bluuebunny/crossref_metadata_2025 huggingface dataset:
Adenosine A3 receptors regulate heart rate, motor activity and body temperature
Authors: Yang J. N. , Wang Y. , Garcia‐Roves P. M. , Björnholm M. , Fredholm B. B.
Published: February 2010

Aim:  To examine the phenotype of mice that lack the adenosine A3 receptor (A3R).

Methods:  We examined the heart rate, body temperature and locomotion continuously by telemetry over several days. In addition, the effect of the adenosine analogue R‐N6‐phenylisopropyl‐adenosine (R‐PIA) was examined. We also examined heat production and food intake.

Results:  We found that the marked diurnal variation in activity, heart rate and body temperature, with markedly higher values at night than during day time, was reduced in the A3R knock‐out mice. Surprisingly, the reduction in heart rate, activity and body temperature seen after injection of R‐PIA in wild type mice was virtually eliminated in the A3R knock‐out mice. The marked reduction in activity was associated with a decreased heat production, as expected. However, the A3R knock‐out mice, surprisingly, had a higher food intake but no difference in body weight compared to wild type mice.

Conclusions:  The mice lacking adenosine A3 receptors exhibit a surprisingly clear phenotype with changes in diurnal rhythm and temperature regulation. Whether these effects are due to a physiological role of A3 receptors in these processes or whether they represent a role in development remains to be elucidated.
Nucleotides and epidermal growth factor induce parallel cytoskeletal rearrangements and migration in cultured adult murine neural stem cells
Authors: Grimm I. , Ullsperger S. N. , Zimmermann H.
Published: January 2010

Aim:  The adult subventricular zone (SVZ) contains neural stem cells that generate neuroblasts migrating to the olfactory bulb (OB) and differentiating into interneurones. The molecular cues controlling essential functions within the neurogenesis pathway such as proliferation, short and long distance migration, functional integration and cell survival are poorly understood. We have previously shown that cultured adult neural stem cells express a considerable variety of nucleotide receptors and that nucleotides and epidermal growth factor (EGF) induce converging intracellular signalling pathways that carry potential for synergism in the control of neural stem cell proliferation and cell survival. Here we investigate the role of EGF and the nucleotides ATP, ADPβS and UTP in neural stem cell migration.

Methods:  Neural stem cells were prepared from adult mice and subjected to adherent culture. Labelling of F‐actin was performed with tetramethylrhodamine isothiocyanate‐phalloidin. Images were processed for quantitative evaluation of fluorescence labelling. Agonist‐induced phosphorylation of AKT and focal adhesion kinase was analysed by quantitative Western blotting. Agonist‐dependent cell migration was assayed using 48‐well microchemotaxis chambers.

Results:  Nucleotides and EGF induce the formation of stress fibres, an increase in the cortical actin cytoskeleton and in cell spreading. This is associated with increased phosphorylation of AKT and focal adhesion kinase. Using microchemotaxis chambers we demonstrate a parallel increase in cell migration.

Conclusion:  Our results suggest that nucleotides and EGF acting as paracrine or autocrine signalling substances can be of relevance for structuring and maintaining the cytoarchitecture of the SVZ and the stream of neuroblasts migrating to the OB.
Efficacy of live attenuated influenza vaccine in children 6 months to 17 years of age
Authors: Belshe Robert B. , Toback Seth L. , Yi Tingting , Ambrose Christopher S.
Published: February 2010

Please cite this paper as: Belshe et al. (2010). Efficacy of live attenuated influenza vaccine in children 6 months to 17 years of age. Influenza and Other Respiratory Viruses 4(3), 141–145.

Background  It has been suggested that live attenuated influenza vaccine (LAIV) may be less effective in older individuals because of prior wild‐type influenza infections. LAIV is currently approved in the United States, South Korea and Hong Kong for individuals 2–49 years of age.

Objective  To examine data from previously published pediatric studies to determine the efficacy of LAIV in various age groups.

Methods  Four studies in which the subject age range exceeded 36 months were identified: one 2‐year study comparing LAIV with placebo and three 1‐year studies comparing LAIV with trivalent inactivated influenza vaccine (TIV). Efficacy against any strain regardless of antigenic similarity to vaccine was analyzed by age; age groups were based on the study design and sample size. A logistic regression model was used to assess whether age, as a continuous variable, was an effect modifier on LAIV efficacy.

Results  The efficacy of LAIV did not vary with age in children aged 15–84 months compared with placebo or in children aged 6 months to 17 years compared with TIV.

Conclusions  The available data from prospective, randomized studies in children does not support the concept that prior repeated exposure to influenza, either through wild‐type infection or vaccination with live, attenuated or inactivated vaccines, reduces the efficacy of LAIV compared with placebo or TIV. The decreased immunologic responses to LAIV reported in older individuals or those with pre‐existing immunity do not appear to translate into reduced protection from influenza in children.
Prymnesium parvum Population Dynamics During Bloom Development: A Role Assessment of Grazers and Virus1
Authors: Schwierzke Leslie , Roelke Daniel L. , Brooks Bryan W. , Grover James P. , Valenti Theodore W. , Lahousse Mieke , Miller Carrie J. , Pinckney James L.
Published: February 2010

Schwierzke, Leslie, Daniel L. Roelke, Bryan W. Brooks, James P. Grover, Theodore W. Valenti, Jr., Mieke Lahousse, Carrie J. Miller, and James L. Pinckney, 2010. Prymnesium parvum Population Dynamics During Bloom Development: A Role Assessment of Grazers and Virus. Journal of the American Water Resources Association (JAWRA) 46(1):63‐75. DOI: 10.1111/j.1752‐1688.2009.00391.x

Abstract:  The toxic haptophyte Prymnesium parvum is a harmful alga known to cause fish‐killing blooms that occur worldwide. In Texas (United States), P. parvum blooms occur in inland brackish water bodies and have increased in frequency and magnitude in recent years. In this study we conducted three consecutive field experiments (Lake Whitney) to investigate the influence of zooplankton and viruses on P. parvum bloom dynamics during the time of year when blooms are still typically active in Texas (early spring). A localized P. parvum bloom developed during our study that involved increasing levels of toxicity (based on Pimephales promelas and Daphnia magna bioassays). Only in our last experiment, during later stages of bloom development and under highly toxic conditions, did the presence of grazers show a statistically significant, negative effect on P. parvum population dynamics. During this experiment, a rotifer‐dominated zooplankton community emerged, composed mostly of Notholca laurentiae, suggesting that this species was less sensitive than other grazers to toxins produced by P. parvum. Microzooplankton may have also been important at this time. Similarly, only our final experiment demonstrated a statistically significant, negative effect of viruses on P. parvum. This exploratory study, resulting in observed impacts on P. parvum populations by both grazers and virus, enhances our understanding of P. parvum ecology and highlights direction for future studies on resistance of zooplankton to prymnesin toxins and algal‐virus interactions.
Factors Influencing Allelopathy and Toxicity in Prymnesium parvum1
Authors: Granéli Edna , Salomon Paulo S.
Published: February 2010

Granéli, Edna and Paulo S. Salomon, 2010. Factors Influencing Allelopathy and Toxicity in Prymnesium parvum. Journal of the American Water Resources Association (JAWRA) 46(1):108‐120.

Abstract:  Some microalgae are able to kill or inhibit nutrient‐competing microalgae, a process called allelopathy. Inhibiting or killing competitors enable these species to monopolize limiting resources, such as nitrogen and phosphorus. Prymnesium parvum is known to produce such allelopathic compounds, substances that seem identical to the ichthyotoxins identified from this species. Biotic and abiotic environmental factors influence not only growth rates but also toxin/allelopathic compounds production by P. parvum cells. Toxin production, as well as allelopathy, including grazer deterrence, increases dramatically in light, temperature, or nutrient stressed P. parvum cells. Correspondingly, toxicity and allelopathy may decrease, or cease completely, if cells are grown with high amounts of N and P in balanced proportions. However, even under nutrient (N and P) sufficient conditions, P. parvum is able to produce toxins/allelopathic compounds, with negative effects on other phytoplankton species or grazers, if cells densities of P. parvum are high relative to other species. This negative effect might shift the plankton community to more toxin resistant species. Filtrates from nutrient‐deficient P. parvum cultures have almost the same strong negative effect on grazers and other phytoplankton species as when Prymnesium cells are grown together with the target organisms. Eutrophication, the increased input of N and P to aquatic ecosystems, besides increasing nutrient concentrations, is usually provoking unbalanced N:P condition for the optimal growth of phytoplankton, deviating from the Redfield ratio, i.e., the phytoplankton cellular nitrogen to phosphorus ratio, N:P = 16:1 (by atoms) or 7.2:1 (by weight). Eutrophication thus both enhances P. parvum growth and increases production of toxins and allelopathic compounds. Supplying N‐deficient or P‐deficient P. parvum cells with the deficient nutrient reduces toxicity to less than half within 24 h after additions. As P. parvum is mixotrophic, uptake of dissolved or particulate organic N (DON or PON) can also reduce toxicity and allelopathy in the same manner as addition of inorganic N to N‐starved cells. In conclusion, P. parvum, by increasing its toxicity and allelopathic ability under poor environmental conditions, outcompetes the co‐occurring phytoplankton species.
Influence of Prey and Nutritional Status on the Rate of Nitrogen Uptake by Prymnesium parvum (haptophyte)1
Authors: Lindehoff Elin , Granéli Edna , Glibert Patricia M.
Published: February 2010

Lindehoff, Elin, Edna Granéli, and Patricia M. Glibert, 2010. Influence of Prey and Nutritional Status on the Rate of Nitrogen Uptake by Prymnesium parvum (Haptophyte). Journal of the American Water Resources Association (JAWRA) 46(1):121‐132. DOI: 10.1111/j.1752‐1688.2009.00396.x

Abstract:  We studied how the specific nitrogen (N) uptake rates of nitrate (NO3−), urea, and the amino acids, glutamic acid and glycine, by Prymnesium parvum were affected by (1) the change from N‐deficient status to N‐sufficient status of the P. parvum cells, (2) presence of prey from a natural Baltic Sea plankton community, and (3) the composition of prey as affected by additions of terrestrial originated dissolved organic matter (DOM) or inorganic nutrients. Nitrogen‐deficient P. parvum (16 μM NO3− and 4 μM PO4−, molar N:P ratio of 4:1) were mixed with a natural Baltic plankton community and given PO43− and (1) NO3− (control) or (2) high molecular weight DOM, >1 kDa concentrated from sewage effluent (+DOM), in a molar N:P ratio of 9‐10:1. With additions of 15N‐enriched substrates, rates of N uptake from NO3−, urea, and the amino acids glycine and glutamic acid were measured every 24 h for 72 h. Initial N‐deficient P. parvum were highly toxic (3.7 ± 0.9 × 10−4 mg Sap equiv/cell) and toxic allelochemicals were released into the medium causing the natural plankton community to lyse. Rates of N uptake differed between the “control” and the “+DOM” treatments over time; total (sum of the N substrates measured) absolute uptake rates (ρcell, fmol N/cell/h) at ambient culture conditions were significantly higher (ANOVA, p < 0.05) in the more toxic “control” treatments compared with the “+DOM” treatments after 48 h. In the “control” treatment, the total ρcell increased significantly (ANOVA, p < 0.01) from time 0 to 48 h, while in the “+DOM” treatment there was no significant increase. Released organic nutrients from the lysed plankton cells may have increased uptake rates of amino acids and urea by P. parvum. All uptake rates declined in all treatments by 72 h. Total dissolved N uptake rates at ambient culture conditions were estimated to make up about 10% of the N P. parvum are potentially capable of ingesting from particulate prey.
Multicell Homogenization of One-Dimensional Periodic Structures
Authors: Gonella Stefano , Ruzzene Massimo
Published: February 2010

Much attention has been recently devoted to the application of homogenization methods for the prediction of the dynamic behavior of periodic domains. One of the most popular techniques employs the Fourier transform in space in conjunction with Taylor series expansions to approximate the behavior of structures in the low frequency/long wavelength regime. The technique is quite effective, but suffers from two major drawbacks. First, the order of the Taylor expansion, and the corresponding frequency range of approximation, is limited by the resulting order of the continuum equations and by the number of boundary conditions, which may be imposed in accordance with the physical constraints on the system. Second, the approximation at low frequencies does not allow capturing bandgap characteristics of the periodic domain. An attempt at overcoming the latter can be made by applying the Fourier series expansion to a macrocell spanning two (or more) irreducible unit cells of the periodic medium. This multicell approach allows the simultaneous approximation of low frequency and high frequency dynamic behavior and provides the capability of analyzing the structural response in the vicinity of the lowest bandgap. The method is illustrated through examples on simple one-dimensional structures to demonstrate its effectiveness and its potentials for application to complex one-dimensional and two-dimensional configurations.
Iron-Corroding Methanogen Isolated from a Crude-Oil Storage Tank
Authors: Uchiyama Taku , Ito Kimio , Mori Koji , Tsurumaru Hirohito , Harayama Shigeaki
Published: January 2010

Microbiologically influenced corrosion of steel in anaerobic environments has been attributed to hydrogenotrophic microorganisms. A sludge sample collected from the bottom plate of a crude-oil storage tank was used to inoculate a medium containing iron (Fe
0
) granules, which was then incubated anaerobically at 37°C under an N
2
-CO
2
atmosphere to enrich for microorganisms capable of using iron as the sole source of electrons. A methanogen, designated strain KA1, was isolated from the enrichment culture. An analysis of its 16S rRNA gene sequence revealed that strain KA1 is a
Methanococcus maripaludis
strain. Strain KA1 produced methane and oxidized iron much faster than did the type strain of
M. maripaludis
, strain JJ
T
, which produced methane at a rate expected from the abiotic H
2
production rate from iron. Scanning electron micrographs of iron coupons that had been immersed in either a KA1 culture, a JJ
T
culture, or an aseptic medium showed that only coupons from the KA1 culture had corroded substantially, and these were covered with crystalline deposits that consisted mainly of FeCO
3
.
Alkaloid Cluster Gene
ccsA
of the Ergot Fungus
Claviceps purpurea
Encodes Chanoclavine I Synthase, a Flavin Adenine Dinucleotide-Containing Oxidoreductase Mediating the Transformation of
N
-Methyl-Dimethylallyltryptophan to Chanoclavine I
Authors: Lorenz Nicole , Olšovská Jana , Šulc Miroslav , Tudzynski Paul
Published: January 2010

Ergot alkaloids are indole-derived secondary metabolites synthesized by the phytopathogenic ascomycete
Claviceps purpurea
. In wild-type strains, they are exclusively produced in the sclerotium, a hibernation structure; for biotechnological applications, submerse production strains have been generated by mutagenesis. It was shown previously that the enzymes specific for alkaloid biosynthesis are encoded by a gene cluster of 68.5 kb. This ergot alkaloid cluster consists of 14 genes coregulated and expressed under alkaloid-producing conditions. Although the role of some of the cluster genes in alkaloid biosynthesis could be confirmed by a targeted knockout approach, further functional analyses are needed, especially concerning the early pathway-specific steps up to the production of clavine alkaloids. Therefore, the gene
ccsA
, originally named
easE
and preliminarily annotated as coding for a flavin adenine dinucleotide-containing oxidoreductase, was deleted in the
C. purpurea
strain P1, which is able to synthesize ergot alkaloids in axenic culture. Five independent knockout mutants were analyzed with regard to alkaloid-producing capability. Thin-layer chromatography (TLC), ultrapressure liquid chromatography (UPLC), and mass spectrometry (MS) analyses revealed accumulation of
N
-methyl-dimethylallyltryptophan (Me-DMAT) and traces of dimethylallyltryptophan (DMAT), the first pathway-specific intermediate. Since other alkaloid intermediates could not be detected, we conclude that deletion of
ccsA
led to a block in alkaloid biosynthesis beyond Me-DMAT formation. Complementation with a
ccsA
/
gfp
fusion construct restored alkaloid biosynthesis. These data indicate that
ccsA
encodes the chanoclavine I synthase or a component thereof catalyzing the conversion of
N
-methyl-dimethylallyltryptophan to chanoclavine I.
Genes Involved in
Cronobacter sakazakii
Biofilm Formation
Authors: Hartmann Isabel , Carranza Paula , Lehner Angelika , Stephan Roger , Eberl Leo , Riedel Kathrin
Published: January 2010

Cronobacter
spp. are opportunistic food-borne pathogens that can cause severe and sometimes lethal infections in neonates. In some outbreaks, the sources of infection were traced to contaminated powdered infant formula (PIF) or contaminated utensils used for PIF reconstitution. In this study, we investigated biofilm formation in
Cronobacter sakazakii
strain ES5. To investigate the genetic basis of biofilm formation in
Cronobacter
on abiotic surfaces, we screened a library of random transposon mutants of strain ES5 for reduced biofilm formation using a polystyrene microtiter assay. Genetic characterization of the mutants led to identification of genes that are associated with cellulose biosynthesis and flagellar structure and biosynthesis and genes involved in basic cellular processes and virulence, as well as several genes whose functions are currently unknown. In two of the mutants, hypothetical proteins ESA_00281 and ESA_00282 had a strong impact on flow cell biofilm architecture, and their contribution to biofilm formation was confirmed by genetic complementation. In addition, adhesion of selected biofilm formation mutants to Caco-2 intestinal epithelial cells was investigated. Our findings suggest that flagella and hypothetical proteins ESA_00281 and ESA_00282, but not cellulose, contribute to adhesion of
Cronobacter
to this biotic surface.
Cleavage of Zearalenone byTrichosporon mycotoxinivoransto a Novel Nonestrogenic Metabolite
Authors: Vekiru Elisavet , Hametner Christian , Mitterbauer Rudolf , Rechthaler Justyna , Adam Gerhard , Schatzmayr Gerd , Krska Rudolf , Schuhmacher Rainer
Published: January 2010

Zearalenone (ZON) is a potent estrogenic mycotoxin produced by severalFusariumspecies most frequently on maize and therefore can be found in food and animal feed. Since animal production performance is negatively affected by the presence of ZON, its detoxification in contaminated plant material or by-products of bioethanol production would be advantageous. Microbial biotransformation into nontoxic metabolites is one promising approach. In this study the main transformation product of ZON formed by the yeastTrichosporon mycotoxinivoranswas identified and characterized by liquid chromatography-tandem mass spectrometry (LC-MS/MS) and LC-diode array detector (DAD) analysis. The metabolite, named ZOM-1, was purified, and its molecular formula, C18H24O7, was established by time of flight MS (TOF MS) from the ions observed atm/z351.1445 [M-H]−and atm/z375.1416 [M+Na]+. Employing nuclear magnetic resonance (NMR) spectroscopy, the novel ZON metabolite was finally identified as (5S)-5-({2,4-dihydroxy-6-[(1E)-5-hydroxypent-1-en-1-yl]benzoyl}oxy)hexanoic acid. The structure of ZOM-1 is characterized by an opening of the macrocyclic ring of ZON at the ketone group at C6′. ZOM-1 did not show estrogenic activity in a sensitive yeast bioassay, even at a concentration 1,000-fold higher than that of ZON and did not interact with the human estrogen receptor in anin vitrocompetitive binding assay.
Identification of Pathogenic
Vibrio
Species by Multilocus PCR-Electrospray Ionization Mass Spectrometry and Its Application to Aquatic Environments of the Former Soviet Republic of Georgia
Authors: Whitehouse Chris A. , Baldwin Carson , Sampath Rangarajan , Blyn Lawrence B. , Melton Rachael , Li Feng , Hall Thomas A. , Harpin Vanessa , Matthews Heather , Tediashvili Marina , Jaiani Ekaterina , Kokashvili Tamar , Janelidze Nino , Grim Christopher , Colwell Rita R. , Huq Anwar
Published: January 2010

The Ibis T5000 is a novel diagnostic platform that couples PCR and mass spectrometry. In this study, we developed an assay that can identify all known pathogenic
Vibrio
species and field-tested it using natural water samples from both freshwater lakes and the Georgian coastal zone of the Black Sea. Of the 278 total water samples screened, 9 different
Vibrio
species were detected, 114 (41%) samples were positive for
V. cholerae
, and 5 (0.8%) samples were positive for the cholera toxin A gene (
ctxA
). All
ctxA
-positive samples were from two freshwater lakes, and no
ctxA
-positive samples from any of the Black Sea sites were detected.
Plasmid pAMI2 of
Paracoccus aminophilus
JCM 7686 Carries
N
,
N
-Dimethylformamide Degradation-Related Genes Whose Expression Is Activated by a LuxR Family Regulator
Authors: Dziewit Lukasz , Dmowski Michal , Baj Jadwiga , Bartosik Dariusz
Published: January 2010

N
,
N
-Dimethylformamide (DMF), a toxic solvent used in the chemical industry, is frequently present in industrial wastes. Plasmid pAMI2 (18.6 kb) of
Paracoccus aminophilus
JCM 7686 carries genetic information which is crucial for methylotrophic growth of this bacterium, using DMF as the sole source of carbon and energy. Besides a conserved backbone related to pAgK84 of
Agrobacterium radiobacter
K84, pAMI2 carries a three-gene cluster coding for the protein DmfR, which has sequence similarities to members of the LuxR family of transcription regulators, and two subunits (DmfA1 and DmfA2) of
N
,
N
-dimethylformamidase, an enzyme of high substrate specificity that catalyzes the first step in the degradation of DMF. Genetic analysis revealed that these genes, which are all placed in the same orientation, constitute an inducible operon whose expression is activated in the presence of DMF by the positive transcription regulator DmfR. This operon was used to construct a strain able to degrade DMF at high concentrations that might be used in the biotreatment of DMF-containing industrial wastewaters. To our knowledge, this is the first study to provide insights into the genetic organization and regulation as well as the dissemination in bacteria of genes involved in the enzymatic breakdown of DMF.
Detection and Quantification of Infectious Avian Influenza A (H5N1) Virus in Environmental Water by Using Real-Time Reverse Transcription-PCR
Authors: Dovas C. I. , Papanastassopoulou M. , Georgiadis M. P. , Chatzinasiou E. , Maliogka V. I. , Georgiades G. K.
Published: January 2010

Routes of avian influenza virus (AIV) dispersal among aquatic birds involve direct (bird-to-bird) and indirect (waterborne) transmission. The environmental persistence of H5N1 virus in natural water reservoirs can be assessed by isolation of virus in embryonated chicken eggs. Here we describe development and evaluation of a real-time quantitative reverse transcription (RT)-PCR (qRT-PCR) method for detection of H5N1 AIV in environmental water. This method is based on adsorption of virus particles to formalin-fixed erythrocytes, followed by qRT-PCR detection. The numbers of hemagglutinin RNA copies from H5N1 highly pathogenic AIV particles adsorbed to erythrocytes detected correlated highly with the infectious doses of the virus that were determined for three different types of artificially inoculated environmental water over a 17-day incubation period. The advantages of this method include detection and quantification of infectious H5N1 AIVs with a high level of sensitivity, a wide dynamic range, and reproducibility, as well as increased biosecurity. The lowest concentration of H5N1 virus that could be reproducibly detected was 0.91 50% egg infective dose per ml. In addition, a virus with high virion stability (
Tobacco mosaic virus
) was used as an internal control to accurately monitor the efficiency of RNA purification, cDNA synthesis, and PCR amplification for each individual sample. This detection system could be useful for rapid high-throughput monitoring for the presence of H5N1 AIVs in environmental water and in studies designed to explore the viability and epidemiology of these viruses in different waterfowl ecosystems. The proposed method may also be adapted for detection of other AIVs and for assessment of their prevalence and distribution in environmental reservoirs.
Mucosal Adhesion Properties of the ProbioticLactobacillus rhamnosusGG SpaCBA and SpaFED Pilin Subunits
Authors: von Ossowski Ingemar , Reunanen Justus , Satokari Reetta , Vesterlund Satu , Kankainen Matti , Huhtinen Heikki , Tynkkynen Soile , Salminen Seppo , de Vos Willem M. , Palva Airi
Published: January 2010

Lactobacillus rhamnosusGG is a well-established Gram-positive probiotic strain, whose health-benefiting properties are dependent in part on prolonged residence in the gastrointestinal tract and are likely dictated by adherence to the intestinal mucosa. Previously, we identified two pilus gene clusters (spaCBAandspaFED) in the genome of this probiotic bacterium, each of which contained the predicted genes for three pilin subunits and a single sortase. We also confirmed the presence of SpaCBA pili on the cell surface and attributed an intestinal mucus-binding capacity to one of the pilin subunits (SpaC). Here, we report cloning of the remaining pilin genes (spaA,spaB,spaD,spaE, andspaF) inEscherichia coli, production and purification of the recombinant proteins, and assessment of the adherence of these proteins to human intestinal mucus. Our findings indicate that the SpaB and SpaF pilin subunits also exhibit substantial binding to mucus, which can be inhibited competitively in a dose-related manner. Moreover, the binding between the SpaB pilin subunit and the mucosal substrate appears to operate through electrostatic contacts and is not related to a recognized mucus-binding domain. We conclude from these results that it is conceivable that two pilin subunits (SpaB and SpaC) in the SpaCBA pilus fiber play a role in binding to intestinal mucus, but for the uncharacterized and putative SpaFED pilus fiber only a single pilin subunit (SpaF) is potentially responsible for adhesion to mucus.
Evolutionary Dynamics of Clustered Irregularly Interspaced Short Palindromic Repeat Systems in the Ocean Metagenome
Authors: Sorokin Valery A. , Gelfand Mikhail S. , Artamonova Irena I.
Published: January 2010

Clustered regularly interspaced short palindromic repeats (CRISPRs) form a recently characterized type of prokaryotic antiphage defense system. The phage-host interactions involving CRISPRs have been studied in experiments with selected bacterial or archaeal species and, computationally, in completely sequenced genomes. However, these studies do not allow one to take prokaryotic population diversity and phage-host interaction dynamics into account. This gap can be filled by using metagenomic data: in particular, the largest existing data set, generated from theSorcerer IIGlobal Ocean Sampling expedition. The application of three publicly available CRISPR recognition programs to the Global Ocean metagenome produced a large proportion of false-positive results. To address this problem, a filtering procedure was designed. It resulted in about 200 reliable CRISPR cassettes, which were then studied in detail. The repeat consensuses were clustered into several stable classes that differed from the existing classification. Short fragments of DNA similar to the cassette spacers were more frequently present in the same geographical location than in other locations (P, <0.0001). We developed a catalogue of elementary CRISPR-forming events and reconstructed the likely evolutionary history of cassettes that had common spacers. Metagenomic collections allow for relatively unbiased analysis of phage-host interactions and CRISPR evolution. The results of this study demonstrate that CRISPR cassettes retain the memory of the local virus population at a particular ocean location. CRISPR evolution may be described using a limited vocabulary of elementary events that have a natural biological interpretation.
Real-Time Microsensor Measurement of Local Metabolic Activities in
Ex Vivo
Dental Biofilms Exposed to Sucrose and Treated with Chlorhexidine
Authors: von Ohle Christiane , Gieseke Armin , Nistico Laura , Decker Eva Maria , deBeer Dirk , Stoodley Paul
Published: January 2010

Dental biofilms are characterized by structural and functional heterogeneity. Due to bacterial metabolism, gradients develop and diverse ecological microniches exist. The aims of this study were (i) to determine the metabolic activity of microorganisms in naturally grown dental biofilms
ex vivo
by measuring dissolved oxygen (DO) and pH profiles with microelectrodes with high spatial resolution and (ii) to analyze the impact of an antimicrobial chlorhexidine (CHX) treatment on microbial physiology during stimulation by sucrose in real time. Biofilms were cultivated on standardized human enamel surfaces
in vivo
. DO and pH profiles were measured in a flow cell system in sterile human saliva, after sucrose addition (10%), again after alternative treatment of the sucrose exposed biofilms with CHX (0.2%) for 1 or 10 min or after being killed with paraformaldehyde (4%). Biofilm structure was visualized by vitality staining with confocal microscopy. With saliva as the sole nutrient source oxygen consumption was high within the superficial biofilm layers rendering deeper layers (>220 μm) anoxic. Sucrose addition induced the thickness of the anaerobic zone to increase with a concurrent decrease in pH (7.1 to 4.4). CHX exposure reduced metabolic activity and microbial viability at the biofilm surface and drove metabolic activity deeper into the biofilm. CHX treatment led to a reduced viability at the biofilm surface with minor influence on overall biofilm physiology after 1 min; even after 10 min there was measurable respiration and fermentation inside the biofilm. However, the local microenvironment was more aerated, less acidogenic, and presumably less pathogenic.
Sequence Analysis of
Leuconostoc mesenteroides
Bacteriophage Φ1-A4 Isolated from an Industrial Vegetable Fermentation
Authors: Lu Z. , Altermann E. , Breidt F. , Kozyavkin S.
Published: January 2010

Vegetable fermentations rely on the proper succession of a variety of lactic acid bacteria (LAB).
Leuconostoc mesenteroides
initiates fermentation. As fermentation proceeds,
L. mesenteroides
dies off and other LAB complete the fermentation. Phages infecting
L. mesenteroides
may significantly influence the die-off of
L. mesenteroides
. However, no
L. mesenteroides
phages have been previously genetically characterized. Knowledge of more phage genome sequences may provide new insights into phage genomics, phage evolution, and phage-host interactions. We have determined the complete genome sequence of
L. mesenteroides
phage Φ1-A4, isolated from an industrial sauerkraut fermentation. The phage possesses a linear, double-stranded DNA genome consisting of 29,508 bp with a G+C content of 36%. Fifty open reading frames (ORFs) were predicted. Putative functions were assigned to 26 ORFs (52%), including 5 ORFs of structural proteins. The phage genome was modularly organized, containing DNA replication, DNA-packaging, head and tail morphogenesis, cell lysis, and DNA regulation/modification modules.
In silico
analyses showed that Φ1-A4 is a unique lytic phage with a large-scale genome inversion (∼30% of the genome). The genome inversion encompassed the lysis module, part of the structural protein module, and a cos site. The endolysin gene was flanked by two holin genes. The tail morphogenesis module was interspersed with cell lysis genes and other genes with unknown functions. The predicted amino acid sequences of the phage proteins showed little similarity to other phages, but functional analyses showed that Φ1-A4 clusters with several
Lactococcus
phages. To our knowledge, Φ1-A4 is the first genetically characterized
L. mesenteroides
phage.
Soil Resources Influence Spatial Patterns of Denitrifying Communities at Scales Compatible with Land Management
Authors: Enwall Karin , Throbäck Ingela N. , Stenberg Maria , Söderström Mats , Hallin Sara
Published: January 2010

Knowing spatial patterns of functional microbial guilds can increase our understanding of the relationships between microbial community ecology and ecosystem functions. Using geostatistical modeling to map spatial patterns, we explored the distribution of the community structure, size, and activity of one functional group in N cycling, the denitrifiers, in relation to 23 soil parameters over a 44-ha farm divided into one organic and one integrated crop production system. The denitrifiers were targeted by thenirSandnirKgenes that encode the two mutually exclusive types of nitrite reductases, the cd1heme-type and copper reductases, respectively. The spatial pattern of the denitrification activity genes was reflected by the maps of the abundances ofnirgenes. For the community structure, only the maps of thenirScommunity were related to the activity. The activity was correlated with nitrate and dissolved organic nitrogen and carbon, whereas the gene pools for denitrification, in terms of size and composition, were influenced by the soil structure. For thenirScommunity, pH and soil nutrients were also important in shaping the community. The only unique parameter related to thenirKcommunity was the soil Cu content. However, the spatial pattern of thenirKdenitrifiers corresponded to the division of the farm into the two cropping systems. The different community patterns, together with the spatial distribution of thenirS/nirKabundance ratio, suggest habitat selection on thenirS- andnirK-type denitrifiers. Our findings constitute a first step in identifying niches for denitrifiers at scales relevant to land management.
Functional Genomic Study of Exogenous
n
-Butanol Stress in
Escherichia coli
Authors: Rutherford Becky J. , Dahl Robert H. , Price Richard E. , Szmidt Heather L. , Benke Peter I. , Mukhopadhyay Aindrila , Keasling Jay D.
Published: January 2010

n
-Butanol has been proposed as an alternative biofuel to ethanol, and several industrially used microbes, including
Escherichia coli
, have been engineered to produce it. Unfortunately,
n
-butanol is more toxic than ethanol to these organisms. To understand the basis for its toxicity, cell-wide studies were conducted at the transcript, protein, and metabolite levels to obtain a global view of the
n
-butanol stress response. Analysis of the data indicates that
n
-butanol stress has components common to other stress responses, including perturbation of respiratory functions (
nuo
and
cyo
operons), oxidative stress (
sodA
,
sodC
, and
yqhD
), heat shock and cell envelope stress (
rpoE
,
clpB
,
htpG
,
cpxR
, and
cpxP
), and metabolite transport and biosynthesis (
malE
and
opp
operon). Assays using fluorescent dyes indicated a large increase in reactive oxygen species during
n
-butanol stress, confirming observations from the microarray and proteomics measurements. Mutant strains with mutations in several genes whose products changed most dramatically during
n
-butanol stress were examined for increased sensitivity to
n
-butanol. Results from these analyses allowed identification of key genes that were recruited to alleviate oxidative stress, protein misfolding, and other causes of growth defects. Cellular engineering based on these cues may assist in developing a high-titer,
n
-butanol-producing host.
Roles of Siderophores, Oxalate, and Ascorbate in Mobilization of Iron from Hematite by the Aerobic Bacterium
Pseudomonas mendocina
Authors: Dehner Carolyn A. , Awaya Jonathan D. , Maurice Patricia A. , DuBois Jennifer L.
Published: January 2010

In aerobic, circumneutral environments, the essential element Fe occurs primarily in scarcely soluble mineral forms. We examined the independent and combined effects of a siderophore, a reductant (ascorbate), and a low-molecular-weight carboxylic acid (oxalate) on acquisition of Fe from the mineral hematite (α-Fe
2
O
3
) by the obligate aerobe
Pseudomonas mendocina
ymp. A site-directed Δ
pmhA
mutant that was not capable of producing functional siderophores (i.e., siderophore
−
phenotype) did not grow on hematite as the only Fe source. The concentration of an added exogenous siderophore (1 μM desferrioxamine B [DFO-B]) needed to restore wild-type (WT)-like growth kinetics to the siderophore
−
strain was ∼50-fold less than the concentration of the siderophore secreted by the WT organism grown under the same conditions. The roles of a reductant (ascorbate) and a simple carboxylic acid (oxalate) in the Fe acquisition process were examined in the presence and absence of the siderophore. Addition of ascorbate (50 μM) alone restored the growth of the siderophore
−
culture to the WT levels. A higher concentration of oxalate (100 μM) had little effect on the growth of a siderophore
−
culture; however, addition of 0.1 μM DFO-B and 100 μM oxalate restored the growth of the mutant to WT levels when the oxalate was prereacted with the hematite, demonstrating that a metabolizing culture benefits from a synergistic effect of DFO-B and oxalate.
Molecular Indicators Used in the Development of Predictive Models for Microbial Source Tracking
Authors: Ballesté Elisenda , Bonjoch Xavier , Belanche Lluís A. , Blanch Anicet R.
Published: January 2010

A number of chemical, microbial, and eukaryotic indicators have been proposed as indicators of fecal pollution sources in water bodies. No single one of the indicators tested to date has been able to determine the source of fecal pollution in water. However, the combined use of different indicators has been demonstrated to be the best way of defining predictive models suitable for determining fecal pollution sources. Molecular methods are promising tools that could complement standard microbiological water analysis. In this study, the feasibility of some proposed molecular indicators for microbial source tracking (MST) was compared (names of markers are in parentheses): host-specific
Bacteroidetes
(HF134, HF183, CF128, and CF193),
Bifidobacterium adolescentis
(ADO),
Bifidobacterium dentium
(DEN), the gene
esp
of
Enterococcus faecium
, and host-specific mitochondrial DNA associated with humans, cattle, and pigs (Humito, Bomito, and Pomito, respectively). None of the individual molecular markers tested enabled 100% source identification. They should be combined with other markers to raise sensitivity and specificity and increase the number of sources that are identified. MST predictive models using only these molecular markers were developed. The models were evaluated by considering the lowest number of molecular indicators needed to obtain the highest rate of identification of fecal sources. The combined use of three molecular markers (ADO, Bomito, and Pomito) enabled correct identification of 75.7% of the samples, with differentiation between human, swine, bovine, and poultry sources. Discrimination between human and nonhuman fecal pollution was possible using two markers: ADO and Pomito (84.6% correct identification). The percentage of correct identification increased with the number of markers analyzed. The best predictive model for distinguishing human from nonhuman fecal sources was based on 5 molecular markers (HF134, ADO, DEN, Bomito, and Pomito) and provided 90.1% correct classification.
Stress-Induced Hsp70 Gene Expression and Inactivation of
Cryptosporidium parvum
Oocysts by Chlorine-Based Oxidants
Authors: Bajszár George , Dekonenko Alexander
Published: January 2010

Our research on the mechanisms of action of chlorine-based oxidants on
Cryptosporidium parvum
oocysts in water revealed a dual-phase effect: (i) response to oxidative stress, which was demonstrated by induced expression of the Hsp70 heat shock gene, and (ii) oocyst inactivation as a result of long-term exposure to oxidants. The relative biocidal effects of sodium hypochlorite (bleach) and electrolytically generated mixed oxidant solution (MOS) on
C. parvum
oocysts were compared at identical free chlorine concentrations. Oocyst inactivation was determined by quantitative reverse transcription-PCR (qRT-PCR) amplification of the heat-induced Hsp70 mRNA and compared with tissue culture infectivity. According to both assays, within the range between 25 and 250 mg/liter free chlorine and with 4 h contact time, MOS exhibits a higher efficacy in oocyst inactivation than hypochlorite. Other RNA-based viability assays, aimed at monitoring the levels of β-tubulin mRNA and 18S rRNA, showed relatively slow decay rates of these molecules following disinfection by chlorine-based oxidants, rendering these molecular diagnostic viability markers inappropriate for disinfection efficacy assessment.
Entry of
Spiroplasma citri
into
Circulifer haematoceps
Cells Involves Interaction between Spiroplasma Phosphoglycerate Kinase and Leafhopper Actin
Authors: Labroussaa Fabien , Arricau-Bouvery Nathalie , Dubrana Marie-Pierre , Saillard Colette
Published: January 2010

Transmission of the phytopathogenic mollicutes, spiroplasmas, and phytoplasmas by their insect vectors mainly depends on their ability to pass through gut cells, to multiply in various tissues, and to traverse the salivary gland cells. The passage of these different barriers suggests molecular interactions between the plant mollicute and the insect vector that regulate transmission. In the present study, we focused on the interaction between
Spiroplasma citri
and its leafhopper vector,
Circulifer haematoceps
. An
in vitro
protein overlay assay identified five significant binding activities between
S. citri
proteins and insect host proteins from salivary glands. One insect protein involved in one binding activity was identified by liquid chromatography-tandem mass spectrometry (LC-MS/MS) as actin. Confocal microscopy observations of infected salivary glands revealed that spiroplasmas colocated with the host actin filaments. An
S. citri
actin-binding protein of 44 kDa was isolated by affinity chromatography and identified by LC-MS/MS as phosphoglycerate kinase (PGK). To investigate the role of the PGK-actin interaction, we performed competitive binding and internalization assays on leafhopper cultured cell lines (Ciha-1) in which His
6
-tagged PGK from
S. citri
or purified PGK from
Saccharomyces cerevisiae
was added prior to the addition of
S. citri
inoculum. The results suggested that exogenous PGK has no effect on spiroplasmal attachment to leafhopper cell surfaces but inhibits
S. citri
internalization, demonstrating that the process leading to internalization of
S. citri
in eukaryotic cells requires the presence of PGK. PGK, regardless of origin, reduced the entry of spiroplasmas into Ciha-1 cells in a dose-dependent manner.
Monitoring the Effects of Chiral Pharmaceuticals on Aquatic Microorganisms by Metabolic Fingerprinting
Authors: Wharfe Emma S. , Winder Catherine L. , Jarvis Roger M. , Goodacre Royston
Published: January 2010

The effects of the chiral pharmaceuticals atenolol and propranolol onPseudomonas putida,Pseudomonas aeruginosa,Micrococcus luteus, andBlastomonas natatoriawere investigated. The growth dynamics of exposed cultures were monitored using a Bioscreen instrument. In addition, Fourier-transform infrared (FT-IR) spectroscopy with appropriate chemometrics and high-performance liquid chromatography (HPLC) were employed in order to investigate the phenotypic changes and possible degradation of the drugs in exposed cultures. For the majority of the bacteria studied there was not a statistically significant difference in the organism's phenotype when it was exposed to the different enantiomers or mixtures of enantiomers. In contrast, the pseudomonads appeared to respond differently to propranolol, and the two enantiomers had different effects on the cellular phenotype. This implies that there were different metabolic responses in the organisms when they were exposed to the different enantiomers. We suggest that our findings may indicate that there are widespread effects on aquatic communities in which active pharmaceutical ingredients are present.
Novel Antibiotic-Free Plasmid Selection System Based on Complementation of Host Auxotrophy in the NAD
De Novo
Synthesis Pathway
Authors: Dong Wei-Ren , Xiang Li-Xin , Shao Jian-Zhong
Published: January 2010

The use of antibiotic resistance genes in plasmids causes potential biosafety and clinical hazards, such as the possibility of horizontal spread of resistance genes or the rapid emergence of multidrug-resistant pathogens. This paper introduces a novel auxotrophy complementation system that allowed plasmids and host cells to be effectively selected and maintained without the use of antibiotics. An
Escherichia coli
strain carrying a defect in NAD
de novo
biosynthesis was constructed by knocking out the chromosomal quinolinic acid phosphoribosyltransferase (QAPRTase) gene. The resistance gene in the plasmids was replaced by the QAPRTase gene of
E. coli
or the mouse. As a result, only expression of the QAPRTase gene from plasmids can complement and rescue
E. coli
host cells in minimal medium. This is the first time that a vertebrate gene has been used to construct a nonantibiotic selection system, and it can be widely applied in DNA vaccine and gene therapy. As the QAPRTase gene is ubiquitous in species ranging from bacteria to mammals, the potential environmental biosafety problems caused by horizontal gene transfer can be eliminated.