The structure-function analysis of Obg-like GTPase proteins along the evolutionary tree from bacteria to humans.

Obg proteins belong to P-loop guanine triphosphatase (GTPase) that are conserved from bacteria to humans. Like other GTPases, Obg cycles between guanine triphosphate (GTP) bound "on" state and guanine diphosphate (GDP)-bound "off" state, thereby controlling various cellular processes. Different members of this group have unique structural characteristics; a conserved glycine-rich N-terminal domain known as obg fold, a central conserved nucleotide binding domain, and a less conserved C-terminal domain of other functions. Obg is a ribosome dependent GTPase helps in ribosome maturation by interacting with several proteins of the 50S subunit of the ribosome. Obg proteins have been widely considered as a regulator of cellular functions, helping in DNA replication, cell division. Apart from that, this protein also takes part in various stress adaptation pathways like a stringent response, sporulation, and general stress response. In this particular review, the structural features of ObgE have been highlighted and how the structure plays important role in interacting with regulators like GTP, ppGpp that are crucial for executing biological function has been orchestrated. In particular, we believe that Obg-like proteins can provide a link between different global pathways that are necessary for fine-tuning cellular processes to maintain the cellular energy status.

Effect of a single amino acid substitution G98D in a ribosome-associated essential GTPase, CgtA, on the growth and morphology of Vibrio cholerae.

CgtA, a highly conserved 50S ribosome-associated essential GTPase, acts as a repressor of the stringent stress response under nutrient-rich growth conditions to suppress basal levels of the alarmone ppGpp in V. cholerae. To further explore the in vivo functionality of CgtA, we introduced an amino acid substitution, i.e., Gly98Asp, in a conserved glycine residue in the N-terminal domain. The constructed V. cholerae mutant was designated CgtA(G98D). Comparison of cell sizes of the CgtA(G98D)mutant with its isogenic wild-type (Wt) strain N16961 under different phases of growth by Transmission Electron Microscopy (TEM) and statistical analysis suggests that CgtA may control the cell size of V. cholerae. The cell length is significantly reduced, corresponding to the delayed growth in the mid-logarithmic phase. The differences in the cell length of CgtA(G98D) and Wt are indistinguishable in the late logarithmic phase. During the stationary phase, marked by higher OD600, a sub-population of CgtA(G98D) cells outnumbered the Wt cells lengthwise. CgtA(G98D) cells appeared slenderer than Wt cells with significantly reduced cell width. However, the centerline curvature is preserved in CgtA(G98D) cells. We propose that in addition to its multitude of intracellular roles, CgtA may influence the cell size of V. cholerae.

GTP Binding is Necessary for the Activation of a Toxic Mutant Isoform of the Essential GTPase ObgE.

Even though the Obg protein is essential for bacterial viability, the cellular functions of this universally conserved GTPase remain enigmatic. Moreover, the influence of GTP and GDP binding on the activity of this protein is largely unknown. Previously, we identified a mutant isoform of ObgE (the Obg protein of Escherichia coli) that triggers cell death. In this research we explore the biochemical requirements for the toxic effect of this mutant ObgE* isoform, using cell death as a readily accessible read-out for protein activity. Both the absence of the N-terminal domain and a decreased GTP binding affinity neutralize ObgE*-mediated toxicity. Moreover, a deletion in the region that connects the N-terminal domain to the G domain likewise abolishes toxicity. Taken together, these data indicate that GTP binding by ObgE* triggers a conformational change that is transmitted to the N-terminal domain to confer toxicity. We therefore conclude that ObgE*-GTP, but not ObgE*-GDP, is the active form of ObgE* that is detrimental to cell viability. Based on these data, we speculate that also for wild-type ObgE, GTP binding triggers conformational changes that affect the N-terminal domain and thereby control ObgE function.

Characterization of mitochondrion-targeted GTPases in Plasmodium falciparum.

Ribosome assembly is critical for translation and regulating the response to cellular events and requires a complex interplay of ribosomal RNA and proteins with assembly factors. We investigated putative participants in the biogenesis of the reduced organellar ribosomes of Plasmodium falciparum and identified homologues of two assembly GTPases - EngA and Obg that were found in mitochondria. Both are indispensable in bacteria and P. berghei EngA is among the 'essential' parasite blood stage proteins identified recently. PfEngA and PfObg1 interacted with parasite mitoribosomes in vivo. GTP stimulated PfEngA interaction with the 50S subunit of Escherichia coli surrogate ribosomes. Although PfObg1-ribosome interaction was independent of nucleotide binding, GTP hydrolysis by PfObg1 was enhanced upon ribosomal association. An additional function for PfObg1 in mitochondrial DNA transactions was suggested by its specific interaction with the parasite mitochondrial genome in vivo. Deletion analysis revealed that the positively-charged OBG (spoOB-associated GTP-binding protein) domain mediates DNA-binding. A role for PfEngA in mitochondrial genotoxic stress response was indicated by its over-expression upon methyl methanesulfonate-induced DNA damage. PfEngA had lower sensitivity to an E. coli EngA inhibitor suggesting differences with bacterial counterparts. Our results show the involvement of two important GTPases in P. falciparum mitochondrial function, with the first confirmed localization of an EngA homologue in eukaryotic mitochondria.

Structural insights of Mycobacterium GTPase-Obg and anti-sigma-F factor Usfx interaction.

An essential protein for bacterial growth, GTPase-Obg (Obg), is known to play an unknown but crucial role in stress response as its expression increases in Mycobacterium under stress conditions. It is well reported that Obg interacts with anti-sigma-F factor Usfx; however, a detailed analysis and structural characterization of their physical interaction remain undone. In view of above-mentioned points, this study was conceptualized for performing binding analysis and structural characterization of Obg-Usfx interaction. The binding studies were performed by surface plasmon resonance, while in silico docking analysis was done to identify crucial residues responsible for Obg-Usfx interaction. Surface plasmon resonance results clearly suggest that N-terminal and G domains of Obg mainly contribute to Usfx binding. Also, binding constants display strong affinity that was further evident by intermolecular hydrogen bonds and hydrophobic interactions in the predicted complex. Strong interaction between Obg and Usfx supports the view that Obg plays an important role in stress response, essentially required for Mycobacterium survival. As concluded by various studies that Obg is crucial for Mycobacterium survival under stress, this structural information may help us in designing novel and potential inhibitors against resistant Mycobacterium strains.

Development, validation and field evaluation of a quantitative real-time PCR able to differentiate between field Mycoplasma synoviae and the MS-H-live vaccine strain.

A quantitative PCR (qPCR) able to differentiate between field Mycoplasma synoviae and MS-H vaccine strain was developed, validated and evaluated. It was developed using nucleotide differences in the obg gene. Analytical specificity and sensitivity assessed using DNA from 194 M. synoviae field samples, three different batches of MS-H vaccine and from 43 samples representing four other avian Mycoplasma species proved to be 100%. The detection limit for field M. synoviae and MS-H vaccine strain was 102-3 and 102 colony-forming units PCR equivalents/g trachea mucus, respectively. The qPCR was able to detect both, field M. synoviae and MS-H vaccine strain in ratios of 1:100 determined both using spiked and field samples. One hundred and twenty samples from M. synoviae-infected non-vaccinated birds, 110 samples from M. synoviae-vaccinated birds from a bird experiment and 224 samples from M. synoviae negative (serology and PCR) birds were used to determine the relative sensitivity and specificity using a previously described M. synoviae PCR as reference. The relative sensitivity and specificity for field M. synoviae were 95.0% and 99.6%, respectively, and 94.6% and 100% for the MS-H-live vaccine, respectively. Field validation and confirmation by multi locus sequence typing revealed that the qPCR correctly distinguished between MS-H and field M. synoviae. Evaluation of the differentiating M. synoviae qPCR in three commercial flocks suggested transmission of MS-H-live vaccine from vaccinated to non-vaccinated flocks at the same farm. Furthermore, it showed evidence for the colonization with field M. synoviae in MS-H-vaccinated flocks.

Two conserved amino acids of juxtaposed domains of a ribosomal maturation protein CgtA sustain its optimal GTPase activity.

CgtA is a highly conserved ribosome binding protein involved in ribosome biogenesis and associated with stringent response. It is a 55 KDa GTPase protein consisting of GTPase, Obg and C-terminal domains. The function of the latter two domains was not clear and despite the importance, the mode of action of CgtA is still largely unknown. Knocking out of CgtA gene is lethal and mutations lead to growth, sporulation and developmental defects in bacteria. It was found that a growth defect and pinhole size colony morphology of Bacillus subtilis was associated with a Gly92Asp point mutation on the Obg domain of its CgtA protein, instead of its GTPase domain. CgtA is an important and essential protein of the deadly diarrhea causing bacteria Vibrio cholerae and in order to investigate the mode of action of the V. cholerae CgtA we have utilized this information. We measured the GTPase activity of V. cholerae CgtA (CgtAvc) protein in the presence of purified ribosome. Our results showed 5-fold increased GTP hydrolysis activity compared to its intrinsic activity. Then we explored the GTPase activity of the mutated CgtAvc (Gly98Asp) located at the Obg domain, which reduced the GTP hydrolysis rate to half. The double point mutations (Gly98Asp, and Tyr194Gly) encompassing another conserved residue, Tyr194, located at the diagonally opposite position in the GTPase domain largely restored (about 82%) the reduced GTPase activity, revealing a fine-tuned inter-domain movement readily associated with the GTPase activity of CgtA and thus maintaining the proper functioning of the CgtA protein.

Conformational dynamics and ribosomal interactions of Bacillus subtilis Obg in various nucleotide-bound states: Insights from molecular dynamics simulation.

Obg, a GTPase, binds to the premature 50S ribosomal subunit and facilitates recruitment of rproteins and rRNA processing to form the mature 50S subunit. This binding depends on nucleotide-induced conformational changes (GDP/GTP). However, the mechanism by which Obg undergoes conformational changes to associate with the premature 50S subunit is unknown. Therefore, 1000 ns molecular dynamics simulations were conducted to investigate this mechanism. Visualization of the simulated trajectory showed that in GDP and GTP-bound states, the C-domain moved towards the SwI region, while in GTP-Mg2+ and ppGpp-bound states, the C-domain shifted towards the N-tails. Further, positioning these conformations of Obg on the 50S subunit suggests possible mechanisms by which the GTP-Mg2+ bound state is responsible for recruiting rprotein, as well as the impact of the absence of Mg2+ in the GTP-bound state. Furthermore, the study provides insights into the conformational changes that may lead to the dissociation of the GDP-bound state from the 50S subunit and explores the potential role of the ppGpp-bound state in inhibiting 70S ribosome formation. Additionally, RMSF and community network analyses reveal how internal dynamics and intricate connections within Obg affect C-domain motion.

Oat Beta-Glucan Dietary Intervention on Antioxidant Defense Parameters, Inflammatory Response and Angiotensin Signaling in the Testes of Rats with TNBS-Induced Colitis.

Male infertility represents a significant public health concern. There is a negative impact of inflammatory bowel diseases (IBDs) on the male reproductive system. The aim of this study was to investigate whether oat beta-glucan (OBG) with different molar mass can modulate parameters of antioxidant defense and inflammatory response in the testes of adult Sprague-Dawley rats with TNBS-induced colitis and whether the OBG intervention can modulate the inflammatory response in association with the RAS system. Results: higher testicular superoxide dismutase (SOD), glutathione reductase (GR) activities and glutathione (GSH) concentration, and lower testosterone (T) level and glutathione peroxidase (GPx) activity, were observed in rats with colitis than in healthy control ones. TNBS-induced colitis resulted in decreased the angiotensin 1-7 (ANG 1-7) level in the testes of rats fed with low-molar mass OBG compared to control animals. Conclusions: although colitis induced moderate pro-oxidant changes in the gonads, it seems plausible that dietary intervention with different fractions of oat beta-glucans mass may support the maintenance of reproductive homeostasis via the stimulation of the local antioxidant defense system.

MRI-based morphometric characterizations of sexual dimorphism of the cerebrum of ferrets (Mustela putorius).

The present study aimed to characterize cerebral morphology in young adult ferrets and its sexual dimorphism using high-field MRI and MRI-based morphometry. Ex vivo short TR/TE (typical T1-weighted parameter setting for conventional MRI) and T2W (long TR/TE) MRI with high spatial resolution at 7-tesla could visualize major subcortical and archicortical structures, i.e., the caudate nucleus, lentiform nucleus, amygdala and hippocampus. In particular, laminar organization of the olfactory bulb was identifiable by short TR/TE-MRI. The primary and secondary sulci observable in the adult ferret were distinguishable on either short TR/TE- or T2W-MRI, and the cortical surface morphology was reproduced well by 3D-rendered images obtained by short TR/TE-MRI. The cerebrum had a significantly lower volume in females than in males, which was attributed to region-specific volume reduction in the cerebral cortex and subcortical white matter in females. A sexual difference was also detected, manifested by an overall reduction in normalized signal ratios of short TR/TE-MRI in all cerebral structures examined in females than in males. On the other hand, an alternating array of higher and lower short TR/TE-MRI intensity transverse zones throughout the cortex, which was reminiscent of the functional cortical areas, was revealed by maximum intensity projection (MIP) in 3D. The normalized signal ratio of short TR/TE-MRI, but not T2W-MRI in the cortex, was negatively correlated with the density of myelin-basic protein immunoreactive fibers (males, r=-0.440; females, r=-0.481). The present results suggest that sexual differences in the adult ferret cerebrum are characterized by reduced volumes of the cerebral cortex and subcortical white matter in females, and by overall reductions in physiochemical characteristics, as obtained by short TR/TE-MRI, in females. It should be noted that short TR/TE-MRI-based MIP delineated functional cortical areas related to myeloarchitecture in 3D. Such an approach makes possible conventional investigation of the functional organization of the cerebral cortex and its abnormalities using high-field MRI.

Identification and development of Tetra-ARMS PCR-based screening test for a genetic variant of OLA1 (Tyr254Cys) in the human failing heart.

Obg-like ATPase 1 (OLA1) protein has GTP and ATP hydrolyzing activities and is important for cellular growth and survival. The human OLA1 gene maps on chromosome 2, at the locus 1q31, close to the Titin (TTN) gene, which is associated with familial dilated cardiomyopathy (DCM). In this study, we found that expression of OLA1 was significantly downregulated in human failing heart tissue (HF) as compared to in non-failing heart tissues (NF). Moreover, using the Sanger sequencing method, we characterized the human OLA1 gene and screened genetic mutations in patients with heart-failing and non-failing. Among failing and non-failing heart patients, we found a total of 15 mutations, including two transversions, one substitution, one indel, and eleven transition mutations in the OLA1 gene. All the mutations were intronic except for a non-synonymous mutation, 5144A>G, resulting in 254Tyr>Cys in exon 8 of the OLA1 gene. Furthermore, haplotype analysis of these mutations revealed that these single nucleotide polymorphisms (SNPs) are linked to each other, resulting in disease-specific haplotypes. Additionally, to screen for the 254Tyr>Cys point mutation, we developed a cost-effective, rapid genetic screening PCR test that can differentiate between homozygous (AA and GG) and heterozygous (A/G) genotypes. Our results show that this test can be used as a genetic screening tool for human cardiomyopathy. These findings have important implications for the diagnosis and treatment of cardiomyopathy.

Knockdown of Obg-like ATPase 1 enhances sorafenib sensitivity by inhibition of GSK-3ß/ß-catenin signaling in hepatocellular carcinoma cells.

Background: To clarify the molecular mechanism of hepatocellular carcinoma (HCC), conducive to developing an effective HCC therapy. Owing to the severe drug resistance, the clinical use of sorafenib, which is approved for HCC treatment, is limited. The precise molecular mechanisms of sorafenib drug resistance remain unclear. In the current work, we evaluated the role of Obg-like ATPase 1 (OLA1) in sorafenib resistance in HCC. Methods: The survival of HCC patients between OLA1 expression and sorafenib treatment was analyzed by Kaplan-Meier plotter. Cell viability was measured by cell counting kit-8 (CCK-8) and colony formation assays. Cell death was detected by propidium iodide (PI) and trypan blue staining. The mRNA and protein levels were measured by real-time quantitative polymerase chain reaction (RT-qPCR) and western blot (WB), respectively. Results: We found that OLA1 was highly correlated with sorafenib resistance of HCC through a public database. Further study showed that knockdown of OLA1 enhanced cell proliferation inhibition and cell death induced by sorafenib, along with a reduction of proliferation-associated proteins (c-Myc and cyclin D1) and increase of apoptosis-related proteins (cleaved caspase-3 and cleaved PARP) in HCC cells. In addition, knockdown of OLA1 reduced the activation of glycogen synthase kinase 3ß (GSK-3ß)/ß-catenin. Conclusions: Our results proved that OLA1 can be a potential target to enhance sorafenib sensitivity in HCC.

Oryza sativa ObgC1 Acts as a Key Regulator of DNA Replication and Ribosome Biogenesis in Chloroplast Nucleoids.

BACKGROUND: The Spo0B-associated GTP-binding protein (Obg) GTPase, has diverse and important functions in bacteria, including morphological development, DNA replication and ribosome maturation. Homologs of the Bacillus subtilis Obg have been also found in chloroplast of Oryza sativa, but their primary roles remain unknown. RESULTS: We clarify that OsObgC1 is a functional homolog of AtObgC. The mutant obgc1-d1 exhibited hypersensitivity to the DNA replication inhibitor hydroxyurea. Quantitative PCR results showed that the ratio of chloroplast DNA to nuclear DNA in the mutants was higher than that of the wild-type plants. After DAPI staining, OsObgC1 mutants showed abnormal nucleoid architectures. The specific punctate staining pattern of OsObgC1-GFP signal suggests that this protein localizes to the chloroplast nucleoids. Furthermore, loss-of-function mutation in OsObgC1 led to a severe suppression of protein biosynthesis by affecting plastid rRNA processing. It was also demonstrated through rRNA profiling that plastid rRNA processing was decreased in obgc1-d mutants, which resulted in impaired ribosome biogenesis. The sucrose density gradient profiles revealed a defective chloroplast ribosome maturation of obgc1-d1 mutants. CONCLUSION: Our findings here indicate that the OsObgC1 retains the evolutionarily biological conserved roles of prokaryotic Obg, which acts as a signaling hub that regulates DNA replication and ribosome biogenesis in chloroplast nucleoids.

OLA1 is a potential prognostic molecular biomarker for endometrial cancer and promotes tumor progression.

Obg-like ATPase 1 (OLA1) is upregulated in the tumor tissues in different types of cancer. However, the function of OLA1 and its molecular mechanisms in endometrial cancer (EC) remain unknown. The present study aimed to elucidate OLA1 expression level and its biological function in endometrial cancer. The differential expression of OLA1 between EC tissues and non-cancerous tissues was analyzed using The Cancer Genome Atlas database and clinical samples. The association between clinicopathological characteristics and OLA1 expression was analyzed using bioinformatics analysis. Cell proliferation, migration and invasion were analyzed by short interfering RNA-mediated knockdown experiments, Cell Counting Kit-8, 5-Ethynyl-2'-deoxyuridine incorporation, wound healing, Transwell and Boyden assays. The potential signaling pathways associated with OLA1 in endometrial cancer were evaluated by Gene Set Enrichment Analysis. The expression levels of OLA1 in EC tissues were upregulated compared with that in non-cancerous tissues (P<0.001). Furthermore, patients with worse survival were found to have higher OLA1 expression, and increased OLA1 expression in endometrial cancer associated with clinical stage (P<0.01), histological type (P<0.01), histological grade (P<0.01), menstrual status (P<0.01), cancer status (P<0.05) and distant metastasis (P<0.05). In RL95-2 and HEC-1B cell lines, decreased levels of OLA1 inhibited proliferation, invasion and migration, and the TGF-ß signaling pathway, ubiquitin-mediated proteolysis and Wnt signaling pathway may be involved in these mechanisms. The present study revealed that OLA1 could be a potential prognostic indicator and therapeutic target in endometrial cancer, and that the TGF-ß signaling, Wnt signaling and ubiquitin-mediated proteolysis pathways may be regulated by OLA1.

Obg-Like ATPase 1 Enhances Chemoresistance of Breast Cancer via Activation of TGF-ß/Smad Axis Cascades.

Understanding the molecular mechanism of drug resistance helps to identify an effective target for breast cancer therapy. In this study we investigated the regulatory role of Obg-like ATPase 1 which is involved in multiple uses of drug resistance against breast cancer. Paclitaxel resistant cell line (MCF-7-PTR) was developed by a continuous increasing paclitaxel concentration. MTT assay was used to validate either acquired resistant or OLA1 modified cell lines. qRT-PCR, western blotting, apoptosis, and cell cycle assays were executed to evaluate gene and protein expression in cell lines. A series of in vitro assays was performed in the cells with RNAi-mediated knockdown to expound the regulatory function of OLA1 in breast cancer. We demonstrated that OLA1 was highly correlated with either acquired or intrinsic resistance of breast cancer. Further study showed that escalated expression of OLA1 promoted the EMT process in tumor cells through TGF-ß/Smad signaling cascades, resulting in the enhanced expression of anti-apoptosis-related proteins (cleaved caspase3, Bax, Bcl-2) and the strengthening depolymerization of microtubules in tumor cells. Our findings revealed that OLA1 enhanced the anti-apoptotic ability and elucidated a regulatory role of OLA1 in promoting chemotherapy resistance of breast cancer. Chemo-sensitivity of the disease can be thus enhanced significantly by knocked down OLA1, which led to the inactivation of the TGF-ß/Smad signaling cascades, polymerized microtubules, and promoted cell apoptosis. Our data suggest that OLA1 may be developed as a potential target to improve chemotherapy of patients with breast cancer.

Attenuation of a Pathogenic Mycoplasma Strain by Modification of the obg Gene by Using Synthetic Biology Approaches.

Mycoplasma species are responsible for several economically significant livestock diseases for which there is a need for new and improved vaccines. Most of the existing mycoplasma vaccines are attenuated strains that have been empirically obtained by serial passages or by chemical mutagenesis. The recent development of synthetic biology approaches has opened the way for the engineering of live mycoplasma vaccines. Using these tools, the essential GTPase-encoding gene obg was modified directly on the Mycoplasma mycoides subsp. capri genome cloned in yeast, reproducing mutations suspected to induce a temperature-sensitive (TS+) phenotype. After transplantation of modified genomes into a recipient cell, the phenotype of the resulting M. mycoides subsp. capri mutants was characterized. Single-point obg mutations did not result in a strong TS+ phenotype in M. mycoides subsp. capri, but a clone presenting three obg mutations was shown to grow with difficulty at temperatures of ≥40°C. This particular mutant was then tested in a caprine septicemia model of M. mycoides subsp. capri infection. Five out of eight goats infected with the parental strain had to be euthanized, in contrast to one out of eight goats infected with the obg mutant, demonstrating an attenuation of virulence in the mutant. Moreover, the strain isolated from the euthanized animal in the group infected with the obg mutant was shown to carry a reversion in the obg gene associated with the loss of the TS+ phenotype. This study demonstrates the feasibility of building attenuated strains of mycoplasma that could contribute to the design of novel vaccines with improved safety.IMPORTANCE Animal diseases due to mycoplasmas are a major cause of morbidity and mortality associated with economic losses for farmers all over the world. Currently used mycoplasma vaccines exhibit several drawbacks, including low efficacy, short time of protection, adverse reactions, and difficulty in differentiating infected from vaccinated animals. Therefore, there is a need for improved vaccines to control animal mycoplasmoses. Here, we used genome engineering tools derived from synthetic biology approaches to produce targeted mutations in the essential GTPase-encoding obg gene of Mycoplasma mycoides subsp. capri Some of the resulting mutants exhibited a marked temperature-sensitive phenotype. The virulence of one of the obg mutants was evaluated in a caprine septicemia model and found to be strongly reduced. Although the obg mutant reverted to a virulent phenotype in one infected animal, we believe that these results contribute to a strategy that should help in building new vaccines against animal mycoplasmoses.

Synthesis, PASS predication, in vitro antimicrobial evaluation and pharmacokinetic study of novel n-octyl glucopyranoside esters.

Octyl ß-d-glucopyranoside (OBG), prepared from d-glucose and octan-1-ol employing MW method, was subjected to direct dimolar valeroylation in pyridine at room temperature (25 °C) with valeroyl chloride. This mainly furnished the corresponding 3,6-di-O-valeroate in 57% yield indicating the regioselectivity at C-6 and C-3 positions. For structural elucidation and to get newer glucopyranosides of potential antimicrobial 3,6-di-O-valeroate was further converted into four novel 2,4-di-O-acyl esters reasonably in good yields. Per-O-acetate and per-O-benzoate of OBG were also prepared for SAR study. PASS predication and in vitro antimicrobial studies established them as better antifungal agent than that of antibacterial. SAR study along with AdmetSAR and SwissADME suggested that incorporation of alkanoyl and aromatic ester groups on octyl glucopyranoside core increase antimicrobial potentiality in very low concentration (10 µgmL-1). Molecular docking revealed that novel 2,4-di-O-tosyl ester and 2,3,4,6-tetra-O-benzoyl ester may act as competitive inhibitors of lanosterol 14-alpha demethylase.

Rice TSV3 Encoding Obg-Like GTPase Protein Is Essential for Chloroplast Development During the Early Leaf Stage Under Cold Stress.

The Spo0B-associated GTP-binding (Obg) proteins are essential for the viability of nearly all bacteria. However, the detailed roles of Obg proteins in higher plants have not yet been elucidated. In this study, we identified a novel rice (Oryza sativa L.) thermo-sensitive virescent mutant (tsv3) that displayed an albino phenotype at 20° before the three-leaf stage while being a normal green at 32° or even at 20° after the four-leaf stage. The mutant phenotype was consistent with altered chlorophyll content and chloroplast structure in leaves. Map-based cloning and complementation experiments showed that TSV3 encoded a small GTP-binding protein. Subcellular localization studies revealed that TSV3 was localized to the chloroplasts. Expression of TSV3 was high in leaves and weak or undetectable in other tissues, suggesting a tissue-specific expression of TSV3 In the tsv3 mutant, expression levels of genes associated with the biogenesis of the chloroplast ribosome 50S subunit were severely decreased at the three-leaf stage under cold stress (20°), but could be recovered to normal levels at a higher temperature (32°). These observations suggest that the rice nuclear-encoded TSV3 plays important roles in chloroplast development at the early leaf stage under cold stress.

A Mutant Isoform of ObgE Causes Cell Death by Interfering with Cell Division.

Cell division is a vital part of the cell cycle that is fundamental to all life. Despite decades of intense investigation, this process is still incompletely understood. Previously, the essential GTPase ObgE, which plays a role in a myriad of basic cellular processes (such as initiation of DNA replication, chromosome segregation, and ribosome assembly), was proposed to act as a cell cycle checkpoint in Escherichia coli by licensing chromosome segregation. We here describe the effect of a mutant isoform of ObgE (ObgE∗) that causes cell death by irreversible arrest of the cell cycle at the stage of cell division. Notably, chromosome segregation is allowed to proceed normally in the presence of ObgE∗, after which cell division is blocked. Under conditions of rapid growth, ongoing cell cycles are completed before cell cycle arrest by ObgE∗ becomes effective. However, cell division defects caused by ObgE∗ then elicit lysis through formation of membrane blebs at aberrant division sites. Based on our results, and because ObgE was previously implicated in cell cycle regulation, we hypothesize that the mutation in ObgE∗ disrupts the normal role of ObgE in cell division. We discuss how ObgE∗ could reveal more about the intricate role of wild-type ObgE in division and cell cycle control. Moreover, since Obg is widely conserved and essential for viability, also in eukaryotes, our findings might be applicable to other organisms as well.

Identification and Characterization of a Chloroplast-Targeted Obg GTPase in Dendrobium officinale.

Bacterial homologous chloroplast-targeted Obg GTPases (ObgCs) belong to the plant-typical Obg group, which is involved in diverse physiological processes during chloroplast development. However, the evolutionarily conserved function of ObgC in plants remains elusive and requires further investigation. In this study, we identified DoObgC from an epiphytic plant Dendrobium officinale and demonstrated the characteristics of DoObgC. Sequence analysis indicated that DoObgC is highly conserved with other plant ObgCs, which contain the chloroplast transit peptide (cTP), Obg fold, G domain, and OCT regions. The C terminus of DoObgC lacking the chloroplast-targeting cTP region, DoObgCΔ1-160, showed strong similarity to ObgE and other bacterial Obgs. Overexpression of DoObgCΔ1-160 in Escherichia coli caused slow cell growth and an increased number of elongated cells. This phenotype was consistent with the phenotype of cells overexpressing ObgE. Furthermore, the expression of recombinant DoObgCΔ1-160 enhanced the cell persistence of E. coli to streptomycin. Results of transient expression assays revealed that DoObgC was localized to chloroplasts. Moreover, we demonstrated that DoObgC could rescue the embryotic lethal phenotype of the Arabidopsis obgc-t mutant, suggesting that DoObgC is a functional homolog to Arabidopsis AtObgC in D. officinale. Gene expression profiles showed that DoObgC was expressed in leaf-specific and light-dependent patterns and that DoObgC responded to wounding treatments. Our previous and present studies reveal that ObgC has an evolutionarily conserved role in ribosome biogenesis to adapt chloroplast development to the environment.