Correction: Plasmids manipulate bacterial behaviour through translational regulatory crosstalk.

[This corrects the article DOI: 10.1371/journal.pbio.3001988.].

Initiation of B-type starch granules in wheat endosperm requires the plastidial α-glucan phosphorylase PHS1.

The plastidial α-glucan phosphorylase (PHS1) can elongate and degrade maltooligosaccharides (MOSs), but its exact physiological role in plants is poorly understood. Here, we discover a specialized role of PHS1 in establishing the unique bimodal characteristic of starch granules in wheat (Triticum spp.) endosperm. Wheat endosperm contains large A-type granules that initiate at early grain development and small B-type granules that initiate in later grain development. We demonstrate that PHS1 interacts with B-GRANULE CONTENT1 (BGC1), a carbohydrate-binding protein essential for normal B-type granule initiation. Mutants of tetraploid durum wheat (Triticum turgidum) deficient in all homoeologs of PHS1 had normal A-type granules but fewer and larger B-type granules. Grain size and starch content were not affected by the mutations. Further, by assessing granule numbers during grain development in the phs1 mutant and using a double mutant defective in both PHS1 and BGC1, we demonstrate that PHS1 is exclusively involved in B-type granule initiation. The total starch content and number of starch granules per chloroplast in leaves were not affected by loss of PHS1, suggesting that its role in granule initiation in wheat is limited to the endosperm. We therefore propose that the initiation of A- and B-type granules occurs via distinct biochemical mechanisms, where PHS1 plays an exclusive role in B-type granule initiation.

Plasmids manipulate bacterial behaviour through translational regulatory crosstalk.

Beyond their role in horizontal gene transfer, conjugative plasmids commonly encode homologues of bacterial regulators. Known plasmid regulator homologues have highly targeted effects upon the transcription of specific bacterial traits. Here, we characterise a plasmid translational regulator, RsmQ, capable of taking global regulatory control in Pseudomonas fluorescens and causing a behavioural switch from motile to sessile lifestyle. RsmQ acts as a global regulator, controlling the host proteome through direct interaction with host mRNAs and interference with the host's translational regulatory network. This mRNA interference leads to large-scale proteomic changes in metabolic genes, key regulators, and genes involved in chemotaxis, thus controlling bacterial metabolism and motility. Moreover, comparative analyses found RsmQ to be encoded on a large number of divergent plasmids isolated from multiple bacterial host taxa, suggesting the widespread importance of RsmQ for manipulating bacterial behaviour across clinical, environmental, and agricultural niches. RsmQ is a widespread plasmid global translational regulator primarily evolved for host chromosomal control to manipulate bacterial behaviour and lifestyle.

Site-specific encoding of photoactivity and photoreactivity into antibody fragments.

Design of biomolecules that perform two or more distinct functions in response to light remains challenging. Here, we have introduced concurrent photoactivity and photoreactivity into an epidermal growth factor receptor (EGFR)-targeting antibody fragment, 7D12. This was achieved by site-specific incorporation of photocaged tyrosine (pcY) for photoactivity and p-benzoyl-ʟ-phenylalanine (Bpa) for photoreactivity into 7D12. We identified a position for installing Bpa in 7D12 that has minimal effect on 7D12-EGFR binding affinity in the absence of light. Upon exposure to 365-nm light, this Bpa-containing 7D12 mutant forms a covalent bond with EGFR in an antigen-specific manner. We then developed a method for site-specific incorporation of pcY and Bpa at two distinct sites in 7D12. Finally, we demonstrated that in the absence of light, this pcY- and Bpa-containing mutant of 7D12 does not bind to EGFR, but irradiation with 365-nm light activates (1) specific binding and (2) covalent bond formation with EGFR.

Interactions between paralogous bacterial enhancer-binding proteins enable metal-dependent regulation of alternative nitrogenases in Azotobacter vinelandii.

All diazotrophic bacteria and archaea isolated so far utilise a nitrogenase enzyme-containing molybdenum in the active site co-factor to fix atmospheric dinitrogen to ammonia. However, in addition to the Mo-dependent nitrogenase, some nitrogen-fixing prokaryotes also express genetically distinct alternative nitrogenase isoenzymes, namely the V-dependent and Fe-only nitrogenases, respectively. Nitrogenase isoenzymes are expressed hierarchically according to metal availability and catalytic efficiency. In proteobacteria, this hierarchy is maintained via stringent transcriptional regulation of gene clusters by dedicated bacterial enhancer-binding proteins (bEBPs). The model diazotroph Azotobacter vinelandii contains two paralogs of the vanadium nitrogenase activator VnfA (henceforth, VnfA1), designated VnfA2 and VnfA3, with unknown functions. Here we demonstrate that the VnfA1 and VnfA3 bEBPs bind to the same target promoters in the Azotobacter vinelandii genome and co-activate a subset of genes in the absence of V, including the structural genes for the Fe-only nitrogenase. Co-activation is inhibited by the presence of V and is dependent on an accessory protein VnfZ that is co-expressed with VnfA3. Our studies uncover a plethora of interactions between bEBPs required for nitrogen fixation, revealing the unprecedented potential for fine-tuning the expression of alternative nitrogenases in response to metal availability.

Evidence of a role for CutRS and actinorhodin in the secretion stress response in Streptomyces coelicolor M145.

CutRS was the first two-component system to be identified in Streptomyces species and is highly conserved in this genus. It was reported >25 years ago that deletion of cutRS increases the production of the antibiotic actinorhodin in Streptomyces coelicolor. However, despite this early work, the function of CutRS has remained enigmatic until now. Here we show that deletion of cutRS upregulates the production of the actinorhodin biosynthetic enzymes up to 300-fold, explaining the increase in actinorhodin production. However, while ChIP-seq identified 85 CutR binding sites in S. coelicolor none of these are in the actinorhodin biosynthetic gene cluster, meaning the effect is indirect. The directly regulated CutR targets identified in this study are implicated in extracellular protein folding, including two of the four highly conserved HtrA-family foldases: HtrA3 and HtrB, and a putative VKOR enzyme, which is predicted to recycle DsbA following its catalysis of disulphide bond formation in secreted proteins. Thus, we tentatively propose a role for CutRS in sensing and responding to protein misfolding outside the cell. Since actinorhodin can oxidise cysteine residues and induce disulphide bond formation in proteins, its over production in the ∆cutRS mutant may be a response to protein misfolding on the extracellular face of the membrane.

Cashew Tree Pollen: An Unknown Source of IgE-Reactive Molecules.

Pollinosis is sub-diagnosed and rarely studied in tropical countries. Cashew tree pollen has been reported as an allergen source although the knowledge of its immunoglobulin E (IgE)-reactive molecules is lacking. Therefore, this work aimed to identify IgE-reactive molecules and provide a proteomic profile of this pollen. From the 830 proteins identified by shotgun analysis, 163 were annotated to gene ontology, and a list of 39 proteins filtered for high confidence was submitted to the Allfam database where nine were assigned to allergenic families. Thus, 12 patients from the northeast of Brazil with persistent allergic rhinitis and aggravation of symptoms during cashew flowering season were selected. Using a 2D-based approach, we identified 20 IgE-reactive proteins, four already recognized as allergens, including a homolog of the birch isoflavone-reductase (Bet v 6). IgE-reactivity against the extract in native form was confirmed for five patients in ELISA, with three being positive for Bet v 6. Herein, we present a group of patients with rhinitis exposed to cashew tree pollen with the first description of IgE-binding proteins and a proteomic profile of the whole pollen. Cashew tree pollen is considered an important trigger of rhinitis symptoms in clinical practice in the northeast of Brazil, and the elucidation of its allergenic molecules can improve the diagnostics and treatment for allergic patients.

Differential temporal release and lipoprotein loading in B. thetaiotaomicron bacterial extracellular vesicles.

Bacterial extracellular vesicles (BEVs) contribute to stress responses, quorum sensing, biofilm formation and interspecies and interkingdom communication. However, the factors that regulate their release and heterogeneity are not well understood. We set out to investigate these factors in the common gut commensal Bacteroides thetaiotaomicron by studying BEV release throughout their growth cycle. Utilising a range of methods, we demonstrate that vesicles released at different stages of growth have significantly different composition, with early vesicles enriched in specifically released outer membrane vesicles (OMVs) containing a larger proportion of lipoproteins, while late phase BEVs primarily contain lytic vesicles with enrichment of cytoplasmic proteins. Furthermore, we demonstrate that lipoproteins containing a negatively charged signal peptide are preferentially incorporated in OMVs. We use this observation to predict all Bacteroides thetaiotaomicron OMV enriched lipoproteins and analyse their function. Overall, our findings highlight the need to understand media composition and BEV release dynamics prior to functional characterisation and define the theoretical functional capacity of Bacteroides thetaiotaomicron OMVs.

O-glycosylation of the transcription factor SPATULA promotes style development in Arabidopsis.

O-linked ß-N-acetylglucosamine (O-GlcNAc) and O-fucose are two sugar-based post-translational modifications whose mechanistic role in plant signalling and transcriptional regulation is still largely unknown. Here we investigated how two O-glycosyltransferase enzymes of Arabidopsis thaliana, SPINDLY (SPY) and SECRET AGENT (SEC), promote the activity of the basic helix-loop-helix transcription factor SPATULA (SPT) during morphogenesis of the plant female reproductive organ apex, the style. SPY and SEC modify amino-terminal residues of SPT in vivo and in vitro by attaching O-fucose and O-GlcNAc, respectively. This post-translational regulation does not impact SPT homo- and heterodimerization events, although it enhances the affinity of SPT for the kinase PINOID gene locus and its transcriptional repression. Our findings offer a mechanistic example of the effect of O-GlcNAc and O-fucose on the activity of a plant transcription factor and reveal previously unrecognized roles for SEC and SPY in orchestrating style elongation and shape.

Discovery of isoflavone phytoalexins in wheat reveals an alternative route to isoflavonoid biosynthesis.

Isoflavones are a group of phenolic compounds mostly restricted to plants of the legume family, where they mediate important interactions with plant-associated microbes, including in defense from pathogens and in nodulation. Their well-studied health promoting attributes have made them a prime target for metabolic engineering, both for bioproduction of isoflavones as high-value molecules, and in biofortification of food crops. A key gene in their biosynthesis, isoflavone synthase, was identified in legumes over two decades ago, but little is known about formation of isoflavones outside of this family. Here we identify a specialized wheat-specific isoflavone synthase, TaCYP71F53, which catalyzes a different reaction from the leguminous isoflavone synthases, thus revealing an alternative path to isoflavonoid biosynthesis and providing a non-transgenic route for engineering isoflavone production in wheat. TaCYP71F53 forms part of a biosynthetic gene cluster that produces a naringenin-derived O-methylated isoflavone, 5-hydroxy-2',4',7-trimethoxyisoflavone, triticein. Pathogen-induced production and in vitro antimicrobial activity of triticein suggest a defense-related role for this molecule in wheat. Genomic and metabolic analyses of wheat ancestral grasses further show that the triticein gene cluster was introduced into domesticated emmer wheat through natural hybridization ~9000 years ago, and encodes a pathogen-responsive metabolic pathway that is conserved in modern bread wheat varieties.

VAL1 acts as an assembly platform co-ordinating co-transcriptional repression and chromatin regulation at Arabidopsis FLC.

Polycomb (PcG) silencing is crucial for development, but how targets are specified remains incompletely understood. The cold-induced Polycomb Repressive Complex 2 (PRC2) silencing of Arabidopsis thaliana FLOWERING LOCUS C (FLC) provides an excellent system to elucidate PcG regulation. Association of the DNA binding protein VAL1 to FLC PcG nucleation regionis an important step. VAL1 co-immunoprecipitates APOPTOSIS AND SPLICING ASSOCIATED PROTEIN (ASAP) complex and PRC1. Here, we show that ASAP and PRC1 are necessary for co-transcriptional repression and chromatin regulation at FLC. ASAP mutants affect FLC transcription in warm conditions, but the rate of FLC silencing in the cold is unaffected. PRC1-mediated H2Aub accumulation increases at the FLC nucleation region during cold, but unlike the PRC2-delivered H3K27me3, does not spread across the locus. H2Aub thus involved in the transition to epigenetic silencing at FLC, facilitating H3K27me3 accumulation and long-term epigenetic memory. Overall, our work highlights the importance of VAL1 as an assembly platform co-ordinating activities necessary for epigenetic silencing at FLC.

Biofortified tomatoes provide a new route to vitamin D sufficiency.

Poor vitamin D status is a global health problem; insufficiency underpins higher risk of cancer, neurocognitive decline and all-cause mortality. Most foods contain little vitamin D and plants are very poor sources. We have engineered the accumulation of provitamin D3 in tomato by genome editing, modifying a duplicated section of phytosterol biosynthesis in Solanaceous plants, to provide a biofortified food with the added possibility of supplement production from waste material.

Mass Spectrometry-Based Method Targeting Ig Variable Regions for Assessment of Minimal Residual Disease in Multiple Myeloma.

Multiple myeloma is a systemic malignancy of monoclonal plasma cells that accounts for 10% of hematologic cancers. With development of highly effective therapies for multiple myeloma, minimal residual disease (MRD) assessment has emerged as an important end point for management decisions. Currently, serologic assays lack the sensitivity for MRD assessment, and invasive bone marrow sampling with flow cytometry or molecular methods has emerged as the gold standard. We report a sensitive and robust targeted mass spectrometry proteomics method to detect MRD in serum, without the need of invasive, sequential bone marrow aspirates. The method detects Ig-derived clonotypic tryptic peptides predicted by sequencing the clonal plasma cell Ig genes. A heavy isotope-labeled Ig internal standard is added to patient serum at a known concentration, the Ig is enriched in a light chain type specific manner, and proteins are digested and analyzed by targeted mass spectrometry. Peptides from the constant regions of the λ or κ light chains, Ig heavy chains, and clonotypic peptides unique to the patient monoclonal Igs are targeted. This technique is highly sensitive and specific for the patient-specific monoclonal Igs, even in samples negative by multiparametric flow cytometry. Our method can accurately and precisely detect monoclonal protein in serum of patients treated for myeloma and has broad implications for management of hematologic patients.

The Vaginal Microbiome as a Tool to Predict rASRM Stage of Disease in Endometriosis: a Pilot Study.

Endometriosis remains a challenge to understand and to diagnose. This is an observational cross-sectional pilot study to characterize the gut and vaginal microbiome profiles among endometriosis patients and control subjects without the disease and to explore their potential use as a less-invasive diagnostic tool for endometriosis. Overall, 59 women were included, n = 35 with endometriosis and n = 24 controls. Rectal and vaginal samples were collected in two different periods of the menstrual cycle from all subjects. Gut and vaginal microbiomes from patients with different rASRM (revised American Society for Reproductive Medicine) endometriosis stages and controls were analyzed. Illumina sequencing libraries were constructed using a two-step 16S rRNA gene PCR amplicon approach. Correlations of 16S rRNA gene amplicon data with clinical metadata were conducted using a random forest-based machine-learning classification analysis. Distribution of vaginal CSTs (community state types) significantly differed between follicular and menstrual phases of the menstrual cycle (p = 0.021, Fisher's exact test). Vaginal and rectal microbiome profiles and their association to severity of endometriosis (according to rASRM stages) were evaluated. Classification models built with machine-learning methods on the microbiota composition during follicular and menstrual phases of the cycle were built, and it was possible to accurately predict rASRM stages 1-2 verses rASRM stages 3-4 endometriosis. The feature contributing the most to this prediction was an OTU (operational taxonomic unit) from the genus Anaerococcus. Gut and vaginal microbiomes of women with endometriosis have been investigated. Our findings suggest for the first time that vaginal microbiome may predict stage of disease when endometriosis is present.

Rheumatic Heart Disease and Myxomatous Degeneration: Differences and Similarities of Valve Damage Resulting from Autoimmune Reactions and Matrix Disorganization.

Autoimmune inflammatory reactions leading to rheumatic fever (RF) and rheumatic heart disease (RHD) result from untreated Streptococcus pyogenes throat infections in individuals who exhibit genetic susceptibility. Immune effector mechanisms have been described that lead to heart tissue damage culminating in mitral and aortic valve dysfunctions. In myxomatous valve degeneration (MXD), the mitral valve is also damaged due to non-inflammatory mechanisms. Both diseases are characterized by structural valve disarray and a previous proteomic analysis of them has disclosed a distinct profile of matrix/structural proteins differentially expressed. Given their relevance in organizing valve tissue, we quantitatively evaluated the expression of vimentin, collagen VI, lumican, and vitronectin as well as performed immunohistochemical analysis of their distribution in valve tissue lesions of patients in both diseases. We identified abundant expression of two isoforms of vimentin (45 kDa, 42 kDa) with reduced expression of the full-size protein (54 kDa) in RHD valves. We also found increased vitronectin expression, reduced collagen VI expression and similar lumican expression between RHD and MXD valves. Immunohistochemical analysis indicated disrupted patterns of these proteins in myxomatous degeneration valves and disorganized distribution in rheumatic heart disease valves that correlated with clinical manifestations such as valve regurgitation or stenosis. Confocal microscopy analysis revealed a diverse pattern of distribution of collagen VI and lumican into RHD and MXD valves. Altogether, these results demonstrated distinct patterns of altered valve expression and tissue distribution/organization of structural/matrix proteins that play important pathophysiological roles in both valve diseases.

Analysis of the coverage capacity of the StreptInCor candidate vaccine against Streptococcus pyogenes.

Streptococcus pyogenes is responsible for infections as pharyngitis, sepsis, necrotizing fasciitis and streptococcal toxic shock syndrome. The M protein is the major bacterial antigen and consists of both polymorphic N-terminal portion and a conserved region. In the present study, we analyzed the in vitro ability of StreptInCor a C-terminal candidate vaccine against S. pyogenes to induce antibodies to neutralize/opsonize the most common S. pyogenes strains in Sao Paulo by examining the recognition by sera from StreptInCor immunized mice. We also evaluated the presence of cross-reactive antibodies against human heart valve tissue. Anti-StreptInCor antibodies were able to neutralize/opsonize at least 5 strains, showing that immunization with StreptInCor is effective against several S. pyogenes strains and can prevent infection and subsequent sequelae without causing autoimmune reactions.

Distinct mitral valve proteomic profiles in rheumatic heart disease and myxomatous degeneration.

Rheumatic heart disease (RHD) affects heart-valve tissue and is the most serious consequence of group A Streptococcus infection. Myxomatous degeneration (MXD) is the most frequent valvopathy in the western world. In the present work, key protein expression alterations in the heart-valve tissue of RHD and MXD patients were identified and characterized, with controls from cadaveric organ donors. Proteins were separated by two-dimensional (2D)-electrophoresis and identified by mass spectrometry. We found 17 differentially expressed protein spots, as compared to control samples. We observed an increased expression of ASAP-2 in the RHD patients' valves, while collagen-VI, haptoglobin-related protein, prolargin, and cartilage oligomeric protein showed reduced expression. Valve tissue of MXD patients, on the other hand, presented lower expression of annexin-A1 and A2, septin-2, SOD (Cu/Zn), and transgelin. Tissue samples from both valvopathies displayed higher expression of apolipoprotein-A1. Biglycan was downexpressed in both diseases. Vimentin and lumican showed higher expression in RHD and lower in MXD. These results suggest that key pathogenetic mechanisms are intrinsically distinct in RHD and MXD.

Novel allergens from ancient foods: Man e 5 from manioc (Manihot esculenta Crantz) cross reacts with Hev b 5 from latex.

SCOPE: Manioc (Manihot esculenta) is a tuber mainly consumed in the Southern Hemisphere and used worldwide by food and chemistry industry. We aimed to recombinantly produce and characterize the first manioc allergen and evaluate its IgE reactivity in sera of Brazilian and Italian patients. METHODS AND RESULTS: The molecule, termed Man e5, was expressed in E. coli, characterized by amino acid analysis, mass spectrometry, circular dichroism, HPLC, and dynamic light scattering. A tertiary structural model of the protein was produced using bioinformatics and susceptibility to pepsin digestion was analyzed in vitro. Based on its high content of charged residues, heat stability, flexibility and lack of secondary structure elements, the allergen was determined a member of the intrinsically disordered protein family. Brazilian patients were selected based on manioc allergy and Italians based on latex allergy and sensitization to Hev b 5.71% of Brazilians and 40% of Italians were in vitro IgE positive to Man e5. Cross-inhibition assays suggest a possible involvement of this allergen in the latex-fruit syndrome. CONCLUSION: Man e5, the first purified allergen from manioc demonstrates IgE cross-reactivity with Hev b 5. Data suggest Hev b 5 might act as primary sensitizer and could therefore lead to allergic manifestations upon manioc consumption without prior exposition.