General Microbiology for BSFT Lecture Module PDF

General Microbiology A Modular Approach for BS Food Technology students (BIOL 015) Bautista, Cano, Reboa, Rodriguez 1 Photo Description: SEM images of novel coronavirus Photo Compliments to: NIAID/Flickr, CC BY 2.0. thru http://science.thewire Bautista, Cano...

General Microbiology A Modular Approach for BS Food Technology students (BIOL 015) Bautista, Cano, Reboa, Rodriguez 1 Photo Description: SEM images of novel coronavirus Photo Compliments to: NIAID/Flickr, CC BY 2.0. thru http://science.thewire Bautista, Cano, Reboa, Rodriguez were faculty from the Department of Biology, College of Science, Polytechnic University of the Philippines. 2  General Instructions Please do not write on this module. Answers to this module should be written in a separate short, white bond paper bearing the subject title, full name, and course, year and section. Follow the format for every activity, especially if the activity will be consisting of more than one page. Do not forget to include the basic information, activity number and the page number. Compile your answers and staple them together. The Authors 3  GENERAL MICROBIOLOGY (BIOL 015) A Modular Approach for BS Food Technology students by Bautista, Cano, Reboa, Rodriguez 4  TABLE OF CONTENTS Page No. MODULE 1: HISTORY AND SCOPE OF MCIROBIOLOGY Lesson 1 Evolution of Microbiology 9 Lesson 2 Origins of Microbiology 9 Lesson 3 Different Theories in Microbiology 10 Lesson 4 Diversity of Microorganism 10 Lesson 5 Impact of Microorganism 11 MODULE 2: MICROBIAL TAXONOMY Lesson 1 Etymology and Definition 13 Lesson 2 Taxonomic Categories 14 Lesson 3 Major Taxonomic Characteristics 14 Lesson 4 Components of Taxonomy 14 Lesson 5 Classification on Hierarchy 15 Lesson 6 Microbial Phylogeny 17 MODULE 3: MICROBIAL DIVERSITY Lesson 1 Definition of Terms 22 Lesson 2 Concept of Functional Diversity 22 Lesson 3 Diversity of Phototropic Bacteria 23 Lesson 4 Morphologically Diverse Bacteria 27 MODULE 4: MICROBIAL CELL STRUCTURE AND FUNCTION Lesson 1 Cell Morphology and Arrangement 30 Lesson 2 Prokaryotic Cell and its Structural Parts 31 Lesson 3 Eukaryotic Cell and its Structural Parts 36 MODULE 5: MICROBIAL GROWTH Lesson 1 Growth Requirements 40 Lesson 2 Physical Requirement 40 Lesson 3 Chemical Requirement 41 5 Lesson 4 Cell Division 41 Lesson 5 Microbial Growth and Quantification 42 Lesson 6 Measuring Number of Microbes 42 MODULE 6: MICROBIAL METABOLISM Lesson 1 Microbial Nutrients 45 Lesson 2 Transporting Nutrients into the Cell 45 Lesson 3 Energy Classes of Microorganism 46 Lesson 4 Catabolism, Fermentation and Respiration 47 Lesson 5 Biosyntheses 48 MODULE 7: MICROBIAL GENETICS Lesson 1 Structure of DNA & RNA 51 Lesson 2 Mutation 56 Lesson 3 Gene Transfer Mechanisms in Prokaryotes 59 MODULE 8: VIRAL GENOMES AND DIVERSITY Lesson 1 Nature of Viruses 62 Lesson 2 Viruses Genome 63 Lesson 3 Taxonomy of Viruses 63 Lesson 4 Viral Replication 64 Lesson 5 Viral Infection 65 MODULE 9: BIOSAFETY AND SECURITY Lesson 1 Biorisk and Biorisk Management 68 Lesson 2 Biosafety Principles and Biosafety Level 71 Lesson 3 Biosecurity 74 MODULE 10: MICROBIAL CONTROL AND DESTRUCTION Lesson 1 Physical Method of Microbial Control 77 Lesson 2 Chemical Method of Microbial Control 79 6 MODULE 11: MICROBIAL APPLICATION IN ENVIRONMENTAL SCIENCE AND INDUSTRY Lesson 1 Properties of Microorganism Useful in the Industry 81 Lesson 2 Primary vs Secondary Metabolites 81 Lesson 3 Major Products for the Health Industry 82 Lesson 4 Major Industrial Products for Food and Beverage 83 Industries 7  GENERAL MICROBIOLOGY Lecture Module 8 MODULE 1: HISTORY AND SCOPE OF MICROBIOLOGY Overview: Microorganisms is considered as ubiquitous for it can found anywhere. It is said that microbes are here on Earth before man was created. As we navigate on this lesson, we will identify what can microbes impart us in life in order for us to fully understand its existence. We will start on the origin and evolution followed by the diversity of the microbes. Followed by the good and detrimental effects of microbes that will give a tremendous impact on both man and biosphere. Learning Outcomes: After successful completion of this module, the learner should be able to: 1. Describe the origins of microbiology 2. Discuss the Germ Theory of Disease 3. Identify the impact of microorganisms Course Material: Microorganism also known as microbes. These are ubiquitous organism that are too small to be seen by the unaided eye. Microorganism represents the major fraction of the Earth’s biomass. Plants and animals are engaged in the world of microbes, their evolution and survival are influenced by microbial activities. Microbiology is a branch of biology that deals with microorganism and their effects on other living things. Evolution of Microbiology Microbial cells first appeared between 3.8 and 4.3 billion years ago First 2 billion years of Earth’s existence, microorganisms are capable to survive without oxygen in the atmosphere. 1 billion years ago, phototrophic microorganisms (organisms that harvest energy from sunlight) occurred. Purple sulfur bacteria and green sulfur bacteria were anoxygenic (non-oxygen producing) were the first phototrophs. Cyanobacteria (oxygenic phototrophs) evolved and began the slow process of oxygenating Earth’s atmosphere, multicellular life forms eventually evolved. Origins of Microbiology Microscope is the ultimate tool in studying microbiology The first known image of microscope and fruiting molds was illustrated by Robert Hooke. Cut from thin slices of cork and observed under the microscope the presence of “tiny little boxes”. He started to formulate the “Cell Theory”. Antoni van Leeuwenhoek constructed single lens microscope and first to observe bacteria by using pepper-water infusion. 9  Louis Pasteur experimented the process of fermentation and sterilization. He disproved the “Spontaneous Generation theory”. He developed a vaccine for rabies. Joseph Lister introduced the aseptic technique in order to kill and prevent from microbial infection of surgical patients. Ignaz Semmelweis introduced the method of hand washing. Robert Koch discovered the causative agent of diseases like anthrax, cholera and tuberculosis. He used his own Koch’s postulate in identifying these diseases. Richard Petri developed a transparent double-sided dish known as “Petri dish”, a standard tool for obtaining pure cultures. Different Theories: 1. Cell Theory by Robert Hooke - “All living organisms are composed of cells” 2. Spontaneous Generation Theory - “Life could arise spontaneously from nonliving matter” 3. Germ Theory of Disease or Koch’s Postulate by Robert Koch - disease agent – man - disease Source: Madigan, M. T. et.al.. Brock Biology of Microorganisms 15th ed. (2019). Pearson Educ. Inc. p. 58. Diversity of Microorganisms 30 Estimated 2x 10 microbial cells on Earth The total amount of nitrogen and phosphorus (essential nutrients for life) within microbial cells is nearly four times that in all plant and animal cells combined Microbes also represent a major fraction of the total DNA in the biosphere (about 31%), and their genetic diversity far exceeds that of plants and animals. Microbes are even abundant in habitats that are too much harsh for other forms of life. All ecosystems are influenced greatly by microbial activities. The metabolic activities of microorganisms can change the habitats in which they live, both chemically and physically, and these changes can affect other organisms. Microbes provide nutritional and other benefits that are essential to human health. 10  Impact of Microorganism 1. Agent of disease - microorganisms are the major cause of human death were infectious disease cause by bacteria and viruses. 2. Agriculture - root nodules of plant contain bacteria that fix molecular nitrogen that can be used by plant - microbes in the rumen of the animal convert cellulose from grass into fatty acids that can be used by animals. 3. Food - cause of foodborne disease and food spoilage - not all microorganisms in foods are harmful. - beneficial microbes are used to improve food safety and to preserve foods such as cheese, yogurt while thru microbial fermentation can produce food like sauerkraut, kimchi, pickles. - colon contains diverse microbial species that assist in the digestion of complex carbohydrates, and that synthesize vitamins and other nutrients essential to host nutrition. 4. Industry - harnessed to produced antibiotics, enzymes, insulin, biofuels and helps clean up waste 5. Environment - used in bioremediation, a process that cleans up toxic wastes and pollutants - decompose or destroy wastes, such as sewage and oil spills. Activity / Assessment: In a separate sheet of paper, write the name, section, module number, title and questions. Then briefly discuss the following questions: 1. Explain the impact of microorganisms in different areas. ______________________________________________________________________ ______________________________________________________________________ ______________________________________________________________________ ______________________________________________________________________ 2. Describe how the cell theory originated. ______________________________________________________________________ ______________________________________________________________________ ______________________________________________________________________ ______________________________________________________________________ 3. Enumerate and discuss the Koch postulate? ______________________________________________________________________ ______________________________________________________________________ ______________________________________________________________________ ______________________________________________________________________ 11 Grading System: Each question is equivalent to 10 points per number a total of 30 points. RUBRICS FOR SCORING ESSAY QUESTION 5 4 3 2 1 The student has full The student has a good The student has a basic The student has some The student has no Level of Understanding understanding of the understanding of the understanding of the understanding of the understanding of question or problem question or problem question or problem question or problem the question or The response reflects a The response reflects The response provides problem. The The response addresses Synthesis of Information complete synthesis of some synthesis of little or no synthesis of response is the question information information information completely Total Score = _________ / 10 x 100 = _________% References: As a reading material, you will be needing a book or an e-book: - Madigan, M. T. et. al.. Brock Biology of Microrganisms (2019) 15th ed. Pearson Education, Inc. - Tortora, G. et.al.. Microbiology: An Introduction. (2013) 11th ed. Pearson Education, Inc. 12 MODULE 2: MICROBIAL TAXONOMY Overview: Taxonomy is the way of identifying different organisms, classifying them into categories, and naming them. All organisms, both living and extinct, are classified into distinct groups with other similar organisms and given a scientific name. Hierarchical classification is one way to help scientists understand, categorize and organize the diversity of life. In this manner, it minimizes confusion and provide reliable means of identifying and naming an organism. Learning Outcomes: After successful completion of this module, the learner should be able to: 1. Differentiate taxonomy from systematics 2. Identify the components of taxonomy 3. Compare and contrast classification from identification 4. Recognize the proper naming of species Course Material: Etymology of Taxonomy: The word taxonomy comes from a From a Greek word taxis - arrangement or order nomos – law nemein - means to distribute or govern Definition: Taxonomy - the science of biological classification. Taxa or Taxon – a group or level of classification or hierarchy categorized at different levels Systematics or phylogeny- the study of diversity of organism and their evolutionary relationship Dichotomous Key- means of assigning an organism to a specific taxonomic category. Taxonomic categories or hierarchy - An ordered group of taxonomic ranks used to classify organisms from general to specific. Domains (Bacteria, Archaea, and Eukarya) Kingdom (contains similar divisions or phyla; most inclusive taxa) Phylum (contains similar classes; equivalent to the Division taxa in botany). 13  Class (contains similar orders) Order (contains similar families) Family (contains similar genera) Genus (contains similar species) Species (specific epithet; lowercase Latin adjective or noun; most exclusive taxa) Major Taxonomic Characteristics: A. Morphological characters D. Molecular characters - General external and internal morphology - Immunological distance - Special structures - Electrophoretic differences - Embryology - DNA hybridization - Karyology and other cytological factors - DNA-RNA sequence B. Physiological characters E. Ecological characters - Metabolic factors - Habitats - Body secretions - Food - Genic sterility factors - Seasonal variation C. Geographic and behavioral characters - Parasite and host Components of Taxonomy: 1. Classification- - taxa are classify based on the similarities in phenotypic (phenetic) characteristics which are expressed in an organism and can be examined visually or can be tested by other means. System of Classification A. Artificial System – share the same characteristics but they are not closely related to one another genetically. B. Natural System – with many of the same characteristics and highly predictive. C. Phylogenetic (Phyletic) System – classifying organism on the basis of descent from a common ancestor e.g. 16S rRNA, DNA base content (G-C ratio), DNA–DNA hybridization, DNA fingerprinting, MLST, RFLP, REP-PCR, Ribotyping, genome analyses. Methods of Classification: a. Phenotypic (Phenetic) Classification System: - groups do not necessarily reflect genetic similarity or evolutionary relatedness. Instead, groups are based on convenient, observable characteristics. e.g. morphology, motility, metabolism, cell chemistry, physiologic, biochemical, pathogenicity, antibiotic sensitivity, serological. b. Genotypic Classification System - considers characteristics of the genome 14  Classification on Hierarchy I. Family encompasses a group of organisms that may contain multiple genera and consists of organisms with a common attribute. II. Genus Grouping similar genera into common families and similar families into common orders is used for classification of plants and animals, higher taxa designations are not useful for classifying bacteria. (i.e., division, class, and order) III. Species groups of populations that can potentially interbreed freely within and among themselves. collection of bacterial strains that share common physiologic and genetic features and differ notably from other microbial species. A. Subspecies are taxonomic subgroups within a species. a. Biotype - a group of organisms having the same or nearly the same genotype b. Serotype - a group of organisms within a species that have the same type and number of surface antigens. c. Genotype may be given to groups below the subspecies level that share specific but relatively minor characteristics. B. Clone is a population of cells derived from a single parent cell and identical. C. Strain - came from pure cultures of the same species are not identical in all ways. Types of Strains: a. Serovar - a strain differentiated by serological means. Strains vary in their antigenic properties. b. Biovar (biotype) - strains that are differentiated by biochemical or other non-serological means. c. Morphovar (morphotype) - a strain which is differentiated on the basis of morphological distinctions. d. Isolate - a pure culture derived from a heterogeneous, wild population of microorganisms. The term isolate is also applicable to eucaryotic microorganisms as well as to viruses. Strain Differentiation Methods- compare phenotypes and that, though useful, are not as precise as genetic homologies in determining evolutionary relationships. 1. Protein Profile 3. Flow Cytometry 2. Immunological Reaction 4. Phage Typing 15 2. Nomenclature - branch of taxonomy concerned with the assignment of names to taxonomic groups in agreement with published rules. -Carolus Linnaeus introduced a formal system of classification dividing living organisms into two kingdoms— Plantae and Animalia. - Every organism is assigned a genus and a species of Latin or Greek derivation by the addition of the appropriate suffix. - naming of microorganisms according to established rules and guidelines set forth in the International Code of Nomenclature of Bacteria (ICNB) or the Bacteriological Code (BC). - The taxonomic classification scheme for prokaryotes is found in Bergey’s Manual of Systematic Bacteriology. - rules governing microbial nomenclature is limited to two taxa, genus and species known as binomial nomenclature. Pointers on How to write the scientific name: 1. Suffixes for order and family are written as -ales and –aceae e.g. Streptococcaceae family type genus is Streptococcus 2. The genus and specific epithet (species), both names are printed underlined or italicized. e.g. Streptococcus pyogenes 3. The genus name is always capitalized in first letter and is always a noun. The species name is lowercase in first letter and is usually an adjective. e.g. Klebsiella pneumoniae 4. The name may be abbreviated by using the uppercase form of the first letter of the genus designation followed by a period (.) and the full species name, which is never abbreviated. e.g. S. aureus Naming of Bacteria based on Cell Arrangement a. Cocci – round c. Spiral Staphylococci – in clusters Spirochete Streptococci – in chains Diplococci – in pairs Sarcinae – packets of four b. Bacilli – rod Diplobacilli – in pairs Streptobacilli – in chain Coccobacilli 3. Identification - the process of determining a particular (organism) belongs to a recognized taxon. The process by which a microorganism’s key features are delineated. 16  Identification method: a. Genotypic characteristics - relate to an organism’s genetic makeup, including the nature of the organism’s genes and constituent nucleic acids. e.g. hair color, height b. Phenotypic characteristics - are based on features beyond the genetic level, including both readily observable characteristics and features that may require extensive analytic procedures to be detected. e.g. skin color Comparison between Genotype from Phenotype https://www.technologynetworks.com/genomics/articles/genotype-vs-phenotype-examples-and-definitions-318446 Methods in Bacterial Identification 1. Microscopic morphology - Gram Staining, Shapes, arrangements, motility 2. Macroscopic morphology – colony appearance, motility 3. Physiological / biochemical characteristics – aerobic, anaerobic, photosynthetic, growth on selective media 4. Chemical analysis – e.g.peptides and lipids in cell membranes 5. Phage Typing – which phage infects the bacterium 6. Serological analysis – what antibodies are produced against the bacterium 7. Pathogenicity – what diseases does the bacterium cause. 8. Genetic & molecular analysis G + C base composition DNA analysis using genetic probes Nucleic acid sequencing & rRNA analysis Microbial Phylogeny most biologists divide all living organisms into 3 domains: Domain Archaea Domain Bacteria Domain Eucarya rRNA sequence data suggests that Archaea & Eucarya may share a more recent common ancestor with each other than with Bacteria. There is often great metabolic and ecological diversity among the members of a group, perhaps reflecting parallel evolution of such things as fermentation pathways, photosynthetic pathways, etc. 17  https://microbe.net/simple-guides/fact-sheet-rrna-in-evolutionary-studies-and-environmental-sampling th Source: Tortora, G. et.al. Microbiology: An Introduction. (2013) 11 ed. p. 280. 1. Phylogeny of domain Archaea - Based primarily on rRNA sequence data, domain Archaea is divided into two phyla: a. Phylum Crenarchaeota Originally containing thermophilic and hyperthermophilic sulfur- metabolizing archaea Recently discovered Crenarchaeota are inhibited by sulfur & grow at lower temperatures b. Phylum Euryarchaeota Contains primarily methanogenic archaea, halophilic archaea, and thermophilic, sulfur-reducing archaea 2. Phylogeny of domain Bacteria - The 2nd edition of Bergey’s Manual of Systematic Bacteriology divides domain Bacteria into 23 phyla. Nine of the more notable phyla a. Phylum Aquiflexa The earliest “deepest” branch of the Bacteria 18  Contains genera Aquiflex and Hydrogenobacter that can obtain energy from hydrogen via chemolithotrophic pathways b. Phylum Cyanobacteria Oxygenic photosynthetic bacteria c. Phylum Chlorobi The “green sulfur bacteria” Anoxygenic photosynthesis Includes genus Chlorobium d. Phylum Proteobacteria The largest group of gram-negative bacteria All major nutritional types are represented: phototrophy, heterotrophy, and several types of chemolithotrophy Sometimes called the “purple bacteria,” although very few are purple; the term refers to a hypothetical purple photosynthetic bacterium from which the group is believed to have evolved Divided into 5 classes: Alphaproteobacteria, Betaproteobacteria, Gammaproteobacteria, Deltaproteobacteria, Epsilonproteobacteria Other medically important Proteobacteria include genera Haemophilus, Vibrio, Camphylobacter, Helicobacter, Rickessia, Brucella A. Photosynthetic genera: - Purple Sulfur Bacteria Chromatium and Thiocapsa (purple sulfur bacteria) Rhodospirillum, Rhodoferax, Rhodobacter (purple non-sulfur bacteria) - Green Sulfur bacteria Chlorobium, Chlorobaculum, “Chlorochromatium” (green sulfur bacteria) Chloroflexus, Heliothrix, Roseiflexus (green non- sulfur bacteria) - Sulfur chemolithotrophs genera Thiobacillus and Beggiatoa - Nitrogen chemolithotrophs (nitrifying bacteria) genera Nitrobacter and Nitrosomonas - Other chemolithotrophs genera Alcaligenes, Methylobacilllus, Burkholderia - Aerobic Anoxygenic Phototrophs genera Roseobacter, Erythrobacter - Other Phototrophic Bacteria genera Heliobacterium, Chloracidobacterium B. Gram Negative Enteric Bacteria - The family Enterobacteriaceae genera Escherichia, Proteus, Enterobacter, Klebsiella,Salmonella, Shigella, Serratia, - The family Pseudomonadaceae genus Pseudomonas e. Phylum Firmicutes “Low G + C gram-positive” bacteria Divided into 3 classes: 19  1. Class I – Clostridia; includes genera Clostridium and Desulfotomaculatum, and others 2. Class II – Mollicutes; bacteria in this class cannot make peptidoglycan and lack cell walls; includes genera Mycoplasma, Ureaplasma, and others 3. Class III – Bacilli; includes genera Bacillus, Lactobacillus, Streptococcus, Lactococcus, Geobacillus, Enterococcus, Listeria, Staphylococcus, and others f. Phylum Actinobacteria “High G + C gram-positive” bacteria Includes genera Actinomyces, Streptomyces, Corynebacterium, Micrococcus, Mycobacterium, Propionibacterium g. Phylum Chlamidiae Small phylum containing the genus Chlamydia h. Phylum Spirochaetes The spirochaetes Characterized by flexible, helical cells with a modified outer membrane (the outer sheath) and modified flagella (axial filaments) located within the outer sheath Important pathogenic genera include Treponema, Borrelia, and Leptospira i. Phylum Bacteroidetes Includes genera Bacteroides, Flavobacterium, Flexibacter, and Cytophyga; Flexibacter and Cytophyga are motile by means of “gliding motility” 3. Phylogeny of domain Eucarya The domain Eucarya is divided into four kingdoms: a. Kingdom Protista protozoa and algae b. Kingdom Fungi fungi (molds, yeast, and fleshy fungi) c. Kingdom Animalia the multicellular animals d. Kingdom Plantae the multicellular plants Activities/Assessments: In a separate sheet of paper, write the name, section, module number, title and questions. Then briefly discuss the following questions: 1. Describe the genotypic from phenotypic characteristics. ______________________________________________________________________ ______________________________________________________________________ ______________________________________________________________________ ______________________________________________________________________ 20  2. Explain how important is the components of taxonomy in microbiology? ______________________________________________________________________ ______________________________________________________________________ ______________________________________________________________________ ______________________________________________________________________ 3. Differentiate Systematics from Taxonomy? ______________________________________________________________________ ______________________________________________________________________ ______________________________________________________________________ ______________________________________________________________________ Grading System: Each question is equivalent to 10 points per number a total of 30 points. RUBRICS FOR SCORING ESSAY QUESTION 5 4 3 2 1 The student has full The student has a good The student has a basic The student has some The student has no Level of Understanding understanding of the understanding of the understanding of the understanding of the understanding of question or problem question or problem question or problem question or problem the question or The response reflects a The response reflects The response provides problem. The The response addresses Synthesis of Information complete synthesis of some synthesis of little or no synthesis of response is the question information information information completely Total Score = _________ / 10 x 100 = _________% References: As a reading material, you will be needing a book or an e-book. Madigan, M. T.. Brock Biology of Microorganisms (2019) 15th ed. Pearson Education, Inc. Tille, Patricia M. Bailey and Scott’s Diagnostic Microbiology (2017) 14th ed. Elsevier Tortora, G. et al.. Microbiology: An Introduction (2013) 11th ed. Pearson Education, Inc. https://www.cliffsnotes.com/study-guides/biology/plant-biology/systematics/types-of- classifications https://microbe.net/simple-guides/fact-sheet-rrna-in-evolutionary-studies-and- environmental-sampling https://www.technologynetworks.com/genomics/articles/genotype-vs-phenotype- examples-and-definitions-318446 21 MODULE 3: MICROBIAL DIVERSITY Overview: Microbial diversity is composed of both phylogenetic and functional diversity. In congruence between phylogeny and functional traits of microorganism which can result from different patterns. Diversity of Phototrophic bacteria and morphologically diverse bacteria and its species were described morphologically and physiologically. Learning Outcomes: After successful completion of this module, the learner should be able to: 1. Differentiate phylogenetic from functional diversity. 2. Identify the different species in phototrophic and morphologic diverse bacteria. 3. Differentiate the identified species from phototrophic and morphologic diverse bacteria. Course Material: Definition of Terms: Phototrophy – the use of light energy is prevalent in the microbial world. Photosynthesis is considered the most important biological process Phototrophs – organisms that carry out photosynthesis Autotrophs – photosynthetic organisms that are capable of growing with carbon dioxide as the sole source of carbon. Photoautotrophs - energy comes from light is used in the reduction of CO2 to organic compounds Photoheterotrophs – phototrophs that use organic carbon as their carbon source Phylogenetic diversity is the component of microbial diversity that deals with evolutionary relationships between microorganisms. Encompasses the genetic and genomic diversity of evolutionary lineages and so can be defined on the basis of either genes or organism. Phylogenetic diversity is defined on the basis of ribosomal RNA gene phylogeny, which is thought to reflect the phylogenetic history of the entire organism. Functional diversity is the component of microbial diversity that deals with diversity in form and function as it relates to microbial physiology and ecology. Result from phylogeny and functional traits of microorganism 1. Gene loss - trait present in the common ancestor of several lineages is subsequently lost in some lineages but retained in others that over evolutionary time became quite divergent. 2. Convergent Evolution - trait has evolved independently in two or more lineages and is not encoded by homologous genes shared by these lineages. 3. Horizontal Gene Transfer - genes that confer a particular trait are homologous and have been exchanged between distantly related lineages. 22 I. Photoautotrophy - is the process by which organisms convert radiant energy into biologically useful energy and synthesize metabolic compounds using only carbon dioxide or carbonates as a source of carbon. Two distinct sets of reactions (1) light reactions that produce ATP; (2) light-independent dark reactions that reduce CO2 to cell material for autotrophic growth. Photosynthesis requires light-sensitive pigment a. chlorophylls—present in plants, algae, and cyanobacteria b. bacteriochlorophylls – present in anoxygenic phototrophs. Oxygenic photosynthesis- the photosynthetic process in cyanobacteria (and chloroplasts). Anoxygenic photosynthesis - O2 is not produced. - Absorption of light energy by chlorophylls and bacteriochlorophylls begins the process of photosynthetic energy conversion, and the net result is chemical energy, ATP. II. Diversity of Phototrophic Bacteria Phototrophic microorganisms, those microbes that conserve energy from light. First phototrophic organisms were anoxygenic phototrophs, organisms that do not generate O2 as a product of photosynthesis. Instead of H2O, these organisms likely to used H2, ferrous iron (Fe2+), or H2S as the electron donor for photosynthesis. Anoxygenic photosynthesis is present in six bacterial phyla: the Proteobacteria, Chlorobi, Chloroflexi, Firmicutes, Acidobacteria, and Gemmatimonadetes. All phototrophic bacteria use chlorophyll-like pigments to harvest energy from light and transfer this energy in cytoplasmic membrane to increase the amount of pigment for better use of light of low intensities for the production of ATP. couple light energy to carbon fixation through a variety of different mechanisms but not all phototrophs fix CO2. Two different types of photosynthetic reaction centers: a. type I reaction centers (FeS-type) b. type II reaction centers (quinone-type, or Q-type) Both types of reaction centers are present in Cyanobacteria whereas only one type or the other is present in anoxygenic phototrophs A. Cyanobacteria Key Genera: Prochlorococcus, Crocosphaera, Synechococcus, Trichodesmium, Oscillatoria, Anabaena - both unicellular and filamentous. - 0.5µm in diameter, as large as 100 µm in diameter. - First oxygen evolving phototrophic organisms on Earth. - oxygenic phototrophs, have both FeS-type and Q-type photosystems. - some can assimilate simple organic compounds such as glucose and acetate if 23  light is present, a process called photoheterotrophy. - have specialized membrane systems called thylakoids that increase the ability of cells to harvest light energy. - cell wall contains peptidoglycan and is structurally similar to that of other Gram-negative bacteria. - have photopigments, fluorescent and emit light when visualized using a fluorescence microscope. - Photopigment produces chlorophyll a, known as phycobilins, function as accessory pigments in photosynthesis. a. Phycocyanin -responsible for the blue-green color of most cyanobacteria b. Phycoerythrin - species producing phycoerythrin are red or brown - exhibit gliding motility - some filamentous cyanobacteria form hormogonia, short, motile filaments that break off from longer filaments to facilitate dispersal in times of stress. - some form Akinetes, cells with thickened outer walls. - many are capable of nitrogen fixation. a. Cyanothece and Crocosphaera -fix nitrogen only at night when photosynthesis does not occur b. Trichodesmium – fix nitrogen during the day c. Nostocales and Stigonematales - facilitate nitrogen fixation by forming specialized cells called heterocysts Five Morphological groups of Cyanobacteria : (1) Chroococcales -unicellular, dividing by binary fission; (2) Pleurocapsales - unicellular, dividing by multiple fission (colonial); (3) Oscillatoriales - filamentous non-heterocystous forms; (4) Nostocales - filamentous, divide along a single axis, and are capable of cellular differentiation; and (5) Stigonematales - morphologically similar to Nostocales except that cells divide in multiple planes, forming branching filaments. Synechococcus and Prochlorococcus the most abundant phototrophs in the oceans Two groups of Marine Nitrogen Fixation: a. Crocosphaera - dominate nitrogen fixation in most of the Pacific Ocean and are widespread in tropical and subtropical habitats b. Trichodesmium - dominant nitrogen-fixer in the North Atlantic Ocean and parts of the Pacific where dissolved iron concentrations are elevated. c. Calothrix and Richelia - form symbiotic associations with diatom found in tropical and subtropical oceans. d. Nodularia and Anabaena- dominate nitrogen fixation in cold waters of the Northern Hemisphere and are often observed in the Baltic Sea 24  B. Purple Sulfur Bacteria Key Genera: Chromatium, Ectothiorhodospira - Purple sulfur bacteria are distinguished by the location of sulfur granules and by their photosynthetic membranes. - anoxygenic phototrophs that use hydrogen sulfide (H2S) as an electron donor for photosynthesis. - found in lakes, marine sediments, and “sulfur springs,” where H2 produced can support the growth of purple sulfur bacteria - also found in microbial mats and salt marsh sediments. - Carotenoid – accessory pigment involved in light harvesting. - under the Q-type system that contain either bacteriochlorophyll a or b, and carry out CO2 fixation by the Calvin cycle. Two Families Purple Sulfur Bacteria: 1. Chromatiaceae - store sulfur granules in the periplasmic space and have vesicular intracellular photosynthetic membrane systems. - stratified lakes containing sulfide and in the anoxic sediments of salt marshes. Example: Genera Chromatium and Thiocapsa 2. Ectothiorhodospiraceae - oxidize H2S to S0 that is deposited outside the cell have lamellar intracellular photosynthetic membrane systems - extremely halophilic (salt loving) or alkaliphilic (alkalinity loving) - found in saline lakes, soda lakes, and salterns, where abundant levels of sulfate Example: Genera Ectothiorhodospira and Halorhodospira C. Purple Non-Sulfur Bacteria Genera: Rhodospirillum, Rhodoferax, Rhodobacter - most metabolically versatile of all microbes. - they are not always purple; these organisms synthesize an array of carotenoids that can give purple bacteria their colors, usually purple, red, or orange. - photoheterotrophs (a condition where light is the energy source and an organic compound is the carbon source), - use a Q-type photosystem, and contain either bacteriochlorophyll a or b - can conserve energy through a variety of metabolic processes - all purple non-sulfur bacteria can fix N2 and will thrive under such conditions, rapidly outcompeting other bacteria Aerobic Anoxygenic Phototrophs Key Genera: Roseobacter, Erythrobacter - Obligatory aerobic heterotrophs use light as a supplemental source of energy to support growth. - Able to photosynthesize when grown on a day/night cycle. - The primary physiological difference with the purple non-sulfur bacteria: *aerobic anoxygenic phototrophs are strict heterotrophs *employ anoxygenic photosynthesis only under oxic conditions as a supplemental source of energy. - contain bacteriochlorophyll a and a Q-type photosystem, but are unable to 25  fix CO2 - rely on organic forms of carbon as their carbon source. - Carotenoids are pigment that give colors of yellow, orange, or pink to cultures D. Green Sulfur Bacteria Key Genera: Chlorobium, Chlorobaculum, “Chlorochromatium” - typically non-motile and strictly anaerobic anoxygenic phototrophic short to long rods. - oxidize hydrogen sulfide (H2S) as an electron donor for autotrophic growth, oxidizing it first to sulfur (S0) and then to sulfate the S0 produced by green sulfur bacteria is deposited only outside the cella - unique means of autotrophy in phototrophic bacteria - contain bacteriochlorophyll c, d, or e and house these pigments in unique structures called chlorosomes - Chlorosomes are oblong bacteriochlorophyll rich bodies bounded by a thin, non- unit membrane and attached to the cytoplasmic membrane in the periphery of the cell - use an FeS-type photosystem’ - live in anoxic, sulfidic, illuminated aquatic environments - tend to have a greater tolerance of H2S than do other anoxygenic phototrophs - found at the greatest depths of all phototrophic microorganisms in lakes or microbial mats, where light intensities are low and H2S levels the highest. - Chlorobaculum tepidum is thermophilic and forms dense microbial mats in high- sulfide hot springs. - C. tepidum also grows rapidly and is amenable to genetic manipulation by both conjugation and transformation and has become the model organism for studying the molecular biology of green sulfur bacteria. - “Chlorochromatium aggregatum” has been used to describe a commonly observed green-colored consortium that is green because the epibionts contain green-colored carotenoids - “Pelochromatium roseum” is brown because its epibionts produce brown- colored carotenoids E. Green Non-sulfur Bacteria Key Genera: Chloroflexus, Heliothrix, Roseiflexus - filamentous (gliding motility), anoxygenic phototrophs of the phylum Chloroflexi. - both aerobic and anaerobic chemoorganotrophs that grow well in the dark by aerobic respiration of a wide variety of carbon sources. - Dehalococcoidetes, a group of dehalogenating bacteria that use halogenated organic compounds as electron acceptors in anaerobic respiration. - Chloroflexus forms thick microbial mats in neutral to alkaline hot springs along with thermophilic cyanobacteria - grow best as photoheterotrophs using simple carbon sources as electron donors such as H2 or H2S. for photosynthesis. - contain bacterioclorophyll a and chlorosomes that contain bacteriochlorophyll c and in this way are similar to green sulfur bacteria. - contain Q-type photosynthetic reaction center that resemble to purple bacteria. 26  F. Other Phototrophic Bacteria Key Genera: Heliobacterium, Chloracidobacterium 1. Heliobacteria - group of phototrophic gram-positive Bacteria found within the phylum Firmicutes - anoxygenic phototrophs that have an FeS-type photosystem and that produce a unique pigment, bacteriochlorophyll g - grow photoheterotrophically using a narrow range of organic compounds including pyruvate, lactate, acetate, or butyrate. Five genera: Heliobacterium, Heliophilum, Heliorestis, Heliomonas, and Heliobacillus. - Rod-shaped or filamentous cells from in bundles - strict anaerobes - grow chemotrophically in darkness by pyruvate fermentation - produce endospores, like Bacillus or Clostridium species - reside in soil, especially paddy (rice) field soils, where their nitrogen fixation activities - found in highly alkaline environments, such as soda lakes and surrounding alkaline soils. III. Morphologically Diverse Bacteria I. Spirochetes - Gram negative, motile, tightly coiled Bacteria, typically slender and flexuous in shape, highly flexible and quite thin (60.5 μm). - corkscrew-like motion that allows cells to burrow through viscous materials or tissues - contains endoflagella which is found in the cell periplasm, with outer sheath - found in aquatic sediments and in animals. - an important human sexually transmitted disease. Spirilla - often confused with the spirochetes - rigid cells, helically curved rod-shaped cells, motile by means of polar flagella - lack the outer sheath, endoflagella, and corkscrew-like motility of spirochetes - Spirulina - long helical filaments that superficially resemble spirochetes Key Genera: Spirochaeta, Cristispira, Treponema, Borrelia, Leptospira 1. Spirochaeta - free-living, anaerobic, and facultative aerobic spirochetes. - common in aquatic environments such as freshwater and sediments, and also in the oceans. a. Spirochaeta plicatilis - large spirochete found in sulfidic freshwater and marine habitats. - 20 or so endoflagella inserted at each pole 27  - arranged in a bundle that winds around the coiled protoplasmic cylinder. b. Spirochaeta stenostrepta - an obligate anaerobe commonly found in H2S-rich black muds. - ferments sugars to ethanol, acetate, lactate, CO2, and H2. 2. Cristispira - found in nature only in the crystalline style of certain molluscs, such as clams and oysters. - lives in both freshwater and marine molluscs - has not been cultured 3. Treponema A. Treponema - Anaerobic or microaerophilic; commensals or pathogens of humans and animals a. T. pallidum - the causal agent of syphilis - not helical but flat and wavy. - thin, measuring only 0.2 μm in diameter - uses dark-field microscopy for suspected syphilitic lesions b. Treponema denticola - common in the human oral cavity associated with gum disease - ferments amino acids such as cysteine and serine, forming acetate as the major fermentation acid, as well as CO2, NH3, and H2S. - common in the rumen, the digestive forestomach of ruminant animals c. Treponema saccharophilum - large, pectinolytic spirochete found in the bovine rumen where it ferments pectin, starch, inulin, and other plant polysaccharides. d. Treponema primitia - can be found in the hindgut of certain termites, fermentation of cellulose causes production of H2 and CO2. - contain an acetogen, which is an important component of the insect’s nutrition e. Treponema azotonutricium - found in the termite hindgut and is capable of nitrogen fixation. 4. Borrelia - animal or human pathogens, causing diseases in cattle, sheep, horses, and birds - the bacterium is transmitted to the animal host from the bite of a tick a. Borrelia burgdorferi - the causative agent of the tickborne Lyme disease, which infects humans and animals. 28  - one of the few known bacteria that has a linear chromosome 5. Leptospira and Leptonema - the two genera contain strictly aerobic spirochetes that oxidize long-chain fatty acids as electron donors and carbon sources. Leptospira - thin, finely coiled, and usually bent at each end into a semi-circular hook - some free-living and many parasitic Two major species of Leptospira: a L. interrogans (parasitic) - parasitic for humans and animals *Rodents are the natural hosts of most leptospiras, although dogs and pigs are also important carriers of certain strains. b L. biflexa (free-living) - *Leptospirosis - a disorder in which the organism localizes in the kidneys and liver, causing nephritis and jaundice Activities / Assessment: In a separate sheet of paper, write the name, section, module number, title and questions. Then briefly discuss the following questions: 1. Differentiate the six Phototrophic Bacteria according to: a. pigment b. habitat c. species/genera d. type of photosystem e. Oxygen type 2. Describe the Morphologically Diverse Bacteria according to: a. Morphology c. Motility e. Disease b. habitat d. Type of flagella Grading System: Five points for each number will be given to each species that will be differentiated or describe. Question #1 has a total points of 30 while in Question # 2 has a total points of 25 Total score of 55 points for this assessment Reference: As a reading material, you will be needing a book or an e-book: Madigan, M. T. et.al.. Brock Biology of Microorganisms. (2019) 15th ed. Pearson Education, Inc. 29 MODULE 4: MICROBIAL CELL STRUCTURE AND FUNCTION Overview: Bacteria are very small to be seen by our naked eye. Do we know how they function? Even we could not see them, by learning the structure of these microorganism and on how it functions, it could help us understand them much better. Learning Outcomes: After successful completion of this module, the learner should be able to: 1. Identify the different microbial structures 2. Determine the function of each structure 3. Compare and contrast prokaryote from eukaryote Course Material: Morphology – deals with the size, shape and arrangement of a living organism. Cell Morphology – the study of the size and shape of the cell. Bacterial Size: less than 3 micrometers (μm) in size. Size of cocci - range from 0.5 to 3 μm Size of bacilli - range from 0.15 to 2 μm (width) to 0.5 to 20 μm (length). Bacterial Shapes: 1. Cocci – spherical and ovoid 2. Bacilli – cylindrical 3. Spiral – curve or loose spiral Bacterial Arrangement: 1. Singly - single 2. Strepto – in chain *Unusual shape 3. Staphylo – in cluster Spirochetes – tightly coiled 4. Diplo – in pairs 5. Sarcinae – three dimensional cubes Arrangement in cocci Arrangement in bacilli a. Singly a. Singly b. Diplococci b. Diplobacilli c. Streptococcus c. Streptobacilli d. Staphylococcus I. Prokaryotic Cell A. Structural Parts 1. Cell Membrane (Cytoplasmic Membrane) - “gatekeeper” for the entrance and exit of dissolved substances 30  Three Major Function of Cytoplasmic Membrane i. cell’s permeability (selective) barrier, preventing the passive leakage of solutes into or out of the cell. ii. anchors several proteins that catalyze a suite of key cell functions. iii. plays a major role in energy conservation and consumption. - energy conservation takes place in: i. mitochondrion (respiration) ii. chloroplast (photosynthesis) a. Bacterial Membrane - phospholipid bilayer containing embedded proteins. - composed of: i. hydrophobic (water-repelling) - fatty acids (inward) ii. hydrophilic (water-attracting) - glycerol molecule containing phosphate (exposed to cytoplasm) - Hopanoids - strengthened by sterol-like molecules that is present in bacteria - Sterols strengthen the membranes of eukaryotic cells where there is an absence of cell wall b. Archaeal Membrane - cytoplasmic membrane of Archaea is structurally similar to those of Bacteria and Eukarya, but the chemistry is somewhat different. Comparison in Cytoplasmic Membrane Bacteria and Eukarya Archaea Structure Same Same Lipid Layer Lipid Bilayer Lipid Monolayer ether bonds between ester linkages bond fatty Chemical Structure glycerol and a acids to glycerol hydrophobic side Hopanoids Present Absent 2. Cell wall - layer outside the cytoplasmic membrane - gives shape and rigidity on the cell - confers structural strength on the cell in order to keep it from bursting due to osmotic pressure. For example, the penicillins and cephalosporins, target bacterial cell wall synthesis, leaving the cell susceptible to osmotic lysis. Since human cells lack cell walls and are therefore not a target of such antibiotics, these drugs are of obvious benefit for treating bacterial infection. Two Major Group: a. Gram positive – thicker and have single type of molecule b. Gram negative – consists of two layers 31 2.1. Bacterial Cell wall 2.1.1. Lysozyme - weakens the peptidoglycan and cause cell lysis - act as a major line of defense against bacterial infection which are present in human secretions including tears, saliva, and other bodily fluids. - destroys pre-existing peptidoglycan, penicillin blocks a key step in its biosynthesis 2.1.2. Peptidoglycan - made up of rigid polysaccharide - not present in Archaea and Eukarya - composed of alternating repeats of two modified glucose residues a. N-acetylglucosamine b. N-acetylmuramic acid - 90% of cell wall in a Gram positive bacteria consist of peptidoglycan and form many layers i. Teichoic acid - embedded in the cell wall and function to bind divalent metal ions, such as Ca2+ and Mg2+, prior to their transport into the cell. ii. Lipoteichoic acid - covalently bonded to membrane lipids rather than to peptidoglycan iii. LPS or lipopolysaccharide - small amount of peptidoglycan in cell wall of Gram negative - Outer membrane is found in second lipid bilayer Two Components of LPS: a. Core polysaccharide b. O specific polysaccharide 2.1.3. Periplasm - space, located between the outer surface of the cytoplasmic membrane and the inner surface of the outer membrane, spans about 15 nm 2.1.4.Porins - channels for the entrance and exit of solutes Two Kinds of Porins: a. Non-specific porins - form water-filled channels through which virtually any very small hydrophilic substance can pass. b. Specific porins- contain a binding site for one or a group of structurally related substances 2.2. Archaeal Cell Wall Methanogens- cell walls of certain methane-producing Archaea a. Pseudomurein and other Polysaccharide Cell Walls The term “murein” is from the Latin word for wall and was an old term for peptidoglycan. 32  Pseudomurein is immune from destruction by both lysozyme and penicillin which are molecules that destroy peptidoglycan. Two Components of Pseudomurein: i. N-acetylglucosamine (also present in peptidoglycan) ii. N-acetyltalosaminuronic acid b. S- layer Paracrystalline surface layer or S layer – most common type of cell wall in Archaea. - consists of interlocking molecule of protein or glycoprotein. - retains periplasmic proteins and prevents their drifting away in gram-negative Bacteria. Example: Methanocaldococcus jannaschii 3. Cell Surface Structures: 3.1 Capsule - organized in a tight matrix that excludes small particles and is tightly attached - readily visible by light microscopy if cells are treated with India ink, which stains the background but not the capsule, 3.2 Slime - more easily deformed and loosely attached, it will not exclude particles and is more difficult to see microscopically * Example of Bacteria with cell surface: Bacillus anthracis and Streptococcus pneumoniae 3.3 Fimbriae, Pili and Hami 3.3.1. Fimbriae - thin (2–10 nm in diameter) filamentous structures made of protein that extend from the surface of a cell - enable cells to stick to surfaces - form pellicles (thin sheets of cells on a liquid surface) or biofilms on solid surfaces. 3.3.2. Pili - typically longer and only one or a few pili are present on the surface of a cell - All Gram negative produce pili while many Gram positive contain pili. - Pili can be receptors for certain types of viruses, they can be easily seen under the electron microscope when they become coated with virus particles Functions of pili: i. Conjugation - facilitating genetic exchange between cells (conjugative or sex pili) ii. Adhesion - adhering of pathogens to specific host tissues that they invade (Type IV and other pili). 33  3.3.3.Hami or hamus - this group have unique attachment structure resembles a tiny grappling hook. - Hami structurally resemble type IV pili except for their barbed terminus, which functions to attach cells both surfaces and to each other. Function of Hami: i. affix cells to a surface to form a networked biofilm ii. preventing cells from being washed away in groundwater flowage. Function of outer surface layers: a. Attachment - Bacterial cell surface polysaccharides assist in the attachment of microorganisms to solid surfaces. - When the opportunity arises, many bacteria will bind to solid surfaces, often forming a thick layer of cells called a biofilm. b. Virulence factors - molecules that contribute to the pathogenicity of a bacterial pathogen c. Preventing dehydration 4. Cell Inclusion: a. Poly-β-hydroxybutyric acid (PHB) b. Glycogen c. Polyphosphate granules d. Elemental sulfur – present in periplasm e. Carbonate f. Magnetosomes Gloeomargarita - organism able to process “biomineralization”. 5. Gas vesicles - conical-shaped structures made of protein. - length:300 -1000 nm; width: 45 -120 nm - Gas vacuoles are irregular bright inclusions seen in light microscopy or transmission electron microscope. Two Proteins in Gas Vesicles: a. GvpA (major protein)-forms the watertight vesicle shell and is a small, hydrophobic, and very rigid protein. b. GvpC (minor protein)- functions to strengthen the shell of the gas vesicle by cross-linking. 6. Endospores - highly differentiated cells that are extremely resistant to heat, harsh chemicals, and radiation. Parts of the endospore: a. exosporium – outermost layer, protein covering b. spore coats – innermost layer, spore-specific protein 34  c. cortex - consists of loosely cross-linked peptidoglycan. Core - inside the cortex which contains the core wall, cytoplasmic membrane, cytoplasm, nucleoid, ribosomes, and other cellular essentials. *Dipicolinic acid - one substance found in endospores but not in vegetative cells. Accumulates in the core that contain large amount of calcium. DPA complex inserts between bases in DNA, which helps stabilize DNA against heat denaturation. *Some bacterial endospores survive heating to temperatures as high as 150°C, although 121°C, the standard for microbiological sterilization kills the endospores of most species. Structures and Features of Endospores: visible by light microscopy as strongly refractile structures. impermeable to most dyes, unstained regions within cells that have been stained with basic dyes such as methylene blue. Special stains and procedures must be used. In the classical endospore-staining protocol, the stain malachite green is used and is infused into the spore with steam. can be seen in electron microscope Function: survival structures and enable the organism to endure unfavorable growth conditions, including but not limited to extremes of temperature, drying, or nutrient depletion - dormant stage of a bacterial life cycle: vegetative cell endospore vegetative cell Example: Bacillus and Clostridia Steps in Vegetative Cell Process: 1. activation - occurs when endospores are heated for several minutes at an elevated but sub-lethal temperature 2. germination - rapid process (occurring in a matter of minutes), is signaled by the loss of refractility of the endospore and loss of resistance to heat and chemicals. 3. outgrowth - involves visible swelling due to water uptake and synthesis of RNA, proteins, and DNA. 7. Cell locomotion: Flagellum (bacteria) or archaellum (archaea) – used for locomotion and these are tiny rotating machines that function to push or pull the cell through a liquid. Bacterial flagella are long, thin appendages (15–20 nm wide, depending on the species) free at one end and anchored into the cell at the other end. 35  Flagella Structure: a. filament – main part, contains flagellin b. hook - consists of a single type of protein and connects the filament to the flagellum motor in the base. c. basal body – the rotor and stator made up to build up this structure. Flagellar Arrangement: a. atrichous - absence of flagella b. monotrichous – presence of flagella at one side c. amphitrichous – presence of flagella at both sides d. lophotrichous – tuft of flagella at one side e. peritrichous – presence of flagella on the entire body Two major types of prokaryotic cell movement: 1. swimming motility – Bacterial movement powered by rotating flagella 2. gliding motility – Gliding bacteria are filamentous or rod-shaped, and the gliding process requires that the cells be in contact with a solid surface. i.e. Filamentous cyanobacteria Gram-negative bacteria Myxococcus xanthus, Cytophaga Flavobacterium II. Eukaryotic Cell and its Parts – structurally more complex and much larger cells. Example: fungi, algae and protozoa and other protist. A. Nucleus- contains the chromosomes B. Nuclear Membrane – contain pores formed from holes where the inner and outer membranes are joined. Cell Division: divide by a process in which the chromosomes are replicated, the nucleus is disassembled, the chromosomes are segregated into two sets, and a nucleus is reassembled in each daughter cell. 2 Genetic states: a. haploid - (one copy of chromosome) b. diploid - (two copies of chromosome) Types of Cell Division: 1. Mitosis- the chromosomes condense, divide, and are separated into two sets, one for each daughter cell 36  2. Meiosis I - homologous chromosomes segregate into separate cells, changing the genetic state from diploid to haploid. Meoisis II - the two haploid cells divide to form a total of four haploid cells called gametes C. Mitochondria – where respiration occurs - The number of mitochondria per cell depends somewhat on the cell type and size - enclosed by a double membrane system: a. outermost mitochondrial membrane is permeable and contains pores that allow the passage of small molecules. b. innermost membrane is less permeable and its structure more closely resembles that of the cytoplasmic membrane of Bacteria. Parts of Mitochondrion: 1. Cristae - formed by invagination of the inner membrane, contain the enzymes needed for respiration and ATP production. - contain transport proteins that regulate the passage of key molecules such as ATP into and out of the matrix 2. Matrix - innermost compartment of the mitochondrion D. Chloroplast - chlorophyll-containing organelles of phototrophic microbial eukaryotes such as the algae and function to carry out photosynthesis. Thylakoids - Chlorophyll and all other components needed for ATP synthesis in chloroplasts are located in a series of flattened membrane discs. - highly impermeable and its major function is to form a proton motive force that results in ATP synthesis. E. Endoplasmic reticulum (ER) - a network of membranes continuous with the nuclear membrane. Two types of endoplasmic reticulum: 1. rough ER - which contains attached ribosomes 2. smooth ER – which does not contain ribosomes F. Golgi complex - products of the ER are chemically modified and sorted into those destined for secretion versus those that will function in other membranous structures in the cell. - glycosylation happened in Golgi complex. G. Lysosomes - fuses with food that enters the cell in vacuoles and then releases digestive enzymes that break down the foods for biosynthesis and energy generation. H. Cytoskeleton – the framework of cell a. microtubules - maintaining cell shape and cell motility by cilia and flagella, moving chromosomes during mitosis. b. microfilaments -smaller than microtubules, about 7 nm in diameter - function in maintaining or changing cell shape, in cell motility. 37  c. intermediate filaments - fibrous keratin proteins that form into fibers 8–12 nm in diameter and function in maintaining cell shape and positioning organelles in the cell. I-J. Flagella and Cilia - present on the surface of many eukaryotic microbes and function as organelles of motility, allowing cells to move by swimming. Cilia - essentially short flagella that beat in synchrony to propel the cell, usually quite rapidly through the medium. Flagella - long appendages present singly or in groups that propel the cell along, more slowly than by cilia, a whip-like motion. ***Dynein a protein attached to the microtubules and uses ATP to drive motility. Assessment: In a separate sheet of paper, write the name, section, module number, title and questions. Then briefly discuss the following questions: 1. Compare and contrast the prokaryotes from eukaryotes. 2. What are the structural reasons for the rigidity that is conferred on the cell wall by the peptidoglycan structure? 3. What is the function of gas vesicles? 4. Describe the structure and function of a bacterial flagellum. 5. How are the mitochondrion and the hydrogenosome similar structurally? Grading System: RUBRICS FOR SCORING ESSAY QUESTION 5 4 3 2 1 The student has full The student has a good The student has a basic The student has some The student has no Level of Understanding understanding of the understanding of the understanding of the understanding of the understanding of question or problem question or problem question or problem question or problem the question or The response reflects a The response reflects The response provides problem. The The response addresses Synthesis of Information complete synthesis of some synthesis of little or no synthesis of response is the question information information information completely Total Score = _________ / 10 x 100 = _________% References: As a reading material, you will be needing a book or an e-book: Engelkirk, P.G.. Burton’s Microbiology for Health Sciences. (2011) 9th ed. Lippincott Williams & Wilkins Madigan, M.T.. Brock Biology of Microorganisms (2019) 15th ed. Pearson Education, Inc. Tille, Patricia M.. Bailey and Scott’s Diagnostic Microbiology (2017) 14th ed. Elsevier Tortora, G. et al.. Microbiology: An Introduction. (2013) 11th ed. Pearson Education, Inc. 38 MODULE 5: MICROBIAL GROWTH Overview: Microbes that are “growing” are increasing in number, accumulating into colonies. Many bacteria survive and grow slowly in nutrient-poor environments by forming biofilms. Learning Outcomes: After successful completion of this module, the learner should be able to: 1. identify the microbial growth requirements. 2. determine how bacteria grow exponentially using generation time; and 3. describe different microbial growth phases. Course Material: Growth Factors may require small amounts of certain organic compounds for growth because they are essential substances that the organism is unable to synthesize from available nutrients. The requirements for microbial growth can be divided into two main categories: physical and chemical. I. Physical Requirements: 1. temperature - Most microorganisms grow well at the temperatures that humans favor. Classified into three primary groups: a. psychrophiles (cold-loving microbes) at 0⁰C. b. mesophiles (moderate-temperature– loving microbes) - 25–40°C, are the most common type of microbe. c. thermophiles (heat-loving microbes) – 50-60⁰C. d. hyperthermophiles - have an optimum growth temperature of 80°C or higher. e. extreme thermophiles – 121⁰C and above. 2. pH – refer to acidity or alkalinity of a solution. - Most bacteria grow best in a narrow pH range near neutrality, between \ pH 6.5 and 7.5. - Very few bacteria grow at an acidic pH below about pH 4. This is why a number of foods, such as sauerkraut, pickles, and many cheeses, are preserved from spoilage by acids produced by bacterial fermentation. - Acidophiles are bacteria that loves acids environment. 3. Osmotic pressure - require water for growth, and their composition is 80–90% water. - High osmotic pressures have the effect of removing necessary water from a cell. - Hypertonic - whose concentration of solutes is higher than in the cell. - Plasmolysis – shrinkage of cell cytoplasm. 39  Types of Halophiles: a. Extreme Halophiles – require high salt concentration b. Obligate Halophiles – require 30% of salt for growth. c. Facultative Halophiles – requires 15% of salt for growth II. Chemical Requirements: 1. Carbon 2. Sulfur, Nitrogen and Phosphorus 3. Trace elements such as iron, copper, molybdenum, and zinc 4. Organic Growth factors 5. Oxygen a. aerobes – microbes that use molecular oxygen b. anaerobes - microbes that do not use oxygen. Types of Oxygen Requirement: 1. Obligate aerobe - organisms that require oxygen to live e.g. Pseudomonas aeruginosa (Gram negative) Mycobacterium tuberculosis (acid-fast) Bacillus (Gram-positive). 2. Facultative anaerobe - ability to continue growing in the absence of oxygen e.g. E. coli and yeast 3. Obligate anaerobe - bacteria that are unable to use molecular oxygen for energy-yielding reactions e.g. Clostridium 4. Aerotolerant anaerobes - cannot use oxygen for growth, but they tolerate it fairly well. e.g. Lactobacilli 5. Microaerophile - they are aerobic; they do require oxygen. They grow only in oxygen concentrations lower than those in air. e.g. Borrelia burgdorferi, a species of spirochaete bacteria that causes Lyme disease in humans, Helicobacter pylori, a species of proteobacteria that has been linked to peptic ulcers and some types of gastritis Cell Division Two Types of Asexual Reproduction in Microbes 1. Binary fission - forms a totally new daughter cell, with the mother cell retaining its original identity. 2. Budding division - forms a totally new daughter cell, with the mother cell retaining its original identity. Generation time - When one cell eventually separates to form two cells, we say that one generation has occurred. - During one generation, all cellular constituents increase proportionally. 40  - Each daughter cell receives a copy of the chromosome(s) and sufficient copies of ribosomes and all other macromolecular complexes, monomers, and inorganic ions to begin life as an independent entity. *Biofilms- an attached polysaccharide matrix containing embedded bacterial cells. Microbial Growth and Quantification Growth - an increase in the number of cells. Culture medium or growth medium is a liquid or gel designed to support the growth of microorganisms. Laboratory cultures of microorganisms are grown in culture media Two broad classes of culture media: 1. Defined media are prepared by adding precise amounts of pure inorganic or organic chemicals to distilled water. Exact composition is known. 2. Complex media are made from digests of microbial, animal, or plant products. Microbial Growth Cycle Four Phases of Growth: a. lag phase - growth begins only after a period of time b. exponential or log phase - cell population doubles at regular intervals c. stationary phase – cells in the population grow while others die d. death phase - growth ceases Exponential growth is a repetitive pattern where the number of cells doubles in a constant time interval. - As one cell divides to become two cells, we express this as 20 -> 21. As two cells become four, we express this as 21 -> 22 and so on. - Bacteria grow quickly in batch culture (enclosed vessel), and cell numbers increase dramatically in a short period of time. - By measuring the rate of cell population increase over time, the growth depicts a certain “growth curve”. - In order to control both specific growth rate and cell density independently, continuous culture, a type of an open system must be done. - The most common type of continuous culture is the chemostat. - In the continuous culture growth vessel, a known volume of sterile medium is added at a constant rate while an equal volume of spent culture medium (which also contains cells) is removed at the same rate. Measuring Number of Microbes Microscopic counting is a quick and easy way of estimating microbial cell numbers. Microscopic counts can be performed either on samples dried on slides or on liquid samples. a. Stained samples to increase contrast between cells and their background 41  b. Liquid samples, counting chambers consisting of a grid with squares of known area etched on the surface of a glass slide are used. A. Direct Measurement of Microbial Growth 1. Plate count - most frequently used method of measuring bacterial populations. Often reported as colony-forming units (CFU). A viable cell is one that is able to divide and form offspring, and in most cell-counting situations. Methods of Viable Plate Count: a. spread plate method - a volume (usually 0.1 ml or less) of an appropriately diluted culture is spread over the surface of an agar plate using a sterile glass spreader. - positions all the colonies on the surface and avoids contact between the cells and melted agar. b. pour plate method – a known volume (usually 0.1-1.0 ml) of culture is pipetted into a sterile Petri plate. (Refer to Figure 5.14 of the book). - colonies will grow within the nutrient agar (from cells suspended in the nutrient medium as the agar solidifies) as well as on the surface of the agar plate. 2. Serial Dilution - to ensure that some colony counts will be within this range, the original inoculum is diluted several times in a process. Refer to Figure 6.16. 3. Filtration - applied frequently to detection and enumeration of coliform bacteria, which are indicators of fecal contamination of food or water. 4. Most Probable Number (MPN) method - the greater the number of bacteria in a sample, the more dilution is needed to reduce the density to the point at which no bacteria are left to grow in the tubes in a dilution series. Uses of MPN: a. microbes being counted will not grow on solid media (such as the chemoautotrophic nitrifying bacteria). b. useful when the growth of bacteria in a liquid differential medium is used to identify the microbes (such as coliform bacteria, which selectively ferment lactose to acid, in water testing). 5. Direct Microscopic Count - a measured volume of a bacterial suspension is placed within a defined area on a microscope slide. Petroff-Hausser cell counter – used for direct microscopic counting. Coulter counter – used for electronic cell counters. B. Indirect Methods by Estimating Bacterial Numbers 1. Turbidity - As bacteria multiply in a liquid medium, the medium becomes turbid, or cloudy with cells. 2. Spectrophotometer (or colorimeter) - instrument used to measure turbidity. 42  3. Metabolic Activity - assumes that the amount of a certain metabolic product, such as acid or CO2, is in direct proportion to the number of bacteria present. 4. Dry Weight - the fungus is removed from the growth medium, filtered to remove extraneous material, and dried in a desiccator. It is then weighed. Activities / Assessments: In a separate sheet of paper, write the name, section, module number, title and questions. Then briefly discuss the following questions: 1. Compare and contrast the physical requirement in microbial growth. 2. Discuss the method of viable plate count. Grading System: In question No. 1, there will be a 10 points per physical requirement. In question No. 2, there will be a 10 points per viable plate count. RUBRICS FOR SCORING ESSAY QUESTION 5 4 3 2 1 The student has full The student has a good The student has a basic The student has some The student has no Level of Understanding understanding of the understanding of the understanding of the understanding of the understanding of question or problem question or problem question or problem question or problem the question or The response reflects a The response reflects The response provides problem. The The response addresses Synthesis of Information complete synthesis of some synthesis of little or no synthesis of response is the question information information information completely Total Score = _________ / 10 x 100 = _________% References: Madigan, M.T. et.al. Brock Biology of Microorganisms 15th ed. (2019). Pearson Education, Inc. Tortora, Funk and Case. Microbiology: An Introduction. (2013). 11th ed. Pearson Education, Inc. 43 MODULE 6: MICROBIAL METABOLISM Overview: Metabolism is the series of biochemical reactions by which the cell breaks down or biosynthesizes various metabolites. In order to grow, cells must incorporate nutrients from the environment, transform them into precursor molecules, and then use them to construct a new cell. Because the metabolic capacities of microbes differ, their nutrient requirements also differ. Learning Outcomes: After successful completion of this module, the learner should be able to: 1. differentiate macronutrients and micronutrients; 2. learn different microbial transport system. 3. discuss the glycolytic pathway Course Material: Microbial Nutrients Cell are primarily composed of elements C, H, O, N, P, and S. These chemical elements are predominant in the cell: 1. C is needed in the largest amount (50% of a cell’s dry weight) 2. O and H are next (combined, 25% of dry weight) 3. N follows (13%). 4. P, S, K, Mg, and Se combine for less than 5% of a cell’s dry weight. All microbes require a core set of nutrients. Macronutrients are required in large amounts Micronutrients are required in minute amounts. e.g. Trace elements as co-factor of certain enzymes Vitamins as growth factors(organic micronutrient) Iron (Fe) plays a major role in cellular respiration Transporting Nutrients into the Cell - The active transport of nutrients into the cell is an energy requiring process driven by ATP (or some other energy-rich compound) or by the proton motive force. - Three classes of transport systems: simple, group translocation, and ABC systems. Each functions to accumulate solutes against the concentration gradient. 1. Simple transport reactions are driven by the energy inherent in the proton motive force. Major transport: a. symport reactions (where a solute and a proton are co-transported in one direction) b. antiport reactions (where a solute and a proton are transported in opposite directions) 44  2. Group translocation differs from simple transport in two important ways: (1) the transported substance is chemically modified during the transport process, and (2) an energy-rich organic compound (rather than the proton motive force) drives the transport event. 3. ABC transport systems - “ABC” standing for ATP-binding cassette- a structural feature of proteins that bind ATP. - transport systems that employ a periplasmic binding protein along with transmembrane and ATP-hydrolyzing components. Energy Classes of Microorganism Classification by Carbon and Energy Source All microorganisms conserve energy from either the oxidation of chemicals or from light. - Chemotrophs - organisms that conserve energy from chemicals. - Chemoorganotrophs use organic chemicals as their electron donors, while chemolithotrophs use inorganic chemicals. - Phototrophic organisms convert light energy into chemical energy (ATP) and include both oxygenic and anoxygenic species. - Heterotroph, its cell carbon is obtained from one or another organic compound. An autotroph, by contrast, uses carbon dioxide (CO2) as its carbon source. Most chemolithotrophs and phototrophs are autotrophs. Autotrophs are also called primary producers because they synthesize new organic matter from inorganic carbon (CO2). Calvin cycle is the major biochemical pathway by which phototrophic organisms incorporate CO2 into cell material. Source: https://microbenotes.com/classification-of-bacteria-on-the-basis-of-nutrition/ 45  Enzymes Enzymes are protein catalysts that increase the rate of biochemical reactions by activating the substrates that bind to their active site. Enzymes are highly specific in the reactions they catalyze, and this specificity resides in the three-dimensional structures of the polypeptide(s) that make up the protein(s). Redox Chemical reactions in the cell are accompanied by changes in energy, expressed in kilojoules. Reactions either release or consume free energy. ∆G0 is a measure of the energy released or consumed in a reaction under standard conditions and reveals which reactions can be used by an organism to conserve energy. Oxidation–reduction reactions require electron donors and electron acceptors. The tendency of a compound to accept or release electrons is expressed by its reduction potential (E0’). The substance oxidized (H2) as the electron donor, and the substance reduced (O2) as the electron acceptor. Redox reactions in a cell often employ redox coenzymes such as NAD+/NADH as electron shuttles. The energy released in redox reactions is conserved in compounds that contain energy- rich phosphate or sulfur bonds. ATP -the prime energy carrier in the cell. Consists of the ribonucleoside adenosine to whi