Introductory Microbiology Lecture 02 - BST 251-3 PDF

Introductory Microbiology (BST 251-3) Dr. Samanthi Udayangani Kumari  Beneficial microbes and their application In fermentation industry In food industry In agriculture In the production of biofuels In bioleaching and microbial minin...

Introductory Microbiology (BST 251-3) Dr. Samanthi Udayangani Kumari  Beneficial microbes and their application In fermentation industry In food industry In agriculture In the production of biofuels In bioleaching and microbial mining In bioremediation of polluted environments In controlling pests In medicine In biotechnology  Fermentation industry Microorganisms yield several industrial products through the process called ‘fermentation’ Convert inexpensive raw materials into expensive industrial chemicals such as alcohols, drugs and solvents Some important products of fermentation – Industrial chemicals Ethanol, Acetone, Isopropanol, butanol, Glycerol, sorbitol, propylene glycol, etc.  Fermentation industry – Organic acids Citric acid, lactic acid, acetic acid, fumaric acid etc. – Enzymes Glucoamylase, amylase, cellulase, lipase, etc. – Amino acids L-lysine, glutamic acid, etc. – Vitamins Riboflavin, vitamin B12, etc. – Pharmaceuticals Penicillin, streptomycin, tetracycline, etc.  Food industry Two kinds of importance in food industry – Can be directly used as foods As a source of protein (single cell protein) – Yeast, Spirulina Source of oil – Mucor, Mortierella Cultivated mushrooms (oyster mushrooms and button mushrooms) – Pleurotus, Agaricus – Useful in giving texture and flavor for certain foods (fermented foods) Curds, yoghurt, bread, cake, etc. – Lactobacillus, Streptococcus, Saccharomyces  Single-cell protein The crude, a refined or edible protein extracted from pure microbial cultures, dead, or dried cell biomass.  Agriculture Play an important role in increasing soil fertility. Recycle elements such as N, P, S, Fe, etc, in soil by degrading various complex organic compounds. Nitrogen fixing bacteria – Convert atmospheric nitrogen into form of utilizable for plants. – Azotobactor, Rhizobium, Azospirillum. Phosphate solubilizing bacteria – Make insoluble phosphate source available for plants by solubilization. – Bacillus, Pseudomonas. Mycorrhizal fungi – Help plants to absorb nutrients. – Glomus.  Production of biofuel Gasohol is a mixture of alcohol and gasoline used for running vehicles Archaea such as Methanococcus, Methanosarcina, Methanospirillum Produce biogas from domestic and agricultural wastes  Bioleaching or microbial mining Certain microbes are capable of oxidizing low grade iron ores like pyrite and separating iron or copper from the crude ore. – Thiobacillus ferrooxidans, Thiobacillus thioxidans – Reduce the cost of purification of the metals from the ores.  Bioremediation Bioremediation is a strategy of pollution alleviation that uses living organisms rather than using chemicals treatment Eco-friendly process of pollution control Several bacteria and fungi are capable of degrading complex pesticides, other toxic chemicals, heavy metals, oils spills, etc. by clearing the environment Eg: Pseudomonas putida for clearing oil spills – Control of heavy metal pollution due to mercury, chromium, zinc, molybdenum, copper etc Bioremediation  Biopesticides The use of microbes to control pests is called biocontrol Biopesticide is meant for the control of insect pests – Nuclear polyhedrosis virus and cytoplasmic polyhedrosis virus are using to control pests – Soil bacterium Bacillus thuringiensis produce Bt toxin to kill insect larvae  Medicine Medical microbiology has given the power to control the majority of the infectious diseases Production of various pharmaceuticals from microbial source is a great development  Applications of Microbiology in Biotechnology Microbiology can quite rightly be called as a modern science with lot of current interest and future scope The recent developments in ‘Genetic Engineering’ and ‘Biotechnology’ were possible only through researches on microorganisms and are sustained by the applications of microbes. The modern subjects such as molecular biology and molecular genetics that form a backbone of biotechnology developed mainly through researches in the field of microbiology Quite rightly microbiology is called the “mother of biotechnology”  Applications of Microbiology in Biotechnology Gene cloning Production of restriction enzymes Production of other enzymes useful in biotechnology Microbes as vectors Agrobacterium mediated gene transfer to plants Recombinant vaccines Gene therapy  Applications of Microbiology in Biotechnology Gene cloning – Microbes are favoured organisms for cloning of genes, as they can be easily cultured in large numbers – Isolating a specific region of DNA and producing millions of identical copies of the DNA within a microbial cell culture – E. coli has been used for cloning innumerable genes – Similarly other organisms such as bacterial genera Streptomyces, Bacillus and Pseudomonas and the unicellular fungus yeast have been exploited for cloning various genes  Applications of Microbiology in Biotechnology Production of restriction enzymes – Microorganisms are the main source of restriction endonucleases used in genetic engineering – DNA should be cut - only the desired portion containing the desired gene – The restriction enzymes produced by various bacteria, such as EcoRI from E. coli are able to cut DNA at specific sequences – So useful in cutting off the desired DNA fragments – Eg : Bam H1, HindIII  Applications of Microbiology in Biotechnology Production of other enzymes useful in biotechnology – Microorganisms are the source of several other enzymes that are most essential for genetic engineering – The most important ones among these are the DNA ligases and polymerases – DNA ligase derived from the bacteriophage T4 is useful in joining DNA fragments together (stitching foreign DNA into a vector DNA molecule) – The DNA polymerase derived from the thermophilic bacterium Thermus aquaticus, useful in DNA amplification  Applications of Microbiology in Biotechnology Microorganisms as vectors – Microorganisms or the plasmids derived from them have been useful as vectors in genetic engineering for transferring genes from one organism to another – The vector carrying the gene of interest can be implanted in an expression host such as E. coli, where the genes can express and produce the gene product – Common vectors pUC, pBR322  Applications of Microbiology in Biotechnology Agrobacterium mediated gene transfer to plants – The bacterial species Agrobacterum tumerfaciens cause tumours in several dicot plant species by a process of transfer of its tumour inducing genes to plants which they naturally infect. – The bacterium bears a plasmid called Ti – plasmid (Tumour inducing plasmid) which enters the plant cells and integrate with plant genome – The biotechnologists have successfully exploited this knowledge for using the agrobacterium plasmid as a vector for the transfer of foreign genes to plants – The disease causing genes are removed from the plasmid and the gene of interest is ligated and this disarmed vector is using for transformation of plants – Useful in producing plants with new traits of pest resistance, drought resistance, improved yield, nutritional quality etc  Applications of Microbiology in Biotechnology Recombinant vaccines – The immunogenic protein (vaccine) is produced in a bacterium (eg; E. coli) by introducing the gene for the protein into the cell of bacterium. – The protein is thus produced is called a recombinant vaccine, because it is derived from the recombinant DNA technology.  Applications of Microbiology in Biotechnology Gene therapy – This is a novel strategy for treating inherited genetic disorders and is one of the most critical of genetic engineering technologies. – Gene therapy involves the process of replacing the defective genes responsible for certain genetic diseases with the correct genes. – For successful gene therapy, the introduced genes should integrate, express and work properly in the recipient.  The harmful microorganisms 1. Infectious pathogens causing diseases 2. Toxin producers 3. Allergens 4. Agents of food spoilage 5. Spoilage of textiles, paper, wooden articles etc  Infectious pathogens causing disease One of the harmful effects of microorganisms is the causation of deadly infectious diseases Among the microbes that human diseases, bacteria have received great attention, and viruses come next in importance There are other pathogenic forms belonging to Fungi, Protozoa, Mycoplasmas and Rickettsias.  Bacterial diseases Bacterial diseases of great severity are  Typhoid –Salmonella typhi  Tuberculosis- Mycobacterium tuberculosis  Leprosy - Mycobacterium leprae  Meningitis - Neisseria meningitis  tetanus - Clostridium tetani  Cholera - Vibrio cholerae  Pneumonia - Streptococcus pneumoniae  Diphtheria - Corynebacterium diptheriae  Antrax - Bacillus anthracis With the discovery of antibiotics, many of the infectious diseases are controlled. However, many of the pathogenic bacteria gain resistance to drugs, major problem in using antibiotics.  Viral diseases Are most difficult to control as there are no antiviral drugs or antibiotics that can inhibit the virus without harming the host cell. Viral diseases eg: AIDS, Hepatitis B, Influenza, Small pox, Measles, Mumps, Chicken pox, Rabies, Yellow fever, Dengue, etc. Control of the diseases through vaccination.  Opportunistic pathogens- Fungi Caused by fungi When a person’s immune system breaks down, several lesser pathogens can cause diseases Fungi include Aspergillus, Mucor, etc. Bacterial sub group member Mycoplasma is also an opportunistic pathogen, not a serious pathogen  Toxin producers Food borne toxicity due to Clostridium botulinum, C. perfringenes, etc. Salmonellosis due to Salmonella spp carried with food. Aflatoxin contamination of food stuffs due to growth of fungi. These microbes do not cause disease, but do cause poisoning of the system.  Allergens Many microbes cause no disease but cause severe allergies Allergy or hypersensitivity is an exaggerated reaction by the human defence system to a foreign agent which is normally harmless. The spores of fungi such as Cladosporium, Alternaria, Fusarium, Aspergillus and Penicillium have been found to cause allergy in several individuals.  Agents of food spoilage Many microorganisms grow in our foods and cause their spoilage Can cause even toxicity Short period of preservation, foods can be refrigerated Chemical food preservatives such as sodium metabisulphite, sodium benzoate, etc. are used for processed foods  5. Spoilage of textiles, paper, paints, wooden articles etc Many microorganism especially fungi are very good producers of enzymes such as cellulases, lignolytic enzymes and pectinases The enzymes cause the degradation of textiles, paper, etc. Humidity should be avoided to control these organisms and as a prevention antifungal compounds can be used, especially in paints and woods Prokaryotic and Eukaryotic Cells  Prokaryotic and Eukaryotic Cells Higher eukaryotes have several organs to carry out specific functions. – E.g. liver, kidney and heart Each organ has specific tissue and each tissue is composed of cells. “Cell is the structural and functional unit of life”. – Contains all essential infrastructure to perform all functions. Based on cellular structure, cells are classified as prokaryotic and eukaryotic cells Most of the prokaryotes are single cells where as eukaryotes are either single cells or part of multicellular tissues system. Both types of cells also have several structural and metabolic differences. Comparison between Prokaryotes and Eukaryotes  Prokaryotes A prokaryotic cell is much simpler and smaller than an eukaryotic cell. It lacks membrane bound organelles including nucleus. Prokaryotic Cell  Outer Flagella A flagellum attached to the bacterial capsule is an essential feature of most of the prokaryotic cell - Especially in the motile bacteria Provides motion/locomotion to the bacteria Guide bacteria in a direction in response to external stimulus: – Chemical stimuli – chemotaxis; positive and negative – Light stimuli – phototaxis Signal sets flagella into rotary motion clockwise or counterclockwise: – Counterclockwise – results in smooth linear direction – run – Clockwise – tumbles Flagellum is a part of cell wall Its motion is regulated by motor proteins present inside the cell Flagellar motion is an energy consuming process and it is managed by an ATPase present at the bottom of the shaft Reduction or suppression of flagellar protein reduces bacterial infectivity and their ability to grow – how some antibiotics work  3 parts: – Filament – long, thin, helical structure composed of protein Flagellin – Hook – curved sheath – Basal body – stack of rings firmly anchored in cell wall Rotates 360o  Arrangements of Flagella Monotrichous A single flagellum at one end Lophotrichous Multiple flagella arising from one or both ends Amphitrichous A single flagellum arises from each end Peritrichous Flagella are randomly dispersed over the cell surface  Periplasmic Flagella Internal flagella, enclosed in the space between the outer sheath and the cell wall peptidoglycan Produce cellular motility by contracting and imparting twisting or flexing motion. Periplasmic flagella  Non-locomotor Appendages Pili are longer and sparser than fimbriae. Fimbriae: Fine, proteinaceous hairlike bristles emerging from the cell surface Involved in adhesion to other cells and surfaces Pili: Rigid tubular structure – pilin protein Found only in Gram negative bacteria Involved in conjugation (a “mating” process)  Surface Layers Bacteria posses 3 barriers to protect the cells from external damage: – Glycocalyx – Cell Wall – Plasma Membrane  Glycocalyx – Outer most layer, made up of high molecular weight polysaccharides. – Impermeable to water/aqueous solvent – Responsible for antigenicity of bacterial cells – Two types: Bacterial capsule – highly organized and tightly attached Slime layer – loosely organized and attached – Functions: Protect cells from dehydration and nutrient loss Inhibit killing by white blood cells by phagocytosis, contributing to pathogenicity Attachment - formation of biofilms  Cell Wall – Responsible for gram staining differences due to their different composition - used in classification of bacterial species. Determine the shape Provides strong structural support – Preventing bursting or collapsing due to osmotic pressure Attachment Can contribute to pathogenicity Can protect the cell from toxic substances Site of action of some antibiotics Cell wall of most bacteria gain their relatively rigid quality from a unique macromolecule –peptidoglycan Peptidoglycan  Gram-Positive Cell Wall Thick, homogeneous sheath of peptidoglycan – 20-80 nm thick – Includes teichoic acid and lipoteichoic acid: function in cell wall maintenance and enlargement during cell division; move cations across the cell envelope; stimulate a specific immune response – Some cells have a periplasmic space, between the cell membrane and cell wall  Gram-Negative Cell Wall Composed of an outer membrane and a thin peptidoglycan layer Outer membrane is similar to cell membrane bilayer structure – Outermost layer contains lipopolysaccharides and lipoproteins (LPS) Lipid portion (endotoxin) may become toxic when released during infections May function as receptors and blocking immune response Contain porin proteins in upper layer – regulate molecules entering and leaving cell – Bottom layer is a thin sheet of peptidoglycan Periplasmic space above and below peptidoglycan  Nontypical Cell Walls Some bacterial groups lack typical cell wall structure, i.e., Mycobacterium and Nocardia – Gram-positive cell wall structure with lipid mycolic acid (cord factor) Pathogenicity and high degree of resistance to certain chemicals and dyes Basis for acid-fast stain used for diagnosis of infections caused by these microorganisms Some have no cell wall, i.e., Mycoplasma – Cell wall is stabilized by sterols – Pleomorphic  Plasma Membrane Bacterial Cytoplasmic Membrane Made of phospholipid bilayer with embedded proteins – fluid mosaic model Acts as a permeability barrier for most molecules – selectively permeable Serves as the location for the transport of molecules in and out of the cell – nutrients and wastes Functions include transport, energy extraction, nutrient processing and synthesis.  Protoplasm/Cytoplasm Dense gelatinous solution within the cell membrane – sugars, amino acids and salts 70-80% water – solvent for materials used in all cell functions Primary site for the cell’s biochemical and synthetic processes.  Chromatin Body/Bacterial Chromosome Single, circular, double stranded DNA molecule that contains all the genetic information required by a cells. Aggregated in a dense area called the nucleoid – DNA is tightly coiled.  Plasmids – Small circular, double-stranded DNA – Free or integrated into the chromosome – Duplicated and passed on to offspring – Not essential to bacterial growth and metabolism – May encode antibiotic resistance, tolerance to toxic metals, enzymes, and toxins – Used in genetic engineering - readily manipulated and transferred from cell to cell  Ribosomes – Made of 60% ribosomal RNA and 40% protein – Consist of two subunits: large and small – Prokaryotic differ from eukaryotic ribosomes in size and number of proteins – Site of protein synthesis  Inclusions and granules – Intracellular storage bodies – Vary in size, number, and content – Bacterial cell can use them when environmental sources are depleted – Examples: glycogen, poly b- hydroxybutyrate, gas vesicles, sulfur and phosphate granules, particles of iron oxide  Cytoskeleton – Many bacteria possess an internal network of protein polymers that is closely associated with the cell wall Bacterial Shapes, Arrangements and Sizes Vary in shape, size, and arrangement, but typically described by one of three basic shapes: – Coccus – spherical – Bacillus – rod Coccobacillus – very short and plump Vibrio – gently curved – Spirillum – helical, comma, twisted rod, Spirochete – spring-like STOP  Eukaryotes A broad spectrum of morphological and functional specializations of cells occurs in the multicellular organisms. However, all Eukaryotic cells conform to a basic structural model. Therefore, the eukaryotic cell is composed of two basic parts: – Cytoplasm – Nucleus  Ultrastructure of Eukaryotes ORGANELLE MAIN FUNCTIONS DIMENSIONS Nucleus Cell division, protein 10 µm diameter synthesis Mitochondrion Respiration pathways 1.0 to 12.5 µm Chloroplast Photosynthetic pathways 5 to 10 µm diameter Lysosome Digestion, recycling & 0.5 to 3.0 µm diameter isolation Golgi apparatus Secretion, reprocessing, Cisternae: 0.5µm thick, lysosome synthesis l-3µm diameter Endoplasmic Support, Golgi apparatus 26 to 56 nm thick Reticulum (ER) synthesis  The Nucleus Largest organelle – often located in the central part of the cytoplasm – Usually spherical occupying up to 75% of the cell volume Enclosed in a double layered nuclear membrane – Each membrane is lipid bilayer with proteins Contains nucleolus and chromatin (DNA) DNA is the genetic material involved in cell division and synthesis of proteins Nucleus – control centre of the cells and contains the blue print for all cellular structure and activities. Nuclear envelope – perforated by pores and facilitate communication between the nucleus and the cytoplasm  The nucleus control protein synthesis by sending molecular messengers in the form RNA – mRNA messenger – Transcription Bound ribosomes are attached to outside membrane network called the endoplasmatic reticulum; Makes proteins destined into membrane and for export from the cell (secretion) Nucleus 10 µm  Functions of Nucleus Main site of DNA in eukaryotic cells Preservation, replication and expression of genetic information It makes RNA for protein synthesis It copies DNA for cell division  Mitochondria Inner matrix Cristae Inter membrane space outer membrane Mitochondrial envelope inner membrane 1.0 to 12.5 µm  “Power house of the cell” as the organelle is actively involved in the generation of ATP to run the cellular activities. Mitochondria is a double layered membrane bound organelle with different structural properties.  Functions of Mitochondria Production of ATP Generation of Reactive Oxygen Species (ROS) in immune cells to kill infectious agents Used to track tree of a family Role in programmed cell death or “apoptosis”  Chloroplasts Grana Thylakoid membrane Frets outer Chloroplast membrane envelope inner membrane Stroma Starch grains  Chloroplasts Chloroplasts are found in plant, algae and other lower invertebrates such as euglena. Contrasting to mitochondria, chloroplast has outer membrane, an inner membrane and then light pigment containing inner most thylakoid membrane. Pigments: Mainly chlorophylls with carotenoids and others Function: Photosynthesis The metabolic pathways are closely associated with the membranes as in the case of the mitochondrion  They are involved in energy reactions They contain extranuclear DNA and characteristic small ribosomes of their own This has led biologists to believe that there may be some similarity in their origins in the cells of eukaryotes.  Lysosomes Membrane-bound organelle Functions: – Site of intracellular digestion – Site of turnover of cellular components Contents: Contains up to 40 different acid Hydrolytic enzymes (pH 5) Distribution: Found in cells involved in extensive phagocytic activities, – e.g. Macrophages and Neutrophil Nature of Lysosomal Enzymes: – Most of these are acid hydrolases and are involved in breaking down a wide range of macromolecules: Proteases Nucleases Phosphatases Phospholipases Sulfatases Beta Glucuronidases These enzymes are inactive in the Cytosol because as the pH of the Cytosol is f 7.2 Sources of Lysosomes: Its membrane is derived from Golgi apparatus while the enzymes are synthesised in the endoplasmic reticulum  Functions of Lysosomal Enzymes Degradation of ingested food material for delivery through vesicular system Degradation of pathogenic bacteria Degradation of old proteins  Endoplasmic Reticulum (ER) The vesicular network starts from nuclear membrane and spread throughout the cytosol constitutes endoplasmic reticulum. There are two different types of endoplasmic reticuli present in the cell: – Rough endoplasmic reticulum (RER) – Smooth endoplasmic reticulum (SER) RER has ribosome attached to it to give a rough appearance whereas SER is devoid of ribosomes. Protein synthesis on ribosome attached to RER are sorted into 3 different categories: – Integral membrane proteins – Proteins for secretion – Protein destined for different organelles  Functions of ER 1. Synthesis of steroid hormone in gonad cells 2. Detoxification 3. Ca2+ sequestration 4. Synthesis of proteins, phospholipids and carbohydrates 5. Protein sorting to different organelles 6. Protein modifications such as glycosylation etc.  Golgi Apparatus Golgi apparatus was first visualized by a metallic stain invented by Camillo Golgi. It is made of flattened, disk like cisternae arranged in a stacked manner to give three distinct zones. – Cis cisternae: Receives material or vesicles from endoplasmic reticulum – Medial cisternae: Actual place where protein are covalently modified with the sugar – Trans Golgi: Sorts vesicle for their destined organelles or plasma membrane  Functions of Golgi Apparatus Protein sorting Protein modifications (Glycosylation) Proteolysis