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Chapter 5 Bacteria structure and physiology Objectives: After reading Chapter Five, you should understand… • The enormous span of time that bacteria have spent on Earth as well as their contribution to it. • The various forms of bacteria and the structures of the cell. • How bacteria reproduce and with what frequency. • The environments (natural and artificial) in which bacteria live. How old is the Earth? What were conditions like on early Earth? Where did energy come from? Were the biochemical subunits we discussed previously present on early Earth? What other factors might have been important for life? How long did it take all of these parameters to fall into place such that microbial life could begin? From 3.5 billion years ago until about 1.5 billion years ago, bacteria (prokaryotes) were the only life forms on Earth. Stromatolites in a tidal pool. Electron microscopy image of a stromatolite (100,000 x magnification). Ancient cyanobacteria began building up stromatolites about three-billion years ago. Microbes and Society, Sigler Spring 2013 1 Bacteria begin producing oxygen Eukaryotes then evolved, and they required: More energy, more nutrients, more space and “nicer” conditions. Bacteria had an advantage…what was it? Bacteria are still the dominant life form today, as they occupy every conceivable niche on Earth. Bacteria structure and physiology General morphology Three major forms Bacilli (bacillus) – rod-shaped – Escherichia coli Cocci (coccus) – spherical – Staphylococcus aureus Spirilla (spirillum) – flexible – Vibrio cholera Microbes and Society, Sigler Spring 2013 2 Escherichia coli Staphylococcus aureus Vibrio cholera These configurations can be useful for identifying clinical bacteria. How do we visualize bacteria? Since bacteria generally do not exhibit coloration in their natural state, staining of bacteria is often necessary in order to visualize them with a microscope. Bacteria cytoplasm (the “insides”) and polar heads of the membrane are negatively charged and will therefore attract a positively-charged stain, like crystal violet or methylene blue. Staining types: Simple staining – easy, one step. Corynebacterium diptheriae Microbes and Society, Sigler Spring 2013 3 Gram staining – developed by Christian Gram (1880s) Performed in multiple steps and separates bacteria into two groups. Gram positive (G+) and Gram negative (G-) G+ bacteria have a thick cell wall (peptidoglycan) that absorbs the stains, while G- bacteria have thinner cell walls that don’t absorb as effectively. Gram stain of E. Streptococcus spp. (G+) coli (G-) and Question: Why do we do the last “safranin” step? Aren’t the bacteria differentiated after the alcohol wash? Microbes and Society, Sigler Spring 2013 4 Surface structures of bacteria Know: Cell wall, cell membrane, glycocalyx, pilus, flagellum, as well as associated structures. Almost all bacteria are encased in a CELL WALL. Contains a tough network of polysaccharide and protein called peptidoglycan. Strength and rigidity – can withstand over 350 pounds of pressure per square inch! Peptidoglycan is found only in bacteria (not fungi, viruses or protists). Gram positive – thick peptidoglycan layer Gram negative – thin peptidoglycan layer (dual membrane layer) The essential functions of peptidoglycan and its confinement to bacteria make it a perfect target for antibiotics meant to rid the body of infections. Certain antibiotics attack infecting organisms by targeting the synthesis of peptidoglycan. e.g. β-lactam antibiotics, such as penicillin, ampicillin, amoxicillin and methicillin. Microbes and Society, Sigler Spring 2013 5 Death of a bacterium after treatment with an antibiotic targeting peptidoglycan synthesis. ? Which will be more susceptible to β-lactam antibiotics, Gram (-) or Gram (+) bacteria? The CELL MEMBRANE is internal to the cell wall and contains a double layer of phospholipids (bilayer). The membrane is the most important boundary between the interior of the cell and the external environment. The cell membrane encloses the CYTOPLASM. The membrane will allow small molecules to pass through, but big molecules cannot pass. Microbes and Society, Sigler Spring 2013 6 Proteins are embedded in the layers and sometimes span the entire thickness of the membrane. These proteins serve two general functions (know the difference): 1. Enzymes that catalyze chemical reactions 2. Porins (membrane-spanning proteins) can carry molecules (e.g. nutrients, antibiotics) across the membrane. Small carbohydrates are found on the outside of the membrane and can serve as recognition sites for host immune defenses (antibodies) e.g. in E. coli O157:H7, the “O157” refers to a membrane surface carbohydrate. A GLYCOCALYX (or “capsule” if rigid and highly structured) can protect the cell from environmental stresses. External mesh of polysaccharides that coat the cell. India Ink capsule stain of Klebsiella pneumoniae showing white capsules (glycocalyx) surrounding purple cells. From: biosci.sierracollege.edu/.../capsule_stain.html 1 µm The saccharides in the glycocalyx are sticky. Why would bacteria want to stick to things? What types of bacteria might benefit from a glycocalyx? Hint: a glycocalyx can help a bacterium evade host defenses. Microbes and Society, Sigler Spring 2013 7 Attachment can also be achieved through a PILUS (plural PILI). Cylindrical rod of helical protein about 1 µm in length and 7 nm thick. E. coli attaching to the lumen of a mouse. From: http://www.pnas.org/content/97/16/F1.medium.gif Pili allow attachment of infectious bacteria to host cells… Example, some therapeutic drugs, such as the antibiotics ampicillin and streptomycin, act to inhibit the formation of the pilus. …and to each other. 1. Pili are important for the exchange of genetic material between two cells. Microbes and Society, Sigler Spring 2013 8 2. Streptococci and other oral bacteria can bind to small pockets between teeth and gums, leading to dental caries. Bacteria will break down sugars, releasing acids that eat away at the tooth enamel = cavity. As tough as tooth enamel is, it’s not indestructible. Acids from foods and bacteria can eat away at it, causing erosion and cavities Where did these sugars come from? Why do the bacteria break them down? Bacteria that penetrate the soft inner tissue of the tooth produce gases that press against nerve ending in the tooth, resulting in a toothache. Do bacteria move? While most bacteria movement is passive, some bacteria can be propelled through liquid media by the action of a FLAGELLUM. Rigid, rotating filament of protein about 20 nm in thickness. Microbes and Society, Sigler Spring 2013 9 Bacteria can have single, tufted or several flagella over the cell surface. A-Monotrichous B-Lophotrichous C-Amphitrichous D-Peritrichous An E. coli flagellum can propel the bacterium about 2000 times its body length in an hour. Equivalent to a person walking 2.25 miles per hour. Schematic of the structure of a flagellum. From: http://www.sedin.org/pics/flglm-lg.jpg Microbes and Society, Sigler Spring 2013 10 Cytoplasmic structures (these are the ones inside the cell) Know: Chromosome, plasmid, ribosome, endospores and associated structures. The cytoplasm is essentially a soup of various compounds such as carbohydrates, lipids and nucleotides. CHROMOSOME – closed loop of DNA Only one chromosome is present in bacteria Contains thousands of genes (4000) How many pairs of chromosomes do humans have? How many genes? What is the function of the chromosome? Some bacteria contain tiny loops of DNA that are separate from the chromosome called PLASMIDS. Contains genes that encode nonessential (under normal conditions) cell functions. 1. Antibiotic resistance 2. Contaminant degradation Microbes and Society, Sigler Spring 2013 11 Plasmids can be swapped between bacteria. What structure can mediate this? This is called gene exchange. RIBOSOMES are made of proteins/nucleic acids and link amino acids together to make polymers called peptides. Eventually, the peptide chains become proteins when they fold in specific configurations. Conceptual drawing of the shape of the ribosome. Microbes and Society, Sigler Spring 2013 12 mRNA Multiple ribosomes translating a mRNA strand (arrow) to make proteins. Can you tell which direction the ribosomes are moving? Growing peptide chain that wil eventually become a protein. Ribosome…you can’t see it. ENDOSPORES (spores) are structures that can confer a high degree of stress tolerance. Members of the genera Bacillus and Clostridium can produce endospores, but not all bacteria have this ability (not E. coli) Endospores A stained preparation of Bacillus subtilis showing endospores (green) and the vegetative cell (red). From: Wikipedia. Vegetative cells Spores contain a chromosome (DNA), two cell membranes, cortex, spore coat, and a surrounding cell wall (exosporium). The cortex gives the spore strength and rigidity, what do you think it is made from? Spores are storage units (like “seeds”) of bacteria. Microbes and Society, Sigler Spring 2013 13 Spore extremes: Spores are likely the most resistant forms of life known. 1995 - Researchers isolated intact spores from a 25 million year-old fossilized bee in a piece of amber. 2001 – Researchers revived Bacillus spores from within salt crystals that were 250 million years old. Some spores can be boiled for two hours or left in alcohol for 20 years and still remain viable. The ability of bacteria to form spores provides a basis for certain forms of bioterrorism. Bacillus anthracis, the bacteria that causes anthrax, is a popular bacterium for these purposes. Microbes and Society, Sigler Spring 2013 14 Bacillus anthracis (vegetative) B. anthracis spores Once in an environment that is suitable for growth, the spores will germinate, become vegetative cells, and potentially infect. Spores enter the body through: Ingestion - Progresses to sepsis (25 – 65% mortality if not treated) Inhalation - Macrophages engulf the inhaled endospores and transport them to lymph nodes. Progresses to meningitis (100%) Cutaneous (skin) contact – Ulcer at site of infection. Rapidly progresses to necrosis. (20%). What route of exposure might terrorist groups choose to use when weaponizing B. anthracis? Growing B. anthracis is easy, but weaponizing it is difficult. Microbes and Society, Sigler Spring 2013 15 Aerosolization is a difficult process. What might make aerosolization difficult? U.S. and Soviet bioweapons specialists discovered that adding silica particles to germ powders made them easier to disperse. Illustration by C. Cain, adapted from S. Jacobsen. By alleviating the clumping, aerosol dissemination would be much easier to achieve. Eliminating clumping is likely the main obstacle preventing more widespread use of B. anthracis as a bioterrorism weapon. If released, it would only take kilogram quantities of spores to cover a 100 square km are and cause 50% mortality. Microbes and Society, Sigler Spring 2013 16 In 1979, an unintentional release of anthrax spores occurred in the former Soviet Union at a biological weapons facility. It was reported that 94 cases of anthrax occurred among citizens living near the facility resulting in 64 deaths. It was estimated that less than one gram of Bacillus anthracis spores were released during this accident. For a detailed report, see http://www.anthrax.osd.mil/documents/library/sverdlovsk.pdf Bacteria reproduction (just a few words, more later) Bacteria reproduce by binary fission. (How do eukaryotic organisms reproduce?) Binary fission results in clones of cells (genetically identical). This process can take as little as 20 minutes for E. coli under good conditions. Bacteria are cultivated in the laboratory on materials called culture media. A culture medium is a solution of nutrients that encourage the growth of microorganisms What nutrients might be contained in culture media? Microbes and Society, Sigler Spring 2013 17 Two types of media exist: Broth – liquid form Gel form – contains agar Agar is a solidifying agent Growth media 1. Many nutrient media are general (nonselective), i.e., they support the growth of many types of bacteria. …but sometimes, one might wish to only grow certain species of bacteria. 2. Selective media contains compounds that inhibit the growth of some bacteria, while promoting the growth of others. E. coli growing on eosin methylene blue agar. Microbes and Society, Sigler Spring 2013 18 …but some bacteria are especially difficult to grow – fastidious bacteria. 3. Special nutrients are added to the medium to support the growth of fastidious bacteria – enriched medium. e.g. blood agar – general medium supplemented with red blood cells. Encourages the growth of streptococci (Streptococcus spp.) Hemolysis 4. Differential media Contains compounds that allow one to visually differentiate between bacteria. e.g. E. coli vs. coliform bacteria. Both types of bacteria live in the guts of warm-blooded animals. Diagnostic ability of the media is based on the activity of two enzymes: galactosidase – blue color when it breaks down galactose. glucuronidase – purple color when it breaks down glucuronide. Microbes and Society, Sigler Spring 2013 19 E. coli (purple) Coliform (blue) Prokaryotic diversity and spectrum The spectrum of prokaryotes How well do we understand the diversity and function of prokaryotes? The vast majority remain unknown. How many do you think have been identified? Photosynthetic bacteria (bacteria, not archaea) How does photosynthesis work and is it an advantage to the bacteria itself? Others? Photosynthesizers are autotrophic – they synthesize their own food (fix carbon) from CO2, and when they die, release it for other organisms (heterotrophs) to use. Autotrophs, such as photosynthetic bacteria, are considered primary producers and are important as the bottom members of the food chain. Microbes and Society, Sigler Spring 2013 20 Cyanobacteria contain green pigments and often become dense in lakes, oceans and swimming pools during cyanobacterial blooms. Satellite photo of a cyanobacteria plume in western Lake Erie, 2003 (left). Glass of lake water containing Microcystis spp. (right). Photosynthetic bacteria are some of the most independent organisms on Earth. Why? They can take carbon (from CO2) and nitrogen (from N2) in the air we breathe and convert it to structural C and N. Why are C and N important for bacteria? …also usable by other organisms when the cyanobacteria die. This unique behavior allows them to inhabit harsh environments. Endolithic cyanobacteria. Microbes and Society, Sigler Spring 2013 21 Archaea Look like bacteria, but are actually not bacteria in the traditional sense. 1. Archaeal cell walls do not contain peptidoglycan 2. Archaeal cell membranes contain unusual lipids. But more interesting…Archaea tend to live in harsh environments – extremophiles 1. An example is the thermoacidophiles, which live under acidic and hot conditions. Sulfolobus acidocaldarius 85o C, pH 1.0 (this is the pH of sulfuric acid) Microbes and Society, Sigler Spring 2013 22 2. The most heat resistant Archaea known is Pyrolobus fumarii. These live near in thermal vents (underwater volcanoes) in the ocean bottom. Distribution of thermal vents in the world’s oceans. Grow well at temperatures up to 113o C, but anything below 90o C is too cold. What makes these Archaea so heat resistant? The key to thermal stability is keeping proteins and membranes operating efficiently. Waxy substances present in membranes and a high proportion of sulfurcontaining proteins help the Archaea to resist the heat. Certain amino acids contain sulfur and these sulfur atoms make strong bonds that keep the proteins from denaturing. Methionine Microbes and Society, Sigler Spring 2013 Cysteine 23 3. Methanogens are a specific type of Archaea. They produce ???? Live in anaerobic environments…remember when we talked about landfills? Methanogens are autotrophs (they don’t use organic carbon). Instead they use hydrogen (H2) and CO2 to gain energy and produce CH4. 4. Another example of Archaea is the extreme halophiles, which live under high salt concentrations such as those in the Great Salt Lake (UT). Many extreme halophiles actually require high concentrations of salt to grow and are not viable in low-salt medium. At least ~9% NaCl with an optimum between 12-23% NaCl Maximum at 32% NaCl. What environments will facilitate such a high salt concentration? Freshwater lake = 0.05% Ocean = 3.5% What conditions will achieve 9% salinity? Hint: the Dead Sea is ~15% salinity Microbes and Society, Sigler Spring 2013 24 Images of the Dead Sea Why does high salinity represent such an extreme environment? Water will move to equilibrate a chemical gradient. In a high salt environment, osmotic forces will pull water out of the cell, dehydrating it if nothing is done to prevent it. …then how do extreme halophiles adapt to such harsh conditions? Extreme halophiles accumulate inorganic ions inside the cell at concentrations that match the salt concentration outside the cell. Sodium, calcium, potassium. Microbes and Society, Sigler Spring 2013 25 In Halobacterium salinarum, a pump in the cell membrane transports high amounts of potassium inside the cell so that the concentrations of potassium is inside and outside of the cell are equal. In this case, potassium serves as the compatible solute. Outside K+ What’s this? Inside What material is this pump made from? With these adaptations, the extreme halophiles have evolved to live in environments where most other organisms die. How does this benefit the halophile? Microbes and Society, Sigler Spring 2013 26