Genome-wide localization of mobile elements: experimental, statistical and biological considerations

BACKGROUND: The distribution and location of insertion elements in a genome is an excellent tool to track the evolution of bacterial strains and a useful molecular marker to distinguish between closely related bacterial isolates. The information about the genomic locations of IS elements is available in public sequence databases. However, the locations of mobile elements may vary from strain to strain and within the population of an individual strain. Tools that allow de novo localization of IS elements and are independent of existing sequence information are essential to map insertion elements and advance our knowledge of the role that such elements play in gene regulation and genome plasticity in bacteria. RESULTS: In this study, we present an efficient and reliable method for linear mapping of mobile elements using whole-genome DNA microarrays. In addition, we describe an algorithm for analysis of microarray data that can be applied to find DNA sequences physically juxtaposed with a target sequence of interest. This approach was used to map the locations of the IS5 elements in the genome of Escherichia coli K12. All IS5 elements present in the E. coli genome known from GenBank sequence data were identified. Furthermore, previously unknown insertion sites were predicted with high sensitivity and specificity. Two variants of E. coli K-12 MG1655 within a population of this strain were predicted by our analysis. The only significant difference between these two isolates was the presence of an IS5 element upstream of the main flagella regulator, flhDC. Additional experiments confirmed this prediction and showed that these isolates were phenotypically distinct. The effect of IS5 on the transcriptional activity of motility and chemotaxis genes in the genome of E. coli strain MG1655 was examined. Comparative analysis of expression profiles revealed that the presence of IS5 results in a mild enhancement of transcription of the flagellar genes that translates into a slight increase in motility. CONCLUSION: In summary, this work presents a case study of an experimental and analytical application of DNA microarrays to map insertion elements in bacteria and gains an insight into biological processes that might otherwise be overlooked by relying solely on the available genome sequence data

Limited functional conservation of a global regulator among related bacterial genera: Lrp in Escherichia, Proteus and Vibrio

Abstract Background Bacterial genome sequences are being determined rapidly, but few species are physiologically well characterized. Predicting regulation from genome sequences usually involves extrapolation from better-studied bacteria, using the hypothesis that a conserved regulator, conserved target gene, and predicted regulator-binding site in the target promoter imply conserved regulation between the two species. However many compared organisms are ecologically and physiologically diverse, and the limits of extrapolation have not been well tested. In E. coli K-12 the leucine-responsive regulatory protein (Lrp) affects expression of ~400 genes. Proteus mirabilis and Vibrio cholerae have highly-conserved lrp orthologs (98% and 92% identity to E. coli lrp). The functional equivalence of Lrp from these related species was assessed. Results Heterologous Lrp regulated gltB, livK and lrp transcriptional fusions in an E. coli background in the same general way as the native Lrp, though with significant differences in extent. Microarray analysis of these strains revealed that the heterologous Lrp proteins significantly influence only about half of the genes affected by native Lrp. In P. mirabilis, heterologous Lrp restored swarming, though with some pattern differences. P. mirabilis produced substantially more Lrp than E. coli or V. cholerae under some conditions. Lrp regulation of target gene orthologs differed among the three native hosts. Strikingly, while Lrp negatively regulates its own gene in E. coli, and was shown to do so even more strongly in P. mirabilis, Lrp appears to activate its own gene in V. cholerae. Conclusion The overall similarity of regulatory effects of the Lrp orthologs supports the use of extrapolation between related strains for general purposes. However this study also revealed intrinsic differences even between orthologous regulators sharing \u3e90% overall identity, and 100% identity for the DNA-binding helix-turn-helix motif, as well as differences in the amounts of those regulators. These results suggest that predicting regulation of specific target genes based on genome sequence comparisons alone should be done on a conservative basis

Wastewater bacteria remediating the pharmaceutical metformin: Genomes, plasmids and products

Metformin is used globally to treat type II diabetes, has demonstrated anti-ageing and COVID mitigation effects and is a major anthropogenic pollutant to be bioremediated by wastewater treatment plants (WWTPs). Metformin is not adsorbed well by activated carbon and toxic N-chloro derivatives can form in chlorinated water. Most earlier studies on metformin biodegradation have used wastewater consortia and details of the genomes, relevant genes, metabolic products, and potential for horizontal gene transfer are lacking. Here, two metformin-biodegrading bacteria from a WWTP were isolated and their biodegradation characterized. Aminobacter sp. MET metabolized metformin stoichiometrically to guanylurea, an intermediate known to accumulate in some environments including WWTPs. Pseudomonasmendocina MET completely metabolized metformin and utilized all the nitrogen atoms for growth. Pseudomonas mendocina MET also metabolized metformin breakdown products sometimes observed in WWTPs: 1-N-methylbiguanide, biguanide, guanylurea, and guanidine. The genome of each bacterium was obtained. Genes involved in the transport of guanylurea in Aminobacter sp. MET were expressed heterologously and shown to serve as an antiporter to expel the toxic guanidinium compound. A novel guanylurea hydrolase enzyme was identified in Pseudomonas mendocina MET, purified, and characterized. The Aminobacter and Pseudomonas each contained one plasmid of 160 kb and 90 kb, respectively. In total, these studies are significant for the bioremediation of a major pollutant in WWTPs today

Studying Gene Expression: Database Searches and Promoter Fusions to Investigate Transcriptional Regulation in Bacteria

<p align="justify">A laboratory project was designed to illustrate how to search biological databases and utilize the information provided by these resources to investigate transcriptional regulation in <em>Escherichia coli. </em>The students searched several databases (NCBI Genomes, RegulonDB and EcoCyc) to learn about gene function, regulation, and the organization of transcriptional units. A fluorometer and GFP promoter fusions were used to obtain fluorescence data and measure changes in transcriptional activity. The class designed and performed experiments to investigate the regulation of genes necessary for biosynthesis of amino acids and how expression is affected by environmental signals and transcriptional regulators. Assessment data showed that this activity enhanced students&rsquo; knowledge of databases, reporter genes and transcriptional regulation.</p

Curriculum Studying Gene Expression: Database Searches and Promoter Fusions to Investigate Transcriptional Regulation in Bacteria †

A laboratory project was designed to illustrate how to search biological databases and utilize the information provided by these resources to investigate transcriptional regulation in Escherichia coli. The students searched several databases (NCBI Genomes, RegulonDB and EcoCyc) to learn about gene function, regulation, and the organization of transcriptional units. A fluorometer and GFP promoter fusions were used to obtain fluorescence data and measure changes in transcriptional activity. The class designed and performed experiments to investigate the regulation of genes necessary for biosynthesis of amino acids and how expression is affected by environmental signals and transcriptional regulators. Assessment data showed that this activity enhanced students ’ knowledge of databases, reporter genes and transcriptional regulation

Supplemental Materials for Studying Gene Expression: Database Searches and Promoter Fusions to Investigate Transcriptional Regulation in Bacteria

(Total pages 53) Appendix I: Questions used to explore student’s knowledge of gene expression and regulatio

Insights into the regulation of malate dehydrogenase: inhibitors, activators, and allosteric modulation by small molecules

Cellular metabolism comprises a complex network of biochemical anabolic and catabolic processes that fuel the growth and survival of living organisms. The enzyme malate dehydrogenase (MDH) is most known for its role in oxidizing malate to oxaloacetate (OAA) in the last step of the tricarboxylic acid (TCA) cycle, but it also participates in the malate-aspartate shuttle in the mitochondria as well as the glyoxylate cycle in plants. These pathways and the specific reactions within them are dynamic and must be carefully calibrated to ensure a balance between nutrient/energy supply and demand. MDH structural and functional complexity requires a variety of regulatory mechanisms, including allosteric regulation, feedback, and competitive inhibition, which are often dependent on whether the enzyme is catalyzing its forward or reverse reaction. Given the role of MDH in central metabolism and its potential as a target for therapeutics in both cancer and infectious diseases, there is a need to better understand its regulation. The involvement of MDH in multiple pathways makes it challenging to identify which effectors are critical to its activity. Many of the in vitro experiments examining MDH regulation were done decades ago, and though allosteric sites have been proposed, none to date have been specifically mapped. This review aims to provide an overview of the current knowledge surrounding MDH regulation by its substrate, products, and other intermediates of the TCA cycle while highlighting all the gaps in our understanding of its regulatory mechanisms

Fostering Student to Student Collaboration Across Institutions in a Protein Centric CURE

Collaboration is an essential component of research because it allows scientists to share expertise and tackle difficult scientific questions. While many undergraduate science courses include inquiry-based or authentic research projects, opportunities for collaboration outside of the classroom are rarely provided. To address these limitations, we developed a set of course activities and assignments to implement student-to-student collaboration between institutions in a protein-centric CURE. The activities were tested in three courses: an upper-level biochemistry class at primarily undergraduate institution, genetics and an introductory biology class at a community college. A survey was created to assess students\u27 perceptions of collaboration and the impact of the activities on their scientific and communication skills. The results indicated that an important component of students\u27 experiences was the assignments designed to guide participants through the collaboration activities. The survey findings suggest that student-to-student collaboration improves science communication skills and motivates students to be more careful with experimental design and data collection. Primary Image: Cross Institutional Student to Student Collaboration in a CURE. The image illustrates a research collaboration between students at a community college and a primarily undergraduate institution while participating in a course-based undergraduate research experience (CURE). Students discussed their hypotheses, shared materials, and analyzed data and experimental observations

Development of the Organonitrogen Biodegradation Database: Teaching Bioinformatics and Collaborative Skills to Undergraduates during a Pandemic

Physical distancing and inaccessibility to laboratory facilities created an opportunity to transition undergraduate research experiences to remote, digital platforms, adding another level of pedagogy to their training. Basic bioinformatics skills together with critical analysis of scientific literature are essential for addressing research questions in modern biology. The work presented here describes a fully online, collaborative research experience created to allow undergraduate students to learn those skills. The research experience was focused on the development and implementation of the Organonitrogen Biodegradation Database (ONDB, z.umn.edu/ondb). The ONDB was developed to catalog information about the cost, chemical properties, and biodegradation potential of commonly used organonitrogen compounds. A cross-institutional team of undergraduate researchers worked in collaboration with two faculty members and a postdoctoral fellow to develop the database. Students carried out extensive online literature searches and used a biodegradation prediction website to research and represent the microbial catabolism of different organonitrogen compounds. Participants employed computational tools such as R, Shiny, and flexdashboard to construct the database pages and interactive web interface for the ONDB. Worksheets and forms were created to encourage other students and researchers to gather information about organonitrogen compounds and expand the database. Student progress was evaluated through biweekly project meetings, presentations, and a final reflection. The ONDB undergraduate research experience provided a platform for students to learn bioinformatics skills while simultaneously developing a teaching and research tool for others