Comparative Genomics of Thiohalobacter thiocyanaticus HRh1T and Guyparkeria sp. SCN-R1, Halophilic Chemolithoautotrophic Sulfur-Oxidizing Gammaproteobacteria Capable of Using Thiocyanate as Energy Source

Abstract

The genomes of Thiohalobacter thiocyanaticus and Guyparkeria (formerly known as Halothiobacillus ) sp. SCN-R1, two gammaproteobacterial halophilic sulfur-oxidizing bacteria (SOB) capable of thiocyanate oxidation via the “cyanate pathway”, have been analyzed with a particular focus on their thiocyanate-oxidizing potential and sulfur oxidation pathways. Both genomes encode homologs of the enzyme thiocyanate dehydrogenase (TcDH) that oxidizes thiocyanate via the “cyanate pathway” in members of the haloalkaliphilic SOB of the genus Thioalkalivibrio. However, despite the presence of conservative motives indicative of TcDH, the putative TcDH of the halophilic SOB have a low overall amino acid similarity to the Thioalkalivibrio enzyme, and also the surrounding genes in the TcDH locus were different. In particular, an alternative copper transport system Cus is present instead of Cop and a putative zero-valent sulfur acceptor protein gene appears just before TcDH. Moreover, in contrast to the thiocyanate-oxidizing Thioalkalivibrio species, both genomes of the halophilic SOB contained a gene encoding the enzyme cyanate hydratase. The sulfur-oxidizing pathway in the genome of Thiohalobacter includes a Fcc type of sulfide dehydrogenase, a rDsr complex/AprAB/Sat for oxidation of zero-valent sulfur to sulfate, and an incomplete Sox pathway, lacking SoxCD. The sulfur oxidation pathway reconstructed from the genome of Guyparkeria sp. SCN-R1 was more similar to that of members of the Thiomicrospira-Hydrogenovibrio group, including a Fcc type of sulfide dehydrogenase and a complete Sox complex. One of the outstanding properties of Thiohalobacter is the presence of a Na + -dependent ATP synthase, which is rarely found in aerobic Prokaryotes.Overall, the results showed that, despite an obvious difference in the general sulfur-oxidation pathways, halophilic and haloalkaliphilic SOB belonging to different genera within the Gammaproteobacteria developed a similar unique thiocyanate-degrading mechanism based on the direct oxidative attack on the sulfane atom of thiocyanate.

Trinuclear copper biocatalytic center forms an active site of thiocyanate dehydrogenase

“…S2C). Proteins homologous to TcDH (sequence identity above 30%) were found in the genomes of more than 50 other species from phylogenetically different bacteria (24).…”

“…The active site includes (i) copper ions, (ii) residues coordinated to these copper ions (for the Cu1 ion, His206, His381, and Asp314; for the Cu2 ion, His135, His528, and Lys103, the latter residue being coordinated to the Cu2 ion in only one of two possible positions; and for the Cu3 ion, His437 and His482), (iii) residues that are not directly coordinated to copper ions but are involved in the formation of a hydrogen bond network or hydrophobic interactions in the active site (Phe436, Lys103), (iv) residues involved in the activation of a reacting water molecule W1 (His136 and Glu288), and (v) water molecules, including an attacking water molecule W1, and ions from the crystallization media (acetate ion in the TcDH1 structure). All of the active site residues, 11 altogether, are conserved in the closest homologs of TcDH (identity >49%) and form a characteristic template that can be used for mapping potential TcDHs in the metagenomes (24).…”

“…The gene coding for TcDH belongs to a distinct region of genome containing, in the case of Tv. paradoxus, a specialized set of genes assumed to participate in copper trafficking (24,37). Proteins coded by these genes may assist in the final assembly of copper ions into the TcDH in the periplasm.…”

Two pathways for thiosulfate oxidation in the alphaproteobacterial chemolithotrophParacoccus thiocyanatusSST

“…Thiohalobacter and Guyparkeria species (Berben et al, 2017;Tsallagov et al, 2019) was detected in the genome of this aphaproteobacterium. TcDH has been hypothesized to attack the sulfane sulfur atom of thiocyanate (SCN -) and directly oxidize SCNto elemental sulfur (S 0 ) and cyanate (CNO -) (Sorokin et al, 2001;Tsallagov et al, 2019).…”

“…Thiohalobacter and Guyparkeria species (Berben et al, 2017;Tsallagov et al, 2019) was detected in the genome of this aphaproteobacterium. TcDH has been hypothesized to attack the sulfane sulfur atom of thiocyanate (SCN -) and directly oxidize SCNto elemental sulfur (S 0 ) and cyanate (CNO -) (Sorokin et al, 2001;Tsallagov et al, 2019). If thiocyanate oxidation in SST proceeds via this pathway, then the sulfur atom produced can be oxidized via the Sox machinery encoded by the 420 genome of this alphaproteobacterium; notably however, no homolog of the cyanate hydratase (cyanase) gene, which is known to convert cyanate to ammonia (NH 3 ) and carbon dioxide (CO 2 ) (Anderson and Little, 1986;Sung and Fuchs, 1988), was detected in the SST genome.…”