Bacterial Cell Division and Growth Curve

Microbial Growth

• Binary fission involves DNA replication and cell elongation, resulting in identical daughter cells.
• The steps of binary fission are: DNA replication, cell elongation, formation of a division septum, and cell separation.
• Cytokinesis in bacteria is directed by the protein FtsZ, which assembles a Z ring to divide the cell.
• Generation time is the time it takes for a bacterial population to double. E. coli doubles every 20 minutes.

Microbial Growth Curve Phases

• Lag Phase: cells synthesize new components.
• Log Phase: cells divide exponentially; gene expression is at its maximum.
• Stationary Phase: the number of viable cells remains constant; endospore formation occurs.
• Death Phase: nutrients deplete, toxic waste accumulates, and cells die. Persister cells can remain alive longer than log phase bacteria.
• Understanding these phases is crucial for describing biochemical characteristics (log phase) and optimizing growth conditions.

Laboratory Methods for Cell Counts

• Direct Cell Count: Uses counting chambers, electronic cell counters (flow cytometry), or membrane filters.
• Plate Count: Involves spread and pour plate techniques to determine colony forming units (CFU).
• Most Probable Number: Tubes are inoculated with different sample amounts to assess bacterial growth.
• Indirect Cell Count: Uses a spectrophotometer, dry weight measurements, or metabolic activity assessments.

Other Cell Division Methods

• Budding: A bud forms on the cell, which then divides.
• Fragmentation: A parent organism splits into fragments, each growing into a new individual.

Biofilms

• Biofilms are complex communities of microorganisms on a surface.
• Microorganisms in Biofilms feed off of each other's released resources. Biofilms are resistant to antimicrobial agents.
• Health risks from biofilms include medical device contamination, wound infections, foodborne illnesses, and dental plaque.
• Biofilms are addressed with physical removal, chemical disinfectants, antibiotics, and prevention via cleaning.

Quorum Sensing

• Quorum sensing is communication between bacteria within a biofilm
• Autoinducers bind to receptors on cells, triggering changes in gene expression and cellular behavior.
• Quorum sensing helps bacteria adapt to changing conditions and compete with other microorganisms.

Oxygen and Carbon Dioxide Requirements for Growth

• Obligate Aerobes: require oxygen to grow.
• Obligate Anaerobes: inhibited or killed by oxygen.
• Facultative Anaerobes: can grow without oxygen but prefer oxygen.
• Aerotolerant Anaerobes: grow evenly with or without oxygen.
• Microaerophiles: require low amounts of oxygen.
• Capnophiles: grow better in higher concentrations of carbon dioxide.

Anaerobes and Oxygen Exposure

• Anaerobes are killed by oxygen because it leads to production of reactive oxygen species (ROS) that damage cells.
• Aerobes produce protective enzymes against ROS, which anaerobes lack.

pH Requirements for Growth

• Minimum pH: the lowest pH at which a microorganism can grow.
  • Acidophiles grow best between pH 0 and 5.5.
• Optimum pH: the pH at which a microorganism grows best.
  • Neutrophiles grow best around neutral pH 7.0.
• Maximum pH: the highest pH at which a microorganism can grow.
  • Alkaliphiles grow best between pH 8 and 10.5.
• Prokaryotes regulate pH using proton pumps to transport H+ across the cell membrane.
• pH regulation is crucial for maintaining cellular processes, as extreme pH can denature proteins.

Temperature Requirements for Growth

• Minimum Temperature: the lowest temperature microorganisms can grow at.
• Optimum Temperature: the temperature at which microorganisms grow best and divide rapidly.
• Maximum Temperature: the highest temperature microorganisms can grow at.
• Microbes rely on their enzymes to influence their preferred temperatures.
• Psychrophiles: grow best between -5°C and 20°C.
• Psychrotrophs: prefer 20°C – 30°C but can grow at low temperatures.
• Mesophiles: grow best between 20°C – 45°C.
• Thermophiles: grow best between 45°C – 80°C.
• Hyperthermophiles: grow at temperatures > 80°C.
• Thermophiles and hyperthermophiles have extra proteins for surviving at high temperatures.

Adaptations to Harsh Environments

• Spore Formation: protects cells from extreme temperatures and dehydration.
• Biofilm Formation: provides nutrition and protection.
• Quorum Sensing: coordinates behaviors and adaptations.
• Horizontal Gene Transfer: allows cells to acquire new adaptations.

Other Growth Requirements

• Barophiles: require high barometric pressure.
• Halophiles: require high salt concentrations.
• Osmophiles: require high osmotic pressure.
• Xerophiles: grow in low humidity.
• Phototrophs: require light.
• Mechanosensitive Channels: help to balance solutes.

Biochemistry of the Genome

• Nucleic acids (DNA and RNA) store and transmit genetic information.
• Nucleotides consist of a nitrogenous base, a 5-carbon sugar, and a phosphate group.
  • The building blocks of nucleic acids.
• Nitrogenous bases: adenine, cytosine, guanine, and thymine in DNA; adenine, cytosine, guanine, and uracil in RNA.
• 5-Carbon Sugar: ribose in RNA, deoxyribose in DNA.
• The sequence of bases determines the genetic code for development, function, and reproduction.
• Pyrophosphate contributes energy during polymerization.

DNA Base Pairing

• Adenine pairs with thymine via 2 hydrogen bonds
• Guanine pairs with cytosine via 3 hydrogen bonds

DNA Double Helix

• The double helix in DNA is antiparallel because the 5’ end aligns with the 3’ end of the opposite strand.
• Major and minor grooves in the DNA backbone allow protein access for replication and transcription regulation.

Ribonucleotides

• Ribonucleotides build RNA.
• Adenine pairs with uracil via 2 hydrogen bonds.
• Uracil replaces thymine in RNA.
• A phosphate group (negative charge) forms a covalent bond with the 3’ carbon.
• The 2’ carbon has an extra hydroxyl (-OH) group.

RNA vs DNA

• Similarities: both are made of nucleotides, store and transmit genetic information, and act as templates for nucleic acid synthesis.
• Differences: RNA is single-stranded, contains uracil instead of thymine. RNA is shorter (<1000 nucleotides), contains ribose instead of deoxyribose. RNA functions in protein synthesis, gene regulation, and catalysis.

Types of RNA Used in Protein Synthesis

• Messenger RNA (mRNA): carries genetic information from DNA to ribosomes via codons. mRNA is made during transcription and is unstable.
• Transfer RNA (tRNA): brings amino acids to the ribosome. tRNA carries an anticodon complementary to mRNA codons and is short.
• Ribosomal RNA (rRNA): catalyses peptide bonds between amino acids, making up ribosomes and demonstrating enzyme activity.

RNA as Hereditary Information

• RNA can carry genetic information like DNA
• Typically in viruses
• Helped with the evolution of life on Earth.

Central Dogma

• DNA contains genetic information transcribed into RNA, then translated into a protein sequence.
• Proteins transport molecules, perform biochemical reactions, and provide support.

Genes, Genotype and Phenotype

• Gene: Codes for a trait.
• Genotype: the combination of genes and alleles.
• Phenotype: the observable characteristics that are expressed.
• Genotype determines phenotype.

Prokaryotic Chromosome Structure

• DNA is circular and double-stranded, folded into a compact structure with the help of histone-like proteins.
• Prokaryotic chromosomes contains less noncoding DNA than eukaryotes.
• The origin of replication is where DNA initiates replication.

Extrachromosomal DNA

• Plasmids aid in breaking down nutrients and resist antibiotics.
• Plasmids with specific genes can be introduced into cells to manipulate genetic makeup through genetic engineering.

Microbial Genetics

• Semiconservative DNA replication creates new DNA molecules with one original strand and one new strand.
• Replication is bidirectional because strands are antiparallel, requiring synthesis in both directions from the origin.
• The leading strand is replicated continuously; the lagging strand is replicated in Okazaki fragments.
• Helicase: breaks hydrogen bonds.
• Primase: synthesizes RNA primers.
• DNA Polymerase: adds nucleotides to the 3’ end.
• Topoisomerase: relieves tension.
• Single-Strand Binding Proteins: stabilize separated strands.
• Exonuclease: proofreads synthesized strands.

Bacteria vs Eukaryotes

• Singular circular vs multiple linear
• One origin point for replication vs multiple
• Rate of replication: 1000 nucleotides per second vs 100 nucleotides per second.
• Telomerase: absent vs present
• Prokaryotes are faster and have less mutations

Plasmid Replication

• The process is unidirectional, starting at an A-T rich region, similar to rolling circle replication.
• DNA ligase joins newly synthesized strands creating 2 complete copies.

RNA Synthesis

• RNA polymerase binds to the promoter, unwinds DNA, and adds nucleotides to the 3’ end.
• Transcription includes initiation, elongation, and termination.

Transcription – Prokaryotes vs Eukaryotes

• Prokaryotes can be monocistronic or polycistronic, while eukaryotes are monocistronic.

Genetic Code

• Set of rules translating DNA to protein, with each codon (3-nucleotide sequence) specifying an amino acid or stop signal.
• The genetic code is almost universal, conserved throughout evolution.

Translation

• Small ribosomal subunit binds to mRNA to find the start codon.
  • Then tRNA brings amino acid in polypeptide chain to bind to the start codon on mRNA.
• tRNA brings amino acids to add to the polypeptide chain.
• A release factor recognizes the stop codon, releasing the completed polypeptide.

Translation: Eukaryotes vs Prokaryotes

• In prokaryotes, transcription and translation occur simultaneously in the cytoplasm, while in eukaryotes, transcription occurs in the nucleus first, then translation can begin.
• Prokaryotes have smaller ribosomes.
• Eukaryotes add a 5’ cap and 3’ polyAtail.

Asexual Prokaryotes Achieve Genetic Diversity

• Mutation: change in the DNA sequence, which can be neutral, beneficial, or harmful.
• Mutagens:
• Chemical mutagens: Chemicals that react with DNA.
• Physical mutagens: Radiation or high energy particles
• Biological mutagens: Viruses of some Bacteria.

UV Radiation

• UV radiation can cause thymine dimers, interfering with normal replication and transcription, leading to errors and mutations.

Mutation Types

• Missense: a single nucleotide change that results in a different amino acid being incorporated into the protein.
• Nonsense: creates a premature stop codon, leading to an incomplete protein.
• Silent: does not change the amino acid sequence due to codon degeneracy.

Mutation Rate Amongst Bacteria

• Scientists expose bacteria to known mutagen and track the rate of mutation that occurs. Scientists can also compare a large number of genomes from one bacteria species and compare their DNA.

DNA Repair Mechanisms

• Mismatch Repair: corrects errors in newly replicated DNA.
• Nucleotide Excision Repair: enzymes recognize and replace damaged strands.
• Direct Repair: enzyme breaks apart pyrimidines in the presence of light.

Horizontal Gene Transfer (HGT)

• Occurs between independent organisms with possible recombination.
• Recombinants are organisms with acquired genetic material from HGT, leading to new traits and increased evolution.

Genetic Recombination

• DNA from 2 sources creates new combinations of genes in their offspring.
• Outcomes: Mix of traits, variation in makeup, increased genetic diversity, and chromosomal abnormalities.

Horizontal Gene Transfer Processes

• Transformation: bacteria takes up DNA from the environment.
• Transduction: a virus carries DNA from one bacterium to another.
• Conjugation: two bacteria connect through a pilus and exchange DNA.

Asexual Gene Transfer

• Results in prokaryotic genetic diversity through mutations and horizontal gene transfer methods.

Transposons

• Small segments of DNA that can jump between locations on the genome.
• Structure: two terminal repeats and a middle region with transposase gene.

Biotechnology

• Biotechnology: Use of microorganisms, cells, or cell components to make a product.

• Genetic engineering: Alter genes in an organism

• Recombinant: Gene inserted into another organism

• Transgenic: Another term for recombinant

• Cloning:

  • Isolate DNA fragment of interest
  • Insert into a cloning vector (plasmid)
  • Introduce recombinant vector into host
  • Grow host cells in culture to amplify DNA

• Restriction endonucleases: Enzymes that cut DNA at specific sites, can be used to create recombinant DNA molecules.

• Plasmids are important in biotechnology as they can be used as cloning vectors.

• Genomic Libraries are collections of DNA fragments that represent the entire genome of an organism. Used to isolate specific genes.

• Gel electrophoresis can be used to separate DNA fragments Based on size. Smaller fragments migrate faster.

• Restriction Fragment Length Polymorphism is an analysis of DNA fragments based on size differences.

• Polymerase Chain Reaction is used to amplify DNA.

• Biotechnology Applications:

  • Medicine
  • Agriculture
  • Industry

Gene Therapy

• Gene therapy mechanisms:

  • Replace mutated gene
  • Inactivate mutated gene
  • Introduce new gene

• Risks:

  • Immune response
  • Insertional mutagenesis

• Benefits:

  • Cure genetic diseases
  • Treat cancer

• Ethical issues include:

  • Informed consent
  • Justice
  • Privacy

• Regulatory Agencies:

  • FDA
  • NIH