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Extracellular lactate was quantified in supernatant by HPLC using a SpectraSERIES P100 pump equipped with a SpectraSystem RI-150 detector and an Aminex HPX-87 column C18 (Bio-Rad, France). The column temperature was 37��C, and eluent (H2SO4, 0.005 N) was used at a flow rate of 0.6 mL min-1. Results Predicted Function and Structure of the Organic Acid Oxidation Region Genes A schematic representation of the DvH organic acid oxidation region consisting of nine open reading frames (ORFs) is represented in Figure ?Figure11, and the corresponding annotations1 are reported in Table ?Table11. Certain ORFs are annotated as the encoding enzymes that are most likely involved in the phosphoroclastic reaction, including DVU3025 (also called por), DVU3029 and DVU3030, which encode PFOR, Pta and Ack, respectively. A sequence analysis of the Pta (DVU3029) revealed the presence of three conserved domains: a catalytic PTA_PTB protein domain, which is found in all Pta, and AAA and DRTGG domains, which are only found in class II enzymes (Campos-Bermudez et al., 2010). DVU3026 encoded a putative lactate permease; however, annotation of the remaining ORFs was unclear: DVU3031 encoded a conserved hypothetical protein consisting of the AAA and DRTGG domains but without a PTA_PTB protein domain, thus excluding putative Pta activity for this protein. DVU3027 and 3028 were annotated as a glycolate oxidase subunit and iron-sulfur cluster-binding protein encoding gene, respectively. However, their amino acid sequences suggested that they corresponded to two subunits of a flavin- and iron sulfur-containing oxidoreductase homolog of the monomeric D-iLDH (Dld-II), which is characterized in Shewanella oneidensis (Pinchuk et al., 2009) as already proposed in Pereira et al. (2011). Despite the low pairwise sequence identity (17% sequence identity, Supplementary Figure S1), DVU3027-28 consisted of the same protein domains and motifs, including the FAD-binding domain (Pfam accession no. PF01565), FAD-linked oxidase domain (PF02913), the 4Fe-4S dicluster domain (PF13183) and CCG domain (PF02754), (Figure ?Figure22). The C-terminal FAD-linked oxidase domain of DVU3027 contained a sequence close to the motif GEHGD and an essential histidine conserved in enzymes that bind lactate (Griffin et al., 1992). DVU3032 and DVU3033 were annotated as a conserved hypothetical protein and iron sulfur cluster-binding protein, respectively. However, their amino-acid sequences shared 26% amino acid sequence identity with the three subunits of the non-flavin iron-sulfur containing oxidoreductase (learn more LldEFG, Supplementary Figure S2) of S. oneidensis (Pinchuk et al., 2009). Moreover, both proteins shared the same multi-domain composition, which is shown in Figure ?Figure22.