Cell cycle synchronization of Cupriavidus necator by continuous phasing measured via flow cytometry

Abstract

The continuous phasing technique was successfully used to obtain a high degree of cell cycle synchrony in cultures of the model organism Ralstonia eutropha JMP 134 (today reclassified into Cupriavidus necator). The responses of the organism were evaluated with flow cytometric determinations of DNA contents and cell size (by fluorescence and forward scatter measurements, respectively, after staining with the DNA-binding dye 4',6-diamidino-2'-phenylindole, DAPI), and cell concentration, after staining with the nucleic acid binding dye LDS-751. The strain was cultivated on a mineral medium with pyruvic acid sodium salt as the limiting carbon and energy source. Famine conditions, and thus cell dormancy, were achieved in every cycle. The best synchronization, according to the determination of DNA contents, was induced with phasing cycle durations of at least 4 h. The method allows the induction of synchrony for an indefinite period if the medium is exchanged rapidly and precisely. The results show that the time required for a complete cell cycle of Cupriavidus necator JMP 134 is independent of the chosen phasing cycle duration, provided that each process cycle lasts at least 3 h which is much longer than the time needed for a single DNA replication cycle. With shorter cycling periods DNA-synthesis is carried out in an uncoupled manner and only weak cell cycle synchrony can be attained. The results also show that DNA-synthesis can only be undertaken by cells when they have exceeded a critical size.