bluuebunny/crossref_metadata_2025 huggingface dataset:
Carbon Monoxide Promotes Lateral Root Formation in Rapeseed
Authors: Cao Ze‐Yu , Xuan Wei , Liu Zhao‐Yang , Li Xin‐Na , Zhao Nan , Xu Peng , Wang Zhe , Guan Rong‐Zhan , Shen Wen‐Biao
Published: June 2007

Carbon monoxide (CO), an odorless, tasteless and colorless gas, has recently proved to be an important bioactive or signal molecule in mammalian cells, with its effects mediated mainly by nitric oxide (NO). In the present report, we show that exogenous CO induces lateral root (LR) formation, an NO‐dependent process. Administration of the CO donor hematin to rapeseed (Brassica napus L. Yangyou 6) seedlings for 3 days, dose‐dependently promoted the total length and number of LRs. These responses were also seen following the application of gaseous CO aqueous solutions of different saturated concentrations. Furthermore, the actions of CO on seedlings were fully reversed when the CO scavenger hemoglobin (Hb) or the CO‐specific synthetic inhibitor zinc protoporphyrin‐IX (ZnPPIX) were added. Interestingly, depletion of endogenous NO using its specific scavenger 2‐(4‐carboxyphenyl)‐4,4,5,5‐tetramethylimidazoline‐1‐oxyl‐3‐oxide potassium salt (cPTIO) or the nitric oxide synthase (NOS) inhibitor NG‐nitro‐l‐arginine methyl ester (l‐NAME), led to the complete abolition of LR development, illustrating an important role for endogenous NO in the action of CO on LR formation. However, the induction of LR development by 200 μmol/L sodium nitroprusside (SNP), an NO donor, was not affected by the presence or absence of ZnPPIX. Furthermore, using an anatomical approach combined with laser scanning confocal microscopy with the NO‐specific fluorophore 4,5‐diaminofluorescein diacetate, we observed that both hematin and SNP increased NO release compared with control samples and that the NO signal was mainly distributed in the LR primordia (LRP), especially after 36 h treatment. The LRP were found to have similar morphology in control, SNP‐ and hematin‐treated seedlings. Similarly, the enhancement of the NO signal by CO at 36 h was differentially quenched by the addition of cPTIO, l‐NAME, ZnPPIX and Hb. In contrast, the induction of NO caused by SNP was not affected by the application of ZnPPIX. Therefore, we further deduced that CO induces LR formation probably mediated by the NO/NOS pathway and NO may act downstream of CO signaling, which has also been shown to occur in animals.
Mutation of Residue Arginine18 of Cytochrome b559α‐Subunit and its Effects on Photosystem II Activities in Chlamydomonas reinhardtii
Authors: Ma Jing‐Jing , Li Liang‐Bi , Jing Yu‐Xiang , Kuang Ting‐Yun
Published: June 2007

It has been known that arginine is used as the basic amino acid in the α‐subunit of cytochrome b559 (Cyt b559) except histidine. However, previous studies have focused on the function of histidine in the activities of photosystem (PS) II and there are no reports regarding the structural and/or functional roles of arginine in PSII complexes. In the present study, two arginine18 (R18) mutants of Chlamydomonas reinhardtii were constructed using site‐directed mutagenesis, in which R18 was replaced by glutamic acid (E) and glycine (G). The results show that the oxygen evolution of the PSII complex in the R18G and R18E mutants was approximately 60% of wild‐type (WT) levels and that, after irradiation at high light intensity, oxygen evolution for the PSII of mutants was reduced to zero compared with 40% in WT cells. The efficiency of light capture by PSII (Fv/Fm) of R18G and R18E mutants was approximately 42%–46% that of WT cells. Furthermore, levels of the α‐subunit of Cyt b559 and PsbO proteins were reduced in thylakoid membranes compared with WT. Overall, these data suggest that R18 plays a significant role in helping Cyt b559 maintain the structure of the PSII complex and its activity, although it is not directly bound to the heme group.
Method for discriminating objectively between patches and non‐patches in a semiarid environment
Authors: Du Toit Justin C.O. , Danckwerts Jock E. , Zacharias Peter J.K.
Published: June 2007

Grass swards that have been grazed by livestock exhibit a patchy structure comprising short and tall components. While such patches are often observed, they are seldom objectively described. This study sought to develop a technique for objectively discriminating between patches (short) and non‐patches (tall) in a semiarid savanna in the Eastern Cape, South Africa. Two independent hypotheses were addressed: (i) vegetation can be categorized into patches and non‐patches by analyzing data comprising a large number of single measurements of sward height; and (ii) there are two distinct grass communities present, each of which comprises a particular range of sward heights. Single and double normal distributions were fitted to height distributions using the maximum likelihood estimation (MLE; hypothesis 1), and species composition data were ordinated using canonical correspondence analysis (CCA) and two‐way indicator species analysis (TWINSPAN; hypothesis 2). In general, the results supported both hypotheses. Patches and non‐patches can be distinguished using MLE unless grazing is patchy at a scale smaller than the lowest scale of sward measurement (here 0.5 m, the diameter of a disc pasture meter). Ordination and classification revealed that – at our site – two distinct grass communities are present, and that patches and non‐patches are characterized by vegetation <6 and 6 cm, respectively.
Crossability and amplified fragment length polymorphism variation among four types of Panicum coloratum L. and meiotic chromosome paring of their F1 hybrids
Authors: Komatsu Toshinori , Ubi Benjamin E. , Enoki Hiroyuki
Published: June 2007

The objective of this study was to determine the genetic relationships among four types (A, B, C and D) of Panicum coloratum L. on the basis of the crossability and the amplified fragment length polymorphism (AFLP) variations among the types, and the meiotic chromosome pairing and fertility in F1 hybrids obtained from crosses between different types. F1 hybrids were obtained from crosses between P. coloratum L. var. makarikariense cv. Burnett (A) and P. coloratum L. PI 404632 (D), and between P. coloratum L. CPI 16794 (B) and P. coloratum L. cv. Solai (C). Other crosses failed to produce F1 hybrid seeds. F1 hybrids between Burnett (A) and PI 404632 (D) had 2n = 36 chromosomes and regular meiotic chromosome pairing with 18 bivalents per cell. They produced approximately 80% stainable pollen and normally set seeds. F1 hybrids between Solai (C) and CPI 16794 (B) had 2n = 45 chromosomes and a mean chromosome pairing of 9.50 univalents, 17.67 bivalents and 0.05 trivalents. The most frequent paring behavior was nine univalents and 18 bivalents, suggesting that Solai has two genomes homologous to those of CPI 16794 and one different genome. The F1 hybrids showed approximately 80% stainable pollen and approximately 20% seed fertility. Cluster analysis of polymorphic AFLP in five plants of each of the four types yielded two major clusters. Burnett (A) and PI 404632 (D) clustered together, while Solai (C) clustered with CPI 16794 (B). The results of hybridization studies and AFLP analysis of the four types of P. coloratum, and meiotic examination of the derived F1 hybrids demonstrate that types A and D are genetically differentiated from types B and C, and are reproductively isolated from these two types.
Community of arbuscular mycorrhizal fungi in Japanese semi‐natural grassland dominated by Pleioblastus chino and Miscanthus sinensis
Authors: Kojima Tomoko , Hayashi Haruo , Saito Masanori
Published: June 2007

For 4 years, from 1999–2002, we studied the flora of arbuscular mycorrhizal (AM) fungi associated with vegetational change due to grazing in a semi‐natural grassland of central Japan. In this grassland in 1999, Pleioblastus chino was distributed widely and was the most dominant plant species, followed by Miscanthus sinensis, which was distributed in part of the grassland. Coverage by the tallgrass M. sinensis decreased with grazing, whereas coverage by P. chino did not change so much during the experimental period. The degrees of AM fungal colonization and spore population in the rhizosphere showed no remarkable yearly changes during the 4 years. Among spores collected from soils, Glomus rubiforme was always dominant. The AM fungal flora in plant roots was examined by polymerase chain reaction (PCR) with taxon‐specific primers. Glomus spp. was detected most frequently. Together, the results implied that the AM fungal community in the semi‐natural grassland in Japan might remain stable despite changes in the coverage of some dominant plant species over time.
Ovule development at anthesis in apricot (Prunus armeniaca) varieties in a Mediterranean climate
Authors: Ruiz D. , Egea J.
Published: June 2007

The stage of ovule development at anthesis has been studied in 2002 and 2003 in 35 apricot varieties and 4 new apricot cultivars grown in a Mediterranean climate. Significant differences were found among varieties as well as a high heterogeneity of the developmental stage of the ovules examined in each variety. In general, a relative delay of the ovule development was observed, the presence of mature ovules at anthesis being negligible. In the 2 years of the study, differences in the developmental stage of primary ovules at anthesis were not significant and correlation to flowering time was not observed, which emphasised the previously reported genetic determination of this trait.
Weak Northern and Strong Tropical Land Carbon Uptake from Vertical Profiles of Atmospheric CO
2
Authors: Stephens Britton B. , Gurney Kevin R. , Tans Pieter P. , Sweeney Colm , Peters Wouter , Bruhwiler Lori , Ciais Philippe , Ramonet Michel , Bousquet Philippe , Nakazawa Takakiyo , Aoki Shuji , Machida Toshinobu , Inoue Gen , Vinnichenko Nikolay , Lloyd Jon , Jordan Armin , Heimann Martin , Shibistova Olga , Langenfelds Ray L. , Steele L. Paul , Francey Roger J. , Denning A. Scott
Published: June 2007

Measurements of midday vertical atmospheric CO
2
distributions reveal annual-mean vertical CO
2
gradients that are inconsistent with atmospheric models that estimate a large transfer of terrestrial carbon from tropical to northern latitudes. The three models that most closely reproduce the observed annual-mean vertical CO
2
gradients estimate weaker northern uptake of –1.5 petagrams of carbon per year (Pg C year
–1
) and weaker tropical emission of +0.1 Pg C year
–1
compared with previous consensus estimates of –2.4 and +1.8 Pg C year
–1
, respectively. This suggests that northern terrestrial uptake of industrial CO
2
emissions plays a smaller role than previously thought and that, after subtracting land-use emissions, tropical ecosystems may currently be strong sinks for CO
2
.
Evolutionary Dynamics of Immune-Related Genes and Pathways in Disease-Vector Mosquitoes
Authors: Waterhouse Robert M. , Kriventseva Evgenia V. , Meister Stephan , Xi Zhiyong , Alvarez Kanwal S. , Bartholomay Lyric C. , Barillas-Mury Carolina , Bian Guowu , Blandin Stephanie , Christensen Bruce M. , Dong Yuemei , Jiang Haobo , Kanost Michael R. , Koutsos Anastasios C. , Levashina Elena A. , Li Jianyong , Ligoxygakis Petros , MacCallum Robert M. , Mayhew George F. , Mendes Antonio , Michel Kristin , Osta Mike A. , Paskewitz Susan , Shin Sang Woon , Vlachou Dina , Wang Lihui , Wei Weiqi , Zheng Liangbiao , Zou Zhen , Severson David W. , Raikhel Alexander S. , Kafatos Fotis C. , Dimopoulos George , Zdobnov Evgeny M. , Christophides George K.
Published: June 2007

Mosquitoes are vectors of parasitic and viral diseases of immense importance for public health. The acquisition of the genome sequence of the yellow fever and Dengue vector,
Aedes aegypti
(
Aa
), has enabled a comparative phylogenomic analysis of the insect immune repertoire: in
Aa
, the malaria vector
Anopheles gambiae
(
Ag
), and the fruit fly
Drosophila melanogaster
(
Dm
). Analysis of immune signaling pathways and response modules reveals both conservative and rapidly evolving features associated with different functional gene categories and particular aspects of immune reactions. These dynamics reflect in part continuous readjustment between accommodation and rejection of pathogens and suggest how innate immunity may have evolved.
Deformation of (Mg,Fe)SiO
3
Post-Perovskite and D'' Anisotropy
Authors: Merkel Sébastien , McNamara Allen K. , Kubo Atsushi , Speziale Sergio , Miyagi Lowell , Meng Yue , Duffy Thomas S. , Wenk Hans-Rudolf
Published: June 2007

Polycrystalline (Mg
0.9
,Fe
0.1
)SiO
3
post-perovskite was plastically deformed in the diamond anvil cell between 145 and 157 gigapascals. The lattice-preferred orientations obtained in the sample suggest that slip on planes near (100) and (110) dominate plastic deformation under these conditions. Assuming similar behavior at lower mantle conditions, we simulated plastic strains and the contribution of post-perovskite to anisotropy in the D″ region at the Earth core-mantle boundary using numerical convection and viscoplastic polycrystal plasticity models. We find a significant depth dependence of the anisotropy that only develops near and beyond the turning point of a downwelling slab. Our calculated anisotropies are strongly dependent on the choice of elastic moduli and remain hard to reconcile with seismic observations.
Nuclear Actin Regulates Dynamic Subcellular Localization and Activity of the SRF Cofactor MAL
Authors: Vartiainen Maria K. , Guettler Sebastian , Larijani Banafshe , Treisman Richard
Published: June 2007

Actin, which is best known as a cytoskeletal component, also participates in the control of gene expression. We report a function of nuclear actin in the regulation of MAL, a coactivator of the transcription factor serum response factor (SRF). MAL, which binds monomeric actin, is cytoplasmic in many cells but accumulates in the nucleus upon serum-induced actin polymerization. MAL rapidly shuttles between cytoplasm and nucleus in unstimulated cells. Serum stimulation effectively blocks MAL nuclear export, which requires MAL-actin interaction. Nuclear MAL binds SRF target genes but remains inactive unless actin binding is disrupted. Fluorescence resonance energy transfer analysis demonstrates that the MAL-actin interaction responds to extracellular signals. Serum-induced signaling is thus communicated to nuclear actin to control a transcriptional regulator.
Culling Prey Promotes Predator RecoveryAlternative States in a Whole-Lake Experiment
Authors: Persson Lennart , Amundsen Per-Arne , De Roos André M. , Klemetsen Anders , Knudsen Rune , Primicerio Raul
Published: June 2007

Many top-predator fish stocks in both freshwater and marine systems have collapsed as a result of overharvesting. Consequently, some of these communities have shifted into seemingly irreversible new states. We showed, for predators feeding on prey that exhibit food-dependent growth, that culling of fish prey may promote predator recovery. We removed old stunted individuals of a prey-fish species in a large, low-productive lake, which caused an increase in the availability of small-sized prey and allowed the predator to recover. The shift in community state has been sustained for more than 15 years after the cull ended and represents an experimental demonstration of an alternative stable state in a large-scale field system. Because most animals exhibit food-dependent growth, shifts into alternative stable states resulting from overcompensating prey growth may be common in nature and may require counterintuitive management strategies.
Remodeled Foundations
Authors: Levin Simon A.
Published: June 2007

Theoretical Ecology Principles and Applications. 3rd ed.
.
Robert M. May and Angela R. McLean, Eds.
. Oxford University Press, Oxford, 2007. 267 pp. $120, £60. ISBN 9780199209989. Paper, $45, £29.95. ISBN 9780199209996.

The contributors offer an up-to-date overview of theories of the structure, function, and dynamics of populations and communities and of applications of these ideas to such issues as fisheries, infectious diseases, food supplies, climate change, and conservation biology.
An Antifungal Agent Inhibits an Aminoacyl-tRNA Synthetase by Trapping tRNA in the Editing Site
Authors: Rock Fernando L. , Mao Weimin , Yaremchuk Anya , Tukalo Mikhail , Crépin Thibaut , Zhou Huchen , Zhang Yong-Kang , Hernandez Vincent , Akama Tsutomu , Baker Stephen J. , Plattner Jacob J. , Shapiro Lucy , Martinis Susan A. , Benkovic Stephen J. , Cusack Stephen , Alley M. R. K.
Published: June 2007

Aminoacyl–transfer RNA (tRNA) synthetases, which catalyze the attachment of the correct amino acid to its corresponding tRNA during translation of the genetic code, are proven antimicrobial drug targets. We show that the broad-spectrum antifungal 5-fluoro-1,3-dihydro-1-hydroxy-2,1-benzoxaborole (AN2690), in development for the treatment of onychomycosis, inhibits yeast cytoplasmic leucyl-tRNA synthetase by formation of a stable tRNA
Leu
-AN2690 adduct in the editing site of the enzyme. Adduct formation is mediated through the boron atom of AN2690 and the 2′- and 3′-oxygen atoms of tRNA's3′-terminal adenosine. The trapping of enzyme-bound tRNA
Leu
in the editing site prevents catalytic turnover, thus inhibiting synthesis of leucyl-tRNA
Leu
and consequentially blocking protein synthesis. This result establishes the editing site as a bona fide target for aminoacyl-tRNA synthetase inhibitors.
Free-Drifting Icebergs: Hot Spots of Chemical and Biological Enrichment in the Weddell Sea
Authors: Smith Kenneth L. , Robison Bruce H. , Helly John J. , Kaufmann Ronald S. , Ruhl Henry A. , Shaw Timothy J. , Twining Benjamin S. , Vernet Maria
Published: June 2007

The proliferation of icebergs from Antarctica over the past decade has raised questions about their potential impact on the surrounding pelagic ecosystem. Two free-drifting icebergs, 0.1 and 30.8 square kilometers in aerial surface area, and the surrounding waters were sampled in the northwest Weddell Sea during austral spring 2005. There was substantial enrichment of terrigenous material, and there were high concentrations of chlorophyll, krill, and seabirds surrounding each iceberg, extending out to a radial distance of ∼3.7 kilometers. Extrapolating these results to all icebergs in the same size range, with the use of iceberg population estimates from satellite surveys, indicates that they similarly affect 39% of the surface ocean in this region. These results suggest that free-drifting icebergs can substantially affect the pelagic ecosystem of the Southern Ocean and can serve as areas of enhanced production and sequestration of organic carbon to the deep sea.
Influence of Phylogeny on Fungal Community Assembly and Ecosystem Functioning
Authors: Maherali Hafiz , Klironomos John N.
Published: June 2007

Ecology seeks to explain species coexistence and its functional consequences, but experimental tests of mechanisms that simultaneously account for both processes are difficult. We used an experimental mycorrhizal plant system to test whether functional similarity among closely related species (phylogenetic conservatism) can drive community assembly and ecosystem functioning. Communities were constructed with the same number of fungal species, but after 1 year of growth, realized species richness was highest where the starting species were more distantly related to each other. Communities with high realized species richness also stimulated plant productivity more than those with low realized species richness. Our findings suggest that phylogenetic trait conservatism can promote coexistence because of reduced competition between distinct evolutionary lineages and enhance ecosystem function because of functional complementarity among those same lineages.
Microplate Fluorescence Assay for Measurement of the Ability of Strains ofListeria monocytogenesfrom Meat and Meat-Processing Plants To Adhere to Abiotic Surfaces
Authors: Gamble Rachel , Muriana Peter M.
Published: June 2007

Listeria monocytogenesis a significant food-borne pathogen that is capable of adhering to and producing biofilms on processing equipment, making it difficult to eliminate from meat-processing environments and allowing potential contamination of ready-to-eat (RTE) products. We devised a fluorescence-based microplate method for screening isolates ofL. monocytogenesfor the ability to adhere to abiotic surfaces. Strains ofL. monocytogeneswere incubated for 2 days at 30°C in 96-well microplates, and the plates were washed in a plate washer. The retained cells were incubated for 15 min at 25°C with 5,6-carboxyfluorescein diacetate and washed again, and then the fluorescence was read with a plate reader. Several enzymatic treatments (protease, lipase, and cellulase) were effective in releasing adherent cells from the microplates, and this process was used for quantitation on microbiological media. Strongly adherent strains ofL. monocytogeneswere identified that had 15,000-fold-higher levels of fluorescence and 100,000-fold-higher plate counts in attachment assays than weakly adherent strains. Strongly adherent strains ofL. monocytogenesadhered equally well to four different substrates (glass, plastic, rubber, and stainless steel); showed high-level attachment on microplates at 10, 20, 30, and 40°C; and showed significant differences from weakly adherent strains when examined by scanning electron microscopy. A greater incidence of strong adherence was observed for strains isolated from RTE meats than for those isolated from environmental surfaces. Analysis of surface adherence amongListeriaisolates from processing environments may provide a better understanding of the molecular mechanisms involved in attachment and suggest solutions to eliminate them from food-processing environments.
Development of a Swine-Specific Fecal Pollution Marker Based on Host Differences in MethanogenmcrAGenes
Authors: Ufnar Jennifer A. , Ufnar David F. , Wang Shiao Y. , Ellender R. D.
Published: June 2007

The goal of this study was to evaluate methanogen diversity in animal hosts to develop a swine-specific archaeal molecular marker for fecal source tracking in surface waters. Phylogenetic analysis of swinemcrAsequences compared tomcrAsequences from the feces of five animals (cow, deer, sheep, horse, and chicken) and sewage showed four distinct swine clusters, with three swine-specific clades. From this analysis, six sequences were chosen for molecular marker development and initial testing. Only onemcrAsequence (P23-2) showed specificity for swine and therefore was used for environmental testing. PCR primers for the P23-2 clonemcrAsequence were developed and evaluated for swine specificity. The P23-2 primers amplified products in P23-2 plasmid DNA (100%), pig feces (84%), and swine waste lagoon surface water samples (100%) but did not amplify a product in 47 bacterial and archaeal stock cultures and 477 environmental bacterial isolates and sewage and water samples from a bovine waste lagoon and a polluted creek. Amplification was observed in only one sheep sample out of 260 human and nonswine animal fecal samples. Sequencing of PCR products from pig feces demonstrated 100% similarity to pigmcrAsequence from clone P23-2. The minimal amount of DNA required for the detection was 1 pg for P23-2 plasmid, 1 ng for pig feces, 50 ng for swine waste lagoon surface water, 1 ng for sow waste influent, and 10 ng for lagoon sludge samples. Lower detection limits of 10−6g of wet pig feces in 500 ml of phosphate-buffered saline and 10−4g of lagoon waste in estuarine water were established for the P23-2 marker. This study was the first to utilize methanogens for the development of a swine-specific fecal contamination marker.
Attachment of Enteric Viruses to Bottles
Authors: Butot S. , Putallaz T. , Croquet C. , Lamothe G. , Meyer R. , Joosten H. , Sánchez G.
Published: June 2007

Storage of water that was deliberately contaminated with enteric viruses in polyethylene terephthalate (PET) bottles led to a rapid decrease of the apparent viral load, thereby hampering the development of samples for a collaborative evaluation of viral detection methods for bottled water. To determine if this decrease was due to spontaneous inactivation or to adhesion, an elution protocol was developed and combined with a rapid and sensitive real-time reverse transcription-PCR-based method to quantify adsorbed norovirus (NV), hepatitis A virus (HAV), and rotavirus (RV) on bottle walls. The NV retention on PET bottle walls after 20 and 62 days reached an average level of 85% and 95% of the recovered inoculum, respectively. HAV and RV also showed adsorption onto PET bottles, reaching 90% and 80%, respectively, after 20 days of storage. NV and RV attachment was demonstrated to be dependent on the presence of autochthonous flora, whereas HAV adsorption was independent of it. Application of the elution and viral detection protocol to 294 commercially available water bottles obtained from 25 different countries did not give any positive result, thereby providing further evidence that the sources used for this product are free from enteric viruses and support for the theory that bottled water is not a vehicle for viral diseases.
Newly Discovered Penicillin Acylase Activity of Aculeacin A Acylase from
Actinoplanes utahensis
Authors: Torres-Bacete Jesús , Hormigo Daniel , Stuart Maribel , Arroyo Miguel , Torres Pedro , Castillón María P. , Acebal Carmen , García José L. , de la Mata Isabel
Published: June 2007

Aculeacin A acylase from
Actinoplanes utahensis
produced by
Streptomyces lividans
revealed acylase activities that are able to hydrolyze penicillin V and several natural aliphatic penicillins. Penicillin K was the best substrate, showing a catalytic efficiency of 34.79 mM
−1
s
−1
. Furthermore, aculeacin A acylase was highly thermostable, with a midpoint transition temperature of 81.5°C.
Distribution and Diversity of Archaeal Ammonia Monooxygenase Genes Associated with Corals
Authors: Beman J.Michael , Roberts Kathryn J. , Wegley Linda , Rohwer Forest , Francis Christopher A.
Published: June 2007

Corals are known to harbor diverse microbial communities of
Bacteria
and
Archaea
, yet the ecological role of these microorganisms remains largely unknown. Here we report putative ammonia monooxygenase subunit A (
amoA
) genes of archaeal origin associated with corals. Multiple DNA samples drawn from nine coral species and four different reef locations were PCR screened for archaeal and bacterial
amoA
genes, and archaeal
amoA
gene sequences were obtained from five different species of coral collected in Bocas del Toro, Panama. The 210 coral-associated archaeal
amoA
sequences recovered in this study were broadly distributed phylogenetically, with most only distantly related to previously reported sequences from coastal/estuarine sediments and oceanic water columns. In contrast, the bacterial
amoA
gene could not be amplified from any of these samples. These results offer further evidence for the widespread presence of the archaeal
amoA
gene in marine ecosystems, including coral reefs.
Production and Secretion Stress Caused by Overexpression of Heterologous α-Amylase Leads to Inhibition of Sporulation and a Prolonged Motile Phase in
Bacillus subtilis
Authors: Lulko Andrzej T. , Veening Jan-Willem , Buist Girbe , Smits Wiep Klaas , Blom Evert Jan , Beekman Aaron C. , Bron Sierd , Kuipers Oscar P.
Published: June 2007

Transcriptome analysis was used to investigate the global stress response of the gram-positive bacterium
Bacillus subtilis
caused by overproduction of the well-secreted AmyQ α-amylase from
Bacillus amyloliquefaciens
. Analyses of the control and overproducing strains were carried out at the end of exponential growth and in stationary phase, when protein secretion from
B. subtilis
is optimal. Among the genes that showed increased expression were
htrA
and
htrB
, which are part of the CssRS regulon, which responds to high-level protein secretion and heat stress. The analysis of the transcriptome profiles of a
cssS
mutant compared to the wild type, under identical secretion stress conditions, revealed several genes with altered transcription in a CssRS-dependent manner, for example,
citM
,
ylxF
,
yloA
,
ykoJ
, and several genes of the
flgB
operon. However, high-affinity CssR binding was observed only for
htrA
,
htrB
, and, possibly,
citM
. In addition, the DNA macroarray approach revealed that several genes of the sporulation pathway are downregulated by AmyQ overexpression and that a group of motility-specific (σ
D
-dependent) transcripts were clearly upregulated. Subsequent flow-cytometric analyses demonstrate that, upon overproduction of AmyQ as well as of a nonsecretable variant of the α-amylase, the process of sporulation is severely inhibited. Similar experiments were performed to investigate the expression levels of the
hag
promoter, a well-established reporter for σ
D
-dependent gene expression. This approach confirmed the observations based on our DNA macroarray analyses and led us to conclude that expression levels of several genes involved in motility are maintained at high levels under all conditions of α-amylase overproduction.
At Least Two Origins of Fungicide Resistance in Grapevine Downy Mildew Populations
Authors: Chen Wei-Jen , Delmotte François , Cervera Sylvie Richard , Douence Lisette , Greif Charles , Corio-Costet Marie-France
Published: June 2007

Quinone outside inhibiting (QoI) fungicides represent one of the most widely used groups of fungicides used to control agriculturally important fungal pathogens. They inhibit the cytochromebc1complex of mitochondrial respiration. Soon after their introduction onto the market in 1996, QoI fungicide-resistant isolates were detected in field plant pathogen populations of a large range of species. However, there is still little understanding of the processes driving the development of QoI fungicide resistance in plant pathogens. In particular, it is unknown whether fungicide resistance occurs independently in isolated populations or if it appears once and then spreads globally by migration. Here, we provide the first case study of the evolutionary processes that lead to the emergence of QoI fungicide resistance in the plant pathogenPlasmopara viticola. Sequence analysis of the complete cytochromebgene showed that all resistant isolates carried a mutation resulting in the replacement of glycine by alanine at codon 143 (G143A). Phylogenetic analysis of a large mitochondrial DNA fragment including the cytochromebgene (2,281 bp) across a wide range of EuropeanP. viticolaisolates allowed the detection of four major haplotypes belonging to two distinct clades, each of which contains a different QoI fungicide resistance allele. This is the first demonstration that a selected substitution conferring resistance to a fungicide has occurred several times in a plant-pathogen system. Finally, a high population structure was found when the frequency of QoI fungicide resistance haplotypes was assessed in 17 French vineyards, indicating that pathogen populations might be under strong directional selection for local adaptation to fungicide pressure.
Metabolic Engineering of
Bacillus subtilis
for Ethanol Production: Lactate Dehydrogenase Plays a Key Role in Fermentative Metabolism
Authors: Romero Susana , Merino Enrique , Bolívar Francisco , Gosset Guillermo , Martinez Alfredo
Published: June 2007

Wild-type
Bacillus subtilis
ferments 20 g/liter glucose in 48 h, producing lactate and butanediol, but not ethanol or acetate. To construct an ethanologenic
B. subtilis
strain, homologous recombination was used to disrupt the native lactate dehydrogenase (LDH) gene (
ldh
) by chromosomal insertion of the
Zymomonas mobilis
pyruvate decarboxylase gene (
pdc
) and alcohol dehydrogenase II gene (
adhB
) under the control of the
ldh
native promoter. The values of the intracellular PDC and ADHII enzymatic activities of the engineered
B. subtilis
BS35 strain were similar to those found in an ethanologenic
Escherichia coli
strain. BS35 produced ethanol and butanediol; however, the cell growth and glucose consumption rates were reduced by 70 and 65%, respectively, in comparison to those in the progenitor strain. To eliminate butanediol production, the acetolactate synthase gene (
alsS
) was inactivated. In the BS36 strain (BS35 Δ
alsS
), ethanol production was enhanced, with a high yield (89% of the theoretical); however, the cell growth and glucose consumption rates remained low. Interestingly, kinetic characterization of LDH from
B. subtilis
showed that it is able to oxidize NADH and NADPH. The expression of the transhydrogenase encoded by
udhA
from
E. coli
allowed a partial recovery of the cell growth rate and an early onset of ethanol production. Beyond pyruvate-to-lactate conversion and NADH oxidation, an additional key physiological role of LDH for glucose consumption under fermentative conditions is suggested. Long-term cultivation showed that 8.9 g/liter of ethanol can be obtained using strain BS37 (BS35 Δ
alsS udhA
+
). As far as we know, this is the highest ethanol titer and yield reported with a
B. subtilis
strain.
Recombinant Expression and Characterization of a Reducing-End Xylose-Releasing Exo-Oligoxylanase from
Bifidobacterium adolescentis
Authors: Lagaert Stijn , Van Campenhout Steven , Pollet Annick , Bourgois Tine M. , Delcour Jan A. , Courtin Christophe M. , Volckaert Guido
Published: June 2007

The family 8 glycoside hydrolase (RexA) from
Bifidobacterium adolescentis
was expressed in
Escherichia coli
. The recombinant enzyme was characterized as a reducing-end xylose-releasing exo-oligoxylanase. Apart from giving insights into this new class of enzymes, knowledge of the RexA enzyme helps to postulate a mechanism for the
B. adolescentis
breakdown of prebiotic xylooligosaccharides.
Virus Attachment to Transparent Exopolymeric Particles along Trophic Gradients in the Southwestern Lagoon of New Caledonia
Authors: Mari Xavier , Kerros Marie-Emmanuelle , Weinbauer Markus G.
Published: June 2007

Viruses on organic aggregates such as transparent exopolymeric particles (TEP) are not well investigated. The number of TEP-attached viruses was assessed along trophic gradients in the southwestern lagoon of New Caledonia by determining the fraction of viruses removed after magnetic isolation of TEP. In order to isolate TEP magnetically, TEP were formed in the presence of magnetic beads from submicrometer precursors collected along the trophic gradients. The mixed aggregates of TEP-beads-viruses were removed from solution with a magnetic field. The percentage of viruses associated with newly formed TEP averaged 8% (range, 3 to 13%) for most of the stations but was higher (ca. 30%) in one bay characterized by the low renewal rate of its water mass. The number of viruses (
N
) attached to TEP varied as a function of TEP size (
d
[in micrometers]) according to the formulas
N
= 100
d
1.60
and
N
= 230
d
1.75
, respectively, for TEP occurring in water masses with short (i.e., <40 days) and long (i.e., >40 days) residence times. These two relationships imply that viral abundance decreases with TEP size, and they indicate that water residence time influences viral density and virus-bacterium interactions within aggregates. Our data suggest that the fraction of viruses attached to TEP is highest in areas characterized by a low renewal rate of the water mass and can constitute at times a significant fraction of total virus abundance. Due to the small distance between viruses and hosts on TEP, these particles may be hot spots for viral infection.
Molecular and Biochemical Characterization of 3-Hydroxybenzoate 6-Hydroxylase from
Polaromonas naphthalenivorans
CJ2
Authors: Park Minjeong , Jeon Yeji , Jang Ho Hee , Ro Hyun-Su , Park Woojun , Madsen Eugene L. , Jeon Che Ok
Published: June 2007

Prior research revealed that
Polaromonas naphthalenivorans
CJ2 carries and expresses genes encoding the gentisate metabolic pathway for naphthalene. These metabolic genes are split into two clusters, comprising
nagRAaGHAbAcAdBFCQEDJI
′-
orf1
-
tnpA
and
nagR2
-
orf2I
″
KL
(C. O. Jeon, M. Park, H. Ro, W. Park, and E. L. Madsen, Appl. Environ. Microbiol. 72:1086-1095, 2006). BLAST homology searches of sequences in GenBank indicated that the
orf2
gene from the small cluster likely encoded a salicylate 5-hydroxylase, presumed to catalyze the conversion of salicylate into gentisate. Here, we report physiological and genetic evidence that
orf2
does not encode salicylate 5-hydroxylase. Instead, we have found that
orf2
encodes 3-hydroxybenzoate 6-hydroxylase, the enzyme which catalyzes the NADH-dependent conversion of 3-hydroxybenzoate into gentisate. Accordingly, we have renamed
orf2 nagX
. After expression in
Escherichia coli
, the NagX enzyme had an approximate molecular mass of 43 kDa, as estimated by gel filtration, and was probably a monomeric protein. The enzyme was able to convert 3-hydroxybenzoate into gentisate without salicylate 5-hydroxylase activity. Like other 3-hydroxybenzoate 6-hydroxylases, NagX utilized both NADH and NADPH as electron donors and exhibited a yellowish color, indicative of a bound flavin adenine dinucleotide. An engineered mutant of
P. naphthalenivorans
CJ2 defective in
nagX
failed to grow on 3-hydroxybenzoate but grew normally on naphthalene. These results indicate that the previously described small catabolic cluster in strain CJ2 may be multifunctional and is essential for the degradation of 3-hydroxybenzoate. Because
nagX
and an adjacent MarR-type regulatory gene are both closely related to homologues in
Azoarcus
species, this study raises questions about horizontal gene transfer events that contribute to operon evolution.
New Enzymatic Method of Chiral Amino Acid Synthesis by Dynamic Kinetic Resolution of Amino Acid Amides: Use of Stereoselective Amino Acid Amidases in the Presence of α-Amino-ε-Caprolactam Racemase
Authors: Yamaguchi Shigenori , Komeda Hidenobu , Asano Yasuhisa
Published: June 2007

d- andl-amino acids were produced froml- andd-amino acid amides byd-aminopeptidase fromOchrobactrum anthropiC1-38 andl-amino acid amidase fromPseudomonas azotoformansIAM 1603, respectively, in the presence of α-amino-ε-caprolactam racemase fromAchromobacter obaeas the catalyst by dynamic kinetic resolution of amino acid amides.
Use of Multilocus Sequence Typing To Investigate the Association between the Presence of
Campylobacter
spp. in Broiler Drinking Water and
Campylobacter
Colonization in Broilers
Authors: Ogden I. D. , MacRae M. , Johnston M. , Strachan N. J. C. , Cody A. J. , Dingle K. E. , Newell D. G.
Published: June 2007

The presence of campylobacters in broiler chickens and throughout the broiler water delivery systems of 12 farms in northeastern Scotland was investigated by sensitive enrichment methods and large-volume filtration.
Campylobacter
presence was independent of the water source and whether the water was treated. The genotypes of
Campylobacter jejuni
isolates recovered from chickens and various locations within the water delivery systems were compared by multilocus sequence typing. Matching strains in shed header tanks and birds were found at 1 of the 12 farms investigated. However, the sequence of contamination or whether the source was within or outside the shed was not determined. Nevertheless, these data provide evidence that drinking water could be associated with broiler infection by campylobacters.
Identification and In Vivo Functional Analysis by Gene Disruption of
ctnA
, an Activator Gene Involved in Citrinin Biosynthesis in
Monascus purpureus
Authors: Shimizu Takeo , Kinoshita Hiroshi , Nihira Takuya
Published: June 2007

Citrinin, a secondary fungal metabolite of polyketide origin, is moderately nephrotoxic to vertebrates, including humans. From the red-pigment producer
Monascus purpureus
, a 21-kbp region flanking
pksCT
, which encodes citrinin polyketide synthase, was cloned. Four open reading frames (ORFs) (
orf1
,
orf2
,
orf3
, and
orf4
) in the 5′-flanking region and one ORF (
orf5
) in the 3′-flanking region were identified in the vicinity of
pksCT. orf1
to
orf5
encode a homolog of a dehydrogenase (similarity, 46%), a regulator (similarity, 38%), an oxygenase (similarity, 41%), an oxidoreductase (similarity, 26%), and a transporter (similarity, 58%), respectively.
orf2
(2,006 bp with four introns) encodes a 576-amino-acid protein containing a typical Zn(II)2Cys6 DNA binding motif at the N terminus and was designated
ctnA
. Although reverse transcriptase PCR analysis revealed that all of these ORFs, except for
orf1
, were transcribed with
pksCT
under citrinin production conditions, the disruption of
ctnA
caused large decreases in the transcription of
pksCT
and
orf5
, together with reduction of citrinin production to barely detectable levels, suggesting that these two genes are under control of the
ctnA
product. Complementation of the
ctnA
disruptant with intact
ctnA
on an autonomously replicating plasmid restored both transcription and citrinin production, indicating that CtnA is a major activator of citrinin biosynthesis.
Acetamide Selection of
Kluyveromyces lactis
Cells Transformed with an Integrative Vector Leads to High-Frequency Formation of Multicopy Strains
Authors: Read Jeremiah D. , Colussi Paul A. , Ganatra Mehul B. , Taron Christopher H.
Published: June 2007

The yeast
Kluyveromyces lactis
has been extensively used as a host for heterologous protein expression. A necessary step in the construction of a stable expression strain is the introduction of an integrative expression vector into
K. lactis
cells, followed by selection of transformed strains using either medium containing antibiotic (e.g., G418) or nitrogen-free medium containing acetamide. In this study, we show that selection using acetamide yields
K. lactis
transformant populations nearly completely comprised of strains bearing multiple tandem insertions of the expression vector pKLAC1 at the
LAC4
chromosomal locus, whereas an average of 16% of G418-selected transformants are multiply integrated. Additionally, the average copy number within transformant populations doubled when acetamide was used for selection compared to G418. Finally, we demonstrate that the high frequency of multicopy integration associated with using acetamide selection can be exploited to rapidly construct expression strains that simultaneously produce multiple heterologous proteins or multisubunit proteins, such as Fab antibodies.
Use of Propidium Monoazide for Live/Dead Distinction in Microbial Ecology
Authors: Nocker Andreas , Sossa-Fernandez Priscilla , Burr Mark D. , Camper Anne K.
Published: June 2007

One of the prerequisites of making ecological conclusions derived from genetic fingerprints is that bacterial community profiles reflect the live portion of the sample of interest. Propidium monoazide is a membrane-impermeant dye that selectively penetrates cells with compromised membranes, which can be considered dead. Once inside the cells, PMA intercalates into the DNA and can be covalently cross-linked to it, which strongly inhibits PCR amplification. By using PCR after PMA treatment, the analysis of bacterial communities can theoretically be limited to cells with intact cell membranes. Four experiments were performed to study the usefulness of PMA treatment of mixed bacterial communities comprising both intact and compromised cells in combination with end-point PCR by generating community profiles from the following samples: (i) defined mixtures of live and isopropanol-killed cells from pure cultures of random environmental isolates, (ii) wastewater treatment plant influent spiked with defined ratios of live and dead cells, (iii) selected environmental communities, and (iv) a water sediment sample exposed to increasing heat stress. Regions of 16S rRNA genes were PCR amplified from extracted genomic DNA, and PCR products were analyzed by using denaturing gradient gel electrophoresis (DGGE). Results from the first two experiments show that PMA treatment can be of value with end-point PCR by suppressing amplification of DNA from killed cells. The last two experiments suggest that PMA treatment can affect banding patterns in DGGE community profiles and their intensities, although the intrinsic limitations of end-point PCR have to be taken into consideration.
EstB-Mediated Hydrolysis of the Siderophore Triacetylfusarinine C Optimizes Iron Uptake of
Aspergillus fumigatus
Authors: Kragl Claudia , Schrettl Markus , Abt Beate , Sarg Bettina , Lindner Herbert H. , Haas Hubertus
Published: June 2007

Aspergillus fumigatus
excretes the fusarinine-type siderophore desferri-triacetylfusarinine C (DF-TafC) to mobilize iron. DF-TafC is a cyclic peptide consisting of three
N

5

-
cis
-anhydromevalonyl-
N

5

-hydroxy-
N

2

-acetyl-
l
-ornithine residues linked by ester bonds; these linkages are in contrast to peptide linkages found for ferrichrome-type siderophores. Subsequent to the binding of iron and uptake, triacetylfusarinine C (TafC) is hydrolyzed, the cleavage products are excreted, and the iron is transferred to the metabolism or to the intracellular siderophore desferri-ferricrocin (DF-FC) for iron storage. Here we report the identification and characterization of the TafC esterase EstB, the first eukaryotic siderophore-degrading enzyme to be characterized at the molecular level. The encoding gene,
estB
, was found to be located in an iron-regulated gene cluster, indicating a role in iron metabolism. Deletion of
estB
in
A. fumigatus
eliminated TafC esterase activity of cellular extracts and caused increased intracellular accumulation of TafC and TafC hydrolysis products in vivo.
Escherichia coli
-expressed EstB displayed specific TafC esterase activity but did not hydrolyze fusarinine C, which has the same core structure as TafC but lacks three
N

2

-acetyl residues. Localization of EstB via enhanced green fluorescent protein tagging suggested that TafC hydrolysis takes place in the cytoplasm. EstB abrogation reduced the intracellular transfer rate of iron from TafC to DF-FC and delayed iron sensing. Furthermore, EstB deficiency caused a decreased radial growth rate under iron-depleted but not iron-replete conditions. Taken together, these data suggest that EstB-mediated TafC hydrolysis optimizes but is not essential for TafC-mediated iron uptake in
A. fumigatus
.
Conservation of PEX19-Binding Motifs Required for Protein Targeting to Mammalian Peroxisomal and Trypanosome Glycosomal Membranes
Authors: Saveria Tracy , Halbach André , Erdmann Ralf , Volkmer-Engert Rudolf , Landgraf Christiane , Rottensteiner Hanspeter , Parsons Marilyn
Published: June 2007

Glycosomes are divergent peroxisomes found in trypanosomatid protozoa, including those that cause severe human diseases throughout much of the world. While peroxisomes are dispensable for both yeast (
Saccharomyces cerevisiae
and others) and mammalian cells in vitro, glycosomes are essential for trypanosomes and hence are viewed as a potential drug target. The import of proteins into the matrix of peroxisomes utilizes multiple peroxisomal membrane proteins which require the peroxin PEX19 for insertion into the peroxisomal membrane. In this report, we show that the specificity of peroxisomal membrane protein binding for
Trypanosoma brucei
PEX19 is very similar to those previously identified for human and yeast PEX19. Our studies show that trafficking is conserved across these distant phyla and that both a PEX19 binding site and a transmembrane domain are required for the insertion of two test proteins into the glycosomal membrane. However, in contrast to
T. brucei
PEX10 and PEX12,
T. brucei
PEX14 does not traffic to human peroxisomes, indicating that it is not recognized by the human PEX14 import mechanism.