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Bacterial Genetics

• The presentation discusses bacterial genetics, prokaryotic and eukaryotic genomes, and genetic components.
• Ravesh Singh is a Scientist and Microbiologist at UKZN, NHLS.

Learning Objectives

• 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
• To understand mutations and repair mechanisms in bacteria

Genomes Overview

• Prokaryotes possess single, circular chromosomes located in the cytoplasm with less DNA than eukaryotic genomes.
• Eukaryotes feature multiple linear chromosomes housed in a membrane-bound nucleus, with larger and more complex genomes.
• Prokaryotes may have extrachromosomal DNA (plasmids).
• Some eukaryotes like mitochondria and chloroplasts carry their own DNA

Prokaryotic Genomes

• The main portion of DNA, along with associated proteins and RNA
• Prokaryotic cells are haploid (single chromosome copy)
• The typical chromosome is a circular molecule of DNA in the nucleoid

Chromosome Structure

• 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.
• The nucleoid is a central subcompartment in the cytoplasm where DNA aggregates

Plasmids

• Small circular, double-stranded DNA
• Stable extra-chromosomal DNA elements that carry nonessential genetic information.
• Plasmids are duplicated and passed on to offspring.
• They replicate independently from the chromosome.
• Plasmids may encode for antibiotic resistance enzymes, and production of toxins and tolerance to toxic metals. -F (fertility) plasmids allow genetic material to be transferred from a donor cell to a recipient.
• R (resistance) plasmids carry genes encoding for resistance to antibiotics
• Plasmids are readily manipulated and transferred from cell to cell for use in genetic engineering.

DNA Replication in Prokaryotes

• Prokaryotic DNA replication, studied in E. coli, occurs at a single origin of replication and proceeds bidirectionally andsemiconservatively.
• The replication consists of 3 steps: Initiation, Elongation, and Termination.

Initiation

• The initiation step involves DnaA protein binding to the origin of replication
• Helicase unwinds DNA strands to form 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.
• The leading strand is synthesized continuously in the 5' to 3' direction
• The lagging strand is synthesized discontinuously in short fragments (Okazaki fragments) joined by DNA ligase.
• RNA primers, synthesized by primase, are required for DNA polymerase initiation.
• Topoisomerase relieves torsional stress caused by unwinding.

Termination and Regulation

• Replication forks converge at a specific site on the chromosome.
• Specific termination proteins can stop replication.
• Daughter chromosomes are separated after replication is terminated.
• Regulation is tightly controlled for accuracy and timeliness.
• Replication initiation is controlled by factors like growth rate and nutrient availability.

Genetic Recombination

• 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) is the transfer of genetic material from parent cell to daughter cell
• Horizontal gene transfer (HGT) involves the transfer of DNA from a donor cell to a recipient cell.
• Exogenous genetic recombination may occur with conjugation, transformation, and transduction.

Transmission Types

• Conjugation requires the attachment of two related species and direct connection via the formation of a bridge that can transport DNA
• Transformation involves the transfer of naked DNA.
• Transduction is DNA transfer is mediated by a bacterial virus.

Conjugation Details

• Transfer of a plasmid or chromosomal fragment from a donor cell to a recipient cell via direct connection.
• In gram-negative bacteria, the cell donor has a fertility plasmid, also know as (F plasmid, F' factor);
• The fertility plasmid allows synthesis of a conjugation (sex) pilus
• Recipient cell is a related species or genus without a fertility plasmid
• Donor transfers fertility plasmid to the recipient through the pilus in F+ and F- cells.

Transformation Details

• Chromosome fragments from a lysed cell are accepted by a recipient cell.
• The genetic code of DNA fragment is acquired by recipient.
• Donor and recipient cells can be unrelated.
• The process is a useful tool in recombinant DNA technology.

Transduction Details

• 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.
• Bacteriophages serve as a carrier of DNA from a donor cell to a recipient cell.

Other Ways Genetics Change

• Transposons special DNA segments that have the capability of moving from one location in the genome to another 'jumping genes'.
• These can move from one chromosome to another in same site, or to a plasmid.
• These can be beneficial or harmful and can cause changes in traits, replacement of damaged DNA and transfer of drug resistance.
• Mutations.

Mutations

• Mutations are a result of natural processes or may be induced
• Spontaneous mutations are heritable changes to the base sequence in DNA
• They result from natural phenomena include radiation or uncorrected replication errors, like UV light creating dimers.

Point Mutation Details

• Point mutations are a 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

• Repair mechanisms attempts to correct mistakes or damage in the DNA
• Mismatch repair involves DNA polymerase, using "proofreading” the new strand while removing mismatched nucleotides.
• Excision repair involves cutting out damaged DNA and replacing it with correct nucleotides.

Bacterial Growth and Metabolism

Learning Objectives

• 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 Overview

• Metabolism refers to all the biochemical reactions that occur in a cell or organism.
• Bacterial metabolism focuses on the chemical diversity substrate oxidations and dissimilation reactions, which generate energy.
• The bacterial cell is a specialized energy transformer.

Heterotrophic Metabolism

• Heterotrophic metabolism is the biologic oxidation of glucose to yield ATP and compounds needed for the bacterial cell for biosynthetic or assimilatory reactions.
• Respiration is a heterotrophic metabolism using oxygen, which generates 38 moles of ATP from 1 mole of glucose, yielding 380,000 cal.
• Fermentation, a heterotrophic metabolism, uses organic compounds rather than oxygen as the terminal electron acceptor.
• Less energy is generated from this incomplete glucose oxidation, supporting anaerobic growth.

Metabolic Cycles

• The Krebs cycle is the oxidative process by which pyruvate is decarboxylated to CO2, yielding 15 moles of ATP (150,000 calories).
• The glyoxylate cycle, a modification of the Krebs cycle in some bacteria, generates acetyl coenzyme A directly from oxidation of fatty acids or other lipid compounds.
• In respiration, ATP is formed through electron transfer reactions within the cytoplasmic membrane driving 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: An organism that obtains carbon in an organic form made by other living organisms
• Autotroph: An organism that uses CO2 (an inorganic gas) as its carbon source
• Autotroph is 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 facilitate molecule crossing faster than possible with 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

• This is the digestion of complex nutrient material into simple, absorbable nutrients.
• It is accomplished through the secretion of exoenzymes into the extracellular environment.

Influences on Microbial Growth

• Include temperature, oxygen requirements, pH, Osmotic pressure, UV light and tolerance to barometric pressure.

Temperature

• 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 15°C; capable of growth at 0 - 20°C
• Mesophiles: Optimum temperature 40°C; Range 10° - 40°C (45); Most human pathogens
• Thermophiles: Optimum temperature 60°C; Capable of growth at 40 - 70°C
• Hyperthermophiles: Archaea that grow optimally above 80°C; Hot-water vents

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 bacteriaare not affected by oxygen, evenly spread along the test tube. -Facultative anaerobe and aerobe: Capable of growth in the absence or presence of oxygen

pH Values

• Acidophiles: Optimum pH is relatively to highly acidic
• Neutrophiles: Optimum pH is ranges about pH 7 (plus or minus)
• Alkaphiles: Optimum pH is relatively to highly basic

Osmotic Pressure

• Bacteria are 80% water and require water to grow
• Sufficiently hypertonic media concentrations cause water loss from the bacteria causing the cell to contract
• Osmosis -fluid leaves bacteria
• Plasmolysis is cell membrane separation.
• Extreme or obligate halophiles need adapted and require high salt concentrations.

UV Light

• Is effective at killing bacteria
• Damages the DNA
• In sufficient quantity can kill organisms
• Low UV range causes mutagenesis
• Endospores are resistant and can survive at high exposures

Barophiles

• Bacteria grow in oceans at moderately high hydrostatic pressures
• Membranes and enzymes depend on pressure to maintain their 3D shape and functional characteristics
• Barotolerants and have is from 100-500.
• Barophilic is 400-500
• Extreme barophilic occur 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, while the host is harmed.
• Non-symbiotic: Includes free-living organisms where relationships are not required for survival.
• Synergism: Members cooperate and share nutrients
• Antagonism: Some members are inhibited or destroyed by others

Biofilms

• Complex relationships among numerous microorganisms
• Develop an extracellular matrix that: Adheres cells, to one another, allows attachment to a substrate, sequesters nutrients, and may protect individual population of bacteria in the biofilm.

Microbial Growth in Bacteria

• Binary fission in prokaryotes involves asexual reproduction
• One cell becomes two, and the basis is for population growth.
• Parent cell enlarges andDuplicates its choromosomes that form a central Septum and divides Cell into two daughter cells

Population Growth

• Generation / doubling time is time required for a complete fission cycle and a measure of the growth rate of an organism.
• Some populations grow a small number of cell to several million in only a few hours!!

Prokaryotic Growth Overview

• It includes:lag phase, logarithmic (log) phase, stationary phase, decline (exponential growth),

Prokaryotic Growth Phases

• Lag phase- No cell division occurs while bacteria adapt to their new environment.
• Logarithmic (log) phase involves Exponential population growth and Human disease symptoms usually develop
• Stationary phase: When reproductive and death rates equalize,

Other Methods Analyzing Growth

• Turbidity
• Direct microscopic count
• Coulter Counting