Whole lifecycle observation of single-spore germinated Streptomyces using a nanogap-stabilized microfluidic chip.

Abstract

Streptomyces is a model bacterium to study multicellular differentiation and the major reservoir for antibiotics discovery. However, the cellular-level lifecycle of Streptomyces has not been well studied due to its complexity and lack of research tools that can mimic their natural conditions. In this study, we developed a simple microfluidic chip for the cultivation and observation of theentire lifecycle of Streptomyces development from the single-cell perspective. The chip consists of channels for loading samples and supplying nutrients, microwell arrays for the seeding and growth of single spores, and air chambers beside the microwells that facilitate the development of aerial hyphae and spores. A unique feature of this chip is that each microwell is surrounded by a 1.5 µm nanogap connected to an air chamber, which provides a stabilized water-air interface. We used this chip to observe the lifecycledevelopment of Streptomyces coelicolor and Streptomyces griseus germinated from single spores, which revealed differentiation of aerial hyphae with progeny spores at micron-scale water-air interfaces and air chambers. Finally, we demonstrated the applicability of this chip in phenotypic assays by showing that the microbial hormone A-Factor is involved in the regulatory pathways of aerial hyphae and spore formation. The microfluidic chip could become a robust tool for studying multicellular differentiation, single-spore heterogeneity, and secondary metabolism of single-spore germinated Streptomyces.