Clustered essential genes, any ideas?

[Koeng]

Hey

I have been annotating a Bacillus genome to only have essential genes showing (I'd like to make a minimal Bacillus cell, so far I have selection systems, planned out cloning steps, vectors do to this with, equipment and bacteria needed, ect). Its taken me quite a while but I finally finished. I took the 253 CDSs from subtiwiki http://subtiwiki.uni-goettingen.de/wiki/index.php/Essential_genes (manually, btw. It took forever... but I haven't learned to program yet so it was the only way) Something weird happened though...

Out of the 374 essential proteins and RNAs (give or take 1 or 2, I haven't looked all the way into the RNA part) 109 of the 374 essentials are in a 202kb region.

That's 29% of all the essential genes in a region 5% of the entire genome... They are also all clustered around dnaA (I read somewhere that dnaA is near to the origin, don't quote me I haven't looked for a while)

My idea (note I have only thought about this for a very short while) of why this is is because during the evolution of this cell messing up essential genes with duplications/ recombination would cause those cells to be less fit for their environment, so the recombination and duplication/mutation occured in other parts of the genome.

Do you guys have any idea of why this occurred? I am thinking of trying to annotate the genomes of Mycoplasma genitalium and E coli to see if they are similar (any programmers out there :) ). Perhaps if I do that and find similar results I'll publish in like F1000 or peerj.... That is if this hasn't already been discovered, which I will surprised if I am the first to find this

-Koeng

[Koeng]

BTW here is a pic of the file

[John Griessen]

On 03/22/2014 03:15 PM, Koeng wrote:
> Do you guys have any idea of why this occurred?

My guess is folding. Protein folding, protein creation from RNA, etc. For some 3D reason.

[Mega [Andreas Stuermer]]

Maybe this island is the remnant of its ancestors. That then duplicated and mutated. 

If you have the minimal genome synthesized at one time, remember that you'll need a marker for the cells. I recommend lux :D 

So the minimal genome bacillus would glow by default (already very cool! but, it's getting even better - wait for it). When you do homologous integration, by knocking in your gene of interest you would knock out luxAB. So you can see that your experiment worked.  :D 

[Lisa Thalheim]

Hey Koeng,

assuming that there is substantial overlap between the categories
"essential genes" and "highly expressed genes", the following may be
part of the answer:

"When the time required to replicate the chromosome exceeds the
duplication time, the dosage of genes near the origin in the cell
increases exponentially with the number of simultaneous replication
rounds. Fast-growing bacteria, such as E. coli or B. subtilis, with
multiple simultaneous replication rounds selectively accumulate highly
expressed genes near the replication origin because of this effect."

as well as

"The directionality of chromosome replication leads to a chronological
asymmetry along the replichore. Since about 1 h separates the beginning
from the end of replication, the favourable conditions leading to
replication start may no longer prevail at the end of the process and
this may result in different mutation rates and compositional biases."

Quoted from "The replication-related organization of bacterial genomes",
http://mic.sgmjournals.org/content/150/6/1609.full.

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[Koeng]

@ Mega I don't have enough money to synthesize a minimal genome :( that's for Craig Venter to do. I am using a positive and negative selection cassette. I'll insert it into regions without essential genes then use long primers (60-100bp) to remove the cassette by homologously recombining near 2 essential genes, deleting the entire region around them (I have planned 52 of these, to delete more than 3mbs). Yea some genes are gonna get expressed more/less, but in the "Combining 2 genome" papers that didn't seem like a huge problem. And it probably is an island from its ancestors, but perhaps adapted for something else...

@ Lisa I think that was just what I was looking for :D Thank you a ton! All of the evidence is in support of that theory. It turns out DNA replication proteins (hence those replication proteins are increasing their own transcription), ribosome proteins, and RNAs is what is mainly there. It looks like most the tRNAs and rRNAs are there actually, then there is fewer and fewer as it gets closer to the terminus. There is still one near the terminus, but it looks like the majority of RNAs are there near the origin, or halfway between the origin and the terminus. Meanwhile the ftsZ ring is 400kb from the terminus, which is pretty nearby in the big scheme of things. I read a paper a while ago (I will try and dig it up) that explained how the ftsZ ring's transcription stayed stable and that when times were good that the chromosomes began replicating more and more and blocked ftsZ ring formation for splitting, and this was an adaptation for quick growth. Since there is some "marker genes" I could use for this analysis (ftsZ, dnaA, ect) I think I will begin looking through slow and fast growing bacterial genomes to find if there is any correlation. Thanks a ton!!!

[Matthew Pocock]

Hiya,

If you want to make a *really* minimal bacillus, you may want to look into L-forms. They lack cell walls, so you can kick out a whole bunch of genes for fatty acid metabolism and cell wall and cell division. L-forms are hyper-competent, so you can get them to pick up almost any DNA of any size.

M

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[Koeng]

Thanks for the L-forms, I never realized that there was Bacillus without cell walls :D 

That would be a great thing to do after I finish making the cells pretty minimal (since if I did it beforehand the media preparation would be more difficult and they would die easier). Thanks for the suggestion!

Perhaps this points to something else: ftsZ ring probably evolved later after the cells acquired the ability to replicate, hence being not near to the "island" of essential genes near the origin