2_Minimal Genome Project

Question: What is the minimum number of protein-coding genes required for life? (at least for bacteria growing under laboratory conditions)

What genes are conserved in all sequenced bacterial genomes?

-> This may be the minimal core set.

Initially: used by Mushegian and Koonin in 1996

• looked for all genes conserved among the 3 complete bacterial genomes available at the time (H. influenzae, M. genitalium, E. coli).

The result: 256 genes in the minimal set

A more recent check of the COG (Clusters of Orthologous Genes) database reveals:

• 63 genes are common to all of life (Archaea, Bacteria, Eukaryotes)

• 91 genes are common to Archaea and Bacteria

• 217 genes are common to Bacteria

• with distantly-related species it is hard to identify orthologs

• some functions can be performed by non-orthologous genes (functional analogs)

• known as NOD (Non-Orthologous gene Displacement)

• results will change as new genome sequences become available

• no evidence that minimal set alone is sufficient for life

• 1999

• Craig Venter + colleagues at TIGR

• They began with two closely-related species of bacteria:

  • Mycoplasma genitalium

    • the smallest genome known

    • 580 Kb, 480 protein-encoding genes

  • M. pneumoniae

    • 816 Kb

    • 480 M. genitalium orthologs + 197 unique genes = 677 total genes

• Used transposable element (TE) insertioaln mutagenesis to randomly knock out genes in both species

• then sequenced DNA flanking the TE to determine where it inserted and which gene was knocked out

• If the TE was inserted within first 80% of a protein-coding region and beyond nucleotide 9 of the coding sequence, it was considered a gene disruption (or a “hit”)

• Only surviving cells could be detected, so “hit” genes must be non-essential

Result/hits:

• 93 genes in M. genitalium

• 150 genes in M. pneumoniae

  (57 had M. genitalium orthologs, 93 were unique)

• For M. genitalium, the directly observed minimal gene number is 480 - 93 = 387. However, this will be an overestimate, because this was not a saturation screen: some genes were not hit just by chance.

• Assume the 197 M. pneumoniae-specific genes are non-essential

• Mutations were recovered in 93 (47%) of these genes

• Assume that only 47% of the non-essential M. genitalium orthologs were hit, then 0.47N = 57 and N = 121, where N is the number of non-essential genes.

• This gives 480 - 121 = 359 essential genes.

• The authors think the number is probably lower and give a final estimate with a range of 265–350 essential genes.

• 111 genes of unknown function were not disrupted, many of these may be required for life.

• Possible problems with the minimal genome estimate

  • Not all 197 M. pneumoniae-specific genes may be non-essential (“new” genes can become essential)

  • Genes were knocked-out individually -> What about synthetic lethals

    
    

• synthetic lethals = two genes can be knocked out individually with no effect, but the cell dies if both genes are knocked out together

• Can a synthetic organism be engineered by combining these 359 (or fewer) genes?

Kobayashi et al. in 2003

• used common laboratory bacterium, Bacillus subtilis (4,100 genes)

• individually knocked-out genes using a targeted, homologous recombination approach

• These results were combined with those of previous studies.

• 271 genes (6% of total) were essential for growth under optimal laboratory conditions

• The remaining 3,830 (94%) were non-essential!

Essential genes were classified into functional categories

• about 50% were involved in DNA/RNA metabolism or protein synthesis

• Over half of the essential protein synthesis genes encode ribosomal proteins

• essential genes of B. subtilis are well-conserved in other bacterial species, and many are also found in Archaea and/or Eukaryotes

• However, not all were completely conserved over all known genomes or even over all known bacterial genomes

• Thus, these essential genes would not show up as part of the minimal genome by the bioinformatic approach

• Of the essential genes, ≈30% were conserved across 54 bacterial species and ≈20% were conserved across 18 Archaea and Eukaryotic species.

• genes were knocked-out individually.

• What about synthetic lethals?

• Can an organism with only 271 genes be engineered?

• the number of essential genes is not strongly correlated with the total number of genes in the genome

• there is a negative correlation between the percentage of essential genes and the total number of genes in the genome.

• In species with few genes, such as M. genitalium, a high proportion of the genes are essential.

• In species with many genes, such as B. subtilis, a low proportion of the genes are essential.

  
  

• More recent work has shown that it is possible to transplant entire bacterial genomes and engineer synthetic life (Gibson et al. 2008, 2010).

• This approach has been used to make a synthetic strain of Mycoplasma that has only 473 genes (Hutchison et al. 2016).

best results:

473 genes