Ch10 11 BIOL251A

• Explain the reasons for studying DNA

• All cells store genetic information as DNA

  • Genetic variation is due to changes in DNA

• Ground-breaking research with Streptococcus pneumonia and bacteriophages led biologists to understand the Flow of genetic information

  • DNA to RNA to Protein 

  • Protein = polypeptide 

  • Genotype to Phenotype

• The proteins involved in bacterial DNA replication make great targets for antibiotics

  • halting DNA replication will kill the bacterial pathogen

• Understand how genetic diversity is occurs in bacterial cells

  • Prokaryotes exchange DNA across species barriers

  • Extra-chromosomal elements contain antibiotic-resistance genes

• Understand and explain the flow of genetic information

- DNA to RNA to Protein 

-protein = polypeptide 

-genotype to phenotype 

• Distinguish between phenotype and genotype and what determines each

Phenotype: -Expressed properties of the genotype - what we can observe ex: yellow glossy looking colony, can break down red blood cells/beta hemolysis 

            -observable characteristics

` May be Influenced by the environment. 

Genotype: The blueprint - Sequence of nucleotides (DNA content); genetic makeup of organisms

The Genotype is determined by the genetic content of an organisms; what genes  are present

Genes are only blueprints - bearers of information

• Genes are only potential properties until expressed

• How changeable/flexible

• Explain the ways that genotypic changes occur in bacteria

-mutations - change in dna sequence/changing of base/ 

 -wild type is common in nature - non mutant 

- a lot of genetic mutations can lead to cancer 

-gene transfer - process by which new donor genes are introduced into a recipient cell 

-three mechanisms figure 11.26 

a). Transformation, the cell takes up DNA directly from the environment. The DNA may remain separate as a plasmid or be incorporated into the host genome.

b). Transduction, a bacteriophage injects DNA that is a hybrid of viral DNA and DNA from a previously infected bacterial cell.

*transduction is transfer of dna via bacteriophage 

c). Conjugation, DNA is transferred between cells through a cytoplasmic bridge after a conjugation pilus draws the two cells close enough to form the bridge. both must be alive at the same time, 2 living cells bump into each other and transfer dna (one has a plasmid to give to the other who does not have a plasmid/feature)

bacteriophage acts as vector 

dna from donor cell injects the dna to receiving cell 

transduction is transfer of dna via bacteriophage 

directly from slides: 

Transformation

The uptake of cell-free (“naked”) DNA from the environment

Competent cells – able to take up DNA

Most bacteria are naturally competent

Naturally occurs between closely related species of bacteria.

DNA may be fragments of chromosomal DNA or plasmids

If the DNA is integrated into genome, the bacterium gains new phenotypic properties

Important mechanisms for acquiring virulence factors and antibiotic resistance

Example: Penicillin-resistant gonorrhea results from transformation.

In the laboratory – process is exploited for genetic engineering

Transduction

Transfer of DNA from one bacterium to another via bacteriophage

Bacteriophage infects bacterial cell, normally packages its own phage DNA during assembly

Occasionally packages fragment of host chromosomal DNA

Transfers host chromosomal DNA to other bacteria

recombination integrates new DNA into chromosome

Now have recombinant strain

don't worry about general or specialized - this tells you the source 

Conjugation

• Explain vertical versus horizontal gene transfer

 -horizontal gene transfer occurs all the time in prokaryotes - acquired from other cells of the same generation, Griffith expt. showed transformation

-vertical generation from different generations, mutations are passed to progeny cells 

• Explain what plasmids are – know the features of plasmids

  •   plasmid - smaller circular dsDNA molecule(s) in many Bacteria and Archaea (1-500 kbp); often more than one plasmid copy/cell

  • do not contain any essential genes 

  • non essential accessory genes

  • genes for antibiotic resistance ****

• What is an R plasmid

  resistance plasmid: a type of plasmid, a small circular DNA molecule found in bacteria, that carries genes providing resistance to one or more antibiotics

• What kind of genes might be carried on plasmids (plasmid encoded traits)

- Plasmids carry a variety of genes that can code for traits such as antibiotic resistance, virulence, and metabolic pathways. 

–Antibiotic resistance

Plasmids can carry genes that make bacteria resistant to antibiotics 

Plasmids can also carry genes that help cells survive their own antibiotic secretions 

Virulence

Plasmids can carry genes that make bacteria more likely to invade and survive in animal systems 

Plasmids can carry genes that produce toxins, such as colicins and subtilisins, that kill other bacteria 

Metabolic pathways

Plasmids can carry genes that help bacteria metabolize compounds 

Plasmids can carry genes that help bacteria expand their nutritional abilities 

Other traits

-carry genes that make bacteria tolerant to heavy metals, such as mercury, cadmium, and silver 

- can carry genes that help bacteria repair DNA damage 

-carry genes that help bacteria fix nitrogen 

Plasmids are extrachromosomal DNA elements that can be found in bacteria and other domains of life. They can spread among bacteria through cell-to-cell contact

• What must plasmids have in order to be transferrable?

  • plasmids must have transfer genes (such as tra genes) &

  •  an origin of replication to be transferred between bacterial cells via conjugation.

    • Origin of Replication (Ori): This is essential for the plasmid to replicate independently inside the bacterial cell.

    • Transfer Genes (tra genes): These genes are part of the conjugative plasmids and encode the machinery needed for transferring the plasmid between bacteria. This includes the formation of a pilus and the mechanism for DNA transfer.

    • Relaxase and Transfer Mechanism: In conjugative plasmids, the relaxase enzyme is involved in the initial separation of the plasmid DNA, and the Type IV secretion system facilitates the transfer of DNA to another bacterium.

    • Mob (Mobilization) Region (for some plasmids): Some plasmids that are not fully conjugative but are mobilizable can still be transferred if they have a mob region. This allows the plasmid to be transferred via conjugation when in the presence of a conjugative plasmid.

• Explain the three different mechanisms of gene transfer in bacteria; know the steps of each mechanism, how are they similar/different and the importance/significance of each of the mechanisms

  • Transformation occurs when a bacterium takes up free DNA from its environment and incorporates it into its own genome, which is what happened in Griffith's experiment. The R strain took up DNA from the heat-killed S strain and became pathogenic.

  • Transduction involves the transfer of genetic material between bacteria through a bacteriophage (virus).

  • Conjugation is the process where bacteria transfer genetic material directly via physical contact, typically through a pilus.

• Explain the experiment that demonstrated gene transfer in bacteria (slide 13)

  • Heat-Killed S Strain: The heat-killed S strain of S. pneumoniae was no longer able to cause disease, but it still contained DNA.

  • Live R Strain: The live, nonpathogenic R strain of S. pneumoniae was harmless on its own.

  • Gene Transfer: When the live R strain and the heat-killed S strain were mixed, some of the DNA from the dead S strain was taken up by the live R strain.

  • Transformation: The R strain incorporated the S strain’s genetic material, which contained the instructions for producing a protective capsule, making the R strain pathogenic (able to cause disease).

  • Result: The transformed R strain now resembled the S strain in appearance and virulence, causing the mice to die.

• Describe and understand the steps of Griffith’s experiment and the significance of the findings

Griffith's experiment showed that a nonpathogenic R strain of S. pneumoniae was transformed into a pathogenic S strain when mixed with heat-killed S strain. Mice injected with this combination died, and the S strain was recovered, indicating that something from the dead S strain had transformed the R strain.

Step 1: Inject mice with live, nonpathogenic R strain of S. pneumoniae (mice survive).

Step 2: Inject mice with heat-killed, pathogenic S strain of S. pneumoniae (mice survive).

Step 3: Inject mice with a mixture of heat-killed S strain and live R strain (mice die).

Step 4: Recover live S strain from the dead mice, showing that the R strain was transformed into the pathogenic S strain.

• Explain the cholera toxin example of transduction

 A patient with cholera suffered severe diarrhea due to a lysogenic phage transferring the cholera toxin gene to Vibrio cholerae. The pathogenic strain arose through horizontal gene transfer (transduction). Treatment: doxycycline

• Explain the Staphylococcus case study

A Type II diabetic patient developed a severe Staphylococcus aureus infection, leading to suspected necrotizing fasciitis (flesh eating) and requiring surgery. CDC analysis revealed virulence genes in prophages, enabling antibiotic-induced horizontal gene transfer.

• Explain the bacillus anthracis case study

  • A student in Botswana studying leather tanning developed flu-like symptoms, including fever, chest pain, and coughing up blood. Suspecting anthrax, the physician ordered tests, which confirmed the diagnosis. The patient showed signs of meningitis and died despite IV penicillin treatment.

  • Anthrax – disease occur when A. anthracis endospores enter the body.

  • Infection typically leads to meningitis (inflammation of the meninges); which is often fatal. The patient likely inhaled the endospores while handling the hides of animals that had been infected.

  • Non-hemolytic Bacillus colonies can be presumptively identified using ‘Red Line Alert Test‘, which is an immunochromatographic test for the detection of surface protein found in Bacillus anthracis vegetative cells.

• Briefly describe the different types of mutations

  • Spontaneous mutations - errors during DNA replication, rare

    • Different types of spontaneous mutations can occur

      • Base substitution – most common

      • Missense - Can occur in gene - wrong amino acid may be incorporated into protein

      • Silent Redundancy in genetic code, mutation might not change amino acid incorporated

      • Nonsense - May generate a stop codon - truncated gene product - shorter than normal

      • Base deletion or addition

      • Results in frameshift mutation

      • Transposition

      • Reversion – change back to the wild-type

  • Induced mutations  - external factors may increase mutation frequency by damaging bases resulting in mispairing 

    • Chemical mutagenesis - chemical mutagens, nucleoside analogs, intercalating agents

    • E.g. nitrous acid, alkalating agents

    • Radiation - UV light or DNA damaging (ionizing) radiation, X-rays, g-rays

    • Mutagens may also be carcinogens, cancer causing agents, not universal

    • A substantial number of cancers are caused by carcinogens 

    • All carcinogens are mutagens

    • Not all mutagens are carcinogens

• Chemical mutagenesis - chemical mutagens, nucleoside analogs, intercalating agents

• Radiation - UV light or DNA damaging (ionizing) radiation, X-rays, g-rays

• Mutagens may also be carcinogens, cancer causing agents, not universal

• All carcinogens are mutagens

• Not all mutagens are carcinogens

• Distinguish between spontaneous and induced mutations

  • Spontaneous mutations are naturally occurring while induced mutations are caused by external factors such as chemical, radiation, or other mutagens 

• What are bacterial mutagenesis assays used for?

  • Simple screening technique that uses bacteria to identify potential carcinogens.

  • Measures the mutagenic potential of chemicals.

  • Ames assay

• Explain the Ames assay and the understand what the results mean

  • A histidine-deficient Salmonella typhimurium strain is grown in normal or mutagen-containing media and plated on histidine-free agar. Mutated bacteria grow, and more colonies in the presence of the mutagen indicate it is mutagenic.

KEY CASE STUDIES TO NOTE: 

Cholera case study 

•  Patient presented with abdominal distress, including severe cramping, nausea, vomiting, and watery diarrhea. He also began to experience intense muscle cramping.

• Diagnosis – Cholera

• Causative agent – Vibrio cholerae

• Treatment - doxycycline

• Gene encoding cholera toxin is introduced to bacteria via a lysogenic phage, which carries the cholera toxin gene

• The toxin activates a chloride transporter to pump chloride ions out of the epithelial cells into the gut lumen. Water then follows the chloride ions, causing the prolific watery diarrhea characteristic of cholera

• Pathogenic strains of V. cholerae result from horizontal gene transfer mechanism of transduction

• V. cholerae that have undergone lysogenic conversion

• Bacteria are now able to produce Cholera toxin

Type II diabetes study 

• Patient suffers from Type II diabetes

• Suffered a scrape on lower leg; cleaned the wound

• Two days later the wounds became more red, swollen, and warm to the touch; soreness was deep in the muscle

• 24 hours later patient developed fever and stiffness in the leg; became weak

• Patient sought medical treatment; leg had rash, blistering, small gas pockets underneath the outermost layer of skin, and some of the skin was gray, with putrid smell of the pus draining from blisters,

• Because of symptoms and the rapidity of progression the physician suspected necrotizing fasciitis.

• Patient was admitted to the hospital and placed on IV broad-spectrum antibiotics

• Patient became worse; drop in bp, shallow breathing, blisters turning black and progressing up leg

• Infected region expanded, surgery was done to remove infected tissue and muscle

• The patient’s infection was determined to be caused by Staphylococcus aureus

• Patient’s S. aureus was analyzed for methicillin resistance

• In the case of MRSA, blood flow to the infected area is limited due to production of bacterial toxins. This also limits the effectiveness of intravenous antibiotics.

• Through genomic analysis by the CDC of the strain isolated from the patient, several important virulence genes were shown to be encoded within pathogenicity islands that were associated with prophages.

• Horizontal transfer of pathogenicity island-encoded virulence factors between strains of S. aureus has been shown to occur through induction of prophage

• can be induced by treatment with antibiotics

Anthrax study

• Patient was a student studying methods of tanning leather hides in Botswana, Africa.

• After three week, patient experiences fever, shills, headache, chest pains, muscle aches, nausea, and other flu-like symptoms, and later was coughing up blood

• Symptoms worsened and patient was taken to the hospital

• Due to the symptoms, and because of patient’s exposure to animal hides, physician suspected patient may have been exposed to anthrax

• Physician ordered chest x-ray, bloodwork, spinal tap

• Patient was put on IV penicillin

• Chest x-ray was consistent with anthrax diagnosis

• A Gram-stain of patient’s blood showed Gram-positive rods in short chains

• Blood and bacteria were present in cerebrospinal fluid (indicating infection had progressed to meningitis)

• Patient died a few days later

• Anthrax – disease occur when A. anthracis endospores enter the body.

• Infection typically leads to meningitis (inflammation of the meninges); which is often fatal. The patient likely inhaled the endospores while handling the hides of animals that had been infected.

• Non-hemolytic Bacillus colonies can be presumptively identified using ‘Red Line Alert Test‘, which is an immunochromatographic test for the detection of surface protein found in Bacillus anthracis vegetative cells.