Development of defined and minimal media for the growth of the hyperthermophilic archaeon Pyrococcus furiosus Vc1

Abstract

The nutritional requirements of Pyrococcus furiosus were determined using extended continuous culture in the absence of elemental sulfur in an anaerobic gaslift bioreactor. Replacement of complex nutrients by maltose, amino acids and vitamins alone supported growth. When the organic components of this defined medium were optimised, equilibrium cell densities of 1010 ml−1 were achieved. Selective feeding and withdrawal revealed requirements for l‐proline, l‐cysteine and d‐biotin. Replacement of maltose by dextrin allowed growth in closed bottles on both the defined and minimal media, again in the absence of elemental sulfur. A simple procedure for the efficient plating of P. furiosus on solid media was also developed.

Random mutagenesis of the hyperthermophilic archaeon Pyrococcus furiosus using in vitro mariner transposition and natural transformation

“…1A). These relatively low plating efficiencies are comparable to previous reports222528. In contrast, we achieved plating efficiencies of up to 82% by incubating plates at 80 °C (Fig.…”

“…Selection of transformants of P. furiosus by natural transformation requires growth on minimal solid medium22. This defined minimal medium is plagued by a relatively low plating efficiency with only 20% of cells forming colonies25. This minimal defined medium contains cellobiose and amino acids as carbon sources, and at elevated temperatures, these can react to form Maillard reaction products that are inhibitory to archaeal growth26.…”

“…Plasmids used in this study are listed in Supplementary Table S2. The strains were grown anaerobically in a defined medium with cellobiose25 as the carbon source and supplemented with 20 μM uracil for COM1 and JFW002 strains22. Medium was prepared as previously described22.…”

Characterization of Ten Heterotetrameric NDP-Dependent Acyl-CoA Synthetases of the Hyperthermophilic ArchaeonPyrococcus furiosus

“…For example, DNA microarray analyses were consistent with the biosynthesis of twelve amino acids (Glu, Arg, Leu, Val, Ile, Ser, Thr, Met, His, Phe, Trp, and Tyr) occurring by conventional pathways [13]. However, growth studies to determine amino acid auxotrophy have given ambiguous results [21–23] and while the genome of P. furiosus does encode genes that could be involved in the biosynthesis of all twenty amino acids, some pathways for amino acids may be incomplete [24]. A more comprehensive understanding of amino acid metabolism in P. furiosus in general is clearly needed before a more definitive correlation between biosynthetic and degradative amino acid pathways can be made and the corresponding roles of the ACS isoenzymes.…”

Significance of polysaccharides in microbial physiology and the ecology of hydrothermal vent environments

“…Ferredoxin is preferentially used over NAD(P) in hyperthermophiles which may be an adaptation related to coenzyme thermolability [Daniel and Danson, 1995 In terms of peptide metabolism, Pyrococcus and Thermo coccus species grow well on complex medium components (e.g., yeast extract, tryptone, peptone). However, there are reports of growth on defined media supplemented with amino acids [Raven, et al, 1992;Raven and Sharp, 1997;Rinker, 1998;and Rinker and Kelly, 1996]. Amino acid metabolism in hyperthermophiles is not well studied.…”