The parABS system is a widely employed mechanism for plasmid partitioning and chromosome segregation in bacteria. ParB binds to parS sites on plasmids and chromosomes and associates with broad regions of adjacent DNA, a phenomenon known as spreading. Although essential for ParB function, the mechanism of spreading remains poorly understood. Using single-molecule approaches, we discovered that Bacillus subtilis ParB (Spo0J) is able to trap DNA loops. Point mutants in Spo0J that disrupt DNA bridging are defective in spreading and recruitment of structural maintenance of chromosomes (SMC) condensin complexes in vivo. DNA bridging helps to explain how a limited number of Spo0J molecules per parS site (~20) can spread over many kilobases and suggests a mechanism by which ParB proteins could facilitate the loading of SMC complexes. We show that DNA bridging is a property of diverse ParB homologs, suggesting broad evolutionary conservation.

This paper describes a novel method for fabricating and sealing high-density arrays of femtoliter reaction chambers. We chemically etch one end of a 2.3 mm diameter glass optical fiber bundle to create an array of microwells. We then use a contact printing method to selectively modify the surface of the material between microwells with a hydrophobic silane. This modification makes it possible to fill the wells with aqueous solution and then seal them with a droplet of oil, forming an array of isolated reaction chambers. Individual β-galactosidase molecules trapped in these reaction chambers convert a substrate into a fluorescent product that can be readily detected because a high local concentration of product is achieved. This binary readout can be used for ultra-sensitive measurements of enzyme concentration. We observed that the percentage of wells showing enzyme activity was linearly dependent on the concentration of soluble β-galactosidase in the picomolar range. A similar response was also observed for streptavidin-β-galactosidase captured by biotinylated beads. These arrays are also suitable for performing single-molecule kinetics studies on hundreds to thousands of enzyme molecules simultaneously. We observed a broad distribution of catalytic rates for individual β-galactosidase molecules trapped in the microwells, in agreement with previous studies using similar arrays that were mechanically sealed. We have further demonstrated that this femtoliter fiber-optic array can be integrated into a PDMS microfluidic channel system and sealed with oil on-chip, creating an easy to use and high-throughput device for single-molecule analysis.

The DNA polymerases involved in DNA replication achieve high processivity of nucleotide incorporation by forming a complex with processivity factors. A model system for replicative DNA polymerases, the bacteriophage T7 DNA polymerase (gp5), encoded by gene 5, forms a tight, 1∶1 complex with Escherichia coli thioredoxin. By a mechanism that is not fully understood, thioredoxin acts as a processivity factor and converts gp5 from a distributive polymerase into a highly processive one. We use a single-molecule imaging approach to visualize the interaction of fluorescently labeled T7 DNA polymerase with double-stranded DNA. We have observed T7 gp5, both with and without thioredoxin, binding nonspecifically to double-stranded DNA and diffusing along the duplex. The gp5/thioredoxin complex remains tightly bound to the DNA while diffusing, whereas gp5 without thioredoxin undergoes frequent dissociation from and rebinding to the DNA. These observations suggest that thioredoxin increases the processivity of T7 DNA polymerase by suppressing microscopic hopping on and off the DNA and keeping the complex tightly bound to the duplex.T he bacteriophage T7 replisome is an elegantly simple, wellstudied model system of DNA replication (1). Replicative DNA synthesis is supported by T7 DNA polymerase (gp5), in a tight, 1∶1 complex with Escherichia coli thioredoxin, which will henceforth be referred to as gp5/trx. By itself, the 80-kDa gp5 synthesizes DNA in a distributive fashion and is capable of adding only a few nucleotides to the 3 0 end of a primer before dissociating from the primer template (2). Association of gp5 with the 12-kDa E. coli host protein thioredoxin results in a stable 1∶1 complex (K D ¼ 5 nM) and drastically increases the processivity of DNA synthesis. This interaction is critical for replication of T7 DNA-the phage cannot reproduce in thioredoxin null strains of E. coli (3, 4). The affinity of gp5/trx for primer-template DNA is increased at least 20-fold compared to that of gp5 alone (5), resulting in a complex that is capable of polymerizing hundreds of nucleotides before dissociation (2).Thioredoxin binds to gp5 via a 71-amino acid loop that resides between two alpha helices in the thumb domain and is called the thioredoxin-binding domain (TBD) (6). This loop is absent from other members of the Pol I family of DNA polymerases, but when inserted into E. coli DNA polymerase I at the homologous site, the resulting chimera displays a thioredoxin-dependent increase in processivity (7). The TBD also functions as a scaffold for the assembly of the replisome. The binding of thioredoxin structures the domain and creates two small solvent exposed loops containing binding sites for the T7 single-stranded DNA-binding protein and the T7 helicase (8, 9). Thioredoxin itself does not bind to DNA, and only one residue on thioredoxin has been implicated in binding to any of the other replisome components other than gp5 (10).Crystal structures of gp5/trx bound to primer-template DNA reveal that the TBD is extended over the dup...

This essay compares the approaches that scientific societies in the ACCESS meta-organization use to implement and assess travel award programs for URM trainees and presents a set of recommendations, including both short- and long-term outcomes assessment in populations of interest and specialized programmatic activities coupled to travel award programs.

Scientific societies aiming to foster inclusion of scientists from underrepresented (UR) backgrounds among their membership often delegate primary responsibility for this goal to a diversity-focused committee. The National Science Foundation has funded the creation of the Alliance to Catalyze Change for Equity in STEM Success (ACCESS), a meta-organization bringing together representatives from several such STEM society committees to serve as a hub for a growing community of practice. Our goal is to coordinate efforts to advance inclusive practices by sharing experiences and making synergistic discoveries about what works. ACCESS has analyzed the approaches by which member societies have sought to ensure inclusivity through selection of annual meeting speakers. Here we discuss how inclusive speaker selection fosters better scientific environments for all and identify challenges and promising practices for societies striving to maximize inclusivity of speakers in their scientific programming.

One sentence summary: This commentary discusses approaches and challenges for bringing personal microbiome and next-generation sequencing experiments into the classroom laboratory. Editor: Beatrix Fahnert † Mark R. Hartman, http://orcid.org/0000-0003-1564-5285 ABSTRACTSequencing and bioinformatics technologies have advanced rapidly in recent years, driven largely by developments in next-generation sequencing (NGS) technology. Given the increasing importance of these advances, there is a growing need to incorporate concepts and practices relating to NGS into undergraduate and high school science curricula. We believe that direct access to sequencing and bioinformatics will improve the ability of students to understand the information obtained through these increasingly ubiquitous research tools. In this commentary, we discuss approaches and challenges for bringing NGS into the classroom based on our experiences in developing and running a microbiome project in high school and undergraduate courses. We describe strategies for maximizing student engagement through establishing personal relevance and utilizing an inquiry-based structure. Additionally, we address the practical issues of incorporating cutting edge technologies into an established curriculum. Looking forward, we anticipate that NGS educational experiments will become more commonplace as sequencing costs continue to decrease and the workflow becomes more user friendly.

Modern genetics relies on cutting-edge sequencing and bioinformatics technologies. A high school experiment that explores current sequencing techniques in the context of race and genetics is described.

Integration host factor (IHF) is a nucleoid-associated protein involved in DNA packaging, integration of viral DNA and recombination. IHF binds with nanomolar affinity to duplex DNA containing a 13 bp consensus sequence, inducing a bend of ~160° upon binding. We determined that IHF binds to DNA Four-way or Holliday junctions (HJ) with high affinity regardless of the presence of the consensus sequence, signifying a structure-based mechanism of recognition. Junctions, important intermediates in DNA repair and homologous recombination, are dynamic and can adopt either an open or stacked conformation, where the open conformation facilitates branch migration and strand exchange. Using ensemble and single molecule Förster resonance energy transfer (FRET) methods, we investigated IHF-induced changes in the population distribution of junction conformations and determined that IHF binding shifts the population to the open conformation. Further analysis of smFRET dynamics revealed that even in the presence of protein, the junctions remain dynamic as fast transitions are observed for the protein-bound open state. Protein binding alters junction conformational dynamics, as cross correlation analyses reveal the protein slows the transition rate at 1 mM Mg2+ but accelerates the transition rate at 10 mM Mg2+. Stopped flow kinetic experiments provide evidence for two binding steps, a rapid, initial binding step followed by a slower step potentially associated with a conformational change. These measurements also confirm that the protein remains bound to the junction during the conformer transitions and further suggest that the protein forms a partially dissociated state that allows junction arms to be dynamic. These findings, which demonstrate that IHF binds HJs with high affinity and stabilizes junctions in the open conformation, suggest that IHF may play multiple roles in the processes of integration and recombination in addition to stabilizing bacterial biofilms.