A research program-linked, course-based undergraduate research experience that allows undergraduates to participate in current research on mycobacterial gene regulation

Undergraduate instructional biology laboratories are typically taught within two paradigms. Some labs focus on protocols and techniques delivered in “cookbook” format with defined experimental outcomes. There is increasing momentum to alternatively employ student-driven, open-ended, and discovery-based strategies, often via course-based undergraduate research experiences (CUREs) using crowd-sourcing initiatives. A fraction of students also participate in funded research in faculty research labs, where they have opportunities to work on projects designed to expand the frontiers of human knowledge. These experiences are widely recognized as valuable but are not scalable, as most institutions have many more undergraduates than research lab positions. We sought to address this gap through our department’s curriculum by creating an opportunity for students to participate in the real-world research process within a laboratory course. We conceived, developed, and delivered an authentic, guided research experience to students in an upper-level molecular biology laboratory course. We refer to this model as a “research program-linked CURE.” The research questions come directly from a faculty member’s research lab and evolve along with that research program. Students study post-transcriptional regulation in mycobacteria. We use current molecular biology methodologies to test hypotheses like “UTRs affect RNA and protein expression levels,” “there is functional redundancy among RNA helicases,” and “carbon starvation alters mRNA 5′ end chemistries.” We conducted standard assessments and developed a customized “Skills and Concepts Inventory” survey to gauge how well the course met our student learning outcomes. We report the results of our assessments and describe challenges addressed during development and execution of the course, including organizing activities to fit within an instructional lab, balancing breadth with depth, and maintaining authenticity while giving students the experience of obtaining interpretable and novel results. Our data suggest student learning was enhanced through this truly authentic research approach. Further, students were able to perceive they were participants and contributors within an active research paradigm. Students reported increases in their self-identification as scientists, and a positive impact on their career trajectories. An additional benefit was reciprocation back to the funded research laboratory, by funneling course alumni, results, materials, and protocols

Defining the Transcriptional and Post-transcriptional Landscapes of Mycobacterium smegmatis in Aerobic Growth and Hypoxia

The ability of Mycobacterium tuberculosis to infect, proliferate, and survive during long periods in the human lungs largely depends on the rigorous control of gene expression. Transcriptome-wide analyses are key to understanding gene regulation on a global scale. Here, we combine 5′-end-directed libraries with RNAseq expression libraries to gain insight into the transcriptome organization and post-transcriptional mRNA cleavage landscape in mycobacteria during log phase growth and under hypoxia, a physiologically relevant stress condition. Using the model organism Mycobacterium smegmatis, we identified 6,090 transcription start sites (TSSs) with high confidence during log phase growth, of which 67% were categorized as primary TSSs for annotated genes, and the remaining were classified as internal, antisense, or orphan, according to their genomic context. Interestingly, over 25% of the RNA transcripts lack a leader sequence, and of the coding sequences that do have leaders, 53% lack a strong consensus Shine-Dalgarno site. This indicates that like M. tuberculosis, M. smegmatis can initiate translation through multiple mechanisms. Our approach also allowed us to identify over 3,000 RNA cleavage sites, which occur at a novel sequence motif. To our knowledge, this represents the first report of a transcriptome-wide RNA cleavage site map in mycobacteria. The cleavage sites show a positional bias toward mRNA regulatory regions, highlighting the importance of post-transcriptional regulation in gene expression. We show that in low oxygen, a condition associated with the host environment during infection, mycobacteria change their transcriptomic profiles and endonucleolytic RNA cleavage is markedly reduced, suggesting a mechanistic explanation for previous reports of increased mRNA half-lives in response to stress. In addition, a number of TSSs were triggered in hypoxia, 56 of which contain the binding motif for the sigma factor SigF in their promoter regions. This suggests that SigF makes direct contributions to transcriptomic remodeling in hypoxia-challenged mycobacteria. Taken together, our data provide a foundation for further study of both transcriptional and posttranscriptional regulation in mycobacteria

Loss of RNase J leads to multi-drug tolerance and accumulation of highly structured mRNA fragments in Mycobacterium tuberculosis

Despite the existence of well-characterized, canonical mutations that confer high-level drug resistance to Mycobacterium tuberculosis (Mtb), there is evidence that drug resistance mechanisms are more complex than simple acquisition of such mutations. Recent studies have shown that Mtb can acquire non-canonical resistance-associated mutations that confer survival advantages in the presence of certain drugs, likely acting as stepping-stones for acquisition of high-level resistance. Rv2752c/rnj, encoding RNase J, is disproportionately mutated in drug-resistant clinical Mtb isolates. Here we show that deletion of rnj confers increased tolerance to lethal concentrations of several drugs. RNAseq revealed that RNase J affects expression of a subset of genes enriched for PE/PPE genes and stable RNAs and is key for proper 23S rRNA maturation. Gene expression differences implicated two sRNAs and ppe50-ppe51 as important contributors to the drug tolerance phenotype. In addition, we found that in the absence of RNase J, many short RNA fragments accumulate because they are degraded at slower rates. We show that the accumulated transcript fragments are targets of RNase J and are characterized by strong secondary structure and high G+C content, indicating that RNase J has a rate-limiting role in degradation of highly structured RNAs. Taken together, our results demonstrate that RNase J indirectly affects drug tolerance, as well as reveal the endogenous roles of RNase J in mycobacterial RNA metabolism.Fil: Martini, María Carla. Worcester Polytechnic Institute; Estados Unidos. Consejo Nacional de Investigaciones Científicas y Técnicas; ArgentinaFil: Hicks, Nathan D.. Harvard University. Harvard School of Public Health; Estados UnidosFil: Xiao, Junpei. Worcester Polytechnic Institute; Estados UnidosFil: Alonso, Maria Natalia. Worcester Polytechnic Institute; Estados Unidos. Consejo Nacional de Investigaciones Científicas y Técnicas; ArgentinaFil: Barbier, Thibault. Harvard University. Harvard School of Public Health; Estados UnidosFil: Sixsmith, Jaimie. Harvard University. Harvard School of Public Health; Estados UnidosFil: Fortune, Sarah M.. Harvard University. Harvard School of Public Health; Estados UnidosFil: Shell, Scarlet S.. Worcester Polytechnic Institute; Estados Unido

mRNA Degradation Rates Are Coupled to Metabolic Status in Mycobacterium smegmatis

The logistics of tuberculosis therapy are difficult, requiring multiple drugs for many months. Mycobacterium tuberculosis survives in part by entering nongrowing states in which it is metabolically less active and thus less susceptible to antibiotics. Basic knowledge on how M. tuberculosis survives during these low-metabolism states is incomplete, and we hypothesize that optimized energy resource management is important. Here, we report that slowed mRNA turnover is a common feature of mycobacteria under energy stress but is not dependent on the mechanisms that have generally been postulated in the literature. Finally, we found that mRNA stability and growth status can be decoupled by a drug that causes growth arrest but increases metabolic activity, indicating that mRNA stability responds to metabolic status rather than to growth rate per se. Our findings suggest a need to reorient studies of global mRNA stabilization to identify novel mechanisms that are presumably responsible.The success of Mycobacterium tuberculosis as a human pathogen is due in part to its ability to survive stress conditions, such as hypoxia or nutrient deprivation, by entering nongrowing states. In these low-metabolism states, M. tuberculosis can tolerate antibiotics and develop genetically encoded antibiotic resistance, making its metabolic adaptation to stress crucial for survival. Numerous bacteria, including M. tuberculosis, have been shown to reduce their rates of mRNA degradation under growth limitation and stress. While the existence of this response appears to be conserved across species, the underlying bacterial mRNA stabilization mechanisms remain unknown. To better understand the biology of nongrowing mycobacteria, we sought to identify the mechanistic basis of mRNA stabilization in the nonpathogenic model Mycobacterium smegmatis. We found that mRNA half-life was responsive to energy stress, with carbon starvation and hypoxia causing global mRNA stabilization. This global stabilization was rapidly reversed when hypoxia-adapted cultures were reexposed to oxygen, even in the absence of new transcription. The stringent response and RNase levels did not explain mRNA stabilization, nor did transcript abundance. This led us to hypothesize that metabolic changes during growth cessation impact the activities of degradation proteins, increasing mRNA stability. Indeed, bedaquiline and isoniazid, two drugs with opposing effects on cellular energy status, had opposite effects on mRNA half-lives in growth-arrested cells. Taken together, our results indicate that mRNA stability in mycobacteria is not directly regulated by growth status but rather is dependent on the status of energy metabolism

The Psp system ofﾠMycobacterium tuberculosisﾠintegrates envelope stress-sensing and envelope-preserving functions

The bacterial envelope integrates essential stress-sensing and adaptive functions; thus, envelope-preserving functions are important for survival. In Gram-negative bacteria, envelope integrity during stress is maintained by the multi-gene Psp response. Mycobacterium tuberculosis was thought to lack the Psp system since it encodes only pspA and no other psp ortholog. Intriguingly, pspA maps downstream from clgR, which encodes a transcription factor regulated by the MprAB-σE envelope-stress-signaling system. clgR inactivation lowered ATP concentration during stress and protonophore treatment-induced clgR-pspA expression, suggesting that these genes express Psp-like functions. We identified a four-gene set – clgR, pspA (rv2744c), rv2743c, rv2742c – that is regulated by clgR and in turn regulates ClgR activity. Regulatory and protein–protein interactions within the set and a requirement of the four genes for functions associated with envelope integrity and surface-stress tolerance indicate that a Psp-like system has evolved in mycobacteria. Among Actinobacteria, the four-gene module occurred only in tuberculous mycobacteria and was required for intramacrophage growth, suggesting links between its function and mycobacterial virulence. Additionally, the four-gene module was required for MprAB-σE stress-signaling activity. The positive feedback between envelope-stress-sensing and envelope-preserving functions allows sustained responses to multiple, envelope-perturbing signals during chronic infection, making the system uniquely suited to tuberculosis pathogenesis

Die SAGAT-Methode: Welche Informationen muss der Towerlotse erfassen?

<div><p>DNA methylation regulates gene expression in many organisms. In eukaryotes, DNA methylation is associated with gene repression, while it exerts both activating and repressive effects in the Proteobacteria through largely locus-specific mechanisms. Here, we identify a critical DNA methyltransferase in <i>M. tuberculosis</i>, which we term MamA. MamA creates N⁶-methyladenine in a six base pair recognition sequence present in approximately 2,000 copies on each strand of the genome. Loss of MamA reduces the expression of a number of genes. Each has a MamA site located at a conserved position relative to the sigma factor −10 binding site and transcriptional start site, suggesting that MamA modulates their expression through a shared, not locus-specific, mechanism. While strains lacking MamA grow normally <i>in vitro</i>, they are attenuated in hypoxic conditions, suggesting that methylation promotes survival in discrete host microenvironments. Interestingly, we demonstrate strikingly different patterns of DNA methyltransferase activity in different lineages of <i>M. tuberculosis</i>, which have been associated with preferences for distinct host environments and different disease courses in humans. Thus, MamA is the major functional adenine methyltransferase in <i>M. tuberculosis</i> strains of the Euro-American lineage while strains of the Beijing lineage harbor a point mutation that largely inactivates MamA but possess a second functional DNA methyltransferase. Our results indicate that MamA influences gene expression in <i>M. tuberculosis</i> and plays an important but strain-specific role in fitness during hypoxia.</p></div

Transcriptional start site (TSS)-mapping reveals a consistent spatial relationship between MamA sites and TSSs of methylation-affected genes.

<p>TSSs in strain H37Rv were mapped by the strategy outlined in (A). mRNA was circularized before random-primed synthesis of cDNA. Dashes indicate the variable 3′ end of an mRNA. Gene-specific primers were then used to amplify and sequence 5′-3′ junctions. Junctions appear as transitions from clean to messy sequence due to the variable 3′ ends. (B) TSS-mapping sequence traces are shown for the four genes whose expression is reproducibly affected by MamA. MamA sites, putative sigma factor −10 binding sites, TSSs and ORFs are shown as indicated in the key.</p

MamA is a DNA methyltransferase that protects <i>lppC</i> from endonucleolytic cleavage.

<p>(A) Southern blotting strategy to assess the status of a PvuII site near the 3′ end of <i>lppC</i>. Genomic DNA was digested with PvuII and analyzed by Southern blot with a probe hybridizing as shown. Fully cleaved DNA generates a 1.8 kb product, while protected DNA produces a 2.1 kb product. (B) DNA from the vaccine strain <i>M. bovis</i> BCG and from <i>M. tuberculosis</i> strains of the Euro-American lineage (Erdmann and CDC1551) is partially protected from PvuII cleavage while DNA from a Beijing lineage strain (HN878) is not. (C) Genetic deletion of <i>mamA</i> abrogates protection of <i>lppC</i>, while deletion of <i>hsdM</i> does not affect protection. (D) Protection is restored by complementation of a <i>ΔmamA</i> strain with an ectopic copy of <i>mamA</i>, but not by empty vector or <i>mamA^E270A</i>. (E) Sequence context of the assayed PvuII site. Underlined bases are predicted to block PvuII if methylated.</p

Deletion of <i>mamA</i> does not grossly affect growth rate or fitness of <i>M. tuberculosis</i> during mouse infection.

<p>(A) The indicated H37Rv-derived strains were normalized at a calculated optical density of 0.01 in Sauton's media and monitored by optical density on the days indicated. Points indicate the mean of triplicate cultures and error bars denote standard deviation. Similar results were obtained in 7H9 medium and by plating for CFU. (B) Mice were infected by the aerosol route with approximately 10,000 CFU of a mixture of unmarked wildtype H37Rv and one of three isogenic <i>mamA</i> mutants marked with kanamycin resistance. Groups of four mice per condition were sacrificed at the indicated time points and the lung burden of total and marked bacilli was determined. The mean proportion of marked bacteria is indicated. Error bars denote standard deviation.</p

Genes with expression differences of 1.5-fold or greater in Δ<i>mamA</i> or complemented strains compared to the wildtype parent (H37Rv) in aerobic growth conditions.

a<p>ANOVA.</p>b<p>Method of Benjamini and Hochberg.</p>c<p>Distance from start of ORF to nearest upstream MamA site.</p>d<p>Distance from start of first ORF in operon (Rv3083) to nearest upstream MamA site.</p>e<p>Distances in wildtype/Δ<i>mamA</i> and complemented strains, respectively; complementation vector integrates in this region.</p>f<p>Complementation vector integrates in this gene.</p