1
Q
describe gene organization in operons and polycistronic mRNA
A
polycistronic = mRNA consisting of a leader followed by one coding region, separated from the second coding region by a spacer
- multiple genes transcribed from a single promoter with distinct start and stop codons for translation of separate peptides
- no poly A tail
- no 5’ cap
2
Q
what are the features of microbial genomes
A
- typically a single dsDNA circular chromosome, usually monoploid
exceptions : vibrio cholerae, 2 circular chromosomes, borrelia burgdorferi, 1 linear chromosome in multiple copies, euryarchaeota are polyploid - plasmids
-> extrachromosomal self-replicating DNA containing non-essential genes
-> organisms may contain multiple different plasmids - > usually circular and smaller than the chromosome
-> borrelia burgdorferi contains 12 linear and 9 circular plasmids
3
Q
describe genome architecture
A
- located in nucleoid
- supercoiling is required, genomes are much larger than cells
- NAPs, nucleoid associated proteins
- histone like proteins in archaea, not in bacteria
-> form nucleosomes similar in structure to those in eukaryotes - transcription and translation can occur simultaneously
4
Q
describe genome structure
A
- groups of genes arranged in operons
->clusters of co-regulated and co-transcribed genes
-> one promoter and regulatory sequences for multiple genes - no introns (except archaea), no alternate splicing
- low amount of non-coding DNA compared to eukaryotes
- genes may overlap
- highly efficient use of genome space
5
Q
describe genome size
A
- range from 112kbp-15Mbp (3-5 Mbp is typical for free living cells)
smallest:
-> nasuia deltocephalinicola - 112kbp, 137 genes, obligate endosymbiont of leafhoppers
-> nanoarchaeum equitans - 490kbp, 530 genes, oligate symbiont of ignicoccus spp
-> mycoplasma genitalium - 580 kbp, 525 genes, free living causes STIs in humans
largest:
-> sorangium cellulosum - 14.7 mbp, 11599 genes, alkaline adaptive free living organism
- genome reduction in obligate endosymbionts and symbionts
6
Q
How do you select for mutant bacteria
How to screen for auxotrophies
A
- ames test
- screen for mutants by looking for a desired phenotype
-> depending on the desired phenotype this can be simple to observe
-> using a method that selects for a phenotype of interest reduces the numbers of colonies to screen
conditional phenotype - they do not grow in the absence of X compound - replica plating
7
Q
what is replica plating
A
- treatment of e.coli cells with mutagen (methyl-nitroguanidine)
- inoculate a plate containing complete growth medium and incubate, both wild type and mutant form colonies
- replica plate, one with complete medium, one without lysine
- complete medium = all stains grow
- medium minus lycine = no growth of lysine auxotroph
8
Q
what are the mechanisms that can result in heritable changes in microbes
A
- mutation
- conjugation
- transformation
- transduction
- genetic engineering
9
Q
what are mutations
A
- occurs naturally and can induce heritable changes (spontaneous mutations)
- monoploid genomes are more likely to retain mutations then diploid/polyploid
-causes of mutations:
-> replication errors - formation of tautomers result in incorrect base during replication, insertions and deletions in repeat regions
-> lesions in DNA due to damage, depurination, ROS - mobile genetic elements
10
Q
what is tautomerization mutations
A
- AT and GC pairs form when keto and amino groups participate in hydrogen bonds
- enol tautomers produce GT base pairs while imino tautomers produce AC base pairs
- temporary enolization of guanine leads to the formation of a GT base pair, if not resolved molecules will contain an AT base pair and a GC to AT transition mutation will have occurred
11
Q
what are insertions and deletion mutations
A
- occurs within regions containing repeats, occur due to displacement of pairing between template and new strand
12
Q
what are point mutations
A
- single base pair substitutions
- transition mutations = pur to pur (A to G), pyr to pyr (T to C)
- transversion mutations = pur and pyr (less common)
- silent = no change in amino acid
- missense = change in amino acid
- nonsense = change to stop codon
13
Q
what is depurination
A
- loss of purine
- can occur spontaneously
- without a proper template the incorrect nucleotide is inserted during DNA replication
- in the next round of DNA replication the strand with the incorrect nucleotide directs synthesis of a complementary strand = mutation
- the strand with the apurinic site can continue to direct synthesis of complementary strands having an incorrect nucleotide
14
Q
what are the effects of mutations
A
- only detectable if they have an impact on phenotype
- missense, nonsense, frameshift = detectable mutations in protein coding genes
lethal mutations = kill
conditional mutations = only expressed under specific environmental conditions
auxotrophies = unable to synthesize required nutrient for growth
neutral mutations = change that has no measurable effect on organisms fitness
beneficial mutations = mutation helps organism
15
Q
how can mutations be induced
A
- mutagens can be used to generate mutations at a higher rate than naturally occurs
base analogs = can be incorporated during replication but alter base pairing (5-bromouracil)
DNA modifying agents = cause changes in a base structure and thereby changes in pairing (methyl nitrosoguanidine)
intercalating agents = insert between bases, result in single bp insertions and deletions (ethidium bromide)
physical agents = cause damage to DNA that must be repaired, which often results in point mutation (radiation, reactive oxygen species)
16
Q
what are the 3 mechanism of horizontal gene transfer
A
- conjugation
- transformation
- transduction
17
Q
what is the fate of DNA acquired via HGT
A
- integration into the recipient genome
-> homologous recombination= involves the presence of homologous regions between genome and acquired DNA
-> site specific recombination= involves mobile genetic elements that include specific enzymes and motifs for insertion
-> passed on via vertical transmission white it is retained - self replicating fragments (plasmids) can replicate and be retained
-> passed on via vertical transmission - fragments that are not self replicating are typically lost
-> linear DNA is sensitive to nucleases and restriction modification systems
-> non replicating circular DNA may not be destroyed but its not retained in a population due to lack of replication
-> CRISPR-Cas systems can recognize and destroy foreign DNA
18
Q
describe conjugation
A
- requires direct cell to cell contact and a conjugative plasmid
- a plasmid that encodes genes for plasmid transfer and cell attachment
- F factor is a well studied conjugative plasmid
- F factor contains insertion sequences that allow for integration into the genome or is can exist stably as a plasmid = episome
- recipient is always an F- cell (does not contain an F factor)
- after conjugation F- cell become F+
19
Q
describe conjugation F- to F+
A
- sex pilus from F+ cell contacts F- cell and retracts bringing the cells close together
- F+ constructs T4SS to allow DNA transfer
- relaxosome nicks one strand at oriT and rolling circle replication occurs
- Tra1 binds 5’ end of nick which is displaced by newly synthesized strand
- Tra1 interacts with T4SS and directs transfer of ssDNA to recipient
- ssDNA is copied and enters recipient generating dsDNA
- end product = 2 F+ cells
20
Q
describe conjugation F- and Hfr
A
- some F+ cells integrate F factor into the genome -> Hfr cells
- these cells can still transfer genetic material to F- cells
- portion of the F factor is transferred (from oriT) and then the chromosome of the organism, and finally the remaining F factor
1.The Hfr cell forms a sex pilus that attaches to the F⁻ cell.
2.The Hfr cell starts transferring part of its chromosomal DNA beginning from the integrated F factor’s origin of transfer (oriT).
Both chromosomal genes and part of the F factor are transferred linearly.
3. Conjugation rarely lasts long enough for the entire chromosome (and full F factor) to transfer.
4.The incoming DNA can recombine with the recipient’s chromosome, introducing new genetic traits (e.g., antibiotic resistance, metabolic abilities).
- full F factor is not transferred, the recipient remains F⁻
- recipient receives chromosomal DNA from donor that can be integrated into its own genome by homologous recombination thereby transferring new genes and new traits
21
Q
describe conjugation F’ and F-
A
- some Hfr cells will deintegrate the F factor
- genes from chromosome will become part of the F factor-> new plasmid is called the F’ plasmid
- transfer of this plasmid via conjugation occurs as before but the recipient will also receive some of the donor’s chromosomal DNA
- these genes do not need to be integrated in the recipient chromosome to be expressed
- if the recipient has copes of these genes in its genome it becomes partially diploid
22
Q
what is transformation
A
- most commonly used method
- the uptake of naked DNA from the environment, typically originates from lysed cells that release DNA into the environment
- can be linear fragments or circular (plasmid)
- observed in diverse ecosystems, biofilms by many bacteria and archaea
- a small fraction of cells in a community may be competent, sometimes at distinct points in cell cycle or due to stressful conditions
- transformation can be induced in the lab
-> techniques to make cells competent include exposure to divalent cations and electric shock, increase membrane permeability
competent = express specific genes that allow it to pick up naked DNA from environment
23
Q
describe transformation in more detail
A
- induction of competence requires the expression of a number of genes
- type lV pili and type ll secretion pathways are involved in the uptake of DNA from the environment
- ssDNA usually enters the cell and the second strand is synthesized
- plasmids may become stable
- linear fragments of DNA typically need to undergo homologous recombination to be maintained in the recipient cell
24
Q
what is transduction
A
- virus mediated transfer of bacterial or archaeal genes to a recipient cell
- viruses can be virulent (lytic cycle) or temperate
lytic cycles = involve infection, followed by immediate replication of the virus and lysis of the host cells once enough virus particles are present
temperate phages = can undergo lysogeny wherein the phage genome is incorporated into the host genome (prophage)
- a lytic cycle can then be triggered under condition of stress
- viruses typically only transfer their own genome but in some cases host DNA can also be included in the virus particles
25
what is generalized transduction
- a fragment of host DNA that is approximately the same size as the viral genome is packaged into a phage particle
- virus assembly only requires incorporation of a certain length of DNA
- incorporation of random fragments of host genome of a specified length
- injection into a susceptible host cell results in transfer of packaged DNA
- this must undergo homologous recombination to be retained in the host
- expression of genes can occur from the fragment until it is degraded
26
what is specialized transduction
- specific portions of host genome are transferred by temperate phages
- related to errors in excision of the viral genome during the lytic cycle
- since phage genome integration occurs at a specific location in the host genome, only the genes flanking this region will be transferred
- the phage itself is non-functional but it can still inject this host DNA into a new bacterium
- retention of these genes requires homologous recombination
27
explain the process of specialized transduction
1. A temperate phage (like λ) inserts its DNA into the bacterial chromosome and becomes a prophage. (Protein: integrase.)
2. Stress (e.g., UV) triggers the prophage to cut itself out so it can enter the lytic cycle.
3. Sometimes the prophage cuts out incorrectly, pulling a nearby bacterial gene with it and leaving out some phage genes. This produces a hybrid (defective) phage genome.
4. The hybrid DNA is packaged into new phage particles. The bacterial cell bursts and releases these phages. (Packaging protein: terminase.)
5. A defective phage injects the hybrid DNA into another bacterium. Because the phage lacks essential genes, it can't complete a normal infection.
6. The bacterial fragment carried by the phage recombines into the recipient’s chromosome (requires host recombination machinery like RecA), so the recipient gains the specific donor gene.
28
explain the CRISPR-Cas system in terms of its function in microbes
CRISPR = clusters regularly interspaced short palindromic repeats
Cas = CRISPR associated sequences
- adaptive immune system found in many bacteria and archaea to protect against bacteriophage infection
- spacer regions consist of viral genome = defense to protect from viral infection
1. virus infects a microbe-> small piece of the viral DNA is cut and inserted into the CRISPR region of the bacterial genome as a new spacer. Enzymes: Cas1 and Cas2.
- spacer acts like a “memory” of past infections.
2. The CRISPR region (containing repeats + spacers) is transcribed into a long RNA molecule.
- This RNA is then cut into small pieces called crRNAs (CRISPR RNAs) — each one carries a unique viral sequence.
3. If the same virus infects again, the crRNA binds to the matching viral DNA.
Cas proteins (like Cas9) cut and destroy the invading DNA, preventing infection.
29
How has the CRISPR-Cas system been adapted for gene editing
- cas9 = proteins encoded by Cas genes
- nuclease that incorporates a guide RNA to identify specific sites in foreign DNA and cuts DNA in both strands at these sites
1. complementary sequence associated with Cas9 targets the region of interest (gRNA)
2. frameshift mutations that occur during DNA repair can silence a gene
3. a donor DNA molecules can be introduced to insert at this site by homologous recombination (replace mutant with correct genome)
30
differences between microbial genomes and gene expression in prokaryotes and eukaryotes
eukaryotes:
- more non coding regions
- transcription and translation occur at different times
- presence of nucleus blocks ribosomes from entering
- linear, x shaped chromosomes
- diploid/polyploid
- introns and exons
- replication fork
- histones present, DNA packaged around them
- vertical gene transfer
prokaryotes:
- less non coding regions
- no introns or exons
- no alternate splicing
- transcription and translation occur together
- single, dsDNA circular
- mRNA has no poly A tail or 5' cap
- genes arranged in operons
- no nucleus , nucleoid present
- monoploid
- plasmids
- replication - origin = bubble
- no histones
- horizontal gene transfer
Bisc 303
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