Growth, Development and Genetic Characteristics of Physarum polycephalum Amoebae Able to Grow in Liquid, Axenic Medium

Abstract

Amoebae from natural isolates of Physarum polycephafum, unlike the plasmodial phase, are unable to grow in axenic medium. A mutant strain of amoebae, CLd-AXE, can be cultured in the liquid, semi-defined medium used for plasmodial culture but lacks some of the properties required for studies of development and gene expression. From crosses of CLd-AXE with wildtype amoebae, new amoebal strains able to grow in axenic medium have been isolated; some of these can also undergo the reversible amoeba-flagellate transformation and apogamic plasmodium development in axenic conditions. Amoebae maintained in active growth in liquid culture for several months showed little change in their properties. Subcultures made with diluted inocula indicated that the same growth rate was achieved even when single amoebae were inoculated in liquid medium. All strains produced colonies with high efficiency when replated on. bacterial lawns. Measurements of DNA content by flow cytometry indicated that the majority of amoebae in liquid cultures were haploid. Homozygous diploid amoebae constructed from one strain grew less well than the haploid cells. Genetic analysis of crosses suggested that amoebae able to grow in liquid axenic medium fell into one major phenotypic class with respect to growth rate, and that mutation at only one or two loci was necessary to allow amoebae to grow in axenic medium. Diploid, heterozygous amoebae constructed by mating a mutant with a wildtype strain were unable to grow in axenic medium, indicating that at least one of the putative axe alleles was recessive.

Cellular events during sexual development from amoeba to plasmodium in the slime mould Physarum polycephalum

“…The isolation of diploid amoebae from mixtures of strains homoallelic for matA was first described by Youngman et al (1 98 1) ; such amoebae have been useful in genetic analysis by allowing dominance and complementation tests on mutants isolated in amoebae (Anderson & Youngman, 1985;Dee et al, 1989). Holt & Huttermann (1 979) observed that nuclear fusion occurred at mitosis in binucleate fusion cells homoallelic for matA, and that cytokinesis gave rise to pairs of uninucleate, presumably diploid daughters.…”

Cloning and characterization of the altA -tubulin gene of Physarum

“…Myxamoebae of the strain LU352 (Dee et al, 1989) were induced to form flagellates as described by Paul et ul. (1992).…”

“…The construction and screening of two Physarum cDNA libraries, ML5 and ML6, are described by Paul et al (1992). The source of RNA for ML6 cDNA synthesis was strain LU352 (Dee et al, 1989), grown axenically (McCullough et al, 1978). ML5 was derived from total myxamoebal RNA, while ML6 was generated using poly(A) + RNA from a culture undergoing the amoeba-flagellate transition.…”

Contact with a solid substratum induces cysts in axenic cultures ofPhysarum polycephalum amoebae: mannitol-induced detergent-resistant cells are not true cysts

“…Cysts are valuable for the storage of genetically defined strains since they survive long periods at 4 "C, frozen at -80 "C, or desiccated on silica gel (Anderson et al, 1983). Strains of amoebae able to grow in liquid axenic medium (Axe strains; Dee et al, 1989) are essential for molecular genetic analyses, including DNA transformation (Burland et al, 1993).…”

“…LU352, an Axe strain of P. polycephalum amoebae able to grow in axenic medium, was used for all experiments (for detailed description and origin, see Dee et al, 1989 biotin, 0.04 g thiamin, H,O to 1 litre; the p H was adjusted to 4.6 with 20% NaOH. Haematin stock solution (0.5 g Sigma bovine haematin or haemin chloride, 10 g NaOH, H,O to 1 litre; stored at 4 "C) was added immediately before use (10 ml 1-l) .…”