Homologs from sulfur oxidation (Sox) and methanol dehydrogenation (Xox) enzyme systems collaborate to give rise to a novel pathway of chemolithotrophic tetrathionate oxidation

Abstract

The SoxXAYZB(CD) -mediated pathway of bacterial sulfur-chemolithotrophy explains the oxidation of thiosulfate, sulfide, sulfur and sulfite but not tetrathionate. Advenella kashmirensis, which oxidizes tetrathionate to sulfate, besides forming it as an intermediate during thiosulfate oxidation, possesses a soxCDYZAXOB operon. Knock-out mutations proved that only SoxBCD is involved in A. kashmirensis tetrathionate oxidation, whereas thiosulfate-to-tetrathionate conversion is Sox independent. Expression of two glutathione metabolism-related proteins increased under chemolithotrophic conditions, as compared to the chemoorganotrophic one. Substrate-dependent oxygen consumption pattern of whole cells, and sulfur-oxidizing enzyme activities of cell-free extracts, measured in the presence/absence of thiol inhibitors/glutathione, corroborated glutathione involvement in tetrathionate oxidation. Furthermore, proteome analyses detected a sulfite:acceptor oxidoreductase (SorAB) exclusively under chemolithotrophic conditions, while expression of a methanol dehydrogenase (XoxF) homolog, subsequently named thiol dehydrotransferase (ThdT), was found to increase 3- and 10-fold during thiosulfate-to-tetrathionate conversion and tetrathionate oxidation respectively. A thdT knock-out mutant did not oxidize tetrathionate but converted half of the supplied 40 mM S-thiosulfate to tetrathionate. Knock-out of another thiosulfate dehydrogenase (tsdA) gene proved that both ThdT and TsdA individually converted ∼ 20 mM S-thiosulfate to tetrathionate. The overexpressed and isolated ThdT protein exhibited PQQ-dependent thiosulfate dehydrogenation, whereas its PQQ-independent thiol transfer activity involving tetrathionate and glutathione potentially produced a glutathione:sulfodisulfane adduct and sulfite. SoxBCD and SorAB were hypothesized to oxidize the aforesaid adduct and sulfite respectively.

Two pathways for thiosulfate oxidation in the alphaproteobacterial chemolithotrophParacoccus thiocyanatusSST

“…Whereas the SST genome annotation did not reveal any tetrathionate hydrolase (tetH) gene, three consecutive ORFs (locus tags DIE28_01705, DIE28_01710 and DIE28_01720), homologous to the xoxF, xoxG and xoxJ genes of the prototypical methanol-oxidizer Methylobacterium extorquens AM1 (Nakagawa et al, 2012;Keltjens et al, 2014;Pol et al, 2014;Skovran and 395 Martinez-Gomez, 2015;Chu and Lidstrom, 2016) were detected. Albeit the xoxF gene of SST was found to have ~75% and 69% translated amino acid sequence identities with the typical PQQbinding XoxF homologs of AM1 and the ThdT variant of XoxF reported from A. kashmirensis (Pyne et al, 2018) respectively, the annotated genome sequence of SST deposited in the GenBank designates the locus DIE28_01705 as a pseudogene, apparently due to a frame shift caused by a 400 missing guanine nucleotide in between positions 1161 and 1162 of the ORF. However, a manual scrutiny of the reads aligned to this region of the SST genome showed that 153 and 173 reads taking part in the assembly contained and lacked the guanine in the above mentioned position respectively, while 36 others had an ambiguous base call in this nucleotide position (notably, proportion of ambiguous base calls across this xoxF ORF was also quite high).…”

“…However, a manual scrutiny of the reads aligned to this region of the SST genome showed that 153 and 173 reads taking part in the assembly contained and lacked the guanine in the above mentioned position respectively, while 36 others had an ambiguous base call in this nucleotide position (notably, proportion of ambiguous base calls across this xoxF ORF was also quite high). As capillary 405 sequencing also failed to resolve this issue unambiguously, we decided to knock this gene out and confirm whether tetrathionate oxidation in SST proceeded via a ThdT-mediated glutathionecoupled mechanism similar to the one described for A. kashmirensis (Pyne et al, 2017(Pyne et al, , 2018.…”

“…In contrast to the alphaproteobacteria, thiosulfate oxidation in most of the beta-and gammaproteobacterial chemolithotrophs proceeds via the formation of tetrathionate-intermediate (S 4 I) (Visser et al, 1996;Bugaytsova and Lindström, 2004;Ghosh et al, 2005;Dam et al, 2007;Rzhepishevska et al, 2007;Kikumoto et al, 2013;Pyne et al, 2017Pyne et al, , 2018. In these bacteria, 85 thiosulfate to tetrathionate conversion is mediated either by (i) doxDA-encoded thiosulfate:quinone oxidoreductase (Rzhepishevska et al, 2007;Kikumoto et al, 2013) that is also present in thermoacidophilic archaea (Müller et al, 2004;), or by (ii) thiosulfate dehydrogenase (Pyne et al, 2018) that is also present in bacteria incapable of further oxidation of tetrathionate to sulfate (Hensen et al, 2006;Denkmann et al, 2012;Frolov et al, 2013;Brito et al, 2015;Orlova et al, 90 2016).…”

“…In contrast to the alphaproteobacteria, thiosulfate oxidation in most of the beta-and gammaproteobacterial chemolithotrophs proceeds via the formation of tetrathionate-intermediate (S 4 I) (Visser et al, 1996;Bugaytsova and Lindström, 2004;Ghosh et al, 2005;Dam et al, 2007;Rzhepishevska et al, 2007;Kikumoto et al, 2013;Pyne et al, 2017Pyne et al, , 2018. In these bacteria, 85 thiosulfate to tetrathionate conversion is mediated either by (i) doxDA-encoded thiosulfate:quinone oxidoreductase (Rzhepishevska et al, 2007;Kikumoto et al, 2013) that is also present in thermoacidophilic archaea (Müller et al, 2004;), or by (ii) thiosulfate dehydrogenase (Pyne et al, 2018) that is also present in bacteria incapable of further oxidation of tetrathionate to sulfate (Hensen et al, 2006;Denkmann et al, 2012;Frolov et al, 2013;Brito et al, 2015;Orlova et al, 90 2016). The S 4 I formed in this way is subsequently oxidized either (i) by the activity of the pyrroloquinoline quinone (PQQ)-binding tetrathionate hydrolase (TetH), as described in Acidithiobacillus species (De Jong et al,1997a;Kanao et al, 2007;Rzhepishevska et al, 2007;van Zyl et al, 2008;Kanao et al, 2013), or (ii) via coupling with glutathione GSH (to form the glutathione:sulfodisulfane adduct GS-S-S-SO 3¯ and sulfite) by the action of another PQQ-binding 95 protein called thiol dehydrotransferase (ThdT, which is a homolog of the XoxF variant of methanol dehydrogenase), followed by the oxidation of GS-S-S-SO 3¯ via iterative typical actions of SoxB and SoxCD, as reported in A. kashmirensis (Pyne et al, 2018).…”

“…Functional dynamics and intersections of the Sox and S 4 I pathways of SST were delineated via physiological studies, whole genome sequencing and analysis, and recombination-based knockout mutagenesis of putative sulfur oxidation genes. Results were then considered in the context of 110 the prototypical Sox and S 4 I pathways of Paracoccus pantotrophus Quentmeier and Friedrich, 2001;Sauve et al, 2007) and Advenella kashmirensis (Ghosh et al, 2005;Dam et al, 2007;Pyne et al, 2017Pyne et al, , 2018 repsectively.…”