Spring 2019BIOL 312: Microbiology

A Town on FireMetagenomic Analysis of Bacterial Communities in Soils Overlying the Centralia, Pennsylvania Mine Fire

Instructor: Dr. Tammy Tobin Susquehanna UniversityE-Mail: tobinjan@susqu.edu

OverviewIn 1962, a surface trash fire ignited an anthracite coal seam in an abandoned strip mine in Centralia, Pennsylvania. Repeated efforts to extinguish the fire failed, and in 1984 Congress responded to the resulting high carbon monoxide levels and frequent land collapses by allocating more than $42 million for relocation efforts. Most of the residents have long since moved, and their homes have been demolished, leaving behind

a ghost town where a coal mining community once thrived (Fig 1).

Figure 1: Left: Centralia, PA prior to the evacuation in 1984. The town had over 1800 residents, several businesses and churches. Below: Old Route 61 through

Centralia (taken in 1997) showing steam, rich in carbon monoxide, venting upward through cracks caused by land collapses.

As a result of this mine fire, surface soil temperatures in affected areas regularly exceed 60°C and soils surrounding the vents are often rich in combustion products such as sulfur and nitrogen that microbial communities can use and transform as a part of their energy-generating processes.

In this case study, you will use information in papers that describe typical geothermal soils and their microbial communities to hypothesize a single bacterial genus that you would expect to find living in Centralia’s fire-affected soils. You will use metagenomic

Metagenomic Analysis of Bacterial Communities in Soils Overlying the Centralia, Pennsylvania Mine Fire 1

analysis to test your hypothesis and then make a presentation that reports your findings and predicts the types of impacts that members of your genus might be having on the Centralia ecosystem.

Metagenomic Analysis of Bacterial Communities in Soils Overlying the Centralia, Pennsylvania Mine Fire 2

GoalsAs a result of participation in these activities, students will be able to:

1. Explain each step in the generation and analysis of Next Gen metagenomic 16S rRNA sequence data.

2. Discuss the basic biology assumptions that underlie sequence analysis (e.g. evolution, structure and function, conservation = function).

3. Evaluate the strengths and weaknesses of the methods employed in Next Gen sequencing, including the impact that data quality has on bioinformatics analysis.

4. Choose and justify the appropriate methods for a specific Next Gen sequencing application. 

5. Apply Next Gen sequencing methodologies to solve their own research questions.

EvaluationThe final evaluation of this project will be based on the successful completion of Team Application Activities and a Final Paper and Presentation.

Figure 2: Steam from “Anthracite Smokers” in Centralia, PA carries dissolved combustion products, such as nitrogen and sulfur, to the surface through soil fractures. As the steam rises it cools and precipitates chemicals

MaterialsRecommended Readings:A Primer on Metagenomics

Computer Resources: Macintosh computer running Java version 7 or higher.

Access to Amazon Web Services EC2 large instance or native install of QIIME 2 https://docs.qiime2.org/2018.2/install/native/#install-qiime-2-within-a-conda-environment .

FigTree

Metagenomics Sequence Resources:Centralia Metagenomics files C02, C09, C10 and C13 are available in the NCBI’s Short Read Archive https://www.ncbi.nlm.nih.gov/sra/SRP082686

Team Application Activities:IntroductionStudents will learn about the history and biogeochemistry of the Centralia Mine Fire environment and will take the GCAT SEEK pre-test.

Team Activity #1Students will work in teams in order to familiarize themselves with metagenomics, LINUX and QIIME, and will propose hypotheses regarding the types of microbial species they expect to see in thermophilic versus mesophilic soils in Centralia.

Team Activity #2Students will use QIIME to test their hypotheses.

Team Activity #3Students will complete their QIIME analysis and begin to prepare their presentations.

Final PresentationEach student team will present their metagenomic findings.

Metagenomic Analysis of Bacterial Communities in Soils Overlying the Centralia, Pennsylvania Mine Fire 3

into the surrounding soils where they can be utilized and transformed by nitrogen and sulfur-cycling bacterial communities.

An Introduction to Next Generation Sequencing and MetagenomicsNext Generation Sequencing and Illumina Sequencing By Synthesis“Next generation (Next Gen) sequencing” is a term that encompasses a variety of DNA sequencing technologies, all of which have a common core approach: they use DNA polymerase to generate thousands or millions of relatively short (compared to traditional sequencing technologies) sequences of a DNA template concurrently. Thus, these sequencing technologies are often referred to as being ‘massively parallel’. They then differ in the manner in which they determine when (and which) base is added to the replicating DNA (that is, in how they actually “read” the sequence). For example, Ion Torrent sequencing uses the tiny pH change that happens each time a new phosphodiester bond is created to determine whether or not a particular base was added.

In this study, DNA was isolated directly from soil samples using a MoBio Powersoil DNA Isolation Kit. Next, a metagenomic 16S rRNA gene library was generated using PCR to amplify a portion of that gene from every bacterial species present in the soil DNA sample. (Figure 3).

Figure 3. Overview of the Polymerase Chain ReactionSequencing was then performed using an Illumina sequencing system as shown in the video (see video now).

Metagenomic Analysis of Bacterial Communities in Soils Overlying the Centralia, Pennsylvania Mine Fire 4

Metagenomic Analysis of Bacterial 16S rRNA genes As you probably recall from your introductory biology classes, protein synthesis (translation) is catalyzed by a structure called the ribosome, a complex structure composed of both proteins and ribosomal RNA (rRNA). Translation begins when the small subunit of the ribosome locates and binds to the 5’ end of the mRNA. Once this has happened, the large ribosomal subunit can attach, and the complete ribosome translocates along the mRNA, catalyzing the formation of peptide bonds between amino acids as they are conveyed to the correct mRNA codon by tRNA.

In order for this process to work correctly, the ribosome must first be able to find the 5’ end of an mRNA and bind to it. That recognition is the job of the 16S rRNA, which contains a 3’ sequence that is complementary to the 5’ end of the mRNA. The 16S rRNA genes have been used extensively for phylogenetic analysis since Carl Woese and George Fox first proposed their use in 1977 (Woese and Fox, 1977). Because of its critical function in translation, the 16S rRNA gene contains highly conserved sequences that can be used to design ‘universal’ PCR primers (primers that work for almost all species), as well as highly variable regions that allow taxon identification based on sequence comparison to known taxa. Well-curated databases, such as the Ribosomal Database Project (Cole, et al. 2003) and the Greengenes database (DeSantis et al 2006) contain regularly updated versions of all of the known 16S rRNA gene sequences, along with their phylogenetic assignments, and are invaluable in this process.

Metagenomic analysis (Handelsman, et al. 1998) is the analysis of genetic samples recovered directly from the environment, without any attempt to isolate the microbes from which they came. This type of analysis allows microbiologists to study the vast numbers of uncultured, or unculturable, microbes in any environment. Current high-profile examples of this type of analysis include the Human Microbiome project, the Earth Microbiome project and the Maternal Microbiome project.

In preparation for this case study, soil was collected from 4 boreholes in Centralia, PA (13.6°C, 34.2°C, 54.2°C and 57.4°C), and genomic DNA was directly isolated from the samples using the MoBio Powersoil Kit. PCR with universal bacterial 16S rRNA V4 region primers was then used to make copies of all of the bacterial 16S rRNA genes in each of these samples. These PCR products were then used as the template for Illumina Sequencing at Michigan State University. You will be using this data to test hypotheses regarding the types of bacteria that live in the hot soils overlying the Centralia, PA mine fire. In the next class period, you will learn a bit about the program that you will be using to perform the analyses.

Metagenomic Analysis of Bacterial Communities in Soils Overlying the Centralia, Pennsylvania Mine Fire 5