Alignment of the c-Type Cytochrome OmcS along Pili of Geobacter sulfurreducens

Immunogold localization revealed that OmcS, a cytochrome that is required for Fe(III) oxide reduction by Geobacter sulfurreducens, was localized along the pili. The apparent spacing between OmcS molecules suggests that OmcS facilitates electron transfer from pili to Fe(III) oxides rather than promoting electron conduction along the length of the pili.There are multiple competing/complementary models for extracellular electron transfer in Fe(III)- and electrode-reducing microorganisms (8, 18, 20, 44). Which mechanisms prevail in different microorganisms or environmental conditions may greatly influence which microorganisms compete most successfully in sedimentary environments or on the surfaces of electrodes and can impact practical decisions on the best strategies to promote Fe(III) reduction for bioremediation applications (18, 19) or to enhance the power output of microbial fuel cells (18, 21).The three most commonly considered mechanisms for electron transfer to extracellular electron acceptors are (i) direct contact between redox-active proteins on the outer surfaces of the cells and the electron acceptor, (ii) electron transfer via soluble electron shuttling molecules, and (iii) the conduction of electrons along pili or other filamentous structures. Evidence for the first mechanism includes the necessity for direct cell-Fe(III) oxide contact in Geobacter species (34) and the finding that intensively studied Fe(III)- and electrode-reducing microorganisms, such as Geobacter sulfurreducens and Shewanella oneidensis MR-1, display redox-active proteins on their outer cell surfaces that could have access to extracellular electron acceptors (1, 2, 12, 15, 27, 28, 31-33). Deletion of the genes for these proteins often inhibits Fe(III) reduction (1, 4, 7, 15, 17, 28, 40) and electron transfer to electrodes (5, 7, 11, 33). In some instances, these proteins have been purified and shown to have the capacity to reduce Fe(III) and other potential electron acceptors in vitro (10, 13, 29, 38, 42, 43, 48, 49).Evidence for the second mechanism includes the ability of some microorganisms to reduce Fe(III) that they cannot directly contact, which can be associated with the accumulation of soluble substances that can promote electron shuttling (17, 22, 26, 35, 36, 47). In microbial fuel cell studies, an abundance of planktonic cells and/or the loss of current-producing capacity when the medium is replaced is consistent with the presence of an electron shuttle (3, 14, 26). Furthermore, a soluble electron shuttle is the most likely explanation for the electrochemical signatures of some microorganisms growing on an electrode surface (26, 46).Evidence for the third mechanism is more circumstantial (19). Filaments that have conductive properties have been identified in Shewanella (7) and Geobacter (41) species. To date, conductance has been measured only across the diameter of the filaments, not along the length. The evidence that the conductive filaments were involved in extracellular electron transfer in Shewanella was the finding that deletion of the genes for the c-type cytochromes OmcA and MtrC, which are necessary for extracellular electron transfer, resulted in nonconductive filaments, suggesting that the cytochromes were associated with the filaments (7). However, subsequent studies specifically designed to localize these cytochromes revealed that, although the cytochromes were extracellular, they were attached to the cells or in the exopolymeric matrix and not aligned along the pili (24, 25, 30, 40, 43). Subsequent reviews of electron transfer to Fe(III) in Shewanella oneidensis (44, 45) appear to have dropped the nanowire concept and focused on the first and second mechanisms.Geobacter sulfurreducens has a number of c-type cytochromes (15, 28) and multicopper proteins (12, 27) that have been demonstrated or proposed to be on the outer cell surface and are essential for extracellular electron transfer. Immunolocalization and proteolysis studies demonstrated that the cytochrome OmcB, which is essential for optimal Fe(III) reduction (15) and highly expressed during growth on electrodes (33), is embedded in the outer membrane (39), whereas the multicopper protein OmpB, which is also required for Fe(III) oxide reduction (27), is exposed on the outer cell surface (39).OmcS is one of the most abundant cytochromes that can readily be sheared from the outer surfaces of G. sulfurreducens cells (28). It is essential for the reduction of Fe(III) oxide (28) and for electron transfer to electrodes under some conditions (11). Therefore, the localization of this important protein was further investigated.     

Role of Geobacter sulfurreducens Outer Surface c-Type Cytochromes in Reduction of Soil Humic Acid and Anthraquinone-2,6-Disulfonate

Deleting individual genes for outer surface c-type cytochromes in Geobacter sulfurreducens partially inhibited the reduction of humic substances and anthraquinone-2,6,-disulfonate. Complete inhibition was obtained only when five of these genes were simultaneously deleted, suggesting that diverse outer surface cytochromes can contribute to the reduction of humic substances and other extracellular quinones.Humic substances can play an important role in the reduction of Fe(III), and possibly other metals, in sedimentary environments (6, 34). Diverse dissimilatory Fe(III)-reducing microorganisms (3, 5, 7, 9, 11, 19-22, 25) can transfer electrons onto the quinone moieties of humic substances (38) or the model compound anthraquinone-2,6-disulfonate (AQDS). Reduced humic substances or AQDS abiotically reduces Fe(III) to Fe(II), regenerating the quinone. Electron shuttling in this manner can greatly increase the rate of electron transfer to insoluble Fe(III) oxides, presumably because soluble quinone-containing molecules are more accessible for microbial reduction than insoluble Fe(III) oxides (19, 22). Thus, catalytic amounts of humic substances have the potential to dramatically influence rates of Fe(III) reduction in soils and sediments and can promote more rapid degradation of organic contaminants coupled to Fe(III) reduction (1, 2, 4, 10, 24).To our knowledge, the mechanisms by which Fe(III)-reducing microorganisms transfer electrons to humic substances have not been investigated previously for any microorganism. However, reduction of AQDS has been studied using Shewanella oneidensis (17, 40). Disruption of the gene for MtrB, an outer membrane protein required for proper localization of outer membrane cytochromes (31), inhibited reduction of AQDS, as did disruption of the gene for the outer membrane c-type cytochrome, MtrC (17). However, in each case inhibition was incomplete, and it was suggested that there was a possibility of some periplasmic reduction (17), which would be consistent with the ability of AQDS to enter the cell (40).The mechanisms for electron transfer to humic substances in Geobacter species are of interest because molecular studies have frequently demonstrated that Geobacter species are the predominant Fe(III)-reducing microorganisms in sedimentary environments in which Fe(III) reduction is an important process (references 20, 32, and 42 and references therein). Geobacter sulfurreducens has routinely been used for investigations of the physiology of Geobacter species because of the availability of its genome sequence (29), a genetic system (8), and a genome-scale metabolic model (26) has made it possible to take a systems biology approach to understanding the growth of this organism in sedimentary environments (23).     

Iron Is Involved in the Maintenance of Circadian Period Length in Arabidopsis

The homeostasis of iron (Fe) in plants is strictly regulated to maintain an optimal level for plant growth and development but not cause oxidative stress. About 30% of arable land is considered Fe deficient because of calcareous soil that renders Fe unavailable to plants. Under Fe-deficient conditions, Arabidopsis (Arabidopsis thaliana) shows retarded growth, disordered chloroplast development, and delayed flowering time. In this study, we explored the possible connection between Fe availability and the circadian clock in growth and development. Circadian period length in Arabidopsis was longer under Fe-deficient conditions, but the lengthened period was not regulated by the canonical Fe-deficiency signaling pathway involving nitric oxide. However, plants with impaired chloroplast function showed long circadian periods. Fe deficiency and impaired chloroplast function combined did not show additive effects on the circadian period, which suggests that plastid-to-nucleus retrograde signaling is involved in the lengthening of circadian period under Fe deficiency. Expression pattern analyses of the central oscillator genes in mutants defective in CIRCADIAN CLOCK ASSOCIATED1/LATE ELONGATED HYPOCOTYL or GIGANTEA demonstrated their requirement for Fe deficiency-induced long circadian period. In conclusion, Fe is involved in maintaining the period length of circadian rhythm, possibly by acting on specific central oscillators through a retrograde signaling pathway.Metals such as iron (Fe), copper (Cu), zinc (Zn), manganese (Mn), molybdenum, and nickel are essential for the various biological processes that govern plant growth and development (Marschner, 1995). For example, Fe is required for DNA synthesis, photosynthesis, nitrogen fixation, hormone synthesis, and electron transport in the respiratory chain (Briat and Lobreaux, 1997). Similarly, Cu is an important component of electron-transfer reactions mediated by proteins such as superoxide dismutase, cytochrome oxidase, and plastocyanin (Clemens, 2001). Zn is a cofactor for many enzymes, and many proteins contain Zn-binding structural domains (Clarke and Berg, 1998). Although only minimal quantities of these micronutrients are required by plants, their limited availability in soils can significantly hinder crop production and affect nutritional quality (Grotz and Guerinot, 2002). In the case of Fe, about 30% of arable land worldwide is considered calcareous, rendering Fe in these soils unavailable to plants (Mori, 1999). Understanding of the fundamental processes involving metal uptake and sequestration has increased in recent years, but how the availability of particular metals interacts with internal signals to govern the growth and development of plants is largely unknown.The daily biological rhythms of many organisms are regulated by a near 24-h circadian clock that is synchronized by environmental changes such as light and temperature (Harmer, 2009; Imaizumi, 2010). The circadian clock regulates diverse aspects of plant growth and development. The operation of the circadian clock in plants can basically be divided into three main parts, input, central oscillator, and output pathways, and each part has its own complex networks. In Arabidopsis (Arabidopsis thaliana), the central oscillator is composed of a network of multiple feedback loops that can be divided into the morning, central, and evening loops (Harmer, 2009). The central feedback loop is composed of the morning-expressed genes CIRCADIAN CLOCK ASSOCIATED1 (CCA1) and LATE ELONGATED HYPOCOTYL (LHY) and the evening-expressed gene TIMING OF CAB EXPRESSION1 (TOC1; Schaffer et al., 1998; Wang and Tobin, 1998; Strayer et al., 2000; Alabadí et al., 2001). Although TOC1 is genetically required for the activation of morning genes (Schaffer et al., 1998; Wang and Tobin, 1998; Strayer et al., 2000), it acts as a repressor and directly regulates the expression of CCA1 and LHY (Gendron et al., 2012; Huang et al., 2012; Pokhilko et al., 2012). In the morning loop, CCA1/LHY form another negative feedback loop with the morning genes PSEUDO-RESPONSE REGULATOR7 (PRR7) and PRR9, with PRR9/PRR7 directly repressing the expression of CCA1 and LHY (Farré et al., 2005; Nakamichi et al., 2010). In the evening loop, TOC1 forms a negative feedback loop with GIGANTEA (GI) by repressing its expression, and GI in turn activates the expression of TOC1 through an unknown component, Y (Huq et al., 2000; Mizoguchi et al., 2005). After receiving input signals in the form of environmental cues, the central oscillator of the Arabidopsis circadian clock generates various rhythmic outputs that control various physiological events (Hotta et al., 2007; de Montaigu et al., 2010).The central oscillator controls a range of important physiological output processes such as flowering, stress and hormone responses, and regulation of nutrient acquisition (Haydon et al., 2011). Although the uptake of nutrition in plants is known to be influenced by light and temperature (Lahti et al., 2005; Baligar et al., 2006), the interaction between nutritional status and the circadian clock is less well studied. The homeostasis of Cu is known to influence the regulation of oscillator genes (Andrés-Colás et al., 2010; Peñarrubia et al., 2010). Arabidopsis under excess Cu or overexpressing Cu transporters COPT1 and COPT3 showed increased Cu accumulation and reduced expression of CCA1, LHY, and circadian clock output genes. Defective developmental phenotypes were also observed in these plants. Spatial and temporal control of Cu homeostasis, therefore, may be important for plant environmental fitness (Andrés-Colás et al., 2010). It has also been reported that disordered circadian rhythm affects Fe homeostasis. Tight regulation of Fe homeostasis to maintain an optimal Fe level in plants has been found to be associated with circadian clock regulators such as TIME FOR COFFEE (TIC) that modulates the expression of the ferritin gene AtFer1 (Duc et al., 2009). The expression of AtFer1 was up-regulated with excess Fe. TIC could repress AtFer1 expression under low-Fe conditions in photoperiodic light and dark cycles (Duc et al., 2009). However, whether Fe status feeds back to regulate the circadian clock is uncertain.Although Fe homeostasis in terms of uptake and translocation has been studied for decades, Fe availability is still an agricultural problem worldwide. Revealing the interplay between Fe homeostasis and internal cues such as modulation of the circadian clock can help increase understanding of their contributions to overall plant development. In this work, we investigated the effect of Fe deficiency on the circadian clock and found that it lengthened the circadian period. Our data suggest that the functional status of chloroplasts under Fe deficiency may play a key role in the lengthened circadian period.