Correction for Tawde and McLaughlin, "Implementing a Virtual Midterm to Identify Unknown Bacteria in a Microbiology Lab Course".

[This corrects the article DOI: 10.1128/jmbe.v22i1.2579.].

A polypeptide model for toxic aberrant proteins induced by aminoglycoside antibiotics.

Aminoglycoside antibiotics interfere with the selection of cognate tRNAs during translation, resulting in the synthesis of aberrant proteins that are the ultimate cause of cell death. However, the toxic potential of aberrant proteins and how they avoid degradation by the cell's protein quality control (QC) machinery are not understood. Here we report that levels of the heat shock (HS) transcription factor σ32 increased sharply following exposure of Escherichia coli to the aminoglycoside kanamycin (Kan), suggesting that at least some of the aberrant proteins synthesized in these cells were recognized as substrates by DnaK, a molecular chaperone that regulates the HS response, the major protein QC pathway in bacteria. To further investigate aberrant protein toxic potential and interaction with cell QC factors, we studied an acutely toxic 48-residue polypeptide (ARF48) that is encoded by an alternate reading frame in a plant cDNA. As occurred in cells exposed to Kan, σ32 levels were strongly elevated following ARF48 expression, suggesting that ARF48 was recognized as a substrate by DnaK. Paradoxically, an internal 10-residue region that was tightly bound by DnaK in vitro also was required for the ARF48 toxic effect. Despite the increased levels of σ32, levels of several HS proteins were unchanged following ARF48 expression, suggesting that the HS response had been aborted. Nucleoids were condensed and cell permeability increased rapidly following ARF48 expression, together suggesting that ARF48 disrupts DNA-membrane interactions that could be required for efficient gene expression. Our results are consistent with earlier studies showing that aberrant proteins induced by aminoglycoside antibiotics disrupt cell membrane integrity. Insights into the mechanism for this effect could be gained by further study of the ARF48 model system.

Implementing a Virtual Midterm to Identify Unknown Bacteria in a Microbiology Lab Course.

Identification of unknown bacteria is an integral module in most introductory Microbiology laboratory courses. This laboratory activity typically involves identifying bacteria based on Gram staining for morphology and gram reaction followed by studying their biochemical characteristics. When instruction moved to online mode earlier this year due to the pandemic, we faced the challenge of moving this hands-on, skill-intensive laboratory activity to remote mode. Here we describe the modular approach we designed to implement the midterm practical of bacterial identification remotely while trying to keep the online format as close to the in-person format as possible in a multi-section synchronous laboratory course. This virtual module was implemented successfully in the summer and fall 2020 semesters.

Loop-Mediated Isothermal Amplification (LAMP) as a Rapid, Affordable and Effective Tool to Involve Students in Undergraduate Research.

Undergraduate research (UR) is a high-impact practice (HIP) to engage undergraduate student in science, technology, engineering and mathematics (STEM), especially from underrepresented groups. UR experiences (UREs) can be integrated into the classroom, making authentic research experiences inclusive and available to all students. However, developing UR pedagogy can be challenging for faculty in resource-limited labs, such as community colleges and small liberal arts colleges. Often molecular biology research methods are expensive, time-consuming and need equipment not readily available or affordable in small schools. Polymerase chain reaction (PCR) is one of the most commonly used techniques in research labs and many UREs. We have investigated loop-mediated isothermal amplification (LAMP) as an inexpensive, accessible alternative to PCR for DNA amplification enabling the identification of microorganisms in the context of UREs. LAMP does not require expensive instrumentation or reagents and uses equipment commonly found in teaching labs. By performing the technique, students learn several key scientific skills that will be useful in their undergraduate or graduate STEM careers. We designed guided independent research experiences for several undergraduates that included the use of LAMP. Students successfully applied the technique to culture samples of common environmental bacteria, including Escherichia coli, Salmonella spp., Staphylococcus aureus, and Enterococcus, and were in addition, able to detect both Salmonella and Enterococcus in directly sampled environmental waters. To highlight the accessibility and affordability of this URE, a simple boiling method was used for DNA preparation from environmental samples. Student response data show positive attitudes toward UR when LAMP is utilized as a research tool to tackle relevant biological questions. The feasibility of using simplified LAMP in UREs points to a potential, more expanded application to public engagement with science and broader and more inclusive interactions with the research community.

Antibiotic Resistance in Environmental Microbes: Implementing Authentic Research in the Microbiology Classroom.

Incorporating Undergraduate Research Experience in Microbiology Classroom. Dr. Mangala Tawde, Associate Professor, Department of Biological Sciences and Geology, Queensborough Community College, CUNY. Undergraduate Research (UR) experience is increasingly being recognized as one of the most transforming experiences students can have in their undergraduate years of education. To make it accessible to all students, incorporating authentic research experiences in the classroom is important and it is a major initiative at Queensborough community college; where we have institutionalized UR as a High Impact Practice. We incorporated an authentic research project into the Microbiology course curriculum for allied health majors. The research project was to isolate and identify antibiotic-resistant microbes from diverse environments. As students are aware of antibiotic resistance being a serious concern in today's medicine, they get interested and are enthusiastically engaged in the research project. Students collect soil samples from various environments and locations of their choice and then they isolate and identify bacteria that may exhibit antibiotic resistance. The microbes isolated from diverse environments are identified based on the 16s rRNA sequence analysis as well as biochemical tests. The research experience is relevant and aligns well with the course curricula, course learning objectives as well as the college's General Education objectives.

Toxic misfolding of Arabidopsis cellulases in the secretory pathway of Pichia pastoris.

Plants produce a large number of cellulases that are either secreted or anchored in the plasma membrane where they likely function in various aspects of cellulose synthesis, modification and degradation during plant growth and development. Very few of these enzymes have been characterized in any detail, however. Here we attempted to produce two Arabidopsis modular cellulases, which contain a catalytic domain belonging to glycoside hydrolase family 9 (GH9) and a carbohydrate binding module (CBM), in the yeast Pichia pastoris. Neither of the intact modular enzymes was detectably produced, although the independently expressed GH9 catalytic domain of one enzyme was secreted when the protein was expressed at low temperature. Expression of intact and truncated cellulases at the standard temperature caused extensive cell lysis, with release of high concentrations of endogenous proteins into the culture medium. Cell lysis appeared to result from misfolding of cellulase proteins within the Pichia secretory pathway. The toxicity of these misfolded cellulases potentially could be exploited to derive host strains with enhanced capability to fold recombinant secretory proteins.