Complete genome sequence of a clinical Bordetella pertussis isolate from Brazil

There has been a resurgence in the number of pertussis cases in Brazil and around theworld. Here, the genome of a clinical Bordetella pertussis strain (Bz181) that wasrecently isolated in Brazil is reported. Analysis of the virulence-associated genesdefining the pre- and post-vaccination lineages revealed the presence of theprn2-ptxS1A-fim3B-ptxP3 allelic profile in Bz181, which is characteristic of thecurrent pandemic lineage. A putative metallo-β-lactamase gene presenting all of theconserved zinc-binding motifs that characterise the catalytic site was identified, inaddition to a multidrug efflux pump of the RND family that could confer resistance toerythromycin, which is the antibiotic of choice for treating pertussis disease.     

Molecular Evolution of the Two-Component System BvgAS Involved in Virulence Regulation in Bordetella

The whooping cough agent Bordetella pertussis is closely related to Bordetella bronchiseptica, which is responsible for chronic respiratory infections in various mammals and is occasionally found in humans, and to Bordetella parapertussis, one lineage of which causes mild whooping cough in humans and the other ovine respiratory infections. All three species produce similar sets of virulence factors that are co-regulated by the two-component system BvgAS. We characterized the molecular diversity of BvgAS in Bordetella by sequencing the two genes from a large number of diverse isolates. The response regulator BvgA is virtually invariant, indicating strong functional constraints. In contrast, the multi-domain sensor kinase BvgS has evolved into two different types. The pertussis type is found in B. pertussis and in a lineage of essentially human-associated B. bronchiseptica, while the bronchiseptica type is associated with the majority of B. bronchiseptica and both ovine and human B. parapertussis. BvgS is monomorphic in B. pertussis, suggesting optimal adaptation or a recent population bottleneck. The degree of diversity of the bronchiseptica type BvgS is markedly different between domains, indicating distinct evolutionary pressures. Thus, absolute conservation of the putative solute-binding cavities of the two periplasmic Venus Fly Trap (VFT) domains suggests that common signals are perceived in all three species, while the external surfaces of these domains vary more extensively. Co-evolution of the surfaces of the two VFT domains in each type and domain swapping experiments indicate that signal transduction in the periplasmic region may be type-specific. The two distinct evolutionary solutions for BvgS confirm that B. pertussis has emerged from a specific B. bronchiseptica lineage. The invariant regions of BvgS point to essential parts for its molecular mechanism, while the variable regions may indicate adaptations to different lifestyles. The repertoire of BvgS sequences will pave the way for functional analyses of this prototypic system.     

Genome-Wide Analysis of the Pho Regulon in a pstCA Mutant of Citrobacter rodentium

The phosphate-specific transport operon, pstSCAB-phoU, of Gram-negative bacteria is an essential part of the Pho regulon. Its key roles are to encode a high-affinity inorganic phosphate transport system and to prevent activation of PhoB in phosphate-rich environments. In general, mutations in pstSCAB-phoU lead to the constitutive expression of the Pho regulon. Previously, we constructed a pstCA deletion mutant of Citrobacter rodentium and found it to be attenuated for virulence in mice, its natural host. This attenuation was dependent on PhoB or PhoB-regulated gene(s) because a phoB mutation restored virulence for mice to the pstCA mutant. To investigate how downstream genes may contribute to the virulence of C. rodentium, we used microarray analysis to investigate global gene expression of C. rodentium strain ICC169 and its isogenic pstCA mutant when grown in phosphate-rich medium. Overall 323 genes of the pstCA mutant were differentially expressed by at least 1.5-fold compared to the wild-type C. rodentium. Of these 145 were up-regulated and 178 were down-regulated. Differentially expressed genes included some involved in phosphate homoeostasis, cellular metabolism and protein metabolism. A large number of genes involved in stress responses and of unknown function were also differentially expressed, as were some virulence-associated genes. Up-regulated virulence-associated genes in the pstCA mutant included that for DegP, a serine protease, which appeared to be directly regulated by PhoB. Down-regulated genes included those for the production of the urease, flagella, NleG8 (a type III-secreted protein) and the tad focus (which encodes type IVb pili in Yersinia enterocolitica). Infection studies using C57/BL6 mice showed that DegP and NleG8 play a role in bacterial virulence. Overall, our study provides evidence that Pho is a global regulator of gene expression in C. rodentium and indicates the presence of at least two previously unrecognized virulence determinants of C. rodentium, namely, DegP and NleG8.     

Identification of a Major QTL That Alters Flowering Time at Elevated [CO2] in Arabidopsis thaliana

Background

The transition from vegetative to reproductive stages marks a major milestone in plant development. It is clear that global change factors (e.g., increasing [CO₂] and temperature) have already had and will continue to have a large impact on plant flowering times in the future. Increasing atmospheric [CO₂] has recently been shown to affect flowering time, and may produce even greater responses than increasing temperature. Much is known about the genes influencing flowering time, although their relevance to changing [CO₂] is not well understood. Thus, we present the first study to identify QTL (Quantitative Trait Loci) that affect flowering time at elevated [CO₂] in Arabidopsis thaliana.

Methodology/Principal Findings

We developed our mapping population by crossing a genotype previously selected for high fitness at elevated [CO₂] (SG, Selection Genotype) to a Cape Verde genotype (Cvi-0). SG exhibits delayed flowering at elevated [CO₂], whereas Cvi-0 is non-responsive to elevated [CO₂] for flowering time. We mapped one major QTL to the upper portion of chromosome 1 that explains 1/3 of the difference in flowering time between current and elevated [CO₂] between the SG and Cvi-0 parents. This QTL also alters the stage at which flowering occurs, as determined from higher rosette leaf number at flowering in RILs (Recombinant Inbred Lines) harboring the SG allele. A follow-up study using Arabidopsis mutants for flowering time genes within the significant QTL suggests MOTHER OF FT AND TFL1 (MFT) as a potential candidate gene for altered flowering time at elevated [CO₂].

Conclusion/Significance

This work sheds light on the underlying genetic architecture that controls flowering time at elevated [CO₂]. Prior to this work, very little to nothing was known about these mechanisms at the genomic level. Such a broader understanding will be key for better predicting shifts in plant phenology and for developing successful crops for future environments.     

Acclimation of Photosynthesis to Elevated CO2 under Low-Nitrogen Nutrition Is Affected by the Capacity for Assimilate Utilization. Perennial Ryegrass under Free-Air CO2 Enrichment

Acclimation of photosynthesis to elevated CO₂ has previously been shown to be more pronounced when N supply is poor. Is this a direct effect of N or an indirect effect of N by limiting the development of sinks for photoassimilate? This question was tested by growing a perennial ryegrass (Lolium perenne) in the field under elevated (60 Pa) and current (36 Pa) partial pressures of CO₂ (pCO2) at low and high levels of N fertilization. Cutting of this herbage crop at 4- to 8-week intervals removed about 80% of the canopy, therefore decreasing the ratio of photosynthetic area to sinks for photoassimilate. Leaf photosynthesis, in vivo carboxylation capacity, carbohydrate, N, ribulose-1,5-bisphosphate carboxylase/oxygenase, sedoheptulose-1,7-bisphosphatase, and chloroplastic fructose-1,6-bisphosphatase levels were determined for mature lamina during two consecutive summers. Just before the cut, when the canopy was relatively large, growth at elevated pCO₂ and low N resulted in significant decreases in carboxylation capacity and the amount of ribulose-1,5-bisphosphate carboxylase/oxygenase protein. In high N there were no significant decreases in carboxylation capacity or proteins, but chloroplastic fructose-1,6-bisphosphatase protein levels increased significantly. Elevated pCO₂ resulted in a marked and significant increase in leaf carbohydrate content at low N, but had no effect at high N. This acclimation at low N was absent after the harvest, when the canopy size was small. These results suggest that acclimation under low N is caused by limitation of sink development rather than being a direct effect of N supply on photosynthesis.     

DYn-2 Based Identification of Arabidopsis Sulfenomes

Identifying the sulfenylation state of stressed cells is emerging as a strategic approach for the detection of key reactive oxygen species signaling proteins. Here, we optimized an in vivo trapping method for cysteine sulfenic acids in hydrogen peroxide (H₂O₂) stressed plant cells using a dimedone based DYn-2 probe. We demonstrated that DYn-2 specifically detects sulfenylation events in an H₂O₂ dose- and time-dependent way. With mass spectrometry, we identified 226 sulfenylated proteins after H₂O₂ treatment of Arabidopsis cells, residing in the cytoplasm (123); plastid (68); mitochondria (14); nucleus (10); endoplasmic reticulum, Golgi and plasma membrane (7) and peroxisomes (4). Of these, 123 sulfenylated proteins have never been reported before to undergo cysteine oxidative post-translational modifications in plants. All in all, with this DYn-2 approach, we have identified new sulfenylated proteins, and gave a first glance on the locations of the sulfenomes of Arabidopsis thaliana.Among the different amino acids, the sulfur containing amino acids like cysteine are particularly susceptible to oxidation by reactive oxygen species (ROS)¹ (1, 2). Recent studies suggest that the sulfenome, the initial oxidation products of cysteine residues, functions as an intermediate state of redox signaling (3 –5). Thus, identifying the sulfenome under oxidative stress is a way to detect potential redox sensors (6, 7).This central role of the sulfenome in redox signaling provoked chemical biologists to develop strategies for sensitive detection and identification of sulfenylated proteins. The in situ trapping of the sulfenome is challenging because of two major factors: (1) the highly reactive, transient nature of sulfenic acids, which might be over-oxidized in excess of ROS, unless immediately protected by disulfide formation (7); (2) the intracellular compartmentalization of the redox state that might be disrupted during extraction procedures, resulting in artificial non-native protein oxidations (8, 9). Having a sulfur oxidation state of zero, sulfenic acids can react as both electrophile and nucleophile, however, direct detection methods are based on the electrophilic character of sulfenic acid (10). In 1974, Allison and coworkers reported a condensation reaction between the electrophilic sulfenic acid and the nucleophile dimedone (5,5-dimethyl-1,3-cyclohexanedione), producing a corresponding thioether derivative (11). This chemistry is highly selective and, since then, has been exploited to detect dimedone modified sulfenic acids using mass spectrometry (12). However, dimedone has limited applications for cellular sulfenome identification because of the lack of a functional group to enrich the dimedone tagged sulfenic acids. Later, dimedone-biotin/fluorophores conjugates have been developed, which allowed sensitive detection and enrichment of sulfenic acid modified proteins (13 –15). This approach, however, was not always compatible with in vivo cellular sulfenome analysis, because the biotin/fluorophores-conjugated dimedone is membrane impermeable (9) and endogenous biotinylated proteins might appear as false positives.More recently, the Carroll lab has developed DYn-2, a sulfenic acid specific chemical probe. This chemical probe consists of two functional units: a dimedone scaffold for sulfenic acid recognition and an alkyne chemical handle for enrichment of labeled proteins (9). Once the sulfenic acids are tagged with the DYn-2 probe, they can be biotinylated through click chemistry (16). The click reaction used here is a copper (I)-catalyzed azide-alkyne cycloaddition reaction (17), also known as azide-alkyne Huisgen cycloaddition (16). With this chemistry, a complex is formed between the alkyne functionalized DYn-2 and the azide functionalized biotin. This biotin functional group facilitates downstream detection, enrichment, and mass spectrometry based identification (Fig. 1). In an evaluation experiment, DYn-2 was found to efficiently detect H₂O₂-dependent sulfenic acid modifications in recombinant glutathione peroxidase 3 (Gpx3) of budding yeast (18). Moreover, it was reported that DYn-2 is membrane permeable, non-toxic, and a non-influencer of the intracellular redox balance (17, 18). Therefore, DYn-2 has been suggested as a global sulfenome reader in living cells (17, 18), and has been applied to investigate epidermal growth factor (EGF) mediated protein sulfenylation in a human epidermoid carcinoma A431 cell line and to identify intracellular protein targets of H₂O₂ during cell signaling (17).Open in a separate windowFig. 1.Schematic views of the molecular mechanism of the DYn-2 probe and the strategy to identify DYn-2 trapped sulfenylated proteins. A, DYn-2 specifically detects sulfenic acid modifications, but no other thiol modifications. B, Biotinylation of the DYn-2 tagged proteins by click reaction. C, Once DYn-2 tagged proteins are biotinylated, a streptavidin-HRP (Strep-HRP) blot visualizes sulfenylation, or alternatively, after enrichment on avidin beads, proteins are identified by mass spectrometry analysis.Here, we selected the DYn-2 probe to identify the sulfenome in plant cells under oxidative stress. Through a combination of biochemical, immunoblot and mass spectrometry techniques, and TAIR10 database and SUBA3-software predictions, we can claim that DYn-2 is able to detect sulfenic acids on proteins located in different subcellular compartments of plant cells. We identified 226 sulfenylated proteins in response to an H₂O₂ treatment of Arabidopsis cell suspensions, of which 123 proteins are new candidates for cysteine oxidative post-translational modification (PTM) events.     

Identification of Novel Cholesterol-binding Regions in Kir2 Channels

Inwardly rectifying potassium (Kir) channels play an important role in setting the resting membrane potential and modulating membrane excitability. We have recently shown that cholesterol regulates representative members of the Kir family and that in the majority of the cases, cholesterol suppresses channel function. Furthermore, recent data indicate that cholesterol regulates Kir channels by specific sterol-protein interactions, yet the location of the cholesterol binding site in Kir channels is unknown. Using a combined computational-experimental approach, we show that cholesterol may bind to two nonanular hydrophobic regions in the transmembrane domain of Kir2.1 located between adjacent subunits of the channel. The location of the binding regions suggests that cholesterol modulates channel function by affecting the hinging motion at the center of the pore-lining transmembrane helix that underlies channel gating either directly or through the interface between the N and C termini of the channel.     

Identification and Analysis of Powdery Mildew‐Responsive miRNAs in Wheat

Wheat powdery mildew, caused by Blumeria graminis f. sp. tritici (Bgt), is one of the most consistently damaging diseases of common wheat worldwide and greatly affects crop productivity. Recently, several plant microRNAs (miRNAs) have been reported as gene expression regulators related to various adverse environments. However, up to now, less is known on the roles of miRNAs in powdery mildew infection response of wheat. In this study, miRNA expression patterns were investigated for identifying Bgt‐responsive miRNAs in wheat leaves using a plant miRNA microarray platform. A total of 79 miRNAs from 24 families were detected in wheat leaves. Among those, seven miRNAs were further validated to be involved in wheat powdery mildew response and two of them have never been reported. In addition, their target expression profiles showed a negative correlation with that of the seven miRNAs in mock‐ and Bgt‐infected samples furtherly proved, which in turn as the robust evidence, that those seven powdery mildew‐responsive miRNAs are highly reliable. These findings could extend the current view about miRNAs as ubiquitous regulators under stress conditions.     

Identification of CO2 Chemoreceptors in Helix pomatia

SYNOPSIS. Gas exchange in pulmonate snails of the family Helicidaeoccurs through a highly vascularized diffusion lung known asthe mantle. The extent of ventilation of the mantle dependsupon the duration and size of opening of an occlusible poreknown as the pneumostome. In Helix aspersa and Helix pomatia,pneumostomal size and frequency of opening are exquisitely sensitiveto CO₂. Respiratory CO₂ chemosensitivity resides in a discreteregion of the subesophageal ganglia. The discharge pattern ofmany neurons in the chemoreceptor area changes during stimulationwith CO₂. However, the electrophysiological response to CO,stimulation alone does not discriminate between CO₂ chemoreceptorcells and CO₂-insensitive neurons active in the pneumostomalresponse to CO₂. We identified a subset of CO₂-sensitive neuronsfrom the larger population of neurons active during CO₂ stimulation.The action potential discharge frequency of CO₂ chemosensoryneurons increased in response to CO₂ stimulation. An increaseddischarge frequency of CO₂-sensitive neurons was associatedwith increased pneumostomal opening, and both the size and thefrequency of pneumostomal opening increased during CO₂ stimulation.Injecting depolarizing current into individual chemosensoryneurons elicited opening of the pneumostome in the absence ofCO₂. Action potential generation in response to CO₂ was independentof synaptic transmission. Removal of individual CO₂-sensitivecells or inhibition of action potential generation in CO₂-sensitivecells reduced or eliminated pneumostomal responses to CO₂. CO₂sensitivity in chemoreceptor cells required extracellular calcium,but not sodium. Substituting barium for calcium supported chemoreceptoractivity. In summary, we have identified respiratory related,chemosensory neurons that are CO₂ sensitive in the absence ofsynaptic input.