Bacterium with a Minimal Genome

[Ygggdrasil]

How many genes does it take to make a living organism? Scientists at the J Craig Venter Institute published a paper today in the journal Science describing the design and synthesis of a bacteria containing the minimal set of genes required for that organism to live. Their work leverages their earlier work figuring out how to chemically synthesize genomes, allowing them to systematically remove genes and other sequences from the bacterial genome until they reach the point where they could not remove any more genes without dramatically affecting the growth and replication of the bacterium. In the end, identified a set of 473 genes that they term the minimal genome.

Although many of these genes are well studied and what one might expect to be required, about 30% of the genes have unknown function. While there are organisms with smaller genomes, most of these are parasitic and rely on their hosts for many biological functions. The synthetic bacteria described in the paper has the smallest genome of any known autonomously replicating organism. The work could provide a nice model system for efforts to computationally model life, and provides a nice platform for designing synthetic genomes with new functions.

Publication: Hutchison et al. (2016) Design and synthesis of a minimal bacterial genome. Science 351: aad6253. doi:10.1126/science.aad6253

Popular press summary: http://www.theatlantic.com/science/...s-shows-how-little-we-know-about-life/475053/

 

[berkeman]

about 30% of the genes have unknown function.

Very interesting. How did they figure out to include that 30% with the unknown function? Did they use some sort of a Monte Carlo variation on many tries?

 

[Ygggdrasil]

Very interesting. How did they figure out to include that 30% with the unknown function? Did they use some sort of a Monte Carlo variation on many tries?

They figured things out mostly by trial-and-error in a semi systematic way (though not via a Monte Carlo method). The key tool is a method that randomly inserts a small DNA sequence into the genome, which, if it inserts into a gene, is likely to disrupt the function of the gene. They can use this method to empirically identify genes, in an unbiased manner, that are nonessential for growth. Of course, one of the main difficulties in interpreting these results is that some genes encode essential functions but are redundant with one or more other genes in the genome. For example, gene A and gene B might redundantly encode the same essential function such that inactivation of gene A leads to a viable bacteria and inactivation of gene B leads to a viable bacteria (leading one to think that the genes are nonessential) but inactivating both gene A and B leads to a non-viable bacteria.

Essentially what the authors did was iterative rounds of identifying nonessential genes to delete, testing different combinations of deletions to find viable combinations, then repeating the experiments to identify more nonessential genes in the smaller genome. This process pared the bacteria down from 901 genes in the starting strain, to 512 genes in an intermediate strain, to 473 genes in their final version.

Initially, they tried to rationally design a minimal genome based the scientific literature (this was a theoretical question that others have investigated), but these efforts did not produce any viable bacteria, necessitating the empirical approach the authors employed.

 

[berkeman]

Beautiful. Thanks

 

[Andy Resnick]

Amazing breakthrough! Does anyone know if the designed genome permits alternative translation or post-translational modification?

 

[mfb]

Well, "designed". They took an existing genome and removed as much as possible. A full bottom-up approach is still some major breakthroughs away. The bacterium needs more than 100 genes with unknown purpose.

I guess that minimal genome will help to understand those genes - you can study what exactly goes wrong if you remove one of those genes, without too much interference.

 

[.Scott]

I expect that the procedure will be repeated with other microbes. Then a comparison can be made among those minimal genomes.

 

[Buzz Bloom]

I have been trying to find out what is in the artificial environment for these minimum gene bacteria. I understand all the amino acids are present, but what else? Can anyone here at the PF cite a reference with this information?

 

[Buzz Bloom]

They took an existing genome and removed as much as possible.

I heard an interview on PBS (Science Friday) with J. Craig Venter, and if I understood him correctly, he said they did some other editing of the genome as well as just removing genes.

 

[Andy Resnick]

The bacterium needs more than 100 genes with unknown purpose.

An erroneous statement. 79 genes were not assigned a specific function, and of those, 24 are classified as 'generic', leaving 65 that have unknown functions. Of those, only 19 are essential- meaning they cannot be removed. 13 of the 19 are of completely unknown function, and as the authors state, these "must represent nearly universal functions and thus can provide biological insights.", possibly representing "fundamentally new processes".

 

[Andy Resnick]

I have been trying to find out what is in the artificial environment for these minimum gene bacteria. I understand all the amino acids are present, but what else? Can anyone here at the PF cite a reference with this information?

It's located here:
http://science.sciencemag.org/content/351/6280/aad6253/suppl/DC1

They cultured the mycoplasma in SP4 media:

https://catalog.hardydiagnostics.com/cp_prod/Content/hugo/SP4Media.htm

SP4 media is not 'defined' media- supplements like fetal bovine serum and yeast extract are 'magic juice' that have essential and unknown growth factor(s).

 

[Buzz Bloom]

SP4 media is not 'defined' media- supplements like fetal bovine serum and yeast extract are 'magic juice' that have essential and unknown growth factor(s).

Hi Andy:

Thank you very much for your prompt and useful citations.

Regards,
Buzz

 

[Ygggdrasil]

Amazing breakthrough! Does anyone know if the designed genome permits alternative translation or post-translational modification?

What do you mean by alternative translation? Are you talking about alternative translation initiation (e.g. in the case of uORFs)? Alternative translation initiation is definitely an important regulatory process in eukaryotes, but I'm not sure whether it occurs in bacteria. Bacteria certainly have many post-translational modifications, but I'm not sure the extent to which these are lost in the minimum genome.

I heard an interview on PBS (Science Friday) with J. Craig Venter, and if I understood him correctly, he said they did some other editing of the genome as well as just removing genes.

Yes. For part of the genome, they performed an additional experiment where they reordered the genes to group them by function. Venter describes this as analogous to the process of defragmenting one's hard drive. After re-arranging the order of these genes, the bacteria grow just as well as the bacterium before re-ordering, showing that gene order and the position of genes in the bacterial genome is not really important for their function.

They performed some other experiments to demonstrate that they could swap in homologous genes from other organisms and change other elements of the genome (e.g. changing all non-AUG start codons to AUGs).

 

[mfb]

An erroneous statement. 79 genes were not assigned a specific function, and of those, 24 are classified as 'generic', leaving 65 that have unknown functions. Of those, only 19 are essential- meaning they cannot be removed. 13 of the 19 are of completely unknown function, and as the authors state, these "must represent nearly universal functions and thus can provide biological insights.", possibly representing "fundamentally new processes".

I got the number from the first post.
Anyway, the number is not relevant - there are essential genes with unknown function.

 

[snorkack]

An erroneous statement. 79 genes were not assigned a specific function, and of those, 24 are classified as 'generic', leaving 65 that have unknown functions.

24+65=89, not 79.

Of those, only 19 are essential- meaning they cannot be removed.

Meaning that 46 more could have been removed.

13 of the 19 are of completely unknown function,

Meaning 6 of the 19 do have a known function.

and as the authors state, these "must represent nearly universal functions and thus can provide biological insights.", possibly representing "fundamentally new processes".

So what were the symptoms of illness when essential genes were removed or damaged?

 

[fredreload]

Not being able to identify 30% of the genes sounds like a problem. Can't they check the protein synthesized by these unknown genes as to figure out what they do? But it seems like they're making progress. If they can eventually identify and self build a viable virus it would be huge progress, but again you can't just build something that took so many years to evolve, sort of like getting a balance between two points, I'd say it'll be easier to run it in a computer simulation program.

P.S. Like the game of life program

 

[fredreload]

Nevermind, you can't predict the phase in which the compound would be in if any mutation is to occur

 

[Ygggdrasil]

Meaning that 46 more could have been removed.

Yes, it's not entirely clear from the publication whether other non-essential genes could have been removed. Removal of many of these genes (which they term quasi-essential) still permits the bacteria to replicate, but at a much slower rate, making it harder to work with the bacteria. From a practical standpoint, the syn3.0 bacteria with a 3 hr doubling time is a much better starting point for future applications stemming from the work than a bacteria with a smaller genome but an 16-hour doubling time (the doubling time of M. genitalium, the naturally occurring bacterium with the smallest genome).

Meaning 6 of the 19 do have a known function.

Some of the unknown genes have similarity to other enzymes (e.g. kinases or hydrolases) but because their substrate its not known, it's unclear what the biological functions of these proteins are.

If they can eventually identify and self build a viable virus it would be huge progress, but again you can't just build something that took so many years to evolve, sort of like getting a balance between two points, I'd say it'll be easier to run it in a computer simulation program.

This paper is a great step toward being able to build a computer simulation of a cell. Unfortunately, as this work points out, there's still more work to be done as it would be difficult to simulate a system when you don't know the function of 30% of the parts.

 

[fredreload]

To do a computer simulation of the bacteria is to eventually check for the mutations and how it adapts to the environment. Thing is there's more than 500 types of known amino acids and if a mutation does occur it would be hard to predict its functionality. And you need to simulate everything to a molecular scale along with its molecular structure depends on how detailed it needs to be, there's also different type of bonds so you need to account for the atomic structure. Depends on how detailed it needs to be it might not be an easy task. It's probably easier to examine a known organism for now

 

[Andy Resnick]

What do you mean by alternative translation? Are you talking about alternative translation initiation (e.g. in the case of uORFs)? Alternative translation initiation is definitely an important regulatory process in eukaryotes, but I'm not sure whether it occurs in bacteria. Bacteria certainly have many post-translational modifications, but I'm not sure the extent to which these are lost in the minimum genome.

Yeah, I was thinking about spliceosome activity (I know extremely little about eukaryotic gene expression, and even less about bacteria).

 

[mfb]

A full computer simulation is even harder than building a genome from scratch: it is not sufficient to understand the function of every piece of the genome, you also have to understand every process connected to it in the cell in detail.

 

[Ygggdrasil]

Yeah, I was thinking about spliceosome activity (I know extremely little about eukaryotic gene expression, and even less about bacteria).

Generation of alternate polypeptide sequences from the same gene via splicing is called alternative splicing (not alternative translation). The spliceosome occurs only in eukaryotes, so these bacteria would not have any splicing. Alternate translation initiation and uORFs are separate regulatory mechanisms from splicing (though they can regulate splicing).

 

[Dr_Zinj]

Interesting report. I wonder if any of those unknown function genes code for proteins, have an enzymatic function, or fold the chromosome into a more productive shape?

 

[Andy Resnick]

24+65=89, not 79.

There is overlap in categories.This is spelled out in detail on page 6, using examples of hydrolases and ABC transporters.

Meaning that 46 more could have been removed.

This is laid out on page 10: some of those 46 genes are needed for growth (i-genes), and deletions of these span a continuum of effects (spelled out on page 3)

Meaning 6 of the 19 do have a known function.

It's not clear (to me)- the way they classify genes, those 6 could be homologous to a transporter, but with unknown substrate.

So what were the symptoms of illness when essential genes were removed or damaged?

This is spelled out on page 3: deletion of a single e-gene resulted in yeast clones that were not viable.

 

[fredreload]

A full computer simulation is even harder than building a genome from scratch: it is not sufficient to understand the function of every piece of the genome, you also have to understand every process connected to it in the cell in detail.

You can always create a molecular scan of the entire bacteria, but you're right, running the processes and the bonding structure of these protein sounds way too complex

 

[mfb]

You can always create a molecular scan of the entire bacteria

In theory. In practice I never saw this realized (even reconstructing the folding of single proteins is a challenging part if they don't crystallize well). And you would still have to model the dynamics.

 

[Ygggdrasil]

Interesting report. I wonder if any of those unknown function genes code for proteins, have an enzymatic function, or fold the chromosome into a more productive shape?

All of the unknown genes appear to code for proteins. Some appear to have enzymatic function, but their substrate is not known.

It has been reported that there are DNA-binding proteins that are important for organizing bacterial chromosomes (e.g. see http://science.sciencemag.org/content/333/6048/1445.long). The protein studied in that paper does not appear to have a detectable homolog in the Mycoplasma mycoides bacteria that the JCVI team studied, though it's possible that other proteins may organize the chromosome in that bacterium. It's definitely an interesting question as to whether such functions are essential in bacteria.