Bacterial Genetics - PDF

Bacterial Genetics Dr Ravesh Singh Scientist: Microbiologist UKZN, NHLS Learning objectives and outcomes To understand the differences between prokaryotic and eukaryotic genomes. To understand the genetic components prokaryotic genomes. To understan...

Bacterial Genetics Dr Ravesh Singh Scientist: Microbiologist UKZN, NHLS Learning objectives and outcomes To understand the differences between prokaryotic and eukaryotic genomes. To understand the genetic components prokaryotic genomes. To understand the function of each genetic component found in prokaryotic genomes. To understand the process of DNA replication on prokaryotes. To understand the mechanisms and process of genetic recombination in prokaryotes. Mutations and repair mechanisms in bacteria. Genomes  Genomes: Organization: Prokaryotes: Have a single, circular chromosome located in the cytoplasm. This chromosome, while large, contains significantly less DNA than eukaryotic genomes. Eukaryotes: Have multiple linear chromosomes housed within a membrane-bound nucleus. Their genomes are typically much larger and more complex.  Genomes Overall: Prokaryotic genomes are smaller, simpler, and more streamlined for efficient function. Eukaryotic genomes are larger, more complex, and offer greater potential for diverse gene expression and regulation, resulting in increased functional and evolutionary flexibility. Additionally: Prokaryotes may possess extrachromosomal DNA elements like plasmids. Some eukaryotes like mitochondria and chloroplasts carry their own DNA. Prokaryotic Genomes Prokaryotic chromosomes Main portion of DNA, along with associated proteins and RNA Prokaryotic cells are haploid (single chromosome copy) Typical chromosome is circular molecule of DNA in nucleoid  Chromosome single, circular, double-stranded DNA molecule contains all the genetic information required by a cell DNA is tightly coiled around a protein dense area called the nucleoid  central subcompartment in the cytoplasm where DNA aggregates  Plasmids Small circular, double-stranded DNA Stable extra-chromosomal DNA elements that carry nonessential genetic information. Duplicated and passed on to offspring. Replicate independently from the chromosome Plasmids May encode for enzymes responsible for antibiotic resistance, production of toxins and tolerance to toxic metals. F plasmids allow genetic material to be transferred from a donor cell to a recipient. R plasmids carry genes encoding for resistance to antibiotics used in genetic engineering readily manipulated & transferred from cell to cell DNA Replication in Prokaryotes  DNA Replication in Prokaryotes Prokaryotic DNA replication, primarily studied in E. coli, occurs at a single origin of replication and proceeds bidirectionally in a semiconservative manner. Consists of 3 steps Initiation, Elongation and Termination. Initiation: DnaA protein binds to the origin of replication. Helicase unwinds the DNA strands, forming a replication fork. Single-stranded binding proteins (SSB) stabilize the single strands.  Elongation: DNA polymerase III synthesizes new DNA strands complementary to the template strands. Leading strand: Synthesized continuously in the 5' to 3' direction. Lagging strand: Synthesized discontinuously in short fragments (Okazaki fragments) joined by DNA ligase. RNA primer: Short RNA primers synthesized by primase are required for DNA polymerase initiation. Topoisomerase: Relieves torsional stress caused by unwinding.  Termination: Replication forks converge at a specific site on the chromosome. Specific termination proteins stop replication. Daughter chromosomes are separated. Regulation: Replication is tightly regulated to ensure its accurate and timely occurrence. Replication initiation is controlled by various factors, including growth rate and nutrient availability.  Genetic Recombination in Prokaryotes  Genetic recombination  occurs when an organism acquires and expresses genes that originated in another organism  Genetic information in prokaryotes can be transferred vertically and horizontally Vertical gene transfer (VGT) transfer of genetic material from parent cell to daughter cell Horizontal gene transfer (HGT) transfer of DNA from a donor cell to a recipient cell Three types Bacterial conjugation Transformation Transduction  DNA Recombination Events  3 means for exogenous genetic recombination in bacteria: 1. Conjugation 2. Transformation 3. Transduction  Transmission of Exogenous Genetic Material in Bacteria conjugation requires the attachment of two related species & formation of a bridge that can transport DNA transformation transfer of naked DNA transduction DNA transfer mediated by bacterial virus 1. Conjugation transfer of a plasmid or chromosomal fragment from a donor cell to a recipient cell via direct connection Gram-negative cell donor has a fertility plasmid (F plasmid, F′ factor) allows the synthesis of a conjugation (sex) pilus recipient cell is a related species or genus without a fertility plasmid donor transfers fertility plasmid to recipient through a pilus F+ and F- Physical Conjugation  2. Transformation Chromosome fragments from a lysed cell are accepted by a recipient cell. genetic code of DNA fragment is acquired by recipient. Donor and recipient cells can be unrelated. Useful tool in recombinant DNA technology.  3. Transduction DNA is transferred from one bacterium to another by a virus. Bacteriophages A virus that infects bacteria. Consist of an outer protein capsid enclosing genetic material. Serves as a carrier of DNA from a donor cell to a recipient cell. Other ways genetics of an organism can change Transposons Mutations Transposons Special DNA segments that have the capability of moving from one location in the genome to another “jumping genes” Can move from one chromosome site to another chromosome to a plasmid plasmid to a chromosome May be beneficial or harmful Changes in traits Replacement of damaged DNA Transfer of drug resistance Mutations Result of natural processes or maybe induced : Spontaneous mutations : Refer to heritable changes to the base sequence in DNA These result from natural phenomena such as radiation or uncorrected errors in replication eg,.UV light  physical mutagen that creates a dimer that cannot be transcribed properly Point Mutation Result of spontaneous or induced mutations. Affects just one base pair in a gene. Base-pair substitutions result in an incorrect base in transcribed mRNA codons Base-pair deletion or insertion results in an incorrect number of bases  Repair Mechanisms Attempt to correct mistakes or damage in the DNA Mismatch repair involves DNA polymerase “proofreading” the new strand removing mismatched nucleotides  Excision repair involves cutting out damaged DNA. replacing it with correct nucleotides. The End Bacterial Growth and Metabolism Dr Ravesh Singh Scientist: Microbiologist UKZN, NHLS Learning objectives and outcomes To know the different metabolic pathways bacteria use. To understand the differences between heterotrophs and autotrophs. To understand nutritional and growth requirements of bacteria. To understand nutritional movement within the bacterial cell. To understands concepts of a microbial growth curve. To know what biofilms are. To know the environmental factors influencing microbial growth To understand the concept of extra-cellular digestion? To understand microbial associations (symbiotic vs non-symbiotic) Metabolism Metabolism refers to all the biochemical reactions that occur in a cell or organism. Bacterial metabolism focuses on the chemical diversity of substrate oxidations and dissimilation reactions (reactions by which substrate molecules are broken down), which normally function in bacteria to generate energy. The bacterial cell is a highly specialized energy transformer. Heterotrophic Metabolism Heterotrophic metabolism is the biologic oxidation of organic compounds, such as glucose, to yield ATP and simpler organic (or inorganic) compounds, which are needed by the bacterial cell for biosynthetic or assimilatory reactions. Respiration Respiration is a type of heterotrophic metabolism that uses oxygen and in which 38 moles of ATP are derived from the oxidation of 1 mole of glucose, yielding 380,000 cal. Fermentation In fermentation, another type of heterotrophic metabolism, an organic compound rather than oxygen is the terminal electron (or hydrogen) acceptor. Less energy is generated from this incomplete form of glucose oxidation, but the process supports anaerobic growth. Krebs Cycle The Krebs cycle is the oxidative process in respiration by which pyruvate (via acetyl coenzyme A) is completely decarboxylated to CO2. The pathway yields 15 moles of ATP (150,000 calories). Glyoxylate Cycle The glyoxylate cycle, which occurs in some bacteria, is a modification of the Krebs cycle. Acetyl coenzyme A is generated directly from oxidation of fatty acids or other lipid compounds. Electron Transport and Oxidative Phosphorylation In the final stage of respiration, ATP is formed through a series of electron transfer reactions within the cytoplasmic membrane that drive the oxidative phosphorylation of ADP to ATP. Bacteria use various flavins, cytochrome, and non-heme iron components as well as multiple cytochrome oxidases for this process. Obtaining Carbon Heterotroph  organism that obtain carbon in an organic form made by other living organisms Autotroph  an organism that uses CO2 (an inorganic gas) as its carbon source  not dependent on other living things  Types of Heterotrophs Saprobes Parasites / pathogens  Nutritional Movement Osmosis: passive movement of water across a semipermeable membrane to equalize solute concentrations on both sides. Facilitated diffusion: Specific transport proteins help certain molecules cross the membrane faster than they would by simple diffusion. Active transport: Requires energy (ATP) to move molecules against their concentration gradient. Endocytosis: Involves the engulfment of materials from the environment into the cell through the plasma membrane - Phagocytosis: Engulfment of large particles - Pinocytosis: Uptake of fluids and dissolved solutes through small vesicles formed from the plasma membrane  Extracellular Digestion digestion of complex nutrient material into simple, absorbable nutrients accomplished through the secretion of enzymes (exoenzymes) into the extracellular environment Environmental Influences on Microbial Growth 1. temperature 2. oxygen requirements 3. pH 4. Osmotic pressure 5. UV light 6. Barophiles  1. Temperatures Minimum temperature lowest temperature that permits a microbe’s growth and metabolism Maximum temperature highest temperature that permits a microbe’s growth and metabolism Optimum temperature promotes the fastest rate of growth and metabolism  Temperature Adaptation Groups  Psychrophiles optimum temperature 15oC capable of growth at 0 - 20oC  Mesophiles optimum temperature 40oC Range 10o - 40oC (45) most human pathogens  Thermophiles optimum temperature 60oC capable of growth at 40 - 70oC Hyperthermophiles Archaea that grow optimally above 80°C hot-water vents  2. Oxygen Requirements Aerobe - requires oxygen Obligate aerobe - cannot grow without oxygen Anaerobe - does not require oxygen Obligate anaerobes die in the presence of oxygen Aerotolerant bacteria - not affected at all by oxygen, evenly spread along the test tube. Facultative anaerobe and aerobe - capable of growth in the absence OR presence of oxygen 2. Oxygen Requirements  3. pH The pH Scale Ranges from 0 - 14 pH below 7 is acidic [H+] > [OH-] pH above 7 is alkaline [OH-] > [H+] pH of 7 is neutral [H+] = [OH-]  3. pH Acidophiles optimum pH is relatively to highly acidic Neutrophiles optimum pH ranges about pH 7 (plus or minus) Alkaphiles optimum pH is relatively to highly basic 4. Osmotic Pressure Bacteria 80% water Require water to grow. Sufficiently hypertonic media at concentrations greater than those inside the cell cause water loss from the cell Osmosis Fluid leaves the bacteria causing the cell to contract Causes the cell membrane to separate Plasmolysis Cell shrinkage Extreme or obligate halophiles Adapted to and require high salt concentrations 5. UV Light Great for killing bacteria Damages the DNA (making little breaks) in sufficient quantity can kill the organisms in a lower range causes mutagenesis Endospores tend to be resistant can survive much longer exposures  6. Barophiles Bacteria in oceans grow at moderately high hydrostatic pressures. membranes and enzymes depend on pressure to maintain their three- dimensional, functional shape Barotolerants Grows at pressures from 100- 500 Barophilic 400-500 Extreme barophilic Higher than 500  Microbial Associations Symbiotic organisms live in close nutritional relationships; Mutualism Obligatory Dependent Both members benefit Commensalism One member benefits Other member not harmed Parasitism Parasite is dependent and benefits Host is harmed  Microbial Associations Non-symbiotic organisms are free-living relationships not required for survival Synergism members cooperate and share nutrients Antagonism some members are inhibited or destroyed by others  Microbial Associations Biofilms Complex relationships among numerous microorganisms. Develop an extracellular matrix which: Adheres cells to one another. Allows attachment to a substrate. Sequesters nutrients. May protect individual population of bacteria in the biofilm.  Microbial Growth in Bacteria Binary fission: Prokaryotes reproduce asexually one cell becomes two basis for population growth Process: parent cell enlarges duplicates its chromosome forms a central septum divides the cell into two daughter cells Population Growth Generation / doubling time  time required for a complete fission cycle  Length of the generation time is a measure of the growth rate of an organism Some populations can grow from a small number of cells to several million in only a few hours!! Prokaryotic Growth Bacterial Growth Curve lag phase no cell division occurs while bacteria adapt to their new environment logarithmic (log) phase Exponential growth of the population occurs Human disease symptoms usually develop stationary phase When reproductive and death rates equalize decline (exponential death) phase accumulation of waste products and scarcity of resources  Other Methods of Analyzing Population Growth Turbidity Direct microscopic count Coulter counting Turbidity Direct Microscopic Count Electronic Counting The End