Search

Broadening access to science, technology, engineering, and mathematics (STEM) professions through the provision of early-career research experiences for a wide range of demographic groups is important for the diversification of the STEM workforce. The size and diversity of the community college system make it a prime educational site for achieving this aim. However, some evidence shows that women and Black, Latinx, and Native American student groups have been hindered in STEM at the community college level. One option for enhancing persistence in STEM is to incorporate the course-based research experiences (CREs) into the curriculum as a replacement for the prevalent traditional laboratory. This can be achieved through the integration of community colleges within extant, multi-institutional CREs such as the SEA-PHAGES program. Using a propensity score-matching technique, students in a CRE and traditional laboratory were compared on a range of psychosocial variables (project ownership, self-efficacy, science identity, scientific community values, and networking). Results revealed higher ratings for women and persons excluded because of their ethnicity or race (PEERs) in the SEA-PHAGES program on important predictors of persistence such as project ownership and science identity. This suggests that the usage of CREs at community colleges could have positive effects in addressing the gender gap for women and enhance inclusiveness for PEER students in STEM.

Bacteriophage EmiRose is a siphovirus infecting Corynebacterium flavescens . The EmiRose genome is 37,431 bp long and composed of 47 protein-coding genes. Based on gene content similarity, EmiRose is not closely related to any previously sequenced bacteriophages in the actinobacteriophage database to date, including other corynebacteriophages. EmiRose is classified as a singleton.

The course-based research experience (CRE) with its documented educational benefits is increasingly being implemented in science, technology, engineering, and mathematics education. This article reports on a study that was done over a period of 3 years to explicate the instructional processes involved in teaching an undergraduate CRE. One hundred and two instructors from the established and large multi-institutional SEA-PHAGES program were surveyed for their understanding of the aims and practices of CRE teaching. This was followed by large-scale feedback sessions with the cohort of instructors at the annual SEA Faculty Meeting and subsequently with a small focus group of expert CRE instructors. Using a qualitative content analysis approach, the survey data were analyzed for the aims of inquiry instruction and pedagogical practices used to achieve these goals. The results characterize CRE inquiry teaching as involving three instructional models: 1) being a scientist and generating data; 2) teaching procedural knowledge; and 3) fostering project ownership. Each of these models is explicated and visualized in terms of the specific pedagogical practices and their relationships. The models present a complex picture of the ways in which CRE instruction is conducted on a daily basis and can inform instructors and institutions new to CRE teaching.

Bacteriophages represent an enormous reservoir of novel genes, many of which are unrelated to existing entries in public databases and cannot be assigned a predicted function. Characterization of these genes can provide important insights into the intricacies of phage–host interactions and may offer new strategies to manipulate bacterial growth and behavior. Overexpression is a useful tool in the study of gene-mediated effects, and we describe here the construction of a plasmid-based overexpression library of a complete set of genes for Waterfoul, a mycobacteriophage closely related to those infecting clinically important strains of Mycobacterium tuberculosis and/or Mycobacterium abscessus. The arrayed Waterfoul gene library was systematically screened in a plate-based cytotoxicity assay, identifying a diverse set of 32 Waterfoul gene products capable of inhibiting the growth of the host Mycobacterium smegmatis and providing a first look at the frequency and distribution of cytotoxic products encoded within a single mycobacteriophage genome. Several of these Waterfoul gene products were observed to confer potent anti-mycobacterial effects, making them interesting candidates for follow-up mechanistic studies.

Bacteriophage EasyJones is a myovirus infecting Mycobacterium smegmatis mc 2 155, with a genome length and gene content similar to those of phages grouped in subcluster C1. Interestingly, EasyJones contains a gene found in a subset of C1 genomes that is similar to the well-characterized immunity repressor of subcluster A1 mycobacteriophage Bxb1.

The bacteriophage population is vast, dynamic, old, and genetically diverse. The genomics of phages that infect bacterial hosts in the phylum Actinobacteria show them to not only be diverse but also pervasively mosaic, and replete with genes of unknown function. To further explore this broad group of bacteriophages, we describe here the isolation and genomic characterization of 116 phages that infect Microbacterium spp. Most of the phages are lytic, and can be grouped into twelve clusters according to their overall relatedness; seven of the phages are singletons with no close relatives. Genome sizes vary from 17.3 kbp to 97.7 kbp, and their G+C% content ranges from 51.4% to 71.4%, compared to ~67% for their Microbacterium hosts. The phages were isolated on five different Microbacterium species, but typically do not efficiently infect strains beyond the one on which they were isolated. These Microbacterium phages contain many novel features, including very large viral genes (13.5 kbp) and unusual fusions of structural proteins, including a fusion of VIP2 toxin and a MuF-like protein into a single gene. These phages and their genetic components such as integration systems, recombineering tools, and phage-mediated delivery systems, will be useful resources for advancing Microbacterium genetics.

Bacteriophages Ilzat and Eleri are newly isolated Siphoviridae infecting Microbacterium foliorum NRRL B-24224. The phage genomes are similar in length, G+C content, and architecture and share 62.9% nucleotide sequence identity.

A challenge for chromosome segregation in all domains of life is the formation of catenated progeny chromosomes, which arise during replication as a consequence of the interwound strands of the DNA double helix. Topoisomerases play a key role in DNA unlinking both during and at the completion of replication. Here we report that chromosome unlinking can instead be accomplished by multiple rounds of site-specific recombination. We show that step-wise, site-specific recombination by XerCD-dif or Cre-loxP can unlink bacterial chromosomes in vivo, in reactions that require KOPS-guided DNA translocation by FtsK. Furthermore, we show that overexpression of a cytoplasmic FtsK derivative is sufficient to allow chromosome unlinking by XerCD-dif recombination when either subunit of TopoIV is inactivated. We conclude that FtsK acts in vivo to simplify chromosomal topology as Xer recombination interconverts monomeric and dimeric chromosomes.

Diverse proteins are known to be capable of forming amyloid aggregates, self-seeding fibrillar assemblies that may be biologically functional or pathological. Well-known examples include neurodegenerative disease-associated proteins that misfold as amyloid, fungal prion proteins that can transition to a self-propagating amyloid form and certain bacterial proteins that fold as amyloid at the cell surface and promote biofilm formation. To further explore the diversity of amyloidogenic proteins, generally applicable methods for identifying them are critical. Here we describe a cell-based method for generating amyloid aggregates that relies on the natural ability of Escherichia coli cells to elaborate amyloid fibrils at the cell surface. We use several different yeast prion proteins and the human huntingtin protein to show that protein secretion via this specialized export pathway promotes acquisition of the amyloid fold specifically for proteins that have an inherent amyloid-forming propensity. Furthermore, our findings establish the potential of this E. coli-based system to facilitate the implementation of high-throughput screens for identifying amyloidogenic proteins and modulators of amyloid aggregation.

The study of chromosome segregation in bacteria has gained strong insights from the use of cytology techniques. A global view of chromosome choreography during the cell cycle is emerging, highlighting as a next challenge the description of the molecular mechanisms and factors involved. Here, we review one of such factor, the FtsK DNA translocase. FtsK couples segregation of the chromosome terminus, the ter region, with cell division. It is a powerful and fast translocase that reads chromosome polarity to find the end, thereby sorting sister ter regions on either side of the division septum, and activating the last steps of segregation. Recent data have revealed the structure of the FtsK motor, how translocation is oriented by specific DNA motifs, termed KOPS, and suggests novel mechanisms for translocation and sensing chromosome polarity.

The bacterial septum-located DNA translocase FtsK coordinates circular chromosome segregation with cell division. Rapid translocation of DNA by FtsK is directed by 8-base-pair DNA motifs (KOPS), so that newly replicated termini are brought together at the developing septum, thereby facilitating completion of chromosome segregation. Translocase functions reside in three domains, alpha, beta and gamma. FtsKalphabeta are necessary and sufficient for ATP hydrolysis-dependent DNA translocation, which is modulated by FtsKgamma through its interaction with KOPS. By solving the FtsKgamma structure by NMR, we show that gamma is a winged-helix domain. NMR chemical shift mapping localizes the DNA-binding site on the gamma domain. Mutated proteins with substitutions in the FtsKgamma DNA-recognition helix are impaired in DNA binding and KOPS recognition, yet remain competent in DNA translocation and XerCD-dif site-specific recombination, which facilitates the late stages of chromosome segregation.

Here, we describe a detailed protocol for generating amyloid aggregates of target amyloidogenic proteins using a bacteria-based system. This system, which we call Curli-dependent amyloid generator (C-DAG), relies on the natural ability of E. coli cells to elaborate surface associated amyloid fibers known as curli that are composed of the amyloidogenic proteins CsgA and CsgB. A bipartite N-terminal signal sequence directs CsgA and CsgB across the inner and outer membranes to the outside of the cell. The transfer of this signal sequence to the N-terminus of heterologous amyloidogenic proteins similarly directs their export to the cell surface, where they assemble as amyloid fibrils. Importantly, protein secretion through the curli export pathway facilitates acquisition of the amyloid fold specifically for proteins that have an inherent amyloid-forming propensity. Thus, C-DAG provides a cell-based alternative to widely used in vitro assays for studying amyloid aggregation that circumvents the need to purify individual proteins of interest. In particular, C-DAG provides a simple method for identifying amyloidogenic proteins and for distinguishing between amyloidogenic and non-amyloidogenic variants of a particular protein. Once the appropriate vectors have been constructed, results can be obtained within 1 week.

Escherichia coli FtsK is a powerful, fast, double-stranded DNA translocase, which can strip proteins from DNA. FtsK acts in the late stages of chromosome segregation by facilitating sister chromosome unlinking at the division septum. KOPS-guided DNA translocation directs FtsK towards dif, located within the replication terminus region, ter, where FtsK activates XerCD site-specific recombination. Here we show that FtsK translocation stops specifically at XerCD-dif, thereby preventing removal of XerCD from dif and allowing activation of chromosome unlinking by recombination. Stoppage of translocation at XerCD-dif is accompanied by a reduction in FtsK ATPase and is not associated with FtsK dissociation from DNA. Specific stoppage at recombinase-DNA complexes does not require the FtsKgamma regulatory subdomain, which interacts with XerD, and is not dependent on either recombinase-mediated DNA cleavage activity, or the formation of synaptic complexes.

The septum-located DNA translocase, FtsK, acts to co-ordinate the late steps of Escherichia coli chromosome segregation with cell division. The FtsK gamma regulatory subdomain interacts with 8 bp KOPS DNA sequences, which are oriented from the replication origin to the terminus region (ter) in each arm of the chromosome. This interaction directs FtsK translocation towards ter where the final chromosome unlinking by decatenation and chromosome dimer resolution occurs. Chromosome dimer resolution requires FtsK translocation along DNA and its interaction with the XerCD recombinase bound to the recombination site, dif, located within ter. The frequency of chromosome dimer formation is approximately 15% per generation in wild-type cells. Here we characterize FtsK alleles that no longer recognize KOPS, yet are proficient for translocation and chromosome dimer resolution. Non-directed FtsK translocation leads to a small reduction in fitness in otherwise normal cell populations, as a consequence of approximately 70% of chromosome dimers being resolved to monomers. More serious consequences arise when chromosome dimer formation is increased, or their resolution efficiency is impaired because of defects in chromosome organization and processing. For example, when Cre-loxP recombination replaces XerCD-dif recombination in dimer resolution, when functional MukBEF is absent, or when replication terminates away from ter.