Zakiya Whatley, Visiting Assistant Professor of Biology

In our last Next Page column of the year, Zakiya Whatley, Visiting Assistant Professor of Biology, provides a recommended read for any budding geneticist; shares which books she\u27s looking forward to reading next for her book group; and tells us what her students will be doing for class this Friday, April 24, 2015, in celebration of National DNA Day

Mutations that Separate the Functions of the Proofreading Subunit of the Escherichia coli Replicase

The dnaQ gene of Escherichia coli encodes the Ɛ subunit of DNA polymerase III, which provides the 3\u27 - 5\u27 exonuclease proofreading activity of the replicative polymerase. Prior studies have shown that loss of Ɛ leads to high mutation frequency, partially constitutive SOS, and poor growth. In addition, a previous study from our laboratory identified dnaQ knockout mutants in a screen for mutants specifically defective in the SOS response after quinolone (nalidixic acid) treatment. To explain these results, we propose a model whereby, in addition to proofreading, Ɛ plays a distinct role in replisome disassembly and/or processing of stalled replication forks. To explore this model, we generated a pentapeptide insertion mutant library of the dnaQgene, along with site-directed mutants, and screened for separation of function mutants. We report the identification of separation of function mutants from this screen, showing that proofreading function can be uncoupled from SOS phenotypes (partially constitutive SOS and the nalidixic acid SOS defect). Surprisingly, the two SOS phenotypes also appear to be separable from each other. These findings support the hypothesis that Ɛ has additional roles aside from proofreading. Identification of these mutants, especially those with normal proofreading but SOS phenotype(s), also facilitates the study of the role of e in SOS processes without the confounding results of high mutator activity associated with dnaQ knockout mutants

Isolation & Characterization of Bacteria in the Built Environment: Measuring The Effect of Pharmaceuticals on Growth

This work reports the isolation and characterization of bacteria from the built environment at Gettysburg College in Gettysburg, PA. Surfaces of a water fountain on campus were swabbed and serially streaked to isolate multiple bacteria on R2A agar. Following multiple rounds of growth, the unknown microbial candidates were narrowed to two visibly­distinct organisms. Morphological characterization and phylogenetic identification based on 16S rDNA sequencing revealed that the isolates were Chryseobactierum hispalense and Microbacterium maritypicum. We report synergistic biofilm formation between Chryseobactierum hispalense and Microbacterium maritypicum. The contamination of drinking water with varying levels of personal care products and pharmaceuticals (PCPPs) is well documented. Additionally, these environmental pollutants and their derivatives affect aquatic life, as illustrated with effect of the antidepressant fluoxetine on mudsnails. To determine if previously reported contaminants affect freshwater bacteria, we assessed both planktonic growth and biofilm formation following exposure to nalidixic acid (non­fluorinated quinolone antibiotic), diphenhydramine (over­the­counter drug Benadryl), and fluoxetine (Prozac)

Probing for Binding Regions of the FtsZ Protein Surface through Site-Directed Insertions: Discovery of Fully Functional FtsZ-Fluorescent Proteins

FtsZ, a bacterial tubulin homologue, is a cytoskeletal protein that assembles into protofilaments that are one subunit thick. These protofilaments assemble further to form a “Z ring” at the center of prokaryotic cells. The Z ring generates a constriction force on the inner membrane and also serves as a scaffold to recruit cell wall remodeling proteins for complete cell division in vivo. One model of the Z ring proposes that protofilaments associate via lateral bonds to form ribbons; however, lateral bonds are still only hypothetical. To explore potential lateral bonding sites, we probed the surface of Escherichia coli FtsZ by inserting either small peptides or whole fluorescent proteins (FPs). Among the four lateral surfaces on FtsZ protofilaments, we obtained inserts on the front and back surfaces that were functional for cell division. We concluded that these faces are not sites of essential interactions. Inserts at two sites, G124 and R174, located on the left and right surfaces, completely blocked function, and these sites were identified as possible sites for essential lateral interactions. However, the insert at R174 did not interfere with association of protofilaments into sheets and bundles in vitro. Another goal was to find a location within FtsZ that supported insertion of FP reporter proteins while allowing the FtsZ-FPs to function as the sole source of FtsZ. We discovered one internal site, G55-Q56, where several different FPs could be inserted without impairing function. These FtsZ-FPs may provide advances for imaging Z-ring structure by superresolution techniques. IMPORTANCE One model for the Z-ring structure proposes that protofilaments are assembled into ribbons by lateral bonds between FtsZ subunits. Our study excluded the involvement of the front and back faces of the protofilament in essential interactions in vivo but pointed to two potential lateral bond sites, on the right and left sides. We also identified an FtsZ loop where various fluorescent proteins could be inserted without blocking function; these FtsZ-FPs functioned as the sole source of FtsZ. This advance provides improved tools for all fluorescence imaging of the Z ring and may be especially important for superresolution imaging

The Prospects of Artificial Endosymbioses

In the engineering of biological systems, it can be said unequivocally that art imitates nature. Nearly all efforts to control human health, the environment, and agriculture involve the appropriation of evolutionary processes. These processes typically originate through incremental changes in the genome that are sustained and promoted through natural selection in descendant lineages. Recombinant DNA technology and more recently genome editing help us imitate these genome-level changes in engineered systems. However, the dramatic evolutionary innovations that are attributed to singular beneficial endosymbioses, in which a mutualist microbial cell inhabits a host’s cell, are also worthy of imitation. For example, researchers are studying how to engineer endosymbiotic bacteria to control mosquito-borne viral diseases, tweak nitrogen-fixing microbes to help crop plants, and treat macular degeneration, just to name a few projects that are under way. (excerpt