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Klebsiella pneumoniae is an opportunistic pathogen, which concerns public health systems worldwide, as multiple antibiotic-resistant strains are frequent. One of its pathogenicity factors is the Type VI Secretion System (T6SS), a macromolecular complex assembled through the bacterial membranes. T6SS injects effector proteins inside target cells. Such effectors confer competitive advantages or modulate the target cell signaling and metabolism to favor bacterial infection. The VgrG protein is a T6SS core component. It may present a variable C-terminal domain carrying an additional effector function. Kp52.145 genome encodes three VgrG proteins, one of them with a C-terminal extension (VgrG4-CTD). VgrG4-CTD is 138 amino acids long, does not contain domains of known function, but is conserved in some Klebsiella, and non-Klebsiella species. To get insights into its function, recombinant VgrG4-CTD was used in pulldown experiments to capture ligands from macrophages and lung epithelial cells. A total of 254 proteins were identified: most of them are ribosomal proteins. Cytoskeleton-associated and proteins involved in the phagosome maturation pathway were also identified. We further showed that VgrG4-CTD binds actin and induces actin remodeling in macrophages. This study presents novel clues on the role of K. pneumoniae T6SS in pathogenesis.

Solid forms can influence the dissolution, stability, and processability of active pharmaceutical ingredients. However, besides the internal arrangement, crystal habit studies are important, since ...

International audience; There is increasing support for water molecules playing a role in signal propagation through G protein-coupled receptors (GPCRs). However, exploration of the hydration features of GPCRs is still in its infancy. Here, we combined site-specific labeling with unnatural amino acids to molecular dynamics to delineate how local hydration of the ghrelin receptor growth hormone secretagogue receptor (GHSR) is rearranged upon activation. We found that GHSR is characterized by a specific hydration pattern that is selectively remodeled by pharmacologically distinct ligands and by the lipid environment. This process is directly related to the concerted movements of the transmembrane domains of the receptor. These results demonstrate that the conformational dynamics of GHSR are tightly coupled to the movements of internal water molecules, further enhancing our understanding of the molecular bases of GPCR-mediated signaling.

Dorsal-ventral patterning of the Drosophila embryo depends on the NFκB superfamily transcription factor Dorsal (Dl). Toll receptor activation signals for degradation of the IκB inhibitor Cactus (Cact), leading to a ventral-to-dorsal nuclear Dl gradient. Cact is critical for Dl nuclear import, as it binds to and prevents Dl from entering the nuclei. Quantitative analysis of cact mutants revealed an additional Cact function to promote Dl nuclear translocation in ventral regions of the embryo. To investigate this dual Cact role, we developed a predictive model based on a reaction-diffusion regulatory network. This network distinguishes non-uniform Toll-dependent Dl nuclear import and Cact degradation, from the Toll-independent processes of Cact degradation and reversible nuclear-cytoplasmic Dl flow. In addition, it incorporates translational control of Cact levels by Dl. Our model successfully reproduces wild-type data and emulates the Dl nuclear gradient in mutant dl and cact allelic combinations. Our results indicate that the dual role of Cact depends on the dynamics of Dl-Cact trimers along the dorsal-ventral axis: In the absence of Toll activation, free Dl-Cact trimers retain Dl in the cytoplasm, limiting the flow of Dl into the nucleus; in ventral-lateral regions, Dl-Cact trimers are recruited by Toll activation into predominant signaling complexes and promote Dl nuclear translocation. Simulations suggest that the balance between Toll-dependent and Toll-independent processes are key to this dynamics and reproduce the full assortment of Cact effects. Considering the high evolutionary conservation of these pathways, our analysis should contribute to understanding NFκB/c-Rel activation in other contexts such as in the vertebrate immune system and disease., Author summary In Drosophila, Toll pathway establishes spatially distinct gene expression territories that define the embryonic dorsal-ventral axis. Toll activation leads to degradation of the IκB inhibitor Cactus, releasing the NFκB superfamily transcription factor Dorsal for nuclear entry. Recently, quantitative analysis of cact mutants revealed that Cact displays an additional function to promote Dl nuclear translocation in ventral regions of the embryo. To understand this novel activity, we developed a predictive theoretical model that shows that the kinetics of Dorsal-Cactus complex formation prior to their recruitment to Toll-signaling complexes is an essential regulatory hub. Cactus controls the balance between the recruitment of these complexes by active Toll receptor and association-dissociation events that generate free Dorsal for direct nuclear import.

Vibrio parahaemolyticus strains recovered from human diarrheal stools (one in 1975 and two in 2001) and environmental sources (four, between 2008 and 2010) were investigated for the presence of virulence genes (trh, tdh, and vpadF), pandemic markers (orf8, toxRSnew), and with respect to their pathogenic potential in two systemic infection models. Based only on the presence or absence of these genetic markers, they were classified as follows: the environmental strains were non-pathogenic, whereas among the clinical strains, the one isolated in 1975 was pathogenic (non-pandemic), and the other two were pathogenic (pandemic). The pathogenic potential of the strains was evaluated in mice and Galleria mellonella larvae infection models, and except for the clinical (pathogenic, non-pandemic) isolate, the others produced lethal infection in both organisms, regardless of their source, serotype, and genotype (tdh, orf8, toxRSnew, and vpadF). Based on mice and larval mortality rates, the strains were then grouped according to virulence (high, intermediate, and avirulent), and remarkably similar results were obtained by using these models: The clinical strain (pathogenic and non-pandemic) was classified as avirulent, and other strains (four non-pathogenic and two pandemic) were considered of high or intermediate virulence. In summary, these findings demonstrate that G. mellonella larvae can indeed be used as an alternative model to study the pathogenicity of V. parahaemolyticus. Moreover, they raise doubts about the use of traditional virulence markers to predict pathogenesis of the species and show that reliable models are indispensable to determine the pathogenic potential of environmental isolates considered non-pathogenic, based on the absence of the long-standing virulence indicators.

One of the most abundant proteins in V. cholerae O1 cells grown under inorganic phosphate (Pi) limitation is PstS, the periplasmic Pi-binding component of the high-affinity Pi transport system Pst2 (PstSCAB), encoded in pst2 operon (pstS-pstC2-pstA2-pstB2). Besides its role in Pi uptake, Pst2 has been also associated with V. cholerae virulence. However, the mechanisms regulating pst2 expression and the non-stoichiometric production of the Pst2 components under Pi-limitation are unknown. A computational-experimental approach was used to elucidate the regulatory mechanisms behind pst2 expression in V. cholerae O1. Bioinformatics analysis of pst2 operon nucleotide sequence revealed start codons for pstS and pstC genes distinct from those originally annotated, a regulatory region upstream pstS containing potential PhoB-binding sites and a pstS-pstC intergenic region longer than predicted. Analysis of nucleotide sequence between pstS-pstC revealed inverted repeats able to form stem-loop structures followed by a potential RNAse E-cleavage site. Another putative RNase E recognition site was identified within the pstA-pstB intergenic sequence. In silico predictions of pst2 operon expression regulation were subsequently tested using cells grown under Pi limitation by promoter-lacZ fusion, gel electrophoresis mobility shift assay and quantitative RT-PCR. The experimental and in silico results matched very well and led us to propose a pst2 promoter sequence upstream of pstS gene distinct from the previously annotated. Furthermore, V. cholerae O1 pst2 operon transcription is PhoB-dependent and generates a polycistronic mRNA molecule that is rapidly processed into minor transcripts of distinct stabilities. The most stable was the pstS-encoding mRNA, which correlates with PstS higher levels relative to other Pst2 components in Pi-starved cells. The relatively higher stability of pstS and pstB transcripts seems to rely on the secondary structures at their 3' untranslated regions that are known to block 3'-5' exonucleolytic attacks.

Modeling expression patterns of Drosophila, in space and time, plays a critical role to understand the development of multicellular organisms. In confocal microscopy, to produce precise quantitative data it is frequently necessary to process and analyze large amounts of digital images. Automatic preprocessing is a crucial step in this scenario, essential to standardize significant features such as orientation, size, position, direction, lighting condition and texture of embryo images. Even though a lot of efforts have been made, a robust embryo standardization strategy is still needed. In this paper, we propose the method Embrystandar. It is designed to remove background artifacts and standardize the direction and orientation of a Drosophila embryo through a sequence of automatic operations. To test its potential for large-scale image processing, Embrystandar was applied in different databases. It showed to be robust and precise, reaching more than 90% success rate.

Dengue is an important mosquito-borne disease and a global public health problem. The disease is caused by dengue virus (DENV), which is a member of the Flaviviridae family and contains a positive single-stranded RNA genome that encodes a single precursor polyprotein that is further cleaved into structural and non-structural proteins. Among these proteins, the non-structural 3 (NS3) protein is very important because it forms a non-covalent complex with the NS2B cofactor, thereby forming the functional viral protease. NS3 also contains a C-terminal ATPase/helicase domain that is essential for RNA replication. Here, we identified 47 NS3-interacting partners using the yeast two-hybrid system. Among those partners, we highlight several proteins involved in host energy metabolism, such as apolipoprotein H, aldolase B, cytochrome C oxidase and glyceraldehyde-3-phosphate dehydrogenase (GAPDH). GAPDH directly binds full-length NS3 and its isolated helicase and protease domains. Moreover, we observed an intense colocalization between the GAPDH and NS3 proteins in DENV2-infected Huh7.5.1 cells, in NS3-transfected BHK-21 cells and in hepatic tissue from a fatal dengue case. Taken together, these results suggest that the human GAPDH-DENV NS3 interaction is involved in hepatic metabolic alterations, which may contribute to the appearance of steatosis in dengue-infected patients. The interaction between GAPDH and full-length NS3 or its helicase domain in vitro as well as in NS3-transfected cells resulted in decreased GAPDH glycolytic activity. Reduced GAPDH glycolytic activity may lead to the accumulation of metabolic intermediates, shifting metabolism to alternative, non-glycolytic pathways. This report is the first to identify the interaction of the DENV2 NS3 protein with the GAPDH protein and to demonstrate that this interaction may play an important role in the molecular mechanism that triggers hepatic alterations.

High-mobility group B (HMGB) proteins have highly conserved, unique DNA-binding domains, HMG boxes, that can bind non-B-type DNA structures, such as bent, kinked and unwound structures, with high affinity. HMGB proteins also promote DNA bending, looping and unwinding. In this study, we determined the role of the Aedes aegypti single HMG-box domain protein AaHMGB; characterized its structure, spatiotemporal expression levels, subcellular localization, and nucleic acid binding activities; and compared these properties with those of its double-HMG-box counterpart protein, AaHMGB1. Via qRT-PCR, we showed that AaHMGB is expressed at much higher levels than AaHMGB1 throughout mosquito development. In situ hybridization results suggested a role for AaHMGB and AaHMGB1 during embryogenesis. Immunolocalization in the midgut revealed that AaHMGB is exclusively nuclear. Circular dichroism and fluorescence spectroscopy analyses showed that AaHMGB exhibits common features of α-helical structures and is more stably folded than AaHMGB1, likely due to the presence of one or two HMG boxes. Using several DNA substrates or single-stranded RNAs as probes, we observed significant differences between AaHMGB and AaHMGB1 in terms of their binding patterns, activity and/or specificity. Importantly, we showed that the phosphorylation of AaHMGB plays a critical role in its DNA-binding activity. Our study provides additional insight into the roles of single- versus double-HMG-box-containing proteins in nucleic acid interactions for better understanding of mosquito development, physiology and homeostasis.

Cyanobacteria tend to become the dominant phytoplankton component in eutrophic freshwater environments during warmer seasons. However, general observations of cyanobacterial adaptive advantages in these circumstances are insufficient to explain the prevalence of one species over another in a bloom period, which may be related to particular strategies and interactions with other components of the plankton community. In this study, we present an integrative view of a mixed cyanobacterial bloom occurring during a warm, rainy period in a tropical hydropower reservoir. We used high-throughput sequencing to follow temporal shifts in the dominance of cyanobacterial genera and shifts in the associated heterotrophic bacteria community. The bloom occurred during late spring-summer and included two distinct periods. The first period corresponded to Microcystis aeruginosa complex (MAC) dominance with a contribution from Dolichospermum circinale; this pattern coincided with high water retention time and low transparency. The second period corresponded to Cylindrospermopsis raciborskii and Synechococcus spp. dominance, and the reservoir presented lower water retention time and higher water transparency. The major bacterial phyla were primarily Cyanobacteria and Proteobacteria, followed by Actinobacteria, Bacteroidetes, Verrucomicrobia, and Planctomycetes. Temporal shifts in the dominance of cyanobacterial genera were associated with physical features of the water (retention time and transparency) and with shifts in the associated heterotrophic bacteria. The MAC bloom was associated with a high abundance of Bacteroidetes, particularly Cytophagales. In the second bloom period, Planctomycetes increased in relative abundance, five Planctomycetes OTUs were positively correlated with Synechococcus or C. raciborskii OTUs. Our results suggest specific interactions of the main cyanobacterial genera with certain groups of the heterotrophic bacterial community; the results suggest that considering biotic interactions may lead to a better understanding of the shifts in cyanobacterial dominance.

Ornithine lipids (OLs) are phosphorus-free lipids found in many bacteria grown under phosphate deprivation, a condition that activates the PhoBR system and leads to phosphate uptake and metabolism. Two OL synthesis pathways have already been described. One depends on OlsB and OlsA acyltransferases to add, respectively, the first and second acyl chains to an ornithine molecule. The other pathway is carried out by OlsF, a bifunctional enzyme responsible for both acylation steps. Although Vibrio cholerae lacks olsBA genes, an olsF homologue (vc0489) was identified in its genome. In this work we demonstrated that V. cholerae produces OLs and expresses vc0489 in response to phosphate depletion, in a PhoBR-dependent manner. In Escherichia coli, under similar condition, vc0489 expression leads to OL accumulation. These results indicate a strong connection between OL synthesis and VC0489 from V. cholerae and, for the first time, a direct regulation of an olsF homologue by the PhoBR system.

Proteins are found in solution as ensembles of conformations in dynamic equilibrium. Exploration of functional motions occurring on micro- to millisecond time scales by molecular dynamics (MD) simulations still remains computationally challenging. Alternatively, normal mode (NM) analysis is a well-suited method to characterize intrinsic slow collective motions, often associated with protein function, but the absence of anharmonic effects preclude a proper characterization of conformational distributions in a multidimensional NM space. Using both methods jointly appears to be an attractive approach that allows an extended sampling of the conformational space. In line with this view, the MDeNM (molecular dynamics with excited normal modes) method presented here consists of multiple-replica short MD simulations in which motions described by a given subset of low-frequency NMs are kinetically excited. This is achieved by adding additional atomic velocities along several randomly determined linear combinations of NM vectors, thus allowing an efficient coupling between slow and fast motions. The relatively high-energy conformations generated with MDeNM are further relaxed with standard MD simulations, enabling free energy landscapes to be determined. Two widely studied proteins were selected as examples: hen egg lysozyme and HIV-1 protease. In both cases, MDeNM provides a larger extent of sampling in a few nanoseconds, outperforming long standard MD simulations. A high degree of correlation with motions inferred from experimental sources (X-ray, EPR, and NMR) and with free energy estimations obtained by metadynamics was observed. Finally, the large sets of conformations obtained with MDeNM can be used to better characterize relevant dynamical populations, allowing for a better interpretation of experimental data such as SAXS curves and NMR spectra.

A putative porin function has been assigned to VCA1008 of Vibrio cholerae. Its coding gene, vca1008, is expressed upon colonization of the small intestine in infant mice and human volunteers, and is essential for infection. In vitro, vca1008 is expressed under inorganic phosphate limitation and, in this condition, VCA1008 is the major outer membrane protein of the bacterium. Here, we provide the first functional characterization of VCA1008 reconstituted into planar lipid bilayers. Our main findings were: 1) VCA1008 forms an ion channel that, at high voltage (~ ± 100mV), presents a voltage-dependent activity and displays closures typical of trimeric porins, with a conductance of 4.28 ± 0.04 nS (n = 164) in 1M KCl; 2) It has a preferred selectivity for anions over cations; 3) Its conductance saturates with increasing inorganic phosphate concentration, suggesting VCA1008 contains binding site(s) for this anion; 4) Its ion selectivity is controlled by both fixed charged residues within the channel and diffusion along the pore; 5) Partitioning of poly (ethylene glycol)s (PEGs) of different molecular mass suggests that VCA1008 channel has a pore exclusion limit of 0.9 nm.

In the study of cyanobacterial communities, molecular methods can be used to detect and quantify toxin-producing strains and assess genotype diversity. These are promising methods for estimating the risk of exposure to the cyanobacterial toxin microcystin (MC), assuming that the abundance of toxic genotypes is related to the MC concentration. This assumption has been tested in many studies, most of which were conducted in temperate and subtropical aquatic environments, and has not always been met. In this study, we collected cyanobacterial samples from a tropical reservoir and tested whether the MC concentration could be explained by variations in the number or ratio of toxin-producing genotypes or by the presence of certain Microcystis genotypes. The genetic diversity of cyanobacteria was determined by cpcBA sequencing, and the proportion of potential MC-producing genotypes was quantified by qPCR using mcyB as the target gene. Cyanobacteria dominated during the entire sampling period (88.4–99.8% of the total phytoplankton biomass), with Microcystis, Anabaena and Cylindrospermopsis occurring as the main genera. Three major Microcystis genotypes were recognized along with other rare or unique genotypes. MCs were detected in all samples (MC-LR, RR and YR), with most concentrations being less than 200 ng L−1. Microcystis was the most abundant group during all the study period and was the main MC-producing genus. Potentially toxic Microcystis cells were present in all samples, varying from 10% to 100% of the total Microcystis. MC concentrations were not correlated with the number or ratio of toxic cells of Microcystis determined by qPCR or with the predominance of certain genotypes. These results indicate the limitations of using molecular methods such as qPCR in MC risk assessment.

Ischaemic preconditioning (IPC) provides myocardial resistance to ischaemia/reperfusion (I/R) injuries. The protection afforded by IPC is not limited to the target tissue but extends to remote tissues, suggesting a mechanism mediated by humoral factors. The aim of the present study was to identify the humoral factors that are responsible for the cardioprotection induced by the coronary effluent transferred from IPC to naive hearts. Isolated rat hearts were submitted to IPC (three cycles of 5 min I/R) before 30-min global ischaemia and 60-min reperfusion. The coronary effluent (Efl_IPC) collected during IPC was fractionated by ultrafiltration in different molecular weight ranges ( 50 kDa) and evaluated for cardioprotective effects by perfusion before I/R in naive hearts. Only the

The evolutionarily conserved Toll signaling pathway controls innate immunity across phyla and embryonic patterning in insects. In the Drosophila embryo Toll is required to establish gene expression domains along the dorsal-ventral axis. Pathway activation induces degradation of the IκB inhibitor Cactus, resulting in a ventral-to-dorsal nuclear gradient of the NFκB effector Dorsal. Here we investigate how cactus modulates Toll signals through its effects on the Dorsal gradient and Dorsal target genes. Quantitative analysis using a series of loss and gain-of-function conditions shows that the ventral and lateral aspects of the Dorsal gradient can behave differently respective to Cactus fluctuations. In lateral and dorsal embryo domains loss of Cactus allows more Dorsal to translocate to the nucleus. Unexpectedly, cactus loss-of-function alleles decrease Dorsal nuclear localization ventrally, where Toll signals are high. Overexpression analysis suggests that this ability of Cactus to enhance Toll stems from mobilization of a free Cactus pool induced by the Calpain A protease. These results indicate that Cactus acts to bolster Dorsal activation, in addition to its role as an NFκB inhibitor, ensuring a correct response to Toll signals.

The evolutionarily conserved Toll signaling pathway controls innate immunity across phyla and embryonic patterning in insects. In the Drosophila embryo Toll is required to establish gene expression domains along the dorsal-ventral axis. Pathway activation induces degradation of the IκB inhibitor Cactus resulting in a nuclear gradient of the NFκB effector Dorsal. Here we investigate howcactusmodulates Toll signals through its effects on the Dorsal gradient and Dorsal target genes. Quantitative analysis using a series of loss and gain-of-function conditions shows that the ventral and lateral aspects of the Dorsal gradient behave differently respective to Cactus fluctuations. Unexpectedly, Cactus favors Dorsal nuclear localization required as response to high Toll signals at the ventral side of the embryo. Furthermore, N-terminal deleted Cactus mimics these effects, indicating that the ability of Cactus to favor Toll stems from mobilization of a free Cactus pool induced by the Calpain A protease. These results indicate that unexplored mechanisms are at play to ensure a correct response to high Toll signals.Summary:The IκB protein Cactus favors high Toll signals, revealing that the ventral and lateral aspects of the Dorsal/NFκB nuclear gradient behave differently respective to Cactus concentrations in the Drosophila embryo.

The evolutionarily conserved Toll signaling pathway controls innate immunity across phyla and embryonic patterning in insects. In the Drosophila embryo, Toll is required to establish gene expression domains along the dorsal-ventral axis. Pathway activation induces degradation of the IκB inhibitor Cactus, resulting in a ventral-to-dorsal nuclear gradient of the NFκB effector Dorsal. Here, we investigate how cactus modulates Toll signals through its effects on the Dorsal gradient and on Dorsal target genes. Quantitative analysis using a series of loss- and gain-of-function conditions shows that the ventral and lateral aspects of the Dorsal gradient can behave differently with respect to Cactus fluctuations. In lateral and dorsal embryo domains, loss of Cactus allows more Dorsal to translocate to the nucleus. Unexpectedly, cactus loss-of-function alleles decrease Dorsal nuclear localization ventrally, where Toll signals are high. Overexpression analysis suggests that this ability of Cactus to enhance Toll stems from the mobilization of a free Cactus pool induced by the Calpain A protease. These results indicate that Cactus acts to bolster Dorsal activation, in addition to its role as a NFκB inhibitor, ensuring a correct response to Toll signals.

Zinc finger proteins are widely found in eukaryotes, representing an important class of DNA-binding proteins frequently involved in transcriptional regulation. Zinc finger motifs are composed by two antiparallel β-strands and one α-helix, stabilized by a zinc ion coordinated by conserved histidine and cysteine residues. In Schistosoma mansoni, these regulatory proteins are known to modulate morphological and physiological changes, having crucial roles in parasite development. A previously described C(2)H(2) zinc finger protein, SmZF1, was shown to be present in cell nuclei of different life stages of S. mansoni and to activate gene transcription in a heterologous system. A high-quality SmZF1 tridimensional structure was generated using comparative modeling. Molecular dynamics simulations of the obtained structure revealed stability of the zinc fingers motifs and high flexibility on the terminals, comparable to the profile observed on the template X-ray structure based on thermal b-factors. Based on the protein tridimensional features and amino acid composition, we were able to characterize four C(2)H(2) zinc finger motifs, the first involved in protein-protein interactions while the three others involved in DNA binding. We defined a consensus DNA binding sequence using three distinct algorithms and further carried out docking calculations, which revealed the interaction of fingers 2-4 with the predicted DNA. A search for S. mansoni genes presenting putative SmZF1 binding sites revealed 415 genes hypothetically under SmZF1 control. Using an automatic annotation and GO assignment approach, we found that the majority of those genes code for proteins involved in developmental processes. Taken together, these results present a consistent base to the structural and functional characterization of SmZF1.

In Drosophila embryonic development, the Bicoid (Bcd) protein establishes positional information of downstream developmental genes like hunchback (hb), which has a strong anterior expression and a sharp on-off boundary in the mid-embryo. The role of Bcd cooperative binding in the positioning of the Hb pattern has been previously demonstrated. However, there are discrepancies in the reported results about the role of this mechanism in the sharp Hb border. Here, we determined the Hill coefficient (nH) required for Bcd to generate the sharp border of Hb in wild-type (WT) embryos. We found that an nH of approximately 6.3 (s.d. 1.4) and 10.8 (s.d. 4.0) is required to account for Hb sharpness at early and late cycle 14A, respectively. Additional mechanisms are possibly required because the high nH is likely unachievable for Bcd binding to the hb promoter. To test this idea, we determined the nH required to pattern the Hb profile of 15 embryos expressing an hb14F allele that is defective in self-activation and found nH to be 3.0 (s.d. 1.0). This result indicates that in WT embryos, the hb self-activation is important for Hb sharpness. Corroborating our results, we also found a progressive increase in the required value of nH spanning from 4.0 to 9.2 by determining this coefficient from averaged profiles of eight temporal classes at cycle 14A (T1 to T8). Our results indicate that there is a transition in the mechanisms responsible for the sharp Hb border during cycle 14A: in early stages of this cycle, Bcd cooperative binding is primarily responsible for Hb sharpness; in late cycle 14A, hb self-activation becomes the dominant mechanism.

RND (resistance-nodulation-division) transporters are found in several Gram-negative bacteria species. These proteins form a complex along with membrane fusion proteins and outer membrane factors. This complex acts as an efflux pump, transporting several different molecules directly from the cytoplasm to the extracellular medium. Although the crystallographic structure of each protein of the complex is known, the complete complex assembly is not yet fully characterized. In 2014, a model for the complete system, based upon a cryoelectron-microscopy map, was proposed. In the present work, we propose an alternative model that also satisfies the volume obtained from the cryoelectron-microscopy assay. In this model, the AcrA helical domains are interleaved to the helical domains of the TolC protein, increasing the diameter of the formed pore. We believe that this model represents a better model for RND-type efflux pump and might contribute to the characterization of this system.

International audience; Describing biological macromolecular energetics from computer simulations can pose major challenges, and often necessitates enhanced conformational sampling. We describe the calculation of conformational free-energy profiles along carefully chosen collective coordinates: “consensus” normal modes, developed recently as robust alternatives to conventional normal modes. In an application to the HIV-1 protease, we obtain efficient sampling of significant flap opening movements governing inhibitor binding from relatively short simulations, in close correspondence with experimental results.

In many cells, motility is mediated by flagellar beating. Protist parasites are capable of highly coordinated motility which contributes to their pathogenicity in mammalian hosts. Understanding the structural aspects of the flagellum may be important to the identification of novel targets for therapeutic intervention. Our group used atomic force microscopy (AFM) to examine the ultrastructure of Trypanosoma cruzi, obtaining valuable information on the organisation of the flagellar sub-structure. AFM images revealed novel flagellar components such as the presence of periodically-spaced protrusions organised along a flagellar furrow and oriented through the major flagellar axis between the axoneme and the paraflagellar rod. The nature and functional role of this structure are still unknown, although the hypothesis that the furrow might physically separate the two distinct domains of the flagellar membrane that comprise the surface of the axoneme and the paraflagellar rod (PFR) has been raised. To test whether the furrow was present or not only in PFR-bearing flagella, different protists containing or lacking the PFR, were analysed by AFM. Analysis of T. cruzi, Trypanosoma brucei and Herpetomonas megaseliae, which present distinct PFRs, showed similar and equivalent furrows along the main axis of their flagella, whereas Crithidia deanei, Giardia lamblia and Tritrichomonas foetus (in which the PFR is reduced or absent) lacked a furrow. Our results strongly suggest that the flagellar furrow is a characteristic feature of PFR-containing flagella and opens new perspectives for its functional role in the definition of sub-domains on the flagellar membrane.

The putative phosphoporin encoded by vca1008 of Vibrio cholerae O1 is expressed in vivo during infection and is essential for the intestinal colonization of infant mice. In vitro, its expression is induced under inorganic phosphate (Pi) limitation in a PhoB/R-dependent manner. In this work we demonstrated that VCA1008 has a strain-specific role in the physiology and pathogenicity of V. cholerae O1. Disruption of vca1008 led to a growth defect, an inability to colonize and a high susceptibility to sodium deoxycholate (DOC; the major bile compound) in the El Tor biotype strain N16961, but did not affect the classical strain O395 in the same way. Furthermore, vca1008 promoter activity was higher in N16961 cells grown under a low Pi supply in the presence of DOC than in the absence of the detergent. In the Pi-limited cells, vca1008 was positively regulated by PhoB, but when DOC was added to the medium, it negatively affected the PhoB-mediated activation of the gene, and enhanced vca1008 expression in a ToxR-dependent manner. These findings reveal for the first time a complex strain-specific interplay between ToxR and PhoB/R systems to control porin genes, as well as the influence of DOC on the expression of PhoB- and ToxR-regulated genes and pathogenesis in pandemic strains of V. cholerae.

The PhoB/PhoR-dependent response to inorganic phosphate (Pi)-starvation in Vibrio cholerae O1 includes the expression of vc0719 for the response regulator PhoB, vca0033 for an alkaline phosphatase and vca1008 for an outer membrane protein (OMP). Sequences with high identity to these genes have been found in the genome of clinical and environmental strains, suggesting that the Pi-starvation response in V. cholerae is well conserved. VCA1008, an uncharacterized OMP involved in V. cholerae pathogenicity, presents sequence similarity to porins of Gram-negative bacteria such as phosphoporin PhoE from Escherichia coli. A three-dimensional model shows that VCA1008 is a 16-stranded pore-forming beta-barrel protein that shares three of the four conserved lysine residues responsible for PhoE anionic specificity with PhoE. VCA1008 beta-barrel apparently forms trimers that collapse into monomers by heating. Properties such as heat modifiability and resistance to denaturation by sodium dodecyl sulfate at lower temperatures permitted us to suggest that VCA1008 is a classical porin, more precisely, a phosphoporin due to its Pi starvation-induced PhoB-dependent expression, demonstrated by electrophoretic mobility shift assay and promoter fusion-lacZ assays.

A proteomic view of G. diazotrophicus PAL5 at the exponential (E) and stationary phases (S) of cultures in the presence of low (L) and high levels (H) of combined nitrogen is presented. The proteomes analyzed on 2D-gels showed 131 proteins (42E+32S+29H+28L) differentially expressed by G. diazotrophicus, from which 46 were identified by combining mass spectrometry and bioinformatics tools. Proteins related to cofactor, energy and DNA metabolisms and cytoplasmic pH homeostasis were differentially expressed in E growth phase, under L and H conditions, in line with the high metabolic rate of the cells and the low pH of the media. Proteins most abundant in S-phase cells were stress associated and transporters plus transferases in agreement with the general phenomenon that binding protein-dependent systems are induced under nutrient limitation as part of hunger response. Cells grown in L condition produced nitrogen-fixation accessory proteins with roles in biosynthesis and stabilization of the nitrogenase complex plus proteins for protection of the nitrogenases from O(2)-induced inactivation. Proteins of the cell wall biogenesis apparatus were also expressed under nitrogen limitation and might function in the reshaping of the nitrogen-fixing G. diazotrophicus cells previously described. Genes whose protein products were detected in our analysis were mapped onto the chromosome and, based on the tendency of functionally related bacterial genes to cluster, we identified genes of particular pathways that could be organized in operons and are co-regulated. These results showed the great potential of proteomics to describe events in G. diazotrophicus cells by looking at proteins expressed under distinct growth conditions.

Cyclin-dependent kinases (CDKs) and their associated regulatory cyclins are central for timely regulation of cell-cycle progression. They constitute attractive pharmacological targets for development of anticancer therapeutics, since they are frequently deregulated in human cancers and contribute to sustained, uncontrolled tumor proliferation. Characterization of their structural/dynamic features is essential to gain in-depth insight into structure-activity relationships. In addition, the identification of druggable pockets or key intermediate conformations yields potential targets for the development of novel classes of inhibitors. Structural studies of CDK2/cyclin A have provided a wealth of information concerning monomeric/heterodimeric forms of this kinase. There is, however, much less structural information for other CDK/cyclin complexes, including CDK4/cyclin D1, which displays an alternative (open) position of the cyclin partner relative to CDK, contrasting with the closed CDK2/cyclin A conformation. In this study, we carried out normal-mode analysis and enhanced sampling simulations with our recently developed method, molecular dynamics with excited normal modes, to understand the conformational equilibrium on these complexes. Interestingly, the lowest-frequency normal mode computed for each complex described the transition between the open and closed conformations. Exploration of these motions with an explicit-solvent representation using molecular dynamics with excited normal modes confirmed that the closed conformation is the most stable for the CDK2/cyclin A complex, in agreement with their experimentally available structures. On the other hand, we clearly show that an open↔closed equilibrium may exist in CDK4/cyclin D1, with closed conformations resembling that captured for CDK2/cyclin A. Such conformational preferences may result from the distinct distributions of frustrated contacts in each complex. Using the same approach, the putative roles of the Thr160 phosphoryl group and the T-loop conformation were investigated. These results provide a dynamic view of CDKs revealing intermediate conformations not yet characterized for CDK members other than CDK2, which will be useful for the design of inhibitors targeting critical conformational transitions.

In this report the ORF PF2001 from the hyperthermophilic archaeon Pyrococcus furiosus was identified and its protein sequence was characterized in silico, revealing characteristics of the conserved domains of the dipeptidyl aminopeptidases, hydrolases of the α/β superfamily, esterases and lipases. In order to understand its function, the ORF PF2001 without the 60 bp of the 5′-terminus, responsible for encoding a signal peptide ( PF2001Δ60 ), was cloned and expressed in Escherichia coli BL21(DE3) pLysS, and the recombinant enzyme was characterized for esterase, lipase and protease activities. The total protein extract from E. coli harboring the plasmid containing the ORF PF2001Δ60 exhibited its highest activity towards the substrate 4-methylumbelliferyl-heptanoate (C7) and lower activities towards 4-methylumbelliferyl-acetate (C2) and 4-methylumbelliferyl-palmitate (C16). The enzyme was thermostable for 120 min at 75 °C and was completely inhibited by 1 mM PMSF. A theoretical structural model was constructed by comparative modeling using as template a prolyl oligopeptidase from Sus scrofa . Although no protease activity was detected a putative catalytic triad (Ser149, Asp233 and His264) with high similarity to the template was identified. The structural characteristics that confer enzymatic specificity to the P. furiosus enzyme are discussed. Taken together the data strongly suggest that ORF PF2001 from P. furiosus is responsible for encoding a novel esterase.

Recently the Generalized Genetic Algorithm has been proposed, based on genetic algorithms and Tsallis statistics. Here, we study molecular preferences of the simple molecules and polypeptides by coupling the Generalized Genetic Algorithm and both a classical force field and a semi-empirical quantum mechanics procedure. The main characteristics of this stochastic algorithm is the capability to find the greatest number of minima for a given energy in an N -dimensional hyper-surface supplying information about the visiting rates for each one of the conformational structures. The main advantage of this method over other global optimization procedures is that this approach provides additional information about visiting frequency of all local and global minima. These characteristics may provide the populations of each conformation without using the (quantum or classical) Monte Carlo procedure.