Overproduction, purification and characterization of the Escherichia coli ferritin

Abstract

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 , 19400) assemble into a spherical protein shell (12 2 1 nm diameter, M , 465000) which sequesters at least 2000 iron atoms in vitro to form an electron-dense iron core of 7.9 -+ 1 nm diameter. Electronmicroscopic and Mossbauer 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. Mossbauer spectroscopy with intact E. coli revealed a novel-high spin Fe(I1) 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.Iron is an essential nutrient for most organisms but it is toxic in excess. One means of resolving this paradox is to sequester surplus iron in an iron-storage protein. Two types of iron-storage protein are known: the well-characterized ferritins of animals and plants and the bacterioferritins of prokaryotes such as Escherichia coli, Aeotobacter vinelandii and Pseudomonas aeruginosa (Andrews et al., 1991). Both types contain 24 subunits which assemble into a spherical protein shell with 4/3/2 symmetry (Mr approximately 500000) and a cavity in which iron can be stored (Ford et al., 1984). However, bacterioferritins differ from ferritins in containing approximately 12 haems/24 subunits and this has led to the suggestion that bacteriofemtin may additionally function as an electron-storage molecule (Stiefel and Watt, 1979).Recently, evidence for the presence of ferritins (as well as bacterioferritins) in prokaryotes has been accumulating. Izuhara et al. (1991) designated fin), which encodes a femtin-like protein (FTN). This has subunit M , 19400 and exhibits 21% amino acid sequence identity (38% similarity) to the ferritins and 17% identity (32% similarity) to E. coli bacterioferritin (BFR). A ferritin ( M , 16700 and M , 400000 for subunit and holomer, respectively) has also been isolated from Bacteroides fragilis and the 30-residue N-terminal amino acid sequence is 43% and 37% identical to the N-terminal regions of human Hchain ferritin and FTN, but only 20% identical to E. coli BFR (Rocha et al., 1992). F...

Regulation ofSalmonella entericaSerovar TyphimuriummntHTranscription by H2O2, Fe2+, and Mn2+

“…It may be related to the fact that MntH is extraordinarily active when expressed, capable of concentrating submicromolar amounts of extracellular Mn 2ϩ to over 10 mM in the cytoplasm (24). The iron content of enterobacteria, by contrast, is typically far lower, in the range of a few hundred micromolar (21,26,33) is present at 10 M in the medium, 10 to 30 times their half-maximal repressive concentrations. The possibility that metal repression is somehow inoperative in earlylog-phase cultures seems unlikely.…”

Fungal ferritins: The ferritin from mycelia of Absidia spinosa is a bacterioferritin

“…There exists some speculation that the roles of the mammalian and bacterio-type ferritins may in fact be somewhat different. This view is supported by the recent isolation of a mammalian-type ferritin from E. coli, an organism which also contains Bfr [13,14] as well as from Helicobacter pylori [15].…”

Ferritin from the obligate anaerobe Porphyromonas gingivalis: purification, gene cloning and mutant studies The GenBank accession number for the sequence reported in this paper is AB016086.

“…The former contain iron, whereas the latter contain haem. Amongst prokaryotes, ferritins have been isolated from Bacteroides fragilis (Rocha et al, 1992), Escherichia coli (Hudson et al, 1993), Helicobacter pylori (Frazier et al, 1993) and Campylobacter jejuni (Wai et al, 1995(Wai et al, , 1996. In addition, ferritin-encoding genes have been found in the genomes of Haemophilus influenzae, Methanobacterium thermo-autotrophicum, Clostridium acetobutylicum, Thermotoga maritima, Archaeoglobus fulgidus, Mycobacterium tuberculosis and Vibrio cholerae (Andrews, 1998).…”

“…1a). All of the fractions in which the ferritin-like particles, exhibiting a spherical structure with an inner electron-dense core, were observed contained a protein with a molecular mass of 18 kDa, which was the expected molecular mass of ferritin (Frazier et al, 1993 ;Hudson et al, 1993 ;Wai et al, 1995 MKISENVTKAINDQIKAEM, which is 58 % identical to the amino-terminal sequence of Bact. fragilis ferritin (Rocha et al, 1992).…”