By Elizabeth Ohneck, PhD
How many genes are necessary for life? We humans have 19,000 – 20,000 genes, while the water flea Daphnia pulex has over 30,000 and the microbe Mycoplasma genitalium has only 525. But how many of these genes are absolutely required for life? Is there a minimum number of genes needed for a cell to survive independently? What are the functions of these essential genes? Researchers from the J. Craig Venter Institute and Synthetic Genomics, Inc., set out to explore these questions by designing the smallest cellular genome that can maintain an independently replicating cell. Their findings were published in the March 25th version of Science.
The researchers started with a modified version of the Mycoplasma mycoides genome, which contains over 900 genes. Mycoplasmas are simplest cells capable of autonomous growth, and their small genome size provides a good starting point for building minimal cells. To identify genes unnecessary for cell growth, the team used Tn5 transposon mutagenesis, in which a piece of mobile DNA is introduced to the cells and randomly “jumps” into the bacterial chromosome, thereby disrupting gene function. If many cells were found to have the transposon inserted into the same gene at any position in the gene sequence, and these cells were able to grow normally, the gene was considered non-essential, since its function was not required for growth; such genes were candidates for deletion in a minimal genome. In some genes, the transposon was only found to insert at the ends of the genes, and cells with these insertions grew slowly; such genes were considered quasi-essential, since they were needed for robust growth but were not necessary for cell survival. Genes which were never found to contain the transposon in any cells were considered essential, since cells that had transposon insertions in these genes did not survive; these essential genes were required in the minimal genome.
The researchers then constructed genomes with various combinations of non-essential and quasi-essential gene deletions using in vitro DNA synthesis and yeast cells. The synthetic chromosomes were transplanted into Mycoplasma capricolum, replacing its normal chromosome with the minimized genome. If the M. capricolum survived and grew in culture, the genome was considered viable. Some viable genomes, however, caused the cells to grow too slowly to be practical for further experiments. The team therefore had to find a compromise between small genome size and workable growth rate.
The final bacterial strain containing the optimized minimal genome, JCVI-syn3.0, had 473 genes, a genome smaller than any autonomously replicating cell found in nature. Its doubling time was 3 hours, which, while slower than the 1 hour doubling time of the M. mycoides parent strain, was not prohibitive of further experiments.
What genes were indispensable for an independently replicating cell? The 473 genes in the minimal genome could be categorized into 5 functional groups: cytosolic metabolism (17%), cell membrane structure and function (18%), preservation of genomic information (7%), expression of genomic information (41%), and unassigned or unknown function (17%). Because the cells were grown in rich medium, with almost all necessary nutrients provided, many metabolic genes were dispensable, aside from those necessary to effectively use the provided nutrients (cytosolic metabolism) or transport nutrients into the cell (cell membrane function). In contrast, a large proportion of genes involved in reading, expressing, replicating, and repairing DNA were maintained (after all, the presence of genes is of little use if there is no way to accurately read and maintain them). As the cell membrane is critical for a defined, intact cell, it’s unsurprising that the minimal genome also required many genes for cell membrane structure.
Of the 79 genes that could not be assigned to a functional category, 19 were essential and 36 were quasi-essential (necessary for rapid growth). Thirteen of the essential genes had completely unknown functions. Some were similar to genes of unknown function in other bacteria or even eukaryotes, suggesting these genes may encode proteins of novel but universal function. Those essential genes that were not similar to genes in any other organisms might encode novel, unique proteins or unusual sequences of genes with known function. Studying and identifying these genes could provide important insight into the core molecular functions of life.
One of the major advancements resulting from this study was the optimization of a semi-automated method for rapidly generating large, error-free DNA constructs. The technique used to generate the genome of JCVI-syn3.0 allows any small genome to be designed and built in yeast and then tested for viability under standard laboratory conditions in a process that takes about 3 weeks. This technique could be used in research to study the function of single genes or gene sets in a well-defined background. Additionally, genomes could be built to include pathways for the production of drugs or chemicals, or to enable cells to carry out industrially or environmentally important processes. The small, well-defined genome of a minimal cell that can be easily grown in laboratory culture would allow accurate modeling of the consequences of adding genes to the genome and lead to greater efficiency in the development of bacteria useful for research and industry.