Topic 5-L2 - Prokaryotic Genetics Flashcards
Prokaryotes do not reproduce sexually.
Simple binary fission produces
genetically identical offspring
Gene names are
italicized – first three letters lower case, end with upper case letter (btuC)
Protein names are the same, but start with an
upper-case letter and are NOT italicized (BtuC).
Mutation -
A heritable change in the DNA sequence of a genome. Includes substitution mutations, insertions, deletions – any change.
Mutant (mutant strain):
An organism whose genome carries a mutation
Wild-type strain: –
Strain isolated from nature and/or one being used as the parental strain in a genetic study. The term “wild-type” can also be applied to a single gene
Genomic locus (plural = loci):
a specific position on the chromosome
Mapped mutations can be described using
nucleotide or amino numbers.
- Convention: WT base or amino acid, then number, then mutant base or amino acid. E.g. HisC (A77K) – residue 77 mutated from an
alanine (A) to a lysine (K).
Deletion mutations shown using the
delta (Δ) symbol (e.g. ΔbtuC)
Phenotype names have three letter (first letter = capital) designations
and strains are shown with a
plus (+) or a minus (–) for that phenotype:
- E.g. His+ strain can make histidine. His- strain is a histidine auxotroph – can’t make histidine.
mutants can be isolated by selection –
mutant grows, parent doesn’t (or grows significantly worse). E.g. antibiotic resistance.
Selection is highly efficient – can identify
single mutant with a desired
phenotype out of millions (or more) of cells
Is it easier to identify mutants that grow better than parent by selection ?
Yes
Using replica plating (plating the same colony on two different plates –
under two different conditions), you can identify
mutants that grow worse than parent (or not at all)
Mutations can be
spontaneous (naturally-occurring “mistakes”) or induced (E.g. using mutagenic chemicals or UV to damage DNA)
Point mutations
(mutations to a single base pair) within a protein
Point mutations can lead to :
- silent mutations
- missense mutation
- nonsense mutation
Silent mutations:
do not change amino acid sequence, different codon, same amino acid
Missense mutations (most common):
lead to a change in that amino acid to a different amino acid
Nonsense mutation:
lead to a change in that amino acid to a stop codon, leading to a premature end to the protein sequence (truncation)
Other mutations are not simple substitutions from one base pair to
another, but instead result in DNA being
added or lost. Insertions or deletions
Deletion mutations (DNA lost) and insertion mutations (DNA added to a specific location) can be as
small as a single bp or can be as large as thousands of bp.
Deletions/insertions within protein coding regions often result in a
frameshift mutation (highly disruptive)
Reversion:
mutant that acquires another mutation to “revert” back to wild-type. Term often applied to phenotype.
- For example – a mutant isolated with a new phenotype. That mutant strain then acquires a second mutation that changes phenotype (reverts) back to wild-type.
Suppressor mutation:
Mutations that compensate for the effects of a prior mutation. Can be to a different gene – “fixes problem” created by
initial mutation.
Horizontal gene transfer –
acquiring new genetic material from
foreign DNA via the environment, a virus (phage) or another organism - plays an even bigger role (on the whole)
Foreign DNA can enter a prokaryotic cell in 3 major ways:
1) Transfor mation
2) Transduction
3) Conjugation
Once inside the cell, this DNA can:
1) Be degraded/lost
2) Replicate as a separate entity (plasmids, phage)
3) Be integrated into the chromosome (recombination, transposition)
Genetic recombination:
Physical exchange of DNA between
genetic elements. One important type is homologous recombination (HR)
Horizontal recombination is an important DNA repair mechanism used to
repair double strand breaks - damaged DNA repaired using a homologous template (other copy of chromosome following DNA replication)
HR also important for
horizontal gene transfer.
Foreign DNA with homology to a region of host chromosome can be inserted into host genome at
that location in place of (or in addition to) the native DNA sequence
Genetic recombination important for genome rearrangements –
deletions, insertions,
inversions of segments of genomic DNA
Homologous recombination
RecA - binds a single-stranded DNA and searches for homologous double-stranded DNA – mediates strand invasion
DNA strand from one source (e.g. chromosome) fused to DNA strand of another (e.g. foreign DNA)
Complex DNA structure that results can be “resolved” in different ways, leading to different combinations of the two DNAs
Depending on nature of homology & how HR plays out there can be different outcomes
(replacement of host gene(s), insertion of foreign gene(s), deletions…)
Transposable elements are
mobile genetic elements found in almost all species.
- Contain transposase gene flanked by inverted repeats.
Transposase enzymes are able to:
Transposition
Transposition
recognize the inverted repeats/cleave DNA to free “transposable element”, cleave another DNA (e.g. chromosomal DNA) & insert transposable element into that DNA.
“Insertion sequence elements” no extra stuff, “transposons” contain extra
genes as well, such as antibiotic-resistance genes
Many transposable elements are
- conservative mechanisms (move from one place to another)
- others work via replicative mechanism - transposon remains at its locus and a copy is produced & inserted elsewhere
_________ are used extensively
in the lab to generate mutant strains
Transposons
Transposable elements can insert
Randomly into genome, in activating genes
Transformation
Process by which free DNA is incorporated into a recipient cell and brings about genetic change
(Can come from many sources)
DNA does not freely cross cell membrane – a cell capable of taking up free DNA is said to be ________. Some bacteria/archaea are naturally ________, others are not.
competent
In naturally competent organisms, competence is often
tightly regulated.
Many bacteria that are not naturally competent can be
made competent artificially in the lab – a common way to transfer DNA into cells
In many competent organisms, DNA from the environment is captured by
pili, which retracts, bringing DNA through outer membrane/cell wall
- competence system
competence system
One strand of DNA typically degraded & other strand passed through cytoplasmic membrane & into cell via a multi-protein competence system
Bacteriophage infections
A bacteriophage (or phage) is a virus that infects a bacterium with Virus’ DNA packaged into virions
Virions
feature protein coats that protect the DNA. Virions bind cells, inject DNA.
Bacteriophage infections has two pathways
- lytic pathway
- lysogenic pathway
lytic pathway,
phage DNA replicated & new particles produced using host resources. Viruses then lyse host cell, released to infect new cell.
lysogenic pathway,
viral DNA integrated into host DNA – prophage. Can be induced, triggering the lytic cycle.
Some phages purely
lytic (only operate via lytic pathway).
Others can operate via the lytic or lysogenic pathway
Transduction
Process in which a virus (phage) transfers DNA from one cell to another.
Two types of transduction
1) Generalized transduction
2) Specialized Transduction
Generalized transduction:
- During the lytic cycle some host cell DNA is accidentally packaged into a viral particle.
- This DNA injected into new cell in place of phage DNA.
Specialized Transduction:
- When a prophage is induced, its DNA is excised from genome & packaged into phage particles.
- Sometimes some neighboring DNA is also packaged by mistake
- This DNA can then be injection into a new cell by that phage particle
Conjugation (mating)
Horizontal gene transfer that requires cell-cell contact
Typically conjugation is mediated by plasmids called
conjugative plasmids – the F plasmid (originally identified in E. coli) has served as a model.
F (fertility) plasmid is large (~100 kbp). Strains with an F plasmid are
called …
F plasmid can be transferred to cells that
….
F+ and are donor cells
lack the plasmid (F-), recipient cells
In conjugation, DNA transfer only from
donor to recipient (unidirectional). Only between F+ and F- cells (two F+ cells won’t mate)
F plasmid encodes many tra(transfer) genes that are involved in the
conjugative transfer process
Some tra genes encode a conjugative pilus – produced by
F+ cells, attach to F- cells only (F plasmid encodes genes that prevent attachment)
In conjugation of F plasmids, pilus attaches and
brings two cells together. Conjugative bridge forms.
- Beginning at oriT , DNA is nicked and single strand is copied
- Copied strand passed to type IV
secretion system, which transfers F plasmid DNA from F+ cell to F- cell through the bridge - F- cell now F+, can act as donor. F+ cell
F+ and F- cells (mating pair) attached via a conjugative pilus remains F+
Conjugative transfer: Hfr strains
F plasmid has insertion sequences & can integrate into chromosome producing an Hfr cell (high frequency recombination).
In Hfr strains conjugation transfer, Transfer to F- cell via a
synonymous mechanism, but can also transfer part of donor’s chromosomal DNA
- Transferred DNA can be incorporated into
recipient strain’s genome by transposition or
recombination - Doesn’t transfer full F plasmid, so recipient
strain remains F-
Much acquired DNA will not be evolutionarily useful and will ultimately
be lost, E.g.:
- Transposon or recombination mediated processes
- Random processes/errors during DNA replication or DNA repair
Genes that provide a selective advantage will be
maintained and can outcompete parental strains that lack this new DNA
Microbial genomes contain a great deal of horizontally-acquired DNA
(can tell by
%GC content different from rest of genome, absence of these
loci in related lineages)
Horizontal gene transfer has huge impacts in all aspects of microbiology. Most notoriously,
infectious disease – new virulence mechanisms, antibiotic resistance
