bluuebunny/crossref_metadata_2025 huggingface dataset:
Crystal structure of
Manduca sexta
prophenoloxidase provides insights into the mechanism of type 3 copper enzymes
Authors: Li Yongchao , Wang Yang , Jiang Haobo , Deng Junpeng
Published: September 2009

Arthropod phenoloxidase (PO) generates quinones and other toxic compounds to sequester and kill pathogens during innate immune responses. It is also involved in wound healing and other physiological processes. Insect PO is activated from its inactive precursor, prophenoloxidase (PPO), by specific proteolysis via a serine protease cascade. Here, we report the crystal structure of PPO from a lepidopteran insect at a resolution of 1.97 Å, which is the initial structure for a PPO from the type 3 copper protein family.
Manduca sexta
PPO is a heterodimer consisting of 2 homologous polypeptide chains, PPO1 and PPO2. The active site of each subunit contains a canonical type 3 di-nuclear copper center, with each copper ion coordinated with 3 structurally conserved histidines. The acidic residue Glu-395 located at the active site of PPO2 may serve as a general base for deprotonation of monophenolic substrates, which is key to the ortho-hydroxylase activity of PO. The structure provides unique insights into the mechanism by which type 3 copper proteins differ in their enzymatic activities, albeit sharing a common active center. A drastic change in electrostatic surface induced on cleavage at Arg-51 allows us to propose a model for localized PPO activation in insects.
BMP inhibition initiates neural induction via FGF signaling and Zic genes
Authors: Marchal Leslie , Luxardi Guillaume , Thomé Virginie , Kodjabachian Laurent
Published: September 2009

Neural induction is the process that initiates nervous system development in vertebrates. Two distinct models have been put forward to describe this phenomenon in molecular terms. The default model states that ectoderm cells are fated to become neural in absence of instruction, and do so when bone morphogenetic protein (BMP) signals are abolished. A more recent view implicates a conserved role for FGF signaling that collaborates with BMP inhibition to allow neural fate specification. Using the
Xenopus
embryo, we obtained evidence that may unite the 2 views. We show that a dominant-negative R-Smad, Smad5-somitabun—unlike the other BMP inhibitors used previously—can trigger conversion of
Xenopus
epidermis into neural tissue in vivo. However, it does so only if FGF activity is uncompromised. We report that this activity may be encoded by FGF4, as its expression is activated upon BMP inhibition, and its knockdown suppresses endogenous, as well as ectopic, neural induction by Smad5-somitabun. Supporting the importance of FGF instructive activity, we report the isolation of 2 immediate early neural targets,
zic3
and
foxD5a
. Conversely, we found that
zic1
can be activated by BMP inhibition in the absence of translation. Finally, Zic1 and Zic3 are required together for definitive neural fate acquisition, both in ectopic and endogenous situations. We propose to merge the previous models into a unique one whereby neural induction is controlled by BMP inhibition, which activates directly, and, via FGF instructive activity, early neural regulators such as Zic genes.
Structures of receptor complexes formed by hemagglutinins from the Asian Influenza pandemic of 1957
Authors: Liu Junfeng , Stevens David J. , Haire Lesley F. , Walker Philip A. , Coombs Peter J. , Russell Rupert J. , Gamblin Steven J. , Skehel John J.
Published: September 2009

The viruses that caused the three influenza pandemics of the twentieth century in 1918, 1957, and 1968 had distinct hemagglutinin receptor binding glycoproteins that had evolved the capacity to recognize human cell receptors. We have determined the structure of the H2 hemagglutinin from the second pandemic, the “Asian Influenza” of 1957. We compare it with the 1918 “Spanish Influenza” hemagglutinin, H1, and the 1968 “Hong Kong Influenza” hemagglutinin, H3, and show that despite its close overall structural similarity to H1, and its more distant relationship to H3, the H2 receptor binding site is closely related to that of H3 hemagglutinin. By analyzing hemagglutinins of potential H2 avian precursors of the pandemic virus, we show that the human receptor can be bound by avian hemagglutinins that lack the human–specific mutations of H2 and H3 pandemic viruses, Gln-226Leu, and Gly-228Ser. We show how Gln-226 in the avian H2 receptor binding site, together with Asn-186, form hydrogen bond networks through bound water molecules to mediate binding to human receptor. We show that the human receptor adopts a very similar conformation in both human and avian hemagglutinin-receptor complexes. We also show that Leu-226 in the receptor binding site of human virus hemagglutinins creates a hydrophobic environment near the Sia-1-Gal-2 glycosidic linkage that favors binding of the human receptor and is unfavorable for avian receptor binding. We consider the significance for the development of pandemics, of the existence of avian viruses that can bind to both avian and human receptors.
Natural and synthetic prion structure from X-ray fiber diffraction
Authors: Wille Holger , Bian Wen , McDonald Michele , Kendall Amy , Colby David W. , Bloch Lillian , Ollesch Julian , Borovinskiy Alexander L. , Cohen Fred E. , Prusiner Stanley B. , Stubbs Gerald
Published: September 2009

A conformational isoform of the mammalian prion protein (PrP
Sc
) is the sole component of the infectious pathogen that causes the prion diseases. We have obtained X-ray fiber diffraction patterns from infectious prions that show cross-β diffraction: meridional intensity at 4.8 Å resolution, indicating the presence of β strands running approximately at right angles to the filament axis and characteristic of amyloid structure. Some of the patterns also indicated the presence of a repeating unit along the fiber axis, corresponding to four β-strands. We found that recombinant (rec) PrP amyloid differs substantially from highly infectious brain-derived prions, both in structure as demonstrated by the diffraction data, and in heterogeneity as shown by electron microscopy. In addition to the strong 4.8 Å meridional reflection, the recPrP amyloid diffraction is characterized by strong equatorial intensity at approximately 10.5 Å, absent from brain-derived prions, and indicating the presence of stacked β-sheets. Synthetic prions recovered from transgenic mice inoculated with recPrP amyloid displayed structural characteristics and homogeneity similar to those of naturally occurring prions. The relationship between the structural differences and prion infectivity is uncertain, but might be explained by any of several hypotheses: only a minority of recPrP amyloid possesses a replication-competent conformation, the majority of recPrP amyloid has to undergo a conformational maturation to acquire replication competency, or inhibitory forms of recPrP amyloid interfere with replication during the initial transmission.
Differing requirements for actin and myosin by plant viruses for sustained intercellular movement
Authors: Harries Phillip A. , Park Jong-Won , Sasaki Nobumitsu , Ballard Kimberly D. , Maule Andrew J. , Nelson Richard S.
Published: September 2009

The actin cytoskeleton has been implicated in the intra- and intercellular movement of a growing number of plant and animal viruses. However, the range of viruses influenced by actin for movement and the mechanism of this transport are poorly understood. Here we determine the importance of microfilaments and myosins for the sustained intercellular movement of a group of RNA-based plant viruses. We demonstrate that the intercellular movement of viruses from different genera [tobacco mosaic virus (TMV), potato virus X (PVX), tomato bushy stunt virus (TBSV)], is inhibited by disruption of microfilaments. Surprisingly, turnip vein-clearing virus (TVCV), a virus from the same genus as TMV, did not require intact microfilaments for normal spread. To investigate the molecular basis for this difference we compared the subcellular location of GFP fusions to the 126-kDa protein and the homologous 125-kDa protein from TMV and TVCV, respectively. The 126-kDa protein formed numerous large cytoplasmic inclusions associated with microfilaments, whereas the 125-kDa protein formed few small possible inclusions, none associated with microfilaments. The dependence of TMV, PVX, and TBSV on intact microfilaments for intercellular movement led us to investigate the role of myosin motors in this process. Virus-induced gene silencing of the
Nicotiana benthamiana
myosin XI-2 gene, but not three other myosins, inhibited only TMV movement. These results indicate that RNA viruses have evolved differently in their requirements for microfilaments and the associated myosin motors, in a manner not correlated with predicted phylogeny.
The human insulin gene is part of a large open chromatin domain specific for human islets
Authors: Mutskov Vesco , Felsenfeld Gary
Published: September 2009

Knowledge of how insulin (INS) gene expression is regulated will lead to better understanding of normal and abnormal pancreatic β cell function. We have mapped histone modifications over the INS region, coupled with an expression profile, in freshly isolated islets from multiple human donors. Unlike many other human genes, in which active modifications tend to be concentrated within 1 kb around the transcription start site, these marks are distributed over the entire coding region of INS as well. Moreover, a region of ≈80 kb around the INS gene, which contains the {tyrosine hydroxylase (TH)–(INS)–insulin-like growth factor 2 antisense (IGF2AS)–insulin-like growth factor 2 (IGF2)} gene cluster, unusually is marked by almost uniformly elevated levels of histone acetylation and H3K4 dimethylation, extending both downstream into IGF2 and upstream beyond the TH gene. This is accompanied by islet specific coordinate expression with INS of the neighboring TH and IGF2 genes. The presence of islet specific intergenic transcripts suggests their possible function in the maintenance of this unusual large open chromatin domain.
Resolving the distinct stages in erythroid differentiation based on dynamic changes in membrane protein expression during erythropoiesis
Authors: Chen Ke , Liu Jing , Heck Susanne , Chasis Joel A. , An Xiuli , Mohandas Narla
Published: September 2009

Erythropoiesis is the process by which nucleated erythroid progenitors proliferate and differentiate to generate, every second, millions of nonnucleated red cells with their unique discoid shape and membrane material properties. Here we examined the time course of appearance of individual membrane protein components during murine erythropoiesis to throw new light on our understanding of the evolution of the unique features of the red cell membrane. We found that the accumulation of all of the major transmembrane and all skeletal proteins of the mature red blood cell, except actin, accrued progressively during terminal erythroid differentiation. At the same time, and in marked contrast, accumulation of various adhesion molecules decreased. In particular, the adhesion molecule, CD44 exhibited a progressive and dramatic decrease from proerythroblast to reticulocyte; this enabled us to devise a new strategy for distinguishing unambiguously between erythroblasts at successive developmental stages. These findings provide unique insights into the genesis of red cell membrane function during erythroblast differentiation and also offer a means of defining stage-specific defects in erythroid maturation in inherited and acquired red cell disorders and in bone marrow failure syndromes.
Whole-genome phylogeny of mammals: Evolutionary information in genic and nongenic regions
Authors: Sims Gregory E. , Jun Se-Ran , Wu Guohong Albert , Kim Sung-Hou
Published: September 2009

Ten complete mammalian genome sequences were compared by using the “feature frequency profile” (FFP) method of alignment-free comparison. This comparison technique reveals that the whole nongenic portion of mammalian genomes contains evolutionary information that is similar to their genic counterparts—the intron and exon regions. We partitioned the complete genomes of mammals (such as human, chimp, horse, and mouse) into their constituent nongenic, intronic, and exonic components. Phylogenic species trees were constructed for each individual component class of genome sequence data as well as the whole genomes by using standard tree-building algorithms with FFP distances. The phylogenies of the whole genomes and each of the component classes (exonic, intronic, and nongenic regions) have similar topologies, within the optimal feature length range, and all agree well with the evolutionary phylogeny based on a recent large dataset, multispecies, and multigene-based alignment. In the strictest sense, the FFP-based trees are genome phylogenies, not species phylogenies. However, the species phylogeny is highly related to the whole-genome phylogeny. Furthermore, our results reveal that the footprints of evolutionary history are spread throughout the entire length of the whole genome of an organism and are not limited to genes, introns, or short, highly conserved, nongenic sequences that can be adversely affected by factors (such as a choice of sequences, homoplasy, and different mutation rates) resulting in inconsistent species phylogenies.
Experimental evidence for the conditions necessary to sustain meandering in coarse-bedded rivers
Authors: Braudrick Christian A. , Dietrich William E. , Leverich Glen T. , Sklar Leonard S.
Published: September 2009

Meandering rivers are common on Earth and other planetary surfaces, yet the conditions necessary to maintain meandering channels are unclear. As a consequence, self-maintaining meandering channels with cutoffs have not been reproduced in the laboratory. Such experimental channels are needed to explore mechanisms controlling migration rate, sinuosity, floodplain formation, and planform morphodynamics and to test theories for wavelength and bend propagation. Here we report an experiment in which meandering with near-constant width was maintained during repeated cutoff and regeneration of meander bends. We found that elevated bank strength (provided by alfalfa sprouts) relative to the cohesionless bed material and the blocking of troughs (chutes) in the lee of point bars via suspended sediment deposition were the necessary ingredients to successful meandering. Varying flood discharge was not necessary. Scaling analysis shows that the experimental meander migration was fast compared to most natural channels. This high migration rate caused nearly all of the bedload sediment to exchange laterally, such that bar growth was primarily dependent on bank sediment supplied from upstream lateral migration. The high migration rate may have contributed to the relatively low sinuosity of 1.19, and this suggests that to obtain much higher sinuosity experiments at this scale may have to be conducted for several years. Although patience is required to evolve them, these experimental channels offer the opportunity to explore several fundamental issues about river morphodynamics. Our results also suggest that sand supply may be an essential control in restoring self-maintaining, actively shifting gravel-bedded meanders.
Differential utilization of T cell receptor TCRα/TCRδ locus variable region gene segments is mediated by accessibility
Authors: Lee Yu Nee , Alt Frederick W. , Reyes Julia , Gleason Megan , Zarrin Ali A. , Jung David
Published: September 2009

T cell receptor (TCR) variable region exons are assembled from germline V, (D), and J gene segments, each of which is flanked by recombination signal (RS) sequences that are composed of a conserved heptamer, a spacer of 12 or 23 bp, and a characteristic nonamer. V(D)J recombination only occurs between V, D, and J segments flanked by RS sequences that contain, respectively, 12(12-RS)- and 23(23-RS)-bp spacers (12/23 rule). Additional mechanisms can restrict joining of 12/23 RS matched segments beyond the 12/23 rule (B12/23). The TCRδ locus is contained within the TCRα locus; TCRα variable region exons are encoded by TRAV and TRAJ segments and those of TCRδ by TRDV, TRDD, and TRDJ segments. On the basis of the 12/23 rule, both TRAV and TRDV gene segments are compatible to rearrange with TRDD gene segments; however, TRAV-to-TRDD joins are not observed in vivo. Absence of TRAV-to-TRDD rearrangement might be explained either by B12/23 restriction or by differential accessibility of the TRDV versus TRAV gene segments for rearrangement to TRDD. We used in vitro substrate analysis to reveal that both TRAV and TRDV 23-RSs mediate rearrangements to the 5′TRDD1 12-RS, demonstrating that B12/23 restriction does not explain these rearrangement biases. However, targeted replacement of TRDD1 and its 12-RSs with TRAJ38 and its 12-RS showed that TRDV gene segments rearrange with the ectopic TRAJ38, whereas TRAV segments do not. Our results demonstrate that sorting of TRAV and TRDV gene segments is determined by differential locus accessibility during T cell development.
Cargo binding induces dimerization of myosin VI
Authors: Phichith Denis , Travaglia Mirko , Yang Zhaohui , Liu Xiaoyan , Zong Alan B. , Safer Daniel , Sweeney H. Lee
Published: September 2009

Although myosin VI has properties that would allow it to function optimally as a dimer, full-length myosin VI exists as a monomer in isolation. Based on the ability of myosin VI monomers to dimerize when held in close proximity, we postulated that cargo binding normally regulates dimerization of myosin VI. We tested this hypothesis by expressing a known dimeric cargo adaptor protein of myosin VI, optineurin, and the myosin VI-binding segment from a monomeric cargo adaptor protein, Dab2. In the presence of these adaptor proteins, full-length myosin VI has ATPase properties of a dimer, appears as a dimer in electron micrographs, and moves processively on actin filaments. The results support a model in which cargo binding exposes internal dimerization sequences within full-length myosin VI. Because, unexpectedly, a monomeric fragment of Dab2 triggers dimerization, it would appear that myosin VI is designed to function as a dimer in cells.
POLITICAL ECONOMY OF INFORMALIZATION
Authors: Slavnic Zoran
Published: September 2009

This article has two main aims. The first is to problematize the dominant view of the informal economy as a sort of separate economy, related primarily to (immigrant) small business and distinct from the so-called formal economy, which for the most part encompasses big companies as well as state economic activities. In contrast, the present article assumes that all economic actors are increasingly ready to adopt informal economic strategies to secure their economical survival. In line with this assumption, the second aim of the article is to contribute to our knowledge of the causes of, as well as the actors within, the current informalization trends that characterize Western economies. The article concludes that the informalization of contemporary advanced economies in general terms is a result of a structural conflict between new economic trends and old regulatory frameworks. These frameworks, with their focus on decommodification, have become too restrictive for new forms of capital accumulation, with their focus on flexible adaptation, which include an increasing demand for the re-commodification of labour. The conflict emerges and intensifies, among other reasons, because of the radically different internal operational logics, agendas and priorities that characterize these two social processes.
Greater efficiency in attentional processing related to mindfulness meditation
Authors: van den Hurk Paul A. M. , Giommi Fabio , Gielen Stan C. , Speckens Anne E. M. , Barendregt Henk P.
Published: September 2009

In this study, attentional processing in relation to mindfulness meditation was investigated. Since recent studies have suggested that mindfulness meditation may induce improvements in attentional processing, we have tested 20 expert mindfulness meditators in the attention network test. Their performance was compared to that of 20 age- and gender-matched controls. In addition to attentional network analyses, overall attentional processing was analysed by means of efficiency scores (i.e., accuracy controlled for reaction time). Better orienting and executive attention (reflected by smaller differences in either reaction time or error score, respectively) were observed in the mindfulness meditation group. Furthermore, extensive mindfulness meditation appeared to be related to a reduction of the fraction of errors for responses with the same reaction time. These results provide new insights into differences in attentional processing related to mindfulness meditation and suggest the possibility of increasing the efficiency in attentional processing by extensive mental training.
DNA replication times the cell cycle and contributes to the mid-blastula transition in Drosophila embryos
Authors: McCleland Mark L. , Shermoen Antony W. , O'Farrell Patrick H.
Published: September 2009

We examined the contribution of S phase in timing cell cycle progression during Drosophila embryogenesis using an approach that deletes S phase rather than arresting its progress. Injection of Drosophila Geminin, an inhibitor of replication licensing, prevented subsequent replication so that the following mitosis occurred with uninemic chromosomes, which failed to align. The effect of S phase deletion on interphase length changed with development. During the maternally regulated syncytial blastoderm cycles, deleting S phase shortened interphase, and deletion of the last of blastoderm S phase (cycle 14) induced an extra synchronous division and temporarily deferred mid-blastula transition (MBT) events. In contrast, deleting S phase after the MBT in cycle 15 did not dramatically affect mitotic timing, which appears to retain its dependence on developmentally programmed zygotic transcription. We conclude that normal S phase and replication checkpoint activities are important timers of the undisturbed cell cycle before, but not after, the MBT.
Epidermal progenitors give rise to Merkel cells during embryonic development and adult homeostasis
Authors: Van Keymeulen Alexandra , Mascre Guilhem , Youseff Khalil Kass , Harel Itamar , Michaux Cindy , De Geest Natalie , Szpalski Caroline , Achouri Younes , Bloch Wilhelm , Hassan Bassem A. , Blanpain Cédric
Published: September 2009

Merkel cells (MCs) are located in the touch-sensitive area of the epidermis and mediate mechanotransduction in the skin. Whether MCs originate from embryonic epidermal or neural crest progenitors has been a matter of intense controversy since their discovery >130 yr ago. In addition, how MCs are maintained during adulthood is currently unknown. In this study, using lineage-tracing experiments, we show that MCs arise through the differentiation of epidermal progenitors during embryonic development. In adults, MCs undergo slow turnover and are replaced by cells originating from epidermal stem cells, not through the proliferation of differentiated MCs. Conditional deletion of the Atoh1/Math1 transcription factor in epidermal progenitors results in the absence of MCs in all body locations, including the whisker region. Our study demonstrates that MCs arise from the epidermis by an Atoh1-dependent mechanism and opens new avenues for study of MC functions in sensory perception, neuroendocrine signaling, and MC carcinoma.
Autophagy promotes synapse development in Drosophila
Authors: Shen Wei , Ganetzky Barry
Published: September 2009

Autophagy, a lysosome-dependent degradation mechanism, mediates many biological processes, including cellular stress responses and neuroprotection. In this study, we demonstrate that autophagy positively regulates development of the Drosophila melanogaster larval neuromuscular junction (NMJ). Autophagy induces an NMJ overgrowth phenotype closely resembling that of highwire (hiw), an E3 ubiquitin ligase mutant. Moreover, like hiw, autophagy-induced NMJ overgrowth is suppressed by wallenda (wnd) and by a dominant-negative c-Jun NH2-terminal kinase (bskDN). We show that autophagy promotes NMJ growth by reducing Hiw levels. Thus, autophagy and the ubiquitin–proteasome system converge in regulating synaptic development. Because autophagy is triggered in response to many environmental cues, our findings suggest that it is perfectly positioned to link environmental conditions with synaptic growth and plasticity.
Staphylococcus aureus synthesizes adenosine to escape host immune responses
Authors: Thammavongsa Vilasack , Kern Justin W. , Missiakas Dominique M. , Schneewind Olaf
Published: September 2009

Staphylococcus aureus infects hospitalized or healthy individuals and represents the most frequent cause of bacteremia, treatment of which is complicated by the emergence of methicillin-resistant S. aureus. We examined the ability of S. aureus to escape phagocytic clearance in blood and identified adenosine synthase A (AdsA), a cell wall–anchored enzyme that converts adenosine monophosphate to adenosine, as a critical virulence factor. Staphylococcal synthesis of adenosine in blood, escape from phagocytic clearance, and subsequent formation of organ abscesses were all dependent on adsA and could be rescued by an exogenous supply of adenosine. An AdsA homologue was identified in the anthrax pathogen, and adenosine synthesis also enabled escape of Bacillus anthracis from phagocytic clearance. Collectively, these results suggest that staphylococci and other bacterial pathogens exploit the immunomodulatory attributes of adenosine to escape host immune responses.
Detection of new protein domains using co-occurrence: application toPlasmodium falciparum
Authors: Terrapon Nicolas , Gascuel Olivier , Maréchal Éric , Bréehélin Laurent
Published: September 2009

Motivation: Hidden Markov models (HMMs) have proved to be a powerful tool for protein domain identification in newly sequenced organisms. However, numerous domains may be missed in highly divergent proteins. This is the case for Plasmodium falciparum proteins, the main causal agent of human malaria.

Results: We propose a method to improve the sensitivity of HMM domain detection by exploiting the tendency of the domains to appear preferentially with a few other favorite domains in a protein. When sequence information alone is not sufficient to warrant the presence of a particular domain, our method enables its detection on the basis of the presence of other Pfam or InterPro domains. Moreover, a shuffling procedure allows us to estimate the false discovery rate associated with the results. Applied to P.falciparum, our method identifies 585 new Pfam domains (versus the 3683 already known domains in the Pfam database) with an estimated error rate <20%. These new domains provide 387 new Gene Ontology (GO) annotations to the P.falciparum proteome. Analogous and congruent results are obtained when applying the method to related Plasmodium species (P.vivax and P.yoelii).

Availability:  Supplementary Material and a database of the new domains and GO predictions achieved on Plasmodium proteins are available at http://www.lirmm.fr/∼terrapon/codd/

Contact:  brehelin@lirmm.fr

Supplementary information:  Supplementary data are available at Bioinformatics online.
LIBRUS: combined machine learning and homology information for sequence-based ligand-binding residue prediction
Authors: Kauffman Chris , Karypis George
Published: September 2009

Motivation: Identifying residues that interact with ligands is useful as a first step to understanding protein function and as an aid to designing small molecules that target the protein for interaction. Several studies have shown that sequence features are very informative for this type of prediction, while structure features have also been useful when structure is available. We develop a sequence-based method, called LIBRUS, that combines homology-based transfer and direct prediction using machine learning and compare it to previous sequence-based work and current structure-based methods.

Results: Our analysis shows that homology-based transfer is slightly more discriminating than a support vector machine learner using profiles and predicted secondary structure. We combine these two approaches in a method called LIBRUS. On a benchmark of 885 sequence-independent proteins, it achieves an area under the ROC curve (ROC) of 0.83 with 45% precision at 50% recall, a significant improvement over previous sequence-based efforts. On an independent benchmark set, a current method, FINDSITE, based on structure features achieves an ROC of 0.81 with 54% precision at 50% recall, while LIBRUS achieves an ROC of 0.82 with 39% precision at 50% recall at a smaller computational cost. When LIBRUS and FINDSITE predictions are combined, performance is increased beyond either reaching an ROC of 0.86 and 59% precision at 50% recall.

Availability: Software developed for this study is available at http://bioinfo.cs.umn.edu/supplements/binf2009 along with Supplementary data on the study.

Contact:  kauffman@cs.umn.edu; karypis@cs.umn.edu
Bifidobacterial enolase, a cell surface receptor for human plasminogen involved in the interaction with the host
Authors: Candela Marco , Biagi Elena , Centanni Manuela , Turroni Silvia , Vici Manuela , Musiani Francesco , Vitali Beatrice , Bergmann Simone , Hammerschmidt Sven , Brigidi Patrizia
Published: September 2009

The interaction with the host plasminogen/plasmin system represents a novel component in the molecular cross-talk between bifidobacteria and human host. Here, we demonstrated that the plasminogen-binding bifidobacterial speciesB. longum,B. bifidum,B. breveandB. lactisshare the key glycolytic enzyme enolase as a surface receptor for human plasminogen. Enolase was visualized on the cell surface of the model strainB. lactisBI07. The His-tagged recombinant protein showed a high affinity for human plasminogen, with an equilibrium dissociation constant in the nanomolar range. By site-directed mutagenesis we demonstrated that the interaction between theB. lactisBI07 enolase and human plasminogen involves an internal plasminogen-binding site homologous to that of pneumococcal enolase. According to our data, the positively charged residues Lys-251 and Lys-255, as well as the negatively charged Glu-252, of theB. lactisBI07 enolase are crucial for plasminogen binding. Acting as a human plasminogen receptor, the bifidobacterial surface enolase is suggested to play an important role in the interaction process with the host.
Anchoring and length regulation of Porphyromonas gingivalis Mfa1 fimbriae by the downstream gene product Mfa2
Authors: Hasegawa Yoshiaki , Iwami Jun , Sato Keiko , Park Yoonsuk , Nishikawa Kiyoshi , Atsumi Tatsuo , Moriguchi Keiichi , Murakami Yukitaka , Lamont Richard J. , Nakamura Hiroshi , Ohno Norikazu , Yoshimura Fuminobu
Published: September 2009

Porphyromonas gingivalis, a causative agent of periodontitis, has at least two types of thin, single-stranded fimbriae, termed FimA and Mfa1 (according to the names of major subunits), which can be discriminated by filament length and by the size of their major fimbrilin subunits. FimA fimbriae are long filaments that are easily detached from cells, whereas Mfa1 fimbriae are short filaments that are tightly bound to cells. However, a P. gingivalis ATCC 33277-derived mutant deficient in mfa2, a gene downstream of mfa1, produced long filaments (10 times longer than those of the parent), easily detached from the cell surface, similar to FimA fimbriae. Longer Mfa1 fimbriae contributed to stronger autoaggregation of bacterial cells. Complementation of the mutant with the wild-type mfa2 allele in trans restored the parental phenotype. Mfa2 is present in the outer membrane of P. gingivalis, but does not co-purify with the Mfa1 fimbriae. However, co-immunoprecipitation demonstrated that Mfa2 and Mfa1 are associated with each other in whole P. gingivalis cells. Furthermore, immunogold microscopy, including double labelling, confirmed that Mfa2 was located on the cell surface and likely associated with Mfa1 fimbriae. Mfa2 may therefore play a role as an anchor for the Mfa1 fimbriae and also as a regulator of Mfa1 filament length. Two additional downstream genes (pgn0289 and pgn0290) are co-transcribed with mfa1 (pgn0287) and mfa2 (pgn0288), and proteins derived from pgn0289, pgn0290 and pgn0291 appear to be accessory fimbrial components.
The cellular level of O-antigen polymerase Wzy determines chain length regulation by WzzB and WzzpHS-2 in Shigella flexneri 2a
Authors: Carter Javier A. , Jiménez Juan C. , Zaldívar Mercedes , Álvarez Sergio A. , Marolda Cristina L. , Valvano Miguel A. , Contreras Inés
Published: September 2009

The lipopolysaccharide O antigen of Shigella flexneri 2a has two preferred chain lengths, a short (S-OAg) composed of an average of 17 repeated units and a very long (VL-OAg) of about 90 repeated units. These chain length distributions are controlled by the chromosomally encoded WzzB and the plasmid-encoded WzzpHS-2 proteins, respectively. In this study, genes wzzB, wzz
pHS-2 and wzy (encoding the O-antigen polymerase) were cloned under the control of arabinose- and rhamnose-inducible promoters to investigate the effect of varying their relative expression levels on O antigen polysaccharide chain length distribution. Controlled expression of the chain length regulators wzzB and wzz
pHS-2 revealed a dose-dependent production of each modal length. Increase in one mode resulted in a parallel decrease in the other, indicating that chain length regulators compete to control the degree of O antigen polymerization. Also, when expression of the wzy gene is low, S-OAg but not VL-OAg is produced. Production of VL-OAg requires high induction levels of wzy. Thus, the level of expression of wzy is critical in determining O antigen modal distribution. Western blot analyses of membrane proteins showed comparable high levels of the WzzB and WzzpHS-2 proteins, but very low levels of Wzy. In vivo cross-linking experiments and immunoprecipitation of membrane proteins did not detect any direct interaction between Wzy and WzzB, suggesting the possibility that these two proteins may not interact physically but rather by other means such as via translocated O antigen precursors.
Role of MexZ and PA5471 in transcriptional regulation of mexXY in Pseudomonas aeruginosa
Authors: Yamamoto Masaki , Ueda Atsuhisa , Kudo Makoto , Matsuo Yasuhiro , Fukushima Jun , Nakae Taiji , Kaneko Takeshi , Ishigatsubo Yoshiaki
Published: September 2009

MexXY, a drug efflux pump in Pseudomonas aeruginosa, confers resistance to aminoglycoside antibiotics. We recently reported that MexZ binds to the promoter region of the mexXY operon. Electrophoretic mobility shift assay (EMSA) using recombinant MexZ and oligonucleotide probes prepared from the intergenic region between mexZ and mexX revealed that MexZ binds to a 20 bp palindromic sequence. Culture of P. aeruginosa in the presence of tetracycline induced higher levels of MexX and MexZ, as measured by immunoblotting and EMSA, than in the absence of antibiotics. When MexZ was expressed by a mexZ expression plasmid, the plasmid-borne MexZ repressed drug-induced MexX production, further confirming that MexZ acts as a repressor of the mexXY operon. PA5471 protein has been reported to be essential for drug-induced MexXY production. Similarly to that report, we observed that plasmid-borne PA5471 induced both MexX and MexZ production in PAO1 cells. Interestingly, interaction between MexZ and PA5471 was observed in a yeast two-hybrid assay. Furthermore, EMSA and in vitro transcription assays revealed that interaction between PA5471 and MexZ reduced MexZ DNA-binding ability, leading to mexXY transcription. These findings contribute to the understanding of the molecular mechanisms underlying the transcriptional regulation of mexZ and mexXY by drug-induced PA5471 expression.
Deletion of Braun lipoprotein gene (lpp) and curing of plasmid pPCP1 dramatically alter the virulence of Yersinia pestis CO92 in a mouse model of pneumonic plague
Authors: Agar Stacy L. , Sha Jian , Baze Wallace B. , Erova Tatiana E. , Foltz Sheri M. , Suarez Giovanni , Wang Shaofei , Chopra Ashok K.
Published: September 2009

Deletion of the murein (Braun) lipoprotein gene,lpp, attenuates theYersinia pestisCO92 strain in mouse models of bubonic and pneumonic plague. In this report, we characterized the virulence of strains from which the plasminogen activating protease (pla)-encoding pPCP1 plasmid was cured from either the wild-type (WT) or the Δlppmutant strain ofY. pestisCO92 in the mouse model of pneumonic infection. We noted a significantly increased survival rate in mice infected with theY. pestispPCP−/Δlppmutant strain up to a dose of 5000 LD50. Additionally, mice challenged with the pPCP−/Δlppstrain had substantially less tissue injury and a strong decrease in the levels of most cytokines and chemokines in tissue homogenates and sera when compared with the WT-infected group. Importantly, theY. pestispPCP−/Δlppmutant strain was detectable in high numbers in the livers and spleens of some of the infected mice. In the lungs of pPCP−/Δlppmutant-challenged animals, however, bacterial numbers dropped at 48 h after infection when compared with tissue homogenates from 1 h post-infection. Similarly, we noted that this mutant was unable to survive within murine macrophages in anin vitroassay, whereas survivability of the pPCP−mutant within the macrophage environment was similar to that of the WT. Taken together, our data indicated that a significant and possibly synergistic attenuation in bacterial virulence occurred in a mouse model of pneumonic plague when both thelppgene and the virulence plasmid pPCP1 encoding theplagene were deleted fromY. pestis.
The enzyme phosphoglucomutase (Pgm) is required by Salmonella enterica serovar Typhimurium for O-antigen production, resistance to antimicrobial peptides and in vivo fitness
Authors: Paterson G. K. , Cone D. B. , Peters S. E. , Maskell D. J.
Published: September 2009

The enzyme phosphoglucomutase (Pgm) catalyses the interconversion of glucose 1-phosphate and glucose 6-phosphate and contributes to glycolysis and the generation of sugar nucleotides for biosynthesis. To assess the role of this enzyme in the biology of the pathogen Salmonella enterica serovar Typhimurium we have characterized a pgm deletion mutant in strain SL1344. Compared to SL1344, SL1344 pgm had impaired growth in vitro, was deficient in the ability to utilize galactose as a carbon source and displayed reduced O-antigen polymer length. The mutant was also more susceptible to antimicrobial peptides and showed decreased fitness in the mouse typhoid model. The in vivo phenotype of SL1344 pgm indicated a role for pgm in the early stages of infection, most likely through deficient O-antigen production. Although pgm mutants in other pathogens have potential as live attenuated vaccine strains, SL1344 pgm was not sufficiently attenuated for such use.
Genetic and biochemical identification of the chorismate mutase from Corynebacterium glutamicum
Authors: Li Pan-Pan , Liu Ya-Jun , Liu Shuang-Jiang
Published: September 2009

Chorismate mutase (CM) catalyses the rearrangement of chorismate to prephenate and is also the first and the key enzyme that diverges the shikimate pathway to either tryptophan (Trp) or phenylalanine (Phe) and tyrosine (Tyr).Corynebacterium glutamicumis one of the most important amino acid producers for the fermentation industry and has been widely investigated. However, the gene(s) encoding CM has not been experimentally identified inC. glutamicum. In this study, thencgl0819gene, which was annotated as ‘conserved hypothetical protein’ in theC. glutamicumgenome, was genetically characterized to be essential for growth in minimal medium, and a mutant deleted ofncgl0819was a Phe and Tyr auxotroph. Genetic cloning and expression ofncgl0819inEscherichia coliresulted in the formation of a new protein (NCgl0819) having CM activity. It was concluded thatncgl0819encoded the CM ofC. glutamicum(CM0819). CM0819 was demonstrated to be a homodimer and is a new member of the monofunctional CMs of the AroQ structural class. The CM0819 activity was not affected by Phe, Tyr or Trp. Two 3-deoxy-d-arabino-heptulosonate 7-phosphate (DAHP) synthases (DS0950 and DS2098, formerly NCgl0950 and NCgl2098) had been previously identified fromC. glutamicum. CM0819 significantly stimulated DAHP synthase (DS2098) activity. Physical interaction between CM0819 and DS2098 was observed. When CM0819 was present, DS2098 activity was subject to allosteric inhibition by chorismate and prephenate. Conserved hypothetical proteins homologous to CM0819 were identified in all knownCorynebacteriumgenomes, suggesting a universal occurrence of CM0819-like CMs in the genusCorynebacterium.
Characterization of the Aspergillus fumigatus phosphomannose isomerase Pmi1 and its impact on cell wall synthesis and morphogenesis
Authors: Fang Wenxia , Yu Xiaoying , Wang Bin , Zhou Hui , Ouyang Haomiao , Ming Jia , Jin Cheng
Published: September 2009

Phosphomannose isomerase (PMI) is an enzyme catalysing the interconversion of mannose 6-phosphate (Man-6-P) and fructose 6-phosphate (Fru-6-P). The reaction catalysed by PMI is the first committed step in the synthesis of mannose-containing sugar chains and provides a link between glucose metabolism and mannosylation. In this study, thepmi1gene was identified to encode PMI in the human fungal pathogenAspergillus fumigatus. Characterization ofA. fumigatusPmi1 expressed inEscherichia colirevealed that this PMI mainly catalysed the conversion of Fru-6-P to Man-6-P and that its binding affinity for Man-6-P was similar to that of yeast PMIs, but different to those of PMIs from bacteria or animals. Loss ofpmi1was lethal unless Man was provided in the growth medium. However, a Δpmi1mutant cell showed a significantly reduced growth rate at a high concentration of Man. Biochemical analysis revealed that both inadequate and replete Man led to an accumulation of intracellular Man-6-P and a reduction in the amount ofα-glucan in the cell wall. Uncoupling of the link between energy production and glycosylation by deletion of thepmi1gene led to phenotypes such as defects in cell wall integrity, abnormal morphology and reduced conidiation. Our results reveal that PMI activity is essential for viability and plays a central regulatory role in both cell wall synthesis and energy production inA. fumigatus.
Presence of the Fps1p aquaglyceroporin channel is essential for Hog1p activation, but suppresses Slt2(Mpk1)p activation, with acetic acid stress of yeast
Authors: Mollapour Mehdi , Shepherd Andrew , Piper Peter W.
Published: September 2009

When grown at pH 4.5,Saccharomyces cerevisiaeacquires a resistance to inhibitory acetic acid levels (∼0.1 M) by destabilizing Fps1p, the plasma membrane aquaglyceroporin that provides the main route for passive diffusional entry of this acid into the cell. Acetic acid stress transiently activates Hog1p mitogen-activated protein (MAP) kinase, which, in turn, phosphorylates Fps1p in order to target this channel for endocytosis and degradation in the vacuole. This activation of Hog1p is abolished with the loss of Fps1p, but is more sustained when cells express an open Fps1p channel refractory to destabilization. At neutral pH, much higher levels of acetate (∼0.5 M) are needed to inhibit growth. Under such conditions, the loss of Fps1p does not abolish, but merely slows, the activation of Hog1p. Acetate stress also activates the Slt2(Mpk1)p cell integrity MAP kinase, possibly by causing inhibition of glucan synthase activity. In pH 4.5 cultures, this acetate activation of Slt2p is strongly enhanced by the loss of Fps1p and is dependent upon the cell surface sensor Wsc1p. Lack of Fps1p therefore exerts opposing effects on the activation of Hog1p and Slt2p in yeast exposed to acetic acid stress.
Truncation in the core oligosaccharide of lipopolysaccharide affects flagella-mediated motility in Pseudomonas aeruginosa PAO1 via modulation of cell surface attachment
Authors: Lindhout Theresa , Lau Peter C. Y. , Brewer Dyanne , Lam Joseph S.
Published: September 2009

In many Gram-negative bacterial species, rough strains producing truncated lipopolysaccharide (LPS) generally exhibit defects in motility compared with smooth strains. However, the role that LPS plays in bacterial motility is not well understood. The goal of this study was to examine the relationship between LPS defects and motility of Pseudomonas aeruginosa. P. aeruginosa wild-type strain PAO1 and three isogenic mutants with defects in the rmlC, migA and wapR genes and producing truncated core oligosaccharide were investigated in terms of motility, attachment to glass and flagella expression. Compared with the wild-type, the three mutants showed significant retardation in both swarming motility on 0.5 % soft-agar plates and swimming motility on 0.3 % soft-agar plates. Moreover, attachment to abiotic surfaces was observed to be stronger in these mutants. The assembly of flagella appeared to be intact in these strains and the ability of individual cells to swim was unaffected. Flagellin proteins prepared from mutants rmlC and rmd, defective in the production of TDP-l-rhamnose and GDP-d-rhamnose, respectively, were compared and a change in molecular mass was observed only in the rmlC mutant. These data indicated that l-rhamnose, and not its enantiomer, d-rhamnose, is incorporated into the flagellin glycan of P. aeruginosa PAO1. The nucleotide-activated sugar precursor TDP-l-rhamnose is therefore shared between LPS biosynthesis and flagellin glycosylation in P. aeruginosa PAO1. Our results suggest that although biochemical precursors are shared by LPS and flagellin glycan biosynthesis, LPS truncations probably alter flagella-mediated motility in P. aeruginosa by modulating cell-surface attachment but not flagella synthesis.
A peroxiredoxin from Mycoplasma hyopneumoniae with a possible role in H2O2 detoxification
Authors: Machado Cláudio X. , Pinto Paulo M. , Zaha Arnaldo , Ferreira Henrique B.
Published: September 2009

Mycoplasma hyopneumoniaeis the causative agent of porcine enzootic pneumonia, which affects pig farms worldwide, causing heavy economic losses. In the infection process, this bacterium is exposed to reactive oxygen species (ROS) from its own metabolism or generated by the host as one of the strategies used to neutralize the pathogen. Although the presence of classical antioxidant enzymes would be expected inM. hyopneumoniae, important genes directly related to protection against ROS, such as superoxide dismutase, catalases and glutathione peroxidase, have not been identified by sequence homology in the genome sequence annotation. Among the few identifiedM. hyopneumoniaegenes coding for proteins possibly involved with suppression of ROS-mediated damage, one (tpx) coding for a peroxiredoxin (MhPrx) has been recognized. The sequence and phylogenetic analyses perfomed in this study indicate that MhPrx is closely related to the atypical 2-Cys peroxiredoxin subfamily, although it has only one cysteine in its sequence. The MhPrx coding DNA sequence was cloned and expressed inEscherichia colito produce a recombinant MhPrx (rMhPrx), which was purified and used to immunize mice and produce an anti-MhPrx polyclonal antiserum. Probing ofM. hyopneumoniaeextracts with this antiserum demonstrated that MhPrx is expressed in all three tested strains (J, 7422 and 7448). Cross-linking assays and size-exclusion chromatography indicate that rMhPrx forms dimers, as has been established for atypical 2-Cys peroxiredoxins. Furthermore, a metal-catalysed oxidation system was used to assay the activity of rMhPrx, showing that it can protect DNA from ROS-mediated damage and may play an essential role during infection.