Field of Science

Chopping bits out of the genome

ResearchBlogging.orgGenerally bacteria genomes tend to be fairly minimal in the amount you can remove from them. Unlike eukaryotes, which can have whole swathes of genome that codes for very little, bacteria, with their limited space for a chromosome, need every gene they can get. They just don't have the space for unnecessary genes.

Streptomyces bacteria, however, have bigger genomes and the luxary to invest in genes which are not strictly necessary for bacterial survival. These are called Secondary metabolite genes (as opposed to the necessary primary metabolites) and they code for genes that form an arsenal of weapons for the Streptomyces to deploy. Most Strep are soil-based, and they need the ability to produce secondary metabolites (such as antibiotics) to fight off invading bacteria, and clear terratory to expand their growth into.

What has recently been done (very ingeniously) is to remove the secondary metabolism genes from the bacterial species Streptomyces avermitilis creating essentially an 'empty' strep bacteria, that can grow and divide but not produce any of the secretory substances that strep are known for. The researchers managed to cut an entire 1.5Mb of DNA right out of the genome - helped by the fact that all the secondary metabolite genes cluster together on one side of the chromosome.

They did this using a common molbio technique, the cre-lox system. "Lox" is an area of DNA and "cre" is the protein that very specifically cuts DNA at the Lox site - it acts like a pair of scissors. They put a Lox site on either end of the DNA that codes for secondary metabolites (using a techinque called recombination in order to attach the lox into the chromosome) along with the DNA for the Cre protein under an inducible promoter. Once the bacteria had grown, they activated Cre production, which then cut the unwanted DNA out of the bacteria. This technique was amazingly successful and is shown diagramatically below (picture from the reference):

The avimitilis now contains no secondary metabolites at all, which makes a wonderful 'empty' system to use for studying how secondary metabolites are made. Genes from other bacteria, or even some of the removed genes, can be added back in, piece at a time, to see how much of the gene is necessary for metabolite production, and which regulatory pathways are the most important. Creating an empty cell also has potential implications for biotechnology, after all when trying to produce antibiotics you want the cell working as hard as possible just to produce your product, not wasting time and resources on other metabolites!

I'm really impressed at this kind of large scale synthetic-biology. It may be an area I end up going into in the near future...


Komatsu M, Uchiyama T, Omura S, Cane DE, & Ikeda H (2010). Genome-minimized Streptomyces host for the heterologous expression of secondary metabolism. Proceedings of the National Academy of Sciences of the United States of America, 107 (6), 2646-51 PMID: 20133795


Alexander said...

I'm a little bit surprised that they used the Cre-Lox system at all. Homologous recombination is an easy tool in bacteria, and they used it to put in the LoxP sites anyway. Why not just cut out the secondary metabolite genes loci and bridge the DSB with a short sequence homologous to both end? No need to introduce LoxP sites and Cre expression at all!
Since after Cre recombination only one of the two LoxP sites remains, it can't be used for integration of foreign DNA at that site. At least that's how I understand it.

Great idea, though! And I'm a little bit surprised that the bacteria actually can tolerate to lose all of the genes at once.

Lab Rat said...

@Alexander: They tried that! Their first attempts at removing the secondary metabolite region were just by trying homologous recombination with a little sequence to recombine with the genome, it turned out to be very ineffective, especially compared with the cre-lox system, which had an insanely high efficiency.

And they also did integrate foreign DNA into the empty bacteria, from related streptomyces species. I'm not sure what site they introduced it into though, it might have been up or downstream of the remaining lox site.

The bacteria tolerated all this loss while kept isolated and on nutritious media, I don't think they'd be as healthy in the wild. These genes are all expressed during exponential phase usually, although I agree it is really amazing that the bacteria were as happy as they were.

Thanks for the comment!