In this work we show that iron response in S. oneidensis
is a rapid process at both molecular and physiological levels. The use of the membrane permeable iron chelator 2,2'-dipyridyl rapidly sequesters both intra- and extra-cellular iron [7
]. As a result, when cells were sampled after one minute of iron depletion, functional and regulatory genes of iron acquisition systems (HugA, TonB1, ExbB1, AlcA, SO4743 and SO2426) were induced greater than six fold. These genes appear to be direct targets of Fur as evidenced by the presence of "Fur boxes" in their promoters. Notably, Fur has a weak affinity for Fe(II) to form a repression complex [36
], allowing for a rapid and sensitive response to a change in iron concentration.
The identification of an anaerobic energy metabolism module for iron experiments conducted under aerobic conditions is intriguing. A straightforward explanation would be that gradually limiting oxygen induces an anaerobic response when bacteria are grown in shake flask cultures. However, we show here that S. oneidensis
continues to grow and cell density continues to increase during iron depletion, a condition which should further limit the concentration of oxygen and thus up-regulate the expression of genes as an anaerobic response. The expression of genes in the anaerobic energy metabolism module does not fit this model. Furthermore, a number of genes involved in aerobic response are induced by iron depletion (Table ), while genes involved in anaerobic response and not co-factored with Fe(II) are not induced by iron depletion. These genes include UbiC, chorismate lyase involved in the initial steps of ubiquinone biosynthesis; Ppc, phosphoenolpyruvate carboxylase synthesizing oxaloacetate; Pta and AceK, enzymes converting pyruvate to acetate during fermentation; GlpK, glycerol kinase synthesizing glycerol-3-phosphate; Tdh, threonine dehydrogenase involved in the supply of reducing potential; and glutamate decarboxylase (SO1769), whose E. coli
counterpart is highly induced by anaerobiosis [37
]. Therefore, an alternative explanation is that an anaerobic energy metabolism module may function as iron-storage proteins to release previously sequestrated iron in their protein products during iron depletion and thereby elevate the intracellular free iron pool. The use of non-essential, iron-cofactor proteins for iron homeostasis has been well documented in E. coli
]. Notably, a microarray study in E. coli
indicates that a large number of genes related to anaerobic energy metabolism (e.g., Hyb and Frd) function in iron storage [10
]. Such a mechanism seems to be present in S. oneidensis
Ten transcriptional regulators were grouped within the module of anaerobic energy metabolism, suggesting that they may be involved in this process. We were able to generate a mutant of one of those genes (SO1415) and to experimentally verify its role in anaerobic energy metabolism. Genome analyses reveal that S. oneidensis
has a large repertoire of transcriptional regulators, e.g., 88 two-component regulatory system proteins that enable the organism to adapt to a diversity of environmental conditions [12
]. Nevertheless, most of the transcriptional regulators remain unstudied. The grouping of transcriptional regulators in anaerobic energy metabolism is an exciting finding; understanding this process is crucial to the potential utilization of Shewanella
species to remediate U.S. Department of Energy's uranium-contaminated sites. In this regard, the rest of the transcriptional factors identified for anaerobic energy metabolism besides SO1415 are worthy of further investigation.
Bioinformatics analyses suggest that genes in the anaerobic energy metabolism module may be directly regulated by Crp. In E. coli
, Crp modulates different biological processes and responds to glucose levels as a global transcriptional factor [38
]. However, in S. oneidensis
, Crp plays a critical role in anaerobic energy metabolism [31
]. The identification of a Crp-binding site in this module provides a reasonable explanation for the function of Crp in multiple branches of anaerobic energy metabolism.
Transcriptomics and genetic studies suggest that protein degradation is involved in iron response. Lack of iron as a protein cofactor may impair the stability of a number of proteins. Induction of heat shock proteins may be necessary to process denatured or misfolded proteins. Intriguingly, no oxidative stress genes, such as Fe-superoxide dismutase (SodB) and genes in the SOS pathway (e.g
., RecA, RpoD, RpoH, LexA, SsB, UmuC and UmuD) [39
], were induced when iron was repleted. This is surprising since the excess of external iron is expected to provoke oxidative stress. It is possible that the concentration of iron used in this study is not sufficient to induce oxidative stress, or that S. oneidensis
had yet not responded to oxidative stress during the period of time examined. Another possibility is that S. oneidensis
employs novel pathways for oxidative stress response.
In E. coli
, the TCA enzymes SdhA and AcnA are controlled by the regulatory cascade of Fur and the small RNA RyhB. Consequently, they are repressed under iron-depleted conditions [10
]. This is not observed in S. oneidensis
(Table ). Indeed, we found that the expression of SdhA and AcnA in S. oneidensis
was regulated neither in the fur
], nor in a strain that over-expresses RyhB (Yang et al., unpublished results), suggesting that SdhA and AcnA are not regulated by Fur and RyhB in S. oneidensis
The regulation of iron acquisition genes by Fur is affirmed both by the presence of "Fur boxes" in the promoters and the abolishment of gene expression in fur
]. This mechanism is well conserved among γ-proteobacteria [1
]. In contrast, it appeared that the regulation of anaerobic energy metabolism and protein degradation modules was largely Fur-independent, as very similar sets of genes also were identified when a fur
mutant was exposed to iron depletion and repletion conditions [25
]. This implies that transcriptional regulators other than Fur are also essential for iron response. Indeed, Fur-independent regulation of gene expression by iron has been observed in E. coli
, V. cholera
, and H. Pylori
]. Nevertheless, we could not completely rule out the possibility of an indirect effect of Fur on anaerobic energy metabolism and protein degradation. It was notable that an earlier study showed that Crp was differentially expressed in a fur
], despite the fact that there was no obvious "Fur box" upstream of the Crp ORF.