Bacterial Genetics Flashcards
Prokaryotic genomes
note the range of genome sizes of some cultured organisms:
- Bacteria:
- Mycoplasma genitalium 0.58Mb (smallest genome of a curable bacterium)
- Streptomyces coelicolor 8.7 Mb (linear chromosome, produces secondary metabolites such as antibiotics)
- Archaea
- Aeropyrum pernix 1.66 Mb (hyperthermophile)
- Haloarcula marismortui 4.27 Mb (extreme halophile)
- Escherichia coli is fairly average: 4.60 Mb
- E. Coli chromosome ~ 1.4mm long = a circle about 0.45mm in diameter
- E.coli cell ~ 4 um (i.e. 0.004mm) long
An E. Coli cell and its DNA to scale:
Genome copies per cell
- E. Coli normally has a single copy of its chromosomes per cell (or two copies when the cell is about to divide)
- But some prokaryotes have multiple copies of the chromosome
- e.g. Cyanobacteria typically have about 10 copies per cell. A Synechosystis cell is about 2um (i.e. 0.002mm) long, and each cell contains DNA with a total length of about 11mm
Packaging the genome
- Bacterial DNA is tightly folded and packed into an irregular DNA structure in the cytoplasm - the nucleoid. DNA packaging is facilitated by nucleoid - associated proteins (NAPs)
- Note: the packaging in archaea is similar to that in eukaryotes (i.e. packaging is facilitated by simple histone-like structure)
Genome size and lifestyle:
Rough correspondence between genome size and complexity of lifestyle. For example:
Mycoplasma genitalium (0.58 Mb) - obligate parasite, small (i.e. 0.2-0.3 um), simple metabolism
Streptomyces coelicolor (8.7 Mb) - soil bacterium with very versatile metabolism, complex structure (branched network of mycelia) and undergoes sporulation
Similarly, if we compare Cyanobacteria with different levels of cellular complexity:
- Prochlorococcus marinus (1.67 Mb), has small simple cells
- Anabaena cylindrica (6.37 Mb), filamentous, multiple cell types
However, other factors also influence genome size:
- Synthesis of DNA is expensive both in terms of energy (ATP) and nutrients (N, C, P)
- Pelagibacter ubique is probably the most abundant heterotrophic bacterium in the oceans but lives in a very nutrient poor habitat
- consequently has streamlined its genome to 1.3 Mb
- the smallest genome known for a free living organism
- Time taken to replicate genome is proportional to genome size
DNA replication
- note: DNA replication starts from a single, defined origin and is bio directional (see YouTube vid from slides)
- One consequence of the bacterial mode of DNA replication - for a given speed of the DNA polymerase, the time taken to replicate the chromosome is directly proportional to the size of the chromosome
- the bigger the chromosome, the longer it takes to replicate
- this is not the case in eukaryotes, where replication proceeds simultaneously at multiple sites along the chromosome
- this is likely to create a strong selective pressure to keep the chromosome small - the time taken to copy a large chromosome is likely to limit the speed at which the cell can replicate
- a short cell replication time gives a strong selective advantage:
- therefore, bacteria under strong selective pressure to eliminate “junk” DNA and unnecessary genes
- This probably explains the dense packing of genes along bacterial chromosomes
Bacterial genome sequencing
modern DNA sequencing technologies such as Oxford Nanopore Technologies’ platform can determine the entire sequence de novo of a bacterial genome in a few days
Identifying open reading frames (ORFs)
- Divide sequence into triplets (codons). Note that any DNA sequence can be read in 6 ways (2 strands, 3 reading- frames on each strands)
- According to the genetic code, 61 of the codons can be translated into one of the twenty amino acids, whereas three codons (TAA, TAG and TGA) represent ‘stop codons’
- ORFs begin with a start codon (ATG, or occasionally GTG) and are followed by a long run of codons before the first stop codon
- Since a stop codon has a 3 in 64 (~1 in 21) chance of occurring at random, then ORFs greater than (say) 40 codons are likely to be significant
- such ORFs are potentially genes
A gene is more than an ORF
Look at notes
Some lessons from prokaryotic genome sequencing:
- Numbers of genes related to life style of the organism
- Dense packing of genes in prokaryotic chromosomes (prokaryotes typically contain 1 gene per 1000 bases, in H. Sapiens contain about 1 gene per 30000 bases)
- Large number of unknown genes (typically 40-60%)
- Genome comparisons reveal evolutionary relationships among prokaryotes
- A possible minimum set of common and essential genes (~300) - these identify the minimal set of genes needed for viability
Limitations of genomics:
- large numbers of unknown genes
- Rela populations of any prokaryotic species are genetically dynamic with DNA changes (single base changes, etc) arising all the time. The published sequence from an organism is only a guide and a “snapshot”
- the whole is more than the sum of the parts. Even if we knew the function of every gene product, it would not begin to tell us how the intact system works.
- Importantly, genomic data allows us to understand evolution through molecular phylogenetics and deduce ancestral genes and gain insight into the gene content and sequence of the last known universal common ancestor (LUCA)
- an allow us to design a synthetic bacterial genomes (e.g. a minimal genome) to create novel cells for biotechnology
Vertical and horizontal transmission of genetic variation:
- A daughter cell may arise that has a change in its genome (vertical)
- prokaryote B may acquire gene from other organisms (may be closely related or very different)
- may acquire genes that become functional components of the genome of that organism
Spontaneous mutations
- either due to damage to DNA bases from
External factors
Chemicals (e.g oxidants) produced in the cell - or errors in DNA replication due to:
- mis-incorporation of bases in the daughter strand (check notes)
- “slippage” between new and template strands
- Tautomerization of bases - rare
Rare tautomerization events
Rare tautomerization events (transition to a different structural isomer)can result in the formation A-C and G-T base pairs during replication
Induced mutations
We can artificially increase mutation frequency
- chemicals that interact with DNA
- e.g. nitrous acid - primarily converts amino groups to Leto groups by oxidative delaminating (i.e. removal of amine group) - C, A, G are converted to uracil (U), hypoxanthine (H) and xanthine (X)
- E.g. intercalating agents such as ethicist bromide - can result in addition or deletion of bases - Radiation - non ionising and ionising
Used in microbial genetics to generate mutations
Non ionising radiation
Purine and pyramiding bases absorb UV radiation
Several effects are known -
e.g. production of pyrimidine dimers: adjacent C and T bases become covalently bound. Results in DNA polymerase being impeded or misreading DNA template
Ionising radiation
X-rays, cosmic rays, gamma rays.
Indirect effect by free radicals (e.g. hydroxyl radical)
Types of DNA mutations
Point mutations
Larger scale mutations
Point mutations that change a single base-pair
Effects:
Outside the coding region
- Could have no effect
- Could change gene expression (e.g. mutation in promoter)
Inside the coding region
- Could make no difference (e.g. TCC and TCA both code for serine)
- Could change an amino acid without affecting the structure and/or function of the protein
- Could modify the structure and/or function of the protein
- Could inactivate the protein completely (e.g. TGC —> TGA produces a STOP codon)
Point mutations that insert or delete a single base pair
- Outside the coding region
- Could have no effect
- Could change the green expression - inside the coding region
- Produces a “frameshift” - every subsequent codon in the ORD is changed. Results in inactivation of the gene - these insertions/deletions (“indels”) may involve more than one base pair
- indels of either one or two bp within an ORF would be equally mutagenic, but indels of three bp (or multiples of three) would potentially be less mutagenic since there is no frameshift
- Frameshift mutants were the basis for Francis Crick’s classic experiment showing that codons are triplets (he showed that genetically combining frameshift mutations in the same gene restored some protein function if the frameshifts added up to a factor of 3 [e.g a delta1 bp’ and a ‘delta2 bp’ ])
Larger scale mutations
- Deletion of a large tract of DNA
Can result in “null mutation” - complete loss of function of one or more genes
Rearrangement of a region of DNA
May or may not be mutagenic depending on break points
Duplication of a region of DNA
May result in duplication of a gene —> opportunity for subsequent evolution of two different, but related gene products (e.g. enzymes that recognise slightly different substrates).
Insertion of DNA elements from elsewhere on genome (i.e. transposons, insertion sequences)
May be mutagenic depending on the site of insertion
Transposons
Check notes
DNA sequence with ability to move = “jumping genes”
IR = inverted repeat e.g.
CTGCAG - - - - - - - - - - - - - - - - - - - - - - - - GACGTC
GACGTC- - - - - - - - - - - - - - - - - - - - - - - - - CTGCAG
Bla encodes B- Lactamase which confers resistance to ampicillin
Detecting mutations
- The classic way is to look for a change in phenotype (a change in the properties of the cell) as an indicator of a change in genotype (change in DNA sequence)
- But of course, ‘neutral’ mutations, or those that generate a very subtle change in phenotype may not be detectable
- Most easily done using storable phenotype e.g. ability/inability to grow on a particular substrate, or resistance/sensitivity to a toxin
E.g. a screen for E. Coli lysine auxotrophs
- E. Coli uses a set of enzymes to synthesis lysine from metabolic precursors
- A mutation in one of the genes coding for one of these enzymes can produce a lysine auxotroph - a mutant strain that can grow only if lysine is supplied in the growth medium
- spread cells on plate containing lysine
- make replica plates
- Identify autotrophs (designated lys-)
Selecting for lys- referents
You can now do the reverse experiment: look for mutations of lys- that restore the wild type phenotype (revenants)
plate out millions of cells from the lys- culture on a plate lacking lysine.
Look for surviving cell coloniesare
- You can now do the reverse experiment: look for mutations of lys- that restore the wild type phenotype
- plate out millions of cells from the lys- culture on a plate lacking lysine
- look for surviving cell colonisers
- Reversion frequency can be increased by treatment with mutagens, but reverants will occur with low frequency anyway (maybe 1 cell in ten million).
- These spontaneous revertants illustrate that mutations are inevitable as cells replicate
- This kind of experiment is the basis for a test for mutagenic chemicals - the Ames test. Treat an auxotrophic culture with the chemical, the more reverants will be obtained.
Most mutations are harmful
It is easier to make a bad mutation than a good one
Experimental demonstration of mutations effects on cell
- Take a bacterial culture.
- Treat cells with a mutagen (e.g. UV light)
- Plate treated and non treated cells on the usual growth medium and count viable cells.
- Compare cell numbers
- Many cells will die: the higher the mutation frequency the lower the survival rate.
- Many of the survivors will have slower growth rates
Some mutations are good - experimental demonstration
- some mutations are good - particularly if you change the rules by changing the growth conditions
experimental demonstration:
- E.g. take a bacterial culture and plate out in the presence of of the antibiotic rifampicin
- [rifampicin inhibits bacterial RNA polymerase by binding to the beta subunit. Resistance can be acquired by point mutations in the gene (rpoB) encoding the beta subunit)
- 1 billion bacterial cells plated onto a medium containing rifampicin.
- 6 cells survived to from colonies - spontaneous resistant mutants
Evolution happening in the lab
- evolution happening in the lab
I.e acquisition of phenotypic changes through natural selection - an increased rate of mutation is usually “bad” in an unchanging environment but can be essential for survival when conditions change
- Experimental demonstration using MNNG (a potent mutagen) and rifampicin
Can mutation rates change without external mutagens
- Some bacteria contain genes that increase mutation rates under certain condition = known as mutator genes
- Conditions are usually complex and changing environments
- these can give a selective advantage under certain conditions
Long term problems caused by mutations
- “Muller’s ratchet”
- imagine we start with a bacterium with the perfect genome for its environment
as the organism replicates, mutations will occur. - Most mutations are harmful - some will result in death and some in slower growth
- at realistic mutation rates, natural selection will not work fast enough to eliminate all harmful mutations from the population
- Result - gradual build-up of harmful mutations declining fitness
This clearly hasn’t happened - bacteria have managed to remain very adapted to their environment.
How do bacteria escape from Muller’s ratchet
- By using repair mechanism to stop mutations happening
- But no repair mechanism is 100% efficient. In fact some are error prone. ,repair mechanisms slow down DNA replication therefore cell division - By horizontal gene transfer
- Mixing genes from different organisms. Allows the possibility of eliminating harmful mutations by combining good genes. Also allows the possibility of combining good traits from different sources
Three mechanisms known for “horizontal” DNA transfer from cell to cell
Transformation
Transduction
Conjugation
Transformation
uptake of naked DNA
Transformation also enables prokaryotes to acquire new plasmids
- some prokaryotes are able to import naked DNA into the cell
- Sometimes the new DNA is incorporated into the chromosome
- This can occur more readily if the new DNA contains sequences that are similar to sequences already present in the genome, since integration can occur through homologous recombination
Transduction
DNA transfer mediated by bacterial viruses (bacteriophages)
Conjugation
DNA transfer from a donor cell to a recipient (requires direct cell to cell contact)
How to detect transformations
- take a mutant strain that is deficient in a particular physiological function due to a mutation in a gene (e.g. an auxotroph)
- take the wild type strain and isolate DNA from it
- Mix mutant cells with wild type DNA and select for the wild type phenotype (e.g. by planting out on minimal medium)
- some cells will survive because they have taken up and incorporated a DNA sequence that complements the mutation
- this only works with the “transformable” prokaryotes. Some are naturally transformable (e.g. Streptococcus, Bacillus, Pseudomonas). Some (e.g. E.coli) become transformable after pre treatment with certain metal ions (e.g. Ca2+)
Check notes
What has happened in the transformants
Check notes
Usually a piece of wildtype DNA is inserted into the mutants chromosome at the correct locus
How is the new DNA sequence incorporated at the correct locus
Check notes
- single strand of donor DNA taken up by the cell
- double strand chromosome
- strand separation and base pairing of homologous sequences
- “crossover” - strands broken and rejoined
Demonstrating transduction in the lab
- Infect a wild type culture with the phage
- isolate progeny phage
- infect an auxotroph culture with the progeny phage
l - ook for survivors on minimal medium
Typically, 1 in 10^7 - 10^8
Transduction in the environment
transduction is a very important mechanism for the spread of prokaryotic genes e.g in the oceans or in the gut where bacteriophages are involved in gene exchange between many different prokaryotes
Conjugation (‘bacterial sex’) - the major mechanism for DNA transfer between prokaryotes
Conjugation plasmids and transposons
Lederberg and Tatum (1946) showed that conjunction requires physical contact between donor and recipient
Check notes Usually a
Conjugation (‘bacterial sex’) - the major mechanism for DNA transfer between prokaryotes
Conjugation plasmids and transposons
Lederberg and Tatum (1946) showed that conjunction requires physical contact between donor and recipient
Check notes
Genes for conjugation are carried on certain plasmids (conjugation plasmids)
Well studied example is the E.coli Fertility (F) plasmid:
- Transfer (tra) genes encode proteins that establish a stable meaning pair and trigger DNA transport from donor to recipient via a specialised transfer pore/channel
- Tn1000 is a transposon
- IS2 and IS3 are insertion sequences
- resistance (R) plasmids are conjugative and carry multiple resistance genes (e.g R100)
- spread of R-plasmids has led to the rapid development of multiple antibiotic resistant strains of pathogenic bacteria
- However, R-plasmids have been detected in old bacterial isolates (pre-dating the clinical use of antibiotics) and have been found in non pathogenic soil bacteria
- R-plasmids have probably been in existence for a long time - a defence mechanism against antibiotic producing soil microbes
Transfer of chromosomal genes by a conjugation
- the E.coli F plasmid is an epitome (a plasmid capable of integrating into the main chromosome)
- Integration is via an insertion (IS) on the plasmid and a homologous sequence on the chromosome)
- F+ strains in which the plasmid is integrated in this way are called Hfr strains (=high frequency of recombination)
Check notes Usually a
Transfer of chromosomal genes by a conjugation
- the E.coli F plasmid is an epitome (a plasmid capable of integrating into the main chromosome)
- Integration is via an insertion (IS) on the plasmid and a homologous sequence on the chromosome)
- F+ strains in which the plasmid is integrated in this way are called Hfr strains (=high frequency of recombination)
Check notes
Interrupted mating as a tool for genetic mapping in E. Coli
E.g. mate an Hfr strain with a multiple autotroph (thr- leu- gal- trp-)
Allow mating for different times, before interrupting mating by mixing cells in blender. Count recombinations for each marker at different times
By combining interrupted mating data from different Hfr strains a detailed genetic mao of E. Coli established long before the era of molecular biology
Summary vertical genetic variation
DNA changes in host
- point mutations
- insertions/deletions
- rearrangements
- duplications
- transposons activity
Horizontal genetic variation summary
DNA acquisition by host
- transformation
- transduction
- conjugation (plasmid or chromosome section)
