To analyze whether MgFur has iron-responsive regulatory functions in
M. gryphiswaldense as well and to clarify the role of Fur in the biomineralization of
magnetosomes, an unmarked fur mutant strain of M. gryphiswaldense was constructed by a
cre-lox-basedmethod (Marx and Lidstrom, 2002), resulting in an unmarked in-frame deletion
of fur. TEM analysis showed that the fur mutant strain RU-1 was still able to produce magnetosomes, although with diameters (28.6± 9.1 nm) and in numbers (40 ± 14.3 per cell) significantly reduced compared to those for the WT (46 ±16.1 magnetosomes per cell and 30.6 nm ± 9.0 nm in diameter)(Mann-Whitney test; P 0.003) (FIG. 2-3).
FIG. 2-3. Transmission electron micrographs of the WT (A) and RU-1 (B). Bar, 100 nm. (C) Magnetite crystal size distribution determined from 200 cells by TEM. (D) Distribution of magnetosome number per cell.
For further characterization, both strains were iron deprived by three passages in LIM supplemented with 10 µM 2,2'-dipyridyl, a medium supporting growth but not magnetite synthesis, until cellular magnetism was nolonger detectable and subsequently inoculated into fresh LIM containing different iron concentrations to reinduce magnetite biomineralization. Under aerobic and anaerobic growth conditions,growth rates of the fur mutant were slightly lower than those of the WT, whereas under microaerobic conditions, RU-1 showedgrowth rates similar to those of the WT at all tested iron concentrations(Table 2-2).
Table 2-2. Growth rate (h-1) and doubling time (in h) of WT and RU-1 grown under different oxygen and iron
concentrationsa
Resulta under the following culture conditions
2% O2 and: 0% O2 and 21% O2 and Strain Parameter
5 µM
Fe citrate Fe citrate 50 µM Fe citrate 250 µM Fe citrate 50 µM Fe citrate 50 µM µ (h-1) 0.169 (± 0.010) (± 0.001) 0.162 (± 0.010) 0.165 (± 0.015) 0.148 (± 0.013) 0.121 WT tD (h) (± 0,24) 4.10 (± 0.02) 4.29 (± 0.26) 4.19 (± 0.68) 4.68 (± 0.60) 5.73 µ (h-1) 0.167 (± 0.003) (± 0.001) 0.158 (± 0.019) 0.149 (± 0.003) 0.124 (± 0.002) 0.090 RU-1 tD (h) (± 0.07) 4.16 (± 0.00) 4.40 (± 0.59) 4.64 (± 0.14) 5.60 (± 0.17) 7.70 a Values are the sample means of at least replicate cultures. Sample standard deviations are in parentheses
However, magnetosome formation in RU-1 became detectable by Cmag only about 3 h after that in the WT (FIG. 2-4B). This delay was also observed with increased extracellular concentrations of ferric citrate (250 µM) (FIG. 2-4D). When RU-1 was cultured under anaerobic conditions, no difference in time course ofmagnetite formation was observed (FIG. 2-4E). In addition, the maximal Cmag values that were reached by RU-1 were significantly smaller than those reached by the WT. These differences weremost pronounced (RU-1 Cmag reached only 40% of WT Cmag) at lowiron concentrations (5 µM). Although Cmag values of thefur mutant increased with extracellular iron concentration toup to 85% of the WT value with 250 µM ferric citrate andunder anaerobic conditions, they never did reach WT levels underany condition tested (FIG. 2-4F).
FIG. 2-4. (A to E) Levels of growth (optical density [OD] at 565 nm) and magnetic response (Cmag) of the WT
and RU-1 grown under different conditions. (F) Relative maximal Cmag of RU-1 grown in LIM, determined from
results shown in panels B to E. Data are from representative experiments done in duplicate. The entire experiment was repeated three times, with comparable results. Values are given as means ± standard deviations.
Consistent with TEM and Cmag data, the total intracellular iron content after microaerobic
growth with 50 µM iron was also reduced in the fur mutantby 50% compared to the level for the WT (FIG. 2-5A). Transcomplementation of the fur mutant by a WT fur allele on pBBR1MCS-2fur resultedin partial restoration of the WT iron content (see FIG. S2-3 inthe supplemental material).
FIG. 2-5. (A) Time courses of total intracellular iron content of the WT and RU-1 during growth in FSM under microaerobic conditions. Values are given as means ± standard deviations (SD) from three independent
replicates. (B) Iron-to-protein ratios of WT and RU-1 nonmagnetic cytoplasmic and membrane-enriched protein fractions. Values are given as means ± SD from two independent replicates.
Unlike in other Magnetospirillum species (Paoletti and Blakemore, 1986; Calugay et al., 2003), no siderophorescould be detected in M. gryphiswaldense under any tested conditionin previous studies. However, although there is no clear genomic indication of siderophore synthesis, we cannot entirely exclude the possibility of the synthesis and use of primary catecholate-likemetabolites as siderophores under some unspecified conditions,as described
for M. magneticum (Calugay et al., 2006). Since fur mutants of several other bacteria (e.g.
Pseudomonas aeruginosa and Shewanella oneidensis) showed increased or constitutive
production of siderophores (Prince et al., 1993; Thompson et al., 2002), we reassessed siderophore production using the CAS decoloration assay with supernatants from cultures grown underdifferent iron concentrations and a modified plate growth CASassay. However, again we were unable to detect siderophore productioneither in the fur mutant or in the WT (data not shown).
As fur mutants of E. coli are known to be susceptible to higheriron concentrations than the WT (Touati et al., 1995), we also checked for increasedsensitivity of M. gryphiswaldense
RU-1 to various metals (Fe, Mn, and Co) at different concentrations. Dose-response assays under microaerobic conditions revealed identical growth yieldsfor the WT and RU-1 between 0 and 500 µM iron as well as 0 and 2 mM manganese (see FIG. S2-4A and B in the supplementalmaterial). Only at very high concentrations of iron (>1,000µM) and manganese (>3 mM) was growth of RU-1 increasinglyinhibited relative to that of the WT. No differences with respectto growth yield could be observed in the presence of Co (5 to100 µM) (data not shown). These data indicate a differentrole for MgFur than for EcFur.
Since growth of M. gryphiswaldense RU-1 was impaired under aerobicconditions, we also compared the sensitivities of the WT andRU-1 strains against the superoxide-producing agent paraquat(Bus et al., 1974). Growth in the presence of 5 µM paraquat resultedin growth yields of the WT being reduced by 40%, whereas growthof RU-1 was inhibited by 60% (see FIG. S2-4C in the supplemental material). At higher paraquat concentrations, both strains were equally inhibited. These results suggest that MgFur might beinvolved in the oxidative stress response of M. gryphiswaldense.