Structural studies of the novel virulence factors BswR and SghA/SghR involved in pathogen virulence regulation

The pathogen-host cross-talking usually involved in different virulence factors, which play crucial roles in the pathogen virulence regulation. Here in our work, we determined crystal structures of three novel virulence factors BswR, SghA, and SghR as well as relevant complex structures. BswR mediates bacterial motilities and biofilm formation, regulates small RNA rsmZ transcription, the molecular basis for the regulation will be proposed. SghA and SghR were identified and characterized to regulate the Agrobacterium infection of plant hosts. SghR is a repressor of SghA, and negatively regulates SghA expression until the environmental signal presence. Detailed structural and functional analyses of SghA-SghR in the regulation of agrobacterial tumorigenesis and the allosteric mechanism of SghR in the regulation of SghA will be presented.​Doctor of Philosophy (SBS

A novel approach based on ferricyanide-mediator immobilized in an ion-exchangeable biosensing film for the determination of biochemical oxygen demand

A novel biochemical oxygen demand (BOD) sensing method employing a ferricyanide (FC) mediator immobilized in an ion-exchangeable polysiloxane was developed. The ion-exchangeable polysiloxane containing alkylammonium groups (PAPS-Cl) was synthesized by sol-gel reaction of 3-(aminopropyl)trimethoxysilane (APTMOS) catalyzed by hydrochloric acid. FC was combined in PAPS-Cl via ion-association and the product was labeled as PAPS-FC, which was employed for a modified glassy carbon electrode. The apparent diffusion coefficient (D-app) of FC on the modified glassy carbon electrode was 9.8 X 10(-11) cm(2) S-1. In a three-electrode system, a linear relationship between the anodic current responses and glucose/glutamate (GGA) concentration was obtained up to 40 mg O-2 L-1 (r = 0.994) when the reaction mixture was incubated for 30 min. Single sensor and piece-to-piece reproducibility were less than 3.8 and 7.7%, respectively The effects of dissolved oxygen, pH, temperature and co-existing substances on the BOD responses were studied. The sensor responses to nine pure organic substances were compared with the conventional BOD5 method and other biosensor methods. Detection results of seawater samples were compared with those obtained from the BOD5 method and showed a good correlation (r = 0.988). (C) 2008 Elsevier B.V. All rights reserved

A dissolved oxygen sensor based on composite fluorinated xerogel doped with platinum porphyrin dye

A new functional fluorinated material taking n-propyltrimethoxysilicane (n-propyl-TriMOS) and 3,3,3-trifluoropropyltrimethoxysilicane (TFP-TriMOS) as precursors was applied to construct a novel dissolved oxygen sensing film. The sensing film was fabricated by dip-coating the functional fluorinated material-doped [meso-tetrakis(pentafluorophenyl) porphyinato] platinum(II) (PtF(20)TPP) onto a glass slide. The oxygen sensing film exhibited a good linear relationship, fast response time, long stability and high sensitivity to dissolved oxygen. In the developed optical oxygen sensor, an LED and a photodiode were composed to construct a back-detection optical system not needing an optical fiber based on fluorescence intensity detection. The smart optical oxygen sensor based on the PtF(20)TPP fluorescence quenching possesses the advantages of portability and low cost and can be applied to the dissolved oxygen in situ monitoring in seawater. Copyright (C) 2009 John Wiley & Sons, Ltd.National Science and Technology Development Project (863 project)[2006AA09Z160]; National Natural Scientific Foundation of China[20775064

BswR controls bacterial motility and biofilm formation in Pseudomonas aeruginosa through modulation of the small RNA RsmZ

Pseudomonas aeruginosa relies on cell motility and ability to form biofilms to establish infections; however, the mechanism of regulation remains obscure. Here we report that BswR, a xenobiotic response element-type transcriptional regulator, plays a critical role in regulation of bacterial motility and biofilm formation in P. aeruginosa. Transcriptomic and biochemical analyses showed that BswR counteracts the repressor activity of MvaT, controls the transcription of small RNA rsmZ and regulates the biogenesis of bacterial flagella. The crystal structure of BswR was determined at 2.3 Å resolution; the monomer comprises a DNA-binding domain with a helix-turn-helix motif in the N terminus and two helices (α6 and α7) with a V-shaped arrangement in the C-terminus. In addition to the contacts between the parallel helices α5 of two monomers, the two helical extensions (α6 and α7) intertwine together to form a homodimer, which is the biological function unit. Based on the result of DNase I protection assay together with structural analysis of BswR homodimer, we proposed a BswR–DNA model, which suggests a molecular mechanism with which BswR could interact with DNA. Taken together, our results unveiled a novel regulatory mechanism, in which BswR controls the motility and biofilm formation of P. aeruginosa by modulating the transcription of small RNA rsmZ.ASTAR (Agency for Sci., Tech. and Research, S’pore)Published versio

A Dynamic Scene Vision SLAM Method Incorporating Object Detection and Object Characterization

Simultaneous localization and mapping (SLAM) based on RGB-D cameras has been widely used for robot localization and navigation in unknown environments. Most current SLAM methods are constrained by static environment assumptions and perform poorly in real-world dynamic scenarios. To improve the robustness and performance of SLAM systems in dynamic environments, this paper proposes a new RGB-D SLAM method for indoor dynamic scenes based on object detection. The method presented in this paper improves on the ORB-SLAM3 framework. First, we designed an object detection module based on YOLO v5 and relied on it to improve the tracking module of ORB-SLAM3 and the localization accuracy of ORB-SLAM3 in dynamic environments. The dense point cloud map building module was also included, which excludes dynamic objects from the environment map to create a static environment point cloud map with high readability and reusability. Full comparison experiments with the original ORB-SLAM3 and two representative semantic SLAM methods on the TUM RGB-D dataset show that: the method in this paper can run at 30+fps, the localization accuracy improved to varying degrees compared to ORB-SLAM3 in all four image sequences, and the absolute trajectory accuracy can be improved by up to 91.10%. The localization accuracy of the method in this paper is comparable to that of DS-SLAM, DynaSLAM and the two recent target detection-based SLAM algorithms, but it runs faster. The RGB-D SLAM method proposed in this paper, which combines the most advanced object detection method and visual SLAM framework, outperforms other methods in terms of localization accuracy and map construction in a dynamic indoor environment and has a certain reference value for navigation, localization, and 3D reconstruction

Structural basis of transcription inhibition by the DNA mimic protein Ocr of bacteriophage T7

Bacteriophage T7 infects Escherichia coli and evades the host restriction/modification system. The Ocr protein of T7 was shown to exist as a dimer mimicking DNA and to bind to host restriction enzymes, thus preventing the degradation of the viral genome by the host. Here we report that Ocr can also inhibit host transcription by directly binding to bacterial RNA polymerase (RNAP) and competing with the recruitment of RNAP by sigma factors. Using cryo electron microscopy, we determined the structures of Ocr bound to RNAP. The structures show that an Ocr dimer binds to RNAP in the cleft, where key regions of sigma bind and where DNA resides during transcription synthesis, thus providing a structural basis for the transcription inhibition. Our results reveal the versatility of Ocr in interfering with host systems and suggest possible strategies that could be exploited in adopting DNA mimicry as a basis for forming novel antibiotics

Insights into the mechanism and regulation of the CbbQO-type Rubisco activase, a MoxR AAA+ ATPase

The vast majority of biological carbon dioxide fixation relies on the function of ribulose 1,5-bisphosphate carboxylase/oxygenase (Rubisco). In most cases the enzyme exhibits a tendency to become inhibited by its substrate RuBP and other sugar phosphates. The inhibition is counteracted by diverse molecular chaperones known as Rubisco activases (Rcas). In some chemoautotrophic bacteria, the CbbQO-type Rca Q2O2 repairs inhibited active sites of hexameric form II Rubisco. The 2.2-Å crystal structure of the MoxR AAA+ protein CbbQ2 from Acidithiobacillus ferrooxidans reveals the helix 2 insert (H2I) that is critical for Rca function and forms the axial pore of the CbbQ hexamer. Negative-stain electron microscopy shows that the essential CbbO adaptor protein binds to the conserved, concave side of the CbbQ2 hexamer. Site-directed mutagenesis supports a model in which adenosine 5'-triphosphate (ATP)-powered movements of the H2I are transmitted to CbbO via the concave residue L85. The basal ATPase activity of Q2O2 Rca is repressed but strongly stimulated by inhibited Rubisco. The characterization of multiple variants where this repression is released indicates that binding of inhibited Rubisco to the C-terminal CbbO VWA domain initiates a signal toward the CbbQ active site that is propagated via elements that include the CbbQ α4-β4 loop, pore loop 1, and the presensor 1-β hairpin (PS1-βH). Detailed mechanistic insights into the enzyme repair chaperones of the highly diverse CO₂ fixation machinery of Proteobacteria will facilitate their successful implementation in synthetic biology ventures.Ministry of Education (MOE)Nanyang Technological UniversityThis work was funded by a Nanyang Technological University startup grant (to O.M.-C.) and by Ministry of Education (MOE) of Singapore Tier 2 grants to O.M.-C. (MOE2016-T2-2-088), Y.-G.G. (MOE2015-T2-1-078), and S.B. (MOE2017-T2-2-089)

Structural basis of σ54 displacement and promoter escape in bacterial transcription.

Gene transcription is a fundamental cellular process carried out by RNA polymerase (RNAP). Transcription initiation is highly regulated, and in bacteria, transcription initiation is mediated by sigma (σ) factors. σ recruits RNAP to the promoter DNA region, located upstream of the transcription start site (TSS) and facilitates open complex formation, where double-stranded DNA is opened up into a transcription bubble and template strand DNA is positioned inside RNAP for initial RNA synthesis. During initial transcription, RNAP remains bound to σ and upstream DNA, presumably with an enlarging transcription bubble. The release of RNAP from upstream DNA is required for promoter escape and processive transcription elongation. Bacteria sigma factors can be broadly separated into two classes with the majority belonging to the σ70 class, represented by the σ70 that regulates housekeeping genes. σ54 forms a class on its own and regulates stress response genes. Extensive studies on σ70 have revealed the molecular mechanisms of the σ70 dependent process while how σ54 transitions from initial transcription to elongation is currently unknown. Here, we present a series of cryo-electron microscopy structures of the RNAP-σ54 initial transcribing complexes with progressively longer RNA, which reveal structural changes that lead to promoter escape. Our data show that initially, the transcription bubble enlarges, DNA strands scrunch, reducing the interactions between σ54 and DNA strands in the transcription bubble. RNA extension and further DNA scrunching help to release RNAP from σ54 and upstream DNA, enabling the transition to elongation

Three distinct 3-methylcytidine (m3C) methyltransferases modify tRNA and mRNA in mice and humans

Chemical RNA modifications are central features of epitranscriptomics, highlighted by the discovery of modified ribonucleosides in mRNA and exemplified by the critical roles of RNA modifications in normal physiology and disease. Despite a resurgent interest in these modifications, the biochemistry of 3-methylcytidine (m3C) formation in mammalian RNAs is still poorly understood. However, the recent discovery of trm141 as the second gene responsible for m3C presence in RNA in fission yeast raises the possibility that multiple enzymes are involved in m3C formation in mammals as well. Here, we report the discovery and characterization of three distinct m3C-contributing enzymes in mice and humans. We found that methyltransferase-like (METTL) 2 and 6 contribute m3C in specific tRNAs and that METTL8 only contributes m3C to mRNA. MS analysis revealed that there is an ∼30–40% and ∼10–15% reduction, respectively, in METTL2 and -6 null-mutant cells, of m3C in total tRNA, and primer extension analysis located METTL2-modified m3C at position 32 of tRNAThr isoacceptors and tRNAArg(CCU). We also noted that METTL6 interacts with seryl-tRNA synthetase in an RNA-dependent manner, suggesting a role for METTL6 in modifying serine tRNA isoacceptors. METTL8, however, modified only mRNA, as determined by biochemical and genetic analyses in Mettl8 null-mutant mice and two human METTL8 mutant cell lines. Our findings provide the first evidence of the existence of m3C modification in mRNA, and the discovery of METTL8 as an mRNA m3C writer enzyme opens the door to future studies of other m3C epitranscriptomic reader and eraser functions.NRF (Natl Research Foundation, S’pore)MOE (Min. of Education, S’pore)Published versio