bluuebunny/crossref_metadata_2025 huggingface dataset:
Rck2 Is Required for Reprogramming of Ribosomes during Oxidative Stress
Authors: Swaminathan Swarna , Masek Tomas , Molin Claes , Pospisek Martin , Sunnerhagen Per
Published: December 2005

Rck2 is a mitogen-activated protein kinase-activated protein kinase in yeast implicated in translational regulation. rck2Δ mutants are mildly sensitive to oxidative stress, a condition that causes dissociation of actively translating ribosomes (polysomes). In rck2Δ cells, polysomes are lost to an even higher degree than in the wild-type upon stress. Cells overexpressing the catalytically inactive rck2-kd allele are highly sensitive to oxidative stress. In such cells, dissociation of polysomes upon stress was instead greatly delayed. The protein synthesis rate decreased to a similar degree as in wild-type cells, however, indicating that in rck2-kd cells, the polysome complexes were inactive. Array analyses of total and polysome-associated mRNAs revealed major deregulation of the translational machinery in rck2 mutant cells. This involves transcripts for cytosolic ribosomal proteins and for processing and assembly of ribosomes. In rck2Δ cells, weakly transcribed mRNAs associate more avidly with polysomes than in wild-type cells, whereas the opposite holds true for rck2-kd cells. This is consistent with perturbed regulation of translation elongation, which is predicted to alter the ratio between mRNAs with and without strong entry sites at ribosomes. We infer that imbalances in the translational apparatus are a major reason for the inability of these cells to respond to stress.
Establishment of Polarity during Organization of the Acentrosomal Plant Cortical Microtubule Array
Authors: Dixit Ram , Chang Eric , Cyr Richard
Published: December 2005

The plant cortical microtubule array is a unique acentrosomal array that is essential for plant morphogenesis. To understand how this array is organized, we exploited the microtubule (+)-end tracking activity of two Arabidopsis EB1 proteins in combination with FRAP (fluorescence recovery after photobleaching) experiments of GFP-tubulin to examine the relationship between cortical microtubule array organization and polarity. Significantly, our observations show that the majority of cortical microtubules in ordered arrays, within a particular cell, face the same direction in both Arabidopsis plants and cultured tobacco cells. We determined that this polar microtubule coalignment is at least partially due to a selective stabilization of microtubules, and not due to a change in microtubule polymerization rates. Finally, we show that polar microtubule coalignment occurs in conjunction with parallel grouping of cortical microtubules and that cortical array polarity is progressively enhanced during array organization. These observations reveal a novel aspect of plant cortical microtubule array organization and suggest that selective stabilization of dynamic cortical microtubules plays a predominant role in the self-organization of cortical arrays.
ENDING RESTRICTIONS TO MIGRATION FROM THE NEW EU MEMBER COUNTRIES: SECTORAL TRADE AND REAL WAGE EFFECTS
Authors: MARQUES HELENA , METCALF HUGH
Published: December 2005

Most EU‐15 countries have kept restrictions to migration from the new member countries but committed to removing them within seven years from the 2004 enlargement. This article predicts the sectoral trade and real wage impact on high‐income, mid‐income, and low‐income members of removing those restrictions, given two extreme scenarios: either all migrants are skilled or all are unskilled. The main effect of skilled migration is the relocation of high‐scale economy, skill‐intensive industries from mid‐income into high‐income countries. The main effect of unskilled migration is the relocation of low‐scale economy, low skill‐intensive industries from low‐income into mid‐income countries. Both high‐income and low‐income members would be better off with skilled migration, but those with mid‐income would benefit from unskilled migration. (JEL F1, F15, F22, J31, L6)
Occurrence and recent long-distance dispersal of deep-sea hydrothermal vent shrimps
Authors: Tokuda Gaku , Yamada Akinori , Nakano Kazuma , Arita Nao , Yamasaki Hideo
Published: December 2005

Deep-sea hydrothermal vents and methane seeps are extreme environments that have a high concentration of hydrogen sulphide. However, abundant unique invertebrates including shrimps of the family Bresiliidae have been found in such environments. The bresiliid shrimps are believed to have radiated in the Miocene (less than 20 Myr); however, the period when and the mechanisms by which they dispersed across the hydrothermal vents and cold seeps in oceans worldwide have not been clarified. In the present study, we collected the deep-sea blind shrimp
Alvinocaris longirostris
from the hydrothermal vent site in the Okinawa Trough and carried out the first investigation of the 18S rRNA gene of a bresiliid shrimp. The phylogenetic analysis revealed that the bresiliid shrimp is situated at an intermediate lineage within the infraorder Caridea and shows monophyly with palaemonid shrimps, which live in shallow sea and freshwater. Furthermore, the mitochondrial cytochrome oxidase I (
COI
) gene sequences were analysed to determine the phylogenetic relationship with known bresiliid shrimps.
A. longirostris
of the Okinawa Trough had two haplotypes of the
COI
gene, one of which was identical to the
Alvinocaris
sp. of the cold seeps in Sagami Bay. These results indicate that a long-distance dispersal of
A. longirostris
occurred possibly within the last 100 000 years.
On the linear and nonlinear acoustic properties of dry and water-confining elasto-plastic interfaces
Authors: Pecorari Claudio , Poznic Milan
Published: December 2005

An experimental investigation into the linear reflection and generation of the second harmonic component following the incidence of an ultrasonic wave onto a dry or water-confining interface between elasto-plastic steel–steel surfaces in contact is presented. The results on dry interfaces show that, although the theoretical models currently used to estimate the stiffness of such interfaces constitute a valid framework, the statistics of asperities in contact requires further development to account for the effects of the elasto-plastic deformation. Similarly, the results with water-confining interfaces indicate that current models not accounting for the liquid-mediated forces between the solid surfaces cannot explain the results presented here. In fact, the experimental evidence is interpreted as suggesting that structural repulsive forces may be responsible for the observed phenomena. Finally, the level of second harmonic generation for these interfaces offers support by the development of inspection techniques which exploit such wave phenomena for the detection of partially closed and dry, or nearly open and water-trapping stress-corrosion surface-breaking cracks.
Haemophilus parasuis invades porcine brain microvascular endothelial cells
Authors: Vanier Ghyslaine , Szczotka Anna , Friedl Peter , Lacouture Sonia , Jacques Mario , Gottschalk Marcelo
Published: December 2005

Haemophilus parasuis, an important swine pathogen, is the aetiological agent of Glässer's disease. It is responsible for cases of polyserositis, meningitis and pneumonia in young pigs. To date, 15 serotypes have been described, although several non-typable isolates are frequently recovered from diseased animals. The pathogenesis ofH. parasuisinfection is poorly understood. To cause meningitis,H. parasuiswould have to cross the blood–brain barrier (BBB), composed of brain microvascular endothelial cells (BMEC). The objective of this study was to investigate the ability ofH. parasuisto interact with porcine brain microvascular endothelial cells (PBMEC). It was demonstrated that the serotype 5 reference strain ofH. parasuis, Nagasaki (originally recovered from a case of meningitis), was able to adhere at very high levels to and, most importantly, invade PBMEC. These capacities were confirmed by electron microscopy.Actinobacillus pleuropnemoniaeserotype 7 (strain WF 83), used as negative control, was not able to adhere to or invade PBMEC. Comparisons of the levels of adhesion and invasion by severalH. parasuisfield strains from different serotypes isolated from cases of either meningitis or pneumonia showed that isolates of serotypes 4 and 5 had a higher invasion capacity than isolates belonging to other serotypes. Inhibition studies demonstrated that PBMEC invasion byH. parasuisrequired rearrangement of actin microfilaments and microtubular cytoskeletal elements but not active bacterial DNA, RNA or protein synthesis. Characterization studies demonstrated that proteinaceous invasin(s) does not seem to play a major role in entry ofH. parasuisinto PBMEC. Intracellular viableH. parasuiswere found in PBMEC up to 6 h after antibiotic treatment. Even at high bacterial doses,H. parasuiswas not toxic to PBMEC. In swine, the invasion of endothelial cells of the BBB may play an important role in the pathogenesis of meningitis caused byH. parasuis.
Low tyrosine content of growth media yields aflagellate Salmonella enterica serovar Typhimurium
Authors: Gray Victoria L. , O'Reilly Michael , Müller Carsten T. , Watkins Ian D. , Lloyd David
Published: December 2005

Identification of Salmonella serotypes is based on flagellar and somatic antigens. The absence of flagella may consequently affect complete identification of the serotype; here it is shown that Salmonella enterica serovar Typhimurium exhibits morphological differences dependent on the peptone constituents of the culture medium. Aflagellate salmonella were produced in certain media where the nutritional ingredient was casein-based peptone or gelatin-based peptone; in gelatin-based peptone, aggregates of salmonella were observed. However, in media containing soy-based peptone as the primary nutrient, salmonella displayed a normal flagellated morphology. Transfer of aflagellate salmonella from nutritionally poor media, with casein- or gelatin-based peptone, into rich nutrient broth allowed flagella synthesis, indicating that the aflagellate form is still able to produce flagella. Amino acid sequencing of the peptones producing aflagellate organisms showed a relatively low tyrosine concentration: only 0·03±0·01 g l−1 for gelatin-based buffered peptone water, compared to 0·21±0·01 for soy-based buffered peptone water. Tyrosine is essential for flagellin, which is the subunit of the salmonella flagellar filament. The addition of 200 μM tyrosine to casein-based peptone media produced flagellate salmonella; 2 mM glucose was needed in addition to tyrosine to achieve a similar morphology in gelatin-based media. Therefore, culture media containing less than 1·20 g tyrosine l−1, and of limited carbohydrate source, when used for serological testing of clinical isolates, may result in an incomplete serological identification.
Self-Folding and Unfolding of Carbon Nanotubes
Authors: Buehler Markus J. , Kong Yong , Gao Huajian , Huang Yonggang
Published: December 2005

Carbon nanotubes (CNTs) constitute a prominent example of nanomaterials. In most studies on mechanical properties, the effort was concentrated on CNTs with relatively small aspect ratio of length to diameters. In contrast, CNTs with aspect ratios of several hundred can be produced with today’s experimental techniques. We report atomistic-continuum studies of single-wall carbon nanotubes with very large aspect ratios subject to compressive loading. It was recently shown that these long tubes display significantly different mechanical behavior than tubes with smaller aspect ratios (Buehler, M. J., Kong, Y., and Guo, H., 2004, ASME J. Eng. Mater. Technol. 126, pp. 245–249). We distinguish three different classes of mechanical response to compressive loading. While the deformation mechanism is characterized by buckling of thin shells in nanotubes with small aspect ratios, it is replaced by a rodlike buckling mode above a critical aspect ratio, analogous to the Euler theory in continuum mechanics. For very large aspect ratios, a nanotube is found to behave like a wire that can be deformed in a very flexible manner to various shapes. In this paper, we focus on the properties of such wirelike CNTs. Using atomistic simulations carried out over a several-nanoseconds time span, we observe that wirelike CNTs behave similarly to flexible macromolecules. Our modeling reveals that they can form thermodynamically stable self-folded structures, where different parts of the CNTs attract each other through weak van der Waals (vdW) forces. This self-folded CNT represents a novel structure not described in the literature. There exists a critical length for self-folding of CNTs that depends on the elastic properties of the tube. We observe that CNTs fold below a critical temperature and unfold above another critical temperature. Surprisingly, we observe that self-folded CNTs with very large aspect ratios never unfold until they evaporate. The folding-unfolding transition can be explained by entropic driving forces that dominate over the elastic energy at elevated temperature. These mechanisms are reminiscent of the dynamics of biomolecules, such as proteins. The different stable states of CNTs are finally summarized in a schematic phase diagram of CNTs.
A Constitutive Model for Strain-Induced Crystallization in Poly(ethylene terephthalate) (PET) during Finite Strain Load-Hold Simulations
Authors: Dupaix Rebecca B. , Krishnan Dwarak
Published: December 2005

Recently, a hyperelastic-viscoplastic constitutive model was developed for PET and the noncrystallizing copolymer PETG (R. B. Dupaix, Ph.D. thesis, MIT, 2003). The materials were found to behave very similarly under monotonic loading conditions and the single constitutive model was able to capture both materials’ behavior. However, differences were observed upon unloading, and it is expected that additional differences would be observed under more complex loading conditions. Here their behavior is investigated under nonmonotonic loading conditions, specifically under load-hold conditions. The model of Dupaix and Boyce (R. B. Dupaix, Ph.D. thesis, MIT, 2003) is modified to include Ahzi’s upper-bound model for strain-induced crystallization [Ahzi et al., Mech. Mater., 35(12), pp. 1139–1148 (2003)]. The crystallization model is adapted to include criteria for the onset of strain-induced crystallization which depend on strain rate and level of deformation. The strain-rate condition prevents crystallization from beginning prior to the deformation process slowing significantly. The level-of-deformation condition delays crystallization until the material has deformed beyond a critical level. The combined model demonstrates differences in behavior between PET and PETG during complex loading situations, indicating its ability to capture the fundamental criteria for the onset of strain-induced crystallization.
Cohesive Layer Modeling of Time-Dependent Debond Growth in Aggressive Environments
Authors: Roy Samit , Wang Yong , Park Soojae , Liechti Kenneth M.
Published: December 2005

The objective of this paper is to model the synergistic bond-degradation mechanisms that may occur at the interface between a fiber-reinforced polymer (FRP) that is adhesively bonded to a substrate and subjected to elevated temperature and humidity. For this purpose, a two-dimensional cohesive-layer constitutive model with a prescribed traction-separation law is constructed from fundamental principles of continuum mechanics and thermodynamics, taking into account strain-dependent, non-Fickian hygrothermal effects as well as diffusion-induced degradation in the cohesive layer. In the interest of solution tractability, a simplified approach is employed where the rate-dependent behavior in the cohesive layer is implemented through the characterization of rate dependence of the maximum stresses and maximum strains in the cohesive layer, rather than through the use of convolution integrals in the free-energy definition. The remainder of the polymeric adhesive outside the cohesive layer is modeled as a nonlinear viscoelastic continuum with time-dependent constitutive behavior. The influence of temperature and moisture concentration on the work-of-separation and on crack growth is derived from first principles. The model is implemented in a test-bed finite element code. Results predicted by the computational model are benchmarked through comparison to experimental data from mixed-mode fracture experiments performed using a moving wedge test.
Damage-Induced Modeling of Elastic-Viscoelastic Randomly Oriented Particulate Composites
Authors: Kim Yong-Rak , Allen David H. , Seidel Gary D.
Published: December 2005

This paper presents a model for predicting the damage-induced mechanical response of particle-reinforced composites. The modeling includes the effects of matrix viscoelasticity and fracture, both within the matrix and along the boundaries between matrix and rigid particles. Because of these inhomogeneities, the analysis is performed using the finite element method. Interface fracture is predicted by using a nonlinear viscoelastic cohesive zone model. Rate-dependent viscoelastic behavior of the matrix material and cohesive zone is incorporated by utilizing a numerical time-incrementalized algorithm. The proposed modeling approach can be successfully employed for numerous types of solid media that exhibit matrix viscoelasticity and complex damage evolution characteristics within the matrix as well as along the matrix-particle boundaries. Computational results are given for various asphalt concrete mixtures. Simulation results demonstrate that each model parameter and design variable significantly influences the mechanical behavior of the mixture.
Fiber Composite Strength Modeling With Extension to Life Prediction
Authors: Wu Edward M. , Kardos John L.
Published: December 2005

This paper focuses on the probability modeling of fiber composite strength, wherein the failure modes are dominated by fiber tensile failures. The probability model is the tri-modal local load-sharing model, which is the Phoenix-Harlow local load-sharing model with the filament failure model extended from one mode to three modes. This model results in increased efficiency in the determination of fiber statistical parameters and in lower cost when applied to (i) quality control in materials (fiber) manufacturing, (ii) materials (fiber) selection and comparison, (iii) accounting for the effect of size scaling in design, and (iv) qualification and certification of critical composite structures that are too large and expensive to test statistically. In addition, possible extensions to proof testing and time-dependent life prediction are discussed and preliminary data are presented.
Computational Micromechanics for High-Temperature Constitutive Equations of Polymer-Matrix Composites With Oxidation Reaction, Damage, and Degradation
Authors: Wang Su Su , Chen Xiaohong
Published: December 2005

The proper determination of high-temperature constitutive properties and damage of polymer-matrix composites (PMC) in an aggressive environment is critical in high-speed aircraft and propulsion material development, structural integrity, and long-term life prediction. In this paper, a computational micromechanics study is conducted to obtain high-temperature constitutive properties of the PMC undergoing simultaneous thermal oxidation reaction, microstructural damage, and thermomechanical loading. The computational micromechanics approach follows the recently developed irreversible thermodynamic theory for polymer composites with reaction and microstructural change under combined chemical, thermal, and mechanical loading. Proper microstructural modeling of the PMC is presented to ensure that reaction activities, thermal and mechanical responses of the matrix, fibers, and fiber-matrix interface are fully addressed. A multiscale homogenization theory is used in conjunction with a finite element representation of material and reaction details to determine continuous evolution of composite microstructure change and associated degradation of the mechanical and physical properties. Numerical examples are given on a commonly used G30-500/PMR15 composite for illustration.
Structure, Regulation, and Putative Function of the Arginine Deiminase System ofStreptococcus suis
Authors: Gruening Petra , Fulde Marcus , Valentin-Weigand Peter , Goethe Ralph
Published: December 2005

Streptococcus suisis an important cause of infectious diseases in young pigs. Little is known about the virulence factors or protective antigens ofS. suis. Recently, we have identified two proteins of the arginine deiminase system (ADS) ofS. suis, which were temperature induced and expressed on the streptococcal surface (N. Winterhoff, R. Goethe, P. Gruening, M. Rohde, H. Kalisz, H. E. Smith, and P. Valentin-Weigand, J. Bacteriol. 184:6768-6776, 2002). In the present study, we analyzed the complete ADS ofS. suis. Due to their homologies to the recently publishedS. gordoniiADS genes, the genes for arginine deiminase, ornithine carbamoyl-transferase, and carbamate kinase, which were previously designatedadiS,octS, andckS, respectively, were renamedarcA,arcB, andarcC, respectively. Our data revealed thatarcA,arcB, andarcCof theS. suisADS are transcribed from an operon (arcABCoperon). Additionally, putative ADS-associated genes were cloned and sequenced which, however, did not belong to thearcABCoperon. These were theflpSgene upstream of thearcABCoperon with homology to theflptranscription regulator ofS. gordoniiand thearcD,arcT,arcH, andargRgenes downstream of thearcABCoperon with high homologies to a putative arginine-ornithine antiporter, a putative dipeptidase ofS. gordonii, a putative β-N-acetylhexosaminidase ofS. pneumoniae, and a putative arginine repressor ofS. gordonii, respectively. The transcriptional start point of thearcABCoperon was determined, and promoter analysis provided evidence that multiple factors contribute to the regulation of the ADS. Thus, a putative binding site for a transcription regulator of the Crp/Fnr family, an ArgR-binding site, and twocis-acting catabolite response elements were identified in the promoter-operator region of the operon. Consistent with this, we could demonstrate that the ADS ofS. suisis inducible by arginine and reduced O2tension and subject to carbon catabolite repression. Furthermore, comparing anarcAknockout mutant in which expression of the three operon-encoded proteins was abolished with the parental wild-type strain showed that thearcABCoperon ofS. suiscontributes to survival under acidic conditions.
Nanoscale Characterization and Determination of Adhesion Forces of
Pseudomonas aeruginosa
Pili by Using Atomic Force Microscopy
Authors: Touhami Ahmed , Jericho Manfred H. , Boyd Jessica M. , Beveridge Terry J.
Published: December 2005

Type IV pili play an important role in bacterial adhesion, motility, and biofilm formation. Here we present high-resolution atomic force microscopy (AFM) images of type IV pili from
Pseudomonas aeruginosa
bacteria. An individual pilus ranges in length from 0.5 to 7 μm and has a diameter from 4 to 6 nm, although often, pili bundles in which the individual filaments differed in both length and diameter were seen. By attaching bacteria to AFM tips, it was possible to fasten the bacteria to mica surfaces by pili tethers. Force spectra of tethered pili gave rupture forces of 95 pN. The slopes of force curves close to the rupture force were nearly linear but showed little variation with pilus length. Furthermore, force curves could not be fitted with wormlike-chain polymer stretch models when using realistic persistence lengths for pili. The observation that the slopes near rupture did not depend on the pili length suggests that they do not represent elastic properties of the pili. It is possible that this region of the force curves is determined by an elastic element that is part of the bacterial wall, although further experiments are needed to confirm this.
Regulation of SpeB inStreptococcus pyogenesby pH and NaCl: a Model for In Vivo Gene Expression
Authors: Loughman Jennifer A. , Caparon Michael
Published: December 2005

For a pathogen such asStreptococcus pyogenes, ecological success is determined by its ability to sense the environment and mount an appropriate adaptive transcriptional response. Thus, determining conditions for analyses of gene expression in vitro that are representative of the in vivo environment is critical for understanding the contributions of transcriptional response pathways to pathogenesis. In this study, we determined that the gene encoding the SpeB cysteine protease is up-regulated over the course of infection in a murine soft-tissue model. Conditions were identified, including growth phase, acidic pH, and an NaCl concentration of <0.1 M, that were required for expression ofspeBin vitro. Analysis of global expression profiles in response to these conditions in vitro identified a set of coregulated genes whose expression patterns showed a significant correlation with that ofspeBwhen examined during infection of murine soft tissues. This analysis revealed that a culture medium that promotes high levels of SpeB expression in vitro produced an expression profile that showed significant correlation to the profile observed in vivo. Taken together, these studies establish culture conditions that mimic in vivo expression patterns; that growth phase, pH, and NaCl may mimic relevant cues sensed byS. pyogenesduring infection; and that identification of other environmental cues that alter expression ofspeBin vitro may provide insight into the signals that direct global patterns of gene expression in vivo.
Characterization and Use of Catabolite-Repressed Promoters from Gluconate Genes in
Corynebacterium glutamicum
Authors: Letek Michal , Valbuena Noelia , Ramos Angelina , Ordóñez Efrén , Gil José A. , Mateos Luís M.
Published: December 2005

The genes involved in gluconate catabolism (
gntP
and
gntK
) in
Corynebacterium glutamicum
are scattered in the chromosome, and no regulatory genes are apparently associated with them, in contrast with the organization of the
gnt
operon in
Escherichia coli
and
Bacillus subtilis
. In
C. glutamicum
,
gntP
and
gntK
are essential genes when gluconate is the only carbon and energy source. Both genes contain upstream regulatory regions consisting of a typical promoter and a hypothetical cyclic AMP (cAMP) receptor protein (CRP) binding region but lack the expected consensus operator region for binding of the GntR repressor protein. Expression analysis by Northern blotting showed monocistronic transcripts for both genes. The expression of
gntP
and
gntK
is not induced by gluconate, and the
gnt
genes are subject to catabolite repression by sugars, such as glucose, fructose, and sucrose, as was detected by quantitative reverse transcription-PCR (qRT-PCR). Specific analysis of the DNA promoter sequences (P
gntK
and P
gntP
) was performed using bifunctional promoter probe vectors containing
mel
(involved in melanin production) or e
gfp2
(encoding a green fluorescent protein derivative) as the reporter gene. Using this approach, we obtained results parallel to those from qRT-PCR. An applied example of in vivo gene expression modulation of the
divIVA
gene in
C. glutamicum
is shown, corroborating the possible use of the
gnt
promoters to control gene expression.
glxR
(which encodes GlxR, the hypothetical CRP protein) was subcloned from the
C. glutamicum
chromosomal DNA and overexpressed in corynebacteria; we found that the level of
gnt
expression was slightly decreased compared to that of the control strains. The purified GlxR protein was used in gel shift mobility assays, and a specific interaction of GlxR with sequences present on P
gntP
and P
gntK
fragments was detected only in the presence of cAMP.
Genetic Characterization of a Single Bifunctional Enzyme for Fumarate Reduction and Succinate Oxidation in
Geobacter sulfurreducens
and Engineering of Fumarate Reduction in
Geobacter metallireducens
Authors: Butler Jessica E. , Glaven Richard H. , Esteve-Núñez Abraham , Núñez Cinthia , Shelobolina Evgenya S. , Bond Daniel R. , Lovley Derek R.
Published: December 2005

The mechanism of fumarate reduction in
Geobacter sulfurreducens
was investigated. The genome contained genes encoding a heterotrimeric fumarate reductase, FrdCAB, with homology to the fumarate reductase of
Wolinella succinogenes
and the succinate dehydrogenase of
Bacillus subtilis
. Mutation of the putative catalytic subunit of the enzyme resulted in a strain that lacked fumarate reductase activity and was unable to grow with fumarate as the terminal electron acceptor. The mutant strain also lacked succinate dehydrogenase activity and did not grow with acetate as the electron donor and Fe(III) as the electron acceptor. The mutant strain could grow with acetate as the electron donor and Fe(III) as the electron acceptor if fumarate was provided to alleviate the need for succinate dehydrogenase activity in the tricarboxylic acid cycle. The growth rate of the mutant strain under these conditions was faster and the cell yields were higher than for wild type grown under conditions requiring succinate dehydrogenase activity, suggesting that the succinate dehydrogenase reaction consumes energy. An orthologous
frdCAB
operon was present in
Geobacter metallireducens
, which cannot grow with fumarate as the terminal electron acceptor. When a putative dicarboxylic acid transporter from
G. sulfurreducens
was expressed in
G. metallireducens
, growth with fumarate as the sole electron acceptor was possible. These results demonstrate that, unlike previously described organisms,
G. sulfurreducens
and possibly
G. metallireducens
use the same enzyme for both fumarate reduction and succinate oxidation in vivo.
The Scc Spirochetal Coiled-Coil Protein Forms Helix-Like Filaments and Binds to Nucleic Acids Generating Nucleoprotein Structures
Authors: Mazouni Khalil , Pehau-Arnaudet Gérard , England Patrick , Bourhy Pascale , Saint Girons Isabelle , Picardeau Mathieu
Published: December 2005

The analysis of the genome of
Leptospira
spp., a group of bacteria of the phylum of spirochetes with several unique evolutionary and morphological features, has allowed the identification of a gene encoding a coiled-coil protein, called Scc, which is completely unrelated to any other eukaryotic or prokaryotic protein. Since coiled-coil proteins are often key elements of the cytoskeleton, we analyzed the protein Scc, which is a 24-kDa protein composed of a N-terminal coiled-coil domain, a proline-rich intermediate domain, and an acidic tail. The gene
scc
is located in an operon which also contains the genes encoding the initiation factor IF3 and the two ribosomal proteins L20 and L35. In this study, we showed that the presence of the coiled-coil domain was responsible for the polymerization of Scc in helix-like structures, in an ATP-independent manner, in both
Escherichia coli
living cells and in vitro. Analysis of the Scc polymers by electron microscopy showed filaments with a width of 6 to 10 nm, similar to that of eukaryotic intermediate filaments. Scc was also found to bind both RNA and double-stranded DNA without detectable sequence specificity. By electron microscopy, we showed that Scc polymer assembly was affected by the presence of nucleic acids, giving rise to rod-shaped structures with a width ranging from 45 to 155 nm. Finally,
Leptospira biflexa
cells depleted in Scc form small colonies, but the morphology of their helicoidal cell body was not affected. These results provide the first insight into a unique DNA binding filament-forming coiled-coil protein that could play an important role in the subcellular architecture of the spirochetal microorganism.
Characterization of the Opposing Roles of H-NS and TraJ in Transcriptional Regulation of the F-Plasmid
tra
Operon
Authors: Will William R. , Frost Laura S.
Published: December 2005

The transfer (
tra
) operon of the conjugative F plasmid of
Escherichia coli
is a polycistronic 33-kb operon which encodes most of the proteins necessary for F-plasmid transfer. Here, we report that transcription from P
Y
, the
tra
operon promoter, is repressed by the host nucleoid-associated protein, H-NS. Electrophoretic mobility shift assays indicate that H-NS binds preferentially to the
tra
promoter region, while Northern blot and transcriptional fusion analyses indicate that transcription of
traY
, the first gene in the
tra
operon, is derepressed in an
hns
mutant throughout growth. The plasmid-encoded regulatory protein TraJ is essential for transcription of the
tra
operon in wild-type
Escherichia coli
; however, TraJ is not necessary for plasmid transfer or
traY
operon transcription in an
hns
mutant. This indicates that H-NS represses transcription from P
Y
directly and not indirectly via its effects on TraJ levels. These results suggest that TraJ functions to disrupt H-NS silencing at P
Y
, allowing transcription of the
tra
operon.
Characterization of Dissimilatory Fe(III) versus NO3−Reduction in the Hyperthermophilic ArchaeonPyrobaculum aerophilum
Authors: Feinberg Lawrence F. , Holden James F.
Published: December 2005

The hyperthermophilic archaeonPyrobaculum aerophilumused 20 mM Fe(III) citrate, 100 mM poorly crystalline Fe(III) oxide, and 10 mM KNO3as terminal electron acceptors. The two forms of iron were reduced at different rates but with equal growth yields. The insoluble iron was reduced when segregated spatially by dialysis tubing, indicating that direct contact with the iron was not necessary for growth. When partitioned, there was no detectable Fe(III) or Fe(II) outside of the tubing after growth, suggesting that an electron shuttle, not a chelator, may be used as an extracellular mediator of iron reduction. The addition of 25 and 50% (vol vol−1) cell-free spent insoluble iron media to fresh media led to growth without a lag phase. Liquid chromatography analysis of spent media showed that cultures grown in iron, especially insoluble iron, produced soluble extracellular compounds that were absent or less abundant in spent nitrate medium. NADH-dependent ferric reductase activity increased approximately 100-fold, while nitrate reductase activity decreased 10-fold in whole-cell extracts from iron-grown cells relative to those from nitrate-grown cells, suggesting that dissimilatory iron reduction was regulated. A novel 2,6-anthrahydroquinone disulfonate oxidase activity was more than 580-fold higher in iron-grown cells than in nitrate-grown cells. The activity was primarily (>95%) associated with the membrane cellular fraction, but its physiological function is unknown. Nitrate-grown cultures produced two membrane-bound,c-type cytochromes that are predicted to be monoheme and part of nitrite reductase and abc1complex using genome analyses. Only one cytochrome was present in cells grown on Fe(III) citrate whose relative abundance was unchanged.
Synthesis, Stability, and Function of Cytadhesin P1 and Accessory Protein B/C Complex of
Mycoplasma pneumoniae
Authors: Waldo Robert H. , Krause Duncan C.
Published: December 2005

The genes MPN141 and MPN142 encode the major adhesin P1 and the cytadherence-related B/C proteins (P90/P40), respectively, in
Mycoplasma pneumoniae
. Using reverse transcriptase PCR we found open reading frames MPN140 to MPN142 constitute a polycistronic transcriptional unit. Cytadherence mutant IV-22 has a frameshift mutation in MPN141 and lacks the P1, B, or C proteins. Recombinant MPN141 and/or MPN142 were introduced into mutant IV-22 by transposon delivery in several configurations, and the levels of the P1, B, and C proteins were assessed by immunoblotting. MPN142 in mutant IV-22 has a wild-type nucleotide sequence, yet the introduction of recombinant MPN141 alone to mutant IV-22, although it restored P1 levels, failed to restore levels of B or C. In contrast, recombinant MPN141 and MPN142 delivered in
cis
or in
trans
were sufficient to restore all three proteins. Taken together, our data indicated that some but not all synthesis of B or C is dependent on coupling to the translation of P1 immediately upstream of MPN142 and demonstrated that proteins B and C are not stable in the absence of P1. The linkage of MPN141 and MPN142 at the levels of transcription, translation, and protein stability, in addition to their previously demonstrated colocalization and the requirement of B and/or C for P1 function, reinforces the conclusion that these proteins constitute a multiprotein complex that functions in receptor binding.
The β-Oxidation Systems ofEscherichia coliandSalmonella entericaAre Not Functionally Equivalent
Authors: Iram Surtaj Hussain , Cronan John E.
Published: December 2005

Based on its genome sequence, the pathway of β-oxidative fatty acid degradation inSalmonella entericaserovar Typhimurium LT2 has been thought to be identical to the well-characterizedEscherichia coliK-12 system. We report that wild-type strains ofS. entericagrow on decanoic acid, whereas wild-typeE. colistrains cannot. Mutant strains (carryingfadR) of both organisms in which the genes of fatty acid degradation (fad) are expressed constitutively are readily isolated. TheS. enterica fadRstrains grow more rapidly than the wild-type strains on decanoic acid and also grow well on octanoic and hexanoic acids (which do not support growth of wild-type strains). By contrast,E. coli fadRstrains grow well on decanoic acid but grow only exceedingly slowly on octanoic acid and fail to grow at all on hexanoic acid. The two wild-type organisms also differed in the ability to grow on oleic acid when FadR was overexpressed. Under these superrepression conditions,E. colifailed to grow, whereasS. entericagrew well. Exchange of the wild-typefadRgenes between the two organisms showed this to be a property ofS. entericarather than of the FadR proteins per se. This difference in growth was attributed toS. entericahaving higher cytosolic levels of the inducing ligands, long-chain acyl coenzyme As (acyl-CoAs). The most striking results were the differences in the compositions of CoA metabolites of strains grown with octanoic acid or oleic acid.S. entericacleanly converted all of the acid to acetyl-CoA, whereasE. coliaccumulated high levels of intermediate-chain-length products. Exchange of homologous genes between the two organisms showed that theS. entericaFadE and FadBA enzymes were responsible for the greater efficiency of β-oxidation relative to that ofE. coli.
Accumulation of Homolanthionine and Activation of a Novel Pathway for Isoleucine Biosynthesis in Corynebacterium glutamicumMcbR Deletion Strains
Authors: Krömer Jens Olaf , Heinzle Elmar , Schröder Hartwig , Wittmann Christoph
Published: December 2005

In the present work, the metabolic consequences of the deletion of the methionine and cysteine biosynthesis repressor protein (McbR) inCorynebacterium glutamicum, which releases almost all enzymes of methionine biosynthesis and sulfate assimilation from transcriptional regulation (D. A. Rey, A. Pühler, and J. Kalinowski, J. Biotechnol.103:51-65, 2003), were studied.C. glutamicumATCC 13032 ΔmcbRshowed no overproduction of methionine. Metabolome analysis revealed drastic accumulation of a single metabolite, which was not present in the wild type. It was identified by isotopic labeling studies and gas chromatography/mass spectrometry asl-homolanthionine {S-[(3S)-3-amino-3-carboxypropyl]-l-homocysteine}. The accumulation of homolanthionine to an intracellular concentration of 130 mM in the ΔmcbRstrain was accompanied by an elevated intracellular homocysteine level. It was shown that cystathionine-γ-synthase (MetB) produced homolanthionine as a side reaction. MetB showed higher substrate affinity for cysteine (Km= 260 μM) than for homocysteine (Km= 540 μM). The cell is able to cleave homolanthionine at low rates via cystathionine-β-lyase (MetC). This cleavage opens a novel threonine-independent pathway for isoleucine biosynthesis via 2-oxobutanoate formed by MetC. In fact, the deletion mutant exhibited an increased intracellular isoleucine level. Metabolic flux analysis ofC. glutamicum ΔmcbRrevealed that only 24% of theO-acetylhomoserine at the entry of the methionine pathway is utilized for methionine biosynthesis; the dominating fraction is either stored as homolanthionine or redirected towards the formation of isoleucine. Deletion ofmetBcompletely prevents homolanthionine accumulation, which is regarded as an important step in the development ofC. glutamicumstrains for biotechnological methionine production.
The Genome Sequence ofMethanosphaera stadtmanaeReveals Why This Human Intestinal Archaeon Is Restricted to Methanol and H2for Methane Formation and ATP Synthesis
Authors: Fricke Wolfgang F. , Seedorf Henning , Henne Anke , Krüer Markus , Liesegang Heiko , Hedderich Reiner , Gottschalk Gerhard , Thauer Rudolf K.
Published: December 2005

Methanosphaera stadtmanaehas the most restricted energy metabolism of all methanogenic archaea. This human intestinal inhabitant can generate methane only by reduction of methanol with H2and is dependent on acetate as a carbon source. We report here the genome sequence ofM. stadtmanae, which was found to be composed of 1,767,403 bp with an average G+C content of 28% and to harbor only 1,534 protein-encoding sequences (CDS). The genome lacks 37 CDS present in the genomes of all other methanogens. Among these are the CDS for synthesis of molybdopterin and for synthesis of the carbon monoxide dehydrogenase/acetyl-coenzyme A synthase complex, which explains whyM. stadtmanaecannot reduce CO2to methane or oxidize methanol to CO2and why this archaeon is dependent on acetate for biosynthesis of cell components. Four sets ofmtaABCgenes coding for methanol:coenzyme M methyltransferases were found in the genome ofM. stadtmanae. These genes exhibit homology tomtagenes previously identified inMethanosarcinaspecies. TheM. stadtmanaegenome also contains at least 323 CDS not present in the genomes of all other archaea. Seventy-three of these CDS exhibit high levels of homology to CDS in genomes of bacteria and eukaryotes. These 73 CDS include 12 CDS which are unusually long (>2,400 bp) with conspicuous repetitive sequence elements, 13 CDS which exhibit sequence similarity on the protein level to CDS encoding enzymes involved in the biosynthesis of cell surface antigens in bacteria, and 5 CDS which exhibit sequence similarity to the subunits of bacterial type I and III restriction-modification systems.
Microarrays Reveal that Each of the Ten Dominant Lineages ofStaphylococcus aureusHas a Unique Combination of Surface-Associated and Regulatory Genes
Authors: Lindsay Jodi A. , Moore Catrin E. , Day Nicholas P. , Peacock Sharon J. , Witney Adam A. , Stabler Richard A. , Husain Sarah E. , Butcher Philip D. , Hinds Jason
Published: December 2005

Staphylococcus aureusis the most common cause of hospital-acquired infection. In healthy hosts outside of the health care setting,S. aureusis a frequent colonizer of the human nose but rarely causes severe invasive infection such as bacteremia, endocarditis, or osteomyelitis. To identify genes associated with community-acquired invasive isolates, regions of genomic variability, and theS. aureuspopulation structure, we compared 61 community-acquired invasive isolates ofS. aureusand 100 nasal carriage isolates from healthy donors using a microarray spotted with PCR products representing every gene from the sevenS. aureussequencing projects. The core genes common to all strains were identified, and 10 dominant lineages ofS. aureuswere clearly discriminated. Each lineage carried a unique combination of hundreds of “core variable” (CV) genes scattered throughout the chromosome, suggesting a common ancestor but early evolutionary divergence. Many CV genes are regulators of virulence genes or known or predicted to be expressed on the bacterial surface and to interact with the host during nasal colonization and infection. Within each lineage, isolates showed substantial variation in the carriage of mobile genetic elements and their associated virulence and resistance genes, indicating frequent horizontal transfer. However, we were unable to identify any association between lineage or gene and invasive isolates. We suggest that theS. aureusgene combinations necessary for invasive disease may also be necessary for nasal colonization and that community-acquired invasive disease is strongly dependent on host factors.
Whole-Genome Plasticity among
Mycobacterium avium
Subspecies: Insights from Comparative Genomic Hybridizations
Authors: Wu Chia-wei , Glasner Jeremy , Collins Michael , Naser Saleh , Talaat Adel M.
Published: December 2005

Infection with
Mycobacterium avium
subsp.
paratuberculosis
causes Johne's disease in cattle and is also implicated in cases of Crohn's disease in humans. Another closely related strain,
M. avium
subsp.
avium
, is a health problem for immunocompromised patients. To understand the molecular pathogenesis of
M. avium
subspecies, we analyzed the genome contents of isolates collected from humans and domesticated or wildlife animals. Comparative genomic hybridizations indicated distinct lineages for each subspecies where the closest genomic relatedness existed between
M. avium
subsp.
paratuberculosis
isolates collected from human and clinical cow samples. Genomic islands (
n
= 24) comprising 846 kb were present in the reference
M. avium
subsp.
avium
strain but absent from 95% of
M. avium
subsp.
paratuberculosis
isolates. Additional analysis identified a group of 18
M. avium
subsp.
paratuberculosis
-associated islands comprising 240 kb that were absent from most of the
M. avium
subsp.
avium
isolates. Sequence analysis of DNA regions flanking the genomic islands identified three large inversions in addition to several small inversions that could play a role in regulation of gene expression. Analysis of genes encoded in the genomic islands reveals factors that are probably important for various mechanisms of virulence. Overall,
M. avium
subsp.
avium
isolates displayed a higher level of genomic diversity than
M. avium
subsp.
paratuberculosis
isolates. Among
M. avium
subsp.
paratuberculosis
isolates, those from wildlife animals displayed the highest level of genomic rearrangements that were not observed in other isolates. The presented findings will affect the future design of diagnostics and vaccines for Johne's and Crohn's diseases and provide a model for genomic analysis of closely related bacteria.
DNA Sequence of a ColV Plasmid and Prevalence of Selected Plasmid-Encoded Virulence Genes among Avian
Escherichia coli
Strains
Authors: Johnson Timothy J. , Siek Kylie E. , Johnson Sara J. , Nolan Lisa K.
Published: December 2005

ColV plasmids have long been associated with the virulence of
Escherichia coli
, despite the fact that their namesake trait, ColV production, does not appear to contribute to virulence. Such plasmids or their associated sequences appear to be quite common among avian pathogenic
E. coli
(APEC) and are strongly linked to the virulence of these organisms. In the present study, a 180-kb ColV plasmid was sequenced and analyzed. This plasmid, pAPEC-O2-ColV, possesses a 93-kb region containing several putative virulence traits, including
iss
,
tsh
, and four putative iron acquisition and transport systems. The iron acquisition and transport systems include those encoding aerobactin and salmochelin, the
sit
ABC iron transport system, and a putative iron transport system novel to APEC,
eit
. In order to determine the prevalence of the virulence-associated genes within this region among avian
E. coli
strains, 595 APEC and 199 avian commensal
E. coli
isolates were examined for genes of this region using PCR. Results indicate that genes contained within a portion of this putative virulence region are highly conserved among APEC and that the genes of this region occur significantly more often in APEC than in avian commensal
E. coli
. The region of pAPEC-O2-ColV containing genes that are highly prevalent among APEC appears to be a distinguishing trait of APEC strains.