MICROBIO 3.1 - BASICS OF BACTERIA

Class Objectives: Bacterial Refresher

• Be able to define and use definitions in sentences and examples.
• Compare and contrast bacteria with eukaryotic host cells.
• List and define features of bacteria structure and morphology.
• Compare and contrast Gram+ and Gram-.
• Learn how Acid Fast differs from Gram+/-.
• Name bacteria based on visual presentation, e.g., Gram+ cocci, Gram- rods.
• List growth requirements of bacteria.
• Identify bacterial growth phases (including identification from graph) and what occurs at each phase.
• Outline the steps of binary fission and describe how it differs from horizontal transfer.
• Compare and contrast the three types of horizontal DNA transfer: conjugation, transduction, transformation.
• Review Establishment of Infectious Disease and provide examples of "Damage", the focus of the module.

Bacterial Names

• Each bacterium is named by its genus, like Escherichia, and species, like coli.
• A strain is a genetic variant or subtype of a microorganism.
• The hyperlink for the "Bacteria Flowchart": https://marian.instructure.com/courses/4159601/files/316625280?wrap=1

Bacteria Structure & Morphology

• Basic components of bacteria include the cell wall, plasma/cytoplasmic membrane, cytoplasm, ribosomes, nucleoid, and pili/fimbriae.
• Capsule is present in some bacteria.
• Gram-positive bacteria have teichoic acid and lipoteichoic acid in their cell wall.
• Gram-negative bacteria have lipopolysaccharide/endotoxin in their outer membrane.
• The morphology of bacteria can be clusters, chains, cocci (spherical), bacilli (rods), or spirochetes (spiral).

Bacteria Cell Anatomy

• Bacteria are haploid, containing a single set of unpaired chromosomes.
• Instead of a nucleus, bacteria have a region called the nucleoid that contains suspended genetic material.
• Genophore (prokaryotic DNA) is double stranded and circular, unlike eukaryotic genetic material.

The Big Four

• The Big Four includes: Gram-positive cocci, Gram-positive rods, Gram-negative cocci, Gram-negative rods

Gram Positive vs. Gram Negative

• Gram-positive bacteria retain purple dye and iodine after an alcohol challenge.
• Gram-negative bacteria do not, and are counterstained with safranin.
• "Staphylo" refers to clusters; "Strepto" refers to chains.

NAG and NAM

• NAG and NAM, found in peptidoglycan, protect against osmotic pressure.
• Murein/peptidoglycan shape determines bacterial shape like rods (bacilli), spheres (cocci), and helices (spirilla).
• Some antibiotics inhibit murein/peptidoglycan synthesis, including penicillins, cephalosporins, & carbapenems (β-lactams).

Outer Membrane & Lipopolysaccharide (LPS)

• Lipid A (endotoxin) anchors LPS to the outer membrane and elicits fever in the host.
• Core polysaccharide is composed of short series of sugars.
• O-antigen consists of hydrophilic carbohydrate chains that exclude hydrophobic compounds.
• Hydrophilic compounds can enter cells via specialized porin channels and passive diffusion.
• Larger molecules such as B12 and iron are translocated in.

Periplasmic Space

• A compartment between two membranes.
• Contains a thin murein/peptidoglycan layer & gel-like solution for facilitating nutrition.
• Contains degradation enzymes to make impermeable molecules smaller and binding proteins for transport.
• Contains B-lactamase enzymes, so Gram- bacteria are more resistant to B-lactam antibiotics.

Acid Fast

• Use a special stain
• Acid-fast bacteria include M. tuberculosis
• Cells walls contain waxes.
• After staining with basic fuchsin, acid-fast bacteria resist decolorization by acid-alcohol.
• Non-acid fast bacteria are counterstained with methylene blue.
• Acid-fast bacteria are red, non-acid-fast cells are blue.

Occupancy & Movement

• Capsule is a slimy outer coating to prevent phagocytosis.
• Flagella are long, helical filaments for motility as well as aid in bacterial chemotaxis.
• Swimming occurs when all the organism is counterclockwise.
• Tumbling is movement that is sometimes counterclockwise, sometimes clockwise.
• Pili (fimbriae) facilitate attachment of cells to other surfaces.

Cytoplasmic Membrane & Transport

• Contains permeases to facilitate the entry of most metabolites.
• Facilitated diffusion is when a substance is carried across the membrane down a concentration gradient and uptake is driven by intracellular use.
• Active transport is the movement of molecules across the cell membrane into a region of higher concentration, assisted by enzymes. Requires energy.
• Group translocation, or phosphorylation-linked transport, is energy-dependent transport of certain sugars, which are chemically altered in the process.

Bacteria: Growth & Genetics

• Growth results in an increase in number via binary fission, forming a colony of millions of cells; generation time varies by species and is affected by environmental factors.
• Genetics: Composed of haploid w/circular dsDNA, pathogenicity islands, extrachromosomal genetic elements (plasmids, bacteriophages), and horizontal DNA transfer.
• Quorum sensing: Bacterial cell communication like virulence factor secretion, biofilm production, sporulation, etc.

Growth Curve

• Tracks the stages of cell population growth.
• Lag Phase: Metabolic activity, no cell division.
• Log Phase: Exponential growth and division.
• Stationary Phase: Proliferation and cell death in steady state.
• Death Phase: Declining population.

Replication

• Binary fission.
• Chromosome replication
• Origin: where replication begins

Growth Requirements

• Carbon, nitrogen, energy source, water, ions.
• Bacteria secrete siderophores (that steal iron from the host.)
• Obligate anaerobes do not use O2 for growth (Ex: Clostridium)
• Obligate aerobes require O2 (Ex: M. tuberculosis)
• Facultative anaerobes grow with or without O2 like Staphylococcus
• Obligate intracellular organism Rely on ATP from host cell (Ex: Chlamydia)
• Semiconservative - Each copy contains 1 parental strand and 1 daughter strand

Bacterial Growth

• Growth: increase in number of bacterial cells.
• Binary fission: asexual reproduction that separates a bacterial cell body into 2 new parts.
• Generates duplicate genetic material and divides into two parts, cytokinesis).
• Generation time: Time it takes for the population to double; rate varies by species and with pH, temperature, and nutrients
• Quorum sensing: Bacterial communication that arises when cell density is too high; moderates virulence factor secretion, biofilm production, and sporulation.
• Pathogenicity islands: distinct regions of some bacterial chromosomes for virulence factors that are absent in non-virulent strains.

Binary Fission

• Asexual & semiconservative- each new chromosome comprises one strand of DNA from the parental chromosome and one complementary daughter strand.
• Origin of replication (OriC)- replication initiator proteins bind.
• Terminus- where DNA replication terminates.
• Replication "bubble" is the space between the strands.
• Two y-shaped replication forks, one on each side of the bubble.
• DNA helicase- enzyme that separate DNA into single strands.
• Synthesis starts at the OriC.
• Synthesized by replisome- contains several enzymes (helicase, primase & DNA polymerase) & creates a replication fork to duplicate both the leading & lagging strand.

Binary Fission Processes

1. Replication initiator proteins bind to OriC.
2. DNA helicase opens OriC & creates two replication forks.
3. DNA is replicated, and daughter strands are created via replisome (i.e. DNA polymerase).
4. Elongation
5. Cleavage furrow develops & cytokinesis.

Bacterial Growth Curve Phases

• Lag Phase: Bacterial cells engage in metabolic activity but not cell division; during this stage, the bacteria acclimate to the growth conditions.
• Logarithmic (exponential) phase:Rapid cell division occurs.
• Stationary phase: curve plateaus because proliferation and cell death are in balance; this steady state is reached when nutrients are running low and/or toxin levels are elevated.
• Death phase: the number of bacteria declines.

Horizontal Gene Transfer

• Process in which an organism transfers genetic material to another organism that is not its offspring.
• Enables bacteria to respond and adapt to their environment much more rapidly by acquiring large DNA sequences from another bacterium in a single transfer.
• Three mechanisms: conjugation, transduction, transformation

Horizontal DNA Transfer

• Conjugation: unidirectional plasmid DNA transfer. Requires high-frequency recombination.
• Transduction: bacteriophage transfers Cell DNA, can be generalized or specialized.
• Transformation: Foreign DNA is picked up.

Conjugation

• Involves the unidirectional transfer of plasmid DNA
• Donor cell has circular F plasmid (F stands for "fertility factor"); the recipient cell, which does not have an F plasmid.
• F+ cell extends sex pilus (aka conjugative pilus) & attaches to F- cell; Brings the two cells in close physical contact.
• One strand of F plasmid unwraps & enters recipient cell.
• Each cell now has single strand of original F plasmid. Each cell synthesizes a complementary strand of plasmid DNA, so that both cells are now F+.
• High-frequency recombination (Hfr) is when plasmid is integrated into bacterial DNA; it takes with it some of the donor cell's DNA.
• Resistance plasmids carry genes that confer antibiotic resistance to bacterial cells; conjugation allows quick dissemination of this trait throughout a colony, ensuring its survival despite antibiotics.

Transduction

• Occurs when bacteriophages transfer DNA between cells.
• A bacteriophage (aka bacterial virus) injects its DNA into the bacterial cell.
• The phage replicates, & the bacterial DNA is fragmented.
• As phage replicates & assembles, some phages will incorporate bacterial DNA fragments.
• Bacterial cell lyses releasing the phages.
• Phages with bacterial DNA can inject donor bacterium's DNA into a new recipient.
• Within new cell, donor DNA combines with recipient's DNA, forming recombinant DNA.

Transformation

• Occurs when bacteria take up foreign DNA & incorporate the material into their own DNA.
• Donor cell with its bacterial DNA exists.
• When bacterium lyses, it releases DNA fragments.
• These fragments can then enter recipient cells.
• Fragments are integrated into recipient DNA.
• Some bacteria (e.g., Neisseria) have surface receptors to facilitate transformation.
• Frequent transformation may facilitate immune system and/or antibiotic evasion.
• Scientists use transformation to study gene expression or inhibition by artificially injecting foreign DNA into eukaryotic cell nucleus (transfection).

Establishment of Infectious Diseases

1. Encounter: The agent meets the host.
2. Entry: The agent enters the host.
3. Spread: The agent spreads from the site of entry.
4. Multiplication: The agent multiplies in the host.
5. Damage: The agent, the host response, or both cause tissue damage.
6. Outcome: The agent or the host wins out, or they learn to coexist.

Bacterial Pathogenisis

• Bacteria produce toxins to break down host tissues to promote their own growth:
  • Facilitating invasion.
  • Releasing nutrients.
  • Resisting destruction.
• Bacteria trigger immune and inflammatory responses as they colonize the host.
• Damage to Host
• Endotoxins: Lipopolysaccharide found in Gram-Negative Strains. -Endotoxin Traits: Part of Cell Wall, Low Toxicity and triggers cytokine release.
• Exotoxins: Traits such as, polypeptides, dimeric A-B, high toxicity and kill/alter cells.

Cytokine Release

• Complement Activation and Inflammation.
• Tissue factor activation/Coagulation.
• TNF, NO, bradykinin/Hypotension.