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The wobble bases of tRNAs that decode split codons are often heavily modified. In bacteria, tRNA^{Glu, Gln, Asp}contains a variety of xnm⁵s²U derivatives. The synthesis pathway for these modifications is complex and fully elucidated only in a handful of organisms, including the Gram-negativeEscherichia coliK12 model. Despite the ubiquitous presence of mnm⁵s²U modification, genomic analysis shows the absence ofmnmCorthologous genes, suggesting the occurrence of alternate biosynthetic schemes for the conversion of cmnm⁵s²U to mnm⁵s²U. Using a combination of comparative genomics and genetic studies, a member of the YtqA subgroup of the radical Sam superfamily was found to be involved in the synthesis of mnm⁵s²U in bothBacillus subtilisandStreptococcus mutans. This protein, renamed MnmL, is encoded in an operon with the recently discovered MnmM methylase involved in the methylation of the pathway intermediate nm⁵s²U into mnm⁵s²U inB. subtilis. Analysis of tRNA modifications of bothS. mutansandStreptococcus pneumoniaeshows that growth conditions and genetic backgrounds influence the ratios of pathway intermediates owing to regulatory loops that are not yet understood. The MnmLM pathway is widespread along the bacterial tree, with some phyla, such as Bacilli, relying exclusively on these two enzymes. Although mechanistic details of these newly discovered components are not fully resolved, the occurrence of fusion proteins, alternate arrangements of biosynthetic components, and loss of biosynthetic branches provide examples of biosynthetic diversity to retain a conserved tRNA modification in Nature.IMPORTANCEThe xnm⁵s²U modifications found in several tRNAs at the wobble base position are widespread in bacteria where they have an important role in decoding efficiency and accuracy. This work identifies a novel enzyme (MnmL) that is a member of a subgroup of the very versatile radical SAM superfamily and is involved in the synthesis of mnm⁵s²U in several Gram-positive bacteria, including human pathogens. This is another novel example of a non-orthologous displacement in the field of tRNA modification synthesis, showing how different solutions evolve to retain U34 tRNA modifications. 

The Microbiology and Cell Science program at the University of Florida compressed two standard 16-week lab courses into five-day versions of the course, which are referred to as bootcamp labs. The bootcamp labs have the same objectives, activities, and assessments as their traditional counterparts. Development of the bootcamp labs was part of a larger effort to increase access to the major, and more broadly STEM, by offering a 2+2 hybrid online transfer program. The results of this mixed-methods study include a direct comparison between bootcamp and traditional lab format as an approach for delivery of a face-to-face lab course. The bootcamp lab cohort has a greater diversity of students, with more women and underrepresented minorities in STEM than the traditional semester-long cohorts. Students in the bootcamp labs have comparable grade outcomes and learning gains to students in traditional lab format. Regression analysis identified GPA, but not lab format, as the most significant predictor of success for students enrolled in lab courses. Qualitative results suggest that the bootcamp format may be a better way than traditional formats to teach microbiology lab. In summary, the results demonstrate that a bootcamp version of a face-to-face microbiology course is just as effective as the traditional semester-long version. This work has broader implications as it supports the bootcamp lab approach as a model in STEM education for increasing access and for overcoming a major barrier to online STEM programs: face-to-face delivery of key lab courses.