The minimal genome

Question 1

What is the concept of the minimal genome?

Answer 1

A hypothetical minimal auto-replicative system

• Aims to strip down a present day bacterium to its minimum essential components pertaining to replication, transcription and translation machinery.
• Understand the basic components of the cell that makes it living.
• Provides a template genome that can be used to recreate life
• A less complex cell that can be reliably modeled and engineered to meet our requirements (aka Synthetic Biology, Synthetic Genomics).
• until recently the search for the minimal gene set was the domain of computer- aided comparative genomics
• the creation of an artificial cell with a minimal genome is far from being straightforward (even though of G. Venter’s “synthetic cell” approach)
• gene sets of completely sequenced organisms is:
  
  • ~ 500 to 10‘000 genes (Prokaryotes)
  • ~ 2’000 to 30’000 genes (Eukaryotes)
• the smallest genome of a free-living organism is the bacteria Pelagibacter ubique with 1’354 protein-coding genes & 35 ncRNA-coding genes (1.3 Mb)

→ minimal gene set that is needed for autonomous cellular life is surprisingly small
(parasitic & symbiotic bacteria have even smaller genomes!

Question 2

What is the classical view on genome evolution in prokaryotes and how is it more realistically?

Answer 2

Classical view on genome evolution:
point mutations accumulate slowly

Bacterial genomes are much more dynamic and can change rapidly:

• rapid loss of large DNA fragments (via homologous recombination)
• integration of horizontally acquired DNA (lateral gene transfer)
• genome rearrangements (transposons, insertion segments, inversions):
  
  • e.g. Inversion via homologous recombination
• Gene duplication:
  
  • e.g. due to “illegitimate crossing over” during replication
• etc

Question 3

Why is it not enough to sequence only a single strain of a species in prokaryotes?

Answer 3

• Bacterial strains belonging to the same species vary considerably in gene content (e.g. different E. coli strains).
• thus sequencing a single strain of a species only reveals an incomplete picture of the species gene content
• The genetic repertoire of a given species (its “pan-genome”) is much larger than the gene content of individual strains
• Example: E. coli
  
  • Pan-genome: 10’131 - 20’000 genes
  • Average E. coli genome: 4’721 genes
  • Core genome: 2’167 genes

Question 4

What is a pan-genome?

Answer 4

The full complement of genes found in all strains of a species

• the pan-genome increases the more sequenced genomes are considered
• codes usually for functions of the cell surface, signal transduction, or pathogenicity
  → thus for functions crucial to conquer rapidly changing environments or niches

Question 5

What is the core-genome?

Answer 5

The gene set found in all genomes of a species

• the more divisions/species/strains are compared, the smaller the “core genome” gets
• these genes are more „stable“ and code for translation and amino acid synthesis

Question 6

What are genomic islands?

Answer 6

• relatively large DNA regions (10 – >100 kb) can integrate into genomes (e.g.: pathogenicity islands)
• by that several genes get integrated at once (via HGT)
• often associated with integrase genes & they have an unusual nucleotide composition (such as GC content or codon usage → hallmarks for foreign origin)

→ primary strategy for gene acquisition in prokaryotes

Question 7

What are Borgs?

Answer 7

• new type of archaeal extrachromosomal element discovered in a metagenomics study
• extraordinarily large (up to 1 Mbp) DNA
• linear extrachromosomal DNA that carry repetitive sequences at the ends and throughout
• their genes were assimilated from methane-oxidizing Methanoperedens archaea

Question 8

What is a bacterial IS elemnt?

Answer 8

Insertion Sequence elements

• Central region encodes 1 or 2 enzymes required for transposition (transposase)
• It is flanked by inverted repeats of characteristic sequence
• The 5’ and 3’ short direct repeats are generated at the target-site DNA during the insertion
• Transposition is catalyzed by one or both of the flanking IS-encoded transposases
• Example: Sulfolobus sp.
  
  • have enormous single-gene- translocation rate
  • → Sulfolobus known for its abundant transposons (Insertion Sequence elements)
  • ~12% of the 3 Mb genome are IS-elements
• a correlation exists between the number of repetitive sequences (e.g. IS-elements) and the rate of single gene translocation
• thus transposons and other repetitive elements are hot-spots for genomic rearrangements
  → play a key role in genome evolution

Question 9

What is Pelagibacter ubique?

Answer 9

One of the most abundant microbes in the ocean

• 0.37 – 0.89 μm long & 0.12 – 0.20 μm in diameter
• 30% of the cell’s volume is taken up by its genome
• the smallest genome (1.3 Mb) of any free living organism
• no duplicated gene copies, no viral genes, and little ncDNA
• genome has been streamlined
• low GC content of only 30%

Question 10

What is Mycoplasma genitalium?

Answer 10

• 0.58 Mb genome
• 470 protein-coding genes
• intracellular parasite in humans
• smallest known genome of an organism capable of independent growth
• lacks a cell wall
• Mycoplasma sp. originate from a Gram positive ancestor via reductive evolution
• evolved by massive genome reduction
• obligate parasites
• lack genomic redundancy

→ Appears to be ideally suited to look for essential genes

Question 11

What is Nanoarchaeum equitans?

Answer 11

• 0.49 Mb genome; 522 protein-coding genes
• exists only in co-culture with Ignicoccus sp. (parasite or symbiont?)
• only 400 nm diameter (smaller than some viruses)
• so far the only representative of the phylum Nanoarchaeota
• lacks genes for the synthesis of amino acids, nucleotides and lipids
• carries ‘split genes‘: → ancestral gene structure of multi domain proteins/RNAs?
• living fossil

Question 12

How can comparative genomics help with finding the minimal genome?

Answer 12

• Exaple:
  
  • comparative genomics: E. coli uses 243 genes for energy metabolism, while H. influenza only 112, and M. genitalium only 31
  • the parasitic M. genitalium can even eliminate essentially all genes of amino acid synthesis pathways
• genes for translation, however, cannot be reduced
• also protein folding genes are resistant to elimination
• comparative genomics: under optimal growth conditions (all essential nutrients present, no stress, no competition) the “minimal genome” consists of ~ 256 genes

→ In fact it is more appropriate to speak of a minimal set of essential functional niches rather than of minimal sets of genes.

• comparison of ~100 genomes revealed that only ~63 genes (!) were not replaced by NOGD and were thus present in all of the investigated genomes
• > 50 thereof are components of translation machinery

Question 13

What is Non-orthologous gene displacement?

Answer 13

Displacement of a gene responsible for a particular biological function in a certain set of species by a non-orthologous (unrelated) gene in a different set of species.

Question 14

What experimental evidence is there regarding the minimal genome?

Answer 14

• Genome-wide analyses of gene knock-outs via transposon insertion, plasmid insertion or antisense RNA
• in these studies > 50% of the genes of the respective organism have been studied
• the experimental approaches revealed a surprisingly similar number for the minimal gene set as comparative genomics did, namely ~300-350 genes
• reason why H. infuenzae and E.coli require more genes unclear; maybe Gram neg. need more genes to build their cell wall, and for transport through it
• Used species: M. genitalium/M. pneumoniae, B. subtilis, H. influenzae, E. coli, S. cerevisiae, C. elegans
• not only the number, also the function of these „minimal gene set“ genes were similar between the experimental (right) and computational (left) studies
• minimal gene set: many genes for information-processing (especially translation), only very few genes with unknown functions

Question 15

What are problems when defining the minimal gene set?

Answer 15

Essential Genes – A conclusive list??

• Different studies came up with a different number of essential genes.
• Computation: Underestimates minimal genes - accounts mainly those genes that have been conserved in evolution.
• Transposon mutagenesis: Overestimates the genes – Classifies genes that slow down growth as essential and essential genes that tolerate mutation as non-essential.
• Most mutants produced are single mutants – synthetic lethality largely not considered

Construction of a single cell with systematic combination of all the mutations in a single strain is beyond the scope of present day technology

What is an essential gene?

Question 16

What are problems with defining an essential gene?

Answer 16

• In these studies random or directed single gene inactivation was performed
• But: simultaneous elimination of two individually non-essential genes may be lethal (aka as ‘synthetic lethality’)
• Conversely, some genes may be individually essential but, in combination with a second disruption, the first becomes tolerated (e.g. toxin–antitoxin genes)
• environmental context of experiment affects the outcome → many ‘minimal cells’ with different ‘minimal genome’ versions.

Question 17

How are the minimal genome and the last universal common ancestor of life (LUCA) related?

Answer 17

• comparative genomics can also contribute to questions of the evolution and LUCA
• LUCA likely was not a single species but a community of organisms that rapidly exchanged genetic material –> the more primitive an organism is, the more unstable is its genome
• minimal gene set, or better ‘minimal set of essential functional niches’ can be used to define the gene-set of LUCA
  (problem: rates of HGT and gene loss unknown)
• it was examined if and how the functional niches in various phylogenetic tree variants are filled
  → LUCA had a simple genome (~600 genes), thus smaller than any now free living bacteria
• interesting detail: LUCA had no DNA polymerase and no DNA helicase → evidence that life did not start with DNA genomes but probably with RNA genomes → RNA world theory
• no ‘Tree of Life‘ but likely more a ‘Shrub of Life‘ or ‘Ring of Life‘

Question 18

What is Buchnera aphidicola?

Answer 18

• a primary endosymbiont of the aphid Cinara cedri
• B. aphidicola has a circular genome of only 416’380 bp (plus a plasmid of 6’045 bp)
• thus genome is smaller than some plant mitochondrial genomes (< 600 kb)
• bacteria live inside special host cells (Bacteriocytes)
• dramatic genome reduction → ~ 75% of its genome was lost during evolution as endosymbiont
• has only 362 genes (35% for translation & transcription)
• lost many biosynthesis genes (nt, cofactors, vitamins) → depends on host
• aphid needs 10 essential amino acids from symbiont
• but: B. aphidicola has lost Trp biosynthesis genes!?
  
  • Trp is synthesized by a secondary symbiont Serratia symbiotica
• B. aphidicola’s massive genome degradation might lead to its replacement by the secondary symbiont in the near future

Question 19

What is Carsonella rudii?

Answer 19

• endosymbiont in aphids
• belongs to γ-proteobacteria
• also lives in bacteriocytes (like organelles C. rudii cells get vertically & maternally transmitted across host generations)
• has a circular genome of only 159’662 bp
• has only 182 ORFs (35% Translation; 18% aa metabolisms)
• thus its genome is only about one third that of the archaeal parasite Nanoarchaeum equitans
• C. rudii has an unusually low GC content of 16.5 %
• host needs amino acids from symbiont
• C. rudii has a very densely packed genome (97.3 % of genome are coding; 90% of the genes are overlapping)
• The genome also lacks many genes for bacterium-specific processes.

→ have they been transferred to the host genome?
→ is C. rudii on its way to become a true organelle?

Question 20

What is Nasuia deltocephalinicola?

Answer 20

• β-proteobacteria
• endosymbiont of Macrosteles quadrilineatus (a leafhopper)
• smallest bacterial genome yet sequenced 112 kb and 137 protein genes
• UGA has been reassigned to Trp codon (occurs very rarely in evolution)
• massive form of genome erosion

Question 21

What is Genome erosion in symbiotic bacteria & relatives?

Answer 21

• tiniest genomes evolve exclusively in maternally inherited symbionts that are obligate mutualists and that have co-diversified with hosts over millions of years
• discovery of these extreme genomes challenges the premise of the minimal genome concept, that there is a lower limit of the genome size of a cellular organism
• tiny genomes show ongoing gene loss, no gene uptake, and an almost complete absence of gene rearrangements

Question 22

What genes are lost?

Answer 22

• Genes are lost in all functional categories
• core genes for central informational processes (replication, transcription, translation) are mostly retained (e.g. ribosomal proteins)
• cell envelope component genes are especially depleted
• DNA repair genes are one of the most depleted functional categories
• Despite this trend, losses of seemingly essential genes have occurred, raising the question of how replication and growth are possible (e.g. oxphos is sometimes missing)

Remarkably: some host genes critical to bacteriocyte function were themselves horizontally transferred into the host genome from bacterial donors!!

Question 23

At which stage should we call an endosymbiont an organelle?

Answer 23

• Protein transport from nuclear-encoded protein into organelles is well known → hallmark for true endosymbiosis
• but no evolutionary intermediate stages of endosymbiosis known (until this report):
  
  • RlpA is a lipoprotein that was acquired by the aphid via HGT (from an unrelated bacteria)
  • expressed solely in the aphid’s maternal Bacteriocytes (mb)
  • RlpA imported into the cytoplasm of Buchnera endsymbiont!!
• Genetically integrated photosynthetic organelles evolved twice via the endosymbiotic uptake of a cyano-bacterium
• After stable establishment in cytoplasm of host: genome reduction (gene loss & gene transfer to the nucleus via EGT)
• import of nuclear-encoded proteins of various phylogenetic origins (EGT & HGT) compensated for essential genes that were lost from the endosymbiont

Is protein import of nuclear-encoded proteins then the decisive event?