Minmal Genome Project

What is the minimum bumber of protein-coding genes required for life ( at least for bacteria growinf under labratroy conditions)

• looked fo all ortholog genes conserved among th 3 complete bacterial genomes available at the time

  • H. influenzae

  • M. genitalium

  • E. coli

• 256 genes in minimal set

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

• some functions can be performed by non-orthologous genes

  • also known as NOD (non orthologous gene displacment)

• results will change as new genome sequences becoma available

• no ecidence that minimal set alone is sufficient for life

• 63 genes are common to all of life

• 91 genes are common to Archaea and Bacteria

• 217 genes are common ro Bacteria

Das COG-Prinzip (Der Dreiecks-Test): Ein Gen gehört erst dann zu einem COG, wenn es ein konsistentes Dreieck bildet:

1. Gen A aus Organismus 1 findet Gen B aus Organismus 2 als besten Treffer.

2. Gen B findet Gen C aus Organismus 3 als besten Treffer.

3. Gen C findet wiederum Gen A aus Organismus 1 als besten Treffer.

Start with closely-related species

• Mycoplasma genitalium

  • smallest genome known (580 kb, 480 protein encoding genes)

• M. pneumoniae (816 Kb, 480 M. genitalium orthologs + 197 unique genes = 677 total genes)

Used TE insetioaln mutagenesis to randomly knock out genes in both species -> sequenced DNA, flanking the TE to determine where it inserted & which gene was knocked out

if TE was inserted within first 80% of protein coding region & beyond nucelotide 9 of the coding sequence it was considered a gene disruption (hit)

only cells that survived could be detected -> hit genes must be non-essential

M.genitalium : 93

M.pneumoniae: 150 (57 had M.genitalium orthologs, 93 unique)

no

480 - 93 =387 would be an overestimation

the screen was not saturated, some genes were not hit ( just by chance)

M. gentialium : 480 genes -> hits 93

M. pneumoniae: 480 + 197 = 677 -> hits 150 (57 +93)

The 197 genes are of course non essentail for M.genitalium but for the hits only 93 were found so we know only 47 % were hit.

In the next step we calculate N with 0.47N =4,793 kp 57 (ortholog hits) and get 121 for N, what is the numver of non-essentail genes

-> 480 - 121 =359 359 essentail genes

the authors estimate this even lower to 265 - 350 genes

• for 111 the function is unknown

• not all 197 M. pneumonia-Specific genes may be non essentail

• genes were knocked out individually. What about synthetic lethals? ( two genes can be knocked out individually with no effect but the cell dies if both genes are knocked out together)

Bcillus subtilis - 4100 genes

• using bacillius subtilis

• knocked out genes using targeted, homologous recombiantion approach

• combined result with the previous studies

result: 217 (6%total) genes were essential for growth under optimal labroratory conditions, reminaing 3830 (94%) not

essential genes were classified into functional categories

• 50 % involved in DNA/RNA metabolism

essential genes were well conserved in other bacterial species (& also Archaea & eucaryotes) -> but not all over all known genomes or even all known bacterial genomes

-> these genes would not show up for bioinformatics approaches

30 % conserved across 54 bacterial

20% conserved across 18 Archaea & Eukaryotic

genes were knocked out individually

snythethic lethals

can organsim with only 271 genes be engineered?

no

-> negative correlation between percentage of essentail genes & the total number of genes in genome

• few genes -> high proportion of essential

• less genes -> los proportion

• 
  

yes

Approach has been used to make synthetic strain of Mycoplasma that has only 472 genes