This project was prepared as part of a BioQUEST faculty development workshop entitled ASM/BioQUEST Bioinformatics Institute at American Society for Microbiology in March 2007. The BioQUEST Curriculum Consortium is committed to the reform of undergraduate biology instruction through an emphasis on engaging students in realistic scientific practices. This approach is sometimes characterized as an inquiry driven approach and is captured in BioQUEST's three P's (problem-posing, problem-solving, and peer-persuasion). As part of this workshop groups of faculty were encouraged to initiate innovative curricular projects. We are sharing these works in progress in the hope that they will stimulate further exploration, collaboration and development. Please see the following links for additional information:

Upcoming events               BEDROCK Problem Spaces

Incorporation of class discussions to strengthen bioinformatic concepts Example: Re-examining phylogenies, a discussion
Authors          Audiences          Overview           Materials          Resources           Future Directions



Ann Matthysse
University of North Carolina at Chapel Hill

Leland (Sandy) Pierson
University of Arizona


Possible Audiences:

Freshman through senior level students that are currently studying bioinformatics as part of a microbiology or microbial genetics course.  


Brief Overview:

To reinforce concepts discussed during lecture on phylogenic analysis of populations. Prior to the class discussion, students would be asked to read a research paper and examine the phylogenetic data presented. Next they would download the original sequence files from NCBI and perform their own phylogenetic analyses and compare their results with those in the original paper. The class discussion would focus not on whether one analysis is correct and the other incorrect, but what are the differences between when the original analysis was performed and today. Examples could include: Changes in the size and complexity of the database. Characters chosen for the analysis (single genes, multiple loci, etc). Differences in the weighting of the characters.  


Project Materials:

Sample papers:

  1. Comparative Genomic Analyses of the Vibrio Pathogenicity Island and Cholera Toxin Prophage Regions in Nonepidemic Serogroup Strains of Vibrio cholerae Manrong Li, Mamuka Kotetishvili, Yuansha Chen, and Shanmuga Sozhamannan Appl Environ Microbiol. 2003 69(3):1728-38.

    Two major virulence factors are associated with epidemic strains (O1 and O139 serogroups) of Vibrio cholerae: cholera toxin encoded by the ctxAB genes and toxin-coregulated pilus encoded by the tcpA gene. The ctx genes reside in the genome of a filamentous phage (CTX ), and the tcpA gene resides in a vibrio pathogenicity island (VPI) which has also been proposed to be a filamentous phage designated VPI . In order to determine the prevalence of horizontal transfer of VPI and CTX among nonepidemic (non-O1 and non-O139 serogroups) V. cholerae, 300 strains of both clinical and environmental origin were screened for the presence of tcpA and ctxAB. In this paper, we present the comparative genetic analyses of 11 nonepidemic serogroup strains which carry the VPI cluster. Seven of the 11 VPI+ strains have also acquired the CTX . Multilocus sequence typing and restriction fragment length polymorphism analyses of the VPI and CTX prophage regions revealed that the non-O1 and non-O139 strains were genetically diverse and clustered in lineages distinct from that of the epidemic strains. The left end of the VPI in the non-O1 and non-O139 strains exhibited extensive DNA rearrangements. In addition, several CTX prophage types characterized by novel repressor (rstR) and ctxAB genes and VPIs with novel tcpA genes were found in these strains. These data suggest that the potentially pathogenic, nonepidemic, non-O1 and non-O139 strains identified in our study most likely evolved by sequential horizontal acquisition of the VPI and CTX independently rather than by exchange of O-antigen biosynthesis regions in an existing epidemic strain.

  2. Phylogenetic Analysis of Anaerobic Psychrophilic Enrichment Cultures Obtained from a Greenland Glacier Ice Core Peter P. Sheridan, Vanya I. Miteva, and Jean E. Brenchley. Appl Environ Microbiol. 2003 Apr;69(4):2153-60

    The examination of microorganisms in glacial ice cores allows the phylogenetic relationships of organisms frozen for thousands of years to be compared with those of current isolates. We developed a method for aseptically sampling a sediment-containing portion of a Greenland ice core that had remained at −9°C for over 100,000 years. Epifluorescence microscopy and flow cytometry results showed that the ice sample contained over 6 × 107 cells/ml. Anaerobic enrichment cultures inoculated with melted ice were grown and maintained at −2°C. Genomic DNA extracted from these enrichments was used for the PCR amplification of 16S rRNA genes with bacterial and archaeal primers and the preparation of clone libraries. Approximately 60 bacterial inserts were screened by restriction endonuclease analysis and grouped into 27 unique restriction fragment length polymorphism types, and 24 representative sequences were compared phylogenetically. Diverse sequences representing major phylogenetic groups including alpha, beta, and gamma Proteobacteria as well as relatives of the Thermus, Bacteroides, Eubacterium, and Clostridium groups were found. Sixteen clone sequences were closely related to those from known organisms, with four possibly representing new species. Seven sequences may reflect new genera and were most closely related to sequences obtained only by PCR amplification. One sequence was over 12% distant from its closest relative and may represent a novel order or family. These results show that phylogenetically diverse microorganisms have remained viable within the Greenland ice core for at least 100,000 years.

  3. Molecular Population Genetics and Phenotypic Diversification of Two Populations of the Thermophilic Cyanobacterium Mastigocladus laminosus. Scott R. Miller,* Michael D. Purugganan, and Stephanie E. Curtis. AEM 72:2793-2800.

    The authors investigated the distributions of genetic and phenotypic variation for two Yellowstone National Park populations of the heterocyst-forming cyanobacterium Mastigocladus (Fischerella) laminosus. The phylogenetic positions of these strains were analyzed along with those of several additional heterocystous cyanobacteria, including a Mastigocladus strain isolated from an Icelandic hot spring (CCMEE 5321) and the thermophile Chlorogloeopsis PCC 6718. The heterocystous cyanobacteria are a monophyletic group, and all analyses were rooted with the outgroup Chroococcidiopsis PCC 7203, which has been suggested to be the closest relative of the heterocystous clade in many previous phylogenies. The tree topology was consistent across maximum likelihood, parsimony, and neighbor-joining methods (Fig. 1). Mastigocladus CCMEE 5321, which is morphologically similar to the Yellowstone strains, was obtained from a similar habitat, and has 98.1% sequence identity at this locus, was inferred with substantial bootstrap support to be the sister taxon of the Yellowstone strains.



Resources and References:

The Biology workbench

The NCBI search site

The Joint Genome Initiative

Intergrated Microbial Genome database

KEGG pathway database
Bacterial metabolic pathways and genes
analysis tools
lots of nice analysis tools
site for mega 3.1 this program does alignments and builds simple trees. It is downloaded to a pc and runs from the pc itself (no internet connection needed).  


Future Directions:

This is the first example of a possible series of discussions aimed at evaluation of primary literature and reinforcement of bioinformatic principles. This approach combines independent thinking and data analysis with in class interactive discussion. These discussions have no single correct outcome and the students will be exposed to the existence of multiple approaches and solutions to problems.