bluuebunny/crossref_metadata_2025 huggingface dataset:
Impaired development of hippocampal mossy fibre synapses in mouse mutants for the presynaptic scaffold protein Bassoon
Authors: Lanore Frederic , Blanchet Christophe , Fejtova Anna , Pinheiro Paulo , Richter Karin , Balschun Detlef , Gundelfinger Eckart , Mulle Christophe
Published: April 2010

Bassoon, a protein highly concentrated at the synaptic active zone, is thought to participate in the organization of the cytomatrix at the site of neurotransmitter release. Bassoon is amongst the first proteins to accumulate at newly formed synaptic junctions, raising the question of the functional role of this protein in the early stages of synaptic development. Here we show that the course of synaptic maturation of hippocampal mossy fibre (MF) synapses (glutamatergic synapses with multiple release sites) is markedly altered during the first 2 weeks of postnatal development in mutant mice lacking the central region of Bassoon (Bsn−/− mice). At postnatal day 7 (P7), Bsn−/− mice display large amplitude MF‐EPSCs with decreased paired pulse ratios, an abnormality which may be linked to deficits in the organization of the presynaptic active zone. Surprisingly, 1 week later, decreased MF‐EPSCs amplitude is observed in Bsn−/− mice, consistent with the inactivation of a subset of synaptic release sites. Finally, at more mature states a decreased posttetanic potentiation is observed at MF‐synapses. These results support the notion that Bassoon is important for organizing the presynaptic active zone during the postnatal maturation of glutamatergic synapses.
Experimental Study of the Performance of Static Seals Based on Measurements of Real Contact Area Using Thin Polycarbonate Films
Authors: Nitta Isami , Matsuzaki Yoshio
Published: April 2010

To clarify the sealing characteristics of metal gasket seals, leakage rates of gas and the real contact area of the seal surfaces were measured under several closing loads. The static seal consisted of a ring-shaped copper gasket and the two steel flanges that held the gasket in place. Gasket widths in the radial direction were 2 mm, 3 mm, and 5 mm. The contact surfaces of the flanges were finished by lathe turning. To determine the leakage flow paths between the gasket and the flanges, the real contact situation between them was observed using a thin polymer film 1 μm in thickness. The results indicated the leakage flow paths on the gasket surface were the radial direction perpendicular to the lathe-turned groove and the circumferential direction along the groove. As the closing loads increased, the leakage flow in the radial direction ceased and only that in the circumferential direction remained. Therefore, the cross-section of the aperture for the leakage flow in the circumferential direction was evaluated from the measured real contact area, and the leakage rates were estimated by assumption of laminar flow. The results agreed well with the measured leakage rates.
A Gas Lubrication Equation for High Knudsen Number Flows and Striated Rough Surfaces
Authors: White James
Published: April 2010

This paper describes the derivation and numerical solution of a lubrication equation appropriate for high Knudsen number flows and certain types of striated rough surfaces. The derivation begins with the compressible form of the lubrication equation together with the nonlinear series form of the Poiseuille flow reported by Fukui and Kaneko (1990, “A Database for Interpolation of Poiseuille Flow Rates for High Knudsen Number Lubrication Problems,” ASME J. Tribol., 112, pp. 78–83.). A multiple-scale analysis is performed on the lubrication equation for a finite-width time-dependent bearing and is limited to either stationary-transverse or longitudinal striated surface roughness of very short length scale. The rough surface averaging that takes place within the multiple-scale analysis includes a fully coupled treatment of the Poiseuille flow. What results is an especially nonlinear lubrication equation with averaged surface roughness effects that is appropriate for high Knudsen number analysis. A rotational transformation is also introduced to provide the roughness averaged lubrication equation in a form that allows analysis of the skewed orientation of a recording head slider with roughness defined relative to the direction of disk motion but with the lubrication equation conveniently expressed in the coordinate system of the slider. A factored-implicit numerical algorithm is described that provides the solution of the roughness averaged lubrication equation. Even though the lubrication equation is highly nonlinear, the numerical scheme is crafted to be fully second-order, time-accurate, and noniterative for tracking the solution in time either to an asymptotic steady-state or in response to a dynamic event. Numerical solutions of several simple geometry bearings are presented that utilize parameters that are typical of the slider-disk interface of current hard disk drives. It is anticipated that the primary benefit of this work may be the ability to accurately and efficiently include the influence of discrete disk data tracks in the air bearing design of very low clearance recording head sliders.
A Simple Method to Calculate Contact Factor Used in Average Flow Model
Authors: Meng Fanming , Wang Q. Jane , Hua Diann , Liu Shuangbiao
Published: April 2010

The average flow model proposed by Patir and Cheng offers a great convenience for the analysis of rough surfaces in lubrication. The contact factor introduced by Wu and Zheng helps to solve a difficulty in local film evaluation using the average flow model. This paper reports a simple method to calculate the contact factor. Method validation is demonstrated by the comparison of the contact factors for Gaussian surfaces obtained with the present method and the fitting formula of Wu and Zheng. The proposed method cannot only easily compute the contact factor values for Gaussian surfaces; it can also be used for those of non-Gaussian and measured surfaces, especially those with unknown probability density distribution of the roughness height.
Minigenome-Based Reporter System Suitable for High-Throughput Screening of Compounds Able To Inhibit
Ebolavirus
Replication and/or Transcription
Authors: Jasenosky Luke D. , Neumann Gabriele , Kawaoka Yoshihiro
Published: April 2010

We describe an
Ebolavirus
minigenome-based system that is suitable for high-throughput screening of compounds able to impair
Ebolavirus
virus replication and/or transcription. The assay is robust (Z′ factor, >0.6) and can be carried out in low-biosafety containment. Results from a pilot screen of 960 compounds are presented.
Incidence and Antimicrobial Susceptibility of
Escherichia coli
and
Klebsiella pneumoniae
with Extended-Spectrum β-Lactamases in Community- and Hospital-Associated Intra-Abdominal Infections in Europe: Results of the 2008 Study for Monitoring Antimicrobial Resistance Trends (SMART)
Authors: Hawser Stephen P. , Bouchillon Samuel K. , Hoban Daryl J. , Badal Robert E. , Cantón Rafael , Baquero Fernando
Published: April 2010

From 2002 to 2008, there was a significant increase in extended-spectrum beta-lactamase (ESBL)-positive
Escherichia coli
isolates in European intra-abdominal infections, from 4.3% in 2002 to 11.8% in 2008 (
P
< 0.001), but not for ESBL-positive
Klebsiella pneumoniae
isolates (16.4% to 17.9% [
P
> 0.05]). Hospital-associated isolates were more common than community-associated isolates, at 14.0% versus 6.5%, respectively, for
E. coli
(
P
< 0.001) and 20.9% versus 5.3%, respectively, for
K. pneumoniae
(
P
< 0.01). Carbapenems were consistently the most active drugs tested.
Characterization of Variation in Rumen Methanogenic Communities under Different Dietary and Host Feed Efficiency Conditions, as Determined by PCR-Denaturing Gradient Gel Electrophoresis Analysis
Authors: Zhou Mi , Hernandez-Sanabria Emma , Guan Le Luo
Published: April 2010

Understanding ruminal methanogens is essential for greenhouse gas mitigation, as well as for improving animal performance in the livestock industry. It has been speculated that ruminal methanogenic diversity affects host feed efficiency and results in differences in methane production. This study examined methanogenic profiles in the rumen using culture-independent PCR-denaturing gradient gel electrophoresis (PCR-DGGE) analysis for 56 beef cattle which differed in feed efficiency, as well as diet (the cattle were fed a low-energy diet or a high-energy diet). The methanogenic PCR-DGGE profiles detected were greatly affected by diet, and the major pattern changed from a community containing predominantlyMethanobrevibacter ruminantiumNT7 with the low-energy diet to a community containing predominantlyMethanobrevibacter smithii,Methanobrevibactersp. AbM4, and/orM.ruminantiumNT7 with the high-energy diet. For each diet, the methanogenic PCR-DGGE pattern was strongly associated with the feed efficiency of the host. Diet-associated bands forMethanobrevibactersp. AbM4 andM.smithiiSM9 and a feed efficiency-related band forM.smithiiPS were identified. The abundance of total methanogens was estimated by determining the numbers of copies of the 16S rRNA genes of methanogens. However, the size of the methanogen population did not correlate with differences in feed efficiency, diet, or metabolic measurements. Thus, the structure of the methanogenic community at the species or strain level may be more important for determining host feed efficiency under different dietary conditions.
Heptahelical Receptors GprC and GprD of
Aspergillus fumigatus
Are Essential Regulators of Colony Growth, Hyphal Morphogenesis, and Virulence
Authors: Gehrke Alexander , Heinekamp Thorsten , Jacobsen Ilse D. , Brakhage Axel A.
Published: April 2010

The filamentous fungus
Aspergillus fumigatus
normally grows on compost or hay but is also able to colonize environments such as the human lung. In order to survive, this organism needs to react to a multitude of external stimuli. Although extensive work has been carried out to investigate intracellular signal transduction in
A. fumigatus
, little is known about the specific stimuli and the corresponding receptors activating these signaling cascades. Here, two putative G-protein-coupled receptors, GprC and GprD, were characterized with respect to their cellular functions. Deletion of the corresponding genes resulted in drastic growth defects as hyphal extension was reduced, germination was retarded, and hyphae showed elevated levels of branching. The growth defect was found to be temperature dependent. The higher the temperature the more pronounced was the growth defect. Furthermore, compared with the wild type, the sensitivity of the mutant strains toward environmental stress caused by reactive oxygen intermediates was increased and the mutants displayed an attenuation of virulence in a murine infection model. Both mutants, especially the Δ
gprC
strain, exhibited increased tolerance toward cyclosporine, an inhibitor of the calcineurin signal transduction pathway. Transcriptome analyses indicated that in both the
gprC
and
gprD
deletion mutants, transcripts of primary metabolism genes were less abundant, whereas transcription of several secondary metabolism gene clusters was upregulated. Taken together, our data suggest the receptors are involved in integrating and processing stress signals via modulation of the calcineurin pathway.
Oral Bacteria as Potential Probiotics for the Pharyngeal Mucosa
Authors: Guglielmetti Simone , Taverniti Valentina , Minuzzo Mario , Arioli Stefania , Stuknyte Milda , Karp Matti , Mora Diego
Published: April 2010

The research described here was aimed at the selection of oral bacteria that displayed properties compatible with their potential use as probiotics for the pharyngeal mucosa. We included in the study 56 bacteria newly isolated from the pharynges of healthy donors, which were identified at the intraspecies level and characterized
in vitro
for their probiotic potential. The experiments led us to select two potential probiotic bacterial strains (
Streptococcus salivarius
RS1 and ST3) and to compare them with the prototype oral probiotic
S. salivarius
strain K12. All three strains efficiently bound to FaDu human epithelial pharyngeal cells and thereby antagonized
Streptococcus pyogenes
adhesion and growth. All were sensitive to a variety of antibiotics routinely used for the control of upper respiratory tract infections. Immunological
in vitro
testing on a FaDu layer revealed different responses to RS1, ST3, and K12. RS1 and ST3 modulated NF-κB activation and biased proinflammatory cytokines at baseline and after interleukin-1β (IL-1β) induction. In conclusion, we suggest that the selected commensal streptococci represent potential pharyngeal probiotic candidates. They could display a good degree of adaptation to the host and possess potential immunomodulatory and anti-inflammatory properties.
Plasmid Transduction Using Bacteriophage Φadh for Expression of CC Chemokines byLactobacillus gasseriADH
Authors: Damelin Leonard H. , Mavri-Damelin Demetra , Klaenhammer Todd R. , Tiemessen Caroline T.
Published: April 2010

Vaginal mucosal microfloras are typically dominated by Gram-positiveLactobacillusspecies, and colonization of vaginal mucosa by exogenous microbicide-secretingLactobacillusstrains has been proposed as a means of enhancing this natural mucosal barrier against human immunodeficiency virus (HIV) infection. We asked whether an alternative strategy could be utilized whereby anti-HIV molecules are expressed within the cervicovaginal milieu by endogenous vaginalLactobacilluspopulations which have been engineeredin situvia transduction. In this study, we therefore investigated the feasibility of utilizing transduction for the expression of two HIV coreceptor antagonists, the CC chemokines CCL5 and CCL3, in a predominant vaginalLactobacillusspecies,Lactobacillus gasseri. Modifying a previously established transduction model, which utilizesL. gasseriADH and its prophage Φadh, we show that mitomycin C induction ofL. gasseriADH transformants containing pGK12-based plasmids with CCL5 and CCL3 expression and secretion cassettes (under the control of promoters P6 and P59, respectively) and a 232-bp Φadh cos site fragment results in the production of transducing particles which contain 8 to 9 copies of concatemeric plasmid DNA. High-frequency transduction for these particles (almost 6 orders of magnitude greater than that for pGK12 alone) was observed, and transductants were found to contain recircularized expression plasmids upon subsequent culture. Importantly, transductants produced CC chemokines at levels comparable to those produced by electroporation-derived transformants. Our findings therefore lend support to the potential use of transduction in vaginalLactobacillusspecies as a novel strategy for the prevention of HIV infection across mucosal membranes.
Lactococcus lactis fabH
, Encoding β-Ketoacyl-Acyl Carrier Protein Synthase, Can Be Functionally Replaced by the
Plasmodium falciparum
Congener
Authors: Du Yu , Gisselberg Jolyn E. , Johnson Jacob D. , Lee Patricia J. , Prigge Sean T. , Bachmann Brian O.
Published: April 2010

Plasmodium falciparum
, in addition to scavenging essential fatty acids from its intra- and intercellular environments, possesses a functional complement of type II fatty acid synthase (FAS) enzymes targeted to the apicoplast organelle. Recent evidence suggests that products of the plasmodial FAS II system may be critical for the parasite's liver-to-blood cycle transition, and it has been speculated that endogenously generated fatty acids may be precursors for essential cofactors, such as lipoate, in the apicoplast. β-Ketoacyl-acyl carrier protein (ACP) synthase III (pfKASIII or FabH) is one of the key enzymes in the initiating steps of the FAS II pathway, possessing two functions in
P. falciparum
: the decarboxylative thio-Claisen condensation of malonyl-ACP and various acyl coenzymes A (acyl-CoAs; KAS activity) and the acetyl-CoA:ACP transacylase reaction (ACAT). Here, we report the generation and characterization of a hybrid
Lactococcus lactis
strain that translates pfKASIII instead of
L. lactis f
abH
to initiate fatty acid biosynthesis. The
L. lactis
expression vector pMG36e was modified for the efficient overexpression of the plasmodial gene in
L. lactis
. Transcriptional analysis indicated high-efficiency overexpression, and biochemical KAS and ACAT assays confirm these activities in cell extracts. Phenotypically, the
L. lactis
strain expressing pfKASIII has a growth rate and fatty acid profiles that are comparable to those of the strain complemented with its endogenous gene, suggesting that pfKASIII can use
L. lactis
ACP as substrate and perform near-normal function in
L. lactis
cells. This strain may have potential application as a bacterial model for pfKASIII inhibitor prescreening.
Abolition of Biofilm Formation in Urinary Tract
Escherichia coli
and
Klebsiella
Isolates by Metal Interference through Competition for Fur
Authors: Hancock Viktoria , Dahl Malin , Klemm Per
Published: April 2010

Bacterial biofilms are associated with a large number of persistent and chronic infections. Biofilm-dwelling bacteria are particularly resistant to antibiotics and immune defenses, which makes it hard if not impossible to eradicate biofilm-associated infections. In the urinary tract, free iron is strictly limited but is critical for bacterial growth. Biofilm-associated
Escherichia coli
cells are particularly desperate for iron. An attractive way of inhibiting biofilm formation is to fool the bacterial regulatory system for iron uptake. Here, we demonstrate that biofilm formation can be impaired by the addition of divalent metal ions, such as Zn(II) and Co(II), which inhibit iron uptake by virtue of their higher-than-iron affinity for the master controller protein of iron uptake, Fur. Reduced biofilm formation of urinary tract-infectious
E. coli
strains in the presence of Zn(II) was observed in microtiter plates and flow chambers as well as on urinary catheters. These results further support that iron uptake is indeed crucial for biofilm formation, and thereby, targeting these uptake systems might be an effective way to eradicate biofilms caused by infectious strains.
Application of Multilocus Sequence Typing To Study the Genetic Structure of Megaplasmids in
Medicago
-Nodulating Rhizobia
Authors: van Berkum Peter , Elia Patrick , Eardly Bertrand D.
Published: April 2010

A multilocus sequence typing (MLST) analysis was used to examine the genetic structure and diversity within the two large extrachromosomal replicons in
Medicago
-nodulating rhizobia (
Sinorhizobium meliloti
and
Sinorhizobium medicae
). The allelic diversity within these replicons was high compared to the reported diversity within the corresponding chromosomes of the same strains (P. van Berkum et al., J. Bacteriol. 188:5570-5577, 2006). Also, there was strong localized linkage disequilibrium (LD) between certain pSymA loci: e.g.,
nodC
and
nifD
. Although both of these observations could be explained by positive (or diversifying) selection by plant hosts, results of tests for positive selection did not provide consistent support for this hypothesis. The strong LD observed between the
nodC
and
nifD
genes could also be explained by their close proximity on the pSymA replicon. Evidence was obtained that some
nodC
alleles had a history of intragenic recombination, while other alleles of this locus had a history of intergenic recombination. Both types of recombination were associated with a decline in symbiotic competence with
Medicago sativa
as the host plant. The combined observations of LD between the
nodC
and
nifD
genes and intragenic recombination within one of these loci indicate that the symbiotic gene region on the pSymA plasmid has evolved as a clonal segment, which has been laterally transferred within the natural populations.
Analysis of the Fine-Scale Population Structure of “
Candidatus
Accumulibacter phosphatis” in Enhanced Biological Phosphorus Removal Sludge, Using Fluorescence
In Situ
Hybridization and Flow Cytometric Sorting
Authors: Kim Jeong Myeong , Lee Hyo Jung , Kim Sun Young , Song Jae Jun , Park Woojun , Jeon Che Ok
Published: April 2010

To investigate the fine-scale diversity of the polyphosphate-accumulating organisms (PAO) “
Candidatus
Accumulibacter phosphatis” (henceforth referred to as “
Ca.
Accumulibacter”), two laboratory-scale sequencing batch reactors (SBRs) for enhanced biological phosphorus removal (EBPR) were operated with sodium acetate as the sole carbon source. During SBR operations, activated sludge always contained morphologically different “
Ca
. Accumulibacter” strains showing typical EBPR performances, as confirmed by the combined technique of fluorescence
in situ
hybridization (FISH) and microautoradiography (MAR). Fragments of “
Ca.
Accumulibacter” 16S rRNA genes were retrieved from the sludge. Phylogenetic analyses together with sequences from the GenBank database showed that “
Ca.
Accumulibacter” 16S rRNA genes of the EBPR sludge were clearly differentiated into four “
Ca.
Accumulibacter” clades, Acc-SG1, Acc-SG2, Acc-SG3, and Acc-SG4. The specific FISH probes Acc444, Acc184, Acc72, and Acc119 targeting these clades and some helpers and competitors were designed by using the ARB program. Microbial characterization by FISH analysis using specific FISH probes also clearly indicated the presence of different “
Ca.
Accumulibacter” cell morphotypes. Especially, members of Acc-SG3, targeted by probe Acc72, were coccobacillus-shaped cells with a size of approximately 2 to 3 μm, while members of Acc-SG1, Acc-SG2, and Acc-SG4, targeted by Acc444, Acc184, and Acc119, respectively, were coccus-shaped cells approximately 1 μm in size. Subsequently, cells targeted by each FISH probe were sorted by use of a flow cytometer, and their polyphosphate kinase 1 (
ppk1
) gene homologs were amplified by using a
ppk1
-specific PCR primer set for “
Ca.
Accumulibacter.” The phylogenetic tree based on sequences of the
ppk1
gene homologs was basically congruent with that of the 16S rRNA genes, but members of Acc-SG3 with a distinct morphology comprised two different
ppk1
genes. These results suggest that “
Ca.
Accumulibacter” strains may be diverse physiologically and ecologically and represent distinct populations with genetically determined adaptations in EBPR systems.
Chromosome Stability and Gene Loss in Cockroach Endosymbionts
Authors: Sabree Zakee L. , Degnan Patrick H. , Moran Nancy A.
Published: April 2010

Insect endosymbiont genomes reflect massive gene loss. Two
Blattabacterium
genomes display colinearity and similar gene contents, despite high orthologous gene divergence, reflecting over 140 million years of independent evolution in separate cockroach lineages. We speculate that distant homologs may replace the functions of some eliminated genes through broadened substrate specificity.
Killing of Bacteria by Copper Surfaces Involves Dissolved Copper
Authors: Molteni Cristina , Abicht Helge K. , Solioz Marc
Published: April 2010

Bacteria are rapidly killed on copper surfaces. However, the mechanism of this process remains unclear. Using
Enterococcus hirae
, the effect of inactivation of copper homeostatic genes and of medium compositions on survival and copper dissolution was tested. The results support a role for dissolved copper ions in killing.
Structure, Function, and Insights into the Biosynthesis of a Head-to-Head Hydrocarbon in
Shewanella oneidensis
Strain MR-1
Authors: Sukovich David J. , Seffernick Jennifer L. , Richman Jack E. , Hunt Kristopher A. , Gralnick Jeffrey A. , Wackett Lawrence P.
Published: April 2010

A polyolefinic hydrocarbon was found in nonpolar extracts of
Shewanella oneidensis
MR-1 and identified as 3,6,9,12,15,19,22,25,28-hentriacontanonaene (compound I) by mass spectrometry, chemical modification, and nuclear magnetic resonance spectroscopy. Compound I was shown to be the product of a head-to-head fatty acid condensation biosynthetic pathway dependent on genes denoted as
ole
(for
ole
fin biosynthesis). Four
ole
genes were present in
S. oneidensis
MR-1. Deletion of the entire
oleABCD
gene cluster led to the complete absence of nonpolar extractable products. Deletion of the
oleC
gene alone generated a strain that lacked compound I but produced a structurally analogous ketone. Complementation of the
oleC
gene eliminated formation of the ketone and restored the biosynthesis of compound I. A recombinant
S. oneidensis
strain containing
oleA
from
Stenotrophomonas maltophilia
strain R551-3 produced at least 17 related long-chain compounds in addition to compound I, 13 of which were identified as ketones. A potential role for OleA in head-to-head condensation was proposed. It was further proposed that long-chain polyunsaturated compounds aid in adapting cells to a rapid drop in temperature, based on three observations. In
S. oneidensis
wild-type cells, the cellular concentration of polyunsaturated compounds increased significantly with decreasing growth temperature. Second, the
oleABCD
deletion strain showed a significantly longer lag phase than the wild-type strain when shifted to a lower temperature. Lastly, compound I has been identified in a significant number of bacteria isolated from cold environments.
Widespread Head-to-Head Hydrocarbon Biosynthesis in Bacteria and Role of OleA
Authors: Sukovich David J. , Seffernick Jennifer L. , Richman Jack E. , Gralnick Jeffrey A. , Wackett Lawrence P.
Published: April 2010

Previous studies identified theoleABCDgenes involved in head-to-head olefinic hydrocarbon biosynthesis. The present study more fully defined the OleABCD protein families within the thiolase, α/β-hydrolase, AMP-dependent ligase/synthase, and short-chain dehydrogenase superfamilies, respectively. Only 0.1 to 1% of each superfamily represents likely Ole proteins. Sequence analysis based on structural alignments and gene context was used to identify highly likelyolegenes. Selected microorganisms from the phylaVerucomicrobia,Planctomyces,Chloroflexi,Proteobacteria, andActinobacteriawere tested experimentally and shown to produce long-chain olefinic hydrocarbons. However, different species from the same genera sometimes lack theolegenes and fail to produce olefinic hydrocarbons. Overall, only 1.9% of 3,558 genomes analyzed showed clear evidence for containingolegenes. The type of olefins produced by different bacteria differed greatly with respect to the number of carbon-carbon double bonds. The greatest number of organisms surveyed biosynthesized a single long-chain olefin, 3,6,9,12,15,19,22,25,28-hentriacontanonaene, that contains nine double bonds.Xanthomonas campestrisproduced the greatest number of distinct olefin products, 15 compounds ranging in length from C28to C31and containing one to three double bonds. The type of long-chain product formed was shown to be dependent on theoleAgene in experiments withShewanella oneidensisMR-1olegene deletion mutants containing native or heterologousoleAgenes expressed intrans. A strain deleted inoleABCDand containingoleAintransproduced only ketones. Based on these observations, it was proposed that OleA catalyzes a nondecarboxylative thiolytic condensation of fatty acyl chains to generate a β-ketoacyl intermediate that can decarboxylate spontaneously to generate ketones.
Molecular Detection and Characterization of Aichi Viruses in Sewage-Polluted Waters of Venezuela
Authors: Alcalá Ana , Vizzi Esmeralda , Rodríguez-Díaz Jesús , Zambrano José L. , Betancourt Walter , Liprandi Ferdinando
Published: April 2010

The circulation of Aichi virus in a major urban area was demonstrated using molecular detection with samples recovered from a major river polluted with sewage discharges in Caracas, Venezuela. Five out of 11 water samples studied were positive, being classified by phylogenetic analysis as genotype B. Analysis of sewage waters appears to be a useful methodology to uncover the presence of a hitherto undetected fecal pathogen in a given geographical area.
The Aminolysis Reaction of
Streptomyces
S9 Aminopeptidase Promotes the Synthesis of Diverse Prolyl Dipeptides
Authors: Arima Jiro , Morimoto Masazumi , Usuki Hirokazu , Mori Nobuhiro , Hatanaka Tadashi
Published: April 2010

Prolyl dipeptide synthesis by S9 aminopeptidase from
Streptomyces thermocyaneoviolaceus
(S9AP-St) has been demonstrated. In the synthesis, S9AP-St preferentially used
l
-Pro-OBzl as the acyl donor, yielding synthesized dipeptides having an
l
-Pro-Xaa structure. In addition, S9AP-St showed broad specificity toward the acyl acceptor. Furthermore, S9AP-St produced cyclo (
l
-Pro-
l
-His) with a conversion ratio of substrate to cyclo (
l
-Pro-
l
-His) higher than 40%.
Enterocin X, a Novel Two-Peptide Bacteriocin from
Enterococcus faecium
KU-B5, Has an Antibacterial Spectrum Entirely Different from Those of Its Component Peptides
Authors: Hu Chih-Bo , Malaphan Wanna , Zendo Takeshi , Nakayama Jiro , Sonomoto Kenji
Published: April 2010

Enterocin X, composed of two antibacterial peptides (Xα and Xβ), is a novel class IIb bacteriocin from
Enterococcus faecium
KU-B5. When combined, Xα and Xβ display variably enhanced or reduced antibacterial activity toward a panel of indicators compared to each peptide individually. In
E. faecium
strains that produce enterocins A and B, such as KU-B5, only one additional bacteriocin had previously been known.
Comparison of Normalization Methods for Construction of Large, Multiplex Amplicon Pools for Next-Generation Sequencing
Authors: Harris J. Kirk , Sahl Jason W. , Castoe Todd A. , Wagner Brandie D. , Pollock David D. , Spear John R.
Published: April 2010

Constructing mixtures of tagged or bar-coded DNAs for sequencing is an important requirement for the efficient use of next-generation sequencers in applications where limited sequence data are required per sample. There are many applications in which next-generation sequencing can be used effectively to sequence large mixed samples; an example is the characterization of microbial communities where ≤1,000 sequences per samples are adequate to address research questions. Thus, it is possible to examine hundreds to thousands of samples per run on massively parallel next-generation sequencers. However, the cost savings for efficient utilization of sequence capacity is realized only if the production and management costs associated with construction of multiplex pools are also scalable. One critical step in multiplex pool construction is the normalization process, whereby equimolar amounts of each amplicon are mixed. Here we compare three approaches (spectroscopy, size-restricted spectroscopy, and quantitative binding) for normalization of large, multiplex amplicon pools for performance and efficiency. We found that the quantitative binding approach was superior and represents an efficient scalable process for construction of very large, multiplex pools with hundreds and perhaps thousands of individual amplicons included. We demonstrate the increased sequence diversity identified with higher throughput. Massively parallel sequencing can dramatically accelerate microbial ecology studies by allowing appropriate replication of sequence acquisition to account for temporal and spatial variations. Further, population studies to examine genetic variation, which require even lower levels of sequencing, should be possible where thousands of individual bar-coded amplicons are examined in parallel.
Formation and Attachment of the Deoxysugar Moiety and Assembly of the Gene Cluster for Caprazamycin Biosynthesis
Authors: Kaysser Leonard , Wemakor Emmanuel , Siebenberg Stefanie , Salas Jose A. , Sohng Jae Kyung , Kammerer Bernd , Gust Bertolt
Published: April 2010

Caprazamycins are antimycobacterials produced by
Streptomyces
sp. MK730-62F2. Previously, cosmid cpzLK09 was shown to direct the biosynthesis of caprazamycin aglycones, but not of intact caprazamycins. Sequence analysis of cpzLK09 identified 23 genes involved in the formation of the caprazamycin aglycones and the transfer and methylation of the sugar moiety, together with genes for resistance, transport, and regulation. In this study, coexpression of cpzLK09 in
Streptomyces coelicolor
M512 with pRHAM, containing all the required genes for dTDP-
l
-rhamnose biosynthesis, led to the production of intact caprazamycins.
In vitro
studies showed that Cpz31 is responsible for the attachment of the
l
-rhamnose to the caprazamycin aglycones, generating a rare acylated deoxyhexose. An
l
-rhamnose gene cluster was identified elsewhere on the
Streptomyces
sp. MK730-62F2 genome, and its involvement in caprazamycin formation was demonstrated by insertional inactivation of
cpzDIII
. The
l
-rhamnose subcluster was assembled with cpzLK09 using Red/ET-mediated recombination. Heterologous expression of the resulting cosmid, cpzEW07, led to the production of caprazamycins, demonstrating that both sets of genes are required for caprazamycin biosynthesis. Knockouts of
cpzDI
and
cpzDV
in the
l
-rhamnose subcluster confirmed that four genes,
cpzDII
,
cpzDIII
,
cpzDIV
, and
cpzDVI
, are sufficient for the biosynthesis of the deoxysugar moiety. The presented recombineering strategy may provide a useful tool for the assembly of biosynthetic building blocks for heterologous production of microbial compounds.
Genetic Detection of Extended-Spectrum β-Lactamase-Containing
Escherichia coli
Isolates from Birds of Prey from Serra da Estrela Natural Reserve in Portugal
Authors: Pinto Luís , Radhouani Hajer , Coelho Céline , Martins da Costa Paulo , Simões Roméo , Brandão Ricardo M. L. , Torres Carmen , Igrejas Gilberto , Poeta Patrícia
Published: April 2010

Extended-spectrum β-lactamase-containing
Escherichia coli
isolates were detected in 32 of 119 fecal samples (26.9%) from birds of prey at Serra da Estrela, and these isolates contained the following β-lactamases: CTX-M-1 (
n
= 13), CTX-M-1 plus TEM-1 (
n
= 14), CTX-M-1 plus TEM-20 (
n
= 1), SHV-5 (
n
= 1), SHV-5 plus TEM-1 (
n
= 2), and TEM-20 (
n
= 1).
Multiplex Identification of Microbes
Authors: Hyman Richard W. , St.Onge Robert P. , Allen Edward A. , Miranda Molly , Aparicio Ana Maria , Fukushima Marilyn , Davis Ronald W.
Published: April 2010

We have adapted molecular inversion probe technology to identify microbes in a highly multiplexed procedure. This procedure does not require growth of the microbes. Rather, the technology employs DNA homology twice: once for the molecular probe to hybridize to its homologous DNA and again for the 20-mer oligonucleotide barcode on the molecular probe to hybridize to a commercially available molecular barcode array. As proof of concept, we have designed, tested, and employed 192 molecular probes for 40 microbes. While these particular molecular probes are aimed at our interest in the microbes in the human vagina, this molecular probe method could be employed to identify the microbes in any ecological niche.
Characterization of a Thermostable Short-Chain Alcohol Dehydrogenase from the Hyperthermophilic Archaeon
Thermococcus sibiricus
Authors: Stekhanova Tatiana N. , Mardanov Andrey V. , Bezsudnova Ekaterina Y. , Gumerov Vadim M. , Ravin Nikolai V. , Skryabin Konstantin G. , Popov Vladimir O.
Published: April 2010

Short-chain alcohol dehydrogenase, encoded by the gene Tsib_0319 from the hyperthermophilic archaeon
Thermococcus sibiricus
, was expressed in
Escherichia coli
, purified and characterized as an NADPH-dependent enantioselective oxidoreductase with broad substrate specificity. The enzyme exhibits extremely high thermophilicity, thermostability, and tolerance to organic solvents and salts.
TaqMan Real-Time PCR Assays To Assess Arbuscular Mycorrhizal Responses to Field Manipulation of Grassland Biodiversity: Effects of Soil Characteristics, Plant Species Richness, and Functional Traits
Authors: König Stephan , Wubet Tesfaye , Dormann Carsten F. , Hempel Stefan , Renker Carsten , Buscot François
Published: April 2010

Large-scale (temporal and/or spatial) molecular investigations of the diversity and distribution of arbuscular mycorrhizal fungi (AMF) require considerable sampling efforts and high-throughput analysis. To facilitate such efforts, we have developed a TaqMan real-time PCR assay to detect and identify AMF in environmental samples. First, we screened the diversity in clone libraries, generated by nested PCR, of the nuclear ribosomal DNA internal transcribed spacer (ITS) of AMF in environmental samples. We then generated probes and forward primers based on the detected sequences, enabling AMF sequence type-specific detection in TaqMan multiplex real-time PCR assays. In comparisons to conventional clone library screening and Sanger sequencing, the TaqMan assay approach provided similar accuracy but higher sensitivity with cost and time savings. The TaqMan assays were applied to analyze the AMF community composition within plots of a large-scale plant biodiversity manipulation experiment, the Jena Experiment, primarily designed to investigate the interactive effects of plant biodiversity on element cycling and trophic interactions. The results show that environmental variables hierarchically shape AMF communities and that the sequence type spectrum is strongly affected by previous land use and disturbance, which appears to favor disturbance-tolerant members of the genus
Glomus
. The AMF species richness of disturbance-associated communities can be largely explained by richness of plant species and plant functional groups, while plant productivity and soil parameters appear to have only weak effects on the AMF community.
Diversity of 16S rRNA Genes within Individual Prokaryotic Genomes
Authors: Pei Anna Y. , Oberdorf William E. , Nossa Carlos W. , Agarwal Ankush , Chokshi Pooja , Gerz Erika A. , Jin Zhida , Lee Peng , Yang Liying , Poles Michael , Brown Stuart M. , Sotero Steven , DeSantis Todd , Brodie Eoin , Nelson Karen , Pei Zhiheng
Published: April 2010

Analysis of intragenomic variation of 16S rRNA genes is a unique approach to examining the concept of ribosomal constraints on rRNA genes; the degree of variation is an important parameter to consider for estimation of the diversity of a complex microbiome in the recently initiated Human Microbiome Project (
http://nihroadmap.nih.gov/hmp
). The current GenBank database has a collection of 883 prokaryotic genomes representing 568 unique species, of which 425 species contained 2 to 15 copies of 16S rRNA genes per genome (2.22 ± 0.81). Sequence diversity among the 16S rRNA genes in a genome was found in 235 species (from 0.06% to 20.38%; 0.55% ± 1.46%). Compared with the 16S rRNA-based threshold for operational definition of species (1 to 1.3% diversity), the diversity was borderline (between 1% and 1.3%) in 10 species and >1.3% in 14 species. The diversified 16S rRNA genes in
Haloarcula marismortui
(diversity, 5.63%) and
Thermoanaerobacter tengcongensis
(6.70%) were highly conserved at the 2° structure level, while the diversified gene in
B. afzelii
(20.38%) appears to be a pseudogene. The diversified genes in the remaining 21 species were also conserved, except for a truncated 16S rRNA gene in “
Candidatus
Protochlamydia amoebophila.” Thus, this survey of intragenomic diversity of 16S rRNA genes provides strong evidence supporting the theory of ribosomal constraint. Taxonomic classification using the 16S rRNA-based operational threshold could misclassify a number of species into more than one species, leading to an overestimation of the diversity of a complex microbiome. This phenomenon is especially seen in 7 bacterial species associated with the human microbiome or diseases.
Caenorhabditis elegans
as an Alternative Model Host for
Legionella pneumophila
, and Protective Effects of
Bifidobacterium infantis
Authors: Komura Tomomi , Yasui Chikako , Miyamoto Hiroshi , Nishikawa Yoshikazu
Published: April 2010

The survival times of
Caenorhabditis elegans
worms infected with
Legionella pneumophila
from day 7.5 or later after hatching were shorter than those of uninfected worms. However, nematodes fed bifidobacteria prior to
Legionella
infection were resistant to
Legionella
. These nematodes may act as a unique alternative host for
Legionella
research.
Species-Specific Type II Restriction-Modification System ofXylella fastidiosaTemecula1
Authors: Matsumoto Ayumi , Igo Michele M.
Published: April 2010

The transformation efficiency ofXylella fastidiosacan be increased by interfering with restriction by the strain-specific type II system encoded by the PD1607 and PD1608 genes. Here, we report results for two strategies:in vitromethylation using M.SssI and isolation of DNA from anEscherichia colistrain expressing the methylase PD1607.
TDP-
l
-Megosamine Biosynthesis Pathway Elucidation and Megalomicin A Production in
Escherichia coli
Authors: Useglio Mariana , Peirú Salvador , Rodríguez Eduardo , Labadie Guillermo R. , Carney John R. , Gramajo Hugo
Published: April 2010

In vivo
reconstitution of the TDP-
l
-megosamine pathway from the megalomicin gene cluster of
Micromonospora megalomicea
was accomplished by the heterologous expression of its biosynthetic genes in
Escherichia coli.
Mass spectrometric analysis of the TDP-sugar intermediates produced from operons containing different sets of genes showed that the production of TDP-
l
-megosamine from TDP-4-keto-6-deoxy-
d
-glucose requires only five biosynthetic steps, catalyzed by MegBVI, MegDII, MegDIII, MegDIV, and MegDV. Bioconversion studies demonstrated that the sugar transferase MegDI, along with the helper protein MegDVI, catalyzes the transfer of
l
-megosamine to either erythromycin C or erythromycin D, suggesting two possible routes for the production of megalomicin A. Analysis
in vivo
of the hydroxylation step by MegK indicated that erythromycin C is the intermediate of megalomicin A biosynthesis.
Low Pore Connectivity Increases Bacterial Diversity in Soil
Authors: Carson Jennifer K. , Gonzalez-Quiñones Vanesa , Murphy Daniel V. , Hinz Christoph , Shaw Jeremy A. , Gleeson Deirdre B.
Published: April 2010

One of soil microbiology's most intriguing puzzles is how so many different bacterial species can coexist in small volumes of soil when competition theory predicts that less competitive species should decline and eventually disappear. We provide evidence supporting the theory that low pore connectivity caused by low water potential (and therefore low water content) increases the diversity of a complex bacterial community in soil. We altered the pore connectivity of a soil by decreasing water potential and increasing the content of silt- and clay-sized particles. Two textures were created, without altering the chemical properties or mineral composition of the soil, by adding silt- and clay-sized particles of quartz to a quartz-based sandy soil at rates of 0% (sand) or 10% (silt+clay). Both textures were incubated at several water potentials, and the effect on the active bacterial communities was measured using terminal restriction fragment length polymorphism (TRFLP) of bacterial 16S rRNA. Bacterial richness and diversity increased as water potential decreased and soil became drier (
P
< 0.012), but they were not affected by texture (
P
> 0.553). Bacterial diversity increased at water potentials of ≤2.5 kPa in sand and ≤4.0 kPa in silt+clay, equivalent to ≤56% water-filled pore space (WFPS) in both textures. The bacterial community structure in soil was affected by both water potential and texture (
P
< 0.001) and was correlated with WFPS (sum of squared correlations [δ
2
] = 0.88,
P
< 0.001). These findings suggest that low pore connectivity is commonly experienced by soil bacteria under field conditions and that the theory of pore connectivity may provide a fundamental principle to explain the high diversity of bacteria in soil.
TbPRMT6 Is a Type I Protein Arginine Methyltransferase That Contributes to Cytokinesis in Trypanosoma brucei
Authors: Fisk John C. , Zurita-Lopez Cecilia , Sayegh Joyce , Tomasello Danielle L. , Clarke Steven G. , Read Laurie K.
Published: April 2010

Arginine methylation is a widespread posttranslational modification of proteins catalyzed by a family of protein arginine methyltransferases (PRMTs). In
Saccharomyces cerevisiae
and mammals, this modification affects multiple cellular processes, such as chromatin remodeling leading to transcriptional regulation, RNA processing, DNA repair, and cell signaling. The protozoan parasite
Trypanosoma brucei
possesses five putative PRMTs in its genome. This is a large number of PRMTs relative to other unicellular eukaryotes, suggesting an important role for arginine methylation in trypanosomes. Here, we present the
in vitro
and
in vivo
characterization of a
T. brucei
enzyme homologous to human PRMT6, which we term TbPRMT6. Like human PRMT6, TbPRMT6 is a type I PRMT, catalyzing the production of monomethylarginine and asymmetric dimethylarginine residues. In
in vitro
methylation assays, TbPRMT6 utilizes bovine histones as a substrate, but it does not methylate several
T. brucei
glycine/arginine-rich proteins. As such, it exhibits a relatively narrow substrate specificity compared to other
T. brucei
PRMTs. Knockdown of TbPRMT6 in both procyclic form and bloodstream form
T. brucei
leads to a modest but reproducible effect on parasite growth in culture. Moreover, upon TbPRMT6 depletion, both PF and BF exhibit aberrant morphologies indicating defects in cell division, and these defects differ in the two life cycle stages. Mass spectrometry of TbPRMT6-associated proteins reveals histones, components of the nuclear pore complex, and flagellar proteins that may represent TbPRMT6 substrates contributing to the observed growth and morphological defects.
Transcriptional Response to Hypoxia in the Aquatic Fungus Blastocladiella emersonii
Authors: Camilo César M. , Gomes Suely L.
Published: April 2010

Global gene expression analysis was carried out with
Blastocladiella emersonii
cells subjected to oxygen deprivation (hypoxia) using cDNA microarrays. In experiments of gradual hypoxia (gradual decrease in dissolved oxygen) and direct hypoxia (direct decrease in dissolved oxygen), about 650 differentially expressed genes were observed. A total of 534 genes were affected directly or indirectly by oxygen availability, as they showed recovery to normal expression levels or a tendency to recover when cells were reoxygenated. In addition to modulating many genes with no putative assigned function,
B. emersonii
cells respond to hypoxia by readjusting the expression levels of genes responsible for energy production and consumption. At least transcriptionally, this fungus seems to favor anaerobic metabolism through the upregulation of genes encoding glycolytic enzymes and lactate dehydrogenase and the downregulation of most genes coding for tricarboxylic acid (TCA) cycle enzymes. Furthermore, genes involved in energy-costly processes, like protein synthesis, amino acid biosynthesis, protein folding, and transport, had their expression profiles predominantly downregulated during oxygen deprivation, indicating an energy-saving effort. Data also revealed similarities between the transcriptional profiles of cells under hypoxia and under iron(II) deprivation, suggesting that Fe
2+
ion could have a role in oxygen sensing and/or response to hypoxia in
B. emersonii
. Additionally, treatment of fungal cells prior to hypoxia with the antibiotic geldanamycin, which negatively affects the stability of mammalian hypoxia transcription factor HIF-1α, caused a significant decrease in the levels of certain upregulated hypoxic genes.