Ferritin mutants of Escherichia coli are iron deficient and growth impaired, and fur mutants are iron deficient

Abstract

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 and bfr::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 the ftnA 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 and fur mutants were confirmed by Mössbauer spectroscopy, which further showed that the low iron contents of ftnA mutants are due to a lack of magnetically ordered ferric iron clusters likely to correspond to FtnA iron cores. In combination with the fur mutation, ftnA and bfr mutations 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.

FIG. 1

Restriction maps of the ftnA (A) and bfr (B) regions of the E. coli chromosome and derived plasmids. The locations and polarities of the ftnA (clockwise) and bfr (anticlockwise) genes are shown in the E. coli physical map (8). Lightly shaded bars represent chromosomal DNA; thin horizontal lines represent plasmid DNA; solid bars represent antibiotic resistance cassettes (kan, kanamycin; spc, spectinomycin), and open bars represent the ftnA or bfr gene. Relevant restriction sites are shown: Ac, AccI; As, AsuII; B, BamHI; Bg, BglI; C, ClaI; D, DraI; H, HindIII; K, KpnI; Ps, PstI; Pv, PvuI; Xb, XbaI; Xh, XhoII. Ac/C and As/Ac denote hybrid restriction sites no longer recognized by the corresponding restriction enzymes, and B* denotes an engineered BamHI site flanking a site-directed internal deletion in the bfr gene.

FIG. 2

Western blots confirming the absence of FtnA and Bfr in the corresponding E. coli mutants. Bacteria were grown aerobically to stationary phase in L broth before being harvested. Whole-cell E. coli proteins (approximately 50 μg per lane) were electrophoresed in SDS-containing 15% polyacrylamide gels, electroblotted, and immunostained with anti-FtnA (A) or anti-Bfr (B) polyclonal serum. Lanes: 1, W3110 (wild type); 2, JRG2951 (bfr::kan); 3, JRG2952 (ftnA::spc); 4, JRG2953 (ftnA::spc bfr::kan). The positions of the immunoreactive polypeptides corresponding to FtnA and Bfr are indicated.

FIG. 3

Iron contents of wild-type and mutant strains of E. coli after aerobic growth. The iron contents (percentage of dry weight) are for E. coli in logarithmic phase (A₆₅₀ = 0.3 to 0.6: open bars in panels A to C) or stationary phase (A₆₅₀ = 1.5 to 3.0: solid bars in panels A to C and E; open and solid bars in panel D). Error bars denote standard deviations. The growth media were L broth (17 μM iron) (A), 8-hydroxyquinoline-extracted L broth (3 μM iron) (B), glucose (0.4%) M9-salts medium (1.8 μM iron) supplemented with either 400 μM sodium citrate (cit) or increasing concentrations of iron citrate (0 to 128 μM) (C), glucose (0.4%) M9-salts medium (1.8 μM iron) with no added iron (open bars) or with 16 μM added iron citrate (solid bars) (D), and L broth (17 μM iron) (E).

FIG. 4

Mössbauer spectra of E. coli. Wild-type and mutant E. coli were grown in minimal medium containing 0.4% glucose and 8 μM ⁵⁷Fe citrate. Fits are superimposed on the experimental data. (A) Mössbauer spectra recorded at 60 K. Component A represents ferric iron, whereas B and C correspond to ferrous iron. (B) Mössbauer spectra recorded at 1.7 K. Component A′ represents nonmagnetic ferric iron, component B′ represents total ferrous iron, and component M (shaded) corresponds to magnetic ferric iron cores. The parameters for components A, A′, B, B′, C, and M are listed in Tables 2 and 3. (C) Mössbauer spectra recorded at 267 K.

FIG. 5

Effects of iron deficiency on the aerobic growth of E. coli wild type and iron storage mutants. The growth medium was 0.4% glucose M9-salts minimal medium (1.8 μM iron). Precultures were grown with 16 μM iron citrate (A) or 1.6 mM sodium citrate (B) and were washed with saline before inoculation at dilutions of 1/100. Error bars represent standard deviations of three cultures: ▵, W3110; ○, JRG2951 (bfr); ◊, JRG2952 (ftnA); □, JRG2953 (ftnA bfr).

FIG. 6

Effects of the iron chelator DTPA on the aerobic growth of E. coli wild-type and iron-storage mutants. Details are as for Fig. 5A except where stated. (A) Effects of DTPA (5 μM). (B) Reversal of the effects of 5 μM DTPA by iron citrate. W3110 is indicated by solid symbols, and JRG2953 (ftnA bfr) is indicated by open symbols, with iron citrate concentrations as follows: ◊ and ⧫, 0.5 μM; ▵ and ▴, 1.5 μM; □ and ■, 5 μM; and ○ and ●, 16 μM. (C) Effect of low-iron preculture on subsequent growth in 5 μM DTPA: precultures were grown with 1.6 mM sodium citrate instead of 16 μM iron citrate, as in Fig. 5B. (D) Comparison of log-phase (open symbols) and stationary-phase (solid symbols) preculture on subsequent growth in 5 μM DTPA: ▴ and ▵, W3110; ■ and □, JRG2953 (ftnA bfr).

FIG. 7

Western blot analysis of FtnA in MC4100 (fur⁺) and H1941 (Δfur). Strains were grown aerobically to stationary phase in L broth before being harvested. Whole-cell E. coli proteins (approximately 50 μg per lane) were electrophoresed in SDS-containing 15% polyacrylamide gels, electroblotted, and immunostained with anti-FtnA polyclonal serum.

FIG. 8

Effects of intracellular and extracellular iron on hydrogen peroxide toxicity. (A) Washed suspensions of W3110 grown to stationary phase in 0.4% glucose M9-salts medium with 1.6 mM sodium citrate or 16 μM iron citrate, to produce inocula with low (0.002%) or high (0.018%) intracellular iron contents, respectively, were diluted 100-fold in L broth containing 250 μM H₂O₂ (solid lines) or no H₂O₂ (broken lines): low-iron inocula (□); high-iron inocula (▴). (B) Low-iron inocula (as above) of W3110 were diluted 100-fold in fresh sodium citrate-containing glucose M9-salts medium with 50 μM H₂O₂ (solid lines) or no H₂O₂ (broken lines) and 16 μM iron citrate (▴) or no iron citrate (□).

FIG. 9

Effects of hydroperoxides on the growth of ferric uptake regulation and iron storage mutants. Stationary-phase inocula grown in L broth were diluted 20-fold into microtiter plates containing 200 μl of L broth with 5 mM H₂O₂ (A), 2 mM tert-butyl hydroperoxide (B), 30 μM cumen hydroperoxide (C), or no additions (D). Aerobic growth at 37°C and 400 rpm was monitored with an iEMS microtiter plate reader. Strains: ▴, H1941 (fur); ●, JRG3236 (fur bfr); ◊, JRG3238 (fur ftnA); and □, JRG3240 (fur ftnA bfr).

FIG. 10

Schematic representation of iron metabolism in E. coli under iron-sufficient and -deficient conditions. The regulatory role of Fur is indicated by the thin arrows and by the +ve (activation) and −ve (repression) signs. Iron flux is indicated by the thick arrows. The abundances of ferritin, Fur, intracellular Fe(II), and iron-containing proteins are indicated schematically. Three intracellular sources of iron are shown: free iron, FtnA iron, and iron released from intracellular iron proteins.