Laboratories

Systems Microbiology

Outline of Research and Education

Escherichia coli is undoubtedly one of the most studied organisms in the world. A vast amount of accumulated biological knowledge and methodologies makes this organism one of the ideal platforms to analyze cells at the systems level. Our lab is one of the leading groups performing post-genomic, systems and synthetic analyses using E. coli as a model system.

Major Research Topics

Genetic interactions

Normally cell systems can tolerate many kinds of perturbation, e.g. environmental changes 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 reliable ways to identify and characterize cellular pathways. To determine the cellular network system in E. coli, we are performing high-throughput systematic genetic interaction studies using double-gene knockout strains. Fig. 1

Bar-code analysis

If each single gene knockout strain has a specific tag, and if we have a way to distinguish their tags from a single cell, then mixed cultures of all the deletion strains can be analyzed simultaneously to monitor population dynamics under competitive growth conditions. For this purpose, we developed a new single gene knockout mutant library carrying 20nt DNA sequences as a bar-code. To validate our approach, we are currently analyzing population changes during growth in a liquid medium for up to three weeks by monitoring the bar-code frequency of each of the deletion strains using deep sequencing methods. Fig. 2.

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 DNA-conjugation. 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 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 genome-sized DNA molecules within constructed vectors and establish transfer systems to conjugate them into target micro-organisms. Fig. 3.

References

  1. R. Takeuchi et al., BMC microbiology, 14, 171, 2014
  2. T. Conway et al., mBio, 5, 2014
  3. W. Aoki et al., Scientific reports, 4, 4722, 2014
  4. H. T. Yong et al., Genes Genet Syst, 88, 233-240 ,2013
  5. Z. Tian et al., BMC systems biology, 7 Suppl 6:S1 ,2013
  6. Baba T, et al., Mol Syst Biol, 2, 0008, 2006
  7. Arifuzzaman M, et al., Genome Res, 16, 686-691, 2006
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 metablites, 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 samplings and the Y axis represents population ratio of all deletion strains.
Fig.3
Fig.3 Wide host-range incP family plasmid RP4 can deliver large plasmid DNA by cross-species conjugation.
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