Escherichia coli contains at least two iron storage proteins, a ferritin (FtnA) and a bacterioferritin (Bfr). To investigate their specific functions, the corresponding genes (ftnA and bfr) were inactivated by replacing the chromosomal ftnA and bfr genes with disrupted derivatives containing antibiotic resistance cassettes in place of internal segments of the corresponding coding regions. Single mutants (ftnA::spc andbfr::kan) and a double mutant (ftnA::spc bfr::kan) were generated and confirmed by Western and Southern blot analyses. The iron contents of the parental strain (W3110) and the bfr mutant increased by 1.5- to 2-fold during the transition from logarithmic to stationary phase in iron-rich media, whereas the iron contents of theftnA and ftnA bfr mutants remained unchanged. The ftnA and ftnA bfr mutants were growth impaired in iron-deficient media, but this was apparent only after the mutant and parental strains had been precultured in iron-rich media. Surprisingly, ferric iron uptake regulation (fur) mutants also had very low iron contents (2.5-fold less iron than Fur+ strains) despite constitutive expression of the iron acquisition systems. The iron deficiencies of the ftnA andfur mutants were confirmed by Mössbauer spectroscopy, which further showed that the low iron contents of ftnAmutants are due to a lack of magnetically ordered ferric iron clusters likely to correspond to FtnA iron cores. In combination with thefur mutation, ftnA and bfrmutations produced an enhanced sensitivity to hydroperoxides, presumably due to an increase in production of “reactive ferrous iron.” It is concluded that FtnA acts as an iron store accommodating up to 50% of the cellular iron during postexponential growth in iron-rich media and providing a source of iron that partially compensates for iron deficiency during iron-restricted growth. In addition to repressing the iron acquisition systems, Fur appears to regulate the demand for iron, probably by controlling the expression of iron-containing proteins. The role of Bfr remains unclear.

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...

The bacterioferritin (BFR) of Escherichia coli is an iron-sequestering haemoprotein composed of 24 identical polypeptide chains forming an approximately spherical protein shell with a central iron-storage cavity. BFR and BFR-A, a variant with a 14-residue C-terminal extension, have been amplified (120-fold and 50-fold, respectively), purified by a new procedure and characterized. The overproduced BFR exhibited properties similar to those of natural BFR, but the iron content (25-75 non-haem Fe atoms/molecule) was 13-39-fold lower. Two major assembly states of BFR were detected, a 24-subunit protein (tetracosamer) and a novel haem-containing subunit dimer. BFR-L subunits assembled into tetracosamers having the same external-surface properties as BFR, presumably because their C-terminal extensions project into and occupy about 60% of the central cavity.As a result, BFR-A failed to take up iron under conditions that allowed incorporation into BFR in vitro. The haem content of BFR-A (1-2 haemshetracosamer) was lower than that of BFR (3.5-10.5 haems/tetracosamer) and this, together with a difference in the visible spectra of the two haemoproteins, suggested that the C-terminal extensions in BFR-A perturb the haem-binding pockets. A subunit dimer form of BFR-A was not detected. A combination of Mossbauer spectroscopy and electron diffraction showed that the BFR loaded with iron in vitro has a ferrihydrite-like iron core, whereas the in-vivo loaded protein has an amorphous core.

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