Course 7.341

Back to the Berkmen Main Webpage 7.341 NOT JUST A BAG OF ENZYMES: DNA DYNAMICS IN THE TINY BACTERIAL CELL MIT Advanced Undergraduate Seminar Fall 2005

Instructors: Melanie Berkmen and Lyle Simmons Time	 	Mondays, 11 am – 1 pm Room	 	68-151 Organizer	Bob Horvitz http://web.mit.edu/biology/www/undergrad/adv-ugsem.html

Course summary:

Bacteria were among the first organisms to inhabit the earth, and they will probably be the last. While some bacteria are the causative agents of diseases such as anthrax and cholera, other bacteria play helpful roles, e.g., in plant development and antibiotic production. Studies of bacteria have been important for our understanding of central biological principles. For example, all cells possess mechanisms that ensure that each daughter cell inherits a full complement of genes after cell division. In humans, improper chromosome segregation may lead to cancer or other diseases. In bacteria, failed chromosome segregation results in death. Many bacteria must also segregate relatively small DNA molecules called plasmids, which can encode antibiotic-resistance and virulence genes. In this course, we will investigate the molecular mechanisms by which bacteria ensure the faithful segregation of their chromosomal and plasmid DNAs. For example, to ensure proper DNA segregation, some bacteria use an apparatus similar to that used for mitosis in eukaryotic cells. If a chromosome inadvertently gets trapped in the cell division plane, a DNA pump is assembled at the site to help move the DNA to its correct destination. Fluorescence microscopy has played a pivotal role in studying the protein and DNA choreography involved in plasmid and chromosome maintenance. We will visit an MIT research laboratory focused on bacterial chromosome dynamics, and students will experience first-hand several fluorescent microscopic techniques used in this type of research. In addition, the class will tour Novartis Institutes for Biomedical Research in Cambridge and meet the Novartis project leader in microbiology and infectious diseases. Goals and Format

In this class, you will learn about many of the current topics concerning DNA dynamics in bacteria. You will also gain familiarity with many useful biological methods and an appreciation for how scientists conduct controlled experiments. Perhaps most importantly, you will learn how to read, discuss, and critically analyze scientific papers.

Each week we will discuss two papers. Everyone will read both papers prior to class. During each class period, each student will actively participate in the presentation and interpretation of the background, methods, results, and conclusions. At the end of each class, the instructors will briefly describe the relevant background and techniques required for understanding the papers covered in the next class period. Requirements

1. Basic knowledge of biology and molecular biology. 2. An eagerness to learn and share your ideas openly with the class. Attendance and active participation are required for each class. Should a student miss a class due to extreme circumstances, the student must contact the instructors ahead of time. The student will complete a written assignment regarding the papers missed in class. 3. Completion of the assigned written course work. Assignments

Each week assignment Read and thoroughly analyze the assigned papers. Write down a list of questions (minimum 3) regarding the assigned papers. These questions should be directly emailed to the instructors at least 1 hour prior to class.

Paper review (due Oct. 24, 2005) Assume you are a professor and you have agreed to review a research paper that we will provide. You will provide a short summary of the work, its major conclusions, and whether the paper supports those conclusions. You will decide whether the paper should be accepted/rejected for publication or whether it requires revisions or additional experiments or controls. Your review (2 pages, double spaced) should indicate the paper’s contributions to the field, strengths, weaknesses, and novelty.

Research proposal (outline due Nov. 28, 2005 & proposal due Dec. 5, 2005) Assume you are a graduate student and you have to pass your qualifying exam. You will need to write a proposal (4 pages, double spaced) describing a set of experiments that follow up on a topic or paper discussed in class. Your proposal will focus on the major questions being explored, the experimental design you will use, the most relevant controls to be included, and what types of results you might expect. Students will hand in an outline one week prior to the proposal due date. Students must discuss the topic with the instructors before the outline is turned in. Course Outline and Syllabus Week 1 (Sept. 13, 2005): Introduction and background

Instructors and students will introduce themselves, their backgrounds, and reasons for participating in the course. Instructors will present the overall goals, requirements and assignments for the course. The instructors will review techniques for reading and analyzing the primary literature. A brief overview of bacterial cell biology, plasmids, and chromosome partitioning will be provided. Students and instructors will brainstorm for possible mechanisms that could be used to ensure faithful DNA partitioning. Lastly, the instructors will briefly discuss the topic for the following week’s papers. Week 2 (Sept. 20, 2005) Anthrax: bacilli, spores, and toxins

Anthrax is a highly infectious disease caused by the bacterium Bacillus anthracis. Anthrax most commonly occurs in domestic livestock such as cattle and sheep, but can also infect humans. We will discuss Robert Koch’s landmark paper published in 1876 showing that B. anthracis causes anthrax. This paper also documents the transition of the bacterium from a dormant spore in the soil to a rod-shaped bacillus during infection in the animal. Over 100 years later, it was discovered that in order to cause disease, B. anthracis requires the presence of particular extrachromosomal DNA elements called plasmids. We will discuss the evidence for plasmid-mediated toxin production.

Research papers: Koch, R. The etiology of anthrax, based on the life history of Bacillus anthracis. Beitrage zur Biologie der Pflanzen. 1876 (2): 277-310. In Milestones in Microbiology: 1556 to 1940, translated and edited by Thomas D. Brock, ASM Press. 1998, p89-95. Mikesell P, Ivins BE, Ristroph JD, Dreier TM. Evidence for plasmid-mediated toxin production in Bacillus anthracis. Infect Immun. 1983 Jan;39(1):371-6.

'''Week 3 (Sept. 27, 2005) - Plasmids encoding virulence factors

Plasmids are extrachromosomal DNA elements that replicate in bacteria and are maintained independently of the chromosome. Many naturally occurring plasmids encode antibiotic resistance genes that can be passed onto other bacteria to make a population of cells resistant to antibiotic treatment. Similarly, many pathogenic bacteria acquire virulence factors from a plasmid. In the absence of the virulence conferring plasmid these “pathogenic” bacteria are benign. In this section we investigate the plasmids that turn Bacillus thuringiensis into an insect pathogen. Research papers:

Wilcks A, Smidt L, Okstad OA, Kolsto AB, Mahillon J, Andrup L. Replication mechanism and sequence analysis of the replicon of pAW63, a conjugative plasmid from Bacillus thuringiensis. J. Bacteriol. 1999 May;181(10):3193-200.

Levinson BL, Kasyan KJ, Chiu SS, Currier TC, Gonzalez JM Jr. Identification of beta-exotoxin production, plasmids encoding beta-exotoxin, and a new exotoxin in Bacillus thuringiensis by using high-performance liquid chromatography. J. Bacteriol. 1990 Jun;172(6):3172-9.

Week 4 (Oct. 4, 2005) - How bacteria get addicted: plasmid toxin-antitoxin systems Plasmids encode mechanisms to ensure that each new cell will carry a copy of the plasmid DNA. Plasmid addiction modules are one of many such mechanisms. These modules regulate programmed cell death by expressing a stable toxin that will kill the cell if the unstable antitoxin (or antidote) is not present. Bacteria become “addicted” to plasmids encoding this module, because cells are rapidly killed in the absence of the plasmid.

Research papers:

Gerdes K, Bech FW, Jorgensen ST, Lobner-Olesen A, Rasmussen PB, Atlung T, Boe L, Karlstrom O, Molin S, von Meyenburg K. Mechanism of postsegregational killing by the hok gene product of the parB system of plasmid R1 and its homology with the relF gene product of the E. coli relB operon. EMBO. J. 1986 Aug;5(8):2023-9.

Smith AS, Rawlings DE. The poison-antidote stability system of the broad-host range Thiobacillus ferrooxidans plasmid. pTFFC2. Mol. Microbiol. 1997 Dec;26(5):961-70.

Week 5 (Oct. 18, 2005) - Plasmid partitioning by the ParA and ParB proteins As we have learned during the previous class period, some plasmids encode addiction modules that require newborn cells to contain the plasmid to avoid rapid cell death from a killing factor. Other plasmids encode partitioning factors, which allow for increased plasmid stability (a.k.a. plasmid inheritance) through an active mechanism of segregating plasmid DNA into cells prior to cell division. Here we will discuss the P1 plasmid partitioning system that involves a ParA ATPase, a DNA-binding protein ParB, and a cis-acting centromere-like site (parS) on the plasmid.

Research papers: Martin KA, Friedman SA, Austin SJ. Partition site of the P1 plasmid. Proc. Natl. Acad. Sci. U S A. 1987 Dec;84(23):8544-7.

Davis MA, Austin SJ. Recognition of the P1 plasmid centromere analog involves binding of the ParB protein and is modified by a specific host factor. EMBO. J. 1988 Jun;7(6): 1881-8.

'''Week 6 (Oct. 25, 2005) Paper Review Due - Visualizing DNA dynamics and partitioning inside the tiny bacterial cell (includes a visit to an MIT lab with a fluorescence microscope)'''

Research paper (1 only): Erdmann N, Petroff T, Funnell BE. Intracellular localization of P1 ParB protein depends on ParA and parS. Proc. Natl. Acad. Sci. U S A. 1999 Dec 21;96(26):14905-10. Week 7 (Nov. 1, 2005) Plasmid segregation by actin-like filaments

Partitioning of plasmid R1 requires the ATPase ParM, the DNA binding protein ParR, and the cis-acting centromere-like site parC. The ATPase activity of ParM is essential for plasmid partition. The role of this ATPase activity remained mysterious until the recent discovery that ParM, an ancient actin homolog, is capable of forming stunning actin-like filaments that span the length of the cell. In this system, ParM polymerization into an actin-like filament generates the force required for directional segregation of plasmids to opposite sides of the cell.

Research papers: van den Ent F, Moller-Jensen J, Amos LA, Gerdes K, Lowe J. F-actin-like filaments formed by plasmid segregation protein ParM. EMBO J. 2002 Dec 16;21(24):6935-43.

Moller-Jensen J, Jensen RB, Lowe J, Gerdes K. Prokaryotic DNA segregation by an actin-like filament. EMBO. J. 2002 Jun 17;21(12):3119-27.

Week 8 (Nov. 8, 2005) - On the move: bacterial chromosomal choreography 

It was once thought that the DNA in bacteria was placed randomly inside the cell. The development of state-of-the-art fluorescent tagging systems led to the discovery that different regions of the chromosome are localized to specific positions in the bacterial cell. Furthermore, time-lapse microscopy has allowed researchers to trace the movements of the chromosome during the cell cycle. This same technique has now been applied to Vibrio cholerae, the causative agent of cholera. This bacterium is unusual because it contains two chromosomes. How does this bacteria ensure that both chromosomes are properly inherited?

Research papers: Webb CD, Graumann PL, Kahana JA, Teleman AA, Silver PA, Losick R. Use of time-lapse microscopy to visualize rapid movement of the replication origin region of the chromosome during the cell cycle in Bacillus subtilis. Mol. Microbiol. 1998 Jun; 28(5): 883-92.

Fogel MA, Waldor MK. Distinct segregation dynamics of the two Vibrio cholerae chromosomes. Mol. Microbiol. 2005 Jan;55(1):125-36.

Week 9 (Nov. 15, 2005) - Tour Novartis Institutes for Biomedical Research in Cambridge and meet the Novartis project leader in microbiology and infectious diseases

Week 10 (Nov. 22, 2005) - Structure Maintenance of Chromosomes Protein (SMC): the great chromosome compacter

The bacterial chromosome is approximately 1000-fold longer than the length of the cell. Without proper chromosome organization and compaction, chromosome partitioning would obviously be a nightmare. The Structural Maintenace of Chromosomes (SMC) proteins are present in all eukaryotes and in many bacteria. SMCs play essential roles in chromosome condensation, cohesion, and segregation in eukaryotes. We will discuss how SMCs are also essential in bacteria for their similar roles in chromosome condensation and segregation.

Research papers: Britton RA, Lin DC, Grossman AD. Characterization of a prokaryotic SMC protein involved in chromosome partitioning. Genes Dev. 1998 May 1;12(9):1254-9.

Soppa J, Kobayashi K, Noirot-Gros MF, Oesterhelt D, Ehrlich SD, Dervyn E, Ogasawara N, Moriya S. Discovery of two novel families of proteins that are proposed to interact with prokaryotic SMC proteins, and characterization of the Bacillus subtilis family members ScpA and ScpB. Mol. Microbiol. 2002 Jul;45(1):59-71.

Week 11 (Nov. 29, 2005) Proposal Topic and Outline Due - RacA: A protein that anchors chromosomes to the cell poles during sporulation

Immediately after 9/11, a number of anthrax spore-laced letters were mailed to the U.S. Congress causing an increase in interest in spore biology. A bacterial spore is formed from an asymmetric cell division, which divides the cell into a forespore and a mother cell. The forespore, which will develop into a mature spore, is approximately 10-times smaller than the typical bacterial cell, and thus presents a unique problem for chromosome partitioning. We will discuss the exciting discovery of the RacA chromosome partitioning protein. RacA anchors the chromosome to the ends (poles) of the developing cell, thus ensuring that the tiny forespore receives a chromosome.

Research papers: Ben-Yehuda S, Rudner DZ, Losick R. RacA, a bacterial protein that anchors chromosomes to the cell poles. Science. 2003 Jan 24;299(5606):532-6.

Ben-Yehuda S, Fujita M, Liu XS, Gorbatyuk B, Skoko D, Yan J, Marko JF, Liu JS, Eichenberger P, Rudner DZ, Losick R. Defining a centromere-like element in Bacillus subtilis by identifying the binding sites for the chromosome-anchoring protein RacA. Mol. Cell. 2005 Mar 18;17(6):773-82.

Week 12 (Dec. 6, 2005) Final Proposal Due - Push it out, shove it out, way out: the SpoIIIE DNA pump 

During sporulation, only ~1/3 of the chromosome is initially captured in the tiny forespore compartment after cell division (see above figure for Week 11, Nov. 29th). The rest of the chromosome is caught in the mother cell and must be pumped into the forespore. The SpoIIIE protein is the DNA pump assembled in the cell septum that moves the DNA from the mother cell into the forespore so that each spore will inherit an entire chromosome. How does the SpoIIIE protein get to the correct location? How does the cell determine which direction to the pump the DNA?

Research papers: Wu LJ, Errington J. Septal localization of the SpoIIIE chromosome partitioning protein in Bacillus subtilis. EMBO J. 1997 Apr 15;16(8):2161-9.

Sharp MD, Pogliano K. Role of cell-specific SpoIIIE assembly in polarity of DNA transfer. Science. 2002 Jan 4;295(5552):137-9.

Week 13 (Dec. 13, 2005)- MreB: the bacterial cytoskeletal protein (and ancient actin homolog) required for replication origin movement

Many bacteria encode a genetic program that allows for the “transformation,” or uptake of DNA, from their surroundings. We will discuss how this program allows the spread of critical bacterial characteristics, like antibiotic or heavy metal resistance, from bacterium to bacterium. At least 20 proteins are required for the uptake and transfer of DNA into the cell. Using a combination of fluorescence microscopy and single-molecule experiments with laser tweezers, it has recently been discovered that the DNA uptake machinery is localized to the cell poles.

Research paper: Gitai Z, Dye NA, Reisenauer A, Wachi M, Shapiro L. MreB actin-mediated segregation of a specific region of a bacterial chromosome. Cell. 2005 Feb 11; 120(3): 329-41.

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