For B. subtilis
, it has been well established that the alternative sigma factor σB
governs the transcription of general stress proteins and the regulation of σB
activity through a partner-switching mechanism (25
). This mechanism is most likely employed in B. cereus
, in which proteins encoded by the sigB
cluster are known to be involved in the regulation of σB
activity, with the exception that the role of orf4
is currently unclear (46
). Our data showed indistinguishable σB
activations upon 42°C heat stress for the wild-type strain and the orf4
-null mutant, suggesting that orf4
is not involved in the regulation of σB
activity (Fig. ). However, significant orf4
induction upon 42°C heat stress and 2.5% salt treatment implies orf4
induction relative to environmental stress (Fig. ). This postulation is supported by the fact that orf4
is a known member of the B. cereus
SigB regulon (47
). Notably, 4% ethanol exposure caused only a slight increase in Orf4 expression (Fig. ). This result is consistent with a previous study (46
) that showed less σB
induction by ethanol exposure than by salt and heat stress.
Two miniferritin/Dps genes are found in B. subtilis
and many other Bacillus
). Dps2/MrgA miniferritin is induced by peroxide via modification of the Per transcription factor by an Fe2+
-catalyzed reaction (31
), and Dps1/DpsA miniferritin is induced by general stress (heat, salt, and ethanol stress, and glucose starvation), which is σB
dependent during exponential growth (7
). The fact that Orf4 is induced by general stress but not by H2
treatment indicates that the regulation of orf4
is similar to that of Dps1/DpsA (Fig. ).
Although Orf4 shows approximately a 30% identity and 50% similarity with bacterioferritins (21
), Orf4 has some distinctive features in comparison with bacterioferritins. For example, unlike findings for most of the bacterioferritin homologs, which readily form tetracosamers, even in the absence of iron (20
), our results showed that rOrf4 has a predominant dimeric form and a polymer form (Fig. ), but the proportions of the dimeric and polymer forms varied slightly in different preparations. Interestingly, a similar observation was reported in overproduced E. coli
Bfr (bacterioferritin), which comprised a dimeric form and a tetracosameric form in various proportions (6
). The physiological significance of the dimeric form in Bfr is assumed to be participation in the release mechanism of iron stores, or alternatively it may have a function distinct from that of the tetracosameric form, e.g., electron transfer (6
). Consistent with these assumptions, our data showed a series of reactions, including ferrous oxidation and induction of rOrf4 assembly, in which conformational change must occur, and the entry of ferric iron into the nucleation core within the polymer after the addition of FeCl2
elucidates the necessity of iron for rOrf4 assembly and deaggregation through the interchangeable Fe state (Fig. and ).
Formation of the rOrf4 polymer can be facilitated rapidly by the addition of ferrous iron (Fig. ), and the amount of iron-induced rOrf4 polymer depends on the quantity of FeCl2. In contrast, the automated rOrf4 polymer can be formed at a relatively slow rate from dimeric rOrf4. The molecular mass of automated-assembly rOrf4 of ~669 kDa is distinct from that of bacterioferritins, whose molecular masses are estimated to be around 450 to 480 kDa. In accordance with this observation, transmission electron microscopy imaging revealed a filament structure in the automated rOrf4 polymer, which is different from the typical roughly spherical structure of bacterioferritins (Fig. ). The automated-assembly rOrf4 still retained the capacity to incorporate iron (data not shown).
Ferric irons were detected at a λ310 nm only in the fractions corresponding to rOrf4 polymers, but no absorbance was detected in the dimeric rOrf4 fractions (Fig. ). This result implies that rOrf4 is able to oxidize ferrous iron efficiently, thereby incorporating mineralized ferric iron into the rOrf4 polymer, as do bacterioferritins. However, rOrf4 exhibited a reaction that was most likely monophasic (Fig. ), as opposed to the biphasic kinetics of iron oxidation/incorporation in bacterioferritins (10
). Therefore, the rOrf4 polymer with the filament structure probably employed a distinct mechanism to coordinate storage of ferric irons within the intramolecular space compared to the storage of ferric iron in the nanocages of bacterioferritin homologs (10
Organisms develop complex strategies to protect cells from injury from oxidants upon oxidative stress. Dps-like proteins play important roles in protecting DNA, usually by the formation of Dps-DNA complexes (12
) and reduce the DNA degradation caused by iron-dependent free-radical generation by the Fenton reaction (53
). Orf4 showed homology (approximately a 27% identity and 40% similarity) with DpsA from the Synechococcus
strains PCC7947 and PCC6031 (21
) and was found to be able to protect DNA from degradation by the oxidative damage caused by H2
treatment through direct DNA binding in a nonspecific manner (Fig. ). Thus, Orf4 has a DNA binding property homologous with that of Dps. The DNA binding of Dps has been attributed to the presence of lysine-rich residues, resulting in a positively charged N terminus or requiring C-terminal extension (13
). Three lysine residues (Lys10
, and Lys18
) are situated at the N terminus of Orf4; however, evidence is still needed to identify whether these residues are involved in DNA binding.
Although our in vitro system demonstrated that rOrf4 can bind DNA and sequester iron, the cell protection provided by orf4
against oxidative stress resulted only in a 1.5- to 2-fold decrease in viability in the WT708 strain compared with that of the wild-type strain, which is not as significant as that of other essential antioxidation genes, including sigB
, and katA
in B. cereus
). We can interpret this result as wild-type B. cereus
expressing a barely detectable level of Orf4 without environmental stress during the logarithmic phase and as the H2
treatment failing to induce Orf4; therefore, the Orf4 level in the wild-type strain would be expected to be only slightly higher than that in WT708, which did not express Orf4. As a certain Orf4 level is required for cell protection upon environmental stress, the relatively smaller difference in Orf4 expression satisfactorily explains the small difference in cell viability. Moreover, the cell survival rates of WT710 and WT711 were elevated upon 1.5 mM t-BOOH treatment or 50°C heat stress, as cells preloaded with Orf4 constitutively expressed pRF305; this result further supports that the Orf4 level is critical to cell protection under oxidative stress.
We demonstrated in this study that Orf4 possesses the properties of ferroxidase activity and iron sequestration and prevents DNA from oxidative degradation in vitro and that Orf4 displays the ability to increase cell viability during environmental stress in vivo. In conclusion, Orf4 was characterized as a Dps-like bacterioferritin that is regulated in much the same way as Dps1/DpsA. These results suggest that Orf4 may have a distinct role in iron metabolism in response to environmental stress, rather than exerting direct activity against specific oxidative stress.