bluuebunny/crossref_metadata_2025 huggingface dataset:
Polymer to polymer to polymer pattern transfer: Multiple molding for 100nm scale lithography
Authors: Mele Elisa , Di Benedetto Francesca , Persano Luana , Cingolani Roberto , Pisignano Dario
Published: April 2006

We demonstrate a multiple molding procedure based on the combination of replica molding, in situ patterning of an ultraviolet curable epoxy resist, micromachining by elastomeric elements, and nanoimprinting lithography. The pattern, with features down to the 100nm scale, is sequentially transferred to several different polymers, allowing one to realize high-resolution organic molds for imprinting compounds of lower glass-transition temperature. The intimate integration of soft and nanoimprinting lithographies enables a combined, multistep mechanical patterning, which can be very useful for a great range of applications for molecular lithography and devices.
Parameter dispersion characterization for arrays of HfC-coated emitters on poly-Si substrate
Authors: Nicolaescu D. , Nagao M. , Filip V. , Tanoue H. , Kanemaru S. , Itoh J.
Published: April 2006

HfC-coated poly-Si field-emission arrays (FEAs) were produced, and the emission statistics were characterized using a model parameter extraction. The poly-Si, chosen as a substrate, allows the integration of FEAs with control circuits and the scaling up of the fabrication process to larger dimensions, as required by field-emission display applications. The Ar ion sputtering process was used for emitter sharpening, and structures with HfC coatings with different thicknesses were realized. The array field-emission model is built up using equations that describe the electron emission from individual emitters. A general analytical relationship for computing the electric field on the emitter apex was derived. Nonuniform distribution functions within the array for the tip radius and work function are considered. A modified Fowler-Nordheim diagram was used, the emission data current-voltage I-V being represented as ln(I∕V3) as a function of 1∕V. A fast and accurate model parameter extraction is performed using a nonlinear extraction algorithm, considered over the entire voltage range for the measurements. The Ar ion sputtering process produces uniform emitters with smooth surfaces, increasing slightly the tip radius. The HfC coating lowers the work function and makes the emitter tips more blunt. An optimum thickness of the HfC layer is shown to exist. The number of active emitters responsible for most of the emission current is derived from the model and compared with direct counts.
Theoretical analysis of triple junction field emission for a type of cold cathode
Authors: Chung M. S. , Hong S. C. , Cutler P. H. , Miskovsky N. M. , Weiss B. L. , Mayer A.
Published: April 2006

The field enhancement due to the dielectric is theoretically investigated for a simple triple junction of metal-dielectric vacuum. The electric potential near the junction is obtained as a function of geometry, dielectric constant, and position. It is found that the key factor to determine the field enhancement is the ratio of the angles subtended by the dielectric and vacuum portions. This enhancement is attributed to the increase of the surface charge density on the metal around the junction by the polarization charge of dielectric. The image field is also found to decrease or increase according to the geometry of the constituents. The current results give rise to a concept in field emission.
Response to Comment on "PDK1 Nucleates T Cell Receptor-Induced Signaling Complex for NF-κB Activation"
Authors: Lee Ki-young , Shim Jae-Hyuck , Hayden Matthew S. , Luehrmann Jan-Schulze , Ghosh Sankar
Published: April 2006

In their comment, Gruber
et al
. report constitutive phosphorylation of protein kinase C θ at threonine residue 538. However, they fail to note that, consistent with our results, a number of other groups have previously reported inducible phosphorylation of Thr-538 in T cells. Although the physiological relevance of this discrepancy is unclear, substantial differences in experimental conditions and reagents may account for the conflicting observations.
Cobalt-Base High-Temperature Alloys
Authors: Sato J. , Omori T. , Oikawa K. , Ohnuma I. , Kainuma R. , Ishida K.
Published: April 2006

We have identified cobalt-base superalloys showing a high-temperature strength greater than those of conventional nickel-base superalloys. The cobalt-base alloys are strengthened by a ternary compound with the L1
2
structure,

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Co
3
(Al,W), which precipitates in the disordered γ face-centered cubic cobalt matrix with high coherency and with high melting points. We also identified a ternary compound,

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Ir
3
(Al,W), with the L1
2
structure, which suggests that the Co-Ir-Al-W–base systems with

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(Co,Ir)
3
(Al,W) structures offer great promise as candidates for next-generation high-temperature materials.
Comment on "PDK1 Nucleates T Cell Receptor-Induced Signaling Complex for NF-κB Activation"
Authors: Gruber Thomas , Freeley Michael , Thuille Nikolaus , Heit Isabelle , Shaw Stephen , Long Aideen , Baier Gottfried
Published: April 2006

We observe that protein kinase C θ (PKCθ) is phosphorylated on the activation loop at threonine 538 (Thr-538) before T cell activation. Our results are inconsistent with the conclusions of Lee
et al
. (Reports, 1 April 2005, p. 114) that the Thr-538 phosphorylation of PKCθ is regulated by T cell receptor activation. Other mechanisms, such as autophosphorylation of Thr-219, might orchestrate the cellular function of PKCθ in T cells.
Evolution of Hormone-Receptor Complexity by Molecular Exploitation
Authors: Bridgham Jamie T. , Carroll Sean M. , Thornton Joseph W.
Published: April 2006

According to Darwinian theory, complexity evolves by a stepwise process of elaboration and optimization under natural selection. Biological systems composed of tightly integrated parts seem to challenge this view, because it is not obvious how any element's function can be selected for unless the partners with which it interacts are already present. Here we demonstrate how an integrated molecular system—the specific functional interaction between the steroid hormone aldosterone and its partner the mineralocorticoid receptor—evolved by a stepwise Darwinian process. Using ancestral gene resurrection, we show that, long before the hormone evolved, the receptor's affinity for aldosterone was present as a structural by-product of its partnership with chemically similar, more ancient ligands. Introducing two amino acid changes into the ancestral sequence recapitulates the evolution of present-day receptor specificity. Our results indicate that tight interactions can evolve by molecular exploitation—recruitment of an older molecule, previously constrained for a different role, into a new functional complex.
Darwinian Evolution Can Follow Only Very Few Mutational Paths to Fitter Proteins
Authors: Weinreich Daniel M. , Delaney Nigel F. , DePristo Mark A. , Hartl Daniel L.
Published: April 2006

Five point mutations in a particular β-lactamase allele jointly increase bacterial resistance to a clinically important antibiotic by a factor of ∼100,000. In principle, evolution to this high-resistance β-lactamase might follow any of the 120 mutational trajectories linking these alleles. However, we demonstrate that 102 trajectories are inaccessible to Darwinian selection and that many of the remaining trajectories have negligible probabilities of realization, because four of these five mutations fail to increase drug resistance in some combinations. Pervasive biophysical pleiotropy within the β-lactamase seems to be responsible, and because such pleiotropy appears to be a general property of missense mutations, we conclude that much protein evolution will be similarly constrained. This implies that the protein tape of life may be largely reproducible and even predictable.
Sexual Conflict via Maternal-Effect Genes in ZW Species
Authors: Miller Paige M. , Gavrilets Sergey , Rice William R.
Published: April 2006

In female heterogamous (ZW) species, sex-linked genes coding for maternal products that are packaged into the egg open a unique arena for genetic conflict that does not occur in male heterogamous (XY) species. Z-linked maternal-effect alleles that help sons and harm daughters are expected to go to fixation, as are W-linked alleles that help daughters and harm sons. This conflict differs from known cases of meiotic drive, because sex-specific ontogeny, physiology, and gene expression greatly simplify the genetic interactions that lead to sexual conflict. Selection on maternal-effect genes may substantially alter the evolution of ZW compared with XY systems.
Platelet-Derived Serotonin Mediates Liver Regeneration
Authors: Lesurtel Mickael , Graf Rolf , Aleil Boris , Walther Diego J. , Tian Yinghua , Jochum Wolfram , Gachet Christian , Bader Michael , Clavien Pierre-Alain
Published: April 2006

The liver can regenerate its volume after major tissue loss. In a mouse model of liver regeneration, thrombocytopenia, or impaired platelet activity resulted in the failure to initiate cellular proliferation in the liver. Platelets are major carriers of serotonin in the blood. In thrombocytopenic mice, a serotonin agonist reconstituted liver proliferation. The expression of 5-HT2A and 2B subtype serotonin receptors in the liver increased after hepatectomy. Antagonists of 5-HT2A and 2B receptors inhibited liver regeneration. Liver regeneration was also blunted in mice lacking tryptophan hydroxylase 1, which is the rate-limiting enzyme for the synthesis of peripheral serotonin. This failure of regeneration was rescued by reloading serotonin-free platelets with a serotonin precursor molecule. These results suggest that platelet-derived serotonin is involved in the initiation of liver regeneration.
Losartan, an AT1 Antagonist, Prevents Aortic Aneurysm in a Mouse Model of Marfan Syndrome
Authors: Habashi Jennifer P. , Judge Daniel P. , Holm Tammy M. , Cohn Ronald D. , Loeys Bart L. , Cooper Timothy K. , Myers Loretha , Klein Erin C. , Liu Guosheng , Calvi Carla , Podowski Megan , Neptune Enid R. , Halushka Marc K. , Bedja Djahida , Gabrielson Kathleen , Rifkin Daniel B. , Carta Luca , Ramirez Francesco , Huso David L. , Dietz Harry C.
Published: April 2006

Aortic aneurysm and dissection are manifestations of Marfan syndrome (MFS), a disorder caused by mutations in the gene that encodes fibrillin-1. Selected manifestations of MFS reflect excessive signaling by the transforming growth factor–β (TGF-β) family of cytokines. We show that aortic aneurysm in a mouse model of MFS is associated with increased TGF-β signaling and can be prevented by TGF-β antagonists such as TGF-β–neutralizing antibody or the angiotensin II type 1 receptor (AT1) blocker, losartan. AT1 antagonism also partially reversed noncardiovascular manifestations of MFS, including impaired alveolar septation. These data suggest that losartan, a drug already in clinical use for hypertension, merits investigation as a therapeutic strategy for patients with MFS and has the potential to prevent the major life-threatening manifestation of this disorder.
Phylogeny of the Ants: Diversification in the Age of Angiosperms
Authors: Moreau Corrie S. , Bell Charles D. , Vila Roger , Archibald S. Bruce , Pierce Naomi E.
Published: April 2006

We present a large-scale molecular phylogeny of the ants (Hymenoptera: Formicidae), based on 4.5 kilobases of sequence data from six gene regions extracted from 139 of the 288 described extant genera, representing 19 of the 20 subfamilies. All but two subfamilies are recovered as monophyletic. Divergence time estimates calibrated by minimum age constraints from 43 fossils indicate that most of the subfamilies representing extant ants arose much earlier than previously proposed but only began to diversify during the Late Cretaceous to Early Eocene. This period also witnessed the rise of angiosperms and most herbivorous insects.
New Dust Belts of Uranus: One Ring, Two Ring, Red Ring, Blue Ring
Authors: de Pater Imke , Hammel Heidi B. , Gibbard Seran G. , Showalter Mark R.
Published: April 2006

We compared near-infrared observations of the recently discovered outer rings of Uranus with Hubble Space Telescope results. We find that the inner ring, R/2003 U 2, is red, whereas the outer ring, R/2003 U 1, is very blue. Blue is an unusual color for rings; Saturn's enigmatic E ring is the only other known example. By analogy to the E ring, R/2003 U 1 is probably produced by impacts into the embedded moon Mab, which apparently orbits at a location where nongravitational perturbations favor the survival and spreading of submicron-sized dust. R/2003 U 2 more closely resembles Saturn's G ring, which is red, a typical color for dusty rings.
The Dark Side of Night Lighting
Authors: Hill David
Published: April 2006

Ecological Consequences of Artificial Night Lighting
.
Catherine Rich and Travis Longcore, Eds.
. Island, Washington, DC, 2005. 478 pp. $65, £46.50. ISBN 1-55963-128-7. Paper, $29.95, £21.50. ISBN 1-55963-129-5.

Through a series of chapters that focus on individual taxa, the contributors discuss the proven and possible effects of photopollution and consider ways in which these effects might be mitigated.
The Phosphopantetheinyl Transferase Superfamily: Phylogenetic Analysis and Functional Implications in Cyanobacteria
Authors: Copp J. N. , Neilan B. A.
Published: April 2006

Phosphopantetheinyl transferases (PPTs) are a superfamily of essential enzymes required for the synthesis of a wide range of compounds including fatty acid, polyketide, and nonribosomal peptide metabolites. These enzymes activate carrier proteins in specific biosynthetic pathways by the transfer of a phosphopantetheinyl moiety to an invariant serine residue. PPTs display low levels of sequence similarity but can be classified into two major families based on several short motifs. The prototype of the first family is the broad-substrate-range PPT Sfp, which is required for biosynthesis of surfactin inBacillus subtilis. The second family is typified by theEscherichia coliacyl carrier protein synthase (AcpS). Facilitated by the growing number of genome sequences available for analyses, large-scale phylogenetic studies were utilized in this research to reveal novel subfamily groupings, including two subfamilies within the Sfp-like family. In the present study degenerate oligonucleotide primers were designed for amplification of cyanobacterial PPT gene fragments. Subsequent phylogenetic analyses suggested a unique, function-based PPT type, defined by the PPTs involved in heterocyst differentiation. Evidence supporting this hypothesis was obtained by sequencing the region surrounding the partialNodularia spumigenaPPT gene. The ability to genetically classify PPT function is critical for the engineering of novel compounds utilizing combinatorial biosynthesis techniques. Information regarding cyanobacterial PPTs has important ramifications for the ex situ production of cyanobacterial natural products.
Inhibition of Epsilon-Poly-
l
-Lysine Biosynthesis in
Streptomycetaceae
Bacteria by Short-Chain Polyols
Authors: Nishikawa Masanobu , Ogawa Ken'ichi
Published: April 2006

Antimicrobial epsilon-poly-
l
-lysine (ePL) is secreted by
Streptomycetaceae
bacteria, and the mechanism of ePL biosynthesis remains to be elucidated. We previously reported that an unknown ePL derivative accumulates in the culture medium of ePL-producing bacteria when glycerol is added to the culture medium (Nishikawa and Ogawa, Appl. Environ. Microbiol.
68:
3575-3581, 2002). In this study, by using matrix-assisted laser desorption ionization—time of flight mass spectrometry and nuclear magnetic resonance, we identified the unknown derivative as the ester formed between the hydroxyl group of a glycerol molecule and the terminal carboxyl group of an ePL molecule. When a short-chain aliphatic polyol, such as ethylene glycol, propanediol, or butanediol, was added instead of glycerol, a corresponding ePL-polyol monoester accumulated in the culture medium of ePL-producing bacteria. ePL esterification was accompanied by ePL synthesis in intact cells and a cell-free system, but no esterification of exogenous ePL was observed. ePL-polyol esters were formed during lysine polymerization. The number of lysine residues of ePL-polyol esters decreased with increasing polyol concentration. Taken together, these results indicate that ePL synthesis is inhibited by polyols via esterification and that ePL elongation occurs via the incorporation of lysine monomers into the carboxyl terminus of ePL.
rRNA and Poly-β-Hydroxybutyrate Dynamics in Bioreactors Subjected to Feast and Famine Cycles
Authors: Frigon Dominic , Muyzer Gerard , van Loosdrecht Mark , Raskin Lutgarde
Published: April 2006

Feast and famine cycles are common in activated sludge wastewater treatment systems, and they select for bacteria that accumulate storage compounds, such as poly-β-hydroxybutyrate (PHB). Previous studies have shown that variations in influent substrate concentrations force bacteria to accumulate high levels of rRNA compared to the levels in bacteria grown in chemostats. Therefore, it can be hypothesized that bacteria accumulate more rRNA when they are subjected to feast and famine cycles. However, PHB-accumulating bacteria can form biomass (grow) throughout a feast and famine cycle and thus have a lower peak biomass formation rate during the cycle. Consequently, PHB-accumulating bacteria may accumulate less rRNA when they are subjected to feast and famine cycles than bacteria that are not capable of PHB accumulation. These hypotheses were tested with
Wautersia eutropha
H16 (wild type) and
W. eutropha
PHB-4 (a mutant not capable of accumulating PHB) grown in chemostat and semibatch reactors. For both strains, the cellular RNA level was higher when the organism was grown in semibatch reactors than when it was grown in chemostats, and the specific biomass formation rates during the feast phase were linearly related to the cellular RNA levels for cultures. Although the two strains exhibited maximum uptake rates when they were grown in semibatch reactors, the wild-type strain responded much more rapidly to the addition of fresh medium than the mutant responded. Furthermore, the chemostat-grown mutant culture was unable to exhibit maximum substrate uptake rates when it was subjected to pulse-wise addition of fresh medium. These data show that the ability to accumulate PHB does not prevent bacteria from accumulating high levels of rRNA when they are subjected to feast and famine cycles. Our results also demonstrate that the ability to accumulate PHB makes the bacteria more responsive to sudden increases in substrate concentrations, which explains their ecological advantage.
Distribution of Environmental Mycobacteria in Karonga District, Northern Malawi
Authors: Chilima Benson Z. , Clark Ian M. , Floyd Sian , Fine Paul E. M. , Hirsch Penny R.
Published: April 2006

The genusMycobacteriumincludes many species that are commonly found in the environment (in soil and water or associated with plants and animals), as well as species that are responsible for two major human diseases, tuberculosis (Mycobacterium tuberculosis) and leprosy (Mycobacterium leprae). The distribution of environmental mycobacteria was investigated in the context of a long-term study of leprosy, tuberculosis,Mycobacterium bovisBCG vaccination, and the responses of individuals to various mycobacterial antigens in Karonga District, northern Malawi, where epidemiological studies had indicated previously that people may be exposed to different mycobacterial species in the northern and southern parts of the district. A total of 148 soil samples and 24 water samples were collected from various locations and examined to determine the presence of mycobacteria. The detection method involved semiselective culturing and acid-fast staining, following decontamination of samples to enrich mycobacteria and reduce the numbers of other microorganisms, or PCR with primers specific for the mycobacterial 16S rRNA gene, using DNA extracted directly from soil and water samples. Mycobacteria were detected in the majority of the samples, and subsequent sequence analysis of PCR products amplified directly from soil DNA indicated that most of the products were related to known environmental mycobacteria. For both methods the rates of recovery were consistently higher for dry season samples than for wet season samples. All isolates cultured from soil appeared to be strains ofMycobacterium fortuitum.This study revealed a complex pattern for the environmental mycobacterial flora but identified no clear differences between the northern and southern parts of Karonga District.
Quantitative Real-Time PCR Analysis of FecalLactobacillusSpecies in Infants Receiving a Prebiotic Infant Formula
Authors: Haarman Monique , Knol Jan
Published: April 2006

The developing intestinal microbiota of breast-fed infants is considered to play an important role in the priming of the infants' mucosal and systemic immunity. Generally,BifidobacteriumandLactobacilluspredominate the microbiota of breast-fed infants. In intervention trials it has been shown that lactobacilli can exert beneficial effects on, for example, diarrhea and atopy. However, theLactobacillusspecies distribution in breast-fed or formula-fed infants has not yet been determined in great detail. For accurate enumeration of different lactobacilli, duplex 5′ nuclease assays, targeted on rRNA intergenic spacer regions, were developed forLactobacillus acidophilus,Lactobacillus casei,Lactobacillus delbrueckii,Lactobacillus fermentum,Lactobacillus paracasei,Lactobacillus plantarum,Lactobacillus reuteri, andLactobacillus rhamnosus. The designed and validated assays were used to determine the amounts of differentLactobacillusspecies in fecal samples of infants receiving a standard formula (SF) or a standard formula supplemented with galacto- and fructo-oligosaccharides in a 9:1 ratio (OSF). A breast-fed group (BF) was studied in parallel as a reference. During the 6-week intervention period a significant increase was shown in total percentage of fecal lactobacilli in the BF group (0.8% ± 0.3% versus 4.1% ± 1.5%) and the OSF group (0.8% ± 0.3% versus 4.4% ± 1.4%). TheLactobacillusspecies distribution in the OSF group was comparable to breast-fed infants, with relatively high levels ofL. acidophilus,L. paracasei, andL. casei. The SF-fed infants, on the other hand, contained moreL. delbrueckiiand lessL. paracaseicompared to breast-fed infants and OSF-fed infants. An infant milk formula containing a specific mixture of prebiotics is able to induce a microbiota that closely resembles the microbiota of BF infants.
Evidence for Outcrossing via the Buller Phenomenon in a Substrate Simultaneously Inoculated with Spores and Mycelium of
Agaricus bisporus
Authors: Callac Philippe , Spataro Cathy , Caille Aurélie , Imbernon Micheline
Published: April 2006

In
Agaricus bisporus
, traditional cultivars and most of the wild populations belong to
A. bisporus
var.
bisporus
, which has a predominantly pseudohomothallic life cycle in which most meiospores are heterokaryons (
n
+
n
). A lower proportion of homokaryotic (
n
) meiospores, which typify the heterothallic life cycle, also are produced. In wild populations, pseudohomothallism was thought previously to play a major role, but recent analyses have found that significant outcrossing also may occur. We inoculated a standard substrate for
A. bisporus
cultivation simultaneously with homokaryotic mycelium from one parent and spores from a second parent. Culture trays produced numerous sporocarps that could theoretically have resulted from five different reproductive modes (pseudohomothallism, selfing or outcrossing via heterothallism, and selfing or outcrossing via the Buller phenomenon [i.e., between a homokaryon and a heterokaryon]). Most or all of the sporocarps resulted from outcrossing between the inoculated homokaryon and the inoculated heterokaryotic spores (or mycelia that grew from them). These data broaden our understanding of population dynamics under field conditions and provide an outcrossing method that could be used in commercial breeding programs.
Natural Electrotransformation of Lightning-Competent
Pseudomonas
sp. Strain N3 in Artificial Soil Microcosms
Authors: Cérémonie Hélène , Buret François , Simonet Pascal , Vogel Timothy M.
Published: April 2006

The lightning-competent
Pseudomonas
sp. strain N3, recently isolated from soil, has been used to study the extent of natural electrotransformation (NET) or lightning transformation as a horizontal gene transfer mechanism in soil. The variation of electrical fields applied to the soil with a laboratory-scale lightning system provides an estimate of the volume of soil affected by NET. Based on the range of the electric field that induces NET of
Pseudomonas
strain N3, the volume of soil, where NET could occur, ranges from 2 to 950 m
3
per lightning strike. The influence of DNA parameters (amount, size, and purity) and DNA soil residence time were also investigated. NET frequencies (electrotransformants/recipient cells) ranged from 10
−8
for cell lysate after 1 day of residence in soil to 4 × 10
−7
with a purified plasmid added immediately before the lightning. The electrical field gradient (in kilovolts per cm) also played a role as NET frequencies ranging from 1 × 10
−5
at 2.3 kV/cm to 1.7 × 10
−4
at 6.5 kV/cm.
Reevaluation of the Life Cycle of
Tuber magnatum
Authors: Paolocci Francesco , Rubini Andrea , Riccioni Claudia , Arcioni Sergio
Published: April 2006

Tuber
spp. are ectomycorrhizal ascomycetes that produce ascocarps known as truffles. Basic aspects of
Tuber
biology have yet to be fully elucidated. In particular, there are conflicting hypotheses concerning the mating system and the ploidy level of the mycorrhizal and truffle hyphae. We used polymorphic microsatellites to compare the allelic configurations of asci with those from the network of the surrounding hyphae in single
Tuber magnatum
truffles. We then used these truffles to inoculate host plants and evaluated the microsatellite configurations of the resulting mycorrhizal root tips. These analyses provide direct evidence that
T. magnatum
outcrosses and that its life cycle is predominantly haploid. In addition to its scientific significance, this basic understanding of the
T. magnatum
life cycle may have practical importance in developing strategies to obtain and select nursery-produced mycorrhizal plants as well as in the management of artificial plantations of this and other
Tuber
spp.
A Mono-2-Ethylhexyl Phthalate Hydrolase from a
Gordonia
sp. That Is Able To Dissimilate Di-2-Ethylhexyl Phthalate
Authors: Nishioka Tuguhiro , Iwata Makoto , Imaoka Takuya , Mutoh Maiko , Egashira Yoshihiro , Nishiyama Takashi , Shin Takashi , Fujii Takao
Published: April 2006

Gordonia
sp. strain P8219, a strain able to decompose di-2-ethylhexyl phthalate, was isolated from machine oil-contaminated soil. Mono-2-ethylhexyl phthalate hydrolase was purified from cell extracts of this strain. This enzyme was a 32,164-Da homodimeric protein, and it effectively hydrolyzed monophthalate esters, such as monoethyl, monobutyl, monohexyl, and mono-2-ethylhexyl phthalate. The

K
m

and
V
max
values for mono-2-ethylhexyl phthalate were 26.9 ± 4.3 μM and 18.1 ± 0.9 μmol/min · mg protein, respectively. The deduced amino acid sequence of the enzyme exhibited less than 30% homology with those of
meta
-cleavage hydrolases which are serine hydrolases but exhibited no significant homology with the sequences of serine esterases. The pentapeptide motif GXSXG, which is conserved in serine hydrolases, was present in the sequence. The enzymatic properties and features of the primary structure suggested that this enzyme is a novel enzyme belonging to an independent group of serine hydrolases.
Development and Characterization of Stable Sediment-Free Anaerobic Bacterial Enrichment Cultures That Dechlorinate Aroclor 1260
Authors: Bedard Donna L. , Bailey Jessica J. , Reiss Brandon L. , Jerzak Greta Van Slyke
Published: April 2006

We have developed sediment-free anaerobic enrichment cultures that dechlorinate a broad spectrum of highly chlorinated polychlorinated biphenyls (PCBs). The cultures were developed from Aroclor 1260-contaminated sediment from the Housatonic River in Lenox, MA. Sediment slurries were primed with 2,6-dibromobiphenyl to stimulate Process N dechlorination (primarilymetadechlorination), and sediment was gradually removed by successive transfers (10%) to minimal medium. The cultures grow on pyruvate, butyrate, or acetate plus H2. Gas chromatography-electron capture detector analysis demonstrated that the cultures extensively dechlorinate 50 to 500 μg/ml of Aroclor 1260 at 22 to 24°C by Dechlorination Process N. Triplicate cultures of the eighth transfer without sediment dechlorinated 76% of the hexa- through nonachlorobiphenyls in Aroclor 1260 (250 μg/ml) to tri- through pentachlorobiphenyls in 110 days. At least 64 PCB congeners, all of which are chlorinated on both rings and 47 of which have six or more chlorines, were substrates for this dechlorination. To characterize the bacterial diversity in the enrichments, we used eubacterial primers to amplify and clone 16S rRNA genes from DNA extracted from cultures grown on acetate plus H2. Restriction fragment length polymorphism analysis of 107 clones demonstrated the presence ofThauera-likeBetaproteobacteria,Geobacter-likeDeltaproteobacteria,Pseudomonasspecies, variousClostridiales,Bacteroidetes,Dehalococcoidesof theChloroflexigroup, and unclassifiedEubacteria. Our development of highly enriched, robust, stable, sediment-free cultures that extensively dechlorinate a highly chlorinated commercial PCB mixture is a major and unprecedented breakthrough in the field. It will enable intensive study of the organisms and genes responsible for a major PCB dechlorination process that occurs in the environment and could also lead to effective remediation applications.
Emergence of Distinct Genotypes of
Cryptosporidium parvum
in Structured Host Populations
Authors: Tanriverdi Sultan , Markovics Alex , Arslan M. Özkan , Itik Aysel , Shkap Varda , Widmer Giovanni
Published: April 2006

Cryptosporidium parvum
is an apicomplexan parasite that infects humans and ruminants.
C. parvum
isolated from cattle in northeastern Turkey and in Israel was genotyped using multiple polymorphic genetic markers, and the two populations were compared to assess the effect of cattle husbandry on the parasite's population structure. Dairy herds in Israel are permanently confined with essentially no opportunity for direct herd-to-herd transmission, whereas in Turkey there are more opportunities for transmission as animals range over wider areas and are frequently traded. A total of 76
C. parvum
isolates from 16 locations in Israel and seven farms in the Kars region in northeastern Turkey were genotyped using 16 mini- and microsatellite markers. Significantly, in both countries distinct multilocus genotypes confined to individual farms were detected. The number of genotypes per farm was higher and mixed isolates were more frequent in Turkey than in Israel. As expected from the presence of distinct multilocus genotypes in individual herds, linkage disequilibrium among loci was detected in Israel. Together, these observations show that genetically distinct populations of
C. parvum
can emerge within a group of hosts in a relatively short time. This may explain the frequent detection of host-specific genotypes with unknown taxonomic status in surface water and the existence of geographically restricted
C. hominis
genotypes in humans.
An Efficient Method of Selectable Marker Gene Excision by Xer Recombination for Gene Replacement in Bacterial Chromosomes
Authors: Bloor Alexandra E. , Cranenburgh Rocky M.
Published: April 2006

A simple, effective method of unlabeled, stable gene insertion into bacterial chromosomes has been developed. This utilizes an insertion cassette consisting of an antibiotic resistance gene flanked by
dif
sites and regions homologous to the chromosomal target locus.
dif
is the recognition sequence for the native Xer site-specific recombinases responsible for chromosome and plasmid dimer resolution: XerC/XerD in
Escherichia coli
and RipX/CodV in
Bacillus subtilis
. Following integration of the insertion cassette into the chromosomal target locus by homologous recombination, these recombinases act to resolve the two directly repeated
dif
sites to a single site, thus excising the antibiotic resistance gene. Previous approaches have required the inclusion of exogenous site-specific recombinases or transposases in
trans
; our strategy demonstrates that this is unnecessary, since an effective recombination system is already present in bacteria. The high recombination frequency makes the inclusion of a counter-selectable marker gene unnecessary.
Cloning and Sequencing of the
ompA
Gene of
Enterobacter sakazakii
and Development of an
ompA
-Targeted PCR for Rapid Detection of
Enterobacter sakazakii
in Infant Formula
Authors: Mohan Nair Manoj Kumar , Venkitanarayanan Kumar S.
Published: April 2006

Enterobacter sakazakii
is an emerging, infant formula-borne pathogen that causes severe meningitis, meningoencephalitis, sepsis, and necrotizing enterocolitis in neonates and infants, with a high fatality rate. Traditional detection methods take up to 7 days to identify
E. sakazakii
. The outer membrane protein A gene (
ompA
), along with its flanking sequences from
E. sakazakii
(ATCC 51329), was cloned in the pGEM-T Easy vector and sequenced. Comparison of the nucleotide and deduced amino acid sequences of the
ompA
gene with other sequences available in the GenBank database revealed a high degree of homology with
ompA
genes of other gram-negative bacteria belonging to the
Enterobacteriaceae
. Based on regions of the
ompA
gene unique to
E. sakazakii
, two primers were synthesized to develop and optimize an
E. sakazakii
-specific PCR. The PCR amplified a 469-bp DNA product from all
E. sakazakii
strains tested but not from other bacteria. Experiments to determine the sensitivity of the PCR indicated that it could detect as few as 10
3
CFU/ml of
E. sakazakii
bacteria in infant formula directly and 10
−1
CFU/ml after an 8-h enrichment step. We conclude that this PCR, combined with enrichment culturing, has the potential to be used as a rapid tool for detecting the presence of
E. sakazakii
in infant formula.
Engineered Biosynthesis of a Novel Amidated Polyketide, Using the Malonamyl-Specific Initiation Module from the Oxytetracycline Polyketide Synthase
Authors: Zhang Wenjun , Ames Brian D. , Tsai Shiou-Chuan , Tang Yi
Published: April 2006

Tetracyclines are aromatic polyketides biosynthesized by bacterial type II polyketide synthases (PKSs). Understanding the biochemistry of tetracycline PKSs is an important step toward the rational and combinatorial manipulation of tetracycline biosynthesis. To this end, we have sequenced the gene cluster of oxytetracycline (
oxy
and
otc
genes) PKS genes from
Streptomyces rimosus
. Sequence analysis revealed a total of 21 genes between the
otrA
and
otrB
resistance genes. We hypothesized that an amidotransferase, OxyD, synthesizes the malonamate starter unit that is a universal building block for tetracycline compounds. In vivo reconstitution using strain CH999 revealed that the minimal PKS and OxyD are necessary and sufficient for the biosynthesis of amidated polyketides. A novel alkaloid (WJ35, or compound 2) was synthesized as the major product when the
oxy
-encoded minimal PKS, the C-9 ketoreductase (OxyJ), and OxyD were coexpressed in CH999. WJ35 is an isoquinolone compound derived from an amidated decaketide backbone and cyclized with novel regioselectivity. The expression of OxyD with a heterologous minimal PKS did not afford similarly amidated polyketides, suggesting that the
oxy
-encoded minimal PKS possesses novel starter unit specificity.
Toxicity and Mode of Action of
Bacillus thuringiensis
Cry Proteins in the Mediterranean Corn Borer,
Sesamia nonagrioides
(Lefebvre)
Authors: González-Cabrera Joel , Farinós Gema P. , Caccia Silvia , Díaz-Mendoza Mercedes , Castañera Pedro , Leonardi Maria Giovanna , Giordana Barbara , Ferré Juan
Published: April 2006

Sesamia nonagrioides
is one of the most damaging pests of corn in Spain and other Mediterranean countries. Bt corn expressing the
Bacillus thuringiensis
Cry1Ab toxin is being grown on about 58,000 ha in Spain. Here we studied the mode of action of this Cry protein on
S. nonagrioides
(binding to specific receptors, stability of binding, and pore formation) and the modes of action of other Cry proteins that were found to be active in this work (Cry1Ac, Cry1Ca, and Cry1Fa). Binding assays were performed with
125
I- or biotin-labeled toxins and larval brush border membrane vesicles (BBMV). Competition experiments indicated that these toxins bind specifically and that Cry1Aa, Cry1Ab, and Cry1Ac share a binding site. Cry1Ca and Cry1Fa bind to different sites. In addition, Cry1Fa binds to Cry1A's binding site with very low affinity and vice versa. Binding of Cry1Ab and Cry1Ac was found to be stable over time, which indicates that the observed binding is irreversible. The pore-forming activity of Cry proteins on BBMV was determined using the voltage-sensitive fluorescent dye DiSC
3
(5). Membrane permeability increased in the presence of the active toxins Cry1Ab and Cry1Fa but not in the presence of the nonactive toxin Cry1Da. In terms of resistance management, based on our results and the fact that Cry1Ca is not toxic to
Ostrinia nubilalis
, we recommend pyramiding of Cry1Ab with Cry1Fa in the same Bt corn plant for better long-term control of corn borers.
Thiamine-Auxotrophic Mutants of
Pseudomonas fluorescens
CHA0 Are Defective in Cell-Cell Signaling and Biocontrol Factor Expression
Authors: Dubuis Christophe , Rolli Joëlle , Lutz Matthias , Défago Geneviève , Haas Dieter
Published: April 2006

In the biocontrol strain
Pseudomonas fluorescens
CHA0, the Gac/Rsm signal transduction pathway positively controls the synthesis of antifungal secondary metabolites and exoenzymes. In this way, the GacS/GacA two-component system determines the expression of three small regulatory RNAs (RsmX, RsmY, and RsmZ) in a process activated by the strain's own signal molecules, which are not related to
N
-acyl-homoserine lactones. Transposon Tn
5
was used to isolate
P. fluorescens
CHA0 insertion mutants that expressed an
rsmZ-gfp
fusion at reduced levels. Five of these mutants were
gacS
negative, and in them the
gacS
mutation could be complemented for exoproduct and signal synthesis by the
gacS
wild-type allele. Furthermore, two thiamine-auxotrophic (
thiC
) mutants that exhibited decreased signal synthesis in the presence of 5 × 10
−8
M thiamine were found. Under these conditions, a
thiC
mutant grew normally but showed reduced expression of the three small RNAs, the exoprotease AprA, and the antibiotic 2,4-diacetylphloroglucinol. In a gnotobiotic system, a
thiC
mutant was impaired for biological control of
Pythium ultimum
on cress. Addition of excess exogenous thiamine restored all deficiencies of the mutant. Thus, thiamine appears to be an important factor in the expression of biological control by
P. fluorescens
.
Very Low Ethanol Concentrations Affect the Viability and Growth Recovery in Post-Stationary-Phase
Staphylococcus aureus
Populations
Authors: Chatterjee Indranil , Somerville Greg A. , Heilmann Christine , Sahl Hans-Georg , Maurer Hans H. , Herrmann Mathias
Published: April 2006

Pharmaceuticals, culture media used for in vitro diagnostics and research, human body fluids, and environments can retain very low ethanol concentrations (VLEC) (≤0.1%, vol/vol). In contrast to the well-established effects of elevated ethanol concentrations on bacteria, little is known about the consequences of exposure to VLEC. We supplemented growth media for
Staphylococcus aureus
strain DSM20231 with VLEC (VLEC
+
conditions) and determined ultramorphology, growth, and viability compared to those with unsupplemented media (VLEC
−
conditions) for prolonged culture times (up to 8 days). VLEC
+
-grown late-stationary-phase
S. aureus
displayed extensive alterations of cell integrity as shown by scanning electron microscopy. Surprisingly, while ethanol in the medium was completely metabolized during exponential phase, a profound delay of
S. aureus
post-stationary-phase recovery (>48 h) was observed. Concomitantly, under VLEC
+
conditions, the concentration of acetate in the culture medium remained elevated while that of ammonia was reduced, contributing to an acidic culture medium and suggesting decreased amino acid catabolism. Interestingly, amino acid depletion was not uniformly affected: under VLEC
+
conditions, glutamic acid, ornithine, and proline remained in the culture medium while the uptake of other amino acids was not affected. Supplementation with arginine, but not with other amino acids, was able to restore post-stationary-phase growth and viability. Taken together, these data demonstrate that VLEC have profound effects on the recovery of
S. aureus
even after ethanol depletion and delay the transition from primary to secondary metabolite catabolism. These data also suggest that the concentration of ethanol needed for bacteriostatic control of
S. aureus
is lower than that previously reported.
Cultivation of Denitrifying Bacteria: Optimization of Isolation Conditions and Diversity Study
Authors: Heylen Kim , Vanparys Bram , Wittebolle Lieven , Verstraete Willy , Boon Nico , De Vos Paul
Published: April 2006

An evolutionary algorithm was applied to study the complex interactions between medium parameters and their effects on the isolation of denitrifying bacteria, both in number and in diversity. Growth media with a pH of 7 and a nitrogen concentration of 3 mM, supplemented with 1 ml of vitamin solution but not with sodium chloride or riboflavin, were the most successful for the isolation of denitrifiers from activated sludge. The use of ethanol or succinate as a carbon source and a molar C/N ratio of 2.5, 20, or 25 were also favorable. After testing of 60 different medium parameter combinations and comparison with each other as well as with the standard medium Trypticase soy agar supplemented with nitrate, three growth media were highly suitable for the cultivation of denitrifying bacteria. All evaluated isolation conditions were used to study the cultivable denitrifier diversity of activated sludge from a municipal wastewater treatment plant. One hundred ninety-nine denitrifiers were isolated, the majority of which belonged to theBetaproteobacteria(50.4%) and theAlphaproteobacteria(36.8%). Representatives ofGammaproteobacteria(5.6%),Epsilonproteobacteria(2%), andFirmicutes(4%) and one isolate of theBacteroideteswere also found. This study revealed a much more diverse denitrifying community than that previously described in cultivation-dependent research on activated sludge.
Assessment of the Roles of LuxS,
S
-Ribosyl Homocysteine, and Autoinducer 2 in Cell Attachment during Biofilm Formation by
Listeria monocytogenes
EGD-e
Authors: Challan Belval Sylvain , Gal Laurent , Margiewes Sylvain , Garmyn Dominique , Piveteau Pascal , Guzzo Jean
Published: April 2006

LuxS is responsible for the production of autoinducer 2 (AI-2), which is involved in the quorum-sensing response of
Vibrio harveyi
. AI-2 is found in several other gram-negative and gram-positive bacteria and is therefore considered a good candidate for an interspecies communication signal molecule. In order to determine if this system is functional in the gastrointestinal pathogen
Listeria monocytogenes
EGD-e, an AI-2 bioassay was performed with culture supernatants. The results indicated that this bacterium produces AI-2 like molecules. A potential ortholog of
V. harveyi luxS
, lmo1288, was found by performing sequence similarity searches and complementation experiments with
Escherichia coli
DH5α, a
luxS
null strain. lmo1288 was found to be a functional
luxS
ortholog involved in AI-2 synthesis. Indeed, interruption of lmo1288 resulted in loss of the AI-2 signal. Although no significant differences were observed between Lux1 and EGD-e with regard to planktonic growth (at 10°C, 15°C, 25°C, and 42°C), swimming motility, and phospholipase and hemolytic activity, biofilm culture experiments showed that under batch conditions between 25% and 58% more Lux1 cells than EGD-e cells were attached to the surface depending on the incubation time. During biofilm growth in continuous conditions after 48 h of culture, Lux1 biofilms were 17 times denser than EGD-e biofilms. Finally, our results showed that Lux1 accumulates more
S
-adenosyl homocysteine (SAH) and
S
-ribosyl homocysteine (SRH) in culture supernatant than the parental strain accumulates and that SRH, but not SAH or AI-2, is able to modify the number of attached cells.
The Locus Coding for the 3-Nitrobenzoate Dioxygenase of
Comamonas
sp. Strain JS46 Is Flanked by IS
1071
Elements and Is Subject to Deletion and Inversion Events
Authors: Providenti Miguel A. , Shaye Rachel E. , Lynes Krista D. , McKenna Neil T. , O'Brien Jason M. , Rosolen Sarah , Wyndham R. Campbell , Lambert Iain B.
Published: April 2006

In
Comamonas
sp. strain JS46, 3-nitrobenzoate (3Nba) is initially oxidized at the 3,4 position by a dioxygenase, which results in release of nitrite and production of protocatechuate. The locus coding for the 3Nba dioxygenase (designated
mnb
, for
m-n
itro
b
enzoate) was mobilized from strain JS46 using a plasmid capture method, cloned, and sequenced. The 3Nba dioxygenase (MnbA) is a member of the phthalate family of aromatic oxygenases. An open reading frame designated
mnbB
that codes for an NAD(P)H-dependent class IA aromatic oxidoreductase is downstream of
mnbA
. MnbB is tentatively identified as the oxidoreductase that transfers reducing equivalents to MnbA in strain JS46. The
mnb
locus is flanked by IS
1071
elements. The upstream element is interrupted by a novel insertion sequence designated IS
Csp1
, and the transposase genes of the flanking insertion elements are transcribed in the direction opposite the direction of
mnbA
transcription. Spontaneous deletion of
mnb
occurs because of homologous recombination between the directly repeated flanking IS
1071
elements. In addition, in ∼0.007 to 0.2% of any population of JS46 cells growing on 3Nba, alternative orientations of
mnb
relative to the flanking IS
1071
elements are detected. These alternative forms are the result of inversions of
mnb
and the flanking IS
1071
elements. Inversions appear to occur because of homologous recombination between the inverted repeats that flank the IS
1071
elements.