Microbiology Skriptum PDF

cfljwn V orlesung 2024 MIKROBIOLOGIE S ommersemester Isabella Moll Skriptum  Microbiology Microbiology is the scientific study of microorganisms, those be...

cfljwn V orlesung 2024 MIKROBIOLOGIE S ommersemester Isabella Moll Skriptum  Microbiology Microbiology is the scientific study of microorganisms, those being unicellular, multicellular, or acellular. Microbiology encompasses numerous sub-disciplines including virology, bacteriology, protistology, mycology, immunology, and parasitology. Microbes are everywhere…. Bacteria… - are the origin of all forms of life - are phylogenetically more diverse than plants or animals - compose the majority of biomass on earth - grow everywhere if water is available, mainly under the earth’s surface - are essential for the food chain - affect climate - cause diseases - are symbionts with animals, human and other microbes - are simply essential for us! How do Microbes impact human life? Pathogenic microorganisms have greatly affected human populations throughout our existence Infectious diseases were the leading cause of death in 1900, whereas they are less significant today. Causes of death Infectious diseases: the leading killers Microorganisms impact human life and health Most microbes are beneficial, not harmful!  - Products of Industrial Microbiology o wine - Modern Agriculture o The mutualistic symbiosis between Nitrogen-fixing bacteria and leguminous plants. o The cow as an example of a ruminant animal - Industrial Microbiology - Recombinant DNA Technology: o Synthesis of Insulin Milestones of Microbiology Antoni van Leeuwenhoek A "natural philosopher" How it all began! (1632-1723) He was fascinated by small things and started making microscopes and was already able to see bacteria, which he imaged and traced Louis Pasteur ‘Father of Microbiology’ (1822-1895) The Defeat of Spontaneous Generation: Pasteur’s Swan-Necked Flask Experiment He was the first to demonstrate that infectious diseases are caused by microbes, disproved the concept of spontaneous generation (the idea that microbes could appear out of nothing), developed the process of pasteurization (as well as being its namesake), and developed some of the world’s first vaccines: splenic fever (Bacillus anthracis) rabies (Rabies lyssavirus) Robert Koch ‘Father of Modern Bacteriology’ (1843-1910) Koch’s Postulates for Proving Cause and Effect in Infectious Diseases He discovered that bacteria can cause disease and proposed a universal method to test this. For his work on the infection disease tuberculosis, he received the Nobel Prize. Because of Koch’s work, anthrax, tuberculosis, and cholera are now manageable Robert Koch diseases.  The diversity of microorganisms The phylogenetic tree Three domains of organisms: the Bacteria and the Archaea, and the Eukarya. In each domain, only a few groups of organisms are plotted. Hyperthermophiles are prokaryotes that reach their optimal growth at a temperature of 80 °C or higher. The groups highlighted in red are macroorganisms. All other organisms in the phylogenetic tree are microorganisms. Ribosomal RNA (rRNA) gene sequencing and phylogeny 1) Cells, either from a pure culture or a sample taken from nature, are disrupted. 2) The gene encoding the rRNA is amplified by the polymerase chain reaction (PCR) method. 3) The gene is sequenced. 4) The sequences obtained are strung together by computer. An algorithm calculates pairwise comparisons and generates a tree 5) that analyzes the differences in rRNA sequence between organisms. In the example given, the sequence differs in the following: Organism 1 versus Organism 2, three changes; 1 versus 3, two changes; 2 versus 3, four changes. Thus, organisms 1 and 3 are more closely related than organisms 2 and 3 or 1 and 2. The size of microorganisms Ø: 0,2µm - 50µm The ratio of surface area to volume in cells. As the size of the cell increases, the ratio of surface area to volume decreases. The metabolism of a cell is inversely proportional to its size. Formula: Surface divided by volume Biggest bacterium: Thiomargarita magnifica: >9000µm  The hallmarks of cellular life Morphology Representative cell shapes (morphologies) in prokayotes. Next to each drawing you can see a phase-contrast image of a model organism with the respective morphology. Structure of a Bacteria Prokaryotic vs. eukaryotic cell Characteristic Prokaryote Eukaryote Chromosome Single circular Paired linear Chromosome location Nucleoid (no membrane) Nucleus (membrane present) Nucleolus Absent Present Extrachromosomal DNA Plasmid Mitochondria and Chloroplast Site of Respiration Cell membrane Mitochondria Ribosomes 30S & 50S /70S 40S & 60S 780S in cytoplasm (70S in organelles) Locomotion Rotating flagella and gliding Undulating flagella and cilia amoeboid movement Pili Sex or attachment Absent Cell wall Peptidoglycan layer Usually absent  Gram staining Hans Christian Joachim Gram (1853-1938) - Staining of bacteria with a basic stain, like crystal violet - The iodine solution is added to form a crystal violet iodine complex; all cells continue to appear blue - The organic solvent such as acetone or ethanol, extracts the blue dye complex from the lipid-rich, thin walled gram negative bacteria to a greater degree than from the lipid poor, thick walled, grampositive bacteria. The gram negative bacteria appear colorless and gram positive bacteria remain blue. - counter stain (safranin): The red dye safranin stains the decolorized gramnegative cells red/pink; the gram-positive bacteria remain blue. Cell wall structure Gram-positive: have a thick peptidoglycan layer, but no outer membrane Gram-negative: have only a thin peptidoglycan layer, but an outer membrane Murein / Peptidoglycan The backbone for the peptidoglycan is formed by N-acetylglucoseamine (NAG) and N-acetylmuramic acid (NAM) The peptidoglycan is actually a very fine mesh of the components. Protects and maintains the shape of the cell. Without this net, bacteria would only occur in an isotonic solution. In a hypertonic or hypotonic solution, they would burst due to the osmotic pressure. Synthesis Penicillin-binding proteins (PBPs) Catalyze the polymerization of NAG and NAM (transglycosylation) and the crosslink between the peptide bridges (transpeptidation). It is called penicilin-binding protein because its structure looks like penicilin. Penicilin works by integrating itself precisely into this binding site, thus interrupting peptidoglycan synthesis and preventing the bacteria from multiplying. Lysozyme also breaks precisely this bond. Archaea: Pseudopeptidoglycan and S-Layer Similarity to the structure of peptidoglycan, especially the peptide crosslinks, in this case between N-acetyltalosaminuronic acid residues (NAT) in place of muramic acid residues. NAG, N-acetylglucosamine. The S-layer of the prokaryote Aquaspirillum serpens is shown (a member of the Bacteria); this S-layer, like many of the S-layers found in the Archaea, exhibits hexagonal symmetry.  Outer membrane of Gram-negative bacteria The gram-negative envelope contains hydrophobic lipopolysaccharides. Ca2 + ions are required to maintain stability of the lipopolysaccharide layer. This membrane is asymmetrical. Gram-positive bacteria Thick cell wall (20-80 nm) of several peptidoglycan layers with Teichoic acids: polymers of the cell and membrane - Polymers made of glycerophosphat and ribitolphosphat moieties. - Interaction of bacteria with eukaryotic host cell Cytoplasmic membrane Symetric bilayer, „fluid mosaic model“, Phospholipid Bilayer Membrane Homeoviscous Adaptation: adaptation of composition of lipid bilayer to environmental conditions to maintain membrane fluidity. Three major import mechanisms: - Passive diffusion o Small molecules and water - Facilitated diffusion o through channels - Active transport o selectively with energy input Import Passive diffusion Especially for water, glycerol, ethanol, CO2 and O2. An example for facilitated diffusion are proteins → aquapurine Transport Simple transport: Ion transport by specific transporters in the inner membrane Group translocation: phosphotransferase system, e.g.: glucose, mannose, mannitol, fructose - Specific: requires selective transporter. - Substrate is chemically modified - requires energy (phosphoenolpyruvate) - Accumulation in the cytoplasm Further ultramicroscopic characteristics of bacteria Surface structures: Flagella can occur at only one pole as well as on the entire surface of the bacterium they are anchored inside the cell where they are driven by a motor the rotation cannot be changed in speed but only in direction Peritrichous - flagella distributed over the entire cell surface Monotrichous and polar - a flagellum at the pole of the bacterium Lophotrichous and polar - several flagella at one pole of the bacterium Amphitrichous and polar - there is a flagellum at both poles of the bacterium Chemotaxis movement pattern of a bacterium Fimbriae Fimbriae serve to mechanically attach and anchor cells to surfaces. This adhesion takes place at specific receptors of the host cell surfaces (e.g. mucous membranes of the intestinal, respiratory or urinary tract) and enables the colonization of the host and thus its infection by the pathogen. Conjugation: Pili are used to transfer genetic material between bacteria. Capsules Capsules are mostly composed of polysaccharides (building blocks e.g. glucose, rhamnose, uronic acids and other sugar derivatives). The capsule of species of Bacillus is composed of protein (poly-Dglutamic acid). Capsule-forming strains grow on solid culture media with large, slimy colonies. Capsules are not necessary for growth, but are very important for survival under unfavorable conditions and determine virulence characteristics in many pathogenic forms. Inclusion body: Sulfur globules Sulfur-oxidizing bacteria can convert intracellular sulfur that is stored sulfur globules, which are strongly refractive under the light microscope.  Some gram-positive bacteria can form endospores Bacteria do not divide in the middle, but in a small area of the cell and the mother cell also surrounds the spore  The hallmarks of cellular life The machine and the coding functions of the cell. In order for a cell to self-replicate, (1) there must be a sufficient amount of energy as well as precursorsfor the synthesis of new macromolecules, (2) genetic material must be replicated so that each cell can obtain a copy after cell division, and (3) gene expression must occur (the processes of transcription and translation) to form the proper amount of proteins and other macromolecules necessary to create a new cell. Information is stored in the DNA A distinction is made here between RNA and DNA. These consist of the pyrimidine bases: cytosine (DNA & RNA), thymine (DNA), uracil (RNA), and the purine bases: Adenine (DNA & RNA) and guanine (DNA & RNA). The RNA consists of a ribose, a phosphorus residue & a base. The DNA consists of a deoxyribose, a phosphorus residue and a base. RNA is predominantly single-stranded, which makes it much more unstable. It is assumed that RNA was the original variant of information storage. DNA is present as a double helix. The two strands run antiparallel. On the outside is the sugar-phosphate backbone. On the inside there are base pairs which are arranged in a ladder are ladder-shaped. A & T and C & G always pair up via hydrogen bonds. A & T form 2 hydrogen bonds and G & C form 3 hydrogen bonds. Therefore, A&T rich regions can be better cleaved. This is also where the transcription mechanism. One turn of the helix contains 10 base pairs and is 3.4nm long. This structure is used to protection of the genetic information. But RNA can also form secondary structures for protection. The 5' & 3' ends were named after the C atoms on which the phosphate or the hydroxyl group is located. Supercoiled DNA and DNA Gyrase In bacteria the chromosome is a circular one. The entire chromosome is organized in 100 minutes. Time is to transfere the entire chromosome from on cell to another cell. DNA is negative supercoiled. Gyrase: introduces negative supercoiling, DNA Topoisomerase: removes supercoiling Replication of Circular DNA During replication there is a origin of replication. It is a specific sequence on the chromosome. Two strands of replication an each side of the fork DNA replication The DNA-polymerase catalyses the nucleophilic attack and thus the phospho-di-ester linkage Structure of the DNA chain and the mechanism of growth by attachment of deoxyribonucleoside triphosphate at the 3' end of the chain. Growth proceeds from the 5'- phosphate to the 3'-hydroxyl end. DNA polymerase catalyzes the tethering reaction. The four deoxyribonucleosides that serve as precursors are deoxythymdine triphosphate (dTTP), deoxyadennosine triphosphate (dATP), deoxyguanosine triphosphate (dGTP), and deoxycytidine triphosphate (dCTP). After insertion of a nucleotide, the two end phosphates of the triphosphate are cleaved off as pyrophosphate (PPi) In contrast to the leading strand, on which synthesis is continuous, the DNA fragments (Okazaki- Fragments) must be joined on the lagging strand. (a) DNA polymerase III synthesizes the DNA in the 5′→3′ direction, it moves in the direction of the RNA primer of a previously synthesized fragment on the next strand. (b) When the DNA polymerase III reaches this fragment, it is replaced by Pol I. (c) DNA polymerase I continues to synthesize DNA while the RNA primer is removed from the preceding fragment. (d) The DNA ligase joins the two fragments. (e) The finished product, a complementary DNA double strand. Processes at the replication fork of the nucleoid Note the polarity and the antiparallel nature of the DNA strands. The helicase unwinds the DNA and the primase adds the RNA primers. The replisome consists of two copies of DNA polymerase III, helicase and primase (which together form the primosome) and many copies of the single-stranded DNA binding protein. Just upstream of the replisome, DNA gyrase removes the over-spiralization in the DNA to be replicated. Note how the downstream strand forms a loop to keep the components of the replisome away from each other and ensure that they continue to move forward unimpeded. Transcription (DNA → RNA) Steps in RNA synthesis The initiation and termination sites are specific nucleotide sequences on the DNA. The sigma factor allows RNA polymerase to recognize the initiation sites (the promoter). The sigma factor is released during elongation. The RNA polymerase moves down the DNA chain, resulting in a temporary opening of the double helix and transcription of one of the DNA strands. When a termination site has been reached, then chain growth ceases and the mRNA and polymerase are released. The RNA polymerase The RNA polymerase consists of five subunits with the designations β, β′, α, ω and σ, whereby α is present in two copies. The subunits interact and form the active enzyme, called the RNA polymerase holoenzyme, to which the sigma factor is not bound as tightly as the others. This leads to the formation of the RNA polymerase core enzyme α2ββ′ω. The core enzyme synthesizes the RNA alone, while the sigma factor recognizes the appropriate site on the DNA, the promoter, to start RNA synthesis (the sigma factor dissociates from the holoenzyme after a short RNA has been synthesized). Termination Termination of transcription. Reverse repeat sequences followed by a stretch of T’s in transcribed DNA lead to the formation of a stem-loop structure in RNA, which can result in the termination of transcription.  Translation (RNA → Protein) Catalysed by the ribosome Ribosome are divided into two subunits. In pokaryotes, the small UE is 30S in size and the large UE is 50S in size, making a total of 70S. Adapter molecule: charged tRNA The RNA is converted into proteins. Three nucleotides each code for one amino acid. This is translated by ribosomes. In contrast to eukaryotic mRNAs, prokaryotic mRNAs can have several open reading frames in addition to one --> polycistronic RNA. The ribosome starts at a start codon (AUG-methionine). The adapter molecule that carries out the transcription is the tRNA. This is loaded with an amino acid and reads the codon with the anticodon. Possible reading frames of an mRNA An internal sequence of an mRNA is shown. (a) The encoded amino acids in the correct reading frame (referred to as the "0 reading frame"). (b) The amino acids that would be encoded by this region of the mRNA in the "-1 reading frame". (c) The amino acids that would be encoded if the ribosome reads the "+1 reading frame". The Genetic Code The sequence of nucleotides in the mRNA molecule is read in consecutive groups of three. RNA is a linear polymer of four different nucleotides, so there are 43 = 64 possible combinations of three nucleotides. However, only 20 different amino acids are commonly found in proteins. Thus the code is redundant and some amino acids are specified by more than one triplet. And three codonsspecify the stop of protein synthesis. Translation process  Amino acids, the building blocks of proteins Structure of polypeptides Primary Structure Secondary Structure Tertiary Structure Quaternary Structure  Bacterial growth: Binary Fission Bacteria grow by dividing into two parts. DNA replication takes place first. This is followed by cell elongation, whereby the cell grows in length. At the beginning of division, a septum forms, through which the cells continue to be connected. The separated cell walls are then completed. Exponential Growth: one cell becomes two, two become four, etc. 10 generations = 1000 cells. N=N0 * 2FtsZ is the protein that accumulates in the middle of the bacterium during septum formation & thus enables the two daughter cells to be cut off. The FtsZ ring is bent by GTP hydrolysis and then also pulled together. Growth of a population in a discontinuous Lag Phase = phase before exponential growth Log Phase = each cell in a population grows and divides Stationary Phase = growth stops because of nutrient limitations and the accumulation of toxic compounds produced by the cells during growth – growing and dying bacteria are present concomitantly Death Phase = the number of dying bacteria exceeds the number of growing ones How do you determine the number of bacteria? Viable count: a sample is taken from the colony at each time point, diluted, the solution is added to a medium and incubated overnight, the next day new colonies are formed which can then be counted and multiplied by the number of dilution steps → number of cells in one milliliter of the original colony Optical density: new medium appears clear, after adding the bacteria the liquid becomes increasingly cloudy, this turbidity can be determined with a photometer.. Dead cells also influence the result of the optical density. Total number of cells: Sample is dripped into a chamber containing a grid, the cells in the grid can then be counted, in order to distinguish between living and dead cells, they are stained Abiotic parameters affecting growth of microorganisms - Temperature - Osmolarity - pH - Oxygen supply - Water activity (Humidity) Growth temperature How do thermophiles and hyperthermophiles manage to survive at high temperatures? Their enzymes and other proteins are much more heat-resistant than those of mesophiles and actually develop their functional optimum at high temperatures. Surprisingly, studies of several thermostable enzymes have shown that their amino acid sequence from the more heat-sensitive forms of enzymes that catalyse the same reactions in mesophiles. Apparently, the substitution of amino acids in only a few places in the enzyme enables the protein to be heat resistant. The heat resistance of the "hot proteins" is also due to a larger number of ionic bonds between basic and acidic amino acids, and the often hydrophobic interior of the protein. pH and growth Despite bacteria can grow at different pH levels, the intracellular pH (pHi) is generally close to neutral! This can be achieved by - enzyme-catalyzed reactions that consume protons: eg.: decarboxylation of amino acids, such as glutamate, arginine - dedicated proton translocating efflux pump In addition to these mechanisms for maintaining pHi cells often deploy specific protective systems that help cope with acid stress. - Modification of the lipid composition of the cytoplasmic membrane to reduce the permeability to protons - cyclopropane fatty acids are produced at higher levels under acid stress and serve to protect against acid pH by decreasing membrane permeability to protons Osmolarity Compatible solutes When an organism is transferred from a medium with high water activity to one with low water activity, it can only obtain water from its environment by increasing its internal solute concentration, which can be achieved either by pumping dissolved substances from the environment into the cell or by synthesizing a solute. Such solutes must not inhibit macromolecules within the cell and are referred to as compatible solutes. In general, these substances are highly water-soluble sugars, alcohols or amino acid derivatives. Glycine betaine, an analog of the amino acid glycine, is widely used in halophilic bacteria. Other common compatible solutes are sugars such as sucrose and trehalose, dimethylsulfonium propionate, which is produced by marine algae, and glycerol, which is produced by several organisms, including xerophilic fungi, which grow at the lowest water potential of all known organisms. In the extremely halophilic archaea, such as Halobacterium, KCl is the compatible solute. Water activity (Moisture) Bacteria require a very high moisture to be able to grow. How high the water activity of an organism of an organism is indicated by the a w value. This ranges between 0 - 1. Gram-negative bacteria: 1 - 0.95 Gram-positive bacteria: below 0.95 Oxygen availability and growth Aerobic, anaerobic, facultative, microaerophilic and aerotolerant anaerobic growth (redox color resazurin as redox indicator, pink when oxidized and colorless when reduced). (a) Oxygen permeates only a short distance in the tube, so obligate aerobe growth occurs only on the surface. (b) Anaerobesthat are sensitive to oxygen grow better the further away from the surface. (c) Facultative aerobe grow everywhere, but better on the surface because they can breathe there. (d) Microaerophiles grow away from the strongest oxic zone. (e) Aerotolerant anaerobes grow everywhere in the tube. However, they do not grow better on the surface because these organisms can only ferment. How to prevent microbial gowth - Heat sterilization - Pasteurization - Ionizing radiation - Filtration - Chemical growth control - Desinfectants Heat sterilization (under pressure) The autoclave A large ‘pressure cooker’ In a chamber under pressure and heat everything is sterilized and all bacteria are killed. This treatment is not always useful, as Gram-positive bacteria, for example, can form spores that would not be killed by this method. For spores we have to do multiple autoclaving circles UV radiation Biosafety cabinet with shortwave UV light when not in use, sterilizing microbiological contaminants from irradiated surfaces. Filtration Is used for liquids. Different filters with different pore sizes can be used to filter out bacteria of different sizes. Lot of sugars Chemical growth control Example: - Phenols destroy all enzymes - Alcohols (60-85%) can be used to disinfect surfaces. - Hydrogen peroxide used as an oxidizing agent used for medical instruments Antibiotics: Natural compounds from different bacterial species (Penicilline) There are three types of antibiotics: Depending on their effect on the bacterial culture, antibacterial agents can be classified as bacteriostatic, bacteriocidal or bacteriolytic. Bacteriostatic agents are often inhibitors of protein synthesis and act by binding to the ribosomes. The binding is relatively weak and after removal, growth can often resume. Bacteriocidal agents - such as formaldehyde- bind firmly to their target site in the cell, are not removed by dilution and by definition kill the cell. However, the dead cells are not destroyed and the total cell count, which is reflected in the turbidity of the culture remains constant. Bacteriolytic agents kill the cells by lysing them, which naturally has an effect on the live cell count and the total bacterial count. An example of this is penicillin that affects synthesis of the cell wall. Determination of the minimum inhibitory concentration (MIC), the minimum bactericidal concentration (MBC) and the sensitivity (with a diffusion test) Problem: Antibiotic resistances Appearance of multi-drug resistant pathogens * Untreatable with conv. Antibiotics (ignore the key in the picture)  Mechanisms of antimicrobial drug resistance? Metabolism Schematic representation of anabolism and catabolism, highlighting the key role of ATP and the proton motive force in coupling these processes. Monomers can originate as pre-formed nutrients from the environment or from catabolic pathways such as glycolysis and the citric acid cycle. In many cases, monomers must be synthesized from intermediates of catabolism or from nutrients found in nature. These biosyntheses are also reactions of anabolism. Nutrient requirements for growth of microorganisms - Macronutriens: o C/N/P/S/H/K/Mg/Ca/Na/Fe - Micronutrients: o Trace elements Macronutriens Carbon, Hydrogen, Oxygen, Nitrogen, Phosphorus, Sulfur, Potassium, Magnesium, Sodium, Calcium, Iron Macromolecular composition of the cell Macromolecule percent of dry weight Proteins 55 Lipids 9,1 Polysaccharides 5,0 Lipopolysaccharides 3,4 DNA 3,1 RNA 20,5  Micronutriens - Vitamins These are usually contained in the media and therefore do not need to be added separately when breeding a colony. Distilled water is simply used as the medium. Culture media for bacteria A distinction is made between defined media and complex media. Defined media: specially composed for a specific strain Complex media: contain different macro- and micronutrients so that several different different organisms can grow. Nutrition and Metabolism of Microorganisms - Energy: ADP+ Pi → ATP - Metabolism: Production of the precursors of macromolecules (sugars, amino acids, fatty acids, etc.) - Enzymes: metabolic catalysts Energy storage during fermentation and respiration - Phosphoenolpyruvate - ADP - ATP - Glucose-6-Phosphat - Acetyl-CoA - Acetylphosphat Redox: electron-transfer from the reducing agent to the oxidant The electron tower. The redox pairs are arranged in such a way that the strongest reducing agents (negative reduction potentials) are at the top of the tower and the strongest oxidizing agents (positive reduction potentials) are at the bottom of the tower. When electrons are released from the top of the tower, they can be "collected" by the acceptors at different levels. The further down the electrons have fallen before they are caught, the greater the difference in reduction potential between the electron donor and electron acceptor, and the more energy is released. For illustration purposes, the differences in released energy are listed on the left side when a single electron donor, H2, reacts with one of the three electron acceptors, fumarate, nitrate and oxygen. Redox-Coenzyme Nicotinamidadenin-dinucleotid The oxidized version: NAD+ Reduced version: NADH Both variants occur in a coupled redox reaction. Schematic representation of an oxidation- reduction reaction. The oxidized and reduced forms of the coenzyme nicotinamide adenine dinucleotide, NAD+ and NADH, are involved in this reaction. 1. NAD+ binds with a substrate to the active center of an enzyme. 2. an enzyme-substrate complex is formed 3. the oxidized substrate and an NADH are released from the complex after the reaction. 4. the NADH can now bind with a substrate (electron acceptor) to another enzyme. 5) This results in another enzyme-substrate complex 6. after the reaction, the enzyme now releases the NAD+ and the reduced substrate 7. the cycle can then start again. Energy-rich compounds Energy storage during fermentation and respiration. (a) In fermentation, ATP synthesis occurs as a result of substrate-level phosphorylation; a phosphate group is attached to an intermediate in the bio-chemical pathway and is eventually transferred to ADP to form ATP. (b) During respiration, the cytoplasmic membrane releases some of the energy of the proton motor force for the formation of ATP from ADP and inorganic phosphate (Pi); this process is called oxidative phosphorylation. The coupling of the proton motor force to ATP synthesis occurs with the cooperation of a membrane protein-enzyme complex called ATP synthase (ATPase).  Glycolysis via the Embden-Meyerhof-Parnas pathway Anaerobic part of glycolysis. Glucose is absorbed & converted to glucose-6-phosphate by adding an ATP and splitting off an ADP → is converted to fructose-6-phosphate→ by adding an ATP and splitting off an ADP to fructose-1,6- phosphate→ this produces 2 glyceraldehyde-3-phosphates → see diagram→ 2 pyruvate are produced Homofermentative → glucose becomes lactate, only one end product is formed, glucose → lactate + 2 ATP Examples: Streptococcus, Enterococcus Heterofermentative → glucose becomes lactate and another metabolic product e.g. ethanol, glucose → lactate + ethanol + ATP Example: Lactobacillus, Leuconostoc Bacterial fermentation pathways - Alcoholic - Homolactic - Herterolactic - Propionic acid - Mixed acid - Butyric acid - Butanol - Caporate - Homoacetogenic - Methanogenic  The citric acid cycle or TCA cycle (tricarboxylic acid cycle) Energy gain via aerobic respiration The TCA cycle begins when the 2-carbon compound acetyl-CoA (formed from pyruvate) combines with the 4- carbon compound oxaloacetate to form the 6-carbon compound citrate. Through a series of oxidations and transformations, the 6-carbon compound is eventually transformed back to the 4-carbon compound oxaloacetate, which then begins another cycle, binding the next acetyl-CoA molecule. The final balance of fuel (NADH/FADH) for the electron transport chain and CO2 formed by the TCA cycle, is listed. NADH and FADH fuel the electron transport chain complexes. Generation of the proton motive force (PMF) by chemiosmosis Bacteria possess membrane-bound electron transport chains that all result in net proton translocation across the cytoplasmic membrane during oxidation-reduction reactions. During respiration, electrons are released from NADH and FADH2 and eventually are transferred to O 2, forming H2O according to the following overall reactions: NADH + H+ + ½O2 → NAD+ +H2O FADH2 + ½O2 → FAD +H2O Cytoplasmic membrane: site of generation and use of the proton motive force - ATP-Synthesis (oxidative phosphorylation) - Locomotion - Transport Aerobic respiration: The citric acid cycle Energy gain via aerobic respiration 1. Glycolyse 2. Citric acid cycle 3. balance sheet  Different forms of metabolism Chemoorganotrophic bacteria require organic compounds to produce energy. Chemolithotrophic metabolism is used by bacteria that generate energy by the oxidation of inorganic molecules such as hydrogen (H2), hydrogen sulfide (H2S), and reduced metals for biosynthesis or energy conservation via aerobic or anaerobic respiration Phototrophic metabolismus uses light as energy source to produce ATP. Here carbon is either gained from CO2 or other organic compounds Chemoorganotrophic metabolism: Aerobic respiration In aerobic respiration, electrons from glycolysis & the citrate cycle are channeled into oxidative phosphorylation → ATP synthesis Chemoorganotrophic metabolism: Anaerobic respiration - anoxic →no oxygen present - oxic → oxygen present  Chemolithotrophic metabolism Most of the chemolithotrophic bacteria use the Calvin-BensonBassham pathway for incorporation of carbon dioxide Ammonia oxidation and electron flow in ammoniaoxidizing bacteria. The reactants and reaction products are highlighted. Cytochrome c (cyt c) in the periplasm of the cell is a different isoform of cytochrome c from the membrane-bound form. AMO: ammonia monooxygenase; HAO: hydroxylamine oxido reductase; Q: ubiquinone. Oxidation of nitrite to nitrate by nitrifying bacteria. The reaction partners and reaction products of this reaction sequence are highlighted. NOR: nitrite oxidoreductase. Phototrophic metabolismus Cyanobacteria Oxygenic photosynthesis in cyanobacteria generally uses water as an electron donor and produces oxygen as a by-product. Carbon dioxide is reduced to form carbohydrates via the Calvin cycle. In most forms the photosynthetic machinery is embedded into folds of the cell membrane, called thylakoids. Purple sulfur bacteria Anoxygenic photosynthesis does not use water as reducing agent, and so does not produce oxygen. Instead, hydrogen sulfide is oxidized to produce granules of elemental sulfur. They use bacteriochlorophyll and carotenoids to obtain the light energy for photosynthesis. In most forms the photosynthetic machinery is embedded in the inner membrane. Purple non-sulfur bacteria obtain their energy from sunlight and carbon from organic material and not carbon dioxide. Phototrophic metabolism How can bacteria adapt to environmental changes? Bacteria must be able to perceive their environment. They must be able to move in the direction of food sources. They must also be able to adapt to their environment. Regulation of gene expression Mechanisms that are used for regulation of gene expression. The product of a gene A is an enzyme A, which in this case is synthesized constitutively and carries out its reaction. Enzyme B is also synthesized constitutively, but its activity can be inhibited. The synthesis of the product of gene C can be prevented by control at the level of translation. The synthesis of the product of gene D can be prevented by control at the level of transcription. Control of mRNA synthesis (Transcription) In prokaryotes, the mRNA polymerase is composed of a core enzyme and a sigma subunit. The core enzyme itself consists of 4 subunits. β-subunit, β'-subunit, α-CTD & α-NTD. The α subunits are responsible for the interaction with the DNA sequence. The sigma factor is also important for the interaction with the DNA. Transcription 1. the mRNA polymerase is brought to the promoter with the help of the sigma factor. Here, the α-subunits also interact with the promoter. 2. this leads to an open complex. This means that a “bubble” is formed in an area of the DNA. → Double helix is opened. 3. transcription can now take place at the open site. 4. at the start of synthesis, the sigma factor is released because it is only needed to initiate transcription. After release, it can contribute to a new transcription. 5. synthesis continues until termination. This is protein-independent. By forming a hairpin & a poly-U-tail, the polymerase can be moved away from the DNA. After transcription, prokaryotic RNAs have a triphosphate 5'-end to protect the mRNA. Functionally comparable to the CAPS of eukaryotic RNAs. In the 5' region, a non-translated region → 5'UTR is located directly after the triphosphate. Before the start codon is the Shine-Dalgarrio sequence, which interacts with the ribosome & therefore serves for the recruitment of the ribosomes in the start region. Between Start & Stop is the open reading frame→ ORF1. After the stop codon (UAG, UAA, UGA) is the 3' untranslated region. In contrast to eukaryotes, there are polycistronic mRNAs in pokaryotes. This means that there can be several open reading frames on one mRNA. Proteins that bind to the promoter A simple way to control transcription is to use proteins that bind directly to the promoters. An operon sequence leads to the synthesis of an RNA that codes for several genes. The advantage of such an arrangement is that genes can be switched on and off simultaneously, → they are coregulated. It also indicates that the genes are functionally connected. lac-Operon Lac operon thus means→ all genes occurring in this sequence (operon) are required to absorb lactose (lacY), to break it down (lacZ) & to modify/degrade it (lacA). lacZ encodes the β-galactosidase (cleaves lactose into glucose and galactose) lacY encodes the permease for lactose uptake lacA encodes the galactoside acetyltransferase lacI is the repressor These individual genes can be switched on and off depending on the presence of lactose. This works thanks to a repressor (with lactose it would be lacI). These dimerize & bind to palindromic sequences. If no lactose is present, the repressor binds to the lac operator. Now the RNA polymerase can bind, but cannot transcribe any further. Induction If lactose is present, it binds directly to the repressor and allosteric regulation occurs. As a result, the repressor can no longer bind to the lac operator or falls away from it. The repressor does not always bind 100% to the operator. This ensures that, for example, lacY is still transcribed in lactose despite the absence of lactose. Without lacY, no lactose could be taken up by the cell. The growth phase and the total proteins increase in relation to each other. The blue line represents β-galactosidase. This shows how little of it is present in the cell without lactose. It only increases rapidly when lactose is added. The same mechanism can also be used to react to the presence of a molecule. Repression Arg-Operon argC , argB and argH encode enzymes of the arginine biosynthesis pathway The genes of the arg operon code for the enzymes involved in arginine synthesis. If enough arginine is available, the cell does not need to synthesize any new enzymes. When arginine is present, it immediately binds to the repressor. This complex then binds to the arg operator and stops transcription. The growth phase and the total proteins increase in relation to each other. In the absence of arginine, the enzymes for arginine biosynthesis are transcribed. As soon as arginine is present, synthesis ceases. A protein does not only have to serve as a repressor, but can also be used as an activator Induction by an activator Maltose operon malE, malF and malG encode subunits of the ABC- transporter required for maltose uptake The activator binding sites recruit the RNA polymerase. A maltose activator protein is required for binding. In the absence of maltose, the RNA polymerase cannot bind to the DNA and therefore cannot code for the ABC transporter. Only when maltose is present can maltose, maltose activator protein & RNA polymerase bind to the activator binding site and carry out transcription. The growth phase and total proteins increase in relation to each other. The blue line shows how little of it is present in the cell without maltose. Only when maltose is added does the concentration of expression products increase rapidly.  What happens if bacteria grow in the presence of two sugars? Diauxic growth …is the phenomenon whereby a population of microbes, when presented with two carbon sources, exhibits bi-phasic exponential growth intermitted by a lag-phase of minimal growth. The uptake of various sugars must be regulated. When a bacterium grows in a solution of glucose, lactose & mannose, it will absorb the nutrients in the above order. This uptake must be coregulated. One way to do this would be: Catabolite repression If glucose is present, the phosphotransferase system takes up and phosphorylates the glucose via group translocation (uptake of a group of sugars & phosphorus transfer). This enzyme is also a regulator protein. catabolite activator protein (CAP) acts as a glucose sensor. It activates transcription of the operon, but only when glucose levels are low. CAP senses glucose indirectly, through the "hunger signal" molecule cAMP. If glucose is present, then cAMP is low. different σ-factors There are seven different sigma factors. The sigma factors bring the polymerases to the promoter region. σ-70 is a promoter for the housekeeping genes, regulates genes that are always needed. σ-S is a promoter for stationary phase & stress factors σ-H is a promoter that is switched on during heat stress The advantage of regulating gene expression of sigma factors is that whole groups of genes can be controlled. However, this type is slower & more general compared to repressor/activator regulation. The highest affinity of the sigma factors is σ70, as this is required for general transcription. How are σ-factors exchanged? Stringent response! ‚Alarmone‘ (p)ppGpp or ‚the magic spot‘? In 1969, Cashel and Gallant identified a ‘magic spot’ (MS) that appeared on a chromatogram (left) made from bacteria that had been starved a key nutrient. This magic spot was later identified as ppGpp and shown to influence the expression of over 500 genes in response to stress. An innovative crystallization technique now has allowed researchers to determine the structure of ppGpp in complex with bacterial RNA polymerase and DksA (upper right). If there are too few amino acids, the EF-tu cannot bring a charged tRNA to the ribosome and then a tRNA without amino acids sits there. The ribosome stops as a result. This makes it a signal for RelA. RelA is the synthetase that creates → ppGpp from GTP/GDP + ATP. This removes the σ-70 factor from the polymerase & downregulates all synthesis pathways that are under this regulation. The amino acid biosynthesis is switched on by ppGpp. Other proteins are broken down into their individual parts to create new amino acids.  σ-factors in E. coli In response to amino acid starvation, RelA catalyses the synthesis of ppGpp. Together with DnaK suppressor (DksA), ppGpp directs transcription initiation at particular gene promoters through direct interaction with RNA polymerase. In part, ppGpp and DksA act by promoting the interaction of RNA polymerase with alternative σ-factors (σ*), such as σE. Signal Transduction and Two-Component Regulatory Systems This describes how signals from the environment can enter the cell and be transported further. Sensor Kinases and Response Regulators The response regulator can be almost anything. Here, for example, a repressor protein. Function: As soon as a molecule binds to a sensor kinase, the sensor kinase is phosphorylated at the cytoplasmic membrane. The sensor kinase has two domains on the cytoplasmic side. One domain is a histidine→ this is phosphorylated, and the other domain is the active center that carries out the phosphorylation. The released phosphorus residue now reacts with the response regulator. Only under these conditions can the response regulator then bind to the operator. → Allosteric effect. The response regulator has autophosphatase activity & can therefore dephosphorylate itself. Examples  Bacterial communication: Quorum sensing Presence of other bacteria Some actions of bacteria are designed in such a way that they only make sense if a whole colony of bacteria performs the same action. Example: Glow of dwarf squid It is not the squid that glows, but an organ colonized by bacteria. → Vibrio fischeri (Gram-negative). These joint actions can only be carried out at high densities. This happens with the release of certain molecules. luxICDABE→ operon for biofluorescence luxI→ codes for the LuxI synthetase, which synthesizes the acyl homoserine lactone, these molecules simply diffuse outwards, every bacterium in the colony does this simultaneously. Only when a certain concentration is reached can they diffuse back into the cell. Then they bind to an activator protein, LuxR can then bind to the promoter & the whole operon is transcribed. This imitates light. PAO1 QS systems involved in virulence gene expression Transcriptional Attenuation: amino acid biosynthesis Termination of transcription by small open reading frames upstream of the gene. Mainly used in the synthesis pathways of amino acids. Excess tryptophan: transcription terminated: Leading sequence is read through quickly, translation continues, a hairpin is formed→ Termination of transcription. Tryptophan deficiency, transcription continues: Leading sequence, no tryptophan present, ribosome stops, an anti-terminator loop forms behind the ribosome, The genes can be transcribed.  Regulation of protein synthesis / Translation The advantage of carrying out the regulation only during translation is that this mechanism can be activated much faster. This allows the cell to react more quickly to its environment. 1. Start codon 2. SD 3. SD AUG 4. rProtein S1 5. Secondary structures 6. Sequences surrounding the TIR 7. Translational repressors 8. Riboregulation 9. Other control mechanisms Translation control starts at initiation. The first ribosome that recognizes the start codon comes in the form of the 30S subunit. Binds to the mRNA using a sequence SD & aSD, just before the start codon. The first tRNA is positioned at the AUG by initiation factors. When the initiation factors are released, structural changes occur and the 50S unit binds to the 30S subunit and thus encloses the mRNA. → 70S initiation complex The translation of a particular mRNA can be inhibited, depending on whether a protein or a molecule is present, the structure of the binding site can change & the ribosome can no longer bind. A protein can also bind directly to the ribosome site and thus block the ribosome. Regulation by mRNA structure RNA thermometer zipper for heat shock control in Escherichia coli The sigma factor for heat stress is transcriptionally regulated. Its ribosome binding site and the mRNA form an inhibitory structure under low temperature. → Ribosome cannot bind (left figure) At a temperature of 42° this secondary structure melts and the ribosome can recognize and bind it.  Riboregulation: Regulation by small regulatory RNAs Instead of a secondary structure, a small RNA can also interact with the translation initiation region and either block translation (negative effect) or form an alternative structure (positive effect) Genes encoding trans-encoded antisense sRNAs (red) are located separate from the genes encoding their target RNAs (blue) and only have limited complementarity. Trans-encoded sRNA can act negatively by base pairing with the 5’ UTR and blocking ribosome binding (left panel). Trans-encoded sRNA can act positively by preventing the formation of an inhibitory structure, which sequesters the ribosome binding site (RBS) (right panel). Riboswitch: small molecule sensing RNAs Thiamine pyrophosphate (TPP) is a thiamine (vitamin B1) derivative and functions as a coenzyme for decarboxylase enzymes, e.g. such as pyruvate dehydrogenase, a-ketoglutarate dehydrogenase, transketolase, thus it is a key factor for carbon metabolism. The TPP riboswitch consensus sequence, like the coenzyme itself, appears in all three domains of life. The riboswitch controls both transcription and translation. Structures in the mRNA that either open or close the translation region. If the signal molecule is not present, then the ribosome binding site is open and translation can take place. If the signal molecule is present &and binds, an alternative secondary structure is formed and the ribosome binding site closes, so that translation is no longer possible. Enzyme B is also synthesized constitutively, but its activity can be inhibited. The enzyme is formed but modified or activated or inactivated by binding a molecule.  Post-translational Regulation: Allosteric feedback inhibition at the enzyme level Feedback inhibition of enzyme activity. The activity of the first enzyme in the pathway is inhibited by the end product, thereby controlling the formation of the end product. If an allostric factor binds, the substrate cannot bind→ Inhibition: Inhibition of the enzymatic reaction. If the allosteric factor is not present, the substrate can bind→ Activity: The enzymatic reaction takes place. Post-translational Regulation: Covalent modification of enzymes Assimilation of ammonia Glutamine synthetase is progressively adenylated when concentration of nitrogen is high Regulation of glutamine synthetase by covalent modification. (a) When cells are cultured in a medium rich in bound nitrogen, glutamine synthetase (GS) is covalently modified by stepwise adenylation. Up to twelve adenyl (AMP) groups can be added. When the medium becomes nitrogen-depleted, these groups are removed and ADP is formed. (b) Adenylated GS subunits are catalytically inactive, therefore the overall GS activity decreases as more subunits are adenylated.  Viruses and Phages - no living entities - require the machinery of the host cell to replicate - obligate intracellular parasites - infect all organisms on earth - source for genetic diversity The diversity of viruses and their hosts Is extremely high. Hosts for viruses and diversity of viruses. (a) The preference of viruses for hosts, organized by cellular domains (left) and for different major groups of eukaryotes (right). (b) Scale diagrams of some viruses. The sizes of viral genomes Comparative genomics. Shown is the genome size of selected viroids, viruses and prokaryotic cells. Bacteriophage phi6 infects Pseudomonas; phage G infects Bacillus; all other bacteriophages infect Escherichia coli.  The diversity of viral genomes The Baltimore classification of viral genomes. Seven classes of viral genomes are known. These genomes can consist of either DNA (a) or RNA (b), and can be either single-stranded (ss) or doublestranded (ds). The example above is a bacterial virus and the example below an animal virus; classes V and VI are exceptions here, as they only infect eukaryotic cells. The path that each viral genome takes to finally be transcribed into mRNA is indicated by arrows; the replication strategy is also shown. The genomes of viruses can consist of either DNA or RNA and some use DNA and RNA as genomic material at different points in their life cycle. However, only one type of nucleic acid is present in the virion of each virus type. This can be single-stranded (ss), double-stranded (ds). Some viral genomes are ring-shaped, but most are linear. Viruses are divided into different classes based on their genomes. The genome can be composed of: - double strand THEN - Single-stranded DNA - Double-stranded RNA - Positive single-stranded RNA - Negative single-stranded RNA - Retroviruses Viruses are not organisms, they merely consist of a nucleic acid and a protein to protect the genome. They therefore need either bacteria or eukaryotic cells in order to reproduce. In addition, they only have very small genomes, which means that they have to compact all their information to a very high degree. There are various mechanisms by which phages and viruses “persuade” the host cell to replicate only the virulent genome. Bacteriophages Bacteriophage Receptors Examples of receptors used by different bacteriophages to infect Escherichia coli. With the exception of MS2, all bacterial viruses shown here are DNA phages.  The Replication Cycle of a Lytic Bacteriophage Bacteriophage T4 infecting Escherichia coli Attachment of the bacteriophage T4 to an Escherichia coli cell and subsequent infection. The three transmission electron microscopic tomograms shown here and the schematic drawings besides represent, from left to right: the initial attachment of a T4 virion to the carbohydrates of the LPS of the outer membrane; the contact formation with the cell wall via the tail needles; the contraction of the tail’s heath and the injection of the T4 genome. The tail tube penetrates the outer membrane and the T4 lysozyme creates a small opening in the peptidoglycan of Escherichia coli. The one-step growth curve of viral replication After attachment, the infectious capacity of the virus particle disappears, a phenomenon known as eclipse. During the latency phase (composed of eclipse and early maturation phase), the synthesis of the viral nucleic acid and protein takes place. During the maturation phase, the nucleic acid and the protein are assembled into a mature virus and finally released in the release phase. Quantification of bacterial viruses Plaques→ visible lysed bacteria on a sample dish with a bacterial lawn. A "top agar" containing a mixture of permissive host bacteria and various dilutions of the virus suspension is poured onto a "base layer" agar plate. Infected cells are lysed and plaques form in the bacterial lawn. A dilution of a suspension in which the virus material has been mixed with the host bacterium using a small amount of molten agar. The mixture is added to the surface of an agar plate of the appropriate medium. The host bacteria, evenly distributed over the top layer of agar, start to grow and after incubation overnight they form a lawn of spreading cells. Any virion that attaches to a cell and reproduces can cause cell lysis. The released virions spread to neighboring cells in the agar, infect them, replicate and lead to lysis and release again. → Plaques are formed. The size of the plaques depends on various conditions. Basically, however, one can say: the smaller the phages, the larger the plaque. The larger the phages, the smaller the plaque. On the one hand, this is due to the fact that large phages require more time for synthesis & because they can produce fewer offspring at once. In addition, small phages diffuse better. Phage Tita→ Calculation of a diluted solution with phages, which allows conclusions to be drawn about how many phages are present in the original medium.  Timeline of events during lytic phage infection. After injection of the DNA, the early and middle mRNAs are formed, which encode the nucleases, DNA polymerase, new phage-specific sigma factors and various other proteins involved in DNA replication. The late mRNAs encodes structural proteins of the phage virion and the T4 lysozyme, which is required for lysis of the cell and release of new phage particles. The T4 phages have a comparatively large genome with around 170,000 base pairs. In E. coli, the RNA polymerase is brought to the promoters of the DNA with the help of a sigma factor. To enable the phage to attach the polymerase to its genome, it uses its own sigma factor or antisigma factors, which prevent the sigma factor from binding to the bacterium's polymerase. This allows the phage to bind the sigma factor and the polymerase begins to transcribe the phage genome. Similar mechanisms also exist in translation to recruit the ribosomes. The entire propagation pathway of the phage takes →25 minutes from infection to lysis. In addition to the sigma factor, phages also have other components, such as nucleases. It needs these to destroy the host genome. Lysis is carried out by the lysozyme. This cleaves the peptidoglycan, i.e. the cell wall. Phage Lamda: A temperate phage The alternatives after infection are replication and release of a mature virus (lysis) or integration of the virus DNA into the host DNA (lysogeny). The lysogenic cell can also be induced to form and lyse a mature virus. During lysogeny the lambda DNA is integrated into the host chromosome. Integration always takes place at a specific site of the host DNA and the attachment site (att) on the phage. Some of the host genes close to the binding site are indicated: gal- operon, use of galactose; bio-operon, biotin biosyntheses; moa-operon, molybdenum cofactor biosyntheses. A locus-specific enzyme (integrase) is involved in this process and the sitespecific pairing of the complementary ends leads to the integration of the phage DNA. The linear lambda genome can circularize after infection thanks to its cohesive ends. Attachment sites are used to create interfaces in both the lambda genome and the host genome. The genomes can be merged at these interfaces. The new genome can now simply be passed on to the daughter cells during cell division. This lysogenic pathway can be interrupted under certain conditions. Induction then leads to the lysogenically modified cell cutting the lambda genome out of the bacterial genome and re-entering the lytic pathway, resulting in the formation of full phages. This happens under stress situations, e.g. food shortages. The lambda genome (a) Change from linear lambda DNA to a ring-shaped molecule after infection. (b) Genetic and molecular map of lambda. The genes are indicated by letters; att, binding site for the phage to the host chromosome. Regulatory genes: cI, repressor protein; OR, operator right; PR, promoter right; OL, operator left; PL, promoter left; cro, gene for the second repressor; N, antiterminator protein. The regulatory region of lambda is highlighted in yellow, the cohesive ends of the lambda genome (cos) are shown in red. The lambda operator on the right (OR). Both the regulatory protein Cro and the lambda repressor CI bind to OR to fulfill regulatory functions. The Cro protein binds to the three sites in the order site 3, site 2 and then site 1, with the synthesis of CI first and its own synthesis last. The lambda repressor (CI) binds to these sites in the opposite order, synthesizing Cro first and then its own synthesis. The Escherichia coli phage Lambda lysis-lysogeny switch The cI gene product executes the lysis-lysogeny decision by binding to operator sites in the Lambda genome (open circles). CI bound to two operator sites recruits RNA polymerase (solid arrow) for transcription of cI and the anti-phage defense system, rexAB. At high concentrations, CI binds a third operator site in the promoter and inhibits transcription governed by RNA polymerase (dashed line). In the absence of CI, RNA polymerase instead proceeds rightward, transcribing genes involved in the lytic program (represented here as “lysis”). The lysis cassette of λ (B) The topology of the holin S and degradation of the peptidoglycan by the endolysin R. (H) Model for spanin function following the canonical holin- endolysin. Spanins activate after PG degradation and undergo a conformational change that causes fusion of the IM and OM. This removes the topological barrier of the OM at the last step of lysis, which results in the release of phage progeny. S...holin R...lysin Rz, Rz1..spanins  Phi-X174: Phage with a single-stranded DNA genome The small size of φX174 facilitated Fred Sanger’s pioneering work in DNA sequencing and was the very first organism to have the whole genome sequenced in 1977! φ X174 has been used as a model system for the study of prokaryotic DNA replication, gene expression, and morphogenesis, and more recently has become one of the most powerful systems for experimental evolution. The phage has only 5.3Kb. These code for a few proteins. The phage injects a positive single strand, then the second strand is synthesized. Replication only occurs when the double strand is present. Protein A (replication initiation protein) is important for this as it binds to the positive strand and initiates a cut. This creates an RNA primer. Transcription by the host cell can then take place. Phage M13: a filamentous phage This phage does not kill the cell after infection. The phage M13 is a single-stranded DNA phage and belongs to the Inoviridae family. M13 uses Escherichia coli, which produces F-pili, as a host for replication. Reproduction does not damage the host cell. Bacteria can exchange information via conjugation. This happens via the f-pilus. The genes for this are encoded on the f-plasmid. The f-pilus is anchored in the cell wall. The proteins required to build the f-pilus can reach the phage through the anchoring. The phage injects its genome into the cell precisely through this possibility, then the second strand is synthesized. This is followed by expression. Then another single strand is formed, which is synthesized by the protein pV. This is followed by export. During export, the phage envelope is built up and the pV proteins are cleaved off. Phage MS2: a small (+) RNA phage The genome of Bacteriophage MS2 is a positive-sense single-stranded RNA molecule of 3569 nucleotides and encodes four proteins: the major coat protein (CP), the maturation protein (A-protein), the replicase (an RNA polymerase necessary for genome multiplication), and the lysis protein. The phage has an icosahedral capsid that surrounds the + strand linear genome, copied 5’-3’ by viral RNA polymerase (replicase); no DNA intermediate!  How do bacteria defend against viruses? Defense systems Bacteria have many ways to fend off phage infection, such as blocking adsorption, injection, or assembly; or through cell suicide or RM systems. These are all innate mechanisms that defend bacteria against phages in general rather than targeting a particular type of phage. CRISPR is an adaptive immune system. It defends bacteria against specific phages and adapts to recognize new threats. About half of bacteria also have an adaptive immune mechanism called a CRISPR- Cas system, which defends against specific types of phages. This system can adapt, rapidly generating immunity against new phage challengers. The CRISPR immune system CRISPR: "clustered regularly interspaced short palindromic repeats." CRISPR-Cas immunity CRISPR is an adaptive immune system found in bacteria and archaea. Cas proteins store pieces of phage DNA as a memory of infection. Other Cas complexes use these memories as guides to find and destroy matching phage genomes to stop subsequent infection. The protospacer adjacent motif (PAM) prevents CRISPR enzymes from cutting the repeat- spacer array Avoiding self-targeting via PAM sequences Surveillance complexes will only cut DNA targets containing short sequences called PAMs. In the Type II-A system shown here, Cas9 guides the acquisition proteins (Cas1, Cas2, and Csn2) to select new spacers from DNA right next to a PAM (in this case, the sequence "NGG") in the phage genome. In contrast, the repeats in CRISPR arrays do not contain the PAM. Thus, despite the complementarity between the crRNA guide and spacer, Cas9 will not bind or cut within the array.  The diversity of animal viruses The structures and relative sizes of vertebrate viruses of the major taxonomic groups. The genome of the hepadnavirus has a complete DNA strand and a part of the complementary strand. Effects of animal viruses on infected cells Most viruses are lytic and very few are known to cause cancer. Many tumorforming viruses belong to the group of herpes viruses. There are various ways in which viruses infect animal cells. One of the ways would be the transformation of the cell into a tumor cell (e.g. HPV virus). Another pathway is the lytic pathway. There is also the parasitizing infection, which leads to the slow release of the virus. The fourth possibility is the latent pathway, i.e. the virus is present but does not initially damage the cell, but occurs later in the case of lytic infection. DNA viruses can occur with or without an envelope. The Lytic Human Herpesvirus Life Cycle (1) Binding: During primary lytic infection, HHVs bind extracellular host cells receptors using specific envelope viral glycoproteins. (2) Entry: HHVs enters the cell via fusion through receptor mediated endocytosis (2a) or endosome formation (2b). (3) Release and Nuclear Transport: After viral uncoating, both the nucleocapsid and tegument proteins are released into the cytoplasm. The nucleocapsids are transported via cytoskeletal structures or diffusion to the nucleus. (4) Nuclear Entry: The viral genome plus some associated viral proteins, including some tegument proteins, enter the nucleus via nuclear pores and the viral genome circularizes. (5) Gene Expression: Immediately early (IE) viral genes, early (E) viral genes and late (L) viral genes are expressed in a temporal fashion. Each set of mRNAs are transported to the cytoplasm and translated into protein before returning to the nucleus and before initiating the next set of viral genes. (6) DNA Replication: Early viral gene expression initiates viral DNA replication. (7) Packaging: Late viral structural proteins assemble into viral capsids and they are packaged with DNA. (8) Egress: Viral progeny bud through the inner nuclear membrane and enter the intermembrane space. Virions are transported to the nuclear associated endoplasmic reticulum and are transported to the cellular plasma membrane, where they are released via cell fusion, exocytosis or cellular lysis.  Poliovirus Poliovirus, also called polio, is a virus that causes Poliomyelitis. The disease spreads widely and is an acute infectious disease. Viruses often invade the central nervous system, damage the motor nerve cells in the anterior horn of the spinal cord, and lead to flaccid paralysis of the limbs, which is more common in children. Picornaviridae: - positive-sense RNA molecules encoding a single, long open reading frame flanked by lengthy untranslated regions. - cap-independent translation and shut off of host cell translation (cap-dependent) Structure and function of a retrovirus Structure of a retrovirus. Genetic map of a typical retrovirus genome. Each end of the genomic RNA contains direct repeat sequences (R). There are two positive RNAs inside the virus. These are located inside the capsid. This is then surrounded by a membrane. Gag …Group Antigens Pol …reverse transcriptase. Env …envelope protein.  The lifecycle of human retroviruses (A) The retroviral lifecycle begins when the envelope proteins of a mature, infectious virion attach to the appropriate receptor (for HIV-1, CD4) and co-receptor (CCR5 or CXRC4) on the surface of the cell, which facilitates viral membrane fusion with the plasma membrane. Following release into the cytoplasm (B), the intact (or near intact) capsid core is trafficked to nuclear pore complexes (NPCs) on the nuclear envelope. During or shortly after nuclear import (C), capsid uncoating and reverse transcription occur (D). The double-stranded DNA product is subsequently integrated into the host genome by integrase (E). Host machinery transcribes the proviral genome (F) into both the viral genomic RNA (vRNA) and mRNA, which templates the translation of viral proteins both in the cytosol and on the surface of the ER, with co-translational insertion of the transmembrane Env protein occurring in the ER (G). A milieu of viral and host proteins traffics along with vRNA to the plasma membrane where they are packaged into nascent budding particles (H). Following the budding (I) and release of the particle from the plasma membrane (J), the viral protease cleaves Gag resulting in the formation of a mature, infectious particle (K). Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) Coronaviruses (CoVs) are a highly diverse family of enveloped positive-sense single- stranded RNA viruses. SARS-CoV-2 enters host cells through an endocytosis pathway mediated by S protein– angiotensin-converting enzyme 2 (ACE2) interactions. Viral RNA enters the cytoplasm after the entry step, and then ORF1a or ORF1ab is translated by the host ribosome. The viral polyproteins are cleaved into NSPs and assemble themselvesinto the replication and transcription complexes. Subgenomic viral mRNAs (after capping) act as templatesfor viral protein translation. Progeny virions are assembled in the endoplasmic reticulum and Golgi body. Afterwards, the virions are exocytosed to complete the life cycle.  Viroids: unusual small pathogenic RNAs Viroids are small (about 300 nucleotides), single- stranded, circular, non-encapsidated pathogenic RNA molecules. They do not code for proteins and thus depend on plant host enzymes for their replication and other functions. They induce plant diseases by direct interaction with host factors but the mechanism of pathogenicity is still unknown. They can alter the expression of selected plant genes important for growth and development. Prions: infective protein aggregates Prion diseases are rapidly progressive, incurable neurodegenerative disorders caused by misfolded, aggregated proteins known as prions, which are uniquely infectious. Remarkably, these infectious proteins have been responsible for widespread disease epidemics, including kuru (transmissible spongiform encephalopathy, TSE) in humans, bovine spongiform encephalopathy (BSE) in cattle. Although not contagious, prion diseases are potentially infective and might spread by horizontal human-to-human and animal-to-human transmission. Mechanism of action of a prion Nerve cells produce the prion protein (PrPc), which fulfills a normal function in the cell. Abnormally folded prion protein (PrPsc) can catalyze the refolding of PrPc to PrPsc. The PrPsc form is protease-resistant, insoluble and forms aggregates in neuronal cells. This ultimately leads to the destruction of nerve tissue and neurological symptoms.  The Microbiom...the entity of all microorganisms that naturally colonize a multicellular organism (i.e. without triggering disease symptoms)'. Helicobacter: The bacterium surrounds itself with ammonia, which is produced from the breakdown of urea by the enzyme urease. This ammonia coating protects it against stomach acid All these organisms help to keep us healthy. The intestine in particular is densely populated with them, as they help us to break down food and synthesize other molecules. They also help to ensure that our bodies are not colonized by The Human Gastrointestinal Tract and Major Members of Its Microbiota pathogenic bacteria. Biochemical/Metabolic Contributions of Intestinal Microorganisms Intestinal bacteria can, for example, synthesize vitamins→ such as B12. The body cannot synthesize 8 amino acids itself; the body is dependent on either ingesting them with food or receiving them from bacteria. Small Bioactive Molecules Produced by Bacteria in the Large Intestine Kleine Bioaktive Moleküle, können von Bakterien im Dickdarm produziert werden. Tryptamin wird von diesen Bakterien produziert & dient als Neurotransmitter. Fermentation in the Colon of Lean and Obese Mice and Transfer of an Obese Condition by Fecal Transplant Lean mice have more Bacteroidetes. Obese mice have more firmicutes and methanogens. Methanogens produce more H2 which facilitates fermentation and the amount of nutrients aquired by the host. VFA… volatile fatty acids Influence of intestinal microbial flora on behaviour. Mouse offspring of mothers with maternal immune activation (MIA) show autism-like behaviour, which is characterized by fear of exploring the centre of a test field and (b) reduced vocalization. (c) By feeding these affected offspring with the human commensal bacterium Bacteroides fragilis cures these behavioural disorders. Some intestinal bacteria can increase the levels of indole-pyruvate and 4-ethylphenylsulfate, the neuroactive substance that is believed to trigger the autismlike behaviours shown in (a) and (b). Bacteroides fragilis can restore the levels to normal. Physical, chemical and anatomical barriers against infection  Bacteria also synthesize antimicrobial molecules to protect themselves from pathogenic bacteria in their habitat. In this way, they also protect the body from overgrowth. When pathogens try to enter the body, they have to overcome three different types of barriers. - Physical barrier - Chemical barrier - Anatomical barrier The first step of infection: Bacterial Adherence Adherent structures: capsules, fimbriae, pili and flagella. Some adhesins are part of an outer cell structure that may or may not be covalently bound to components of the cell wall. Some particularly strongly pathogenic bacteria form a capsule. The capsule of Bacillus anthracis consists of a polypeptide that contains only the amino acid D-glutamate. Fimbriae. Computer-generated image of a scanning electron micrograph of cells of Salmonella enterica (typhi) cells, showing the numerous fimbriae and the much thicker peritrichously arranged flagella. A cell is approx. 1 µm thick. Biofilm Pseudomonas aeruginosa opportunistic pathogen, forms biofilms in the lung of cystic fibrosis patients. Biofilm matrix: extra-cellular polysaccharides a) Loose association b) Adhesion c) Colonization Free-swimming bacteria attach to the epithelial cells. This initially occurs as loose attachment. Colonization then begins. These colonies can form biofilms. Biofilm→ is produced by bacteria during colonization → mucus, biofilm matrix (polysaccharide matrix). The producing cells are protected in this matrix. Capsules are another variant of bacteria to protect themselves from the immune system or drugs. Adherence proteins → require bacteria to be able to attach properly Lipoteichoic acid→ enables Gram-positive bacteria to attach to epithelium Fimbriae→ Enable binding to the epithelium  Enzyme Virulence Factors of Some Gram-Positive Bacterial Pathogens The activity of some enzymes as virulence factors. (a) Hyaluronidase. (b) Coagulase and streptokinase. Hyaluronidase and streptokinase occur particularly in virulent strains of virulent strains of Streptococcus pyogenes and coagulase in virulent strains of Staphylococcus aureus. Virulence Factors in Salmonella Pathogenicity islands and plasmids Salmonella species infect humans and cause various diseases of the gastrointestinal tract. Salmonella species encode a large number of virulence factors: Type I fimbriae, for the attachment of bacterial cells to tissues of the gastrointestinal tract; antiphagocytic proteins that prevent the bacterial cells from being engulfed by phagocytes of the host; proteins that ensure survival, if the bacteria are phagocytized; Siderophores, which bind iron very well and enable pathogenic bacteria to bypass the host's iron complexation systems; exotoxins and endotoxins. With the exception of endotoxin, almost all of these virulence factors are encoded by genes that are located on mobile DNA and not on the chromosome. Several genes for these virulence factors, are found on the chromosome in gene clusters called Salmonella pathogenicity islands (SPI). Some Exotoxins and Cytotoxins Produced by Human Bacterial Pathogens 3 classes of exotoxins: - AB toxins - cytotoxins - superantigen toxins AB-Toxins Toxins, which consist of two components. The A subunit is the toxic component. The B subunit serves to transport and absorb the active toxin into the host cell. The diphtheria toxin excreted by C. diphtheriae The Activity of Diphtheria Toxin The diphtheria toxin is excreted by C. diphtheriae as a single polypeptide. The B-part of the polypeptide binds specifically to a host cell receptor that is found on many eukaryotic cells, the epidermal growth factor, which binds heparin. After binding, proteolytic cleavage between fragments A and B enables the penetration of fragment A into the host cytoplasm through its cytoplasmic membrane. Fragment A interrupts protein synthesis by blocking the transfer of an amino acid from a tRNA to the growing polypeptide chain in the cytoplasm. The toxic A peptide catalyzes the ADP-ribosylation of elongation factor 2 (EF-2). The altered elongation factor is no longer able to fulfill its role in translation. This leads to the cessation of all protein biosynthesis and subsequently to the death of the cell. The botulinum toxin excreted by Clostridium botulinum Clostridium botulinum: Gram-positive, rod-shaped, anaerobic, spore-forming, motile bacterium with the ability to produce the neurotoxin botulinum. C. botulinum is responsible for foodborne botulism (ingestion of preformed toxin), infant botulism (intestinal infection with toxin-forming C. botulinum), and wound botulism (infection of a wound with C. botulinum). C. botulinum produces heat-resistant endospores that are commonly found in soil and are able to survive under adverse conditions. The Activity of Botulinum Toxin (a) After stimulation of the peripheral nerves and the cranial nerves, acetylcholine (A) is generally released from the vesicles of the neuronal circuit with the motor end plate. Acetylcholine then binds to the specific receptors, leading to contraction. (b) Botulinum toxin prevents the release of acetylcholine (A) from the vesicles, so that there is no stimulus for the muscle fibers. This leads to irreversible muscle relaxation and muscle paralysis. The lack of muscle contraction can lead to death by suffocation if the diaphragm muscles are severely affected. The Tetanus toxin excreted by Clostridium tetani Clostridium tetani: is a rod-shaped, Gram-positive common soil bacterium that can form spores. It is motile by way of various flagellums that surround its body. C. tetani cannot grow in the presence of oxygen. It grows best at temperatures ranging from 33 to 37 °C. C. tetani is the causative agent of tetanus. C. tetani spores are extremely hardy and can be found globally in soil or in the gastrointestinal tract of animals. If inoculated into a wound, C. tetani can grow and produce a potent toxin, tetanospasmin, which interferes with motor neurons, causing tetanus. The Activity of Tetanus Toxin (a) Normally, muscle contraction is reduced by the release of glycine (G) from the inhibitory interneurons. The glycine acts on motor neurons, blocking their stimulation and the release of acetylcholine (A). (b) The tetanus toxin binds to the interneuron and in this way prevents the release of glycine from the vesicles, resulting in the continuous release of acetylcholine in the muscle fibers, triggering an irreversible contraction of the muscles and spastic paralysis. For ease of visualization, the inhibitory interneuron was drawn near the motor endplate, but it is actually located near the spinal cord. The cholera toxin excreted by Vibrio cholerae Vibrio cholerae Gram-negative, facultative anaerobe and comma-shaped bacteria. The bacteria naturally live in brackish or saltwater where they attach to crabs, shrimp, and other shellfish. Can cause cholera, which can be derived from the consumption of undercooked or raw sea food. The Activity of Cholera Toxin Attachment and colonization of the intestinal microvilli by Vibrio cholerae, followed by the production and binding of the A-B enterotoxin through a specific interaction with the GM1 ganglioside of the host cells. The subunit of the A-B toxin binds to GM1; subunit A is transported through the cell membrane into the cell interior. The internalized subunit A activates the adenylyl cyclase. This enzyme activation leads to disruption of the normal sodium ion influx and loss of water into the intestinal lumen, resulting in severe diarrhea. Cholera disease is treated by compensating for the loss of ions and water (i.e. by administering large quantities of fluids). The use of antibiotics can possibly shorten the duration of the disease by preventing the multiplication of V. cholerae; however, this has no influence on the effects of the toxin already produced and released. Cytotoxins (Cytolytic toxins) – the Staphylococcal α-Toxin The α-toxin of staphylococci is a pore-forming cytotoxin that is produced by growing Staphylococcus cells. After it is released as a monomer, seven identical protein subunits oligomerize in the cytoplasmic membrane of the target cells. The oligomer forms a pore, releases the contents of the cell and thus enables the influx of extracellular material and the efflux of intracellular material. In the case of erythrocytes, hemolysis occurs, whereby cell lysis becomes visible. The inset photo top left shows the structure of the αtoxin, as a top view of the pore; the 7 subunits are colored differently. The inset photo top right shows a scanning electron micrograph of S. aureus cells.  Exotoxins vs. Endotoxins Endotoxins The LPS molecules are only moderately toxic, but they induce a strong fever in the host. This weakens the body, but does not trigger an immune response. Lipopolysaccharide (LPS) released by Gram-negative bacteria binds to the LPS-binding protein (LBP) present in body fluids. The LPS/LBP complex then binds to the protein CD14. The LBP is released and the LPS/CD14 complex binds to the receptor TLR-4. This new TLR- 4/CD14/LPS complex triggers a signal transduction that originates from TLR-4 and leads to the activation of the transcription factor NF-kB. This triggers the transcription and subsequent translation of genes for inflammatory cytokines and chemokines such as interleukin 1 (IL-1), IL- 6, IL-8 and TNF-a (tumor necrosis factor alpha) in the cell nucleus. These mediator molecules in turn activate other cells and generate inflammation and the uptake and destruction of pathogens. Basics of Immunology The human immune system uses a two-pronged defense against invading pathogens. Innate immunity, the first of the cooperating lines of defense, is the basic ability of the immune system of multicellular organisms to attack pathogens in general regardless of their identity. In contrast, the adaptive immunity is triggered by exposure to specific pathogens, which cannot be eliminated from the body by the innate defense system alone. Each adaptive immune response is directed against a specific type of invading pathogen.  The blood and lymphatic systems (a) The blood and lymphatic circulation in the body. The main blood vessels and associated organs are shown in red, the most important lymphatic organs and lymphatic ducts are highlighted in green. The most important lymphoid organs are the bone marrow and the thymus. The second most important lymphoid organs are the lymph nodes, the spleen and the MALT. (b) Connections between the blood and lymphatic circulations. Blood flows through the veins to the heart, to the lungs and then through the arteries to the tissues. Lymph flows through the thoracic lymphatic duct into the left subclavian vein of the circulatory system. (c) The exchange of cells between the blood and lymphatic systems in detail. Both the blood and the lymphatic capillaries are closed vessels, but the cells migrate from the blood capillaries to the lymphatic capillaries and back, a process known as extravasation. (d) A secondary lymphoid organ, the lymph node. The schematic diagram shows the main anatomical regions and the immune cells present in each region. The anatomy of MALT and the spleen is analogous to that of the lymph nodes. Major Cell Types Found in Normal Human Blood The diversity of immune cells  Microbial Invasion and the Innate Immune Response (a) Tissue damage, such as a cut with a razor blade, can lead to the invasion of microorganisms and the release of cytokines and chemokines by damaged cells. (b) Phagocytes are directed to the site of infection via the gradient of chemokines and squeeze through dilated capillaries via extravasation (diapedesis). (c) Invading microorganisms are removed by phagocytosis and the tissue returns to a healthy state. Overview of the immune system Pathogens are destroyed by three different mechanisms: (1) Innate immunity is based on the interaction between pathogen-associated molecular patterns (PAMPs), which are found on the surface components of pathogens, and pattern-recognizing molecules, which are found on phagocyte receptors. Adaptive immunity (= acquired immunity) is coordinated by antigenpresenting T cells and is characterized by two distinct effector mechanisms: (2) antibody-mediated immunity is based on soluble, antigen-specific antibody proteins produced by antigenstimulated B cells; (3) cell-mediated (or cellular) immunity is based on the action of antigen-specific T effector cells. Pathogen Associated Molecular Patterns (PAMPs) Pathogen Recognition by Phagocytes The recognition of pathogens by phagocytes. (a) Phagocytes are characterized by pathogen-associated molecular patterns (PAMPs) with pattern recognition receptors (Preformed Pattern Recognition Receptors, PRRs). These interactions activate the phagocytes, which then ingest the pathogens that they destroy; they also activate further phagocytes. (b) Stained scanning electron micrograph of a neutrophil (blue) phagocytosing several cells of a methicillin-resistant Staphylococcus aureus The macromolecules in the pathogens or on their surface exhibit pathogen-associated molecular patterns (PAMPs), which consist of repeating subunits that infectious agents often have in common. PAMPs can be polysaccharides, proteins, nucleic acids or lipids. An example of example of a PAMP is the lipopolysaccharide (LPS), or bacterial flagellin, the double-stranded RNA (dsRNA) of certain viruses and the lipoteichoic acids of gram-positive bacteria. A Toll-type receptor. The membrane-spanning TLR2 interact