bluuebunny/crossref_metadata_2025 huggingface dataset:
Functional dissection of Ctr4 and Ctr5 amino-terminal regions reveals motifs with redundant roles in copper transport
Authors: Beaudoin Jude , Laliberté Julie , Labbé Simon
Published: December 2005

Copper uptake in the fission yeastSchizosaccharomyces pombeis carried out by a heteromeric complex formed by two proteins, Ctr4 and Ctr5. In this study, a stable expression system using integrative plasmids was developed to investigate the respective roles of Ctr4 and Ctr5 in copper transport. It was shown that expression of full-length Ctr4 or truncated Ctr4 containing residues 106–289 was required for localization of Ctr5 to the plasma membrane. Likewise, when the full-length Ctr5 or truncated Ctr5 from residues 44–173 was co-expressed with Ctr4, this protein was visualized at the periphery of the cell. To determine the importance of the Mets motifs (consisting of five methionines arranged as Met-X2-Met-X-Met, where X is any amino acid) of Ctr4 and Ctr5 in the heteroprotein complex, we co-expressed Ctr5 lacking the Mets motif and Cys-X-Met-X-Met sequence with wild-type Ctr4 or its mutant derivatives. Conversely, Ctr4 lacking the Mets motif and Met122was expressed with wild-type Ctr5 or its mutant derivatives. These experiments revealed that the five Mets motifs of Ctr4 and the Ctr4 residue Met122have equally important roles in copper assimilation. Furthermore, the two partially overlapping Mets motifs and the Cys-X-Met-X-Met sequence in Ctr5 have redundant functions in copper transport, with the latter sequence making a greater contribution than the former. Together, the data reveal that co-expression of both Ctr4 and Ctr5 is necessary for the proper function and localization of the heteroprotein complex to the plasma membrane. Once on the cell surface, the N-terminal regions of Ctr4 and Ctr5 can function independently to transport copper; however, the greatest efficiency is achieved when both N termini are present.
Subfamilies of cpmA, a gene involved in circadian output, have different evolutionary histories in cyanobacteria
Authors: Dvornyk Volodymyr
Published: December 2005

ThecpmAgene mediates an output signal in the cyanobacterial circadian system. This gene and its homologues are evolutionarily old, and occur in some non-photosynthetic bacteria and archaea as well as in cyanobacteria. The gene has two functional domains that differ drastically in their level of polymorphism: the N-terminal domain is much more variable than the PurE homologous C-terminal domain. The phylogenetic tree of thecpmAhomologues features four main clades (C1–C4), two of which (C1 and C3) belong to cyanobacteria. These cyanobacterial clades match respective ones in the previously reported phylogenetic trees of the other genes involved in the circadian system. The phylogenetic analysis suggested that the C3 subfamily, which comprises the genes from the cyanobacteria with thekaiBC-based circadian system, experienced a lateral transfer, probably from evolutionarily old proteobacteria about 1000 million years ago. The genes of this subfamily have a significantly higher nonsynonymous substitution rate than those of C1 (2·13×10−10and 1·53×10−10substitutions per nonsynonymous site per year, respectively). It appears that the functional and selective constraints of thekaiABC-based system have slowed down the rate of sequence evolution compared to thecpmAhomologues of thekaiBC-based system. On the other hand, the differences in the mutation rates between the two cyanobacterial clades point to the different functional constraints of the systems with or withoutkaiA.
A novel class of CoA-transferase involved in short-chain fatty acid metabolism in butyrate-producing human colonic bacteria
Authors: Charrier Cédric , Duncan Gary J. , Reid Martin D. , Rucklidge Garry J. , Henderson Donna , Young Pauline , Russell Valerie J. , Aminov Rustam I. , Flint Harry J. , Louis Petra
Published: December 2005

Bacterial butyryl-CoA CoA-transferase activity plays a key role in butyrate formation in the human colon, but the enzyme and corresponding gene responsible for this activity have not previously been identified. A novel CoA-transferase gene is described from the colonic bacteriumRoseburiasp. A2-183, with similarity to acetyl-CoA hydrolase as well as 4-hydroxybutyrate CoA-transferase sequences. The gene product, overexpressed in anEscherichia colilysate, showed activity with butyryl-CoA and to a lesser degree propionyl-CoA in the presence of acetate. Butyrate, propionate, isobutyrate and valerate competed with acetate as the co-substrate. Despite the sequence similarity to 4-hydroxybutyrate CoA-transferases, 4-hydroxybutyrate did not compete with acetate as the co-substrate. Thus the CoA-transferase preferentially uses butyryl-CoA as substrate. Similar genes were identified in other butyrate-producing human gut bacteria from clostridial clusters IV and XIVa, while other candidate CoA-transferases for butyrate formation could not be detected inRoseburiasp. A2-183. This suggests strongly that the newly identified group of CoA-transferases described here plays a key role in butyrate formation in the human colon.
Comparative analysis of proteins with a mucus-binding domain found exclusively in lactic acid bacteria
Authors: Boekhorst Jos , Helmer Quinta , Kleerebezem Michiel , Siezen Roland J.
Published: December 2005

Lactic acid bacteria (LAB) are frequently encountered inhabitants of the human intestinal tract. A protective layer of mucus covers the epithelial cells of the intestine, offering an attachment site for these bacteria. In this study bioinformatics tools were used to identify and characterize proteins containing one type of mucus-binding domain, called MUB, that is postulated to play an important role in the adherence of LAB to this mucus layer. By searching in all protein databases 48 proteins containing at least one of these MUB domains in nine LAB species were identified. These MUB domains varied in size, ranging from approximately 100 to more than 200 residues per domain. Complete MUB domains were found exclusively in LAB. The number of MUB domains present in a single protein varied from 1 to 15. In some cases, orthologous proteins in closely related species contained a different number of domains, indicating that repeats of the domain undergo rapid duplication and deletion. Proteins containing the MUB domain were often encoded by gene clusters that encode multiple extracellular proteins. In addition to one or more copies of the MUB domain, many of these proteins contained other domains that are predicted to be involved in binding to and degradation of extracellular components. These findings strongly suggest that the MUB domain is an LAB-specific functional unit that performs its task in various domain contexts and could fulfil an important role in host–microbe interactions in the gastrointestinal tract.
Escherichia coli mutators: selection criteria and migration effect
Authors: Le Chat Ludovic , Fons Michel , Taddei Francois
Published: December 2005

In silico, it has been shown that mutator alleles that increase mutation rate can be selected for by generating adaptive mutations.In vitroandin vivo, competition between wild-type bacteria and isogenic mutator mutants is consistent with this view. However,in vivo, the gain of the mutator seems to be reduced when migration is allowed.In vitro, the advantage of mutators has been described as frequency-dependent, leading to mutator advantage only when they are sufficiently frequent. Using anin vitrosystem, it is demonstrated that (i) the selection of mutators is frequency-independent, yet depends on at least one mutator bacterium bearing an adaptive mutation (its presence depends on chance, mutation rates and population size of mutator bacteria); (ii) on average, the mutator gain is always equal to the ratio of the adaptive mutation frequency of the mutator versus wild-type; (iii) when migration into an empty niche is allowed, the mutator benefit is reduced if migration occurs after fixation of the adaptive mutation into the wild-type population. It is concluded that in all cases, mutator gain depends directly on the ratio of bacteria carrying a beneficial mutation in mutator versus wild-type lineages.
Phenotypic switching in Cryptococcus neoformans
Authors: Guerrero A. , Jain N. , Goldman D. L. , Fries B. C.
Published: December 2005

Phenotypic switching has been described in serotype A and D strains ofCryptococcus neoformans. It occursin vivoduring chronic infection and is associated with differential gene expression and changes in virulence. The switch involves changes in the polysaccharide capsule and cell wall that affect the yeast's ability to resist phagocytosis. In addition, the phenotypic switch variants elicit qualitatively different inflammatory responses in the host. In animal models of chronic cryptococosis, the immune response of the host ultimately determines which of the switch variants are selected and maintained. The importance of phenotypic switching is further underscored by several findings that are relevant in the setting of human disease. These include the ability of the mucoid colony variant of RC-2 (RC-2 MC) but not the smooth variant (RC-2 SM) to promote increased intracerebral pressure in a rat model of cryptococcal meningitis. Furthermore, chemotherapeutic and immunological antifungal interventions can promote the selection of the RC-2 MC variant during chronic murine infection.
A cysteine/methionine auxotroph of the opportunistic fungus Aspergillus flavus is associated with host-range restriction: a model for emerging diseases
Authors: Scully Lisa R. , Bidochka Michael J.
Published: December 2005

The evolution of host specialization in pathogens is a topic of considerable interest, particularly since it can represent a decisive step in the emergence of infectious diseases. Aspergillus flavus is an opportunistic fungus capable of infecting a wide variety of hosts, including plants, insects and mammals, although with low virulence. Here the derivation of an A. flavus strain that exhibits severe host restriction is reported. This strain exhibited a severe diminution or a complete lack of conidial production on a variety of standard agar media and on various plant species. However, it retained its ability to infect insects from various orders and to re-emerge from and adequately conidiate on the insect cadavers as a culmination of the pathogenic life cycle. This strain, demonstrating insect-dependent conidiation, was discovered to be a cysteine/methionine auxotroph due to an inability to reduce sulfate to sulfite. However, other A. flavus auxotrophs tested for plant and insect host range failed to show insect-dependent conidiation. An association between this specific auxotroph and a decreased host range is shown, emphasizing the role of nutrition in the host–pathogen relationship with respect to host restriction and evolution towards obligate pathogenesis.
Development of a multilocus sequence typing method for analysis of Lactobacillus plantarum strains
Authors: de las Rivas Blanca , Marcobal Ángela , Muñoz Rosario
Published: December 2005

Lactobacillus plantarum is a species of considerable industrial and medical interest. To date, the lack of reliable molecular methods for definite identification at strain level has hindered studies of the population biology of this organism. Here, a multilocus sequence typing (MLST) system for this organism is described, which exploits the genetic variation present in six housekeeping loci to determine the genetic relationship among isolates. The MLST system was established using 16 L. plantarum strains that were also characterized by ribotyping and restriction fragment length polymorphism (RFLP) analysis of the PCR-amplified 16S–23S rDNA intergenic spacer region (ISR). Ribotyping grouped the strains into four groups; however, RFLP analysis of the ISRs showed no differences in the strains analysed. In contrast, MLST had a good discriminatory ability. The sequence analysis of the six genes showed 14 different allelic combinations, with 12 of them represented by only one strain. By using this MLST approach we were able to confirm the identity of two strains deposited in the Spanish Type Culture Collection as different strains. Phylogenetic analysis indicated a panmictic population structure of L. plantarum and split decomposition analysis indicated that recombination plays a role in creating genetic heterogeneity in L. plantarum. As MLST allows precise identification, and easy comparison and exchange of results obtained in different laboratories, the future application of this new molecular method could be useful for the identification of valuable L. plantarum strains.
Steam Pressure Generated in a Spherical Cavity in a Moisture Saturated Polymer Matrix Composite during Rapid Heating
Authors: Muralidharan Vijayanand , Hui Chung-Yuen
Published: December 2005

The time history of steam pressure inside an isolated spherical micro-cavity in a polymer matrix composite is studied by assuming that the chemical potential of water is continuous across the cavity–polymer interface. Steam pressure inside the cavity is due to heating of moisture-saturated composites from its initial temperature to a final temperature. Exact closed form solutions are obtained for the steam pressure developed under infinitely fast heating in an infinite plate. The effect of cavity shape on the induced steam pressure is studied by comparing this solution with a previous result on cracklike cavities.
Amorphous and Semicrystalline Solid Polymers: Experimental and Modeling Studies of Their Inelastic Deformation Behaviors
Authors: Khan Fazeel , Krempl Erhard
Published: December 2005

The study of the inelastic deformation behavior of six amorphous and semicrystalline polymers was performed to develop and verify the capabilities of a constitutive material model. The test conditions consisted of piecewise constant strain rates for loading and unloading. Immediate control mode switching capability permitted using load control for creep and recovery tests. Positive, nonlinear rate sensitivity was observed in all cases for monotonic loading and the prior loading rate was found to have a strong influence on creep, relaxation and strain recovery (emulating creep at zero stress) tests. In particular, a fast prior rate engenders a larger change in the output variable: strain in conditions of creep and stress drop in relaxation. Based on the absence of any distinctive deformation traits, the preponderance of data collected in the experimentation program suggests that both categories of polymers can be modeled using the same phenomenological approach. Modeling of the experimental data is introduced with a uniaxial form of the Viscoplasticity Theory Based on Overstress for Polymers (VBOP). Simulations and model predictions are provided for various loading histories. Additional modifications necessary to extend the theory to finite deformation and inelastic compressibility are then presented. An objective formulation is obtained in the Eulerian framework together with the recently proposed logarithmic spin by Xiao [Xiao, H., Bruhns, O., and Meyers, A., 1997, “Hypoelesticity Model Based Upon the Logarithmic Stress Rate,” J. Elast., 47, pp. 51–68].
Analysis of Interfacial Debonding in Three-Dimensional Composite Microstructures
Authors: Swaminathan Shriram , Pagano N. J. , Ghosh Somnath
Published: December 2005

This paper is aimed at analyzing stresses and fiber-matrix interfacial debonding in three-dimensional composite microstructures. It incorporates a 3D cohesive zone interface model based element to simulate interfacial debonding in the commercial code ABAQUS. The validated element is used to examine the potential debonding response in the presence of fiber–fiber interactions. A two-fiber model with unidirectional fibers is constructed and the effect of relative fiber spacing and volume fraction on the stress distribution in the matrix is studied. In addition, the effect of fiber orientation and spacing on the nature of initiation and propagation of interfacial debonding is studied in a two-fiber model. These results are expected to be helpful in formulating future studies treating optimal fiber orientations and payoff in controlling fiber spacing and alignment.
Post-Curing Effects on Marine VARTM FRP Composite Material Properties for Test and Implementation
Authors: Cain Jason J. , Post Nathan L. , Lesko John J. , Case Scott W. , Lin Yin-Nian , Riffle Judy S. , Hess Paul E.
Published: December 2005

Structural composites are increasingly being utilized in many large naval and civil structures where it is vital that their long-term performance be predictable and their variability definable over the life of the structure. However, these properties may be influenced by the degree of cure of the resin, particularly for room-temperature-cured systems. Thus, this investigation defines the postcure effects on E-glass/vinyl-ester fiber-reinforced polymer (FRP) composites manufactured using the vacuum-assisted resin transfer molding (VARTM) method, which are typical of those used by the US Navy for ship structures. The composites are differentiated by varying levels of postcure temperature and duration, and examined for the effects of advancing cure at various points in the time after postcure. Pseudo-quasi-isotropic [0/+45/90/−45/0]s and angle ply laminate [±45]2s samples from each level of postcure are examined at 1, 10, 30, 100, and 300 days after postcure in order to track strength, stiffness, failure strain, creep, and fatigue performance as functions of time. In parallel, the matrix polymer is inspected using FTIR (Fourier transform infrared spectroscopy) to directly assess the degree of conversion. Dynamic mechanical analysis and shrinkage measurements are also undertaken to assess the Tg and the amount of shrinkage undergone during post-curing, as well as the advancing of the level of cure during the prescribed aging time. Results suggest that the degree of conversion is limited to 80% for the vinyl-ester oligomer and 90–95% for styrene following a postcure of 93°C. It is observed that after 300 days of ambient storage the nonpostcured samples approach the degree of conversion exhibited by those postcured at 93°C, as measured by FTIR. Resin dominated quasi-static properties are greatly affected by the degree of cure, whereas fiber dominated properties are not. Where the degree of cure is comparatively low, viscoelastic properties cause greater changes in creep response as well as influencing fatigue performance.
A Thermodynamic Framework for Describing Solidification of Polymer Melts
Authors: Kannan K. , Rao I. J. , Rajagopal K. R.
Published: December 2005

A thermodynamic framework is presented that can be used to describe the solidification of polymer melts, both the solidification of atactic polymers into an amorphous elastic solid and the crystallization of other types of polymer melts to semi-crystalline elastic solids. This framework fits into a general structure that has been developed to describe the response of a large class of dissipative bodies. The framework takes into account the fact that the natural configuration of the viscoelastic melt and the solid evolve during the process and that the symmetries of these natural configurations also evolve. Different choices are made as to how the material stores energy, produces entropy, and for its latent heat, latent energy, etc., that lead to models for different classes of materials. The evolution of the natural configuration is dictated by the manner in which entropy is produced, how the energy is stored etc., and it is assumed that the constitutive choices are such that the rate entropy production is maximized, from an allowable class of constitutive models. Such an assumption also determines the crystallization kinetics, i.e., provides equations such as the Avrami equation. Using the framework, a model is developed within which the problem of fiber spinning is studied and we find that the model is able to predict observed experimental results quite well.
Modeling Thermo-Oxidative Layer Growth in High-Temperature Resins
Authors: Pochiraju K. V. , Tandon G. P.
Published: December 2005

This paper describes modeling of degradation behavior of high-temperature polymers under thermo-oxidative aging conditions. Thermo-oxidative aging is simulated with a diffusion-reaction model in which temperature, oxygen concentration, and weight-loss effects are considered. A parametric reaction model based on a mechanistic view of the reaction is used for simulating reaction-rate dependence on the oxygen availability in the polymer. Macroscopic weight-loss measurements are used to determine the reaction and polymer consumption parameters. The diffusion-reaction partial differential equation system is solved using Runge-Kutta methods. Simulations illustrating oxidative layer growth in a high-temperature PMR-15 polyimide resin system under isothermal conditions are presented and correlated with experimental observations of oxidation layer growth. Finally, parametric studies are conducted to examine the sensitivity of material parameters in predicting oxidation development.
Buckling Analysis of Delaminated and Stitched Composite Plate System Under Hygrothermal Pressure
Authors: Phoenix S. Leigh , Yavuz A. Kadir , Papoulia Katerina D. , Hui C. Yuen
Published: December 2005

In this study, we develop a model for buckling of a partially delaminated composite plate with transverse stitching to resist out of plane deformations. The model applies to carbon fiber/polyimide matrix composites rapidly heated to around 370 °C, where it is known that steam-induced delamination (the popcorn effect) becomes an issue as the pressures generated approach the tensile strength of the matrix. Thus, a key element is the incorporation of this hygrothermal pressure within the formulation. This complex composite structure is modeled as two adhesively connected, specially orthotropic, rectangular plates, and the delaminations with internal vapor pressure are considered as holes in the adhesive layer. The intact regions of the adhesive layer and the stitches are modeled by continuous and discrete linear mechanical springs, respectively. The energy contributions of each component in the system are expressed in terms of out-of-plane displacements. The boundary conditions are that the system is simply supported along all edges so as to permit a Fourier sine series to approximate the transverse displacements. Application of the energy minimization approach gives a system of algebraic equations to determine the unknown weighting coefficients of the functions describing the transverse deflections of each plate layer. Deformed shapes of the system under axial compressive loads are obtained for different hygrothermally induced pressure conditions so as to show that the model works well. Parametric studies on critical buckling loads are performed for a few stitch and delamination configurations. It is found that stitching through delaminated areas can increase critical buckling loads and alter the sequence of corresponding mode shapes.
Compression of Packed Particulate Systems: Simulations and Experiments in Graphitic Li-ion Anodes
Authors: Yi Y.-B. , Wang C.-W. , Sastry A. M.
Published: December 2005

Increased thermal conductivity, electronic conductivity, and reversible capacity (i.e., reduced irreversible capacity loss, or ICL) have been demonstrably achievable by compression of anodes into higher volume fraction plates, though excessive compression can impair Li-ion battery performance. In our previous study, we correlated conductivity and compression of these materials. Here, we further investigated the effects of friction and deformability of particles on the compressibility of model carbons of Li-ion anodes. First, we implemented a statistically unbiased technique for generating a range of random particulate systems, from permeable to impermeable arrangements, along with a contact model for randomly arranged triaxial ellipsoidal particles, suitable for implementation in finite element analysis of compression of a random, porous system. We then quantified the relationship between interfacial friction and jamming fraction in spherical to ellipsoidal systems and applied these models to correlate maximum stresses and different frictional coefficients, with morphology (obtained by image analysis) of graphite particles in Li-ion anodes. The simulated results were compared with the experiments, showing that the friction coefficient in the system is close to 0.1 and that the applied pressure above 200kg∕cm2(200MPa) can damage the materials in SL-20 electrodes. We also conclude that use of maximum jamming fractions to assess likely configuration of mixtures is unrealistic, at best, in real manufacturing processes. Particles change both their overall shapes and relative orientations during deformation sufficient to alter the composite properties: indeed, it is alteration of properties that motivates post-processing at all. Thus, consideration of material properties, or their estimation post facto, using inverse techniques, is clearly merited in composites having volume fractions of particles near percolation onset.
Differential Implementation of the Viscoelastic Response of a Curing Thermoset Matrix for Composites Processing
Authors: Zobeiry Nima , Vaziri Reza , Poursartip Anoush
Published: December 2005

The ability to manufacture thermoset matrix composite materials into large and complex structures can be significantly enhanced by modeling the behavior of the system during the process. As a result there has been much research on all aspects of the cure of these materials. A particularly important aspect is the development of mechanical properties in the thermoset matrix as it evolves from a low molecular weight material into a fully cross-linked solid. The behavior is generally acknowledged to be viscoelastic, and as both temperature and degree of cure vary with time, the characterization and representation of the behavior is both critical and complex. Many approaches have been suggested and tried, ranging from 1D or 2D implementations of simple linear elastic cure hardening responses (which have been shown to be essentially pseudo-viscoelastic formulations) through to more sophisticated representations of viscoelastic behavior as Prony series of Maxwell elements coded in 3D hereditary integral FE implementations. In this paper we present a differential approach for the viscoelastic representation of a curing thermoset matrix composite undergoing an arbitrary temperature cycle by noting that the viscoelastic response can be represented very well by a Prony series. For this case, we show that a differential approach is equivalent to an integral formulation, but appears to have some significant benefits in terms of extension to more general descriptions (e.g., thermo-viscoelastic behavior), ease of coding and implementation, and perhaps most importantly, computer runtimes. Rather than using a differential approach where the order of the governing differential equation grows very fast with the number of springs or dashpots, we use the stresses in the individual Maxwell elements to capture the complete history of the material and allow for a much simpler formulation. A 1D formulation of this differential approach, including thermo-viscoelasticity, is developed, and results and benchmarks are presented.
Recruitment of
Bacillus subtilis
RecN to DNA Double-Strand Breaks in the Absence of DNA End Processing
Authors: Sanchez Humberto , Kidane Dawit , Castillo Cozar M. , Graumann Peter L. , Alonso Juan C.
Published: December 2005

The recognition and processing of double-strand breaks (DSBs) to a 3′ single-stranded DNA (ssDNA) overhang structure in
Bacillus subtilis
is poorly understood. Mutations in
addA
and
addB
or null mutations in
recJ
(Δ
recJ
),
recQ
(Δ
recQ
), or
recS
(Δ
recS
) genes, when present in otherwise-Rec
+
cells, render cells moderately sensitive to the killing action of different DNA-damaging agents. Inactivation of a RecQ-like helicase (Δ
recQ
or Δ
recS
) in
addAB
cells showed an additive effect; however, when Δ
recJ
was combined with
addAB
, a strong synergistic effect was observed with a survival rate similar to that of Δ
recA
cells. RecF was nonepistatic with RecJ or AddAB. After induction of DSBs, RecN-yellow fluorescent protein (YFP) foci were formed in
addAB
Δ
recJ
cells. AddAB and RecJ were required for the formation of a single RecN focus, because in their absence multiple RecN-YFP foci accumulated within the cells. Green fluorescent protein-RecA failed to form filamentous structures (termed threads) in
addAB
Δ
recJ
cells. We propose that RecN is one of the first recombination proteins detected as a discrete focus in live cells in response to DSBs and that either AddAB or RecQ(S)-RecJ are required for the generation of a duplex with a 3′-ssDNA tail needed for filament formation of RecA.
The Streptococcal Blr and Slr Proteins Define a Family of Surface Proteins with Leucine-Rich Repeats: Camouflaging by Other Surface Structures
Authors: Waldemarsson Johan , Areschoug Thomas , Lindahl Gunnar , Johnsson Eskil
Published: December 2005

Regions with tandemly arranged leucine-rich repeats (LRRs) have been found in many prokaryotic and eukaryotic proteins, in which they provide a remarkably versatile framework for the formation of ligand-binding sites. Bacterial LRR proteins include the recently described Slr protein ofStreptococcus pyogenes, which is related to internalin A ofListeria monocytogenes. Here, we show that strains of the human pathogenStreptococcus agalactiaeexpress a protein, designated Blr, which together with Slr defines a family of internalin A-related streptococcal LRR proteins. Analysis with specific antibodies demonstrated that Blr is largely inaccessible onS. agalactiaegrown in vitro, but surface exposure was increased ∼100-fold on mutants lacking polysaccharide capsule. InS. pyogenes, surface exposure of Slr was not affected in a mutant lacking hyaluronic acid capsule but was increased >20-fold in mutants lacking M protein or protein F. Thus, both Blr and Slr are efficiently camouflaged by other surface structures on bacteria grown in vitro. When Blr and Slr exposed on the bacterial surface were compared, they exhibited only little immunological cross-reactivity, in spite of extensive residue identity, suggesting that their surface-exposed parts have been under evolutionary pressure to diverge functionally and/or antigenically. These data identify a family of immunologically diverse streptococcal LRR proteins that show unexpected complexity in their interactions with other bacterial surface components.
Purification and Characterization of LPXTGase from
Staphylococcus aureus
: the Amino Acid Composition Mirrors That Found in the Peptidoglycan
Authors: Lee Sung G. , Fischetti Vincent A.
Published: December 2005

Bacterial surface proteins are important molecules in the infectivity and survival of pathogens. Surface proteins on gram-positive bacteria have been shown to attach via a transpeptidase, termed sortase, that cleaves an LPXTG sequence found close to the C termini of nearly all surface proteins on these bacteria. We previously identified a unique enzyme (LPXTGase) from
Streptococcus pyogenes
that also cleaves the LPXTG motif with a catalytic activity higher than that of sortase, suggesting that it plays an important role in the attachment process. We have now purified and characterized an LPXTGase from
Staphylococcus aureus
and found that it has both similar and unique features compared to the
S. pyogenes
enzyme. The
S. aureus
enzyme is glycosylated and contains unusual amino acids, like its streptococcal counterpart. Like the streptococcal enzyme, staphylococcal LPXTGase has an overrepresentation of amino acids found in the peptidoglycan, i.e., glutamine/glutamic acid, glycine, alanine, and lysine, and furthermore, we find that these amino acids are present in the enzyme at precisely the same ratio at which they are found in the peptidoglycan for the respective organism. This suggests that enzymes responsible for wall assembly may also play a role in the construction of LPXTGase.
Identification of an ABC Transporter Required for Iron Acquisition and Virulence inMycobacterium tuberculosis
Authors: Rodriguez G. Marcela , Smith Issar
Published: December 2005

Iron availability affects the course of tuberculosis infection, and the ability to acquire this metal is known to be essential for replication ofMycobacterium tuberculosisin human macrophages.M. tuberculosisovercomes iron deficiency by producing siderophores. The relevance of siderophore synthesis for iron acquisition byM. tuberculosishas been demonstrated, but the molecules involved in iron uptake are currently unknown. We have identified two genes (irtAandirtB) encoding an ABC transporter similar to the YbtPQ system involved in iron transport inYersinia pestis. Inactivation of theirtABsystem decreases the ability ofM. tuberculosisto survive iron-deficient conditions. IrtA and -B do not participate in siderophore synthesis or secretion but are required for efficient utilization of iron from Fe-carboxymycobactin, as well as replication ofM. tuberculosisin human macrophages and in mouse lungs. We postulate that IrtAB is a transporter of Fe-carboxymycobactin. TheirtABgenes are located in a chromosomal region previously shown to contain genes regulated by iron and the major iron regulator IdeR. Taken together, our results and previous observations made by other groups regarding two other genes in this region indicate that this gene cluster is dedicated to siderophore synthesis and transport inM. tuberculosis.
Coordinated Regulation of Two Independent Cell-Cell Signaling Systems and Swarmer Differentiation inSalmonella entericaSerovar Typhimurium
Authors: Kim Wook , Surette Michael G.
Published: December 2005

Almost all members of the genusSalmonelladifferentiate and migrate on semisolid surfaces in a coordinated population behavior known as swarming. Important virulence determinants are coupled to swarmer differentiation in several other pathogenic organisms, collectively suggesting that conditions that trigger swarming in the laboratory may fortuitously promote the cells to enter a robust physiological state relevant to the host environment. Here, we present evidence that expression of two independent cell-cell signaling systems are also coupled to swarmer differentiation inS. entericaserovar Typhimurium. Expression of bothpfsandsdiAgenes was up-regulated in the actively migrating swarmers compared to their vegetative counterparts propagated in broth or spread plated on the surface of swim, swarm, and solid media. Accordingly, swarmers produced elevated levels of a universally recognized signaling molecule, autoinducer-2, and exhibited increased sensitivity toN-acyl homoserine lactones (AHLs), signaling molecules thatSalmonelladoes not produce. Expression of therckoperon was concomitantly up-regulated in the swarmers in an SdiA-dependent manner only in the presence of exogenous AHLs. In addition to the previously reported adaptive antibiotic resistance phenotype and global shift in metabolism, this work presents another component of the physiological changes that are specifically associated with swarmer differentiation in serovar Typhimurium and not simply due to growth on a surface.
Regulation of the
Mycobacterium tuberculosis mce1
Operon
Authors: Casali Nicola , White Amy M. , Riley Lee W.
Published: December 2005

In the murine model of infection, a
Mycobacterium tuberculosis mce1
operon mutant elicits an aberrant granulomatous response, resulting in uncontrolled replication and failure to enter a persistent state. In this study, we demonstrate that the
mce1
genes can be transcribed as a 13-gene polycistronic message encompassing Rv0166 to Rv0178. Quantitative reverse transcriptase PCR and immunoblot analyses revealed that the
mce1
genes and proteins are expressed during in vitro growth but are significantly down-regulated in intracellular bacilli isolated from murine macrophages. A homologue of the FadR subfamily of GntR transcriptional regulators, Rv0165c (designated Mce1R), lies upstream and is divergently transcribed from the operon. To investigate whether this gene plays a role in regulation of
mce1
expression, we created an
M. tuberculosis mce1R
deletion mutant. There was no difference in expression of
mce1
operon genes in Δ
mce1R
compared to expression in the wild type during logarithmic growth in vitro. However, in bacilli isolated from murine macrophages, expression of
mce1
genes was significantly higher in Δ
mce1R
. In addition, overexpression of
mce1R
resulted in repression of the
mce1
genes. These data demonstrate that Mce1R is a negative regulator that acts intracellularly to repress expression of the
mce1
operon. We propose that Mce1R facilitates balanced temporal expression of the
mce1
products required for organized granuloma formation, which is both protective to the host and necessary for the persistence of
M. tuberculosis
.
Expression Analysis of Up-Regulated Genes Responding to Plumbagin inEscherichia coli
Authors: Chen Jenn-Wei , Sun Chang-Ming , Sheng Wei-Lun , Wang Yu-Chen , Syu Wan-Jr
Published: December 2005

Plumbagin is found in many medicinal plants and has been reported to have antimicrobial activities. We examined the molecular responses ofEscherichia colito plumbagin by using a proteomic approach to search for bacterial genes up-regulated by the drug. The protein profile obtained was compared with that ofE. coliwithout the plumbagin treatment. Subsequent analyses of the induced proteins by mass spectroscopy identified several up-regulated genes, includingygfZ, whose function has not been defined. Analyses of the 5′-flanking sequences indicate that most of these genes contain a marbox-like stretch, and several of them are categorized as members of themar/soxregulon. Representatives of these genes were cloned into plasmids, and the marbox-like sequences were modified by site-directed mutagenesis. It was proven that mutations in these regions substantially repressed the level of proteins encoded by the downstream genes. Furthermore, plumbagin's early effect was demonstrated to robustly induce SoxS rather than MarA, an observation distinctly different from that seen with sodium salicylate.
High-Affinity Vanadate Transport System in the CyanobacteriumAnabaena variabilisATCC 29413
Authors: Pratte Brenda S. , Thiel Teresa
Published: December 2005

High-affinity vanadate transport systems have not heretofore been identified in any organism.Anabaena variabilis, which can fix nitrogen by using an alternative V-dependent nitrogenase, transported vanadate well. The concentration of vanadate giving half-maximum V-nitrogenase activity when added to V-starved cells was about 3 × 10−9M. The genes for an ABC-type vanadate transport system,vupABC, were found inA. variabilisabout 5 kb from the major cluster of genes encoding the V-nitrogenase, and like those genes, thevupABCgenes were repressed by molybdate; however, unlike the V-nitrogenase genes the vanadate transport genes were expressed in vegetative cells. AvupBmutant failed to grow by using V-nitrogenase unless high levels of vanadate were provided, suggesting that there was also a low-affinity vanadate transport system that functioned in thevupBmutant. ThevupABCgenes belong to a family of putative metal transport genes that include only one other characterized transport system, the tungstate transport genes ofEubacterium acidaminophilum. Similar genes are not present in the complete genomes of other bacterial strains that have a V-nitrogenase, includingAzotobacter vinelandii,Rhodopseudomonas palustris, andMethanosarcina barkeri.
Biochemical and Structural Characterization of an Essential Acyl Coenzyme A Carboxylase from
Mycobacterium tuberculosis
Authors: Gago Gabriela , Kurth Daniel , Diacovich Lautaro , Tsai Shiou-Chuan , Gramajo Hugo
Published: December 2005

Pathogenic mycobacteria contain a variety of unique fatty acids that have methyl branches at an even-numbered position at the carboxyl end and a long
n
-aliphatic chain. One such group of acids, called mycocerosic acids, is found uniquely in the cell wall of pathogenic mycobacteria, and their biosynthesis is essential for growth and pathogenesis. Therefore, the biosynthetic pathway of the unique precursor of such lipids, methylmalonyl coenzyme A (CoA), represents an attractive target for developing new antituberculous drugs. Heterologous protein expression and purification of the individual subunits allowed the successful reconstitution of an essential acyl-CoA carboxylase from
Mycobacterium tuberculosis
, whose main role appears to be the synthesis of methylmalonyl-CoA. The enzyme complex was reconstituted from the α biotinylated subunit AccA3, the carboxyltransferase β subunit AccD5, and the ε subunit AccE5 (Rv3281). The kinetic properties of this enzyme showed a clear substrate preference for propionyl-CoA compared with acetyl-CoA (specificity constant fivefold higher), indicating that the main physiological role of this enzyme complex is to generate methylmalonyl-CoA for the biosynthesis of branched-chain fatty acids. The α and β subunits are capable of forming a stable α6-β6 subcomplex but with very low specific activity. The addition of the ε subunit, which binds tightly to the α-β subcomplex, is essential for gaining maximal enzyme activity.
Rap Phosphatase of Virulence Plasmid pXO1 InhibitsBacillus anthracisSporulation
Authors: Bongiorni Cristina , Stoessel Ricarda , Shoemaker Dorinda , Perego Marta
Published: December 2005

This study shows that theBacillus anthracispXO1 virulence plasmid carries a Rap-Phr system, BXA0205, which regulates sporulation initiation in this organism. The BXA0205Rap protein was shown to dephosphorylate the Spo0F response regulator intermediate of the phosphorelay signal transduction system that regulates the initiation of the developmental pathway in response to environmental, metabolic, and cell cycle signals. The activity of the Rap protein was shown to be inhibited by the carboxy-terminal pentapeptide generated through an export-import processing pathway from the associated BXA0205Phr protein. Deregulation of the Rap activity by either overexpression or lack of the Phr pentapeptide resulted in severe inhibition of sporulation. Five additional Rap-Phr encoding systems were identified on the chromosome ofB. anthracis, one of which, BA3790-3791, also affected sporulation initiation. The results suggest that the plasmid-borne Rap-Phr system may provide a selective advantage to the virulence ofB. anthracis.
Stringent and Relaxed Recognition of
oriT
by Related Systems for Plasmid Mobilization: Implications for Horizontal Gene Transfer
Authors: Jandle Sarah , Meyer Richard
Published: December 2005

The plasmids R1162 and pSC101 have origins of conjugative transfer (
oriT
s) and corresponding relaxases that are closely related. The
oriT
s are made up of a highly conserved core, where DNA is cleaved by the relaxase prior to transfer, and an inverted repeat that differs in size and sequence. We show that in each case the seven base pairs adjacent to the core and within one arm of the inverted repeat are sufficient to determine specificity. Within this DNA there are three AT base pairs located 4 bp from the core. Mutations in the AT base pairs suggest that the relaxase makes essential contacts at these locations to the minor groove of the DNA. The remaining four bases are different for each
oriT
and are both necessary and sufficient for stringent recognition of
oriT
by the pSC101 mobilization proteins. In contrast, the R1162 mobilization proteins have a much more relaxed requirement for the base sequence of this specificity region. As a result, the R1162 mobilization proteins can initiate transfer from a variety of sites, including those derived from the chromosome. The R1162 mobilization proteins could therefore contribute to the horizontal gene transfer of DNA from diverse sources.
Mutational Analysis of the
Myxococcus xanthus
Ω4406 Promoter Region Reveals an Upstream Negative Regulatory Element That Mediates C-Signal Dependence
Authors: Viswanathan Kartik , Viswanathan Poorna , Kroos Lee
Published: December 2005

C signaling plays a key role in coordinating cell movement and differentiation during the multicellular developmental process of
Myxococcus xanthus
. C signaling regulates expression of genes induced after about 6 h into development, when cells are forming mounds. One gene whose expression depends absolutely on C signaling was identified by insertion of a transposable element at site Ω4406 which generated a transcriptional fusion between
lacZ
and an upstream promoter. We have investigated regulation of the Ω4406 promoter. A 5′ deletion revealed a negative regulatory element located between bp −533 and −100 relative to the transcriptional start site. In the absence of this element, the promoter was still developmentally regulated but about fourfold more active. Also, the truncated promoter region retained normal dependence on two developmental regulators, FruA and DevS, but lost its dependence on the C-signaling protein CsgA. We infer that C signaling partially overcomes the negative effect of the upstream element on activity of the Ω4406 promoter. Deletion of downstream DNA between bp 50 and 140 caused a threefold loss in expression, suggesting that a positive regulatory element lies in this region. Additional positive and negative regulatory elements are present in the region from bp −69 to −49, based on the effects of multiple-base-pair mutations. Within this region, a 5-bp element and a C-box-like sequence resemble sequences found in other developmentally regulated
M. xanthus
promoter regions, but the effects of single-base-pair changes in these sequences suggest that each functions uniquely. We conclude that regulation of the Ω4406 promoter involves multiple positive and negative regulatory elements located upstream and downstream of the region typically bound by RNA polymerase.
Genes for Small, Noncoding RNAs under Sporulation Control in
Bacillus subtilis
Authors: Silvaggi Jessica M. , Perkins John B. , Losick Richard
Published: December 2005

The process of sporulation in the bacterium
Bacillus subtilis
is known to involve the programmed activation of several hundred protein-coding genes. Here we report the discovery of previously unrecognized genes under sporulation control that specify small, non-protein-coding RNAs (sRNAs). Genes for sRNAs were identified by transcriptional profiling with a microarray bearing probes for intergenic regions in the genome and by use of a comparative genomics algorithm that predicts regions of conserved RNA secondary structure. The gene for one such sRNA, SurA, which is located in the region between
yndK
and
yndL
, was induced at the start of development under the indirect control of the master regulator for entry into sporulation, Spo0A. The gene for a second sRNA, SurC, located in the region between
dnaJ
and
dnaK
, was switched on at a late stage of sporulation by the RNA polymerase sigma factor σ
K
, which directs gene transcription in the mother cell compartment of the developing sporangium. Finally, a third intergenic region, that between
polC
and
ylxS
, which specified several sRNAs, including two transcripts produced under the control of the forespore-specific sigma factor σ
G
and a third transcript generated by σ
K
, was identified. Our results indicate that the full repertoire of sporulation-specific gene expression involves the activation of multiple genes for small, noncoding RNAs.