Introduction After publication of this work, it was brought to our attention that it has not been tested whether R. palustris CGA009 and its derivative strains can use dimethyl sulfoxide (DMSO) as an electron sink (Luxem et al., 2020). Here, we report the results of additional bioinformatic, physiological, and chemical experiments to test whether R. palustris CGA009 can use DMSO as an electron acceptor. The results are ambiguous. Bioinformatic analysis did not identify any proteins known to reduce DMSO, nor did the physiological experiments demonstrate growth with DMSO as the exclusive electron sink. Still, R. palustris CGA009 cultures do produce the product of DMSO respiration, dimethyl sulfide (DMS), exclusively in the presence of DMSO. Here, we discuss the impacts on our initial data interpretation as well as the implications for future researchers interested in using DMSO as an external electron sink for R. palustris CGA009. Results We began by searching for the presence of proteins with potential DMSO reduction capability in the R. palustris CGA009 genome. DMSO reduction activity has been demonstrated in several clades of the complex-iron–sulfur-molybdenum (CISM) protein superfamily, including the closely related DMSO, trimethylamine n-oxide (TMAO) and biotin sulfoxide (BSO) reductases that together form the DMSO reductase (DMSOR) protein family (Gon et al., 2000; McCrindle et al., 2005; Kappler and Schäfer, 2014; Kappler et al., 2019). These reductases vary in their selectivity for their substrates and in whether they are involved in anaerobic respiratory processes (Iobbi-Nivol et al., 1996; Pollock and Barber, 1997; Gon et al., 2000; McEwan et al., 2002; Ezraty et al., 2005; McCrindle et al., 2005; Kappler et al., 2019). No annotations to DMSO or TMAO reductases were found in the NCBI or KEGG databases for R. palustris CGA009 (accessed April 2020). Because of the broad substrate specificity of many enzymes in the DMSOR family, it is possible that there are enzymes capable of DMSO reduction whose annotations are less precise (Kappler et al., 2019). To identify such enzymes, we used BLAST (blastp) to search for homologs of known DMSO reductases from closely related strains in the R. palustris CGA009 genome. The query sequences included DorA from R. capsulatus 37b4 (Knäblein et al., 1996; Shaw et al., 1996), DorA and TorA from Rhodobacter spheroides 2.4.1 (Mouncey et al., 1997; Laguna, 2010) and Rhodopseudomonas palustris BisB18 (Oda et al., 2008), the other members of the DMSO reductase operons from R. palustris BisB18 and, for completeness, representative members from throughout the CISM superfamily compiled by (Leu et al., 2020) (Table S1). This search resulted in eleven possible hits (Table S1). To assess the likelihood that these hits are putative DMSO reductases, we made a phylogenetic tree for the CISM superfamily (Fig. 1). None of the hits from R. palustris CGA009 grouped with the Type II DMSOR family enzymes, which include the versatile sulfur- and nitrogen-oxide reductase DmsA. Four of the hits clustered as a sister group to known formate dehydrogenases while one formed an outgroup to the clade containing formate dehydrogenases and arsenate, arsenite, selenate and sulfur reductases. The remaining five hits formed a basal outgroup to all clusters within the superfamily, along with xanthine dehydrogenase (Xdha) from Paenibacillus mucilaginosus, which has not, to our knowledge, been observed to reduce DMSO. Only the BisC homologue RPA4653 (WP_011160185) clusters in a clade with proteins known to reduce DMSO, the Type III DMSOR family enzymes. The primary function of BisC, the catalytic subunit of the biotin sulfoxide (BSO) reductase, appears to be the production of biotin and methionine from their respective sulfoxides (Kappler et al., 2019). However, several representatives of the less well characterized BisC/TorZ/MtsZ subgroup of the Type III DMSOR have also been shown to reduce DMSO (Pollock and Barber, 1997; Kappler et al., 2019). It is possible that this could be the enzymatic pathway responsible for DMSO reduction in R. palustris CGA009. We did not measure whether R. palustris CGA009 synthesized RPA4653 in the presence of DMSO, though we note that it was not detected in the previous proteomic analysis during growth on succinate and acetate without DMSO. Given the inconclusive nature of this result, we performed additional physiological and chemical experiments. 1 Fig. (Figure presented.) Maximum-likelihood phylogenetic tree of CISM family proteins from R. palustris CGA009 (red) and reference sequences (black). The tree was inferred using the Geneious (version 2020.2.4) plugin of FastTree (version 2.1.11) based on a MAFFT (version 7.450) alignment of 60 amino acid sequences. Clusters with known or hypothesized functional roles are highlighted. Only one of the putative DMSOR homologs from R. palustris CGA009 (i.e., BLASTp hits; see Methods) clusters with proteins known to have potential DMSO reduction activity, the BisC subgroup of the Type III DMSOR. No putative homologs cluster with the DmsA Type II DMSOR. The scale bar represents amino acid changes and bootstrap support values are shown in grey. Abbreviations: BisC, biotin sulfoxide reductase; DmsA, DMSO reductase; DorA, DMSO reductase; FdhA, formate dehydrogenase; NarB, assimilatory nitrate reductase; NxrA, nitrite oxidoreductase; TorA, trimethylamine N-oxide (TMAO) reductase; XdhA, xanthine dehydrogenase. Amino acid sequences are included in Table S1. Growth on the carbon substrate butyrate, which is more reduced than biomass, requires the presence of an electron sink like nitrate, DMSO, bicarbonate for carbon fixation or the absence of fixed nitrogen for nitrogen fixation (Muller, 1933; Hillmer and Gest, 1977; Richardson et al., 1988; McKinlay and Harwood, 2010a, 2010b; McKinlay and Harwood, 2011). Here, we tested whether the presence of DMSO enables phototrophic growth of R. palustris CGA009 on butyrate.
All Science Journal Classification (ASJC) codes
- Ecology, Evolution, Behavior and Systematics