Leafy green vegetables have been identified as a source of foodborne illnesses worldwide over the past decade. Human enteric pathogens, such as Escherichia coli O157:H7 and Salmonella, have been ...implicated in numerous food poisoning outbreaks associated with the consumption of fresh produce. An understanding of the mechanisms responsible for the establishment of pathogenic bacteria in or on vegetable plants is critical for understanding and ameliorating this problem as well as ensuring the safety of our food supply. While previous studies have described the growth and survival of enteric pathogens in the environment and also the risk factors associated with the contamination of vegetables, the molecular events involved in the colonization of fresh produce by enteric pathogens are just beginning to be elucidated. This review summarizes recent findings on the interactions of several bacterial pathogens with leafy green vegetables. Changes in gene expression linked to the bacterial attachment and colonization of plant structures are discussed in light of their relevance to plant-microbe interactions. We propose a mechanism for the establishment and association of enteric pathogens with plants and discuss potential strategies to address the problem of foodborne illness linked to the consumption of leafy green vegetables.
Bioinformatics skills are increasingly relevant to research in most areas of the life sciences. The availability of genome sequences and large data sets provide unique opportunities to incorporate ...bioinformatics exercises into undergraduate microbiology courses. The goal of this project was to develop a teaching module to investigate the abundance and phylogenetic relationships amongst bacteriophages using a set of freely available bioinformatics tools. Computational identification and examination of bacteriophage genomes, followed by phylogenetic analyses, provides opportunities to incorporate core bioinformatics competencies in microbiology courses and enhance students' bioinformatics skills. The first activity consisted of using PHASTER (PHAge Search Tool Enhanced Release), a bioinformatics tool that identifies bacteriophage sequences within bacterial chromosomes. Further computational analyses were conducted to align bacteriophage proteins, genomes, and determine phylogenetic relationships amongst these viruses. This part of the project was carried out using the Clustal omega, MAFFT (Multiple Alignment using Fast Fourier Transform), and Interactive Tree of Life (iTOL) programs for sequence alignments and phylogenetic analyses. The laboratory activities were field tested in undergraduate directed research, and microbiology classes. The learning objectives were assessed by comparing the scores of pre and post-tests and grading final presentations. Post-tests were higher than pre-test scores at or below p = 0.002. The data suggest in silico phage hunting improves students' ability to search databases, interpret phylogenetic trees, and use bioinformatics tools to examine genome structure. This activity allows instructors to integrate key bioinformatic concepts in their curriculums and gives students the opportunity to participate in a research-directed learning environment in the classroom.Bioinformatics skills are increasingly relevant to research in most areas of the life sciences. The availability of genome sequences and large data sets provide unique opportunities to incorporate bioinformatics exercises into undergraduate microbiology courses. The goal of this project was to develop a teaching module to investigate the abundance and phylogenetic relationships amongst bacteriophages using a set of freely available bioinformatics tools. Computational identification and examination of bacteriophage genomes, followed by phylogenetic analyses, provides opportunities to incorporate core bioinformatics competencies in microbiology courses and enhance students' bioinformatics skills. The first activity consisted of using PHASTER (PHAge Search Tool Enhanced Release), a bioinformatics tool that identifies bacteriophage sequences within bacterial chromosomes. Further computational analyses were conducted to align bacteriophage proteins, genomes, and determine phylogenetic relationships amongst these viruses. This part of the project was carried out using the Clustal omega, MAFFT (Multiple Alignment using Fast Fourier Transform), and Interactive Tree of Life (iTOL) programs for sequence alignments and phylogenetic analyses. The laboratory activities were field tested in undergraduate directed research, and microbiology classes. The learning objectives were assessed by comparing the scores of pre and post-tests and grading final presentations. Post-tests were higher than pre-test scores at or below p = 0.002. The data suggest in silico phage hunting improves students' ability to search databases, interpret phylogenetic trees, and use bioinformatics tools to examine genome structure. This activity allows instructors to integrate key bioinformatic concepts in their curriculums and gives students the opportunity to participate in a research-directed learning environment in the classroom.
The implementation of Course‐Based Undergraduate Research Experiences (CUREs) has made it possible to expose large undergraduate populations to research experiences. For these research experiences to ...be authentic, they should reflect the increasing collaborative nature of research. While some CUREs have expanded with multiple schools across the nation, it is still unclear how a structured extramural collaboration between students and faculty from an outside institution affects student outcomes. In this study, we established three cohorts of students: 1) no‐CURE 2) single institution CURE (CURE) and 3) external collaborative CURE (ec‐CURE) and assessed academic and attitudinal outcomes. The ec‐CURE differs from a regular CURE in that students work with faculty member from an external institution to refine their hypothesis and discuss their data. The sharing of ideas, data and materials with an external faculty allowed students to experience a level of collaboration not typically found in an undergraduate setting. Students in the ec‐CURE had the greatest gains in experimental design, self‐reported course benefits, scientific skills and STEM importance. Importantly this study occurred in a diverse community of STEM disciplinary faculty from 2‐ and 4‐year institutions illustrating that exposing students to structured external collaboration is both feasible and beneficial to student learning.
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.
sp. MET metabolized metformin stoichiometrically to guanylurea, an intermediate known to accumulate in some environments including WWTPs.
MET completely metabolized metformin and utilized all the nitrogen atoms for growth.
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
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
MET, purified, and characterized. The
and
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.
This study examines how the inclusion of structured external collaborations in course-based undergraduate research experiences (CUREs) affects learning outcomes. Students worked with faculty from an ...external institution to refine their hypotheses and discuss their data. In the collaborative CURE cohort, students had greater gains in learning outcomes compared with students in standard CUREs and no-CURE controls.
The implementation of course-based undergraduate research experiences (CUREs) has made it possible to expose large undergraduate populations to research experiences. For these research experiences to be authentic, they should reflect the increasingly collaborative nature of research. While some CUREs have expanded, involving multiple schools across the nation, it is still unclear how a structured extramural collaboration between students and faculty from an outside institution affects student outcomes. In this study, we established three cohorts of students: 1) no-CURE, 2) single-institution CURE (CURE), and 3) external collaborative CURE (ec-CURE), and assessed academic and attitudinal outcomes. The ec-CURE differs from a regular CURE in that students work with faculty member from an external institution to refine their hypotheses and discuss their data. The sharing of ideas, data, and materials with an external faculty member allowed students to experience a level of collaboration not typically found in an undergraduate setting. Students in the ec-CURE had the greatest gains in experimental design; self-reported course benefits; scientific skills; and science, technology, engineering, and mathematics (STEM) importance. Importantly this study occurred in a diverse community of STEM disciplinary faculty from 2- and 4-year institutions, illustrating that exposing students to structured external collaboration is both feasible and beneficial to student learning.
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.
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 approximately 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.
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.
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 >90% 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.
Microbiology courses often include a laboratory activity on the identification of unknown microbes. This activity consists of providing students with microbial cultures and running biochemical assays ...to identify the organisms. This approach lacks molecular techniques such as sequencing of genes encoding 16S rRNA, which is currently the method of choice for identification of unknown bacteria. A laboratory activity was developed to teach students how to identify microorganisms using 16S rRNA polymerase chain reaction (PCR) and validate microbial identities using biochemical techniques. We hypothesized that designing an experimental protocol to confirm the identity of a bacterium would improve students' knowledge of microbial identification techniques and the physiological characteristics of bacterial species. Nitrogen-fixing bacteria were isolated from the root nodules of Medicago truncatula and prepared for 16S rRNA PCR analysis. Once DNA sequencing revealed the identity of the organisms, the students designed experimental protocols to verify the identity of rhizobia. An assessment was conducted by analyzing pre- and posttest scores and by grading students' verification protocols and presentations. Posttest scores were higher than pretest scores at or below p = 0.001. Normalized learning gains (G) showed an improvement of students' knowledge of microbial identification methods (LO4, G = 0.46), biochemical properties of nitrogen-fixing bacteria (LO3, G = 0.45), and the events leading to the establishment of nitrogen-fixing symbioses (LO1&2, G = 0.51, G = 0.37). An evaluation of verification protocols also showed significant improvement with a p value of less than 0.001.
bacteriophage HMSP1-Susan has a genome of 51,963 bp in size, with a GC content of 52.5%. It contains 97 putative coding sequences; 83% of these coding sequences (CDS) encode proteins classified as ...hypothetical or having unknown functions. HMSP1 has limited homology to previously reported viruses and likely represents a new phage that infects this nitrogen-fixing bacterium.
PDF
