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1 2006S Bio153 Lab 2: Prokaryotes and Protists July 4th / July 6th Supporting material in the textbook: Chapters 27 & 28 Introduction: The two groups studied in today’s lab exercise represent the vast majority of organismal diversity. Prokaryotes and protists make up all organisms that are not fungi, animals or plants – and fungi, animals and plants represent a very small portion of the tree of life! Figure 1. The tree of life (based on 16s-RNA sequences). Thus, it is a gross misrepresentation of diversity to allot these vast groups to a single lab exercise. However, keep in mind that the great diversity in prokaryotes is evident primarily in their array of metabolic pathways rather than their morphology, and the internal structure of prokaryotic cells is best seen under an electron microscope. Part 1: Prokaryotes Prokaryotes are an enormously diverse group of organisms with a long evolutionary history. The most obvious differences between prokaryotic and eukaryotic cells (i.e., the cells of protists, animals, fungi and plants) are a) prokaryotic cells are usually much smaller than eukaryotic cells, and b) prokaryotic cells are much simpler in structure than eukaryotic cells (they lack a nucleus, membrane-bound organelles, etc.). There are two major domains among prokaryotes: the Archaea and the Eubacteria (or Bacteria). It is interesting to note that bacteria are thought to be phylogenetically closer to eukaryotes than they are to Archaea – in other words, you are more closely related to a bacterium such as Streptococcus than Streptococcus is to a 2 methanogen! Archaea are difficult to isolate and culture; therefore, we will be looking only at Eubacteria. Gram staining: Species in the domain Eubacteria have cell walls that contain a substance called peptidoglycan. Peptidoglycan is a polymer consisting of sugars and amino acids that strengthen the bacterial cell wall. The Gram stain attaches to peptidoglycan; thus, the amount of stain a bacterial cell holds is a measure of how much peptidoglycan is in the cell wall. Gram-positive bacteria have a thick layer of peptidoglycan and thus retain a lot of stain, making them appear purple (Fig 2a; Fig 3). Gram-negative bacteria have relatively little peptidoglycan in their cell walls, surrounded by a thin outer membrane composed of lipopolysaccharide. This means that they retain relatively little stain and appear pink (Fig 2b; Fig 3). Peptidoglycan accounts for approximately 90% of the dry weight of Gram-positive bacteria, but only 10% of the dry-weight of Gram-negative strains. Many antibiotics work by interfering with the formation of the peptidoglycan layer. (a) (b) Figure 2. Structure of Gram-positive (a) and Gram-negative (b) bacterial cell walls. Figure 3. Gram-positive and Gram-negative bacteria under the compound microscope. 3 Bacterial Morphology: Bacteria exist as single cells or in colonies. They do not show true multicellularity (i.e. lack complex junctions between cells). Bacterial cells are often named for their shape. The four most common shapes are spherical (cocci), rod-shaped (bacilli), comma-shaped (vibrio), and spiral shaped. The species name may also reflect the types of groupings the individual cells make. Diplo- means that the bacterial cells occur in pairs; Strepto- means the cells form chains, and Staphylo- means the cells form clumps. Thus, Staphylococcus aureus (a bacterium commonly occurring on human skin that is one of the causative agents of necrotizing fasciitis, or flesheating disease) grows as a clump of spherical cells. The cause of strep throat is bacterium known as Streptococcus pyogenes or group A beta-hemolytic streptococcus, which occurs as a chain of spherical cells. Cyanobacteria: Cyanobacteria are sometimes called blue-green algae. Although they are often blue-green, they are not algae – algae are eukaryotes (their cells have a nucleus) and are considered the basal group of the plants. Cyanobacteria are among the most abundant organisms on earth, and played an extremely important role in the early history of organismal diversification. All cyanobacteria perform oxygenic photosynthesis. Photosynthesis requires a source of electrons, and organisms that perform oxygenic photosynthesis (cyanobacteria and plants) split water molecules to obtain these electrons, generating oxygen as a byproduct. Before the evolution of eukaryotes, cyanobacteria generated large amounts of oxygen, radically altering the earth’s atmosphere. Although prokaryotes lack membrane-bound organelles, photosynthetic bacteria, such as cyanobacteria, may be filled with tightly packed folds of their outer membrane. The effect of these membranes is to increase the surface area on which photosynthesis can take place. Anabaena is a filamentous cyanobacteria capable of nitrogen fixation, and is common in many freshwater systems. It produces a neurotoxin that is not fatal to humans, but at high concentrations often leads to fish kills. The presence of Anabaena affects water quality, as it causes a bad smell and disagreeable taste. Exercise: • Examine the slides of Gram-positive and Gram-negative bacteria. Note the colour of the cells, their very small size, and the shape of the cells. • Examine the slide of Anabaena, noting the colour, shape and arrangement of the cells. 4 Part 2: Protists The taxonomy of the group informally known as protists is contentious and under constant revision, so we’ll focus on the functional differences among some selected groups – their locomotion, their structure, and their modes of reproduction. The ciliates: The ciliates are unicellular protists of enormous complexity. They are members of the lineage Alveolata (have small sacs called alveoli below the plasma membrane). Ciliates are covered with cilia that propel them through water and aid in feeding. Some cilia are fused into cirri, which can produce a more forceful beat. Others are modified into trichocysts, which can be discharged to catch prey or repel predators. Ciliates are mostly diploid. Most ciliates have 2 types of nuclei – the macronucleus and the micronucleus. The macronucleus has many copies of the genome, and is transcriptionally active. The micronucleus has one copy of the diploid genome, and is important in conjugation. Ciliates reproduce asexually by fission, but can also participate in an exchange of genetic material with another ciliate of an opposite mating type. This is not true sexual reproduction, because no new individual arises from conjugation. During conjugation, two cells join together, dissolving their cell membranes at the site of junction and forming a cytoplasmic bridge. The macronucleus disintegrates and the micronucleus undergoes meiosis. One haploid micronucleus from each cell migrates across the cytoplasmic bridge and fuses with the micronucleus in the other cell. This recombined micronucleus gives rise to a new macronucleus. This periodic genetic rejuvenation is necessary for survival; cell lines that are prevented from undergoing conjugation eventually die out. Paramecium has an oral groove lined with cilia. It rotates on its axis while it swims, sweeping food into its gullet. When it bumps into an object, calcium channels in the cell membrane open, and the cell rapidly depolarizes (loses electrical charge). This reverses to direction of the cilia, and Paramecium swims backwards. Although it feeds mostly on bacteria, it also has trichocysts which it uses to repel predators such as Didinium. Diatoms: Diatoms are members of the group Stramenopila, and are a major component of phytoplankton. They contribute greatly to global photosynthesis and to the carbon cycle in the oceans. Stramenopila all share a 5 distinctive type of flagellum that is covered in hollow “hairs”, but in diatoms, only reproductive cells have flagella. Diatoms have silica-based cell walls (frustules) that fit together like the halves of a petri dish. Individual diatoms are diploid (2N). Reproduction is mostly asexual, with daughter cells each receiving onehalf of the parent cell wall. Because the halves of the cell wall are not the same size, one daughter cell tends to be smaller than the parent. After several generations of asexual reproduction, average size of individuals in the population declines, due to this unequal division. Reduction of body size in the population can trigger sexual reproduction. Amoebozoa: Amoeba proteus This organism derives its name from the Greek God Proteus, who could assume many forms. It is a lobose amoeba, in the lineage Ameobozoa, which includes plasmodial and cellular slime molds. The distinctive feature of members of this group is their ability to move through “cell crawling”. Amoeba proteus is a relatively large protist, and moves using extensions of the cytoplasm called pseudopodia. The edge of the pseudopodium is relatively stiff compared to the liquid interior: transitions from gel (stiffer cytoplasm) to sol (more liquid cytoplasm) to gel account for the extension of the pseudopodium. This cytoplasmic streaming is readily visible under the compound microscope. Food is engulfed and contained within food vacuoles. Contractile vacuoles expel excess water. Cellular and plasmodial slime molds also exhibit “cell crawling”. Cellular slime molds, such as Dictyostelium discoideum, take the form of individual amoeba when food is plentiful. When food is scarce, these individual amoebae aggregate to form a multicellular assembly called a pseudoplasmodium or slug. The slug functions as a single organism in that it has a front and back end, moves and responds to light and temperature gradients. The slug can form a fruiting body with a stalk supporting one or more balls of spores (inactive cells protected by a cell wall). These spores can develop into new amoebae when food becomes plentiful. Plasmodial slime molds are somewhat different in that they form a web-like supercell (a single cell with many diploid nuclei). When food is scarc e, a fruiting stalk forms and nuclei undergo meiosis (2N → 1N) and form spores. These spores 6 are dispersed and germinate to form haploid amoebae. Two haploid amoebae fuse to form a diploid cell that eventually becomes a new supercell. Exercise: • Examine the protist material on display. Look at the display pictures and prepared slides. Make a wet mount of Amoeba proteus and examine under the compound microscope. Make diagrams and study notes.