion mutants. The specific activities of the truncation-fusion mutants AmyKΔC500-587::OP and AmyKΔC492-587::OP were 25.5- and 18.5-fold that of AmyK, respectively. The
k
cat
/

K
m

was increased from 1.0 × 10
5
liters · mol
−1
· s
−1
for AmyK to 30.6 × and 23.2 × 10
5
liters · mol
−1
· s
−1
for AmyKΔC500-587::OP and AmyKΔC492-587::OP, respectively. Comparative analysis of structure models indicated that the higher flexibility around the active site may be the main reason for the improved catalytic efficiency. The proposed terminal truncation and oligopeptide fusion strategy may be effective to engineer other enzymes to improve specific activity and catalytic efficiency.
Environmental Detection of Genogroup I, II, and IV Noroviruses by Using a Generic Real-Time Reverse Transcription-PCR Assay
Authors: Miura Takayuki , Parnaudeau Sylvain , Grodzki Marco , Okabe Satoshi , Atmar Robert L. , Le Guyader Françoise S. 
Published: August 2013

ABSTRACT
Norovirus is the most common agent implicated in food-borne outbreaks and is frequently detected in environmental samples. These viruses are highly diverse, and three genogroups (genogroup I [GI], GII, and GIV) infect humans. Being noncultivable viruses, real-time reverse transcription-PCR (RT-PCR) is the only sensitive method available for their detection in food or environmental samples. Selection of consensus sequences for the design of sensitive assays has been challenging due to sequence diversity and has led to the development of specific real-time RT-PCR assays for each genogroup. Thus, sample screening can require several replicates for amplification of each genogroup (without considering positive and negative controls or standard curves). This study reports the development of a generic assay that detects all three human norovirus genogroups on a qualitative basis using a one-step real-time RT-PCR assay. The generic assay achieved good specificity and sensitivity for all three genogroups, detected separately or in combination. At variance with multiplex assays, the choice of the same fluorescent dye for all three probes specific to each genogroup allows the levels of fluorescence to be added and may increase assay sensitivity when multiple strains from different genogroups are present. When it was applied to sewage sample extracts, this generic assay successfully detected norovirus in all samples found to be positive by the genogroup-specific RT-PCRs. The generic assay also identified all norovirus-positive samples among 157 archived nucleic acid shellfish extracts, including samples contaminated by all three genogroups.
Exoproteome Profiles of Clostridium cellulovorans Grown on Various Carbon Sources
Authors: Matsui Kazuma , Bae Jungu , Esaka Kohei , Morisaka Hironobu , Kuroda Kouichi , Ueda Mitsuyoshi 
Published: August 2013

ABSTRACT

The cellulosome is a complex of cellulosomal proteins bound to scaffolding proteins. This complex is considered the most efficient system for cellulose degradation.
Clostridium cellulovorans
, which is known to produce cellulosomes, changes the composition of its cellulosomes depending on the growth substrates. However, studies have investigated only cellulosomal proteins; profile changes in noncellulosomal proteins have rarely been examined. In this study, we performed a quantitative proteome analysis of the whole exoproteome of
C. cellulovorans
, including cellulosomal and noncellulosomal proteins, to illustrate how various substrates are efficiently degraded.
C. cellulovorans
was cultured with cellobiose, xylan, pectin, or phosphoric acid-swollen cellulose (PASC) as the sole carbon source. PASC was used as a cellulose substrate for more accurate quantitative analysis. Using an isobaric tag method and a liquid chromatography mass spectrometer equipped with a long monolithic silica capillary column, 639 proteins were identified and quantified in all 4 samples. Among these, 79 proteins were involved in saccharification, including 35 cellulosomal and 44 noncellulosomal proteins. We compared protein abundance by spectral count and found that cellulosomal proteins were more abundant than noncellulosomal proteins. Next, we focused on the fold change of the proteins depending on the growth substrates. Drastic changes were observed mainly among the noncellulosomal proteins. These results indicate that cellulosomal proteins were primarily produced to efficiently degrade any substrate and that noncellulosomal proteins were specifically produced to optimize the degradation of a particular substrate. This study highlights the importance of noncellulosomal proteins as well as cellulosomes for the efficient degradation of various substrates.
Marked Synergistic Bactericidal Effects and Mode of Action of Medium-Chain Fatty Acids in Combination with Organic Acids against Escherichia coli O157:H7
Authors: Kim S. A. , Rhee M. S. 
Published: August 2013

ABSTRACT

The aim of this study was to examine the synergistic bactericidal effects of medium-chain fatty acids (MCFAs; caprylic, capric, and lauric acid) and organic acids (OAs; acetic, lactic, malic, and citric acid) against
Escherichia coli
O157:H7 and to identify their underlying mechanism(s) of action.
E. coli
O157:H7 was treated with MCFAs, OAs, or different combinations of MCFAs and OAs. Membrane damage and cell morphology were examined by flow cytometry and transmission electron microscopy, respectively. Combined treatment resulted in an additional log-unit reduction compared with the sum of the reductions obtained after individual treatment. For example, caprylic acid (1.0 mM, or 0.016%) and citric acid (1.0 mM, or 0.012%) alone showed negligible bactericidal effects (0.30- and 0.06-log-unit reductions, respectively); however, a marked synergistic effect (>7.15-log-unit reduction) was observed when the two were combined. Although flow cytometry and microscopic analyses of bacteria treated with individual MCFAs and OAs showed evidence of membrane disruption, the bacteria were still able to form colonies; thus, the cell damage was recoverable. In contrast, cells exposed to combined treatments showed clear membrane disintegration and/or cell death (irreversible damage). The mechanism underlying the antimicrobial effects of combined treatment with MCFAs or OAs may involve disruption of the bacterial membrane, which then facilitates the entry of other antimicrobial compounds into the cytoplasm. The main advantage of combined treatment with very low concentrations of natural antimicrobial compounds is that it is very cost-effective. Thus, this approach may be an alternative to more conventional antimicrobial treatments, such as those currently used in public health, medical centers, and the food industry.