The FtsZ protein, a tubulin-like GTPase, plays pivotal role in prokaryotic cell division. In vivo it localizes to the midcell and assembles into ring-like structure-the Z-ring. Z-ring serves as an essential scaffold recruit all other division proteins generates contractile force for cytokinesis, but its supramolecular structure remains unknown. Electron microscopy (EM) has been unsuccessful detecting due dense cytoplasm of bacterial cells, conventional fluorescence light (FLM) only provided...

Loop-mediated isothermal amplification (LAMP) is a versatile technique for detection of target DNA and RNA, enabling rapid molecular diagnostic assays with minimal equipment. The global SARS-CoV-2 pandemic has presented an urgent need new better methods, colorimetric LAMP utilized in numerous studies detection. However, the sensitivity early reports been below that standard RT-qPCR tests, we sought to improve performance. Here report use guanidine hydrochloride combined primer sets increase...

Significance Bacterial cytokinesis is orchestrated by a macromolecular complex termed the divisome. Central to divisome ring-like, polymeric structure, called FtsZ-ring (Z-ring). The Z-ring may generate constrictive force analogous that provided eukaryotic actomyosin ring drive membrane invagination during cytokinesis. By combining single-molecule imaging with genetic manipulations, we found that, unexpectedly, rate of septum closure in Escherichia coli cells robust many substantial...

Summary In most bacterial cells, cell division is dependent on the polymerization of FtsZ protein to form a ring‐like structure ( Z ‐ring) at midcell. Despite its essential role, molecular architecture ‐ring remains elusive. this work we examine roles two ‐associated proteins, ZapA and ZapB , in assembly dynamics E scherichia coli cells. cells deleted zapA or zapB observed abnormal septa highly dynamic structures. While details these structures are difficult discern under conventional...

Cell elongation in rod-shaped bacteria is mediated by the Rod system, a conserved morphogenic complex that spatially controls cell wall assembly glycan polymerase RodA and crosslinking enzyme PBP2. Using Escherichia coli as model we identified PBP2 variant promotes system function when essential accessory components of machinery are inactivated. This hyperactivates synthesis vivo stimulates activity RodA-PBP2 complexes vitro. Cells with activated synthase also exhibited enhanced...

The prokaryotic tubulin homolog, FtsZ, forms a ring-like structure (FtsZ-ring) at midcell. FtsZ-ring establishes the division plane and enables assembly of macromolecular machinery (divisome). Although many molecular components divisome have been identified their interactions extensively characterized, spatial organization these proteins within is unclear. Consequently, physical mechanisms that drive assembly, maintenance, constriction remain elusive. Here we applied single-molecule based...

ABSTRACT The cell envelope of gram-negative bacteria consists two membranes sandwiching the peptidoglycan (PG) wall. outer membrane (OM) contains integrated beta-barrel proteins and has an leaflet composed lipopolysaccharide (LPS). LPS is transported from inner where it made to OM surface by Lpt system. In polarly elongating alpha-proteobacterium Brucella abortus , transport been localized polar growth zone division site. However, not tracked in live proteobacteria like Escherichia coli that...

RecG and RuvAB are proposed to act at stalled DNA replication forks facilitate restart. To define the roles of these proteins in fork regression, we used a combination assays determine whether RecG, or both capable acting fork. The results show that binds C-terminus single-stranded binding protein (SSB) forming stoichiometric complex 2 monomers per SSB tetramer. This occurs solution bound single stranded (ssDNA). result this is stabilization interaction with ssDNA. In contrast, does not bind...

Cell division in Escherichia coli begins with the polymerization of FtsZ into a ring-like structure, Z-ring, at midcell. All other proteins are thought to require Z-ring for recruitment future site. Here, it is reported that associated ZapA and ZapB form FtsZ-independent structures Upon disruption by antagonist SulA, remained midcell as cloud-like accumulation. Using ZapA(N60Y), variant defective interaction FtsZ, was established these without connection Z-ring. Furthermore, accumulations...

The mammalian mRNA 5′ cap structures play important roles in cellular processes such as nuclear export, efficient translation, and evading innate immune surveillance regulating 5′-mediated turnover. Hence, installation of the proper is crucial therapeutic applications synthetic mRNA. core structure, Cap-0, generated by three sequential enzymatic activities: RNA triphosphatase, guanylyltransferase, N7-guanine methyltransferase. Vaccinia virus capping enzyme (VCE) a heterodimeric that has been...

Commercially synthesized genes are typically made using variations of homology-based cloning techniques, including polymerase cycling assembly from chemically microarray-derived oligonucleotides. Here, we apply Data-optimized Assembly Design (DAD) to the synthesis hundreds codon-optimized in both constitutive and inducible vectors Golden Gate Assembly. Starting oligonucleotide pools, synthesize three simple steps: (1) amplification parts belonging individual assemblies parallel a single...

New antibiotics are needed to combat the growing problem of resistant bacterial infections. An attractive avenue toward discovery such next-generation therapies is identify novel inhibitors clinically validated targets, like cell wall biogenesis. We have therefore developed a pathway-directed whole-cell screen for small molecules that block activity Rod system Escherichia coli This conserved multiprotein complex required elongation and morphogenesis rod-shaped bacteria. It composed synthases...

Abstract Loop-mediated isothermal amplification (LAMP) is a versatile technique for detection of target DNA and RNA, enabling rapid molecular diagnostic assays with minimal equipment. The global SARS-CoV-2 pandemic has presented an urgent need new better methods, colorimetric LAMP utilized in numerous studies detection. However, the sensitivity early reports been below that standard RT-qPCR tests, we sought to improve performance. Here report use guanidine hydrochloride combined primer sets...

Direct methods for determining the fidelity of DNA polymerases are robust, with relatively little sample manipulation before sequencing. In contrast, measuring RNA polymerase and reverse transcriptase fidelities complicated by additional preparation steps that introduce ambiguity error. Here, we describe a sequencing method, termed Roll-Seq, simultaneously individual transcriptases (RT) using Pacific Biosciences single molecule real-time By high rolling-circle activity, Roll-Seq generates...

Bacterial cell division requires the coordinated assembly of more than ten essential proteins at midcell. Central to this process is formation a ring-like suprastructure (Z-ring) by FtsZ protein plan. The Z-ring consists multiple single-stranded protofilaments, and understanding arrangement protofilaments inside will provide insight into mechanism its function as force generator. This information has remained elusive due current limitations in conventional fluorescence microscopy electron...

Bacterial cell division requires the coordinated assembly of more than ten essential proteins at midcell1,2. Central to this process is formation a ring-like suprastructure (Z-ring) by FtsZ protein plan3,4. The Z-ring consists multiple single-stranded protofilaments, and understanding arrangement protofilaments inside will provide insight into mechanism its function as force generator5,6. This information has remained elusive due current limitations in conventional fluorescence microscopy...

ABSTRACT Cell elongation in rod-shaped bacteria is mediated by the Rod system, a conserved morphogenic complex that spatially controls cell wall (CW) assembly. In Escherichia coli , alterations CW synthase component of system called PBP2 were identified overcome other inactivating defects. activity was stimulated suppressors vivo, and purified complexes with these changes showed more robust synthesis vitro. Polymerization actin-like MreB also found to be enhanced cells activated synthase....

Abstract Mining phages for new enzymatic activities continues to be important the development of tools biotechnology. In this study, we used MetaGPA—a method linking genotype phenotype in metagenomic data—to identify deoxycytidine deaminases, a protein family highly associated with cytosine modifications metaviromes. Unexpectedly, subset these deaminases exhibited preference 5-methylcytosine (5mC) over (C) both mononucleotide and single-stranded DNA substrates. methylome sequencing workflow,...

Many stem cells undergo asymmetric division to produce both a self-renewing cell and differentiating daughter cell. We previously reported that in male Drosophila germline (GSCs), preexisting (old) histone H3 is segregated the self-renewed cell, whereas incorporates newly synthesized (new) H3. Here, we show H4 likewise asymmetrically, while histones H2A H2B are symmetrically. Using superresolution imaging, visualize spatially separable old new distributions interphase GSC nuclei on isolated...