General Microbiology Notes (PDF)

Zagazig University Faculty of Pharmacy Microbiology & Immunology Dept. General Microbiology & Immunology notes (Part I) Second Level Term I -1-  INTRODUCTION AND HISTORICAL REVIEW Microbiology is the study of minute o...

Zagazig University Faculty of Pharmacy Microbiology & Immunology Dept. General Microbiology & Immunology notes (Part I) Second Level Term I -1-  INTRODUCTION AND HISTORICAL REVIEW Microbiology is the study of minute or small form of organisms (not seen by unaided or naked eye). Antonie Van Leeuwenhoek  known as “Father of Microbiology” was the first man to see microorganisms  He was able to make a simple microscopes with high magnification (x300) enabled him to observe small swimming organisms in a drop of pool water and called it “animalcules”. The theory of Spontaneous Generation (abiogenesis)  Life is created spontaneously from non-living things or decaying organic matter.  This theory opposes what is known as Germ theory (biogenesis), which recognizes life as progeny of already existing life.  Francesco Redi challenged spontaneous generation concept by showing that maggots on decaying meat came from fly eggs deposited on the meat, and not from the meat itself. -2- Lazzaro Spallanzani  He showed that flasks sealed and then boiled had no growth of microorganisms, and he proposed that air carried germs to culture medium; but he commented that external air might be needed to support the growth of animals already in medium.  John Needham repeated the experiment, but he used cork as stopper- to avoid killing vital spirit. Unfortunately, the boiled broth could still develop microorganisms. This again supported the theory of spontaneous generation.  Francois Appert applied this process (Appertization) for preserving soups and liquids after extensive boiling and sealing. Louis Pasteur “Father of Bacteriology and Immunology"  The French biochemist was able to disprove the theory of spontaneous, by boiling broth and keeping it in “swan-necked flask” open to the air without spoilage.  He Solved the problem of brewery industry –production of vinegar- by heating juice at 50-60oC a process called “Pasteurization”.  He established the germ theory of fermentation: “all fermentative processes are -3-  the results of microbial activity” and specific microorganisms are responsible for different types of fermentation.  He participated with Robert Koch in finding germ theory of disease.  He put the principles of immunology based on Edward Jenner’s work and developed vaccines for anthrax, chickenpox, anthrax, cholera and rabies.  He referred to this procedure as vaccination (vacca = cow in Latin), appreciating the work of Jenner.  Edward Jenner made the greatest contribution in preventive medicine. He noticed that milkmaids who were infected with cowpox became immune to smallpox.  He made infection with material from diseased cow (pustules) inoculated into arms of young boy and after 6 weeks inoculation with smallpox produced no disease. John Tyndall  Tyndall proved the existence of heat-resistant forms of bacteria (endospores) that are not killed by boiling for 5 ½ hours. He devised a method to get rid of these spores that was named after him “Tyndallization” or batch sterilization. -4- Robert Koch 1. He established the relationship between Bacillus anthracis and anthrax (Koch's Postulates)  still used to establish the link between a particular microorganism and a particular disease: o The microorganisms must be present in every case of the disease but absent from healthy individuals o The suspected microorganisms must be isolated and grown in pure culture o The same disease must result when the isolated microorganism is inoculated into a healthy host o The same microorganism must be isolated again from the diseased host 2. Koch developed culture media and techniques for isolation and cultivation of microbes 3. He isolated and identified specific pathogens (anthrax, TB, gonorrhoea, cholera, typhoid fever, etc.) -5- Koch's laboratory  Julius Petri developed cultivation of microorganisms in plates named after him (Petri dish).  Frau Hesse introduced the use of agar (1881) as solidifying agent  Christian Gram developed Gram stain.  Paul Ehrlich introduced chemotherapy, by discovery of the compound (arsphenimine), known as “the Magic Bullet” for treatment of trpanosomiasis and syphilis.  Gerhard Domagk discovered the sulpha drugs  Joseph Lister introduced the concept of aseptic surgery by using phenol in washing and disinfection of hands and surgical instruments and to spray room during operation.  Alexander Fleming discovered the antibiotic penicillin.  Jonas Salk & Albert Sabin prepared vaccines for poliovirus -6-  Classification of Living organisms DEVELOPMENT OF MICORBIOLOGY  Before discovery of microorganisms the living organisms were included in two kingdoms: animal kingdom and plant kingdom.  The distinction between the two kingdoms based on structural and physiological features as follows: Criteria Plant kingdom Animal kingdom Energy source Light (photosynthesis) oxidation of organic matter Chlorophyll present absent Principal reserve material starch glycogen and fat Active movement absent present Cell wall present absent Mode of growth open closed  After discovery of microorganisms, 3rd kingdom (Protista) was proposed by Haeckel in 1866. Kingdom protista  This kingdom includes unicellular or coencytic and multicellular organisms.  Members of this kingdom lacked specialization and differentiation of cell types and tissues.  The distinction from other kingdoms is based on cellular organization.  This kingdom included: protozoa, fungi, algae, and bacteria. Prokaryotes and Eukaryotes  After introduction of Electron Microscope, it was possible to distinguish cells according to their structure into two types, prokaryotic and eukaryotic cells  The prokaryotes were removed from kingdom protista.  Protista was first modified to comprise only eukaryotic organisms: 1. Algae: chlorophyll-containing protists -7-  2. Protozoa: nonphotosynthetic heterotrophic, unicellular eukaryotic organisms that lack cell wall. 3. Fungi: nonphotosynthetic eukaryotic organisms that have cell wall. 4. Slime moulds: share some features with fungi and protozoa. Table 1. differences between prokaryotic and eukaryotic cell. Characteristic Prokaryotic Eukaryotic Size of cell Smaller, typically 0.2-2.0 um Larger, typically 10-100 um in in diameter diameter Nucleus Not true nucleus (nucleoid): No True nucleus: nuclear membrane Nuclear membrane No protein in nucleus Protein in nucleus Single chromosome: circular Even number of chromosomes molecule of DNA(haploid) (diploid) No nucleoli Nucleoli Membrane enclosed Absent Present organelles (e.g. Golgi bodies, mitochondria, chloroplasts) Flagella Simple in structure Complex Cell membrane No carbohydrates and generally Sterols and carbohydrates that lacks sterols serve as receptors present Cytoskeleton absen Present Cytoplasmic streaming Absent Present Ribosomes Smaller size (70s) Larger size (80s); 70s in organelles Chromosome Single circular chromosome; Multiple linear chromosomes arrangement lacks histones with histones Cell division Binary fission Involves mitosis and meiosis Sexual reproduction absent Present Prokaryotic organisms comprised 1. Eubacteria: the true bacteria 2. Cyanobacteria (or blue green algae)  They are photosynthetic prokaryotic microorganisms. -8-   In cyanobacteria, the photosynthetic pigments are located in lamellae under the cytoplasmic membrane.  Unlike other photosynthetic bacteria, which are anaerobic, they possess the same chlorophyll as algae and oxidize water into oxygen  They cannot swim through liquid environments; instead they glide on solid surfaces. 3. Archaebacteria  These are group of prokaryotic microorganisms that are different from eubacteria in DNA, cell wall, and metabolism.  The archaea are diverse, in morphology (cell shape, cell aggregation, gram reaction) and physiology (nutrition, respiration, growth temp, multiplication). Methanogenic bacteria Extremely halophilic Extremely they produce methane bacteria thermophilic bacteria from organic matter grow in presence of anaerobically) high concentrations of sodium chloride)  Archaeal cell wall is different from that of bacteria in that it lacks muramic acid and D-amino acids and therefore is resistant to lysozyme and b-lactam antibiotics.  Archaeal membranes contain polar lipids such as phospholipids, sulfolipids, and glycolipids and also contain nonpolar lipids. Whittaker’s Five-Kingdoms System  It is based on three levels of cellular organization and three main types of nutrition; photosynthesis, absorption, and ingestion. 1. Animalia: multicellular, nonwalled eucaryotes with ingestive nutrition. 2. Plantae: multicellular, walled eucaryotes with photoautotrophic nutrition. 3. Myceteae (Fungi): multicellular and unicellular, walled eucaryotes with absorptive nutrition. -9-  4. Protista: unicellular eukaryotes with various nutritional mechanisms. 5. Monera (Procaryotae): all prokaryotic organisms.  It does not distinguish bacteria from archaea. Woese Six Kingdoms  It replaced the Monera kingdom in Whittaker's five kingdoms by Eubacteria and Archaebacteria. Woese’s Three-Domains  Woese and collaborators (1988) used rRNA studies to group all living organism into three domains:  Bacteria: contain prokaryotic organisms with bacterial rRNA and membrane lipids that contain ester-linked straight-chain fatty acids and cell walls contain muramic acid.  Archaea: contains prokaryotic organisms with 1. archaeal rRNA 2. membrane lipids with ether-linked branched aliphatic chains 3. cell wall lacks muramic acid. 4. distinctive RNA polymerases 5. ribosomes with a different composition and shape than those of bacteria. -10-  Eucarya: contains all eucaryotic organisms (including kingdoms; myceteae, protista, plantae, animalia). Microorganisms Non-cellular cellular Viruses and viroids Prokaryotic Eukaryotic A- Bacteria A- Algae B- Cyanobacteria B- Fungi C- Archaebacteria C- Protozoa D- Slime moulds Viruses  Viruses are submicroscopic infectious agents, which are small packets of nuclear material within a “coat” of protein with only a few enzymes.  They are “obligate intracellular parasites” that replicate only inside living -11-  cells. So they parasitize and cause diseases in all cellular living beings. Viroids  Viroids are naked circular ssRNA molecules which have no enzymes or proteins.  Viroids have no capsids and their RNAs do not act as mRNAs. Prions  Prions are infinitely small proteinaceous infectious particles (PrP) without a nucleic acid.  Proins cause progressive, degenerative central nervous system disorders in animals like bovine spongiform encephalopathy (mad cow disease). TAXONOMIC DIVISIONS OF LIVING ORGANISMS Systematics is the scientific study of organisms for characterizing and arranging them in an orderly manner. Systematics includes: 1. Classification: It is the arrangement or ordering of organisms into groups (taxa) based on similar characteristics and includes species as the smallest and most definitive level of division. 2. The taxa comprising the family, genus, and species are the levels of classification most commonly used for the pathogenic bacteria, protozoa, and fungi. 3. Taxonomy: Taxonomy is the science of biological 4. Identification: this is the arrangement of unidentified organisms to a particular class in a previously made classification. 5. Nomenclature: assigning correct international scientific name to organisms.  The binomial system of nomenclature devised by Carl von Linne (Carolus Linnaeus), the genus name is capitalized while the species is not; both terms are italicized (e.g., Escherichia coli). -12- 6. Phylogeny: it is the study of the evolutionary history of organisms.  The taxonomic ranks (in ascending order) are: species, genus, tribe (sometimes), family, order, class, phylum or divison, kingdom and Domain  The basic taxonomic group of classification is the “species” which is usually defined as a group of organisms whose members can interbreed. A prokaryotic species is a collection of strains that share many stable properties and differ significantly from other groups of strains. IMPORTANT TYPES OF MICROORGANISMS VIRUSES ☼ A virus is an infectious agent that is mainly constructed of two components: 1. A genome consisting of either ribonucleic acid (RNA) or deoxyribonucleic acid (DNA), but not both 2. A protein-containing structure (capsid) composed of subunits called capsomers designed to protect the genome. The genome together with capsid is called nucleocapsid. ☼ Envelope: it is present in many viruses and composed of a protein-containing lipid bilayer ☼ A complete virus particle is called a virion. General properties of viruses 1. Very small in size (~20-300 nm) -13-  2. They are not retained by bacterial filter 3. They cannot be seen by ordinary microscope (need EM) 4. They contain one type of nucleic acid (DNA or RNA) 5. They are obligate intracellular organisms (No intracellular organizations or genetic information for enzymes synthesis) Viral symmetry  In electron micrographs, viruses appear to be constructed of small morphological units, which are spherical, ring, or cylindrical in shape.  The way of arrangement of these units is called viral symmetry that may be  icosahedral (or cubic), helical or complex Cultivation of Viruses  Viruses replicate only inside living cells  methods for viral cultivation include I. Tissue culture Tissue cultures: as a monolayer of cultured cells that can be used for cultivation of viruses. Cell cultures are of three types a- Primary cell culture:  The cells can be subcultured from 2-3 times. They are obtained directly from tissues of living organism, e.g. from the kidney of Rhesus monkey. b- Secondary or Semi-continuous cell culture -14-  These cells can be subcultured for many generations (30-50) without dying out. These are derived from transformed cells. c- Tertiary cells cultures (Continuous cell lines)  These cells are derived from cancer tissue. These cells can be cultured for unlimited number, e.g. Hela cells. II. Chick Embryo: embryonated eggs of chickens provide different types of cells and tissues that support the multiplication of many viruses. III. Animal inoculation  Some viruses cannot be cultivated on chick embryo or tissue cultures. These can be inoculated into whole animal.  Some viruses cannot be cultivated by any of the mentioned methods. Steps of replication of viruses 1. Attachment: Viral proteins on the capsid or phospholipid envelope interact with specific receptors on the host cellular surface. 2. Penetration: Penetration is the passage of the virion from the surface of the cell across the cell membrane and into the cytoplasm. -15- Receptor-mediated endocytosis The infecting virus particle is bound to the host cell surface receptor and the cell membrane invaginates, enclosing the virion in an endocytotic vesicle (endosome). Membrane fusion Some enveloped viruses enter a host cell by fusion of their envelope with the plasma membrane of the cell. 3. Uncoating: The viral capsid is removed and degraded by host enzymes releasing the viral genomic nucleic acid. 4. Replication: transcription or translation of the viral genome is initiated. This stage of viral replication differs greatly between DNA and RNA viruses. This process results in the synthesis of viral proteins and genome. 5. Assembly (maturation): After synthesis of viral genome and proteins, viral proteins are packaged with newly replicated viral genome into new virions that are ready for release from the host cell. Most DNA viruses assemble in the nucleus, whereas most RNA viruses develop in the cytoplasm. 6. Virion release: by lysis or budding.  Naked viruses are released by lysis of the infected host cell  Enveloped viruses are released by budding. -16-  FUNGI  Fungi are non-photosynthetic eukaryotic organism.  All fungi are heterotrophs, requiring organic compounds for energy and carbon.  Fungi are aerobic or facultatively anaerobic The majority of fungi are saprophytes in soil and water, where they primarily decompose plant material.  Fungi include yeasts, moulds, and fleshy fungi. The mycelial fungi or moulds  They grow as a mass of branching interlacing filaments “hyphae” known as mycelium. Hyphae have cell wall with cross-walls perforated, allowing free moving of nuclei and cytoplasm.  The hyphae may be septate or the entire organism may be coencytic (multinucleate mass of continuous cytoplasm within a series of branching tubes). -17-  Cell wall contains chitin.  The hyphae grow by elongating at the tips. Each part of hyphae is capable of growth.  The portion of the mycelium concerned with obtaining nutrients is called the vegetative mycelium and the portion concerned with reproduction is the reproductive or aerial mycelium. Yeasts  They are unicellular fungi.  Yeasts are distinguished from moulds in: a. Cell wall composition (containing glucan and mannan). b. Colonial morphology (Yeasts produce a colony similar to a bacterial colony. c. Their higher optimum temperature for growth (25-30oC) compared with moulds (20-25oC) and faster growth.  Yeasts are typically spherical or oval in shape. -18-  Yeasts grow by budding.  In some species of yeasts buds are produced that fail to be detached resulting in a short chain of cells called a pseudohypha (pseudomycelium).  Yeasts are capable of facultative anaerobic growth.  In absence of oxygen they ferment carbohydrates and produce ethanol and carbon dioxide. This is the basis of the wine making and baking industries. Reproduction of fungi  Fungi reproduce by forming sexual and asexual spores. Asexual spores Sexual spores  They are formed by the aerial  They result from the fusion of nuclei mycelium of one organism. from two opposite strains of the same species of fungus.  When the spores germinate they  Organisms that grow from sexual spores result in organisms that are will have genetic characteristics of both genetically identical to the parent. parent strains. Classification of fungi according to sexual reproduction 1. Class Zygomycotina (Phycomycetes)  Zygomycotina have non-septate mycelium and their asexual spores (sporangiospores) are formed inside a specialized body called sporangium.  Sexual spores (zygospores) result from fusion of two nuclei that are separated from the rest of fungus body by cell wall (cells).  Examples of this class are Rhizpus nigricans and Mucor spp. -19-  2. Class Ascomycotina (Ascomycetes)  Ascomycotina (sac fungi) include moulds with septated hyphae or yeast.  Their sexual spores (ascospores) are formed in an ascus.  The asexual spores (conidiospores or conidia) are formed on specialized hyphae called conidiophores.  Examples of this class are Trichophyton, Microsporum, and Blastomyces species. Ascospores 3. Class Basidiomycotina (Basidiomycetes)  Basidiomycotina or club fungi have septated hyphae.  The sexual spores (usually 4 basidiospores) are formed on the surface of club- shaped organ called basidium.  Asexual spores are formed as conidia.  Example of this group is Cryptococcus neoformans. -20-  Basidispores 4. Class Deuteromycotina (Deuteromycetes) or imperfect fungi  They have no known sexual process.  Examples of this class are Epidermophyton, Sporothrix, and Candida spp. Candida albicans Asexual fungal spores 1. Sporangiospores: spores borne inside a sac or sporangium formed on the top of aerial non-septate hypha called sporangiophore. 2. Conidia: asexual spores formed on the termini or sides of hyphae or conidiophores. The common types of conidia are a) Arthrospores (arthroconidia) are conidia formed by segmentation of filament of a septated mycelium into separate cells. b) Chlamydospores: are conidia formed as enlarged, thick-walled cells within hyphae. c) Blastospores: are blastic spores produced as bud that then separate from the parent cell. -21- Dimorphism  The phenomenon of having two forms by certain fungi is called dimorphism.  For example they may grow as filamentous form in the soil, whereas in a suitable animal host they may reproduce like yeast.  Example of dimorphic fungi is Histoplasma capsulatum that grows at room temperature at 25oC on a suitable culture medium as filaments (mycelial form), but when grow on blood agar at 37oC it will form a single-celled rounded yeast.  Dimorphism is advantage to fungi because it allows them to reproduce more rapidly in adverse conditions. SLIME MOULDS  Slime moulds are characterized by the presence of an amoeboid multinucleate mass of cytoplasm called a “plasmodium” as a stage in their life cycle.  The creeping plasmodium gives walled spores that germinate to produce unflagellated amoebas (myxamoebae), which undergo sexual fusion and grow into typical plasmodia again. -22-  PROTOZOA  Protozoa are unicellular non-photosynthetic chemoheterotrphic eukaryotic microorganisms.  A typical protozoan cell consists of a tough structure (pellicle) covering, a plasma membrane and cytoplasm.  The cytoplasm can be distinguished into an outer layer (ectoplasm) and an inner layer (endoplasm).  Within the cytoplasm there are one or two nuclei, mitochondria, food vacuoles and contractile vacuoles, which pump out excess water. It also has structures for motility.  Protozoa ingest their food as solid particles by phagocytosis or through mouth opening (cytosome). Reproduction  Almost all protozoa are able to reproduce both sexually and asexually.  Asexual reproduction is by splitting into two cells of equal size by binary fission, or by multiple fissions, producing several organisms (schizogony).  Sexual reproduction occurs by fusion of two cells or gametes to give diploid zygote (gametogony).  The trophic and reproducing form of protozoan is called trophozoite.  Under certain adverse environment conditions (lack of food or moisture, adverse temperature changes), many secretes a heavy protective coating around itself, forming a round or oval cyst.  The cyst allows parasitic forms to survive outside the host. Classification of protozoa of clinical importance Sarcodina (amoeba)  These are unicellular, motile by psuedopedia, feed by phagocytosis and reproduce asexually by binary fission. -23-  The organism may exist as trophozoite or cyst e.g. Entamoeba histolytica. Trophozoite cyst Ciliophora  These are unicellular organisms that move by cilia and have two nuclei (macronucleus and micronucleus) e.g. Balantidium coli.  They feed via cytosome and reproduce asexually by binary fission. Trophozoite cyst Mastigophora  These are unicellular organisms, motile by flagella or mastegotes, and reproduce asexually by binary fission e.g. Giardia lamblia Trophozoite cyst Sporozoa  These organisms have no organs of locomotion and produce spores.  Their life cycle is complex involving alternating sexual (by gametogony) and asexual reproduction (by schizogony) in more than one host. -24-  Examples of sporozoa are Plasmodium spp., and Toxoplasma spp. Life cycle of Plasmodium BACTERIA General Properties  Bacteria are heterogenous group of prokaryotic microscopic organisms.  They inhabit diverse environment and perform many functions, many of which are essential for life on earth e.g. recycling of nutrients.  Bacteria are unicellular organisms except for actinomycetes (filamentous bacteria). Actinomycetes  Bacteria have cell wall that contains mucopeptide polymer called murein or peptidoglycan (except Mycoplasma).  Bacteria multiply by binary fission except actinomycetes that form conidiospores.  All bacteria are heterotrophic except for photosynthetic and chemolithotrophic bacteria -25- Morphology of bacteria  Bacteria have three fundamental morphological forms: spherical (coccus), rods (bacillus) and curved or spiral (vibrio, spirillum and spirochaete).  Cocci may be arranged like bunches of groups (Staphylococcus) in chains (Streptococcus) in pairs (diplococci, e.g. pneumococci and Neisseria).  They may exist in groups of four (Micrococcus tetragenous) or in cubical packets of eight (Sarcina).  Bacilli are rods. Some bacilli form threads (Proteus) or grow in chains (Bacillus) or produce club shaped organisms (Corynebacterium). Some bacilli are spindle shaped (fusiforms). Special Types of bacteria Actinomycetes  Actinomycetes grow as branched, long or short filaments of cells.  They divide by binary fission and may or may not produce external spores.  The majority of these organisms are soil and water saprophytes. -26-  Actinomycetes include the following genera: Actinomyces:  The genus Actinomyces contain obligatory anaerobic or microaerophilc that grow in a mass branched filaments (mycelium). Nocardia: Members of this genus are distributed in the soil. Streptomyces:  They grow in long branched filaments, but unlike Actinomyces and Nocardia they form long chains of spores called conidia.  Members of the genus Streptomyces are rarely pathogenic, but they can produce antibiotics. Rickettsiae  These are obligate intracellular parasites, because they lack some important metabolites (NAD) and depend on host cell  They are smaller in size than other bacteria but are still visible under light microscope.  Unlike bacteria their growth is enhanced in the presence of sulfonamide.  They are parasites of arthropods (e.g. fleas, lice ticks, and mites) from which they can infect man and animals. Examples of rickettsiae are R. Prowazekii that causes epidemic typhus. -27-  Chlamydia  The chlamydiae are obligate intracellular parasites that are metabolically defective and cannot synthesize ATP, so described as energy parasites.  They have complex method of reproduction  The chlamydiae are divided into group A (Chlamydia trachomatis) that cause trachoma & conjunctivitis and is inhibited by sulfonamide, and group B (Chlamydia psittacosis) that cause severe pneumonia and is resistant to sulfonamide. Mycoplasmas  Mycoplasmas are bacteria that lack cell wall, so it can acquire different morphological form (pleomorphic).  They have three-layered cell membrane.  They are the smallest organism known that are capable of growth and reproduction outside the living host cells.  They grow as tiny fried egg shaped colonies that cannot be seen by naked eyes. -28-  Mycoplasma are membrane parasites because they parasitize at cell surfaces.  They require cholesterol in their growth media.  They are the causative agents of pleuropneumonia so-called pleuropneumonia like organism (PPLO). Spirochaetes  Spirochaetes are heterogeneous group of slender thin-walled unicellular helical or spiral flexible bacteria that are motile.  The cytoplasm is surrounded by a cytoplasmic membrane and a pepidoglycan layer contributes to cell rigidity and shape.  One family (Treponemataceae) includes human pathogens: the Treponema, the Borrelia, and the Leptospira.  Members of Treponema are actively motile and several flagella wrap around the bacterial cell body.  Unlike other motile bacteria, these flagella do not protrude into the surrounding medium but are enclosed within the bacterial outer membrane.  Pathogenic treponemes cannot be cultivated in laboratory media and are maintained by subculture in susceptible animals. -29-  Mycobacteria  Members of the genus Mycobacterium (fungus-bacterium) possess thick, complex, lipid-rich, waxy cell walls that render them acid-fast (resistant to decolourization by dilute mineral acids or alcohol after staining with hot carbol fuchsin), and resistant to many chemical antimicrobial agents.  Mycobacteria are responsible for tuberculosis and leprosy and include a number of other saprophytic species. Bacterial cell structure  A typical bacterial cell structure consists of a cell wall, a cytoplasmic membrane (cell membrane) and a cytoplasm containing a diffuse nuclear material or nucleoid, ribosomes, and cytoplasmic structures.  Outside many bacterial cells there is a diffuse slime layer or a dense capsule.  Some bacteria possess locomotive organs called flagella, some have short hairy like appendages called fimbriae or pili.  Some types of bacteria form dormant spores under adverse environmental conditions. -30-  The nucleoid  The nucleoid carries the genetic traits of bacteria and consists of a single continuous circular molecule capable of approximately carrying 3000 to 6000 different genes.  It is a single, haploid chromosome. Some prokaryotes e.g. Borrelia and several Streptomyces spp. have linear chromosomes.  It is tightly folded to occupy 25% of the cell volume.  Some bacteria carry additional extra chromosomal but smaller molecules of DNA capable of carrying additional genes (5-160 genes). These are called plasmids.  Examples of plasmids are F-Factors (fertility factors), R-Factors (resistance factors), and Colicin-Factors Cytoplasm  This is an aqueous mixture containing 70 to 80% water in addition to colloidal and soluble materials and some organized structures like the ribosomes.  It contains different cellular materials: nutrients, metabolites, metabolic products, enzymes, structural proteins, and reserve-food Cytoplasmic Structures  Prokaryotic cells lack autonomous plastids, such as mitochondria and chloroplasts  The electron transport enzymes are in the cytoplasmic membrane.  The photosynthetic pigments of photosynthetic bacteria are localized in specialized -31-  membrane arrangements underlying the cell membrane.  In some cyanobacteria, the photosynthetic membranes often form multilayered structures known as thylakoids. The ribosomes  Ribosomes are cellular structures composed of protein and RNA  They are the organelles of protein synthesis.  The bacterial ribosomes measures approximately 100 A o in diameter and are known as 70 S ribosome (S is Swedberg unit, which refers to the sedimentation velocity that reflect the size and density of objects).  When bacterial ribosomes are not engaged in protein synthesis, they dissociate into two ribosomal subunits, 50S and 30S.  The 50s and 30s subunits are scattered throughout the cytoplasm and join together to form a 70s ribosome when protein synthesis begins.  The 30S ribosomal subunit consists of 16S rRNA and 21 proteins, while the 50S ribosomal subunit consists of 23S rRNA, 5S rRNA and 34 proteins. The cell membrane  It is composed of phospholipids and proteins  The phospholipid molecules form a double layer with the hydrophilic parts of molecules oriented outwards and the lipophilic parts inward.  The protein molecules freely moving and partly or completey traversing this layer -32-  forming “fluid mosaic structure”.  Unlike the eukaryotic cell membrane, the membranes of prokaryotes have no sterols except for mycoplasmas that incorporate cholesterol into their membranes when grown in presence of sterol containing media.  Convoluted invaginations of the cytoplasmic membrane form specialized structures called mesosomes, which function in the formation of cross-walls during cell division  The major functions of cytoplasmic membrane are: 1. Selective semi permeability and transport of solutes. 2. Electron transport and oxidative phosphorylation. 3. Excretion of hydrolytic exoenzymes. 4. Having the enzymes and carrier molecules that function in the biosynthesis of DNA, cell wall polymers, and membrane lipids. -33- The Cell Wall and Cell Envelope  In gram-positive bacteria The cell wall is thick layer (30-80nm), to counteract the high internal osmotic pressure (5-20 atm) inside the cell. The cell wall is composed mainly of peptidoglycan (up to 90%) and teichoic acids (up to 50 of dry weight of the wall).  In gram negative bacteria The cell wall is thin layer (5-20 nm) because the internal osmotic pressure of the cell is relatively low (2-5 atm) It is composed of few layers of peptidoglycan (2-5nm thick) and outer membrane made of lipoprotein, phospholipid, and lipopolysaccharide.  The backbone of the bacterial cell wall that gives it its strength is the murein, mucopeptide, or peptidoglycan.  Peptidoglycan is a complex polymer composed polysaccharides and polypeptide.  Its backbone is composed of alternating units of N-acetyl glucosamine and N-acety muramic acid.  A set of idenitical tetra peptide chains of L- alanine, D-glutamate, L-lysine or diaminopimelic acid and D-alanine is attached to N-acety muramic acid.  The terminal aminoacid (D-alanine) in one chain is attached, through peptide linkage, to the dibasic amino acid (number 3) of another chain. -34-  The polysaccharide chains of peptidoglycan are cross-linked with polypeptide chains.  The cell wall contains additional components that differ from gram positive and gram negative bacteria.  Most gram-positive cell wall contains considerable amount of teichoic acid and teichuronic acids.  There are two types of teichoic acids: wall techoic acid, covalently linked to peptidoglycan; and membrane techoic acid (lipotechoic acid) covalently linked to membrane glycolipid.  Teichoic acids constitute major antigen in gram-positive species -35-  In gram-negative bacteria, the other components constitute the outer membrane.  It has bilayered structure; its inner leaflet resembles the cytoplasmic membrane while the phospholipids of the outer leaflet are replaced by lipopolysaccharide (LPS) molecules. As a result, the leaflets of this membrane are asymmetrical  The ability of the outer membrane to exclude hydrophobic molecules is an unusual feature among biological membranes and serves to protect the cell from bile salts.  Because of its lipid nature it excludes hydrophilic molecules. However, the outer membrane has special channels, consisting of protein molecules called porins that permit the passive diffusion of low-molecular-weight hydrophilic compounds like sugars, amino acids, and certain ions.  The lipopolysaccharide of gram-negative cell wall consists of a complex lipid, called lipid A to which is attached a polysaccharide made up of a core and a terminal oligosaccharide.  Lipid A consists of phosphorylated glucosamine disaccharide units to which are attached a number of long-chain fatty acids. ß-hydroxymyristic acid, a C14 fatty acid, is always present and is unique to this lipid. -36-  LPS is extremely toxic to animals and is called endotoxin of gram-negative bacteria because it is firmly bound to the cell and released only when the cells are lysed.  The toxicity is associated with lipid A. the antigenic specificity is due to the terminal oligosaccharide.  The space between the outer and inner membrane is called the periplasmic space that constitutes 20-40% of cell volume.  The cell envelops of acid fast and related bacteria (mycobacteria, nocardia, and corynebacteria) are considerably more complex than other bacteria.  Mycolic acid (long, branched chained fatty acids up to 70C atoms) covalently bound via a polysaccharide to peptidoglycan.  Other mycolic acid containing compounds and other complex lipids form a thick waxy membranous layer outside the peptidoglycan layer. -37- Differences between Gram-positive and gram negative cell wall Characterisitic Gram positive Gram-negative Thickness of cell wall 30-80 nm 5-20 nm Internal osmotic 5-20 atm 2-5 atm pressure Peptidoglycan layer Thick, constitute Thin layer (2-5 nm), 10-20% of most of the cell wall cell wall (up to 90%) Third amino acid in L-lysine Meso-diaminopimelic acid tetrapeptide chain of peptidoglycan Constituent other up to 50% DW of cell Majority of cell wall than PG wall; Teichoic acid constituents: a) lipoprotein, and Teichuronic acid b)phospholipids, and c) lipopolysaccharide Outer membrane Absent Present Growth in presence of Protoplast spheroplast peptidoglycan inhibitors  -38- The role of cell wall  It gives the cell its characteristic shape  It gives mechanical strength to the cell and protection against high internal osmotic pressure  Cell wall acts as bases for classification of bacteria into gram positive, gram negative, and acid fast organisms.  The cell wall plays a role in the cell division.  It is a target for many antimicrobial agents (ß-lacatam antibiotics) and enzymes, (lysozymes).  The cell wall serves as antigenic determinant of the cell surface (e.g somatic antigen or O antigen) and the lipolysaccharide of gram-negative bacteria act as non-specific toxin (endotoxin). Capsule and Slime Layer  Extracellular polymer, which forms a condensed, well-defined layer closely surrounding the cell called the capsule or amorphous loose layer called slime layer (or glycocalyx).  Except for Bacillus anthracis, which form a capsule of poly-D-glutamic acid, the extracellular material is polysaccharide. Functions of capsule  It enhances the virulence of pathogens (by escaping phagocytosis).  The capsule carries the capsular antigen, KAg Flagella  Bacterial flagella are thread-like appendages. They are the organs of locomotion.  Flagella are made of protein called flagellin. Flagella carry the flagellar antigen, H Ag.  According to the arrangement of flagella on their surfaces, bacterial cells can be monotrichous, amphitricous, lophotricous and peritrichous. Those cells -39-  without flagella are described as atrichous. Pili (Fimbriae)  Many gram-negative bacteria have rigid surface appendages called pili or fimbriae  shorter and finer than flagella  The pili are composed of protein called pilin and are the organs of adhesion.  Some bacteria have specialized pili that enables bacteria to undergo conjugation and transfer of genetic material between bacteria.  These are called sex pili and its genetic determinants are carried on transferable plasmids, called fertility factors (F-factor). -40-  Bacterial endospores  The spore is a resting cell highly resistant to desiccation, heat, and chemical agents  In response to adverse environmental conditions (like depletion of nutrition, desiccation, exposure to high temperature, etc.), each cell forms a single internal spore e.g. Bacillus and Clostridium.  When returned to favorable nutritional conditions and activated, the spores germinate to produce a single vegetative cell. Structure of spore The core  It is the spore protoplast. It contains a complete chromosome, all the protein- synthesizing apparatus, and an energy generating system.  The heat resistance of spores is due to their dehydrated state and the presence of of large amount of of calcium dipicolinate in the core The Spore wall  It is the innermost layer surrounding the inner spore membrane. It contains normal peptidoglycan and becomes the cell wall of the germinating vegetative cell. The Cortex  It is the thickest layer of the spore envelope. It contains an unusual type of peptidoglycan.  It is extremely sensitive to lysozyme; its autolysis plays role in spore germination. -41- The Coat  It is composed of a keratin-like protein.  The impermeability of this layer confers on spores their relative resistance to antibacterial chemical agents. The Exosporium is a lipoprotein membrane containing some carbohydrate.  The spores may vary in shape, size and site of formation from one organism to another.  The spore may be oval, spherical or cylindrical in shape.  According to their site of formation inside the cell, spores may be central, terminal, or subterminal. The extreme high-resistance of the spore is due to: 1. Low permeability due to thick cortex and keratin-like structure of coat. 2. Presence of pyridine 2-6 dicarboxylic acid and divalent metals and dipicolinic acid, which give heat resistance character. 3. Low content of water that lead to low metabolic activity. -42- Germination  It occurs when environmental conditions improve  The germination process occurs in three stages: 1. Activation by heat, abrasion, acidity, and compounds containing –SH groups. 2. Initiation  An autolysin rapidly degrades the cortex peptidoglycan. Water is taken up, calcium dipicolinate is released, and many spore constituents are degraded by hydrolytic enzymes. 3. Outgrowth Degradation of cortex and outer layers results in emergence of new vegetative cells Other Bacterial Products Pigments  Some bacteria produce specific pigments when they are cultivated under specific conditions.  These pigments may by  Intracellular: they cannot diffuse into the medium and thus only the cells are coloured  Extracellular: they diffuse into the medium coloring it Toxins  These are poisons which are produced as a result of the growth of the bacteria or may be part of the bodies of the bacteria.  They may be extracelluar (exotoxins) or intracellular (endotoxins).  The lipopolysaccharides of gram-negative bacteria (pyrogens), produce a febrile reaction upon injection in mammals, are bacterialendotoxins  They are dangerous when present in parentals and must be removed.  Endotoxins are inactivated only by heating at temperatures over 300 oC but not by normal sterilization processes -43-  Table 3. Differences between exotoxins and endotoxins. Characteristic Exotoxins Endotoxins Secretion Excreted by living cells, found in Part of the cell, released after cell death high conc. in fluid medium and disintegration Chemical nature Polypeptide lipopolysaccharide, lipid A is responsible for toxicity. Stability to heat Thermolabile, toxicity destroyed Highly thermostable, withstand heat over by heat over 60o C 60o C (destroyed over 300 o C) Antigenicity and Highly antigenic, stimulate form Poorly antigenic, do not stimulate immunogenicity of high-titre antitoxins formation of antitoxins stimulate (antibodies that neutralize toxin formation of antibodies to polysaccharide action) part Toxicity Highly toxic, fatal for lab weakly toxic, fatal for lab animals in animals in µg or less hundreds of µg Specific action Highly specific in its action less specific Toxoid formation Converted into antigenic toxoid Not converted into toxiod by formalin, acid... etc. Fever induction Do not produce fever in host Often produce fever in host Bacterial Reproduction  Bacteria multiply by simple binary fission.  The chromosome duplicates itself and each copy is attached to the CM at the mesosome.  A septum is laid in its middle dividing it into 2 cells that are copies of the mother cell.  The 2 duagther cells may or may not remain close to one another and if they do remain they form cell aggregates that are characteristic of the species.  Bacteria divide very rapidly (some bacteria, like E. coli, divide every 20 minutes) and a single cell isolated in the laboratory can produce millions of bacteria overnight. -44-  NUTRITION AND CULTIVATION OF MICROORGANIMS  Microorganisms require nutrients to build up their cellular components and provide the required energy for their activities.  According to the mechanism of nutrition organisms can be osmotrophic or phagotrophic.  Phagotrophic organisms ingest (engulf) their food and digest it internally  Osmotrophic organisms secrete exoenzymes that digest complex nutrients into simpler absorbable form. Nutritional Requirements 1. Energy requirements  Organisms vary in the way they get their energy requirements.  Phototrophic organisms or phototrophs: Photosynthetic organisms, like plants, algae, and some bacteria can use light energy  Chemotrophic organisms or chemotrophs: they obtain their energy needs from chemical reactions.  Chemoorganotrophs obtain their energy form oxidation of organic compounds  Chemolithotrophs obtain the energy from oxidation of inorganic material.  Different types of bacteria may oxidize specific substances  The most common are the bacteria, which oxidize various organic compounds commonly sugars  Hydrogen bacteria convert hydrogen to water  Methane bacteria oxidize methane to CO2  Nitrifying bacteria oxidize ammonia to nitrite or nitrate  Sulplur bacteria which oxidize H2S to S or to sulphate. 2. Carbon requirements  Autotrophic organisms like plants, can get their carbon needs from inorganic -45-  carbon compounds (CO2).  Heterotrophic organisms obtain their carbon requirements from complex organic compounds.  Combining the energy and carbon requirements, organisms can be classified into four categories: 1. Photoautotrophic organisms: obtaining energy from light and carbon from CO2. 2. Photoheterotrophic organisms: obtaining their energy needs from light and their carbon needs from organic carbon compounds. 3. Chemoautotrophic organisms: obtaining their energy from chemical reactions (oxidation) and their carbon from CO2. 4. Chemoheterotrophic organisms: obtaining energy from chemical reacations and their carbon needs from CO2. 3. Nitrogen requirement  Nitrogen is required for the synthesis of amino acids (and proteins); purines and pyrimidines (and nucleic acid); vitamins and coenzymes.  Nitrogen-fixing organisms: few species of organisms can assimilate gaseous nitrogen and make the gaseous nitrogen depot available for other organisms and these are called. These grow in symbiosis with the roots of legumes  Non-exacting or non-fastidious organisms can use nitrogen in inorganic form, such as ammonium salts and nitrates as the sole source of nitrogen.  These bacteria are simple in their growth requirements (require only a few simple nitrogen compounds) but complex in its metabolism.  Fastidious or exacting organisms are strict in their requirement and need specific materials in their food; e.g. Haemophilus influenzae that requires the “X” factor (haematin), and/or the “V” factor (NAD) to grow. -46-  4. Oxygen requirements  Microorganisms can be classified into four categories with respect to atmospheric oxygen a. Strict or obligate aerobes  Obligate aerobic organisms require atmospheric oxygen for their growth and fail to grow in absence of oxygen since only oxygen can act as a terminal electron acceptor.  These organisms have cytochrome oxidase, catalase and superoxide dismutase that neutralize the reactive oxygen species such as hydrogen peroxide that are toxic. b. Obligate anaerobes (Strict anaerobes)  Obligate anaerobic organisms cannot grow in presence of oxygen and require the removal of oxygen by physical, chemical or biological means for their growth.  These lack cytochrome oxidase, superoxide dismutase and catalase enzymes  In presence oxygen, the toxic H2O2 is accumulated.  Instead they give off electrons to inorganic compound other than oxygen, e.g. nitrate and sulfate (anaerobic respiration) or to organic compound (fermentation). c. Facultative anaerobes  Facultative anaerobic organisms can grow in presence or absence of oxygen.  These organisms can switch their metabolism between fermentation and respiration according to the environmental conditions.  Aerobes and facultative anaerobes are protected from the toxicity resulting from hydrogen peroxide and the more toxic free radical superoxide is avoided by the activity of superoxide-dismutase and catalase enzymes that catalyzes the reaction 2O2  2 H  O2 +H2O2 2H2O2 catalase H2O + O2 d. Microaerophiles  Microaerophilic organisms cannot grow in absence of oxygen but require a low -47-  tension of oxygen.  They have a limited amount of cytochrome oxidase and lacks catalase.  In high oxygen tension, H2O2 accumulates and the cells die.  This happens in the top of a tube of liquid medium in which they grow. Bacteria grow at deeper layers (insufficient oxygen). At the bottom of the tube there is no oxygen and hence they fail do not grow. -48-  5. Minerals and trace elements requirements  Sulfur is a constituent of certain amino acids, e.g. cysteine and coenzymes  Iron is required for the synthesis of cytochromes.  Many enzymatic reactions require certain heavy metal ions such as Mg, Zn. Co, Cu, and Mn.  Sodium, potassium and calcium ions are required for enzymatic reactions.  Large quantities of phosphorous are required for the synthesis of nucleotide (including ATP), nucleic acids and lipids. 6. Growth factors requirements  Growth factors are nutritional elements that the organism need but cannot synthesize it or synthesize it in amounts not sufficient for their normal growth.  These growth factors may be amino acids, complex organic compounds, or vitamins that act as important coenzymes.  Examples: thiamine for Bacillus anthracis, riboflavin for Clostridium tetani, and vitamin K for Bacteroides fragilis. 7. Moisture requirements and water activity  Microorganisms require free water for growth  Water in the environment dissolves foods and wastes, as a medium for biochemical reactions and as a source of hydrogen and oxygen.  The available water surrounding the organisms is expressed as water activity (a w). The water activity (aw) is the relative humidity of the air space in the immediate environment or the partial pressure of water in the solution in relation to that of distilled water at the same temperature (aw) = RH/100 Nutrient broth has thus aw = RH.98.5/100 = 0.985  Microorganisms commonly require water activities above 0.90 in order to grow and multiply and the optimal is 0.95. Some fungi can grow in 0.60.  Moulds can generally grow in the least amount of moisture thus it can be seen on -49-  almost dry surface e.g. on leather and wall paints, while yeasts require more moisture.  Bacteria require much more moisture than fungi and among them some require more moisture than others. Gram-negative bacteria require (aw) more than 0.95.  The water activity of foods can be used to predict how rapidly food spoilage occurs.  Foods with high water activity spoil more rapidly.  The water activities of foods can be artificially lowered, by drying or adding sugars or salts in order to prevent food spoilage Environmental Requirements 1. Temperature requirements  Each organism can survive and grow within a temperature range with an optimum temperature for growth.  The lowest temperature at which the organism can grow is the minimum growth temperature while the maximum temperature above which growth ceases is the maximum growth temperature. Table 5. Types of microorganisms according to optimum growth temperatures Type of MO Growth temperature (oC) Minumun Optimum Maximum Psychrophilic 0 10-15 20 Mesophilic 20 30-40 45 Thermophilic 45 55-60 80  Thermotolerant organisms can survive at high temperature, however, these temperatures are not optimal for their growth. -50-  2. Oxidation reduction potential (Eh)  Anaerobic microorganisms require low Eh in the medium for growth.  This is done by removal of atmospheric oxygen by physical or chemical means, e.g. by including reducing agents, like dextrose, cysteine, thioglycolic acid (as in fluid thioglycolate medium) or myoglobin (as in cooked meat medium).  In anaerobic jars oxygen may be removed by chemical reactions that combines oxygen with the hydrogen liberated from a reaction kit or by evacuation and replacement of air in the jar with N2 or CO2. -51-  Covering solid media with paraffin oil may be an alternative.  Microaerophilic organisms require an atmosphere containing 5-10% CO2. This can be achieved by using CO2 incubators or candle jar. CO2 incubator Candle jar 3. Osmotic pressure  Osmotic pressure is the minimum amount of pressure that must be applied to solution in order to prevent the flow of water across a membrane within the solution.  The osmotic pressure of the medium affects the growth of the microorganisms.  In hypertonic medium the cytoplasmic membrane shrinks, as a result of loss of water, thus separating the rigid cell wall from the cytoplasmic membrane and this hinders growth (plasmolysis).  In hypotonic medium the cell absorbs water and the cytoplasmic membrane expands (plasmoptysis). Unless the cell wall is intact the cell will lyse.  Some organisms can grow at media of high osmotic pressure and these organisms (halophilic or osmophilic).  Some bacteria and many yeasts and moulds are osmophilic i.e. can grow easily in -52-  very hypertonic environments with very low moisture content.  Certain ocean bacteria are halophilic  Some bacteria can tolerate up to 6.5% or 9% sodium chloride, this being a characteristic of the species.  Foods fruit preserves and salted fish are resistant to spoilage by microorganisms because of their high osmotic pressure (hypertonic). 4. The pH requirements  The pH of the medium affects the growth of microorganisms, as it affects the charges on the surface of the cell and transport of nutrients and other functions of the cell.  Most bacteria like pH around neutrality, pH 6-8 (in most culture media, pH 6.8-7.2).  Few bacterial species prefer acid environments, e.g. species of Lactobcillus and Acetobacter  A few can tolerate high pH (9), e.g. Vibrio cholera.  Some organisms like Thiobacillus thioxidans can grow at extremely low pH (conc. H2SO4).  The fungi in general prefer acidic pH (5-6) in their culture media.  Food can be preserved by lowering their pH, as in pickling. -53-  Culture media for growth of microorganisms  For their growth and cultivation, microorganisms require culturing media that satisfy the nutritional and environmental factors.  Different types of culture media are available for cultivation of microorganisms.  According to its chemical composition the culture media can be: a. chemically defined (synthetic), i.e. its exact chemical composition is known b. complex media, the one in which one or more ingredient is not exactly defined.  Based on its physical state culture media can be either liquid media or solid media (semi-solid). According to the purpose of use, culture media can be:  Ordinary (universal or general purpose) media  These culture media allow growth of many types of microorganisms and contains nutrients that satisfy the need of these microorganisms.  Examples of these media are nutrient broth or nutrient agar.  Although these media allow the growth of many types of microorganisms, it cannot satisfy the need of fastidious organisms.  Enriched media  In order to grow fastidious microorganisms, the culture media must contain extra nutrients of complex organic matter like blood, serum, and egg etc. Examples of these media are blood agar, chocolate agar etc.  Selective media  These media allow the growth of few number of organisms while suppressing the others, by including inhibitory agent that differentially inhibit the latter.  Examples are the azide media for selection of streptococci, the addition of bile salts for selection of enteric bacteria, tellurite for selection of diphtheroids, selenite for -54-  selection of Salmonella etc. Enrichment media: liquid selective culture media are used before using selective media in order to increase the number of the targeted microorganisms before their isolation.  Differential media  Besides growing the microorganisms, these media allow the distinction between the different types according to the pattern of growth.  Example of these are the media designed to distinguish between microorganisms according to their ability to ferment certain sugars, by including such sugar and acid base indicator in the medium, so the fermenting organism would change the color of the indicator and so its colonies appears different from the non-fermenters.  The differential medium could be an enriched or selective besides being differential at the same time.  Examples of enriched and differential medium is the blood agar when used for streptococci as it allows their differentiation according to their haemolytic activities into α, β, or γ.  Example of selective and differential media is MacConkey agar that selects gram negative enteric bacteria and differentiates them according to their ability to ferment lactose. MICROBIAL GROWTH AND GROWTH CURVE  Growth can be defined as an orderly increase in all chemical constituents of an organism.  Growth normally results in cellular multiplication except in the coencytic organisms.  In multicellular organisms, cellular multiplication leads to an increase in the size of the individual. In unicellular organisms, it leads to an increase in the number of individuals. -55-  The measurement of growth A. Measurement of cell mass a) The bacteria of a culture are rather uniform in size, weight, opacity, nitrogen content, enzyme content and cellular activity, etc. b) Measurement of one of these properties of a culture is a measure of the number of bacteria in it. 1. Dry weight The dry weight is proportional to the cell mass, which is proportional to the cell number. If the cells are collected, washed several times and then desiccated, their dry weight may be proportional to their number. 2. Total nitrogen contents The nitrogen content in a culture reflects the biopolymer contents and is proportional to cell number since bacteria generally contain about 14% of their dry weight in the form of nitrogen. 3. Protein contents  Protein contents are proportional to cell mass. 4. Particular enzyme  cell has a constant number of molecules of particular enzyme, the quantity of such enzyme reflects the cell number. 5. Overall rate of metabolic processes  The rate of acid production by bacteria or rate of consumption of a sugar, production of a pigment, or a gas may be measured quantitatively and used as a measure of bacterial (or fungal) counts using calibration curves  e.g. “acidimetric” microbiological assays of vitamins and amino acids 6. The incorporation of radioactive material  rate of incorporation of a radiolabelled nutritional element, e.g. specific amino acid is a sensitive method for following growth. 7. The optical measurements  Bacteria in a suspension may absorb, reflect and deflect light rays in proportion to their cell mass and to their numbers. -56-  The amount of light scattered measured by nephelometer; the amount of light transmitted or absorbed (retained) measured by spectrophotometer or a colorimeter and the amount of opacity measured by comparison with standard opacity tubes; may thus be proportional to the number of bacteria in the suspension.  the color of the medium or the bacterial pigments may interfere  The method is so simple and very common in turbidimetric assays of antibiotics, vitamins and amino acids. B. Measurement of cell number Importance  standardization of inocula in microbiological assays  evaluation of sterilization techniques and antimicrobial agents  industrial fermentations  assay of vitamins and amino acids, etc.  Total counts  This determines the number of dead and live cells. a. Counting chamber  A sample of the suspension is placed on the surface of a special slide and is -57-  covered with a special cover slip.  The slide is often called “counting chamber” and has a square depression (micro chamber) in which a specific volume (usually 0.01 ml) of the suspension is enclosed.  The bottom of the chamber is marked in squares of exact dimension.  When the number of bacteria in several squares is determined microscopically, the total number of bacteria in the chamber and in one ml of the suspension could be calculated. b. Coulter counter An electronic device called “Coulter counter” used for enumeration of particles in suspensions can be used to enumerate bacteria. -58-  Viable count  This method counts only live bacteria, which can grow after being cultured in appropriate media.  After a series of known dilutions (Serial dilution technique), a known volume is cultured into appropriate medium.  The developed colonies are counted and the original number of viable bacteria in the sample is worked out and expressed as colony forming units per unit volume (cfu/ml).  Several techniques employing solid media are used, such as the pour plate method, the surface plate counting method and the membrane filter method.  Kinetics of bacterial growth  Bacteria multiply by simple binary fission.  The increase in cell mass or cell number with time is used to express growth rate.  If the amount of growth is plotted against time, a characteristic curve called the growth curve will be obtained. -59-  When the conditions are optimal for growth and multiplication, the cells divide at a constant high rate and the increase in cell mass or number will be exponential. The Generation Time (G)  The time needed for bacterial cells to double their initial number is termed generation time “G”.  Each bacterial species has a characteristic mean generation time that may be short (20 minutes for E. coli) or long (24 hours for very slow growing species such as Mycobacterium tuberculosis).  The generation time “G” describes the rate of growth and is constant for a species (or a strain) under a given set of environmental condition. Bacterial growth curve A- Batch culture  It is a closed system in which nutrients are added once at the beginning.  In a batch culture, the growth curve can be divided into four main phases: 1. Lag phase  During this period, the cell number does not significantly increase, but there is a steady increase in cell size and great metabolic activity. -60- 2. The Logarithmic (Log) or Exponential Phase  After the lag phase, the bacteria begin to divide and their rate of growth begins to increase gradually to reach a constant rate.  The increase in cell number is exponential and there is a linear relationship between time and log the number of cells. The slope of the line represents the growth rate of the culture  Bacteria in the logarithmic phase of growth are highly sensitive to changes in temperature, pH, nutrients, inhibitory agents, or other conditions.  Cells in logarithmic phase are suitable in the microbiological methods of assay of antibiotics and growth factors. 3.The Stationary Phase  Due to the exhaustion of nutrients, the accumulation of toxic products, and the overcrowding of the bacterial population.  (death rate = growth rate). The cells appear uniform in size, spores begin to form, secondary metabolites (e.g. antibiotics and exotoxins) are produced. 4. The Death or Decline Phase  The death rate exceeds the rate of growth.  A small number of survivors may remain for months or even years in culture.  Finally, the culture becomes sterile when all the cells have died  The cells appear heavily granulated and weak. B-Continuous Culture (Open System)  To maintain cells in the exponential phase, fresh medium is continuously introduced to replace exhausted media in a constant state. Such culture is a continuous culture.  This can be avchieved by the chemostat and the turbidostat. The chemostat  This device consists of a culture vessel, with an overflow siphon and a mechanism -61-  for dripping in fresh medium from a reservoir at a regular rate.  The medium in the culture vessel is stirred by a stream of sterile air  Each drop of fresh medium that enters causes a drop of culture to remove out.  The medium is prepared so that one nutrient limits growth.  The vessel is inoculated, and the cells grow, until the limiting nutrient is exhausted, fresh medium from the reservoir allowed to flow in  Under these conditions, the cell concentration remains constant and the growth rate is directly proportional to the flow rate of the medium. The turbidostat  This device resembles the chemostat except that the flow of medium is controlled by a photoelectric mechanism, which measures the turbidity of the culture.  When the turbidity exceeds the chosen level, fresh medium is allowed to flow in -62-  C- Synchronous Culture  They are cultures in which all the cells are approximately at the same stage of the division cycle and so would divide at the same moment  In a growing bacterial culture, cells are growing non-synchronously at any time) i.e. all the cells in the culture do not divide at the same time.  It is important to synchronize the growth of the bacteria to study the properties of the cell during its division cycle e.g. the synthesis of DNA, or the susceptibility to antimicrobial agents.  If we use cells from ordinary cultures, the sample will contain cells at different stages of growth and the result will be only an average value. Synchronization techniques 1. The organism is starved for a limiting nutrient, e.g. glucose or thymine before its restoration. 2. The culture is incubated at non-optimum temperature for a while and then bringing it to the optimum growth temperature. 3. The culture is filtered through a number of layers of filter, which act as sieve. The small cells will be recovered and used as synchronously dividing population. -63-  BACTERIAL METABOLISM  Metabolism is all chemical and biological reactions that take place in the cell.  Metabolism involves catabolism and anabolism  Catabolism is the breakdown of complex material to provide energy and building units for synthesis of new cell material e.g. the breakdown of sugar glucose into lactic acid or carbon dioxide  Anabolism is the assembly of small molecules (building blocks) into more complex ones that are transported to appropriate areas in the cell and form the cell structures, resulting in cell growth. Examples of anabolism are the formation of proteins from amino acids and the formation of DNA from nucleotides.  During the breakdown of many organic molecules energy is released. Some of this energy can be conserved in adenosine triphosphate (ATP) or guanosine triphosphate (GTP)  Microorganisms can extract energy from foods and conserve it in molecules of ATP. This extraction of energy requires the modification of the food  Steps of energy production A. Digestion: it involves the breakdown of large molecules into simpler ones. For example, the digestion of proteins yields amino acids B. Transport of small molecules into the cell. C. Degradation of small organic molecules into simpler molecules. For example, the degradation of glucose gives pyruvate or acetate. Organic molecules are oxidized and the electrons are donated to electron carriers (coenzymes), such as nicotinamide adenine dinucleotide (NAD), or flavine adenine dinucleotide (FAD). Some energy is conserved in molecules of ATP. D. Complete oxidation of pyruvate or acetate to carbon dioxide.  The complete oxidation results in the reduction of additional molecules of NAD -64-  and FAD, which are used in an electron transport system (ETS) that is coupled to the synthesis of ATP.  Most of the energy in organic molecules is conserved in molecules of ATP. Enzymes  Enzymes are biological catalysts that participate in most cellular reactions.  Enzymes speed up chemical reactions without change during the process.  The name of an enzyme is constructed by adding the suffix ase to the name of substrate on which the enzyme acts. For example, the enzyme that catalyses the breakdown of a lipid is called lipase  In order to have a chemical reaction, an intermediate state, called the activated state, must be achieved and this requires energy.  The amount of energy required for a substrate molecule to reach the activated state is called the activation energy, which is expressed in calories per mole  The function of an enzyme is determined by its three- dimensional shape.  Substrate molecules bind to specific sites called active site.   The active site may have a shape that is complementary to the shape of its substrate (“lock and key” model of enzyme activity, where the substrate (key) fits into the active site of the enzyme (lock)  The functioning enzyme molecule (holoenzyme) is composed of protein part (apoenzyme) that determines the specificity of substrate and a non-protein part (prothetic group or coenzyme) that determines the reaction type. -65-  Nutrient transport  Nutrients and energy found in the environment are used by microorganisms for growth and other biological activities.  Living organisms accumulate nutrients and discharge cellular wastes by passive diffusion, facilitated diffusion, and active transport 1. Passive and facilitated diffusion  Molecules pass through the cell membrane from areas of high concentration to areas of low concentration (within concentration gradient).  Cells do not need energy  Diffusion has a limited role in transporting nutrients into the cell.  In facilitated diffusion, carrier proteins transfer nutrient molecules from the environment into the cell and increase the rate of transport into the cell. 2. Active transport  In active transport, energy driven carrier proteins transport material against concentration gradient.  Most of the nutrients that enter the cell are concentrated by active transport.  The cell expends energy to bring nutrients into the cell by hydrolysis of ATP. In this way, the cell can accumulate nutrients inside the cell at concentrations far exceeding that outside the cell. Bioenergetics  Bioenergetics is the field of study that is concerned with the production and use of -66-  energy by living organisms.  Phosphorylated nucleotides such as ATP and GTP are generally used as the source of energy.  ATP is most commonly used by the cell as the “energy currency” for reactions that require energy.  Chemotrophic organisms obtain their energy from the oxidation of molecules.  Chemoorganotrophs use organic molecules as the electron donor  Chemolithotrophs use inorganic molecules such as ammonia, molecular hydrogen, and hydrogen sulfide as their electron donors.  The principal energy storage molecules used in the cell is ATP.  The ATP molecule is rather unstable, and it is susceptible to hydrolysis. When the molecule breaks, it releases free energy in the reaction: ATP + H2 O ADP + Pi + energy  This reaction releases about 7.3 kilocalories (kcal) of energy per mole and can be used to power other reactions.  This property makes ATP the energy currency for the cell. Mechanisms of ATP synthesis Substrate-level phosphorylation  It is the synthesis of ATP when a phosphorylated substrate donates its phosphate group to adenosine diphosphate (ADP).  For example, during the biochemical reaction in which 1,3-diphosphoglycerate is converted into 3-phosphoglycerate -67- Oxidative phosphorylation  It is the synthesis of ATP when electrons are passed from one electron carrier to another in a membrane associated electron transport system (ETS).  The free energy liberated during the transport of electrons from one carrier to another is sufficient to power the synthesis of ATP. Photophosphorylation  It is the synthesis of ATP when electrons from chlorophyll are used.  Similar to oxidative phosphorylation, except that solar energy is used to oxidize light-sensitive pigments such as chlorophylls.  The electrons from the light-sensitive pigment are passed through an electron transport system in much the same way as in oxidative phosphorylation.  The passage of electrons from one carrier to another also results in the release of sufficient free energy to power the synthesis of several ATP molecules. -68-  During ATP synthesis some chemicals are oxidized while others are reduced.  Chemicals that are oxidized can be light-sensitive pigments or food molecules.  chemicals that are reduced can be other organic molecules or coenzymes such as NAD or FAD.  Coenzymes such as NAD, NADP, FAD, and flavine adenine mononucleotide (FMN) are commonly used as electron carriers in metabolic reactions.  For example, prior to oxidative phosphorylation the cell oxidizes chemical substrates, reducing electron carriers such as NAD during the process. These, in turn, carry electrons to an electron transport system where ATP is synthesized. Chemoorganotrophic Metabolism  Most eukaryotic cells and large number of bacteria, carry out chemoorganotrophic metabolism in which organic molecules are oxidized.  The free energy released from this oxidation is partly conserved in molecules of ATP synthesized by substrate level phosphorylation and/or oxidative phosphorylation.  Many organic molecules especially carbohydrates can be used as the electron source for chemoorganotrophic metabolism for the synthesis of ATP.  For example, the oxidation of 1 mole of glucose to CO 2 and water releases approximately 688 kcal.  Some of this energy is conserved and can give 38 moles of ATP. Stages of glucose oxidation 1. Oxidation of glucose to 2 pyruvate molecules  This produces 2 ATP and 2 NADH+ 2H+. 2. Oxidation of the pyruvate to CO2  This produces 8 NADH+ 8 H+ + 2 FADH2 +2 GTP.  This stage is carried out the through the tricarboxylic acid (TCA) cycle (also -69-  known as the citric acid, or the Krebs cycle). 3. Oxidation of NADH+ H+ and FADH2 in an electron transport system (ETS)  This produces 34 ATP.  All three stages together are called respiration. Fermentation  Some microorganisms can carry out only the oxidation of glucose to pyruvate and then a reduction of pyruvate or any other organic molecules by NADH+ H+. This process, called fermentation, results in the production of 2 ATP/glucose.  In the absence of an external electron acceptor (e.g., oxygen or nitrate ions), or if the cell is deficient in some enzymes of the Krebs cycle, the energy metabolism is restricted to fermentation.  In other words, when the final electron acceptor in catabolic oxidation- reduction reactions is organic compound the process is called fermentation. -70-  Respiration  Organisms that can respire oxidize pyruvate to acetate  The electrons released during the oxidation of glucose are carried by electron carriers (e.g. NAD or FAD) to electron transport system on the plasma membrane of prokaryotes.  The electron transport system consists of a sequence of molecules that are reduced and then reoxidized.  The transport of electrons or protons across the plasma membrane can release sufficient free energy to power the synthesis of ATP.  The electrons passing through the electron transport system are used to reduce molecules such as oxygen or inorganic ions called external electron acceptors.  If oxygen is the final electron acceptor, the process is called aerobic respiration.  If other inorganic molecules are the final electron acceptors the process is called anaerobic respiration.  The overall chemical reaction for aerobic respiration of glucose: Glucose (C6H12O6) + 38 ADP + 38 Pi + 6 O2  38 ATP + 6 CO2 + 6 H2O Facultative anaerobes  Chemoorganotrophs that can ferment and respire  These organisms will respire rather than ferment when the oxygen concentration is sufficiently high, where oxygen rather than organic molecule is the final electron and proton acceptor.  A facultative anaerobe consumes carbohydrate more rapidly when it ferments than when it respires. In addition, when an organism ferments, it releases wastes, such as acids, alcohols, and gases. -71-  Cells consume glucose more slowly when they respire, but their growth rate is greater.  Respiration utilizes the carbohydrate more efficiently to produce ATP, leaving much more carbohydrate available for the synthesis of cell material. Chemolithotrophic metabolism  Chemolithotrophs obtain their energy from the oxidation of reduced inorganic compounds and do not carry out fermentations.  For example, Nitrosomonas is an obligately aerobic chemolithotroph that obtain their energy from oxidation of ammonia.  During this oxidation, ammonia is oxidized to nitrite ion and the electrons are passed through an electron transport system that uses FAD instead of NAD, as its first electron carrier.  In this metabolism, ATP is generated when the electrons are passed through the electron transport system, creating a proton motive force that powers the synthesis of ATP.  The final electron acceptor is oxygen, which is reduced to water.  Other bacteria utilize other inorganic substances, such as hydrogen sulfide, molecular hydrogen, ferrous ions, nitrite ions, and sulfur, as the source of electrons of their chemolithotrophic metabolism. -72- Phototrophic metabolism  Phototrophs are organisms that absorb light for the synthesis of ATP.  These organisms utilize light energy to oxidize chlorophyll.  The electrons obtained during this oxidation are passed along various electron carriers in an electron transport system to produce ATP and/or NADPH (reducing power).  Both of these molecules are employed by phototrophic organisms to power anabolic reactions, such as the fixation of carbon dioxide. Biosynthesis (Anabolism)  Biosynthesis includes all cellular reactions in which biological polymers and their precursor molecules are made.  Microbial growth requires the polymerization of biochemical building blocks into protein, nucleic acid, polysaccharides, and lipids.  The building blocks come in the growth medium or are synthesized by the growing cells.  Growth demands a source of metabolic energy  Some metabolic pathways (as glycolysis) can be used in either directions (catabolism or anabolism) according to organism's need these are called amphibolic pathways.  Glucose-6-phosphate, phosphoenolpyruvate, oxaloacetate, and α-ketoglutarate give most biosynthetic end products.  When provided with building blocks and a source of metabolic energy, a cell -73-  synthesizes macromolecules.  Synthesis of nucleic acids and protein is template-directed.  DNA serves as the template for its own synthesis and for the synthesis of RNA. mRNA serves as the template for the synthesis of proteins.  Synthesis of carbohydrate and lipids is determined by enzyme specificities.  When the macromolecules are synthesized, they self-assemble to form the structures of the cell, e.g., ribosomes, membranes, cell wall, flagella etc.  The rate of synthesis and the metabolic pathways must be regulated so that biosynthesis is balanced. Regulation of metabolism  In their normal environment, microbial cells regulate their metabolic pathways, so that no intermediate is made in excess.  The concentration of nutrients in the environment is involved in the metabolic reactions regulation  When a carbon source suddenly becomes abundant, the enzymes required for its catabolism increase in amount and activity.  When a building block (such as an amino acid) suddenly becomes abundant, the enzymes required for its biosynthesis decrease in both amount and activity. Constitutive enzymes These enzymes are generally synthesized inside the microbial cell irrespective of the presence of their substrates -74- Induced enzymes They are enzymes that are not produced by the organism except in the presence of the substrate itself Enzyme induction  The majority of enzymes are produced only when needed and in the amount needed. End product repression  When the product of the enzyme reaches a certain concentration (above what the cell needs) this product represses further synthesis of the enzyme Feedback inhibition and repression  They control the enzymes catalyzing biosynthesis (i.e. anabolism)  In feedback inhibition the end product in the pathway inhibits the action of the first enzyme in that pathway.  In repression the end product inhibits the production of all the enzymes concerned in its biosynthesis. End product inhibition  The product affects the activity of the enzyme. High concentration of the product immediately inhibits the activity of enzyme molecules already synthesized and the rate of product formation decreases. -75-  MICROBIAL GENETICS  The nucleic acids (DNA and/or RNA) are the carrier of genetic material in living organisms.  The genetic material determines the characteristics of the organism through two important functions: 1. ability to duplicate itself and transfer to the progeny, through DNA or chromosomal replication 2. ability to express these genetic traits  through transcription to mRNA and translation into proteins. The Structure of nucleic acids A nucleic acid (NA) consists of  a backbone of alternating phosphate and sugar molecules.  To each sugar is attached a pyrimidine or purine base.  The structure of NA may be also regarded as a polymer of nucleotides. DNA  Composed of purine & pyrimidine bases that bind to the sugar 2-deoxyribose by phosphodiester bridges  nucleotide  purine bases  adenine (A) and guanine (G)  pyrimidine bases  cytosine (C), and thymine (T).  DNA is usually a double helix  consist of two chains of polynucleotides coiled around each other.  adenine (A) is always paired (by 2 hydrogen bonds) with thymine (T)  guanine (G) is always paired (by 3 hydrogen bonds) with cytosine (C) -76-  The two strands are said to be complementary.  The two polynucleotide chains are antiparallel (i.e., sugar-phosphate backbones are oriented in opposite directions).  a major groove and a smaller minor groove are formed by the double helix backbone.  In procaryotes  DNA exists as closed circular, supercoiled molecule associated with basic (histone- like) proteins.  In eucaryotes  DNA is more highly organized; it is associated with basic (histone) proteins and is coiled into repeating units known as nucleosomes. RNA differs from DNA in  RNA composed of o the sugar ribose instead of 2-deoxyribose o uracil (U) instead of thymine (T).  RNA is single strand  coil back on itself  there are 3 types of RNA o Ribosomal (rRNA) o Transfer (tRNA) o Messenger (mRNA) -77-  DNA Replication  The pattern of DNA replication is described as 'semiconservative‘  produce 2 copies  each copy contained one of the original strands and one new strand.  Each strand of DNA is conserved  each strand serve as templates for the production of another strand This pattern of replication is responsible for maintaining (conserving) the proper sequence of bases on DNA molecule.  Replication starts at a point called 'origin replication' by separation of the two strands  The replication origin  specific segment of the DNA molecule consisting of about 245 bp.  Replication fork  the area of the DNA molecule where strand separation occurs and the synthesis of new DNA takes place.  A replicon consists of an origin of replication and the DNA that is replicated from that origin.  The bacterial chromosome has single replicon (one bubble).  Eukaryotes have multiple replicons (several bubbles exist) to efficiently replicate the relatively large molecules within a reasonable time -78-  Polymerization of nucleotides occur in  5' to 3' direction only  for the original strand 3'  5' (Leading strand)  A major challenge in DNA replication is  how to achieve 5'-3' polymerization in the opposite direction from the template strand which is itself is from 5'-3' direction (lagging strand)  The problem is solved by  different modes of polymerization for the two growing strands -79-   DNA replication is carried out using DNA polymerases.  cells in all organisms contain multiple highly specialized DNA polymerases  Bacteria  5 DNA polymerases  I, II, III, IV, V  Yeast  8 DNA polymerases  humans  at least 15 DNA polymerases  The rate of DNA synthesis  750-1000 bp/second in prokaryotes  50-100 bp/second in Eukaryotes Bacterial DNA Polymerases  Polymerase III  main polymerase responsible for DNA replication  To initiate replication DNA polymerase require presence of primer  short strand of RNA to which growing polynucleotide chain is covalently attached -80-   Polymerase I has exonuclease activity to remove mismatched nucleotide & adds correct nucleotide  repair process  Proofreading  removing incorrect nucleotides immediately after they added to growing DNA during replication process.  The proofreading function of DNA polymerase improves fidelity of replication to one error in every 109 -1010 bp  Other polymerase function  not understood till now Steps of DNA replication in E. coli DNA replication is extraordinary complex; at least 30 proteins are required to replicate the E. coli chromosome  replication proceeds at a rate of 750 base pairs per second per replication fork. (1) DnaA protein binds to origin of replication (Ori C)  DNA replication always starts at the replication origin (Ori C) by separation of the two strands. (2) Helicase (dnaB) binds replication fork to unwind the 2 DNA strands  at the same time topoisomerases (DNA gyrase) relieve the tension caused by unwinding process. (3) Single-stranded DNA binding proteins (SSBs)  keep the single stranded region of the template DNA apart. (4) Primase  synthesize small RNA molecule (~10 nucleotides)  act as primer for DNA synthesis. -81- (5) DNA polymerase III synthesizes complementary strand of DNA according to base-pairing rules  direction of DNA synthesis is from 5' to 3' end of newly formed strand  On leading strand synthesis is continuous  on lagging strand discontinuous (Okazaki fragments) are generated  DNA synthesis is bi-directional  2 replication forks in opposite directions from origin of replication (6) DNA polymerase I  removes primer and fills gaps that result from RNA deletion. (7) DNA ligases join DNA fragments to form a complete DNA strand When prokaryotic cell contains more than one DNA structure (chromosome or other genetic elements)  each usually replicates independently DNA (Gene) expression The Genetic Code  DNA base sequence  define amino acid sequence of protein  The 4 bases code for  the 20 amino acid.  Codon represented by 3 bases Provide (43) 64 different codons.  61 of codons specify amino acids  called sense codons. -82-   3 codons  UGA, UAG and UAA do not specify amino acids  called non-sense (stop) codons.  Some amino acids are encoded by more than 1 codon  called degeneracy.  Gene expression consists of two steps (1) DNA Transcription RNA Synthesis ( 3 types) (2) mRNA Translation  protein synthesis Types of RNA 1. Messenger RNA (mRNA)  Carries the message that directs synthesis of proteins.  mRNA  formed by transcription of gene  from DNA to RNA.  transcription done by RNA polymerase  initiates RNA synthesis at a promoter site on the DNA and transcribes DNA until termination codon reached 2. Ribosomal RNA (rRNA)  rRNAs are components of the ribosomes.  They are made from large precursor molecule that is enzymatically cleaved to 16s, 23s and 5s rRNA. 3. Transfer RNA (tRNA)  Carries amino acids during protein synthesis.  tRNAs are used to distinguish different amino acids.  Each tRNA has a triplet of nucleotides called 'anticodon‘  binds to triplet nucleotides on mRNA, called codon during protein synthesis.  Each type of tRNA is covalently bound to one of the 20 aa. -83-   When aa is attached to tRNA  tRNA is said to be charged. Gene and its Structure  The gene is unit of heredity of living organism  It is linear sequence of nucleotides that has a fixed start point and end point  encodes protein, tRNA, or rRNA.  Template strand is the one strand that contains coding information and d i r e c t s R N A s y n t h e s i s.  It has controlling elements (promoters)  regulate its expression.  Genes are not overlapping (With few exceptions).  In prokaryotes  coding sequence is continuous (few bacterial genes are interrupted).  In eukaryotes  most genes have coding sequences (exons) that are interrupted by non-coding sequences (introns). Genes that code for