Laboratories

Systems Microbiology

Prof. Mori
Professor
Hirotada MORI
Assistant Professor
Ai MUTO
Labs HP
http://biodata.naist.jp

Outline of Research and Education

Escherichia coli is undoubtedly one of the most studied organisms in the world. Vast amounts of accumulated biological knowledge and methodologies make this organism one of the ideal platforms to analyze cells at the system level. Our lab is one of the leading groups performing post-genomic, system and synthetic approaches towards understanding the entire cell system of E. coli.

Genetic interactions

Normally cell systems can tolerate many kinds of perturbation, e.g. environmental stresses and genetic mutations. In E. coli, most single gene knockout strains do not exhibit substantial phenotypic changes. This characteristic is called “robustness” and is caused by the function of a network of compensatory backup systems. This is one of the main reasons why the computational design of a cell system has been unsuccessful so far. Genetic interaction analysis is one of the most powerful and reliable ways to identify and characterize cellular networks. To identify the complex cellular network structure in E. coli, we are performing high-throughput systematic genetic interaction studies using double-gene knockout strains as shown in Fig. 1.

Novel method for population dynamics by Bar-code strains

To monitor each strain’s growth in a bar-coded single gene knockout strain library, named ASKA bar-coded collection. Each mutant has different 20nt DNA sequence as a molecular bar-code. Using a mixed culture of an entire set of knockout strains, we are now performing population analysis during the long-term stationary phase and sub-lethal concentration of antibiotics and determined each of strains behavior during stress conditions by deep-sequencing to elucidate the interaction between cells in the mixed culture as shown in Fig. 2. This new resource will accelerate population analysis in a variety of conditions.

Genome size design and cross-species transfer of DNA by conjugation

We have developed a very efficient method to construct double knockout strains using F plasmid based conjugal transfer system. The F (incF) plasmid has a narrow host-range but incP and incW plasmid families have much wider host-ranges. We are expanding our conjugation vector system from the F plasmid system to the incP and incW plasmids to enable the transfer of large DNA molecules from E. coli into other microbes. Our long-term goal is to design and construct bacterial genome-size DNA molecules and transfer large size genomes into the target micro-organisms to engineer cells as shown in Fig. 3.

Major Research Topics

  1. Genetic interaction networks
  2. Quantitative metabolic network analysis
  3. Development of artificial chromosome and cross-species transfer systems of huge DNA

References

  1. Baba T, et al., Mol Syst Biol, 2, 0008, 2006
  2. A. Typas et al., Nat Methods 5, 781-787, 2008
  3. T. Conway et al., mBio, 5, 2014
  4. R. Takeuchi et al., BMC microbiology, 14, 171, 2014
  5. Y. Otsuka et al., Nucleic acids research 43, D606-617, 2015
  6. K. Nakahigashi et al., DNA Res 23, 193-201, 2016
  7. E. H. Morales et al., Nat Commun 8, 15320, 2017
  8. L. Maier et al., Nature 555, 623-628, 2018
Fig.1
Fig.1 (A) The concept of synthetic lethal/sickness analysis: Red circles represent essential metabolites for cells. If cells have redundant routes to produce essential metabolites, double deletion methods may identify such redundant steps of genes (enzymes). (B) The conjugation method to generate double knockout strains by combining single knockout strains.
Fig.2
Fig.2 The X axis shows time points of sampling and the Y axis represents population ration of all deletion strains.
Fig.3
Fig.3 Wide host-range incP family plasmid RP4 can deliver large DNA fragment by cross-species conjugation.
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