Characterization of the transcriptionally active form of dephosphorylated DctD complexed with dephospho-IIAGlc.

Bacterial enhancer-binding proteins (bEBPs) acquire a transcriptionally active state via phosphorylation. However, transcriptional activation by the dephosphorylated form of bEBP has been observed in DctD, which belongs to Group I bEBP. The formation of a complex between dephosphorylated DctD (d-DctD) and dephosphorylated IIAGlc (d-IIAGlc) is a prerequisite for the transcriptional activity of d-DctD. In the present study, characteristics of the transcriptionally active complex composed of d-IIAGlc and phosphorylation-deficient DctD (DctDD57Q) of Vibrio vulnificus were investigated in its multimeric conformation and DNA-binding ability. DctDD57Q formed a homodimer that could not bind to the DNA. In contrast, when DctDD57Q formed a complex with d-IIAGlc in a 1:1 molar ratio, it produced two conformations: dimer and dodecamer of the complex. Only the dodecameric complex exhibited ATP-hydrolyzing activity and DNA-binding affinity. For successful DNA-binding and transcriptional activation by the dodecameric d-IIAGlc/DctDD57Q complex, extended upstream activator sequences were required, which encompass the nucleotide sequences homologous to the known DctD-binding site and additional nucleotides downstream. This is the first report to demonstrate the molecular characteristics of a dephosphorylated bEBP complexed with another protein to form a transcriptionally active dodecameric complex, which has an affinity for a specific DNA-binding sequence.IMPORTANCEResponse regulators belonging to the bacterial two-component regulatory system activate the transcription initiation of their regulons when they are phosphorylated by cognate sensor kinases and oligomerized to the appropriate multimeric states. Recently, it has been shown that a dephosphorylated response regulator, DctD, could activate transcription in a phosphorylation-independent manner in Vibrio vulnificus. The dephosphorylated DctD activated transcription as efficiently as phosphorylated DctD when it formed a complex with dephosphorylated form of IIAGlc, a component of the glucose-phosphotransferase system. Functional mimicry of this complex with the typical form of transcriptionally active phosphorylated DctD led us to study the molecular characteristics of this heterodimeric complex. Through systematic analyses, it was surprisingly determined that a multimer constituted with 12 complexes gained the ability to hydrolyze ATP and recognize specific upstream activator sequences containing a typical inverted-repeat sequence flanked by distinct nucleotides.

An archaeal transcription factor EnfR with a novel 'eighth note' fold controls hydrogen production of a hyperthermophilic archaeon Thermococcus onnurineus NA1.

Thermococcus onnurineus NA1, a hyperthermophilic carboxydotrophic archaeon, produces H2 through CO oxidation catalyzed by proteins encoded in a carbon monoxide dehydrogenase (CODH) gene cluster. TON_1525 with a DNA-binding helix-turn-helix (HTH) motif is a putative repressor regulating the transcriptional expression of the codh gene cluster. The T55I mutation in TON_1525 led to enhanced H2 production accompanied by the increased expression of genes in the codh cluster. Here, TON_1525 was demonstrated to be a dimer. Monomeric TON_1525 adopts a novel 'eighth note' symbol-like fold (referred to as 'eighth note' fold regulator, EnfR), and the dimerization mode of EnfR is unique in that it has no resemblance to structures in the Protein Data Bank. According to footprinting and gel shift assays, dimeric EnfR binds to a 36-bp pseudo-palindromic inverted repeat in the promoter region of the codh gene cluster, which is supported by an in silico EnfR/DNA complex model and mutational studies revealing the implication of N-terminal loops as well as HTH motifs in DNA recognition. The DNA-binding affinity of the T55I mutant was lowered by ∼15-fold, for which the conformational change of N-terminal loops is responsible. In addition, transcriptome analysis suggested that EnfR could regulate diverse metabolic processes besides H2 production.

Reconfigurable neuromorphic computing block through integration of flash synapse arrays and super-steep neurons.

Neuromorphic computing (NC) architecture inspired by biological nervous systems has been actively studied to overcome the limitations of conventional von Neumann architectures. In this work, we propose a reconfigurable NC block using a flash-type synapse array, emerging positive feedback (PF) neuron devices, and CMOS peripheral circuits, and integrate them on the same substrate to experimentally demonstrate the operations of the proposed NC block. Conductance modulation in the flash memory enables the NC block to be easily calibrated for output signals. In addition, the proposed NC block uses a reduced number of devices for analog-to-digital conversions due to the super-steep switching characteristics of the PF neuron device, substantially reducing the area overhead of NC block. Our NC block shows high energy efficiency (37.9 TOPS/W) with high accuracy for CIFAR-10 image classification (91.80%), outperforming prior works. This work shows the high engineering potential of integrating synapses and neurons in terms of system efficiency and high performance.

Unraveling the Pathobiological Role of the Fungal KEOPS Complex in Cryptococcus neoformans.

The KEOPS (kinase, putative endopeptidase, and other proteins of small size) complex has critical functions in eukaryotes; however, its role in fungal pathogens remains elusive. Herein, we comprehensively analyzed the pathobiological functions of the fungal KEOPS complex in Cryptococcus neoformans (Cn), which causes fatal meningoencephalitis in humans. We identified four CnKEOPS components: Pcc1, Kae1, Bud32, and Cgi121. Deletion of PCC1, KAE1, or BUD32 caused severe defects in vegetative growth, cell cycle control, sexual development, general stress responses, and virulence factor production, whereas deletion of CGI121 led to similar but less severe defects. This suggests that Pcc1, Kae1, and Bud32 are the core KEOPS components, and Cgi121 may play auxiliary roles. Nevertheless, all KEOPS components were essential for C. neoformans pathogenicity. Although the CnKEOPS complex appeared to have a conserved linear arrangement of Pcc1-Kae1-Bud32-Cgi121, as supported by physical interaction between Pcc1-Kae1 and Kae1-Bud32, CnBud32 was found to have a unique extended loop region that was critical for the KEOPS functions. Interestingly, CnBud32 exhibited both kinase activity-dependent and -independent functions. Supporting its pleiotropic roles, the CnKEOPS complex not only played conserved roles in t6A modification of ANN codon-recognizing tRNAs but also acted as a major transcriptional regulator, thus controlling hundreds of genes involved in various cellular processes, particularly ergosterol biosynthesis. In conclusion, the KEOPS complex plays both evolutionarily conserved and divergent roles in controlling the pathobiological features of C. neoformans and could be an anticryptococcal drug target. IMPORTANCE The cellular function and structural configuration of the KEOPS complex have been elucidated in some eukaryotes and archaea but have never been fully characterized in fungal pathogens. Here, we comprehensively analyzed the pathobiological roles of the KEOPS complex in the globally prevalent fungal meningitis-causing pathogen C. neoformans. The CnKEOPS complex, composed of a linear arrangement of Pcc1-Kae1-Bud32-Cgi121, not only played evolutionarily conserved roles in growth, sexual development, stress responses, and tRNA modification but also had unique roles in controlling virulence factor production and pathogenicity. Notably, a unique extended loop structure in CnBud32 is critical for the KEOPS complex in C. neoformans. Supporting its pleiotropic roles, transcriptome analysis revealed that the CnKEOPS complex governs several hundreds of genes involved in carbon and amino acid metabolism, pheromone response, and ergosterol biosynthesis. Therefore, this study provides novel insights into the fungal KEOPS complex that could be exploited as a potential antifungal drug target.

Transition of Dephospho-DctD to the Transcriptionally Active State via Interaction with Dephospho-IIAGlc.

Exopolysaccharides (EPSs), biofilm-maturing components of Vibrio vulnificus, are abundantly produced when the expression of two major EPS gene clusters is activated by an enhancer-binding transcription factor, DctD2, whose expression and phosphorylation are induced by dicarboxylic acids. Surprisingly, when glucose was supplied to V. vulnificus, similar levels of expression of these clusters occurred, even in the absence of dicarboxylic acids. This glucose-dependent activation was also mediated by DctD2, whose expression was sequentially activated by the transcription regulator NtrC. Most DctD2 in cells grown without dicarboxylic acids was present in a dephosphorylated state, known as the transcriptionally inactive form. However, in the presence of glucose, a dephosphorylated component of the glucose-specific phosphotransferase system, d-IIAGlc, interacted with dephosphorylated DctD2 (d-DctD2). While d-DctD2 did not show any affinity to a DNA fragment containing the DctD-binding sequences, the complex of d-DctD2 and d-IIAGlc exhibited specific and efficient DNA binding, similar to the phosphorylated DctD2. The d-DctD2-mediated activation of the EPS gene clusters' expression was not fully achieved in cells grown with mannose. Furthermore, the degrees of expression of the clusters under glycerol were less than those under mannose. This was caused by an antagonistic and competitive effect of GlpK, whose expression was increased by glycerol, in forming a complex with d-DctD2 by d-IIAGlc. The data demonstrate a novel regulatory pathway for V. vulnificus EPS biosynthesis and biofilm maturation in the presence of glucose, which is mediated by d-DctD2 through its transition to the transcriptionally active state by interacting with available d-IIAGlc. IMPORTANCE Transcription regulation by bacterial two-component systems is achieved by a response regulator upon its transition to the transcriptionally active form via kinase activity of its cognate sensor under specific conditions. A well-known response regulator, DctD, is converted to its phosphorylated form when DctB senses ambient dicarboxylic acids. Phospho-DctD induces expression of its regulon, including the gene clusters for biosynthesis of exopolysaccharides (EPSs), the essential constituents of biofilm matrix. In the absence of dicarboxylic acids, however, DctD-mediated induction of these EPS gene clusters and biofilm maturation was observed if glucose was supplied. This suggests that dephospho-DctD could play a role in activating the transcription of target genes. A component of glucose-phosphotransferase system, IIAGlc, was present in a dephosphorylated state in the presence of glucose. Dephospho-DctD formed a complex with dephospho-IIAGlc and was converted to a transcriptionally active state. These findings suggest the other response regulators could also have alternative pathways of activation independent of phosphorylation.

Overexpression of Rice OsS1Fa1 Gene Confers Drought Tolerance in Arabidopsis.

Small peptides and proteins play critical regulatory roles in plant development and environmental stress responses; however, only a few of these molecules have been identified and characterized to date because of their poor annotation and other experimental challenges. Here, we present that rice (Oryza sativa L.) OsS1Fa1, a small 76-amino acid protein, confers drought stress tolerance in Arabidopsis thaliana. OsS1Fa1 was highly expressed in leaf, culm, and root tissues of rice seedlings during vegetative growth and was significantly induced under drought stress. OsS1Fa1 overexpression in Arabidopsis induced the expression of selected drought-responsive genes and enhanced the survival rate of transgenic lines under drought. The proteasome inhibitor MG132 protected the OsS1Fa1 protein from degradation. Together, our data indicate that the small protein OsS1Fa1 is induced by drought and is post-translationally regulated, and the ectopic expression of OsS1Fa1 protects plants from drought stress.

Vibrio vulnificus induces the death of a major bacterial species in the mouse gut via cyclo-Phe-Pro.

BACKGROUND: A foodborne pathogen, Vibrio vulnificus, encounters normal microflora inhabiting the gut environments prior to causing fatal septicemia or gastroenteritis and should overcome the barriers derived from the gut commensals for successful infection. Its interactions with gut commensals during the infection process, however, have not yet been understood. In the present study, the effect of V. vulnificus on the community structures of gut microbiota in mice was examined. RESULTS: Analyses of microbiota in the fecal samples of mice that died due to V. vulnificus infection revealed the decreased abundance of bacteria belonged to Bacteroidetes, notably, the species Bacteroides vulgatus. In vitro coculturing of the two bacterial species resulted in the decreased survival of B. vulgatus. The antagonistic effect of V. vulnificus against B. vulgatus was found to be mediated by cyclo-Phe-Pro (cFP), one of the major compounds secreted by V. vulnificus. cFP-treated B. vulgatus showed collapsed cellular morphology with an undulated cell surface, enlarged periplasmic space, and lysed membranes, suggesting the occurrence of membrane disruption. The degree of membrane disruption caused by cFP was dependent upon the cellular levels of ObgE in B. vulgatus. Recombinant ObgE exhibited a high affinity to cFP at a 1:1 ratio. When mice were orally injected with cFP, their feces contained significantly reduced B. vulgatus levels, and their susceptibility to V. vulnificus infection was considerably increased. CONCLUSIONS: This study demonstrates that V. vulnificus-derived cFP modulates the abundance of the predominant species among gut commensals, which made V. vulnificus increase its pathogenicity in the hosts. Video abstract.

Transcription activation of two clusters for exopolysaccharide biosynthesis by phosphorylated DctD in Vibrio vulnificus.

NtrC-mediated production of exopolysaccharides (EPS), essential components for Vibrio vulnificus biofilms, is highly increased in the presence of dicarboxylic or tricarboxylic acids. Gel-shift assays showed that regulation of the EPS-gene cluster I (EPS-I cluster) by NtrC was direct via binding of phosphorylated NtrC (p-NtrC) to the regulatory region of the EPS-I cluster. In contrast, p-NtrC did not bind to the EPS-II and EPS-III clusters, suggesting that NtrC regulation was not direct and another transcription factor belonging to an NtrC-regulon might play a role in activating their transcription. A candidate transcription factor, DctD, of which expression was induced by NtrC, activated the expression of the EPS-II and EPS-III clusters via direct binding to their upstream regions. Under growth conditions with either dicarboxylic or tricarboxylic acids, the expression of NtrC was induced and the transcription of dctD was activated. Furthermore, DctD exhibited higher transcriptional activity under the conditions with dicarboxylic acids than with tricarboxylic acids. Therefore, this study demonstrates that under dicarboxylate-rich conditions, both the abundance and activity of DctD were markedly induced, which activates the expression of two EPS clusters to maximize biosynthesis of EPS facilitating biofilm maturation in V. vulnificus.

Role of DegQ in differential stability of flagellin subunits in Vibrio vulnificus.

Biofilm formation of Vibrio vulnificus is initiated by adherence of flagellated cells to surfaces, and then flagellum-driven motility is not necessary during biofilm maturation. Once matured biofilms are constructed, cells become flagellated and swim to disperse from biofilms. As a consequence, timely regulations of the flagellar components' expression are crucial to complete a biofilm life-cycle. In this study, we demonstrated that flagellins' production is regulated in a biofilm stage-specific manner, via activities of a protease DegQ and a chaperone FlaJ. Among four flagellin subunits for V. vulnificus filament, FlaC had the highest affinities to hook-associated proteins, and is critical for maturating flagellum, showed the least susceptibility to DegQ due to the presence of methionine residues in its DegQ-sensitive domains, ND1 and CD0. Therefore, differential regulation by DegQ and FlaJ controls the cytoplasmic stability of flagellins, which further determines the motility-dependent, stage-specific development of biofilms.

Differential regulation of a placental SAM68 and sFLT1 gene pathway and the relevance to maternal vitamin D sufficiency.

OBJECTIVE: The goal of this study was to determine if an axis of placental gene expression associated with early onset and severe preeclampsia (EOSPE) was operative in term pregnancy and correlated with vitamin D sufficiency. METHODS: qPCR analysis of NKX2-5, SAM68, sFLT1 and membrane bound VEGFR1/FLT1 mRNA expression was conducted in placentas from 43 subjects enrolled in a vitamin D3 pregnancy supplementation trial. Pair-wise rank order correlations between patient-specific gene expression levels were calculated, and their relationship to maternal 25(OH)D status was assessed by a two-sample Wilcoxon test. Additionally, we probed the mechanistic link between SAM68 and sFLT1 using siRNA depletion in a human trophoblast cell line model. RESULTS: Positive and highly significant correlations were found between SAM68 vs. sFLT1 and SAM68 vs. FLT1 expression levels, as were significant and differential correlations between the expression of these genes and perinatal 25(OH)D status. The variability when stratified by race/ethnicity was qualitatively distinct from those previously observed in EOSPE. Mechanistic studies confirmed a functional role for SAM68 protein in the regulation of sFLT1 expression. NKX2-5 expression was not significantly correlated with sFLT1 or SAM68 expression in these samples, suggesting that its expression may be significant at earlier stages of pregnancy or be restricted to pathological settings. CONCLUSIONS: These data further support our overarching hypothesis that SAM68 expression is a key determinant of VEGFR1 isoform expression in the placenta, and provide additional insights into how this gene pathway may be differentially deployed or modified in normal and pathological pregnancies.

Second heart field-specific expression of Nkx2-5 requires promoter proximal interaction with Srf.

The cardiac homeobox transcription factor Nkx2-5 is a major determinant of cardiac identity and cardiac morphogenesis. Nkx2-5 operates as part of a complex and mutually reinforcing network of early transcription factors of the homeobox, GATA zinc finger and MADS domain families to initiate the program of cardiac development and differentiation, particularly in outflow tract precursor cells in the second heart field (SHF). We have now found evidence for another aspect of cardiac transcription factor cooperativity between Nkx2-5 and the cardiac enriched MADS domain transcription factor Srf. Specifically, Srf interaction with an evolutionarily conserved binding site in the Nkx2-5 CpG island-like proximal promoter is required for cardiac specific expression mediated by an SHF enhancer, and for combinatorial activation of these elements by cardiac transcription factors. These results provide further insight into cooperative gene regulation during cardiogenesis at the level of promoter-enhancer interactions.

Multi-Factor Regulation of the Master Modulator LeuO for the Cyclic-(Phe-Pro) Signaling Pathway in Vibrio vulnificus.

LeuO plays the role of a master regulator in the cyclic-L-phenylalanine-L-proline (cFP)-dependent signaling pathway in Vibrio vulnificus. cFP, as shown through isothermal titration calorimetry analysis, binds specifically to the periplasmic domain of ToxR. Binding of cFP triggers a change in the cytoplasmic domain of ToxR, which then activates transcription of leuO encoding a LysR-type regulator. LeuO binds to the region upstream of its own coding sequence, inhibiting its own transcription and maintaining a controlled level of expression. A five-bp deletion in this region abolished expression of LeuO, but a ten-bp deletion did not, suggesting that a DNA bending mechanism is involved in the regulation. Furthermore, binding of RNA polymerase was significantly lower both in the deletion of the ToxR binding site and in the five-bp deletion, but not in the ten-bp deletion, as shown in pull-down assays using an antibody against RNA polymerase subunit α. In summary, multiple factors are involved in control of the expression of LeuO, a master regulator that orchestrates downstream regulators to modulate factors required for survival and pathogenicity of the pathogen.

Role of Flagellin-Homologous Proteins in Biofilm Formation by Pathogenic Vibrio Species.

The pathogenic bacterium Vibrio vulnificus exhibits the ability to form biofilm, for which initiation is dependent upon swimming motility by virtue of a polar flagellum. The filament of its flagellum is composed of multiple flagellin subunits, FlaA, -B, -C, and -D. In V. vulnificus genomes, however, open reading frames (ORFs) annotated by FlaE and -F are also present. Although neither FlaE nor FlaF is involved in filament formation and cellular motility, they are well expressed and secreted to the extracellular milieu through the secretion apparatus for flagellar assembly. In the extrapolymeric matrix of V. vulnificus biofilm, significant levels of FlaEF were detected. Mutants defective in both flaE and flaF formed significantly decreased biofilms compared to the wild-type biofilm. Thus, the potential role of FlaEF during the biofilm-forming process was investigated by exogenous addition of recombinant FlaEF (rFlaEF) to the biofilm assays. The added rFlaE and rFlaF were predominantly incorporated into the biofilm matrix formed by the wild type. However, biofilms formed by a mutant defective in exopolysaccharide (EPS) biosynthesis were not affected by added FlaEF. These results raised a possibility that FlaEF specifically interact with EPS within the biofilm matrix. In vitro pulldown assays using His-tagged rFlaEF or rFlaC revealed the specific binding of EPS to rFlaEF but not to rFlaC. Taken together, our results demonstrate that V. vulnificus FlaEF, flagellin-homologous proteins (FHPs), are crucial for biofilm formation by directly interacting with the essential determinant for biofilm maturation, EPS. Further analyses performed with other pathogenic Vibrio species demonstrated both the presence of FHPs and their important role in biofilm formation.IMPORTANCE Flagellar filaments of the pathogenic Vibrio species, including V. vulnificus, V. parahaemolyticus, and V. cholerae, are composed of multiple flagellin subunits. In their genomes, however, there are higher numbers of the ORFs encoding flagellin-like proteins than the numbers of flagellin subunits required for filament assembly. Since these flagellin-homologous proteins (FHPs) are well expressed and excreted to environments via a flagellin transport channel, their extracellular role in the pathogenic Vibrio has been enigmatic. Their biological significance, which is not related with flagellar functions, has been revealed to be in maturation of biofilm structures. Among various components of the extracellular polymeric matrix produced in the V. vulnificus biofilms, the exopolysaccharides (EPS) are dominant constituents and crucial in maturation of biofilms. The enhancing role of the V. vulnificus FHPs in biofilm formation requires the presence of EPS, as indicated by highly specific interactions among two FHPs and three EPS.

Increased Innate Lymphoid Cell 3 and IL-17 Production in Mouse Lamina Propria Stimulated with Giardia lamblia.

Innate lymphoid cells (ILCs) are key players during an immune response at the mucosal surfaces, such as lung, skin, and gastrointestinal tract. Giardia lamblia is an extracellular protozoan pathogen that inhabits the human small intestine. In this study, ILCs prepared from the lamina propria of mouse small intestine were incubated with G. lamblia trophozoites. Transcriptional changes in G. lamblia-exposed ILCs resulted in identification of activation of several immune pathways. Secretion of interleukin (IL)-17A, IL-17F, IL-1ß, and interferon-γ was increased, whereas levels of IL-13, IL-5, and IL22, was maintained or reduced upon exposure to G. lamblia. Goup 3 ILC (ILC3) was found to be dominant amongst the ILCs, and increased significantly upon co-cultivation with G. lamblia trophozoites. Oral inoculation of G. lamblia trophozoites into mice resulted in their presence in the small intestine, of which, the highest number of parasites was detected at the 5 days-post infection. Increased ILC3 was observed amongst the ILC population at the 5 days-post infection. These findings indicate that ILC3 from the lamina propria secretes IL-17 in response to G. lamblia, leading to the intestinal pathology observed in giardiasis.

Polar landmark protein HubP recruits flagella assembly protein FapA under glucose limitation in Vibrio vulnificus.

How motile bacteria recognize their environment and decide whether to stay or navigate toward more favorable location is a fundamental issue in survival. The flagellum is an elaborate molecular device responsible for bacterial locomotion, and the flagellum-driven motility allows bacteria to move themselves to the appropriate location at the right time. Here, we identify the polar landmark protein HubP as a modulator of polar flagellation that recruits the flagellar assembly protein FapA to the old cell pole, thereby controlling its activity for the early events of flagellar assembly in Vibrio vulnificus. We show that dephosphorylated EIIAGlc of the PEP-dependent sugar transporting phosphotransferase system sequesters FapA from HubP in response to glucose and hence inhibits FapA-mediated flagellation. Thus, flagellar assembly and motility is governed by spatiotemporal control of FapA, which is orchestrated by the competition between dephosphorylated EIIAGlc and HubP, in the human pathogen V. vulnificus.

Nkx2-5 Second Heart Field Target Gene Ccdc117 Regulates DNA Metabolism and Proliferation.

The cardiac transcription factor Nkx2-5 is essential for normal outflow tract (OFT) and right ventricle (RV) development. Nkx2-5-/- null mouse embryos display severe OFT and RV hypoplasia and a single ventricle phenotype due to decreased proliferation of Second Heart Field (SHF) cells, a pool of cardiac progenitors present in anterior pharyngeal arch mesoderm at mid-gestation. However, definition of the precise role of Nkx2-5 in facilitating SHF expansion is incomplete. We have found that Nkx2-5 positively and directly regulates a novel target gene, Ccdc117, in cells of the SHF at these stages. The nuclear/mitotic spindle associated protein Ccdc117 interacts with the MIP18/MMS19 cytoplasmic iron-sulfur (FeS) cluster assembly (CIA) complex, which transfers critical FeS clusters to several key enzymes with functions in DNA repair and replication. Loss of cellular Ccdc117 expression results in reduced proliferation rates associated with a delay at the G1-S transition, decreased rates of DNA synthesis, and unresolved DNA damage. These results implicate a novel role for Nkx2-5 in the regulation of cell cycle events in the developing heart, through Ccdc117's interaction with elements of the CIA pathway and the facilitation of DNA replication during SHF expansion.

Grain width 2 (GW2) and its interacting proteins regulate seed development in rice (Oryza sativa L.).

BACKGROUND: Seed size has been extensively studied in crop plants, as it determines crop yield. However, the mechanism of seed development remains elusive. In this study, we explored the mechanism of seed development in rice (Oryza sativa L.), and identified proteins affecting seed size. RESULTS: Proteomic analysis showed that glyceraldehyde 3-phosphate dehydrogenase, chitinase 14 (CHT14), and phosphoglycerate kinase (PGK) accumulated to high levels in the seeds of the natural japonica rice mutant Oochikara, which carries a loss-of-function mutation in the grain width 2 (GW2) gene; GW2 encodes a RING-type E3 ubiquitin ligase. In vitro pull-down and ubiquitination assays showed that CHT14 and PGK directly interacted with GW2 but were not ubiquitinated by GW2. Immunoblot analysis revealed that protein disulfide isomerase-like 1-1 accumulated to high levels in young developing seeds of the gw2 mutant compared with the wild type. Histochemical ß-glucuronidase staining showed strong expression of GW2 in leaf and root tissues but weak expression in leaf sheaths and internodes. In addition, transformation of the green fluorescent protein (GFP) gene under the control of the GW2 promoter in rice revealed GFP expression in the aleurone layer of seeds. CONCLUSIONS: Collectively, these results suggest that GW2 regulates seed size through direct interactions with proteins involved in carbohydrate metabolism by modulating their activity or stability and controlling disulfide bond formation in various proteins during seed development. Additionally, GW2 participates in vegetative as well as reproductive growth, and protects the seed from pathogen attack.

Potential Survival and Pathogenesis of a Novel Strain, Vibrio parahaemolyticus FORC_022, Isolated From a Soy Sauce Marinated Crab by Genome and Transcriptome Analyses.

Vibrio parahaemolyticus can cause gastrointestinal illness through consumption of seafood. Despite frequent food-borne outbreaks of V. parahaemolyticus, only 19 strains have subjected to complete whole-genome analysis. In this study, a novel strain of V. parahaemolyticus, designated FORC_022 (Food-borne pathogen Omics Research Center_022), was isolated from soy sauce marinated crabs, and its genome and transcriptome were analyzed to elucidate the pathogenic mechanisms. FORC_022 did not include major virulence factors of thermostable direct hemolysin (tdh) and TDH-related hemolysin (trh). However, FORC_022 showed high cytotoxicity and had several V. parahaemolyticus islands (VPaIs) and other virulence factors, such as various secretion systems (types I, II, III, IV, and VI), in comparative genome analysis with CDC_K4557 (the most similar strain) and RIMD2210633 (genome island marker strain). FORC_022 harbored additional virulence genes, including accessory cholera enterotoxin, zona occludens toxin, and tight adhesion (tad) locus, compared with CDC_K4557. In addition, O3 serotype specific gene and the marker gene of pandemic O3:K6 serotype (toxRS) were detected in FORC_022. The expressions levels of genes involved in adherence and carbohydrate transporter were high, whereas those of genes involved in motility, arginine biosynthesis, and proline metabolism were low after exposure to crabs. Moreover, the virulence factors of the type III secretion system, tad locus, and thermolabile hemolysin were overexpressed. Therefore, the risk of foodborne-illness may be high following consumption of FORC_022 contaminated crab. These results provided molecular information regarding the survival and pathogenesis of V. parahaemolyticus FORC_022 strain in contaminated crab and may have applications in food safety.

Cyclo-(l-Phe-l-Pro), a Quorum-Sensing Signal of Vibrio vulnificus, Induces Expression of Hydroperoxidase through a ToxR-LeuO-HU-RpoS Signaling Pathway To Confer Resistance against Oxidative Stress.

Vibrio vulnificus, an opportunistic human pathogen, produces cyclo-(l-Phe-l-Pro) (cFP), which serves as a signaling molecule controlling the ToxR-dependent expression of innate bacterial genes, and also as a virulence factor eliciting pathogenic effects on human cells by enhancing intracellular reactive oxygen species levels. We found that cFP facilitated the protection of V. vulnificus against hydrogen peroxide. At a concentration of 1 mM, cFP enhanced the level of the transcriptional regulator RpoS, which in turn induced expression of katG, encoding hydroperoxidase I, an enzyme that detoxifies H2O2 to overcome oxidative stress. We found that cFP upregulated the transcription of the histone-like proteins vHUα and vHUß through the cFP-dependent regulator LeuO. LeuO binds directly to upstream regions of vhuA and vhuB to enhance transcription. vHUα and vHUß then enhance the level of RpoS posttranscriptionally by stabilizing the mRNA. This cFP-mediated ToxR-LeuO-vHUαß-RpoS pathway also upregulates genes known to be members of the RpoS regulon, suggesting that cFP acts as a cue for the signaling pathway responsible for both the RpoS and the LeuO regulons. Taken together, this study shows that cFP plays an important role as a virulence factor, as well as a signal for the protection of the cognate pathogen.