The bacterial genome Flashcards
1
Q
How big are bacterial genomes?
A
Most prokaryontic genomes are 1 - 3 Mb large, however some are as large as 13 Mb (e.g. Sorangium cellulosum).
2
Q
What is the main canonical frontier between cell world and virosphere?
A
Translation
3
Q
What is special about the Tupanvirus?
A
It encodes largest translational apparatus within the known virosphere.
In this translation-associated gene set, only the ribosome is lacking.
4
Q
What is special about the genome of pandoraviruses?
A
- Most complex viruses (genomes reach 2.5 Mb)
- Large fraction of the pan-genome codes for proteins without homologs in cells or other viruses
–> De novo gene creation could contribute to pandoravirus genomes
5
Q
What is the traditional view of the bacterial genome?
A
- Bacterial genome is haploid
- the bacterial chromosome is a circular DNA molecule
- extrachromosomal DNA (pasmids) is circular and contain non-essential genes
- Bacterial chromosome is located in the “nucleoid”
–> Recent and ongoing genome projects challenge this view.
6
Q
What is the nucleoid?
A
= region within the cytosol of bacteria that contains most of the DNA; can be easily visualized by staining methods
7
Q
What is the definition of a bacterial chromosome?
A
de facto definition:
A chromosome is a DNA replicon that codes for house-keeping genes that are essential for the survival of the bacteria.
- Large DNA replicons are referred to as bacterial chromosomes
- Smaller ones are called extra-chromosomal elements, plasmids, or small chromosomes
- what if a small replicon is dispensable under lab conditions, but is crucial in the “real” world → should it be called plasmid or small chromosome?
- and how should one name replicons that are essential for life only under certain environmental conditions? Dispensable chromosomes?
–> all quite imprecise definitions
8
Q
How does DNA Replication in bacteria work in general?
A
- Starts an origin of replication (ori)
- Is bidirectional
9
Q
How many oris do the following organisms have:
E. coli, S. cerevisiae, H. sapiens
A
- E. coli: 1 ori
- S. cerevisiae: 300 oris (1 per 40 kb DNA)
- H. sapiens: 20’000 oris (1 per 150 kb DNA)
10
Q
What problems does the replication of linear (bacterial) chromosomes pose?
A
- DNA replication of the 3‘ ends
- DNA polymerase doesn’t start de novo but can only extend an already existing strand (RNA primer)
BUT: RNA primer gets subsequently degraded → gap in the DNA strand
Eukarya solve this problem via telomerase
11
Q
What proteins are collaborating at the replication fork?
A
- DNA helicase (brown mitten)
- Single-strand binding protein (4 black balls)
- RNA primase (green bell)
- DNA polymerase (orange donut)
12
Q
What strategies can solve the problems associated with the replication of linear (bacterial) chromosomes?
A
Eukarya: solve this problem via telomerase
Bacteria:
- Borellia: ends of DNA double strand are covalently connected via hairpin loops
- Streptomyces: Special proteins are covalently connected to 5’ end → it is assumed that these proteins prime the terminal replication → Also known as Invertron Telomer
13
Q
What is an Invertron Telomer and what does it do?
A
DNA polymerase interacts with the 5’-terminal protein (TP) and catalyzes the formation of a covalent bond between the TP and a dNTP. The dNTP bound to the TP has a free 3’-OH group which acts as the primer for chain elongation.
14
Q
What is the general anatomy of a bacterial genome?
A
- Condensation to a bacterial chromosome is necessary to fit larger genome into smaller cell
- Done with supercoiling: happens when additional helical turns are introduced (positive supercoiling) or removed (negative supercoiling) within a circular DNA double strand
- DNA adopts the B-form helix
- Wide and accessible major groove, narrower minor groove
15
Q
How does the structure model of the E. coli nucleoid look like?
A
- it is assumed that the E. coli genome is attached to a protein core structure from which about 12-80 supercoil loops emerge
- this genome organization is RNase-sensitive
- protein core consists of: DNA Gyrases & DNA Topoisomerases (needed for negative supercoiling & for relaxing positive supercoils; energy dependent process)
- most abundant proteins are the HU-proteins: 60-100 bp DNA are wrapped around one HU dimer (analogous to eukaryal histone proteins); ~60’000 HU molecules /cell → cover around 1/5 of the E. coli genome
16
Q
How was the supercoil-loop model validated?
A
- radioactive radiation was used to introduce nicks into DNA and the effects on supercoiling were determined
- Supercoiling is monitored by TMP (Trimethylpsoralen) binding (binds better to relaxed DNA)
- if E. coli chromosome is not organized in supercoiled-loop-domains, then one nick should relax the entire chromosome
→ one would expect an “all or nothing” effect - However, a gradual, linear increase in TMP binding (and thus in DNA relaxation) was observed
–> supports supercoil-loop model
17
Q
When can a bacteria be polyploid?
A
- In general the assumption that bacteria are haploid organisms is an oversimplification
- during exponential growth (especially in fast growing bacteria) > 4x sequence copies
close to the replication origins compared to the “ends” of the replicon (rRNA genes most often located close to the ori) - some species always carry multiple copies of their genome per cell (usually nearly identical copies)
- most of the times the advantage of polyploidy remains unclear, but most of the time it is a safeguard against mutations by gene conversion
18
Q
Give three examples of bactreia that are polyploid.
A
Deinococcus radiodurans:
- extreme resistant towards radiation and desiccation
- survives radioactive radiation of 17‘000 Gy –> 1’700x amount a human would die at
- has 5-8 copies of its chromosome/cell → oligoploid
- most likely needed to repair DNA strand breaks via homologous recombination
- Cells even survived low earht orbit (1 year outside ISS) and do not exhibit any morphological damage
- nano-sized particles over the surface of LEO-returned cells
- space-returned cells revealed pronounced outer membrane associated events with numerous vesicles
- metabolites, proteins and mRNAs were extracted from space-exposed cells
- proteome & transcriptome: multi-faceted response (e.g. UvrABC endonuclease excision repair upregulate; increased catalase & putrescine to cope with ROS)
Haloferax volcanii: halophilic archaeon
- Upon phosphate starvation, H. volcanii degrades its own gDNA to use the phosphate
- Ribosome concentration remains constant, thus rRNA is not used as P source
- Hypothesis: DNA might have evolved initially as storage polymer and only later gained its function as genetic material
- Can have over 30 genome copies/cell
Achromatium oxaliferum:
- Bacteria with multiple compartements, each containing own genomes
- Inside compartments, chromosomes might be independently replicated and use intracellular genen transfer to increase diversity
- Intermediate evolutionary state between uni- and multicellular life
- allow for the generation of “experimental” versions of functioning proteins or RNAs
19
Q
What is gene conversion?
A
Asymmetrical homologous recombination resulting in one allele “overwriting” another
20
Q
How does the E. coli genome look like?
A
- Genome is extremely compact
- possible advantage: faster replication time (e.g. during favorable environmental conditions).
- Only 11% of the E. coli genome consists of non-protein-coding DNA (H. sapiens > 90%)
- Both DNA strands are coding for genes
21
Q
How is the complexity of an organism most likely determined?
A
Speculation:
the non-coding part of genomes is responsible for the increased complexity of e.g. mammals compared to bacteria
22
Q
What is an operon?
A
- A characteristic hallmark of prokaryal genomes
- Quite frequently operons consist of genes that are involved in the same metabolic pathway
- e.g. Lactose operon or Tryptophan operon
- There are some bacteria that have operons that comprise of functinally unrelated genes
- e.g. Methanocossus jannaschii or Aquifex aeolicus
23
Q
How does the gene order in bacterial genomes look like?
A
- Gene order in bacteria is very dynamic and not conserved
- Only weak similarity in gene order between bacterial phyla
-
But: Related operons have identical gene order (genes that work together, stay together)
- Reason: Horizontal gene transfer promotes clustering of genes that work together, since this increases chance to be fixed in the recipient genome
- Gene orientation often conserved
- Reason: To avoid head-on-collisions of the replication and transcription machineries
24
Q
What is special about bacterial RNA transciption?
A
- Bacterial transcripts are typically uncapped
- Also some bacterial RNAs are capped:
- Bacterial coenzyme-cap added during initiation
- Bacterial cap provides RNA stability (other roles?)
25
What is the general genome structure of a bacterial genome?
* Chromosomes are more than a random collection of genes (more than „beads-on-a-string“)
* Genes are not randomly distributed between the leading and the lagging strand of the DNA
* in general, more genes are located at the leading strand → reason: Replication and transcription machineries pass over them in the same direction
* crucial genes are more frequently found close to the ori (e.g. rRNA genes)
* Have accessory elements
→ the bacterial genome consists of a core of genes (**endo-genome**) and an individual set of accessory elements (free or integrated into the main chromosome), which is also referred to as **exo-genome**
26
What are accesory elements?
* very frequently genomic differences found due to insertions/deletions in different isolates of the same species
* these differences usually due to DNA elements of a few kb up to 200 kb
* Integrated accessory elements:
* Transposons
* Retrons
* Prophages
* Plasmids
* Pathogenicity islands:
* Segment in genomes of pathogenic bacteria, that codes for virulence genes (e.g. toxins, pili, host cell adsorption, invasion factors)
* are mobile elements due to their association with insertion sequences (IS) and transposons
* Evolutionary “reason“ for accessory elements: most likely advantageous for genetic flexibility/plasticity
* Also called exo-genomes
27
What is a plasmid in general?
* often, but not always circular
* carry genes that are usually not found on the chromosome, and are not crucial for survival of the organism (under “normal” conditions)
* can also integrate into the chromosome
28
What different types of plasmids exist?
* **Resistance**: Antibiotic resistance
* **Fertility**: Conjugation and DNA transfer between bacteria
* **Killer**: Synthesis of toxins that kill other bacteria
* **Degradative**: Enzymes for metabolism of unusual molecules
* **Virulence**: Pathogenicity
29
What kind of chromosomes/plasmids do the following organisms have:
## Footnote
**E. coli, V. cholerae, Borellia burgdorferi**
**E. coli**:
* 4.6 Mb chromosome
* no large plasmids (max. several kb long), not essential
**V. cholerae**:
* two circular DNA molecules: 2.96 Mb and 1.07 Mb
* 71% of genes on larger molecule.
* Smaller DNA carries plasmid-like genes (essential and non-essential) e.g. integron (genes needed to integrate phages or other plasmids)
* → smaller DNA molecule could be ‘mega-plasmid’, that has been picked up during evolution
**Borellia burgdorferi**:
* several linear plasmids: harbor essential genes (Purine synthesis)
30
What was the initial assumption on plasmid segragation?
Replicated DNA associates with membrane and is carried along passively as the cell elongates
→ wrong: Chromosome migration much faster than cell elongation
31
Why is plasmid segragation so important?
Especially important for low copy plasmids (and single copy genomes)
→ active partitioning mechanisms needed
32
What genes are involved in the segragation of plasmids in bacteria and yeast?
parA, parB and parC
**parA**: ATPase that binds to bacterial "centromer" aka parC
**parB**: protein that binds directly to parC and recruits parA
33
How does the partioning system from E. coli plasmid R1 work?
34
How does the segregation of bacterial chromosomes work in general?
* Often same system as plasmids
* Bacterial chromosomes have an ordered configuration in the cell and are not like a “bowl of spaghetti”.
* One or several DNA loci are specifically positioned in the cell
* Dynamic, “mitotic-like” mechanism for bacterial chromosome segregation
35
How does the dynamic, “mitotic-like” mechanism for bacterial chromosome segregation work?
* parA establishes dynamic cytoskeleton-like structure:
* Fors double curved structures
* After cell division: parA extends from septum (new pole) → over time it shrinks towards new pole → until only 2 punctated foci on both poles
36
Why are plasmids not lost?
* Plasmids get lost with a frequency of only \< 10-6 to 10-7 per cell division (true even for single copy plasmids!)
* Why does a bacteria not eliminate a plasmid, when it is no longer needed (e.g. when there is no antibiotic in the media)?
* e.g. Plasmid-encoded suicide system (Toxin-Antitoxin System):
Cell can only survive, if they keep the plasmid. Plasmid carries an operon that encodes a stable toxin and an unstable antitoxin. In case of plasmid loss, the antitoxin gets degraded and the toxin kills the cell.
37
What is the Toxin-Antitoxin (TA) System?
* TA systems are genetic elements occurring in almost all prokaryotes (bacteria and archaea)
* Initially found on plasmids
* Recently many TA systems found on chromosomes
* In all eight TA classes, the toxin is a protein
* Toxin-Antitoxin-pairs do not necessarily function on the protein/protein level (e.g. antitoxin inhibits mRNA of toxin)
* Type II is the most abundant and best understood class
* Particularly abundant in pathogenic and free-living bacteria (e.g. E.coli and M. tuberculosis) but not in host-associated, parasitic bacteria
38
Name four examples of TA systems.
**parD operon on E. coli plasmid R1:**
* kid (killer determinant) is toxin, inhibits replication
* kis (killer suppression) is antitoxin
**ccd Operon on E. coli plasmid F:**
* ccdB is toxin, inhibits Topoisomerase II
* ccdA is antitoxin
**RelBE sytsem (chromosome-encoded):**
* RelE is toxin, inhibits translation by cleaving mRNAs on the ribosome
* RelB ist antitoxin
**hok/sok system on plasmid R1 (Type I):**
* hok (host killing) mRNA is stable and encodes a 52 AA long peptide, that destroys cell wall
* sok (supressor of killing) is a 64 nt long unstable antisense RNA, that prevents translation of hok
39
What are chromosome-encoded TA systems for?
* Not fully understood yet
* Role in stress response, persistence
* Environmental stress has to be sensed and signal needs to be transduced into the cell
* → change in gene expression
* e.g. low [amino acid] → high [(p)ppGpp] (is an alarmone)
→ activation of Lon protease → degrade r-proteins but also antitoxins
* Example: MazE – MazF system
40
What is the MazEF TA system and how does it work?
* chromosome-encoded TA system
* MazF is toxin and inhibits translation
* Antitoxin MazE
* form a hexameric complex --\> inactive
* MazF:
* Is an endonuclease
* Cleaves ACA sequences in mRNAs close to start codons and in 16S rRNA close to 3‘-end
* cut produces leaderless mRNAs and truncated ribosomes
* does not create large pools of leaderless transcripts or specialized ribosomes
* „Stress-ribosome“ (carrying truncated 16S rRNA) selectively translate leaderless mRNAs in vivo
* Is reversible: RtcB can reattach leader sequence
* Is programmed cell deatch in bacteria that is stress activated --\> nutrient starvation, antibiotic stress, heat shock etc.
41
What is mazEF-mediated programmed cell death (PCD) in bacteria?
* upon nutrient starvation (or antibiotic stress, heat shock, etc…) mazEF-mediated PCD activated
* PCD aids in endurance of the population during stress; surviving minority scavenges nutrients from dead cells → “nutritional-altruism”
* PCD facilitates a “multicellular-like” behavior of bacterial populations
42
How does translation initiation in bacteria work?
* The 3’-end of the 16S rRNA that is free to bind with the mRNA includes the sequence 5′–ACCUCC–3′ (**Anti-Shine-Dalgarno sequence**)
* The complementary sequence, 5′–GGAGGU–3′ (**Shine-Dalgarno sequence**), can be found in whole or in part in many bacterial mRNA.
* 1159 E. coli protein genes investigated:
* in average the SD-antiSD duplex is 6.3 nt long
* maximal SD-antiSD helix length is 12-13 nt
43
What was the former species definition and what was the problem with it?
**Former species definition**:
* Members of a species share identical or very similar structural properties
* Robert Koch (1880): used staining and biochemical tests to define species
* Problem:
* bacterial strains of one species exist that possess dramatically different properties.
* e.g. pathogenic and completely non-pathogenic E. coli
**20th century: novel species definition** that relies on a more evolutionary perspective
* Members of a species can mate and reproduce.
* _BUT_: this definition is difficult to apply to prokarya, who can exchange genetic material quite easily and frequently
44
What are the machanisms for gene flow between prokaryotes?
**Conjugation**:
* Two bacteria come into physical contact and one bacterium (donor) transfers DNA to the second bacterium (recipient)
* Transferred DNA can be:
* copy of some or possibly all of the donor cell's chromosome
* segment of chromosomal DNA integrated in a plasmid --\> episome transfer
**Transduction**:
* Involves transfer of a small segment of DNA from donor to recipient via a bacteriophage
**Transformation**:
* Recipient cell takes up from its environment a fragment of DNA released from a donor cell
45
What are so called O-islands?
* extra DNA in a pathogenic strain that is distributed in different positions within the genome
* Example: comparison of the E. coli lab strain K12 with the pathogenic strain O157:H7
* former has a genome size of 4.6 Mb and the latter 5.5 Mb
* the extra DNA in the pathogenic strain is distributed in ~200 positions within the genome
* O-islands contain 1387 genes, including toxin genes and other pathogenicity factors
* but also K12 has 234 gene segments, that are absent in the pathogenic strain and which encode 528 genes
46
What is lateral gene transfer?
* = horizontal gene transfer.
* Exchange of genetic material between different species
* 12.8% of the E.coli genome originates from lateral gene transfer
* Gene transfer not only between bacteria, but also between bacteria and archaea, or bacteria and eukarya
* Even between vertebrates: e.g. Teleost fishes and the parasite Lamprey (Neunauge)
47
What is molecular phylogeny and how did it affect the prokaryal species concept?
* within eukarya kin relations can be deduced from comparison of gene sequences.
* within prokarya this is more difficult due to the horizontal gene transfer.
* thus, prokaryal phylogenetic trees need to be re-evaluated that have been established in the pre-genomic era.
* in the 1970ies, Carl Woese compared 16S rRNA sequences to study the prokaryal taxonomy.
* rRNA turned out to be an ideal chronometer since it is central for the cell metabolism, is highly conserved, but also contains regions of variability
* a group of prokaryotes (formerly known as archaebacteria) did not fit to bacteria based on the rRNA sequences → Archaea
* 1990 C. Woese postulated, that three rather than two domains of life exist.
48
How does "the" bacterial genome look like?
49
What is Mycoplasma genitalium?
* Gram-neg. bacteria
* 1995 whole genome sequenced (shot-gun sequencing)
* 2nd ever sequenced bacterial genome
* Parasite in primate genital and respiratory tracts
* small genome: 582.970 bp
* 470 predicted coding regions
* no cell wall
50
What is Genome Transplantation?
* Put genome from one species into the cell of another (and eliminate host genome)
* Example:
* Genom donor: Mycoplasma mycoides (1.08 Mb)
* Genome aceptor: Mycoplasma capricolum (1.01 Mb)
51
What is the strategy for whole genome synthesis?
**Splitting up the genome**
* 101 cassettes (5-7 kb), individually synthesized, sequenced
* Watermarks introduced
* Aminoglycoside resistance gene introduced in cassette 89
52
What is the Gibson isothermal assembly?
**In vitro recombination**
* Five stage assembly:
* Stage A–C: in vitro recombination → Amplification in E.coli
* Stage D and E: TAR (transformation associated recombination) cloning in yeast
53
How was Synthetic genome design of the syn1.0 genome done?
* based on two laboratory strains of Mycoplasma mycoides
* To differentiate between the synthetic and the natural genome they inserted 4 watermark (WM) sequences
* They succeeded in chemically synthesizing & assembling a 1.08 Mb large genome of M. mycoides
* Transplanted into M. capricolum to create new M. mycoides cells
* Cells with synthetic genome have same morphology as M. mycoides wt control
* Proteome analysis looked the same for WT and syn1.0
54
How was syn3.0 synthesized from syn1.0?
* guided by transposon insertion mutagenesis (to reveal essential genes in the syn1.0 genome) they eliminated non-essential genes and intergenic regions to construct syn3.0
* synthesized the syn3.0 genome as 8 overlapping segments (to avoid design flaws)
* whole-genome synthesis workflow in \< 3 weeks (2 orders of magnitude faster than 2008)
* almost all genes involved in reading and expressing of the genome had to be retained from the syn1.0 genome
* Doubling time of syn3.0 cells is 3x slower (178 min) [but still much faster than M. genitalium (16 h)]
55
What are the advantages and drawbacks of synthetic/artificial life?
**Negative**:
* We must be aware of bioerror and bioterror
* --\> new lab standards needed ensuring no gene exchange with the wild
**Positive**:
* The jump from a 0.58 to 1.08 Mb synthetic genome is encouraging
* possibility to test billions of genome combinations
* --\> researchers could select important products such as pharmaceuticals, fuels, chiral chemicals and novel materials
56
What is the aim of synthetic biology (or synthetic genomics) and what are possible approaches?
* _Aim_: predictably bioengineer organisms that perform beneficial functions - from producing antibiotics to purifying contaminated water
* _Apporaches_:
* **Rational design:**
* Characterize many biological components to generate a library of modules that can be assembled within an organism to give predictable, reliable outcomes
* **Direct evolution**:
* Genetic mutations of unknown impact are introduced into target of interest, generating a library of mutants that is screened for desired characteristics
* Iterative rounds of the process produce mutants with optimized traits
* Different opinions on which method is more effective
57
How can we get from natural genomes to synthetic genomes? (Steps)
