Chapter 8 - Microbial Genetics and Antibiotic Resistance Flashcards
What is a genotype?
An organisms genetic makeup (DNA)
What is a phenotype?
The expressed properties that are encoded in the genotype
What is vertical gene transfer?
The flow of genetic information from one generation to the next (parent to offspring)
What is semiconservative replication?
Each new double-stranded DNA molecule contains one original strand and one new strand
What is the process of transcription in prokaryotes?
Initiation: RNA polymerase binds to the promoter, and DNA unwinds at the beginning of a gene
Elongation: RNA is synthesized by complementary base pairing of free nucleotides with the nucleotide bases on the template strand of DNA
Termination: Transcription reaches the terminator, RNA and RNA polymerase are released, and the DNA helix re-forms
What is mRNA?
Messenger RNA
Synthesized during transcription
Carries coded information for making proteins to the ribosomes
What is rRNA?
Ribosomal RNA
Forms part of the ribosomes
What is tRNA?
Transfer RNA
Binds to and carries correct amino acids and adds them to the polypeptide chain
What is the process of translation?
tRNA carrying first amino acid is paired with the start codon on the mRNA
- First tRNA moves to the P site and a tRNA carrying the second amino acid enters the A site
First amino acid joins the second by a peptide bond
Ribosome moves along the mRNA unitl the second tRNA is in the P site
- Next codon to be translated is brought into the A site
- First tRNA now occupies the E site
Second amino acid joins to the third by another peptide bond, and the first tRNA is released from the E site
Ribsoome continues moving along the mRNA. and new amino acids are added to the polypeptide
When ribosome reaches a stop codon, the polypeptide is released
The last tRNA is released, and the ribosome comes apart
- Released polypeptide folds into a protein
What is a promoter?
The starting site on a DNA strand for transcription of RNA by RNA polymerase
What is a sense (coding) strand?
Strand of DNA that encodes a protein
What is an anti-sense (complementary) strand?
DNA strand that does not encode a protein but is complementary to the coding strand
What are the differences between prokaryotic and eukaryotic DNA?
Prokaryote:
- Single circular chromosome in nucleoid
- Small
- Typically single copy
- Asexual reproduction - binary fission
- No introns in DNA
- Sometimes overlap control/operons
Eukaryote:
- More than 1 chromosome in nucleus
- Diploid (2 copies)
- Linear
- Histone packing
- Sexual reproduction - mitosis/meiosis
What are plasmids?
Piece of extra-chromosomal DNA found in prokaryotes
Circular and supercoiled
Easily transferred
No standard size - can be large or small
Non-essential genes
Collect genes that are deeemed valuable - antibiotic resistance, selective advantages, genetic flexibility, toxins, etc.
What is an inducible operon, an example of one, and how does it work?
Starts off, transcription must be turned on
Ex: lac operon
Repressor active, operon off - normal state of inducible operon is off
- lacI gene is transcribed and translated into a repressor protein
- Repressor binds to the operator, which physically blocks RNA polymerase from transcribing
Repressor inactive, operon on - lactose enters the cell and is converted into allolactose
- Inducer molecule, allolactose, binds to the repressor protein, which inactivates the repressor - can no longer block transcription
- Structural genes are transcribed, leading to the production of enzymes needed for lactose catabolism
What is a repressible operon, an example of one, and how does it work?
Start in on position, gets turned off
Ex: trp operon
Repressor inactive, operon on
- Repressor is inactive, so transcription and translation proceed, leading to the synthesis of tryptophan
Repressor active, operon off
- When the corepressor tryptophan binds to repressor protein, the activated repressor binds with the operator, preventing transcription from the operon
What is recombination?
New chromosome with a genotype different from that of the parent results from the combination of genetic material from two organisms
This new arrangement of genes is usually accompanied by new chemical or physical properties
What are the 3 types of recombination?
General recombination
- Reciprocal exchange of DNA between a pair of DNA sequences
- Anywhere on chromosome
- Typified by the exchanges occurring in bacterial transformation, bacterial recombination, and bacterial transduction
Site-specific recombination
- The integration of a viral genome into the bacterial chromosome
Replicative recombination
- The movement of genetic elements as they switch position from one place on the chromosome to another
What is natural selection?
The surivial and reproduction of bacteria with a new genotype that is favored by natural environments
Results in huge diversity of microbes
What is a mutation?
Permanent change in the base sequence of DNA
Genotypic change can result in phenotypic change
What are the 3 main categories of mutations?
Base substitution - single base in the DNA sequence is replaced with a different base
Insertion - an extra base is added in the DNA sequence
Deletion - a base is removed from the original DNA sequence
What are the 4 types of mutations that result from base substitutions, insertions, or deletions?
Nonsense - generates a stop codon that wasn’t there before
Missense - causes a change in amino acid
Silent - nucleotide changes but the amino acid doesn’t change
Frameshift - changes the codon reading frame
What is a spontaneous mutation?
A mutation that occurs randomly because of mistakes made during DNA replication
Occur in the absence of mutation-causing agents
What are mutagens?
Agents in the environments, like chemicals and radiation, that directly or indirectly bring about mutations
What are the 3 types of chemical mutagens?
Cause base substitutions
Nucleoside analogs
- Mimic a nucleoside and is put in instead of a normal one
- As next strand replicates, it puts in a new base
Nucleotide-altering chemicals
- Change the actual structure of the nucleotide - cleave off important pieces to change base
Frameshift mutagens
- Puts itself into double stranded DNA so it becomes incorporated into the DNA
What are the 2 types of radiation mutagens?
Cause large-scale mutations
Ionizing radiation
- Form free radicals that cause single or double strand breaks in DNA
Nonionizing radiation
- Cause cross-links between adjacent pyrimidies (dimers)
- Exposure to UV light causes adjacent thymines to form a dimer, which disrupts their normal base pairing
What is mutation rate?
The probability that a gene will mutate when a cell divides
How do mutagens affect mutation rates?
Increase the rate of mutation from the spontaneous rate (1 in 10^6) to 1 in 10^5 - 10^3
How does positive (direct) selection of mutants work?
Detection of mutant cells by rejection of the unmutated parent cells
Expose bacterial cells to an environmental change, and those who are mutated to survive in environment will live and those who are unmutated will die, indicating which are mutants
How does negative (indirect) selection work?
Selects a cell that cannot perform a certain function
Uses replica plating
What is the process for replica plating?
Bacteria culture spread on nutrient agar plate (both mutant and non-mutant will grow) this is MASTER PLATE
Master plate pressed onto sterile velvet - picks up some cells of every colony
Velvet pressed on two agar plates, one nutrient agar and one with the environmental conditions you are testing for mutation
Replica plates are then incubated, allowing cells to grow and form colonies
Results: Prototrophs grow on both types of media, auxotrophs only grow on nutrient agar
What is an auxotroph?
A mutant microbe that has a nutritional requirement that is absent in the parent
How would you select for antibiotic resistant bacteria?
Positive (direct) selection
Plate bacteria on a medium containing antibiotic
The few cells that have an antibiotic-resistant mutation will grow and form colonies, whereas the normal susceptible bacteria will not grow
What is a carcinogen?
Substances that cause cancer in animals/humans
What is the process for an Ames test, and what is it used for?
Uses bacteria to assess the mutagenic potential of chemical compounds
Based on observation that exposure of mutant bacteria to mutagenic substances may cause new mutations that reverse the effect of original mutation (reversions)
- Specifically measures reversion of histidine auxotrophs of Salmonella to histidine-synthesizing cells
Steps:
- 2 cultures are prepared of Salmonela that have lost ability to syntehsize histidine
- Suspected mutagen is added to experimental sample only
- Rat liver extract (activator) is added to both samples
- Each sample is poured onto a plate of medium lacking histidine and are incubated for 2 days
- Only bacteria whose histidine-dependent phenotype has mutated back to histidine-synthesizing will grow into colonies
- Test plates will show an increase in the number of histidine-synthesizing revertants if the test chemical is indeed a mutagen and potential carcinogen
What is horizontal gene transfer?
Passage of genes to other microbes of the same generation
What is crossing over?
Cell picks up foreign DNA and some of it inserts into the cell’s chromosome
Some of the genes carried by the chromosome will be shuffled
What are the 3 mechanisms for horizontal gene transfer?
Transformation
Transduction
Conjugation
What is transformation?
“Naked” DNA enters into another cell and can provide new genes to the cell
Recipient cell takes up donor DNA that is in the environment after another cell dies
- Donor DNA aligns with complementary bases
- Recombination occurs between donor DNA and recipient DNA
What was Griffith’s experiment and what did it prove?
Demonstrated genetic transformation
Experiment:
- Living encapsulated bacteria injected into mouse – mouse died – colonies of all encapsulated bacteria were isolated from dead mouse
- Living nonencapsulated bacteria injected into mouse – mouse lived – a few colonies of nonencapsulated bacteria were isolated from mouse; phagocytes destroyed nonencapsulated bacteria
- Heat-killed encapsulated bacteria injected into mouse – mouse remained healthy – no colonies were isolated from mouse
- Living nonencapsulated and heat-killed encapsulated bacteria injected into mouse – mouse died – colonies of encapsulated bacteria were isolated from dead mouse
What is transduction?
By a bacterial virus, DNA passed from one bacteria to another in a bacteriophage and then incorporated into the host DNA
Phage injects its DNA
- Phage enzymes degrade host DNA
- Cell synthesizes new phages that incorporate phage DNA and, mistakenly, some host DNA
- Transducing phage injects donor DNA
- Donor DNA is incorporated into recipient’s chromosome by recombination
What is conjugation?
Plasmids transferred via a pilus
Donor cell attaches to a recipient cell with its pilus
- Pilus may draw cells together
- One strand of F plasmid DNA transfers to the recipient
- The recipient synthesizes a complementary strand to become an F+ cell with a pilus; the donor synthesizes a complementary strand, restoring its complete plasmid
How do plasmids contribute to gene transfer?
Conjugation - donor bacterium transfer a plasmid to recipient through pilus
- Plasmids contain genes that have been collected because they are beneficial, these genes are then incorporated into the recipient
Transformation - plasmids can be released into the environment when bacteria die and lyse, allowing other bacteria to take up the plasmids and incorporate its genetic material
How do plasmids contribute to resistance?
Plasmids often contain genes that provide resistance to antibiotics
Plasmids can move between bacteria through horizontal gene transfer, which allows different bacteria to take up these genes - spreads resistance rapidly
What are 5 types of genes that plasmids carry?
Antibiotic resistance: resistance factors are plasmids that contain genes for antibiotic resistance
Virulence genes: some plasmids encode proteins that enhance pathogenicity of a bacterium
Metabolic genes: dissimilation plasmids code for enzymes that trigger catabolism of certain unusual sugars and hydrocarbons
Fertility genes: F factor is a conjugative plasmid that carries genes for sex pili and for the transfer of the plasmid to another cell
Bacteriocin genes: Some plasmids contain genes for synthesis of bacteriocins, which are toxic proteins that kill other bacteria
Are plasmids essential for survival?
Not necessarily essential for the normal survival of bacteria, but they provide significant advantages under certain conditions, such as antibiotic presence or some environmental stresses
Can enhance bacterial adaptability and survival in different environments, but bacteria can survive without them in normal living conditions
What are transposons and how do they contribute to the movement of genes within and between organisms?
What:
- Segments of DNA that can move from one region of DNA to another
- Contain a gene for an enzyme (transposase) for cutting and resealing DNA (cut piece of DNA out of one place in chromosome and insert in another)
- Simple transposons only carry essential elements
- Complex transposons carry other genes (i.e. selective advantage such as antibiotic-resistant gene)
How:
- Transposon is excised from original location and inserted into a new site in the genome - can disrupt genes at new site and deactivate them
- May be carried between cells on plasmid or viruses, so they can spread from one organism to another through horizontal gene transfer
How do genetic mutation and recombination contribute to natural selection?
Genetic mutation and recombination create diversity in future cell generations
Diversity provides the raw material for evolution, and natural selection is its driving force
Natural selection will act on diverse populations to ensure the survivale of those fit for that particular environment
What are the 6 ways in which DNA is manipulated through recombinant DNA technology?
Restriction enzymes
- Cut DNA at specific sites in a specific way
- DNA from another place can be incorporated into cut DNA and sealed with ligase to create new DNA sequence
Vector
- Usually plasmids or phages that we use to transfer DNA
PCR
- Amplify and synthesize genes
Inserting foreign DNA into cells
- Transformation, electroporation, protoplast fusion
- Gene gun and microinjection
Libraries (cDNA and genomic)
- Genomic - take one organisms genome and chop it up with restriction enzymes and seal it into vectors, which you can then try and pull genes out
- cDNA – express genes and get mRNA and create a library in the same way as genomic
Selection of a clone
How does natural selection for antibiotic resistance work?
Starting point is a large bacterial population mainly consisting of bacteria that are susceptible to antibiotics and a couple of bacteria that are antibiotic-resistant by chance
- A bactericidal antibiotic is added, which kills most of the susceptible bacteria in the population, which the resistant bacteria survives
- Only the resistant will continue to proliferate in the presence of the antibiotic and increases in number over time
- End result is a population of mainly resistant bacteria
Selection can occur at any site in the body to which the antibiotic reaches
- Antibiotic can select for resistance genes and mechanisms in both pathogenic bacteria and in commensal bacteria living in the body that have nothing to do with the infection in question
What is the selective pressure for antibiotic resistance?
In presence of antimicrobial, microbes are either killed or, if they carry resistance genes, survive
Survivors will replicate, and their progeny will quickly become the dominant type throughout the microbial population
How does mutation impact antibiotic resistance?
Most microbes reproduce by dividing every few hours, allowing them to evolve rapidly and adapt quickly to the new environmental conditions
During replication, mutations arise and some of these mutations may help and individual microbe survive exposure to an antimicrobial
What are the 4 mechanisms of antibiotic resistance?
Blocking entry – slow or prevent entry of drug into the cell
Inactivation of antibiotic by enzymes – production of enzyme that destroys or deactivates drug
Alteration of target molecule – alter target of drug so it binds less effectively
- If antibiotic is supposed to bind to a specific target, but the target’s conformation is changed, it prevents binding
Efflux of antibiotic – pump antimicrobial drug out of cell before it can act
What are the 7 contributing factors to development of antibiotic resistance?
Overuse
Inappropriate prescribing
Societal pressures
Inadequate diagnostics – “just-in case” or too broad
Hospital use – fertile environment
Agricultural use
Limited availability of new antibiotics
What are the 4 ways to combat the development of antibiotic resistant bacteria?
Preventing infections, preventing the spread of resistance
- Proper individual hygiene to prevent infection so antibiotics are not needed
Tracking by CDC
Improving antibiotic prescribing/stewardship
- Only prescribing when absolutely necessary
- Not asking doctor for antibiotics when they’re not needed
- Not taking antibiotics from previous illness “just in case”
Developing new drugs and diagnostic tests
