week 6+7 – the dynamic bacteria genome Flashcards
1
Q
Bacterial Chromosomes are Dynamic
chatgpt
A
bacterial chromosomes are not static blueprints but highly fluid, modular systems” due to:
Inversions (e.g., of promoter regions – phase variation).
Variable number tandem repeats (VNTRs).
Transposable elements and IS elements.
Plasmids and conjugative elements.
2
Q
Bacterial Chromosomes are Dynamic
this dynamic nature supports..
chatgpt
A
rapid phenotypic switching in response to environmental change (like adhesion or motility).
3
Q
Horizontal Gene Transfer in Bacteria
transformation
chatgpt
A
Transferred Material: Naked DNA fragments from environment
Mediating Agent: Competence proteins (Com)
Key Features: Requires natural competence; DNA must have sequence similarity for recombination
Biological Significance: Environmental adaptation; potential for DNA repair or gene acquisition
Example: Streptococcus pneumoniae DNA uptake
4
Q
Horizontal Gene Transfer in Bacteria
Conjugation
chatgpt
A
Transferred Material: Plasmids (sometimes chromosomal segments)
Mediating Agent: Conjugative plasmid (e.g. F)
Key Features: Requires direct cell-cell contact via pilus; major AMR driver
Biological Significance: Main driver of antimicrobial resistance (AMR) transfer across species
Example: F plasmid or RK2 in E. coli
5
Q
Horizontal Gene Transfer in Bacteria
Transduction
chatgpt
A
Transferred Material: Chromosomal or plasmid DNA
Mediating Agent: Bacteriophages (viruses)
Key Features: Phage accidentally packages host DNA; generalised or specialised types
Biological Significance : Major route for virulence factor transfer (toxins, capsules, etc.)
Example: Phage P1 or λ transferring gal operon
6
Q
Counter-Intuitive Bacterial Phenomena
Highlight key examples:
chatgpt
A
Phase variation and inversion-based ON/OFF switching.
Use of toxin-antitoxin systems in plasmid maintenance.
Non-uniform gene expression in clonal colonies.
Bacterial “hedge-betting” strategies for survival.
“These are not intuitive behaviors in genetically identical populations but are common due to selective pressures.”
7
Q
Spread of Antimicrobial Resistance (AMR)
chatgptt
A
HGT enables rapid dissemination of AMR genes across species boundaries.”
Link integrons, plasmids, and transposons to this spread.
Add: “Transposons often carry multiple resistance genes and can hop onto conjugative plasmids, facilitating inter-species transfer.”
8
Q
Major Driver of Pan-Genome Expansion
chatgpt
A
Pan-genome = core genome + accessory genes (strain-specific).
HGT is a primary driver, especially through:
Conjugation (broad host-range plasmids like RK2).
Transduction (including generalised and specialised).
Transformation of environmental DNA.
9
Q
DNA Surveillance and Repair
bacterial genomic maintenance
chatgpt
A
The LexA regulon and RecA-SOS response are central to bacterial DNA damage sensing and repair.”
Note that transcription-coupled repair (TCR) uses RNA polymerase stalling to detect lesions.
10
Q
Microbial Arms Race
chatgpt
A
Mention the co-evolutionary battle between bacteria and bacteriophages.
Add CRISPR-Cas systems as a bacterial adaptive immune system, and that phages have evolved anti-CRISPRs.
Mention how restriction-modification systems form another defense line.
Add: “This ongoing conflict drives innovation in both microbial offense and defense.”
11
Q
DNA code
A
- Largely static
- Some mutations
12
Q
Bacteria DNA
A
- Don’t regard DNA code as static
- Have mechanisms that ensure that the DNA sequence is constantly changing and constantly being triaged
- One of the big reasons for bacterial survival
- Programmed instability
13
Q
Genetic differences within different cells in the same colony
causes
A
2 main causes:
1. Inversion of segments at specific loci
2. Short tracts that differ in length at specific loci
14
Q
Genetic differences within different cells in the same colony
1. Inversion of segments at specific loci
A
- There are certain loci (position) where a short segment of DNA can either be in one orientation or the other
- For promoter to work has to be pointing to the genes
15
Q
Genetic differences within different cells in the same colony
1. Inversion of segments at specific loci
- induce it?
A
Get a synapse at the end
An enzyme that can make a double stranded break is going to cut at the edge
When it makes a double stranded break
Inversion occurs
There is a separate locus which is responsible for making an enzyme that causes the inversion
* Can have an enzyme that biases in one direction
16
Q
Genetic differences within different cells in the same colony
1. Inversion of segments at specific loci
- Gene for fimbriae in salmonella
A
o Can freeze DNA with the promoter sequence in one direction or to bias it in the other direction
o Why does salmonella do this
Should cling onto the gut of the person or not
Can calculate whether it is worthwhile to stay or if it is better to go
* Bacterial hedge betting
17
Q
Genetic differences within different cells in the same colony
1. Inversion of segments at specific loci
- Gene for fimbriae in salmonella
ensures….
A
heterogeneity in a population
18
Q
Genetic differences within different cells in the same colony
1. Inversion of segments at specific loci
- Gene for fimbriae in salmonella
all or nothing regulation
A
o Two states: on or off
o Can regulate the enzyme
19
Q
Genetic differences within different cells in the same colony
1. Inversion of segments at specific loci
Gene for fimbriae in salmonella
- Contrasts with regulation by…
A
by repressors and activators
o Repressors and activators allow a graded response (little or large impact)
o These do not
Promoter either reads the gene or doesn’t
On or off
20
Q
Genetic differences within different cells in the same colony
1. Inversion of segments at specific loci
example: Bacteroides fragilis
A
polysaccharide biosynthesis
21
Q
Genetic differences within different cells in the same colony
2. Short tracts that differ in length at specific loci
A
- Variation due to the no. of repeats at a loci (position)
o When DNA replicated DNA polymerase makes mistakes so get more or less repeats
o Loci in chromosome where you have a variable no. of repeats - Homopolymer tracts of different lengths (e.g. G)
- Repeated two base sequences (e.g. CA)
22
Q
Genetic differences within different cells in the same colony
2. Short tracts that differ in length at specific loci
found in?
A
- ONLY FOUND IN PARTICULAR. BACTERIA AT SPECIFIC LOCATIONS
o Particularly prevalent in:
Bacteria that infect mammalian hosts
Genes that encode cell surface proteins
23
Q
Genetic differences within different cells in the same colony
2. Short tracts that differ in length at specific loci
evidence
A
- The genome sequence of the food-borne pathogen Campylobacter jejuni reveals hypervariable sequences
o Open reading frame
o Black dots
o Where sequences differ at certain loci
Difficulties with sequencing - Got around this by cloning DNA
24
Q
Genetic differences within different cells in the same colony
2. Short tracts that differ in length at specific loci
evidence - Campylobacter jejuni
what at certain loci?
A
o Found that there was hypervariability at certain loci
- Specifically, in genes that code the products on the surface of the bacterium, there is enormous variability
o Real life application binding
- Sequence variation is generated during replication by replication error (slipped strand mispairing)
25
TRANSPOSABLE ELEMENTS
- Small blocks of sequence that jump from one location to another
o Allows bacteria to access a range of phenotypes
- DNA sequences that can move from one location to another within the genome
26
TRANSPOSABLE ELEMENTS
Insertion sequences
Insertion sequences (IS elements) are the simplest mobile genetic element
o 1-3kb in length
o Encode only the transposase protein and a regulatory protein
o The transposase is required for transposition
o Contain an inverted repeat sequence at their termini that is required for transposition
27
TRANSPOSABLE ELEMENTS
IS elements
how
- Double strand cut and resealed in another order
- Enzyme recognises the ends (where you get the cut)
o Signature sequences at the ends
28
TRANSPOSABLE ELEMENTS
IS elements
distance?
- Insertion sequences can jump from one location to another over long distances
29
TRANSPOSABLE ELEMENTS
IS elements
- Why have bacteria evolved these sequences
o We don’t know?
o to speed up evolution?
30
TRANSPOSABLE ELEMENTS
Insertion sequences are...
mutagenic
31
TRANSPOSABLE ELEMENTS
Insertion sequences can be...
polar
- also can prevent expression of other genes in a operon (premature termination of transcription)
32
TRANSPOSABLE ELEMENTS
Insertion sequences and outward promoters
- Tn insertions can lead to the expression of nearby genes
- Can contain an outward reading promoter (Pout)
o Using these a mobile promoters
o Exist to create variation and carry info?
33
TRANSPOSABLE ELEMENTS
Insertion sequences and outward promoters
why is it inverted?
o If was a direct repeat the protein wouldn’t recognise
o Inverted repeats force it into a conformation that is a clear signal
o Spontaneous mutation in inverted repeat?
o Would not recognise the IR as for transposase to engage needs to IRS
o The insertion sequence wont go anywhere
34
TRANSPOSONS
Transposons are transposable elements that have one or more genes unrelated to transposition
35
TRANSPOSONS
transported with?
- These additional genes are transposed with the transposable element
36
TRANSPOSONS
length
- Transposons are several kb in length
37
TRANSPOSONS
many carry..
- Many transposons carry antibiotic resistance genes
o Big segment of DNA that is flanked by two partially functional insertion sequences
o Example of a mobile genetic element (can change position from one to another)
38
INTEGRONS
- DNA elements found in some transposons
o Comes from transposons transposing into transposons
o E.g. Tn21 family (class II)
- Site specific-recombination system
- Captures gene cassettes
39
INTEGRONS
encodes...
- Encode integrase / specific attachment site / strong promoter
40
INTEGRONS
able to aquire...
multiple Ab resistance genes
41
INTEGRONS
can be found in...
- Can be found in plasmids and chromosomes too (Horizontal gene transfer)
Vibrio cholerae super-integrons / 100s genes
42
What is the major difference between an insertion element and a transposon?
- Insertion elements don’t have passengers
o Just the driver (transposase) who moves them around
- Composite transposons can carry passengers
o Antibiotic resistance cassettes
- Integrons have loads of passengers
43
STATIC GENOME
: Each gene resides at a fixed chromosomal position
44
STATIC GENOME
whole genome sequencing has shown
- Discovery of transposable elements (1970s) upset this classical picture
- Whole genome sequencing has shown us that:
o Individual bacterial genomes are more fluid than initially thought
Genetic material of bacteria subject to large alteration
* i.e. insertions/inversions/deletions/transpositions
even more dynamic due to horizontal gene transfer
45
BACTERIAL PLASMIDS
origin of replication
Bacteria only have one origin of their (circular) chromosome
46
BACTERIAL PLASMIDS
replication
- Bidirectional replication
o Starts at a specific position
Need to ensure cell division is coordination with chromosome replication
47
BACTERIAL PLASMIDS
replication forks
- Two replication forks
48
BACTERIAL PLASMIDS
mechanism of replication
binding of initiator protein
synthesis of RNA primers + unwinding of DNA by helicase and SS DNA binding proteins
formation of two replication forks
49
BACTERIAL PLASMIDS
size
- Usually small (a few Kb to a few hundred Kb) circular DNA molecules with their own origin of replication
o Some bacteria have large “megaplasmids”
o Some bacteria have linear plasmids
50
BACTERIAL PLASMIDS
copies
- Number of copies of different plasmids varies
o High copy number plasmids: 200 copies per cell
o Low copy number plasmids: 1-2 copies per cell
51
BACTERIAL PLASMIDS
found in?
- Found in most bacterial species
52
BACTERIAL PLASMIDS
replicate alongside..
main chromosome
53
BACTERIAL PLASMIDS
- Coupling of chromosome copy to...
cell division
o Not with plasmids
o Bacterial chop and change
54
BACTERIAL PLASMIDS
properties of plasmids
1) Replication origin (essential)
2) Passenger genes
3) Active partition function
4) Conjugation functions
55
BACTERIAL PLASMIDS
properties of plasmids
1) Replication origin (essential)
o Directs replication
o Essential
o Some host-specific, some broad host range
o Incompatibility groups
56
BACTERIAL PLASMIDS
properties of plasmids
2) Passenger genes
o Any gene but likely to be something that gives an advantage to the host bacterium
o E.g. genes for antibiotic resistance, ampR determinant (pratical) resistance to metals, ability to degrade complex molecules (e.g. tol plasmids)
57
BACTERIAL PLASMIDS
properties of plasmids
2) Passenger genes
examples
ColE1
trait: bacteriocin with kills E coli
orginal source: e coli
58
BACTERIAL PLASMIDS
properties of plasmids
3) Active partition function
o Ensures that when cell divides
o Each daughter cell receives plasmids
o Some partition systems involve actin like filaments
o Some involve toxin-antitoxin couples where the antitoxin is unstable
59
BACTERIAL PLASMIDS
properties of plasmids
3) Active partition function
Active partitioning using filaments
plasmid pairing
plasmid partitioning
cell division
- Drives progeny up
60
BACTERIAL PLASMIDS
properties of plasmids
3) Active partition function
Active partitioning using toxin-antitoxin pair:
cell divison
plasmid inherited cell survies
plasmid not inherited - toxin kills cell
61
BACTERIAL PLASMIDS
properties of plasmids
3) Active partition function
Active partitioning using toxin-antitoxin pair:
couples
- Plasmids carry toxic anti-toxic couples
o These couples are involved inn active partitioning
62
BACTERIAL PLASMIDS
properties of plasmids
3) Active partition function
Active partitioning using toxin-antitoxin pair:
which is more stable
- Toxin more stable than antitoxin
- The antitoxic degrades faster than the toxin
- So the cell that hasn’t received the plasmid as some stage (after cell division) the cell will die
o This ensures that plasmids that don’t inherit will die
63
BACTERIAL PLASMIDS
properties of plasmids
4) Conjugation functions
o Ensures that plasmid can transfer from one bacterium to another
o THIS IS AN EXAMPLE OF HORIZONTAL GENE TRANSFER (HGT)
64
BACTERIAL PLASMIDS
properties of plasmids
4) Conjugation functions
Conjugative plasmids
- Many large plasmids encode genes that enable the transfer of the plasmid between cells
o DNA is transferred through a pilus (The anatomy of pilus determines function and comes in lots of shapes) that connects the two cells
o The transfer process is called conjugation and the recipient is a transconjugant
65
BACTERIAL PLASMIDS
properties of plasmids
4) Conjugation functions
Conjugative plasmids: occurs?
- Conjugation occurs in and between bacterial species and also occurs between bacteria and plants
66
BACTERIAL PLASMIDS
properties of plasmids
4) Conjugation functions
Non-Conjugative plasmids
- Most small plasmids are nonconjugative
- They are maintained in both daughters cells following cell division
- They can not transfer to another cell on their own
- Have been employed in gene cloning technology as vectors fro introducing and maintaining DNA in bacteria
67
BACTERIAL PLASMIDS
properties of plasmids
Plasmids in the lab
- Genetic engineering
- Recombineering
- Promoter activity assays
- Complementation
o Mutational analysis
- Overexpression
o Protein purification
68
BACTERIAL PLASMIDS
the F plasmid
F =
fertility factor
69
BACTERIAL PLASMIDS
the F plasmid features:
OriC
Origin of replication
OriT
Origin of transfer
Tra region
Genes required for conjugative transfer (large region)
IS elements
Important for F-plasmid conjugative function
70
BACTERIAL PLASMIDS
the F plasmid: mating pair formation
- F plasmid encodes ~35 transfer genes (tra)
- Encodes functions for conjugation
- Polycistronic operon / co-ordinately regulated
- Many genes encode for the assembly of mating pair formation apparatus
- Complex structure / similar secretion apparatus must span IM/PG/OM of the donor
71
BACTERIAL PLASMIDS
the F plasmid: mating pair formation
function
1) Contact recipient
2) Bring donor and recipient together
3) Create cytoplasmic bridge
72
BACTERIAL PLASMIDS
the F plasmid: Conjugation apparatus
Pilus (Top Center):
Pilus (Top Center):
Forms physical contact between donor (F⁺) and recipient (F⁻) cells.
Retracts to bring cells close for DNA transfer.
73
BACTERIAL PLASMIDS
the F plasmid: Conjugation apparatus
Relaxosome (Bottom):
Includes the relaxase (TraI), which nicks the F plasmid at the oriT site.
5’ end of the DNA is covalently bound to relaxase and shuttled through the T4SS
74
BACTERIAL PLASMIDS
the F plasmid: Conjugation apparatus
ATPase Activity (Cytoplasmic Base):
Several ATPases (e.g., TraC, TraD) hydrolyze ATP to drive pilus assembly and DNA transfer.
Energetically expensive process — tightly regulated.
75
BACTERIAL PLASMIDS
the F plasmid: Conjugation apparatus
Membrane-Spanning Mating Pair Formation Complex:
Composed of multiple Tra proteins forming a Type IV secretion system (T4SS).
Transfers the relaxase-DNA complex across the donor membrane.
76
BACTERIAL PLASMIDS
the F plasmid: Conjugation apparatus
function summary
BACTERIAL PLASMIDS
the F plasmid: Conjugation apparatus
77
BACTERIAL PLASMIDS
the F plasmid:
Transfer of the F plasmid
- Contact between donor cell (F+) and recipient (F-)
- F-pilus retracts
o Cells in close proximity
- F plasmid DNA is nicked
- Rolling-circle DNA replication starts
- A single stranded linear branch is transferred to the recipient (leading strand)
- Lagging strand synthesis takes place in recipient
- Replication complete / F strand becomes circular
78
BACTERIAL PLASMIDS
the F plasmid:
Transfer of the F plasmid
5 steps
1) conjugation occurs F+ and F- cell
2) one strand of the F factor is nicked by an endonuclease and moves across the conjugation tube
3) the DNA complement is synthesized on both single strands
4) movement across conjugation tube is completed; DNA synthesis is completed
5) ligase closes circles; conjugants seperate
79
BACTERIAL PLASMIDS
the F plasmid:
Transfer of the F plasmid
simplified
1) pilus attachment and retraction
2) pore formation
3) mating pair stabilisation
4) DNA-cleavage at oriT
5) DNA transfer
6) DNA supercoiling
80
BACTERIAL PLASMIDS
Cognitive transfer – why its important
- Conjugative transfer is carried out by plasmids
- Importance for HGT
- Widespread antibiotic resistance is mainly due to the spread of transposons that contain one ore more antibiotic genes
o If the transposon inserts into a conjugative plasmid this facilitates the transfer of the transposon into a new strain or even new bacterial species
Carrying the passenger genes
81
BACTERIAL PLASMIDS
Other conjugative plasmids (RK2)
- Broad host range (cf F plasmid)
- R plasmid (ApR, TcR, KmR)
- Size / 2 tra regions / IS and Tn
- Transfer function is highly regulated
82
BACTERIAL PLASMIDS
Other conjugative plasmids (RK2):
RK2 mpf:
- Smaller (0.1uM)
- Rigid / inflexible
- Mating pair formation (mpf) apparatus
o Encoded by a variety of systems
- Related to protein export systems
o T4 secretion systems
83
BACTERIOPHAGE
what
- Simply viruses which infect bacteria
- Important role in molecular biology
- DNA transfer/HGT
84
BACTERIOPHAGE
structure:
- Nucleic acid enclosed by protein coat
- RNA/DNA (ds / ss)
- Many morphologies
o Simple/small
o Filamentous
o Heads and tails
85
BACTERIOPHAGE
how many?
Untrigintillion = estimate of number of phages, quintillion = number of phage attacks on bacteria per second, unvigintillion = phages in the human gut, 1 million = phase in an drop of seawater, ~2000-3000 = fully annotated genomes, ~12 fully characterized.
86
BACTERIOPHAGE
phage morphologies
- DNA goes in the head
- Fixed sized
o Limits the size of bacterial genome
87
BACTERIOPHAGE
Lytic infection cycle
1) Attachment
2) Entry of phage and degradation of host DNA
3) Synthesis of viral genomes and proteins (Some bacteria survival (resistant to other viruses) because viral DNA remains within them)
4) Assembly (phage assembly)
5) Release
88
BACTERIOPHAGE
cycles: can split into
lytic cycle and lysogenic cycle
89
BACTERIOPHAGE
lysogenic cycle
The phage DNA integrates into the bacterial chromosome → becomes a prophage.
Passed on vertically through cell division.
Can carry virulence genes (e.g., cholera toxin, diphtheria toxin).
If triggered by stress, the prophage exits the genome and enters the lytic cycle (e.g., via the SOS response).
90
BACTERIOPHAGE
lytic cycle and lysogenic cycle
when host in trouble:
- When host is in trouble/under threat
- Time to get out
- Viral dna synthesises and leaves host
91
BACTERIOPHAGE
integration
- Catalysed by integrase
- Site-specific recombination
92
BACTERIOPHAGE
genetic structure
relatively compact and highly organized, reflecting the phage’s efficiency and specialization
The genetic economy of phages reflects selective pressure for rapid replication and efficient packaging, with modular genomes shaped by horizontal gene exchange.
93
BACTERIOPHAGE
Lysogenic cycle: summary
Key Feature: Phage DNA integrates, forms prophage
Relevance to HGT: Can transfer virulence factors via specialised transduction
94
BACTERIOPHAGE
Lytic cycle: summary
Key Feature: Phage replicates, host lyses
Relevance to HGT: Can transfer random genes via generalised transduction
95
BACTERIOPHAGE
A bacteriophage genome typically includes:
Structural Genes
Packaging Genes
DNA Replication Genes
Lysis Genes
Integration/Excision Genes
Regulatory Genes
Anti-host Genes
Cargo (optional)
96
BACTERIOPHAGE
Excision
- Excision is in response to stress
o E.g. induction of the SOS response
97
BACTERIOPHAGE
Bacteriophage and their ‘war’ with bacteria
- Bacteriophages are the most abundant viruses on the planet
- The majority of free-living bacterial species are thought to be infected by phages
- It is thought that phages evolved shortly after the emergence of bacteria
o Billions of years ago
- And hence the arms race between bacteria and phages is considered almost as old as bacteria themselves
RESTRICTION ENZYMES
98
BACTERIOPHAGE
Discovery of Restriction-modification systems
Researchers observed that phage infection success varied depending on the bacterial host strain.
In the 1950s, a phenomenon called host-controlled modification was identified:
A phage could infect and replicate efficiently only in the same host strain.
When moved to a new strain, infection was mostly unsuccessful (e.g., 1 in 10,000).
Rare surviving phages adapted to the new host and lost the ability to infect the original host.
99
BACTERIOPHAGE
Discovery of Restriction-modification systems:
led to the concept of restriction-modification:
Bacteria restrict foreign DNA using restriction enzymes.
They protect their own DNA through methylation.
100
BACTERIOPHAGE
Discovery of Restriction-modification systems:
- What have bacteria developed
o Facing the abundance and diversity of phages
o Bacteria have developed multiple lines of defence that can collectively be referred to as the “prokaryotic immune system’
o Early research on bacteria defence systems mainly focused on restriction modification (R-M) and abortive infection (Abi) systems
o It is now recognised that prokaryotic immunity is much more complex than previously perceived
101
BACTERIOPHAGE
Diversity of defence systems
- Antiphage defense systems can be divided into those that target viral nucleic acids, abortive infection systems, and other types of systems
- Bacteria have antiviral defence systems led my enzymes
o Wont be asked detail of mechanisms in exam
102
BACTERIOPHAGE
Diversity of defence systems:
Clustered regularly interspaced short palindromic repeats:
- CRISPR
o Cluster of regularly interspaced palindromic repeats
- Cas
o CRISPR-associated genes and proteins
- Present in genomes of 40% bacteria and 85% archaea
103
BACTERIOPHAGE
Diversity of defence systems:
Stages of CRISPR-Cas immunity
provide adaptive immunity in bacteria and archaea through three key stages
1) adaptation
2) expression
3) interference
104
BACTERIOPHAGE
Diversity of defence systems:
Stages of CRISPR-Cas immunity
1) adaptation
Foreign DNA (e.g., phage) is recognized and a short sequence (spacer) is integrated into the CRISPR array.
key components
Cas1, Cas2
105
BACTERIOPHAGE
Diversity of defence systems:
Stages of CRISPR-Cas immunity
2. Expression
The CRISPR array is transcribed into a long pre-crRNA, then processed into individual crRNAs.
key components
Cas genes, RNase III
106
BACTERIOPHAGE
Diversity of defence systems:
Stages of CRISPR-Cas immunity
3. Interference
crRNAs guide Cas proteins to complementary foreign DNA, which is then cleaved and destroyed.
key components
crRNA + Cas nuclease
107
BACTERIOPHAGE
Some things to know about CRISPR-Cas
1) Just one of many defence systems
2) Sometimes found in ‘defence islands’
3) Recognition of ‘prey’ DNA doesn’t always trigger cleavage
4) Just as evolution has developed CRISPR-Cas for biological functions, biotechnologists are developing them for commercial applications
108
BACTERIOPHAGE
Viruses have evolved functions to overcome bacterial defences against viruses
- Viruses can use RNA decoys to thwart CRISPR defences
- Can wrap up bacterial defence systems
- Phages overcome bacterial immunity via diverse anti-defence proteins
109
HGT in bacteria
3 principal mechanisms for horizontal transfer between bacteria:
1. Transformation (naked DNA)
2. Conjugation (plasmids)
3. Transduction (viruses)
110
HGT in bacteria
3 principal mechanisms for horizontal transfer between bacteria:
1. Transformation (naked DNA)
- If a bacterium is able to take up DNA fragments from the environment (dead ect.)
- We say it is naturally competent
- This is due to competence genes (com genes)
N.B. lab e coli strains are not naturally competent (need to overcome how to get DNA over cell wall)
111
HGT in bacteria
3 principal mechanisms for horizontal transfer between bacteria:
1. Transformation (naked DNA)
Natural transformation / competence in bacteria
- Acquisition of naked DNA from the extracellular environment
- Requires the synthesis of specialised proteins (~20 to 50)
- Not all bacteria can take up DNA fragments from environment
- Those that can, can because they have competence genes
112
HGT IN BACTERIA
3 principal mechanisms for horizontal transfer between bacteria:
1. Transformation (naked DNA)
Natural transformation / competence in bacteria
EXAMPLES
gram negative
- Haemophilus
gram positive
- bacillus
113
HGT IN BACTERIA
3 principal mechanisms for horizontal transfer between bacteria:
1. Transformation (naked DNA)
in gram positive
Stages in DNA uptake:
1. Binding of dsDNA to surface
2. Double strand cleavage
3. Conversion to ssDNA (1 strand destroyed
o Uptake into cytoplasm
o Within minutes/ ~185nt/s
114
HGT IN BACTERIA
3 principal mechanisms for horizontal transfer between bacteria:
1. Transformation (naked DNA)
in gram positive
steps
binding
fragmentation
transpirt and degradation
115
HGT IN BACTERIA
3 principal mechanisms for horizontal transfer between bacteria:
1. Transformation (naked DNA)
in gram negative
Gram -ve have inner and outer membrane
- DNA uptake system: Neisseria gonorrhoaea
- DNA uptake sequence (DUS): 10 bp (GCCGTCTGAA)
o Genome enriched for DUS
116
HGT IN BACTERIA
3 principal mechanisms for horizontal transfer between bacteria:
1. Transformation (naked DNA)
in gram negative
Stages in DNA uptake
1. Binding of ds DNA to surface
2. Uptake into periplasm (across outer membrane)
3. Conversion to ssDNA (strand degradation)
o Crosses CM
o Enters cytoplasm
117
HGT IN BACTERIA
3 principal mechanisms for horizontal transfer between bacteria:
1. Transformation (naked DNA)
in gram negative
STEPS
binding
uptake into periplasm
transport and degresation
118
HGT IN BACTERIA
3 principal mechanisms for horizontal transfer between bacteria:
1. Transformation (naked DNA)
gene products
- Need gene products to take up DNA
o Bacteria have evolved these gene products
119
HGT IN BACTERIA
3 principal mechanisms for horizontal transfer between bacteria:
1. Transformation (naked DNA)
The fate of transforming DNA
- ssDNA from <1 to 5kb
- homologous recombination event
- DNA must contain 20-200bp of DNA with high similarity (barrier)
120
HGT IN BACTERIA
3 principal mechanisms for horizontal transfer between bacteria:
1. Transformation (naked DNA)
The fate of transforming DNA: supercoiling
- Supercoiling (negative)
- Local region of unwinding moves
o Flickering bubble
- DNA needs to be single stranded to be noticed by flickering bubble and therefore taken in (incorporated into bacterial chromosome --> must contain some similarities to the bacterial DNA)
- Very rarely there will be a transfer
121
HGT IN BACTERIA
3 principal mechanisms for horizontal transfer between bacteria:
1. Transformation (naked DNA)
incompetent bacteria
Can induce competence
- Ca2+ induced competence
o Divalent cation promote DNA binding to bacteria
o Heat shock induces DNA uptake
- Electroporation
o A short pulse of high voltage electricity
o Causes temporary permeability of the outer bacterial surface
o Allows DNA to enter without killing the bacteria
Fundamental techniques that have aided the development of recombinant DNA technologies (using plasmids)
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HGT IN BACTERIA
3 principal mechanisms for horizontal transfer between bacteria:
2. Conjugation (plasmids)
Bacterial has to physically couple
Only one plasmid to give
* HOWEVER THIS PLASMID CAN BE V. LARGE (e.g. 100k bases)
* Viral transduction has a size limit due to size of head
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HGT IN BACTERIA
3 principal mechanisms for horizontal transfer between bacteria:
3. Transduction (viruses)
- Creates virus and can go anywhere (50k bases)
- Potential to be more potent
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HGT IN BACTERIA
3 principal mechanisms for horizontal transfer between bacteria:
3. Transduction (viruses)
bacteriophage: how viruses are assembled
- Assemble head (empty)
o Problem of putting 50k bases of DNA
o Pump it in (room for error)
- Gatekeeper protein checks sequences being pumped in
o Sometimes host DNA is packed in
o End up with viral particle that contains host DNA
- Error gives rise for transduction (generalised transduction - head gets wrong part of DNA)
- These is how sequences are transduced from one bacteria to another without physical contact
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HGT IN BACTERIA
3 principal mechanisms for horizontal transfer between bacteria:
3. Transduction (viruses)
definition
- Definition: gene transfer mediated through carriage of DNA in a bacteriophage particle
o This is generalised transduction
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HGT IN BACTERIA
3 principal mechanisms for horizontal transfer between bacteria:
3. Transduction (viruses)
The fate of transducing DNA fragments
1. Stable integration into the recipient chromosome
o < 10%; via homologous recombination
2. Remaining free (non-integrated) in cytoplasm
o >90%; often circularised
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HGT IN BACTERIA
3 principal mechanisms for horizontal transfer between bacteria:
3. Transduction (viruses)
- Abortive transduction:
o Transduced DNA stable but non-replicating
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HGT IN BACTERIA
Lysogenic infection cycle
integration
- Catalysed by integrase
- Site-specific recombination
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HGT IN BACTERIA
Lysogenic infection cycle
Excision
- Reverse reaction
- Catalysed by integrase but also requires Xis (excisionase)
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HGT IN BACTERIA
Lysogenic infection cycle
Excision (imprecise)
Imprecise excision occurs when a prophage (phage DNA integrated into the bacterial chromosome during lysogeny) exits the chromosome incorrectly, taking adjacent bacterial genes with it.
rare, illegitimate excision
-> defective phage
- This makes a transducing phage
o Specialised transduction
When an accident occurs
- Size constraint/head
o BIG LIMITATION WITH VIRAL TRANSDUCTION
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Lysogenic infection cycle
Excision (imprecise)
how it happens
In the lysogenic cycle, a temperate phage (like λ) integrates its genome into a specific site on the bacterial chromosome.
Under stress (e.g., UV light), the prophage may be induced to excise and enter the lytic cycle.
Normally, precise excision restores the host genome and frees intact phage DNA.
But sometimes, excision is sloppy → part of the host bacterial DNA (e.g., nearby toxin gene) is mistakenly included with the phage DNA.
The resulting phage particle carries hybrid DNA (phage + bacterial genes).
When this phage infects another bacterium, it can transfer those host genes — a process called specialised transduction.
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Lysogenic infection cycle
Excision (imprecise)
key points
Only occurs at specific sites (e.g., near the prophage insertion site).
Can transfer toxin genes, antibiotic resistance, or metabolic genes.
Major mechanism for spreading virulence factors in bacteria like E. coli or Vibrio cholerae.
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HGT IN BACTERIA
3 principal mechanisms for horizontal transfer between bacteria:
What is the significance of these mechanisms?
The mobilome:
- The elements of the mobilome have selfish tendencies
o Want to pass on their own genes
o Survival
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HGT IN BACTERIA
3 principal mechanisms for horizontal transfer between bacteria:
What is the significance of these mechanisms?
Bacteriophage
- Lytic/lysogenic/prophage
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HGT IN BACTERIA
3 principal mechanisms for horizontal transfer between bacteria:
What is the significance of these mechanisms?
transposons
- Insertion elements
- Composite transposons
- Conjugative transposons
- Integrons
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HGT IN BACTERIA
3 principal mechanisms for horizontal transfer between bacteria:
What is the significance of these mechanisms?
Natural competence
- Environmental DNA uptake
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HGT IN BACTERIA
3 principal mechanisms for horizontal transfer between bacteria:
What is the significance of these mechanisms?
disaster waiting to happen?
- Resistance to antimicrobials
- Drivers of resistance is horizontal gene transfer
- AMR
- Mobilome acts selfishly — promotes its own survival and transmission
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HGT IN BACTERIA: IMPORTANCE
- For the microbial world
* Accelerated evolution: HGT allows bacteria to rapidly acquire new traits (e.g., antibiotic resistance, virulence factors) without waiting for slow mutations.
* Adaptability: Enables survival in harsh environments (e.g., uptake of resistance genes, metabolic pathways).
* Genetic diversity: Even in asexual organisms, HGT creates variation and facilitates evolution across species boundaries.
* Community interactions: Promotes cooperation and competition through gene sharing, impacting microbial ecosystems.
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HGT IN BACTERIA: IMPORTANCE
- For the selfish DNA
* Survival and propagation: Mobile genetic elements like plasmids, transposons, and bacteriophages use HGT to spread themselves.
* Self-promotion: Many carry genes that improve their host’s survival chances (e.g., antibiotic resistance), ensuring their own transmission.
* Hijacking of host systems: Selfish DNA elements exploit bacterial machinery to replicate and spread — sometimes to the host’s benefit, sometimes not.
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HGT IN BACTERIA: IMPORTANCE
- For humankind
* Medical challenges: HGT is a major driver of antimicrobial resistance (AMR) — a global health threat.
* Biotech and genetic engineering: Understanding and harnessing HGT mechanisms (e.g., transformation, electroporation) has led to recombinant DNA technology, GMOs, and synthetic biology.
* Pathogen evolution: Helps predict and track emerging diseases, as virulence factors can jump between species.
* Environmental and industrial uses: Engineered microbes can clean up pollutants (bioremediation), produce drugs, or aid in agriculture — often enabled by HGT tools.
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HGT IN BACTERIA: IMPORTANCE
- For gaia (Earth as a living system):
* Genetic flow across the biosphere: HGT contributes to the interconnectedness of life — genes move not just vertically (parent to offspring) but horizontally (across species).
* Microbial stewardship: Microbes regulate key planetary processes (e.g., nitrogen cycle, carbon fixation), and HGT helps them adapt and maintain these cycles.
* Dynamic balance: By enabling rapid adaptation, HGT helps microbial communities respond to environmental changes — stabilizing ecosystems.
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HGT IN BACTERIA: IMPORTANCE
- For the central dogma of molecular biology
* Challenges the traditional view: The central dogma (DNA → RNA → Protein) assumes a fixed genome, but HGT shows genomes can change dynamically and unexpectedly.
* Expands the framework: DNA can come from external sources, be integrated, and become functional — a layer of complexity not originally considered.
* Gene flow, not just gene expression: It redefines what we mean by "inheritance" and "identity" at the molecular level.
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BACTERIAL CHROMOSOME INTEGRITY
Replication errors (mismatches)
Radiation damage (thymine dimers)
Attack by electrophilic chemicals
Oxidative stress
Unwound DNA more susceptible to chemical attack
Most susceptible to mutations
* Non-template strand each base at some point exposed to environment
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BACTERIAL CHROMOSOME INTEGRITY
e.g. UV
Two T on top of each other on one strand
- When exposed to UV these T cross link
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BACTERIAL CHROMOSOME INTEGRITY
DNA repair in bacteria
o Direct reversal of chemical damage (the adaptive response, Ada)
o Mismatch repair (mut gene products)
o Base excision repair (mut gene products)
o Nucleotide excision repair (UvrA, UvrB and UvrC)
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BACTERIAL CHROMOSOME INTEGRITY
DNA repair in bacteria
conserved?
- These mechanisms of repair are highly conserved from bacteria to humans
o Highlights their importance
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BACTERIAL CHROMOSOME INTEGRITY
DNA repair in bacteria:
Excision repair
1. Find damage
2. Cut both side of the damage
3. Remove the damage
4. Copy the undamaged strand to make a patch
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BACTERIAL CHROMOSOME INTEGRITY
DNA repair in bacteria:
locating damage
- ~4,000,000 bp in a bacterial genome
- Not trivial
- Many different forms of damage
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BACTERIAL CHROMOSOME INTEGRITY
DNA repair in bacteria:
locating damage:
- How do you design an enzyme to recognise damage?
o DNA dependent RNA polyermase is transcribing genes
o If it comes to a lesion not able to copy it -> spots errors
o If DNA dependent RNA polymerase stalls it signals to the transcription coupled repair factor which repairs
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BACTERIAL CHROMOSOME INTEGRITY
DNA repair in bacteria:
Two strategies for DNA repair:
1. Global DNA repair – screens and looks for errors
2. Transcription-coupled repair - DNA dependent RNA polymerase stalls and brings in the transcription-coupled repair factor
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BACTERIAL CHROMOSOME INTEGRITY
DNA repair in bacteria:
different mechanisms deal with...
chemical damage and DNA mismatches
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BACTERIAL CHROMOSOME INTEGRITY
DNA repair in bacteria:
DSB or SSB more serious?
- Double strand breaks are far more serious and require the help of RecA and induction of the SOS system, which is regulated by the LexA
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BACTERIAL CHROMOSOME INTEGRITY
DNA repair in bacteria:
DSB causes
- Double stranded breaks are caused by severe chemical or radiation damage and also by clashes between the transcription and replication apparatus
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BACTERIAL CHROMOSOME INTEGRITY
DNA repair in bacteria:
DSB require
require the help of RecA and induction of the SOS system, which is regulated by the LexA
o lexA is a transcriptional repression
repress the genes encoding repair finctions when they are not needed
repression is reversed by irreversible proteolysis
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BACTERIAL CHROMOSOME INTEGRITY
DNA repair in bacteria:
DSB repair
- The main pathway is by homologous recombination
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BACTERIAL CHROMOSOME INTEGRITY
DNA repair in bacteria:
DSB repair:
homologous recombination
- The 3’ end is one of the broken strands exchanges with their equivalent sequence on the homologous chromosome forming a D-loop
- The 3’ end is extended and then displaced by DNA helicase
- This allows it to anneal to the other broken end
- Any gaps are filled by DNA polymerase and DNA ligase
o Equipment to do this is induced by the SOS system
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BACTERIAL CHROMOSOME INTEGRITY
DNA repair in bacteria:
DSB repair:
homologous recombination
STEPS
1. DSB
2. end resection
3. strand invasion and DNA synthesis
(D loop)
4. strand displacement annealing
5. DNA synthesis ligation
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BACTERIAL CHROMOSOME INTEGRITY
DNA repair in bacteria:
if cant repair
- Bacteria try to repair their DNA but if they fail they hit the auto-destruct button
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BACTERIAL CHROMOSOME INTEGRITY
DNA repair in bacteria:
monitor SOS regulon activity
- Prophage has worked out clever ways to monitor SOS regulon activity
