Wednesday, October 1, 2008

What a man can make …….


Genome Transplantation in Bacteria: Changing One Species to Another
Carole Lartigue, John I. Glass, Nina Alperovich, Rembert Pieper, Prashanth P. Parmar, Clyde A. Hutchison III, Hamilton O. Smith, and J. Craig Venter 2007. Science 317, 632.

Complete Chemical Synthesis, Assembly, and Cloning of a Mycoplasmagenitalium Genome.
Daniel G. Gibson, Gwynedd A. Benders, Cynthia Andrews-Pfannkoch, Evgeniya A. Denisova, Holly Baden-Tillson, JayshreeZaveri, Timothy B. Stockwell, AnushkaBrownley, David W. Thomas, Mikkel A. Algire, Chuck Merryman, Lei Young, Vladimir N. Noskov, John I. Glass, J. Craig Venter, Clyde A. Hutchison III, Hamilton O. Smith 2008Published Online January 24, 2008 Science DOI: 10.1126/science.1151721

In these two papers, researchers from J. Craig Venter Institute in Rockville Maryland showed us the possibility of changing the entire genetic information of a living microorganism to create a new artificial organism.
In first paper they used two closely related Mycoplasmas: Mycoplasma mycoides large colony (LC) and M. capricolum. They used mycoplasma because of its specificity. Mycoplasmas do not have a cell wall, which makes them resistant to some therapeutic antibiotics like B-lactams. This also makes them more susceptible for uptake of different substances from the environment. One also cannot underestimate the biological importance of Mycoplasmas as pathogenic microorganisms, causing some nasty diseases in human and animals. Another interesting fact is that Mycoplasmas, compared to other bacteria, have relatively small genome, ranging from 0.6 to 1.4Mb. Up to now, 12 Mycolasma genomes have been completely sequenced and 11 more are in progress.
Small genome size and lack of the cell wall makes them ideal candidates for the genome “switching” experiment.
So, Carole Lartigue with coworkers took the large colony of M. mycoides and prepared intact genomic DNA in the way usually used for PFGE (pulsed field gel electrophoresis). All proteins were removed by proteinase treatment. This purified DNA was used for transformation of M. capricolum strain. After a few days some transformants grew on selective media. These transformants had genetic markers specific for donor strain but no sign of host specific markers was detected. That means that it is possible to replace a whole genome, at least using closely relative bacteria.
Cloning large genomic fragments is not a technical problem. Some bacterial artificial chromosome (BAC) clones already contain inserts of about 300kb in size. Such clones can be easily introduced into E. coli cells by electroporation. Using some specific recombination systems it could be possible to join two or three large clones inside E. coli cells to generate single DNA molecule, then isolate such a genome and introduce it into a new bacterial host.
But there is another way to build an entire genome. In second of the presented papers, the authors described this alternative way. They split the whole Mycoplasma genitalium genome into 101 cassettes, each of about 5000-7000bp. Those cassettes were chemically synthesized by three companies. All cassettes have specific overlapping sequences so they can be stuck together. The in vitro recombination event yielded intermediate assembliesof approximately 24 kb, 72 kb ("1/8 genome"), and 144 kb ("1/4genome"), which were all cloned as bacterial artificial chromosomes(BACs) in The complete synthetic genomewas assembled by transformation-associated recombination (TAR)cloning in the yeast Saccharomycescerevisiae, then isolatedand sequenced. This method allowed construction of an entire “artificial” bacterial genome.
Mycoplasmas have small genomes, but we already know some bacteria with even smaller genomes. The amphid endosymbiont Buchnera aphidicola genome consists of ~422kb, but a psyllidendosymbiont, Carlsonella ruddii, has even smaller genome (~160kb). Such small genomes are specific for pathogenic and especially endosymbiotic style of life. These bacteria reduce their genomes by deleting genes that are not necessary, like some anabolic pathways. On the other hand, they keep genes that are useful for themselves and for their hosts. These genes encode basic functions like replication, transcription and translation, as well as some of the biosynthetic pathways encoding for amino acids, cofactors and other essential compounds that their host cannot obtain from their diet. Surprisingly, also lots of genes encoding transport (uptake) systems have been eliminated.
Studying such small genomes we can learn which genes are really core genes and cannot be removed from the genome and which genes are not necessary. Furthermore, we can induce such big reductions in larger bacterial genomes. Analysis of these data as well as data obtained from analysis of the eukaryotic organelle (mitochondria and plastids) genomes could be used to construct an artificial endosymbiont.
Perhaps, it could be possible to make a new endosymbiont,which could be specific for some body tissue like liver or pancreas. They could live inside the cell,producingspecific proteins like insulin, clotting factors or other factors deficient in genetic diseases. This is fantasy but who knows the future…….

Additional articles:
S. G. E. Andersson 2006. TheBacterial World Gets Smaller. Science 13, pp.: 259 – 260.

A. I. Nilsson,S. Koskiniemi,S. Eriksson,E. Kugelberg,J. C. D. Hinton,D. I. Andersson 2005, Bacterial genome size reduction by experimental evolution. Proc. Natl. Acad.Sci. 102, pp.: 12112-12116.

Quanzhou Tao and Hong-Bin1998 Zhang 1998 Cloning and stable maintenance of DNA fragmentsover 300 kb in Escherichia coli with conventional plasmid-based vectors. NAR 26, 21, pp.: 4901-4909.

dr Jaroslaw Krol

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