Figure 5. Modeled structure of Fpx. A domain-swapped homo- dimeric model was generated. The two polypeptide chains are shown in white and gray, and helices in the white chain are labeled. Binuclear metals are shown as black spheres. Loops connecting the helices aB and aC are indicated by arrows. doi:10.1371/journal.pone.0034825.g005 physiological role as a hydroperoxide scavenger, particularly in the presence of high levels of hydroperoxides. We demonstrated here that the fpx gene disruptant of H. thermophilus was unable to grow aerobically, whereas growth under anaerobic and microaerobic conditions was comparable to that of the WT strain. The observed growth profiles are consistent with the above hypothesis. The absence of an fpx gene in A. aeolicus, a microaerobic species related to H. thermophilus, also suggests the dispensability of Fpx for the detoxification of low amounts of ROS under microaerobic conditions. By homology modeling, domain-swapping was predicted to occur in Fpx. Moreover, domain-swapping was also observed in the crystal structure of SulE and P. furiosus Rbr [23,24,32]. All three of these homologs are harbored by thermophiles. It was reported previously that domain-swapped SulE is highly stable and is difficult to be dissociated. Hence, we speculate that a domain-swapped confor- mation may contribute to the structural stability of Rbr-like proteins in thermal environments. To confirm this possibility, the amino acid sequences of 507 proteins annotated as Rbr-like proteins were compared and analyzed. It was reported that the length of the loop connecting helices aB and aC of Rbr-like proteins determines whether domain-swapping occurs, with shorter loop resulting in domain swapping (Fig. S2) [23]. In the phylogenetic tree, the majority of the homologs possessed by thermophiles (groups II, III, and V) are predicted to be domain-swapped Rbr-like proteins (Fig. 6), whereas group VI including the probable non-domain- swapped homologs is composed of mainly mesophiles. In the constructed phylogenetic tree, groups I, II, and III are composed of Rprs and the single-domain Rbr-like proteins, including Fpx. Rpr is an Rbr-like protein that was originally isolated from C. acetobutylicum [12,26,33] and has atypical domain composition characterized by an Rdx-like domain at the N- terminus and a ferritin-like domain at the C-terminus. Thus, although both Rpr and typical Rbr proteins possess two domains, these proteins are predicted to have different evolutionary lineages. On the other hand, as typical Rbrs are only distributed in limited areas of the phylogenetic tree (groups IV to VII), the common ancestor of Rbr-like proteins not expected to be a two-domain- type protein. In addition, as suggested by the term ‘‘evolutionary tinkering’’, numerous proteins may have evolved by the re- An Essential Antioxidant in H. thermophilus combination of small and simple proteins [34]. Considering this process of molecular evolution, a protein that has a simpler domain composition is more likely to be an ancestral protein. Therefore, typical Rbr is predicted to have arisen through the fusion of an Rdx-like domain and the ancestor of single-domain Rbr-like proteins contained in group III. In a similar manner, Rpr is predicted to have arisen from the fusion of an Rdx-like domain and the ancestor of single-domain Rbr-like proteins in group II. If these hypotheses are correct, Fpx can be considered to be an ancestral type of Rbr. As a number of small ferritin-like superfamily (FLSF) proteins have been identified and investigated recently, the phylogenetic relationship between FLSF proteins has received increased attention [35–37]. In conclusion, this study has demonstrated the function of Fpx as an NADPH-dependent peroxidase that is indispensable for the aerobic growth of H. thermophilus, and provides new insights into the distribution and enzymatic properties of Rbr-like peroxidases. Further structural and phylogenetic studies on the Rbr-like proteins are expected to uncover the specificity for both redox partners and substrates, and the molecular evolution of Rbr-like peroxidases, respectively. The genetic and transcriptional analyses of the fpx gene disruptant and other mutants are currently underway in our laboratory for revealing the detailed physiological role of Fpx in H. thermophilus. Materials and Methods Bacterial strains, media, and plasmids H. thermophilus TK-6 (IAM 12695, DSMZ 6534) was cultivated at 70uC with CO2 as the sole carbon source in a 100-mL vial in an inorganic medium [15] for both aerobic and microaerobic conditions. For anaerobic (denitrification) conditions, the medium was supplemented with 4 g L21 sodium nitrate. The head space gas in the vial was replaced with gas mixtures of H2:O2:CO2 (75:10:15, v/v) for aerobic conditions, H2:O2:N2:CO2 (75:2:8:15, v/v) for microaerobic conditions, and H2:N2:CO2 (75:10:15, v/v) for anaerobic conditions. For solid culture, liquid medium containing 10 mg L21 of CuSO4?5H2O was solidified with 1.0% (w/v) of GELRITE (Wako Pure Chemical Industries, Osaka, Japan) [38]. Escherichia coli strains JM109 and BL21 (DE3) were used as cloning hosts for derivatives of pUC19 and pET21c (Novagen, Madison, WI), respectively. E. coli strains were routinely cultivated in Luria-Bertani (LB) medium at 37uC. When required, antibiotics were added to the medium at the following concentra- tions: 50 mg mL21 kanamycin and 100 mg mL21 ampicillin for E. coli; and 500 mg mL21 kanamycin for H. thermophilus [31]. Construction of plasmids for heterologous expression and homologous recombination For heterologous protein expression, the gene encoding Fpx (fpx, hth_1222, GenBank Accession Number BAI69676.1) was ampli- fied by PCR using the chromosomal DNA of H. thermophilus as a template with primer pairs, FpxE-F (59-TAAGGAGCTCTA- CATATGAAGAGTTTAGCAGG-39), containing an NdeI site (underlined) at the initiation codon, and FpxE-R (59- GGCTCAAGCTTTAGGCTTTTAG-39), containing an HindIII site (italics) after the stop codon. The nucleotide sequence of the obtained PCR product was confirmed on both strands. The PCR fragment was digested with NdeI and HindIII, and then ligated into NdeI-HindIII-digested pET21c. The resultant plasmid was designated pET-Fpx. For disruption of the fpx gene, the plasmid pFGD was constructed as follows: a 2-kb fragment carrying the fpx gene with KpnI sites located in the middle of the coding sequence was amplified from H. thermophilus chromosomal DNA by PCR PLoS ONE | www.plosone.org 5 April 2012 | Volume 7 | Issue 4 | e34825