Chemically defined media for cultivation of Naegleria gruberi

Abstract

Cultivation of amebae of the axenic strain of Naegleria gruberi, NEG-M, was achieved in media consisting entirely of chemically defined components. A complete medium that contains 31 components allows growth with yields up to 5 x 106 amebae per ml. A minimal medium gives lower yields but defines 22 components that are essential for continuous cultivation: 11 amino acids, 6 vitamins, hematin, guanosine, D-glucose, Mg2+, and inorganic phosphate. These media allow precise studies of the metabolism and differentiation of this unusual eukaryote.

Intermediary Metabolism in Protists: a Sequence-based View of Facultative Anaerobic Metabolism in Evolutionarily Diverse Eukaryotes

“…The results from these studies indicated the presence of lipid biosynthetic pathways and pointed towards the absence of a purine biosynthetic pathway and an inability to synthesise de novo sufficient quantities of several amino acids (Fulton et al 1984; Raederstorff and Rohmer 1987). The sequence of the mitochondrial genome (deposited in Genbank with accession number NC_002573) indicates the presence and function of a typical mitochondrial respiratory chain.…”

“…Prior to genome sequencing, little was known about the central metabolism of Naegleria , although a minimal medium had been described (Fulton et al 1984) and the sterol composition and biochemistry of growing amoebae had been subject to some analysis (Raederstorff and Rohmer 1987). The results from these studies indicated the presence of lipid biosynthetic pathways and pointed towards the absence of a purine biosynthetic pathway and an inability to synthesise de novo sufficient quantities of several amino acids (Fulton et al 1984; Raederstorff and Rohmer 1987).…”

Naegleria: A Research Partner For Cell and Developmental Biology1

“…Little is known about the metabolism of Naegleria [reviewed in 471. The definition of 22 required nutrients for Naegleria gruberi NEG-MD [30] provides information about biosynthetic capabilities as well as pathways that may be absent, and also raises questions that challenge further work, such as the reason for an absolute requirement for hemin. Studies of Naegleria relationships using allozymes are revealing a diversity of standard enzymes that these cells possess, from aconitase to UPDglucose pyrophosphorylase [see 57 and citations therein].…”

“…Using NEG-M we worked to develop chemically defined and minimal media; by the time our work was complete the strain had evolved to grow well in these media, whereas NEG-M would not. Since the chemically defined medium was called medium MD, the derivative strain is NEG-MD [30]; it, like its parent NEG-M, is 2X. Recently we have isolated a 1X strain from NEG-MD; this new strain, NEG-H, does not grow in the defined medium (C. F., unpubl.…”

“…We have tried without success to obtain transformation using several heterologous genes, and currently are focusing on a homologous marker, the gene for OMP decarboxylase, the last step in uracil synthesis. Our studies with chemically defined media show that strain NEG does not require uracil (and therefore has this enzyme) but that its growth is improved by uracil (so it can use exogenous uracil as a source of this pyrimidine) [30]. We have cloned this Naegleria gene by functional complementation of OMP decarboxylase deficient mutants of E .…”