Environmental Microbiology (2011) doi:10.1111/j.1462-2920.2011.02626.x Minireview Genomic insights into microbial iron oxidation and iron uptake strategies in extremely acidic environmentsemi_2626 1..15 Violaine Bonnefoy1,2* and David S. Holmes3 tiple iron uptake systems. This could be an adaption 1Laboratoire de Chimie Bactérienne, UPR-CNRS 9043, allowing them to respond to different iron con- Institut de Microbiologie de la Méditerranée, Marseille, centrations via the use of a multiplicity of different France. siderophores. Both Leptospirillum spp. and Acidithio- 2Aix-Marseille Université, Marseille, France. bacillus spp. are predicted to synthesize the acid 3Center for Bioinformatics and Genome Biology, stable citrate siderophore for Fe(III) uptake. In addi- Fundación Ciencia para la Vida and Depto. de Ciencias tion, both groups have predicted receptors for Biologicas, Facultad de Ciencias Biologicas, siderophores produced by other microorganisms, Universidad Andres Bello, Santiago, Chile. suggesting that competition for iron occurs influenc- ing the ecophysiology of acidic environments. Little is known about the genetic regulation of iron oxidation Summary and iron uptake in acidophiles, especially how the use This minireview presents recent advances in our of iron as an energy source is balanced with its need to understanding of iron oxidation and homeostasis in take up iron for metabolism. It is anticipated that inte- acidophilic Bacteria and Archaea. These processes grated and complex regulatory networks sensing dif- influence the flux of metals and nutrients in pristine ferent environmental signals, such as the energy and man-made acidic environments such as acid mine source and/or the redox state of the cell as well as the drainage and industrial bioleaching operations. Acido- oxygen availability, are involved. philes are also being studied to understand life in extreme conditions and their role in the generation of Introduction biomarkers used in the search for evidence of existing or past extra-terrestrial life. Iron oxidation in acido- Scope of this minireview philes is best understood in the model organism This minireview focuses on iron-oxidizing microorganisms Acidithiobacillus ferrooxidans. However, recent func- that inhabit acidophilic environments. There are a number tional genomic analysis of acidophiles is leading to a of other reviews and papers that discuss issues related to deeper appreciation of the diversity of acidophilic iron- life in extremely acidic conditions that will not be covered oxidizing pathways. Although it is too early to paint a here, including the occurrence and composition of acido- detailed picture of the role played by lateral gene philic communities (Gonzalez-Toril et al., 2003; Rawlings transfer in the evolution of iron oxidation, emerging and Johnson, 2007; Johnson, 2008; Demergasso et al., evidence tends to support the view that iron oxidation 2010), resistance to low pH and metal homeostasis arose independently more than once in evolution. (Baker-Austin and Dopson, 2007; Franke and Rensing, Acidic environments are generally rich in soluble iron 2007; Dopson, 2010), reduced inorganic sulfur com- and extreme acidophiles (e.g. the Leptospirillum pounds (RISCs) energetics (Holmes and Bonnefoy, 2007; genus) have considerably fewer iron uptake systems Quatrini et al., 2009; Bonnefoy, 2010) and genomics and compared with neutrophiles. However, some acido- metabolic reconstruction (Tyson et al., 2004; Valenzuela philes have been shown to grow as high as pH 6 and, in et al., 2006; Holmes and Bonnefoy, 2007; Jerez, 2007; the case of the Acidithiobacillus genus, to have mul- 2008; Quatrini et al., 2007a; 2009; Valdes et al., 2008; Siezen and Wilson, 2009; Bonnefoy, 2010; Denef et al., Received 17 June, 2011; accepted 16 September, 2011. *For corre- spondence. E-mail [email protected]; Tel. (+33)4911641 2010) including genomics of over 50 acidophiles (Carde- 46; Fax (+33)491718914. nas et al., 2010). © 2011 Society for Applied Microbiology and Blackwell Publishing Ltd 2 V. Bonnefoy and D. S. Holmes Acidophiles and their environments hydroxides (as low as 10-18 M at pH 7.0), rendering it difficult to access by biological systems. In contrast, under We define ‘extreme acidophiles’ as those organisms acidic conditions Fe(II) is stable even in the presence of whose growth optimum is lower than pH 3. Most acido- atmospheric oxygen. This provides an opportunity for philes belong to the eubacterial or archaeal kingdoms microorganisms to use Fe(II) as an electron donor and as (prokaryotes) (Johnson, 1998; 2007; 2009; Hallberg and a source of energy that is not readily available to micro- Johnson, 2001; Johnson and Hallberg, 2003), although organisms living in circum-neutral environments. unicellular eukaryotes have also been detected in some However, extremely acidic environments can also chal- acidic environments (e.g. Lopez-Archilla et al., 2001; lenge microorganisms with the highest levels of soluble Amaral Zettler et al., 2002; Baker et al., 2009; Johnson, iron and the threat that this imposes on life via the reaction 2009; Cid et al., 2010). of Fe(II) with oxygen generating free radicals (Fenton Acidophilic microorganisms inhabit pristine environ- reaction) that damage macromolecules and cause cell ments such as volcanic, geothermal areas and acid rock death (Touati, 2000). drainage (ARD) (Lopez-Archilla et al., 2001; Gonzalez- Toril et al., 2003). They are also found in acidic environ- ments of anthropogenic origin such as bioleaching heaps Phylogeny of iron oxidizers and acid mine drainage (AMD) (Rawlings, 2002; 2007; Baker and Banfield, 2003; Johnson and Hallberg, 2003; The ability to oxidize iron is widely distributed in acido- Johnson, 2007; 2009; Norris, 2007; Schippers, 2007; philic Bacteria and Archaea (Fig. 1) (Weber et al., 2006; Schippers et al., 2010). Acidophiles are found over a wide Emerson et al., 2010; Hedrich et al., 2011). Until now, the range of temperatures and include psychrotolerants (but majority of known acidophilic iron oxidizers are found in not psychrophiles) that can grow down to 4°C (Harrison, the Nitrospira class as well as in Gram-positive (Firmicute 1982; Johnson et al., 2001; Dopson et al., 2007; Hallberg and Actinobacteria) and archaeal (Crenarchaeota et al., 2010), mesophiles (up to 40°C), moderate thermo- and Euryarchaeota) phyla. In Proteobacteria, only five philes (between 40°C and 55°C) and extreme thermo- acidophiles are encountered (‘Ferrovum myxofaciens’, philes (above 55°C) (Hallberg and Johnson, 2001; Norris, Acidiferrobacter thiooxydans,‘Thiobacillus prosperus’, 2007; Schippers, 2007; Johnson, 2009). Many acidophilic Acidithiobacillus ferrivorans and At. ferrooxidans) prokaryotes are obligate chemolithoautotrophs obtaining (Hedrich et al., 2011). It is not yet known whether this their energy and electrons from the oxidation of ferrous distribution reflects real evolutionary trends or merely iron [Fe(II)] and RISCs (Hallberg and Johnson, 2001; results from an incomplete analysis due to biases of Rawlings, 2005; 2007; Johnson and Hallberg, 2008; sampling that have relied mainly on culture-based approaches. Johnson, 2009) and their carbon by fixing CO2 from the atmosphere (Rawlings, 2007; Johnson and Hallberg, Widespread distribution of iron oxidation capabilities has also been observed in microorganisms from circum- 2008). Some also fix atmospheric N2 (Johnson, 2007; Rawlings, 2007). Heterotrophs have also been detected neutral pH environments. However, as opposed to the that scavenge organic carbon compounds produced by acidophiles, there seems to be an enrichment of iron the autotrophs (Hallberg and Johnson, 2001; Rawlings, oxidizers in the Proteobacteria (Emerson et al., 2010). 2002; Johnson and Hallberg, 2003; Johnson, 2007; 2009; This minireview will focus mainly on iron oxidation in Norris, 2007; Schippers, 2007; Nancucheo and Johnson, acidic environments. Microbial iron oxidation in circum- 2010; Schippers et al., 2010). neutral environments has been reviewed recently (Emerson et al., 2010; Bird et al., 2011) and will not be discussed here, except for comparison with acidophilic Problems of high iron load in acidic conditions pathways. Acidic environments provide a special opportunity and at the same time an unusual challenge for life. Natural Fe(II) iron oxidation geomicrobiological processes and industrial operations, Bioenergetic problems of iron oxidation such as bioleaching, convert insoluble metal sulfides into water-soluble metal sulfates that include extraordinarily When growing on Fe(II), autotrophs are confronted with high concentrations of soluble iron that can reach values a crucial bioenergetic problem. The reduction potential of as high as 160 g l-1, a concentration about 1016 higher the Fe(II)/Fe(III) couple is +0.78 V which is just below than typically found in circum-neutral environments. In that of O2/H2O (1.12 V) at the pH of the external medium oxygen (O2)-saturated environments at neutral pH, Fe(II) (pH 2), consequently, there is little energy available from is rapidly oxidized to ferric iron [Fe(III)]. Thus, iron pre- the oxidation of Fe(II) (Ingledew, 1982; Bird et al., 2011). dominantly occurs in the ferric form as poorly soluble iron Also, since the reduction potential of NAD+ (-0.32 V at © 2011 Society for Applied Microbiology and Blackwell Publishing Ltd, Environmental Microbiology Iron oxidation and iron homeostasis in acidophiles 3 Fig. 1. An inferred phylogenetic tree of acidophilic iron-oxidizing Bacteria