Large-scale preparation of fully deuterated cell components. Ribosomes from Escherichia coli with high biological activity.
Some applications of NMR and of neutron scattering require fully deuterated biological material which should be highly active and available in large quantities. These requirements are hardly compatible since full deuteration is achieved easily only if cells are grown in minimal media. This condition used in standard batch fermentation results in both low yields and reduced activities of the biological mass. Here we report a method which combines the apparently incompatible requirements taking advantage of a recent observation according to which the appearance of growth inhibiting extracellular products could be prevented. The method was applied for growing Escherichia coli cells, strain MRE600rif (resistance against high doses of rifampicin is used as selection marker) on partially deuterated media (76% and 84% D2O) with glucose as carbon source and on deuterated acetate and succinate with 100% D2O when full deuteration was to be achieved. The essential point for preserving the log-phase character of the cells is that the cultivation is carried out at substrate limiting conditions thus keeping the growth rate at low levels (for glucose the growth rate, mu < or = 0.35 h-1, for acetate/succinate mu < or = 0.1 h-1) which avoids the accumulation of the substrate or of by-products in the medium. Our data suggest that acetate is a main extracellular component for accompanying or triggering the transition from logarithmic growth to stationary phase of E. coli cells cultivated on glucose as carbon source. The cells were first grown in fed-batch to high cell densities (above 50 g wet cells/l) under conditions of substrate limitations. A steady-flow fermentation followed keeping the growth rate at about mu of 0.1 h-1. Cells were harvested in kg quantities, the extracted ribosomes showed a normal complement of proteins, contained intact rRNA and were fully active. The ribosomal protein and rRNA fractions could be efficiently reconstituted to highly active particles. In the case of full deuteration a matching point of 120% (tentative D2O scale) was achieved. The reported method facilitates the preparation of deuterated biological material for applications in NMR and neutron scattering analysis.
Vanatalu
K
K
Laboratory of Molecular Genetics, Estonian Academy of Sciences, Tallinn.
Paalme
T
T
Vilu
R
R
Burkhardt
N
N
Jünemann
R
R
May
R
R
Rühl
M
M
Wadzack
J
J
Nierhaus
K H
KH
D000085:Acetates / Q000378:metabolism; D019342:Acetic Acid; D001426:Bacterial Proteins / Q000737:chemistry; D002455:Cell Division; D003903:Deuterium / Q000032:analysis* / Q000378:metabolism; D004926:Escherichia coli / Q000737:chemistry / Q000254:growth & development / Q000378:metabolism*; D005285:Fermentation; D005947:Glucose / Q000378:metabolism; D007553:Isotope Labeling; D009682:Magnetic Resonance Spectroscopy; D012269:Ribosomal Proteins / Q000737:chemistry*; D012270:Ribosomes / Q000737:chemistry*; D013386:Succinates / Q000378:metabolism; D019802:Succinic Acid
D000085:Acetates; D001426:Bacterial Proteins; D012269:Ribosomal Proteins; D013386:Succinates; D019802:Succinic Acid; D003903:Deuterium; D005947:Glucose; D019342:Acetic Acid