Bacterial Structure and Growth, and Genetics Flashcards
Functions of Bacterial Envelope
Protect cells from mechanical disruption and from bursting caused by hypertonicity.
Metabolic process
Mediate attachment to human cell surfaces (disease)
Chromosome segregation
Electron transport system (mitochondria)
Endotoxin
Peptidoglycan Structure
Major Cell Wall components: peptidoglycan and teichoic acid
Peptidoglycan composed of glycan chains (NAG-NAM) cross-linked by peptide chains
Peptidoglycan is resistant to most mammalian enzymes except lysozyme (in tears)
Penicillin blocks the formation of cross-links
Differences between Gram Positive (+) and Gram Negative (-) Envelope
Gram+
2 Layers:
Inner cytoplasmic membrane
Thick Peptidoglycan layer (60-100%) (Cell Wall)
Low lipid content
No endotoxin
No porin channel
Gram - 3 Layers: Inner cytoplasmic membrane Thin Peptidoglycan layer (5-10% peptidoglycan) Outer Membrane with LPS
High lipid content (b/c outer membrane)
Endotoxin (LPS) (composed of 3 parts: Lipid A is embedded in membrane, core polysaccharide, then O side chain formed by linked sugars)
Porin channel (selective)
Gram Stain Procedure
1) Heat Fixation: Denatures microbial proteins, fixing microbe to slide
2) Primary Stain: Crystal Violet dye
—Gram+ stains Purple
—Gram- stains Purple
3) Mordant (Iodine): sets the stain; increases the crystal violet affinity; omission of this step causes pink stain, regardless of cell type.
—Gram+ remains purple
—Gram- remains Purple
4) Decolorize with Ethanol:
—Gram+ remains Purple
—Gram- becomes Colorless
5) Counterstain (Safranin Dye)
—Gram+ remains Purple
—Gram- stains Pink
Aerobic vs. Anaerobic Bacteria
Catalase and Superoxide Dismutase
Catalase Test
Aerobic bacteria
Require O2 and metabolize by respiration
Cannot ferment
Contain Catalase and Superoxide Dismutase
Anaerobic bactera
O2 inhibits or kills them
Ferment in absence of O2
No catalase or superoxide dismutase
Catalase Test
Add hydrogen peroxide to samples. Bubble formation = O2, indicating positive result.
Catalase:
Converts hydrogen peroxide to water and O2
(Hydrogen peroxide was formed by adding electrons or protons to O2)
Superoxide Dismutase (SOD):
Protects oxygen-metabolizing cells against harmful effects of superoxide free radicals
Converts superoxide anion to water and hydrogen peroxide.
Both hydrogen peroxide and superoxide anion are very toxic to cells.
Antibiotic Targets of Peptidoglycan Synthesis
Beta-Lactam Antibiotics:
Inhibit Last Step in Peptidoglycan Synthesis (Transpeptidation step) = the final cross-linking between peptide side chains, mediated by transpeptidase (penicillin-binding protein; PBPs).
Bacitracin:
Inhibits release of the muropeptide subunits of peptidoglycan from the lipid carrier molecule that carries the subunit to the outside of the membrane.
Cycloserine:
Inhibits synthesis of D-Ala, which is required for the synthesis of the NAG-pentapeptide.
Vancomycin:
Recognizes and binds to the two D-Ala residues on the end of the peptide chains and prevents the synthesis of long polymers of NAM-NAG and the cross-linking of them.
Bacterial Growth Curve
Lag Phase:
—Growth is not detectable. The cells are active in adjusting the levels of vital cellular constituents necessary for growth in the new medium.
Log (Exponential) Phase:
—Constant, maximal growth rate. The generation time is constant.
—Penicillin works only in this phase b/c this is when cell walls are being synthesized.
Stationary Phase:
—When a required nutrient becomes exhausted or the concentration of toxic waste products becomes too high, growth stops.
Sporulation
Clostridium and Bacillus:
—The only medically important bacteria species that form spores.
—Clostridium: terminal endospores.
—Bacillus: central endospores.
Spores survive adverse conditions.
Germination under appropriate environmental conditions.
Sterilization requires High Heat and Pressure.
Prokaryotes vs. Eukaryotes Differences
and
Potential targets for antibiotics?
Size: Avg size 1-10 micron; 10-100 micron Nucleus: Nucleoid (no membrane); Membrane-bound Chromosomes: Single circular loop of naked DNA; Linear, arranged with Histones Organelles: Absent Present, various functions Ribosomes: Present, small: 70S Present, large: 80S Cell wall: Present Absent
Potential targets for antibiotics:
Ribosomes and Cell wall
Factors that Contribute to High Frequency of Mutations in Bacteria
Rapid Growth
Selections
Haploidy
—Thus, mutations are dominant, rather than selective
DNA Replication in Bacteria
Replication begins @ origin of replication.
Two replication forks proceed in opposite directions until they meet at the replication termination site (ter).
Cell division by Binary Fission
Doubling time for E. coli = 20 min
DNA Replication = 40 min
Daughter strand in E. coli begins replication before cell division is complete.
Mycobacterium doubling time = 24 hours.
—Thus, culturing M. tuberculosis takes too long for the purposes of diagnosis.
Bacterial Chromosoms are Circular:
—Otherwise, if they were linear, they would require a telomere, which is present in human cells to protect the ends of the DNA and facilitate replication of the ends.
Prokaryotic Replication Fork
DNA Gyrase:
—Responsible for supercoiling bacteria DNA, to get it back into the nucleoid region of the cell after synthesis.
—Quinolone antibiotics target DNA Gyrase. (Quinolones, e.g. floxocin and nalidixic acid)
RNA Polymerase:
—Function is lagging strand synthesis.
—Rifampin targets RNA Polymerase
Binary Fission
Binary Fission
1) Begins with DNA synthesis at origin of replication.
2) Parent cell enlarges: Volume, Cell membrane, and Cell wall.
3) Notches develop in cell wall as chromosome is replicated and attached.
—Cell Wall is responsible for segregating and separating the cell into two. (In humans, the centrosomes and spindles do this.)
4) Septum grows inward to divide cell and chromosome moves towards center.
5) Septum is completed, membrane repaired, and cells either separate or remain together (diplo- or tetrad- configuration)
Ribosomes Targeted by Antibiotics
Ribosomes Targeted by Antibiotics
Small (30s) Subunit:
—Aminoglycosides: Block initiation of translation and cause misreading of mRNA
—Tetracyclines: Block attachment of tRNA to the ribosome
—Streptogramins: Interfere with certain steps of protein synthesis
Large (50s) Subunit:
—Macrolides: Prevent the continuation of protein synthesis. *Erythromycin
—Chloramphenicol: Prevents peptide bonds from being formed
—Lincosamides: Prevent continuation of protein synthesis
—Oxazolidinones: Interfere with initiation of protein synthesis
Ribosomes in Prokaryotes vs. Eukaryotes
Ribosome: 70s (Pro), 80s (Euk)
Small subunit: 30s (Pro), 40s (Euk)
Large subunit: 50s (Pro), 60s (Euk)
Transcription and Translation in Bacteria vs. Humans
Transcription and Translation are coupled in bacteria. This cannot occur in human cells because those two process occur at different locations.
Causes of Mutation
Causes of Mutation:
—Spontaneous mutation:
-Errors in DNA replication
-Spontaneous lesions
-Transposable genetic elements (insertion sequence, IS)
—Induced mutation:
-Chemicals
-Ultraviolet radiation induces Thymine dimerization. Hence why sun exposure causes skin cancers.
-X-rays induce single- and double-stranded breaks
Transversion Mutation
Single Base Changes:
—Transversion: Purine and Pyrimidine base exchange
—Transition: Purine to Purine or Pyrimidine to Pyrimidine base change
Transition Mutation
Single Base Changes:
—Transversion: Purine and Pyrimidine base exchange
—Transition: Purine to Purine or Pyrimidine to Pyrimidine base change
Replacement Mutation
Types of Mutations in Nucleotide Sequence
Replacement: One base change. Note: This type of mutation cannot cause a frameshift.
Microdeletion: Removal of a single base pair
Microinsertion: Addition of a single base pair
Deletion: Removal of segment of many base pairs
Insertion: Addition of segment of many base pairs
Inversion: Change the direction of a DNA segment
Duplication: Addition of a redundant DNA segment
