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TRANSCRIPT
MOBILOME
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• < 2% of the human genome encodes genes• Genes identical between different mammals: we share >98% of genes
with chimpanzees and >85% with mice• Evolution relies on changes in non-coding regions of genomes —>
particularly in regulatory sequences to alter gene expression• The majority of the genetic information in humans (and most mammals)
consists of transposable elements (TEs)• Regulatory sequences often originate from TEs• TEs are repetitive genetic sequences that once had or still have the
ability to transpose = to mobilize and insert elsewhere in the genome• TEs are diverse across species and often species-specific
In mammals
Senft & Macfarlan 2021 2
Senft & Macfarlan 2021
Barbara McClintock discovered transposable elements (TEs) in 1950s by attributing pigment changes in maize to TE transposition —> proposed TEs controlling elements regulate developmentTEs can be divided into 2 classes:
DNA transposons excise and insert a DNA intermediate when they transpose: ’cut and paste’Retrotransposons reverse transcribe RNA intermediates prior to integration: ’copy and paste’
DNA transposons are few and inactive in most mammals vs retrotransposons are abundantRetrotransposons are classified by whether they contain long terminal repeats (LTRs)Most LTR-containing retrotransposons in mammals are endogenous retroviruses (ERVs)Frequent recombination between LTRs leaves behind many solo LTRs in the genomeRetrotransposons lacking LTRs include autonomous long interspersed elements (LINEs) and non-autonomous short interspersed elements (SINEs), which require LINE-derived proteins for their mobilizationHumans encode additional primate-specific composite elements called SINE variable-number tandem-repeat Alu (SVA) elements
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Genetic Elements, introM
adigan et al. 2020
E. coli strain K-12 Resistance plasmid R100
Madigan et al. 2020
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• Mobilome: the sum total of all mobile genetic elements (MGEs) in a genome
• Best to think of MGEs as a continuum rather than a neat categorization
• MGEs represent a highly heterogeneous group of elements with blurred differences
• This continuum of MGEs within an individual bacterial species, never mind a community as a whole, is highly varied
• MGEs can be inherited vertically
• MGE’s central role in HGT means that even within an individual species there is great heterogeneity
256 shades of grey
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The mobilome, defined as all mobile genetic elements (MGEs) of the microbiome, influences the composition of microbial communities and the spread of antimicrobial resistance genes and virulence factors via horizontal gene transfer (HGT)
• Plasmids• Insertion sequences• Integrative conjugative elements• Mobilisable genetic elements • Integrons and gene cassettes • Bacteriophages
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• Bacteria undergoing rapid evolutionary change often contain relatively large numbers of mobile elements, especially insertion sequences, simple transposable elements whose genes encode only transposition
• Recombination among identical elements generates chromosomal rearrangements such as deletions, inversions, or translocations —> genomic diversity upon which natural selection can act
• Chromosomal rearrangements that accumulate in bacteria during stressful growth conditions are often flanked by repeats or insertion sequences
• The functions conferred by mobilome extend beyond antibiotic resistance and are quite diverse, including digestion of most classes of carbohydrates, mercury and metals resistance, virulence and catabolism used in bioremediation
• Tight evolutionary connections between MGEs and defense systems —> recombinases, transposases and integrases, endonucleases part of the Toxin-Antitoxin system and Restriction-Modification system and CRISPR-Cas system
Mobilome importance
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https://youtu.be/86JCMM5kb2A
Homology-dependent double strand break repair
https://youtu.be/31stiofJjYw
Non-homologous end joining
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Recap, I
Recombination, I• Recombination is the physical exchange of DNA
between genetic elements (structures that carry genetic information) after HGT
• Homologous recombination, a process that results in genetic exchange between homologous DNA sequences from 2 different sources
• Homologous DNA sequences are those that have nearly the same sequence
• Bases can pair over an extended length of the two DNA molecules to facilitate exchange
• RecA protein, SOS repair system is the key to homologous recombination (DNA replication when ss stalls) —> activate polymerases —> translasion
• RecA is essential in nearly every homologous recombination pathway
Madigan et al. 2020
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Recombination, IIM
adigan et al. 2020
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1. Endonuclease cuts DNA in the middle of a strand —> nicking one strand of the donor DNA molecule
2. Nicked strand is separated from the other strand by proteins with helicase activity and binds single-strand binding protein + RecA
3. Base pairing with the complementary sequence in the recipient DNA molecule —> displaces the other strand of the recipient DNA molecule (strand invasion)
4. Heteroduplex according to spatial orientation: patches/splices
5. Resolvases resolve the heteroduplex by promoting ds DNA
https://youtu.be/MnYppmstxIs
What is CRISPR?
Recap, II
• Antiviral system in Bacteria & Archaea• CRISPR contain short repeats of constant DNA sequence alternating with short variable DNA
sequences, spacers• Spacers are pieces of viral or other foreign DNA and function as “memory bank” of past viral
encounters• Cas (CRISPR-associated) proteins have endonuclease activity for the defense against foreign
DNA and incorporate new spacers into CRISPR region
CRISPR, the clustered regularly interspaced short palindromic repeats
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CRISPR, immunizationM
adig
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t al.
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• When a virus injects its DNA, the Cas proteins of a CRISPR region may recognize specific DNA sequences known as protospacer adjacent motifs (PAMs)
• Cas protein cleaves viral DNA at a locus near PAM (termed the protospacer) and inserts the short DNA region into the CRISPR region of the chromosome, where it becomes a spacer —> confers “genetic memory”
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• Any viral DNA:crRNA duplexes formed are cleaved by Cas endonuclease activity
• Invading DNA is degraded in a process called interference
• With part of its genome destroyed an invading virus cannot proceed to replicate
• Immunization occurs when virus has been inactivated by environmental factors (e.g. UV radiation) or when the host’s restriction enzyme system cleaves the invading DNA prior infection’s begin
CRISPR, interferenceM
adig
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t al.
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• Pre-CRISPR RNA (pre-crRNA) and contains an array of RNA sequences complementary to both the repeat and spacer regions
• Cas proteins process the transcript into individual spacer RNAs by targeting the repeat regions
• crRNAs+Cas surveillance for complementary incoming viral DNAs
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https://youtu.be/FhcZLqvs5yg
Recap, III
Rolling Circle Mechanism Plasmid Replication
Rolling circle replication• Not a Semiconservative replication only one strand is transcribed, off of negative strand of the
replicative form in fX174
• In the synthesis in of the fX174 genome, the rolling circle facilitates the continuous production of positive strands from the replicative form
• The positive strand of the latter is nicked by A protein and the 3’ end of the exposed DNA is used to prime synthesis of a new strand
• Only the negative strand serves as a template
• When the growing viral strand reaches unit length (5386 residues for fX174), the A protein cleaves it and then ligates the two ends of the newly synthesized single strand to give a ssDNA circle
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Madigan et al. 2020
Horizontal Gene Transfer (HGT), I
• Gene transfer from one cell to another by means other than the vertical process
• In prokaryotic cells, 3 HGT mechanisms: transformation, transduction, conjugation
• HGT can be detected in genomes once the genes have been annotated:
a. Presence of genes that encode proteins typically found only in distantly related species
b. Presence of a stretch of DNA whose guanosine/cytosine (GC) content or codon bias differs significantly from that of the rest of the genome
• Lateral gene transfer
• Prokaryotic cells are actively exchanging genes in nature
• HGT “fine-tuning” an organism’s genome to a particular ecological situation or habitat
Madigan et al. 2020
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Horizontal Gene Transfer (HGT), II
1. Transformation, in which free DNA released from one cell is taken up by another
2. Transduction, in which DNA transfer is mediated by a virus
3. Conjugation, in which DNA transfer requires cell-to-cell contact and a conjugative plasmid in the donor cell
• DNA transfer typically occurs in only one direction: donor —> recipient
Madigan et al. 2020
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General routes of horizontal gene transfer within natural communities
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Outer membrane vesicles and DNA packaged into virus-like particles although their contribution to overall horizontal gene transfer is unknown
• A schematic outlining the stages through which DNA must go on its journey from donor to recipient bacteria
• The process begins with DNA in a potential donor cell becoming available and ends when this DNA becomes a functional part of a recipient cell’s genome
Thomas & Nielsen, 2005
The process of horizontal gene transfer
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Thomas & Nielsen, 2005
Donor and Selection of recipient
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Thomas & Nielsen, 2005
Uptake and Recipient
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Horizontal Gene Transfer (HGT), III• Fate of transferred DNA:
1.It may be degraded by the recipient cell’s restriction enzymes or other DNA destruction systems
2. It may replicate by itself (but only if it possesses its own origin of replication, plasmid or phage genome)
3.It may recombine with the recipient cell’s chromosome
• CRISPR–Cas systems in archaea and bacteria for defense against mobile genetic elements (viruses, plasmids and transposons)
• MGEs are key contributors to the evolution of different types of CRISPR–Cas types (6 different types)
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24Frost et al., 2005
Horizontal Gene Transfer (HGT), IV
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Transduction
Frost et al., 2005
• DNA genome (yellow) of a temperate phage inserts into chromosome (dark blue) as a prophage
• It later replicates, occasionally packaging host DNA alone (generalized transduction) or with its own DNA (specialized transduction)
• It lyses the cell, and infects a naive recipient cell in which the novel DNA recombines into the recipient host cell chromosome (red)
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Conjugation
Frost et al., 2005
• Large, low copy number conjugative plasmids (orange) and integrated conjugative elements (ICEs) use a protein structure, pilus, to establish a connection with the recipient cell and to transfer themselves (Gram-negative)
• A copy of a small, multi-copy plasmid/defective genomic island /a copy of entire chromosome can be transferred to a naive cell —> either insert into the chromosome or replicate independently if compatible with the resident plasmids (light green)
• Conjugative transposons and plasmids of Gram-positive bacteria do not use pili
Overview of plasmids and conjugative transfer in the horizontal spread of genes
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In the donor cell: a, integration of the plasmid into the chromosome by recombination between insertion sequence elements; b, movement of a transposable element through a circular intermediate from the chromosome to the plasmids; c, initiation of rolling-circle replication at the mating-pair apparatus.
In the recipient cell: d, recircularization; e, attack by restriction endonucleases (scissors); f, replication; g, integration into the chromosome by an illegitimate Campbell recombination; h, recombination between transferred chromosomal DNA and the resident chromosome.
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TranspositionFrost et al., 2005
• Transposons (pink) integrate into new sites on the chromosome or plasmids by non-homologous recombination
• Integrons (dark green) use similar mechanisms to exchange single gene cassettes (brown)
Transformation, I• Genetic transfer process by which free DNA is incorporated into a recipient cell and
brings about genetic change
• Several organisms are naturally transformable
• Because the DNA in prokaryotic cells is present as a large single molecule, when a cell is gently lysed, its DNA pours out
• Bacterial chromosomes break easily because of their extreme length (if linearized, the Bacillus subtilis chromosome would be 1700 μm long, ~4.2 Mb, fragment of 10 kb ~ 1000 bases per genes —> 10 genes)
• A single cell incorporates only one or at most a few DNA fragments, so only a small proportion of the genes of one cell can be transferred to another in a single transformation event
• COMPETENCE involves approximately 20 to 50 proteins
• Naturally transformable bacteria develop time-limited competence in response to specific environmental conditions such as altered growth conditions, nutrient access, cell density (by quorum sensing) or starvation
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• The steps involved in this process include the release of extracellular DNA into the environment and the uptake of DNA into the cytoplasm of the recipient bacterial cell that has developed a regulated physiological state of competence
• Following uptake, for the transferred DNA to persist it must integrate into the bacterial genome through homologous recombination or by sequence-independent, illegitimate recombination
• Plasmids that succeed in reconstituting a replication-proficient form do not need to integrate into the host genome
Thomas & Nielsen, 200530
Transformation, II
• Persistence of extracellular DNA in the environment determines the bacterial exposure time and transformation frequency
• DNA sequence integrity (e.g. metilation state, breaks, etc..) • In soil and sediments, ~ 1 μg extracellular DNA per g material • In fresh and marine water ~ 0.03 to 88 μg dissolved DNA per liter • The uptake of DNA by competent bacteria occurs rapidly in vitro, at about 100
bp per second in Streptococcus pneumoniae and 60 bp per second in Acinetobacter baylyi
• Selectivity and non selective DNA uptake • For homologous recombination: incoming DNA must contain regions
between 25 to 200 bp in length of high similarity to the recipient genome• 0.1% of internalized DNA fragments are successfully recombined in A. baylyi,
whereas up to 25–50% of the internalized DNA fragments are recombined in Bacillus subtilis and S. pneumoniae
• A log-linear decrease in recombination frequencies with increasing sequence divergence
Is all DNA, good DNA?
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Competence, I• Ability of a cell to take up DNA and be transformed, is genetically determined
• Competence is regulated: special proteins play a role in DNA uptake and processing
• Competence-specific proteins: membrane associated DNA-binding protein, a cell wall autolysin, and various nucleases
• In Streptococcus, 100% of the cells can become competent, but only for a brief period during the growth cycle
• Competence in V. cholerae is controlled not only by quorum sensing but also by chitin sensing and catabolite repression
• Acinetobacter, Bacillus, Streptococcus, Staphylococcus, Haemophilus, Campylobacter, Haemophilus, Helicobacter, Neisseria, Pseudomonas, Deinococus and Thermus are naturally competent and easy to transform others are not.
• Natural competence provides a nutritional advantage, as free DNA is rich in carbon, nitrogen, and phosphorus (DNA as a source of nutrients or genetic information)
• If E. coli are treated with high concentrations of Ca2+ and then chilled —> competent for dsDNA
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Bacillus subtilis competence
• Natural competence in B. subtilis is regulated by quorum sensing (cell abundance behavior), only 20% of population are competent for hours
• Cells produce and excrete a small peptide during growth, and the accumulation of this peptide to high concentrations induces the cells to become competent
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Competence, II• Streptococcus pneumoniae (the cause of bacterial pneumonia) cell can bind ~ 10 ds
DNA of 10–15 kbp each
• ds-fragments are taken up —> ssDNA (~8 kb)
• DNA fragments in the mixture compete with each other for uptake and thus the probability of a transformant taking up DNA that confers an advantage or a selectable marker decreases
• During transformation, DNA is bound at the cell surface by a DNA-binding protein resembles a pilus that is able to pull the DNA into the periplasm of a gram-negative bacterium or through the thick cell wall of a gram-positive bacterium
• Competence-specific protein binds the donor DNA, for protection —> RecA —> integration into recipient genome
Madigan et al. 2020
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• Plasmids• Integrative conjugative elements• Insertion sequences, Transposons• Integrons and gene cassettes • Bacteriophages• Mobilisable genetic elements
Mobile genetic elements (MGEs) of the microbiome
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Length: less than a kilobase to megabases
Main function: they are highly heterogeneous elements and the simplest just encode their own replication functions. Some also encode conjugation functions. They commonly contain cargo DNA that encodes functions for survival in different environments, for example, antibiotic-resistance genes and virulence factors
HGT mechanism: conjugation, transduction, and transformation
Resistance plasmid R100
Plasmids, I
Plasmids are replicons that are distinct from chromosomal DNA found in bacteria and archaea
Madigan et al. 2020
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• Most Bacteria and Archaea contain a single circular chromosome containing all (or most) of the organism’s genes (Euk, linear DNA)
• Plasmids are circular or linear double-stranded DNA molecules, one or more copies
• Plasmids replicate separately from the chromosome
• Plasmid ~1 kbp to more than 1 Mbp; 5% of total genomes
• Thousands of different plasmids are known, and >300 different plasmids from strains of E. coli
A. Enzymes that replicate chromosomal DNA also replicate plasmids. Some of the genes encoded on a plasmid function to direct the initiation of plasmid replication and to partition replicated plasmids between daughter cells
B. Virulent genes, antibiotic and metal resistance, other special metabolism
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Plasmids, II Recombination
DNA transfer starts at oriT (origin of transfer) site
tra, transfer
oriV, replication start site
Insertion sequences
Madigan et al. 2020
Integrative conjugative elements (ICEs), also called conjugative transposons
Length: 18 kilobases and upwardsMain function: they are highly heterogeneous elements that have the capability of inserting into bacterial genomes and transferring by conjugation between bacteriaThey commonly contain cargo DNA that encodes functions for survival in different environments, for example, antibiotic-resistance genes and virulence factorsHGT mechanism: conjugation, GREAT DIVERSITY ACCORDING TO WHAT IS IS THE LEADER
Integrative conjugative
elements
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Recombination
DNA transfer starts at oriT (origin of transfer) site
tra, transfer
oriV, replication start site
Insertion sequences
• An integrative and conjugative element (ICE) is integrated into one site in the host chromosome and is bounded by specific sequences on the right (attR) and left (attL)
• Excision yields a covalently closed circular molecule as a result of recombination between attL and attR to yield attP (in the ICE) and attB (in the host chromosome
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Integrative and conjugative element life cycle, I
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Integrative and conjugative element life cycle, II
• An ICE-free cell can serve as a potential recipient• During conjugation, the donor and recipient are brought in close contact, and a single
DNA strand is transferred to the new host through the action of rolling circle replication• Following transfer, DNA polymerase in the recipient synthesizes the complementary
strand to regenerate the double-stranded, circular form• A recombination event between attP and attB results in integration into host chromosome
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Integrative and conjugative element life cycle, III
Madigan et al. 2020
• F plasmid & E.coli chromosome carry several copies of mobile genetic elements called insertion sequences (IS)
• IS regions of sequence homology between chromosomal and F plasmid DNA—> homologous recombination —> diverse Hfr (given the place of insertion in chromosome and direction)
• When a recipient cell is encountered, conjugation is triggered
• DNA strand usually breaks during transfer, only part of the donor chromosome is typically transferred
• ICE < 3% of total genome in E. coli
• Rolling cycle replication
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Insertion sequences, IS are short transposable elements containing genes that code for proteins involved in transposition
Length: kilobasesMain function: the simplest code for proteins involved in transposition only. They often have cargo genes that encode functions for survival in different environments, for example, antibiotic-resistance genes and virulence factors
HGT mechanism: they can be spread by transposing to conjugative elements and by transformation and transduction
Insertion sequencesM
adigan et al. 2020
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Transposons, I• Transposons are transposable elements
• Transposons >> IS elements
• Inverted repeats (IR) at both ends and transposase
• Transposase recognizes the inverted repeats and moves the segment of DNA flanked by them from one site to another—> transposase recognizes, cuts, and ligates the DNA during transposition
• Any DNA that lies between the two inverted repeats is moved and is part of the transposon
• Antibiotic resistance genes (Tn5 kanamycin resistance; Tn10, tetracycline resistance)
• Conjugative transposons contain tra genes and can move between bacterial species by conjugation as well as transpose from place to place within a single bacterial genome
• Bacteriophage Mu, which is both a virus and a transposon (mutagen)
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Madigan et al. 2020
• Transposons are a broad class of MGE that propagate by inserting the element into new locations in the host genome
• Dedicated recombinase, variously referred to as transposase, integrase or site-specific recombinase
• Transposases are diverse enzymes that catalyse the integration of the transposon at a new location in the genome
• Some transposases directly move the element whereas others insert a copy of the element at a new location, in some cases via an RNA intermediate
• Transposition processes are often associated with DNA replication and repair, and sometimes reverse transcription, to allow propagation of the element
• High frequency of transposition from 1 to 103 and 1 to 107 per cell cycle• Transposon strong driver of genome evolution mediate a variety of large-
scale chromosomal changes
Transposons, II
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Transposons, III
Family 1: NitSJ-C1 from Nitrosopumilus sp. SJ; Family 2: AciBoo-C1 from Aciduliprofundum boonei T469; Family 3: HenMar-C1 from Henriciella marina DSM 19595; Family 4: MetMaz1FA1A3-C1 from Methanosarcina mazei
• A casposon, a member of a distinct superfamily of archaeal and bacterial self-synthesizing transposons
• Employ a recombinase (casposase) homologous to the Cas1 endonuclease
• Gave rise to the adaptation module of CRISPR-Cas systems and CRISPR repeats themselves
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Integrons and gene cassettes
Length: from 0.5 to hundreds of kilobases Main function: mobilise gene cassettes that are associated with a variety of functions (including antimicrobial-resistance genes and virulence factors)HGT mechanism: site-specific recombination
• Gene cassettes can be moved between integrons (through an intermediate form of circular DNA molecule) and assembled in large arrays
• Integrons themselves can in turn be mobilised via the action of composite transposons, conjugative elements, plasmids, or by transformation
• Integrons are genetic elements that can capture gene cassettes (mobile DNA containing a recombination site) through the activity of an enzyme called integrase
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Integrons
The basic integron platform consists of the following: • intI, a gene for the integron integrase• Pc, an integron-carried promoter• attI, the integron-associated recombination site• Gene cassettes, sequentially inserted into an array via recombination between attI
and the cassette associated recombination sites, attCGene cassettes normally contain a single open reading frame (ORF) (arrow) expressed from the Pc promoter
Gill
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Integrons, II
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• In 1994, Jörg Hacker and colleagues coined the phrase “pathogenicity island” (PAI) (Blum et al., Infect Immun. 62, 606–614, 1994) to describe a region of 70 kb of DNA in uropathogenic E. coli's chromosome that encoded virulence factors (including α-hemolysin), yet was not present in nonpathogenic E. coli
• Chromosomal rearrangements due to insertion sequences —> the evolution of several bacterial pathogens, increasing their pathogenic potential
• Horizontal transfer of transposable elements and prophages —> presence of chromosomal islands or genomic islands—> gene clusters for specialized non essential functions for survival —> virulence factors
Pathogenicity Island
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Dob
rint e
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4• Chromosomal islands are presumed to be of “foreign” origin: often flanked by
inverted repeats —> that the whole region was inserted into the chromosome by transposition at some point
• Chromosomal islands are found in some strains of a particular species but not in other
• G+C DNA content is usually quite different from the remainder of the genome, indicating horizontal gene transfer as a mechanism of acquisition (base composition and codon bias)
• Site of insertion is often associated with tRNA genes, which are sites of integration of foreign DNA and attachment sites for bacteriophages
• Finally, PAIs are often unstable DNA regions and heterogeneous
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Bacteriophages (phages) are viruses that replicate within bacteria and archaea
Length: a few to hundreds of kilo bases
Main function: replicate and destroy (lytic phages) or integrate DNA into host genome
(lysogenic phages)
HGT mechanism: transduction
Bacteriophages, IM
adigan et al. 2020
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• Lytic cycle: the enzymes responsible for DNA packaging into virions sometimes package host DNA accidentally, transducing particle cannot lead to viral lytic infection
• Upon lysis of the cell, transducing particles are released along with normal virions that contain the virus genome
• During following infections a small proportion of the population receives transducing particles that inject the DNA they packaged from the previous host bacterium
• DNA cannot replicate but it can recombine with the DNA of the new host, small # of defective particles low probability transduction for any given gene (low frequency)
Madigan et al. 2020
Bacteriophages, II
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Length: less than a kilobase to megabasesMain function: they are highly heterogeneous elements that do not contain enough genetic information for independent conjugative transfer but can utilise the transfer functions of conjugative plasmids or ICEsThey can exist as plasmids or as integrative elements; the latter are sometimes called integrative and mobilisable elements (IMEs)They commonly contain cargo DNA that encodes functions for survival in different environments, for example, antibiotic-resistance genes and virulence factorsHGT mechanism: conjugation
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Mobilisable genetic elements
X axis represents the effect of the trait on the fitness of the host bacteriaY axis represents the effect of the trait on the fitness of the host bacteria's neighborsTraits kept within the cell (‘private’ traits, governing the parasitism–mutualism axis) Traits secreted outside the cell (‘public’ traits, introducing Hamilton's social space, which govern the helping–harming axis; Hamilton, 1964; West et al., 2007)
General classification of social traits carried by MGEs and their effect on the bearer or a social neighbors
Rankin et al. 2011
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