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Genomics of Microorganisms > The bacterial genome > Flashcards

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).

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2
Q

What is the main canonical frontier between cell world and virosphere?

A

Translation

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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.

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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

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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.

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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

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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

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8
Q

How does DNA Replication in bacteria work in general?

A
  • Starts an origin of replication (ori)
  • Is bidirectional
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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)
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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

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11
Q

What proteins are collaborating at the replication fork?

A
  1. DNA helicase (brown mitten)
  2. Single-strand binding protein (4 black balls)
  3. RNA primase (green bell)
  4. DNA polymerase (orange donut)
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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
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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.

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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
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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
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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

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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
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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
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19
Q

What is gene conversion?

A

Asymmetrical homologous recombination resulting in one allele “overwriting” another

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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
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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

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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
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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
Q

What is the general genome structure of a bacterial genome?

A
  • 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
Q

What are accesory elements?

A
  • 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
Q

What is a plasmid in general?

A
  • 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
Q

What different types of plasmids exist?

A
  • 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
Q

What kind of chromosomes/plasmids do the following organisms have:

E. coli, V. cholerae, Borellia burgdorferi

A

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
Q

What was the initial assumption on plasmid segragation?

A

Replicated DNA associates with membrane and is carried along passively as the cell elongates

→ wrong: Chromosome migration much faster than cell elongation

31
Q

Why is plasmid segragation so important?

A

Especially important for low copy plasmids (and single copy genomes)

→ active partitioning mechanisms needed

32
Q

What genes are involved in the segragation of plasmids in bacteria and yeast?

A

parA, parB and parC

parA: ATPase that binds to bacterial “centromer” aka parC

parB: protein that binds directly to parC and recruits parA

33
Q

How does the partioning system from E. coli plasmid R1 work?

A
34
Q

How does the segregation of bacterial chromosomes work in general?

A
  • 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
Q

How does the dynamic, “mitotic-like” mechanism for bacterial chromosome segregation work?

A
  • 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
Q

Why are plasmids not lost?

A
  • 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
Q

What is the Toxin-Antitoxin (TA) System?

A
  • 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
Q

Name four examples of TA systems.

A

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
Q

What are chromosome-encoded TA systems for?

A
  • 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
Q

What is the MazEF TA system and how does it work?

A
  • 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
Q

What is mazEF-mediated programmed cell death (PCD) in bacteria?

A
  • 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
Q

How does translation initiation in bacteria work?

A
  • 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
Q

What was the former species definition and what was the problem with it?

A

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
Q

What are the machanisms for gene flow between prokaryotes?

A

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
Q

What are so called O-islands?

A
  • 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
Q

What is lateral gene transfer?

A
  • = 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
Q

What is molecular phylogeny and how did it affect the prokaryal species concept?

A
  • 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
Q

How does “the” bacterial genome look like?

A
49
Q

What is Mycoplasma genitalium?

A
  • 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
Q

What is Genome Transplantation?

A
  • 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
Q

What is the strategy for whole genome synthesis?

A

Splitting up the genome

  • 101 cassettes (5-7 kb), individually synthesized, sequenced
  • Watermarks introduced
  • Aminoglycoside resistance gene introduced in cassette 89
52
Q

What is the Gibson isothermal assembly?

A

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
Q

How was Synthetic genome design of the syn1.0 genome done?

A
  • 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
Q

How was syn3.0 synthesized from syn1.0?

A
  • 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
Q

What are the advantages and drawbacks of synthetic/artificial life?

A

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
Q

What is the aim of synthetic biology (or synthetic genomics) and what are possible approaches?

A
  • 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
Q

How can we get from natural genomes to synthetic genomes? (Steps)

A

Genomics of Microorganisms (6 decks)

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