Overproduction, purification and characterization of the Escherichia coli ferritin.
Recent studies have indicated that Escherichia coli possesses at least two iron-storage proteins, the haem-containing bacterioferritin and ferritin. The ferritin protein has been amplified 600-fold to 11-14% of total cell protein in a bfr mutant and purified to homogeneity with an overall yield of 13%. The cellular ferritin content remained relatively constant throughout the growth cycle and amplification was accompanied by a 2.5-fold increase in cellular iron content. The isolated ferritin contained 5-20 non-haem iron atoms/holomer and resembled the eukaryotic ferritins rather than the prokaryotic bacterioferritins in containing no haem. The 24 subunits of this ferritin (M(r) 19,400) assemble into a spherical protein shell (12 +/- 1 nm diameter, M(r) 465,000) which sequesters at least 2000 iron atoms in vitro to form an electron-dense iron core of 7.9 +/- 1 nm diameter. Electron-microscopic and Mössbauer spectroscopic studies with iron-loaded ferritin showed that the core can be either crystalline (ferrihydrite) or amorphous, depending on the absence or presence of phosphate, respectively. Mössbauer spectroscopy with intact E. coli revealed a novel-high spin Fe(II) component which is enhanced in bacteria amplified for ferritin but not in the parental strain. Western blotting showed that ferritin and bacterioferritin are immunologically distinct proteins. E. coli is thus an organism containing both a ferritin and a bacterioferritin and the relative roles of the two iron-storage proteins are discussed in this study.
Hudson
A J
AJ
Krebs Institute for Biomolecular Research, Department of Molecular Biology and Biotechnology, University of Sheffield, England.
Andrews
S C
SC
Hawkins
C
C
Williams
J M
JM
Izuhara
M
M
Meldrum
F C
FC
Mann
S
S
Harrison
P M
PM
Guest
J R
JR
D000595:Amino Acid Sequence; D001426:Bacterial Proteins; D015153:Blotting, Western; D002453:Cell Cycle; D003460:Crystallization; D018360:Crystallography, X-Ray; D003470:Culture Media; D003573:Cytochrome b Group / Q000737:chemistry* / Q000378:metabolism; D004591:Electrophoresis, Polyacrylamide Gel; D004926:Escherichia coli / Q000378:metabolism*; D005293:Ferritins / Q000096:biosynthesis* / Q000737:chemistry* / Q000302:isolation & purification / Q000378:metabolism; D007501:Iron / Q000378:metabolism*; D008854:Microscopy, Electron; D008969:Molecular Sequence Data; D008970:Molecular Weight; D009154:Mutation; D015204:Spectroscopy, Mossbauer
D001426:Bacterial Proteins; D003470:Culture Media; D003573:Cytochrome b Group; D005293:Ferritins; C018032:bacterioferritin; D007501:Iron