The dynamics of intracellular polysulfides during growth of R. pomeroyi DSS-3 is similar to E. coli
It is reported that polysulfides are key intermediates in the microbial oxidation of sulfide to sulfate (Berg et al., 2014). Inorganic polysulfides (H2Sn) are extremely pH and redox sensitive and thus difficult to measure. Here we used polysulfides-sensitive probe psGFP to monitor the dynamic changes of intracellular polysulfides level. The psGFP probe was constructed by introducing a pair of cysteine residues near the GFP chromophore whose spatial distance designed to allow the formation of internal –Sn- (n > 3) bonds but not –S2- (disulfide) bonds (Hu et al., 2019). The bonds altered the internal equilibrium of GFP chromophore, reflected by increasing the 408 nm excitation peak and decreasing the 488 nm peak. Hence, the 408/488 nm ratio was used to evaluate the response of psGFP to polysulfides. (Wan et al., 2017).
The pBBR-MCS2 vector was used to construct recombinant plasmid pBBR-psGFP, and then the recombinant plasmid was transformed into R. pomeroyi DSS-3 for expression. We firstly monitored the dynamics of polysulfides during a growth cycle in R. pomeroyi DSS-3 harboring psGFP gene. (Han et al. 2008). The endogenous polysulfide level was relatively low in early log phase. It gradually increased and reached the maximum in early stationary phase, and then began to decrease (Fig. 1). The dynamic change is quite similar to that of E. coli during a growth cycle (Hu et al., 2019). For most heterotrophic bacteria, the endogenous polysulfide content of cells in stationary phase is higher than that of cells in log phase (Hou et al., 2018). These results reveal that the content of intracellular polysulfides is related to the growth phase in R. pomeroyi DSS-3.
Utilization of different sulfur sources and sulfide/polysulfides production by R. pomeroyi DSS-3
Different sulfur sources (NaHS, cysteine and dimethyl trisulfide) were added to resuspended cells to detect the intermediate polysulfides. When added NaHS to a final concentration of 600 µM, the 408/488 nm ratio rose from 0.95 to1.55, which is much more significant than cysteine and dimethyl trisulfide (DMTS)-treated groups (Fig. 2). This indicates that R. pomeroyi DSS-3 harbors sulfide-oxidizing enzymes and these enzymes can convert sulfide to zero valent sulfur such as polysulfides. (Holmkvist et al. 2011). (Holmkvist et al. 2011).
We also analyzed the end products from sulfide oxidation. Similarly, NaHS, cysteine and methionine were added to resuspended cells. (Otte et al., 1999). Sulfite was not detectable in all the groups, and thiosulfate was the main products, probably because small amount of sulfite produced was oxidized to sulfate rapidly (Fig. 3).
Ruegeria pomeroyi DSS-3 uses SQR and Fcc to oxidize sulfide to polysulfide.
Sulfide:quinone oxidoreductase (SQR and flavocytochrome c-sulfide dehydrogenase (Fcc) are two enzymatic systems that catalyze the oxidation of sulfide to zero valent sulfur S0. SQR, an ancient membrane-bound flavoprotein, is a common enzyme for sulfide oxidation detoxification in various organisms (Hu et al., 2018). SQR oxidizes sulfide to elemental S and donates electrons to the ubiquinone pool (Nguyen et al. 2022). Fcc contains a small FccA cytochrome c subunit and a larger FccB flavoprotein subunit which can bind sulfide (Lü et al., 2017). Unlike SQR, Fcc is a soluble periplasmic enzyme that donates electrons to the cytochrome c pool. Fcc is mainly present in phototrophic bacteria and chemolithotrophic SOB, less widespread than SQR. The physiological role of Fcc remains debatable. If indeed the Fcc oxidizes sulfide in vivo, both green and purple sulfur bacteria apparently have alternative sulfide-oxidizing enzymes such as SQR that may be more efficient in conserving energy. (Kamyshny et al. 2010). However, it is also possible that Fcc is advantageous for the cells especially at very low sulfide concentrations. (Leavitt et al. 2013).
To improve H2S removal rate, we overexpressed SQR or FccB to generate genetically engineered R. pomeroyi DSS-3 and compared the H2S removal rate of each strain. As shown in Fig. 4, R. pomeroyi DSS-3 (RpFccB) has the fastest H2S degradation rate, but the changes are not significant. This may suggest that individual SQR or FccB can not oxidize H2S efficiently, but they have synergistic effect and work together to oxidize H2S at high rate. (Leloup et al. 2009). To study the functions of RpSQR, it was expressed in E. coli BL21 heterologously. Comparing with E. coli BL21 without any sulfur-oxidizing genes, E.coli BL21 (RpSQR) can hardly oxidize H2S (Fig. 5). The result shows that the function of RpSQR is very weak, which is consistent with the stronger function of FccB in Fig. 4. The Fcc is nonfunctional in E. coli due to the lack of maturation proteins for c-type cytochrome in E. coli under aerobic conditions (Lü et al., 2017).