Growth physiology, genomics, and proteomics of Desulfurivibrio dismutans sp. nov., an obligately chemolithoautotrophic, sulfur disproportionating and ammonifying haloalkaliphile from soda lakes

Abstract

Elemental sulfur disproportionation combined with obligate autotrophy is a unique type of sulfur-based anaerobic metabolism known in a limited number of bacteria, primarily found among the members of Desulfobacterota phylum. Until recently, the only characterized alkaliphilic representative of this group was Desulfurivibrio alkaliphilus, originally isolated as an H₂-dependent sulfur reducer. In this study, we describe the properties of a novel species within this genus, Desulfurivibrio dismutans strain AMeS2, which was originally enriched and isolated from a soda lake sample as an autotrophic elemental sulfur disproportionating bacterium. Similar to D. alkaliphilus AHT 2^T, D. dismutans AMeS2 is an obligately alkaliphilic and moderately salt-tolerant autotrophic bacterium. In contrast to known neutrophilic sulfur disproportionating bacteria, it is capable of disproportionating sulfur without Fe(III). It can also grow by dissimilatory sulfur reduction to sulfide or nitrate reduction to ammonium (DNRA) with formate (but not with H₂) as the electron donor. The addition of formate to sulfur-disproportionating AMeS2 culture significantly increased the sulfur-reducing activity but did not completely abolish the oxidative branch of sulfur disproportionation. Genome analysis confirmed the presence of dissimilatory sulfur oxidation and dissimilatory sulfur and nitrate reduction machineries in the strain. S⁰ disproportionation occurs by means of cytoplasmic dissimilatory sulfite reductase (Dsr) donating electrons to, and periplasmic polysulfide reductase (PsrABC) receiving electrons from the menaquinone pool. Nitrate reduction to ammonium (DNRA) occurs by the combined action of a membrane formate dehydrogenase FdnGHI, periplasmic nitrate reductase, and octaheme c ammonifying nitrite reductase. Autotrophic growth is enabled by the Wood-Ljungdahl pathway (WLP). The genome also encodes proteins that presumably connect the oxidative branch of sulfur disproportionation with the carbon (WLP) cycle. Comparative proteomics of cells grown by sulfur disproportionation and formate-dependent DNRA demonstrated overexpression of the genes encoding Psr and rDSR at sulfur-disproportionating conditions, confirming their key role in this process. On the contrary, the genes encoding DNRA proteins are upregulated in the presence of nitrate. Thus, genomic and proteomic analyses revealed the pathways for energy conservation in a new representative of Desulfurivibrio growing at DNRA and under the thermodynamically challenging conditions of sulfur disproportionation.

Keywords: alkaliphiles; extremophiles; nitrate reduction; proteomics; reversed sulfate reduction; soda lakes; sulfur disproportionation.

Figure 1

Phylogenetic position of Desulfurivibrio dismutans AMeS2^T (in bold) based on sequence analyses of a concatenated alignment of 120 single-copy conserved bacterial protein markers (names of uncultured clusters are given according to the Genome Taxonomy Database Release 09-RS220) (Parks et al., 2020). The trees were built using the IQ-TREE 2 program (Minh et al., 2020) with ultrafast bootstrap approximation (Minh et al., 2013) as well as an approximate likelihood-ratio test for branches (Anisimova and Gascuel, 2006). The bootstrap consensus tree is shown with values placed at the nodes. Bar, 0.10 changes per position.

Figure 2

Macro- (a) and micro- (b–d) morphology of strain AMeS2 grown at 0.6 M total Na⁺ and pH 10. (a) Difference in polysulfide formation between sulfur disproportionating (left, with domination of S₃²⁻) and in sulfur + formate culture (right, with domination of S₅²⁻); (b) Phase contrast microphotograph of cells in sufur-disproportionating culture; (c) Total electron microscopy showing flagellation; (d) Thin section electron microscopy showing large nucleoid region (N) and periplasmic compartment (P).

Figure 3

Growth dynamics and activity of resting cells of strain AMeS2 grown under different conditions. All incubations were performed in carbonate/bicarbonate buffer at pH 10 and 0.6 M total Na⁺. (a,b) Autotrophic growth and sulfur products formation at sulfur disproportionating conditions or on sulfur + 50 mM formate, respectively; (c) Comparison of the rates of S⁰ disproportionation (S₈; bars with solid outline) and formate oxidation by S⁰ (S₈ + f; bars with dashed outline) in AMeS2 resting cells (cell protein = 0.4 mg mL⁻¹) pregrown at three different conditions: sulfur disproportionating, sulfur + 50 mM formate and 10 mM nitrate + 50 mM formate. The rates are shown as the amount of reduced (as total sulfanes; blue bars) and oxidized (sulfate; orange bars) sulfur products formed by the resting cells, incubated under each condition (S₈ or S₈ + f). The results are the mean of three replicate experiments for cultures and two for cell suspensions.

Figure 4

Growth and product formation of strain AMeS2 in ammonifying conditions (at pH 9.5 and 0.6 M total Na⁺) with 50 mM formate as the electron donor with either 10 mM nitrate or 2 mM × 5 mM nitrite as the electron acceptors. The medium was reduced with 0.2 mM sulfide. The arrow indicates a second addition of 5 mM nitrite. The initially added ammonium (2 mM) was subtracted from the measured values. The results are the mean of three replicate experiments with nitrate and two with nitrite.

Figure 5

Influence of Na⁺ (carbonate buffer, pH 10) and pH (at 0.6 M total Na⁺) on sulfidogenic activity of strain AMeS2 at sulfur-disproportionating conditions in growing culture and resting cells. Sulfidogenic activity is shown as amount of total sulfanes formed by growing cultures (in red) or the grown cultures (resting cells, in blue). Results of duplicate experiments.

Figure 6

VulcanoPlot showing the AMeS2 gene expression during the growth by sulfur disproportionation and nitrate reduction with formate (DNRA). Positive fold change values indicate that the genes are upregulated during sulfur disproportionation, while negative values indicate that the genes are upregulated during DNRA. Horizontal dashed line indicates p = 0.001. Red and green dashed lines indicate log(2) fold change difference between two experiments equal to 2/−2 (i.e., the fold change = 4/−4). Green circles: upregulated (their expression level is above 2) during S⁰ disproportionation genes, red circles: upregulated (their expression level is above 2) at formate oxidation by nitrate genes. Large circles: the genes with p < 0.001 and the fold changes above 4 or below −4. The numbers in the circles are in the following format: the locus tag (protein rank by riBAQ calculated for S⁰ disproportionation culture/protein rank by riBAQ calculated for formate plus nitrate culture). Colored flags to the left of the proteins indicate that the genes encoding these proteins are part of the gene cluster of the same color, shown below the Volcano plot. Five gene clusters with highly regulated genes encoding the proteins playing significant roles in sulfur disproportionation or DNRA are shown. Pink: octaheme c nitrite reductase; blue: Psr; green: AprAB—QmoABC; yellow: Sat—DsrAB; light violet: octaheme c nitrite reductase. The genes of the proteins presumably not part of these enzyme complexes are in gray. The details are given in the main text.

Figure 7

Sulfur metabolism of AMeS2. The mechanisms of S⁰ transfer, oxidation, and reduction, the possible connection of S⁰ oxidation and the WLP, and the spots and the sites of ATP synthesis are shown. Protein designations are shown in green; substrates and products are displayed in black; and nucleotide-phosphates and pyrophosphate are depicted in purple. The reduced/oxidized sulfur atom is in red. The electron flux is in the red dotted line. Qmo/Sat proteins are represented by orange shapes, Psr—green shapes, Dsr—purple shapes, ATP synthase—blue shapes, and sulfur transferases—white, while persulfurated sulfur transferases are in yellow. The details are given in the main text.