Metabolites, proteins and mRNA were measured comprehensively and quantitatively and hints about the robustness of metabolic systems were revealed.

Ishii, N., Nakahigashi, K., Baba, T., Robert, M., Soga, T., Kanai, A., Hirasawa, T., Naba, M., Hirai, K., Hoque, A., Ho, PY., Kakazu, Y., Sugawara, K., Igarashi, S., Harada, S., Masuda, T., Sugiyama, N., Togashi, T., Hasegawa, M., Takai, Y., Yugi, K., Arakawa, K., Iwata, N., Toya, Y., Nakayama, Y., Nishioka, T., Shimizu, K., Mori, H. and Tomita, M. (2007)
Multiple high-throughput analyses monitor the response of E. coli to perturbations,Science, 316(5824), 593-7

“Let’s try to explore all the metabolic reactions in a tiny single cell using advanced biotechnology. It’ll be one of the most exhaustive analysis of a single cell organism.”

said Masaru Tomita, director of IAB, by assembling a team of specialists of the metabolome, proteome, transcriptome. The aim of the team was to measure and understand a large number of energy metabolism components in the E. coli cell. This study quantitatively evaluated the robustness of the metabolic system revealing how E. coli uses multiple strategies to maintain its metabolism under control.

All cells, from bacteria to humans, transform energy sources such as glucose into ATP, an energy-storing molecule, in a process known as energy metabolism. This is one of the most basic cell activities, and is controlled by multiple genes. E. coli is a commonly used bacteria in biological research and it is believed that basic functions of the cell can be better understood by studying energy metabolism.

For this work the team grew wild strains of E. coli at different growth rates (rate of cell doubling) to study the effect on metabolism. Another set of experiments was performed to examine the function of individual metabolic enzymes by using 24 different strains of E. coli in which a single enzyme-encoding gene had been removed (knocked-out) and growing them at the same fixed growth rate. Quantitative data for thousands of metabolites, proteins and RNAs was then collected using state-of-the-art analytical methods and technologies. Furthermore, the team accurately measured the amount of the main 130 metabolites, 57 proteins and 85 RNAs in the central energy metabolic pathways of the cell.
In addition, the researchers also evaluated the amount of traffic or activity in the different pathways studied using a methods call metabolic flux analysis.

The results show that most enzymes in the pathways studied are dispensable on an individual basis even though they all play important roles in the whole process. Even more remarkable the amounts of most metabolites didn’t change very significantly.

These results can be attributed to the presence of more than one gene having the same function in the cell and also to the presence of bypasses that allow to reroute metabolic reactions around the blocked pathway and maintain levels of crucial components. Some strains widely changed the amount of proteins and RNAs, but those strains had duplicate mutation; that is, the strain mutated to use unusual pathway instead of knocked out gene.

Increases in growth rate logically require higher energy requirements. The quantity of multiple RNAs and proteins that make up the cells energy factory changed significantly, by adjusting to the the variable energy requirments although the amounts of metabolites themselves didn’t vary largely, probably demonstrating both the importance of stable levels of metabolites as well as their stable production/consumption ratio at varying growth rates.

In summary, the IAB research team quantitatively demonstrated for the first time how E. coli uses various strategies to overall stability of its energy metabolism. This breakthrough was published in the prestigious American scientific research magazine “Science”.
   
This project made heavy use of original technologies developed at IAB for the analysis of metabolites using a combination of capillary gel electrophoresis and mass spectrometry called “CE-MS” that can measure thousands of metabolites at a time. The researchers also used original technology to analyze proteins. Together these methods of large-scale analysis can be used in different fields of research. For example, the system can be applied to identify cancer-cell specific metabolic pathways to facilitate the development of anticancer agents. The system can also be applied for the improvement of the metabolic function of industrially useful microorganism’s such as bacteria that produce bioethanol or bioplastics.

The members of the research team are already at work on these next steps. The elucidation of the mysteries of cellular life and its numerous applications has just begun.

Fig1. Heat map of changes in the cellular components. Blue represents decrease, yellow represents no change, and red represents increase. RF: Reference sample (Wildtype, growth rate 0.2h-1), GR: Wildtype grown at several Growth Rate, KO: Knock-Out mutant strain. Metabolites (anion, cation, nucleotide) were analyzed using CE-MS, proteins were analyzed with LC-MS, and mRNA concentration was analyzed using quantitative PCR.  

 Comment in:   Science. 2007 Apr 27;316(5824):550-1.