Chlorobaculum tepidum Outer Membrane Vesicles May Transport Biogenic Elemental Sulfur

Abstract

Outer membrane-derived vesicles (OMVs) have been studied in different phyla of Gram-negative bacteria, most extensively in the Pseudomonadota, where they have been shown to participate in diverse biological and environmental processes. To date, the production of OMVs has not been reported in the Chlorobiaceae within the phylum Chlorobiota. Chlorobaculum. tepidum is the model organism for the Chlorobiaceae that synthesizes and consumes insoluble extracellular sulfur (S(0)) globules by an unknown mechanism. Here, we report evidence implicating outer membrane vesicles in biogenic S(0) globule synthesis. We demonstrate that Cba. tepidum secretes OMVs in the extracellular milieu, and that OMV concentration and size vary with growth conditions, particularly sulfide concentration. A core of 31 proteins involved in diverse biological processes such as cell wall biogenesis, inorganic ion transport and metabolism were found to be shared between OMVs, extracellular S(0) globules and Cba. tepidum intact cells. Multiple analytical methods indicated that OMVs contain S(0) and that OMVs and biogenic S(0) globules share protein and polysaccharide signatures, including lipooligosaccharides. Together these lines of evidence indicate that Cba. tepidum's OMVs are one component of sulfur transport between cells and extracellular sulfur globules.

Keywords: Chlorobaculum tepidum; Chlorobiaceae; NanoIR spectroscopy; Raman spectroscopy; outer membrane vesicles; proteomics; sulfur globules.

Figure 1.

The effect of Cba. tepidum growth phase on extracellular particle concentration in cultures and protein concentration in purified particle preparations. See Fig. S1A for details on purification. A) Extracellular particles concentration from different growth phases. B) Total protein concentration of particles purified at different growth phases. Error bars represent the standard error, n = 3 with * indication p < 0.05 and ** indicating p < 0.01 (t-test, two tailed, unequal variance).

Figure 2.

Transmission electron microscopy (TEM) images of Cba. tepidum’s OMVs. A) and B) TEM images show spherical OMVs budding out of rod-shaped Cba. tepidum after 8h and 14h of growth, respectively (white arrows). C) and D) TEM analysis of OMVs purified via serial ultracentrifugation from a 14 h old Cba. tepidum culture.

Figure 3.

Analysis of Cba. tepidum extracellular particles for hallmark characteristics of OMVs including size (A), protein (B), LPS or LOS (C), KDO (D).

Figure 4.

Comparative proteome analysis of Cba. tepidum whole cells, OMVs from cultures grown on sulfide (HS-OMV) or sulfide + thiosulfate (PF7-OMV), and biogenic sulfur globules (S(0) glob.). A) Venn diagram indicating the distribution of 1,447 proteins across sample group (n = 3 samples per group). B) Principal component analysis score plot of samples based on label free quantification. The first and the second principal components (PC) account for 80.6% and 7.3% of the variability in the data, respectively. Loadings for PC1 and PC2 are displayed (blue dashed arrows) for proteins grouped by PSORTb predicted localization: Cytoplasmic, Unknown, Inner Membrane (IM), Outer Membrane (OM), Periplasmic (Peri), and Extracellular (ExC).

Figure 5.

OMVs concentration and size vary with increased sulfide (HS) concentration. A) OMV concentrations measured after 14 h of growth with different concentrations of sulfide as the sole electron donor. B) MRSP-based size distribution of particles purified from the cultures in panel A (see Fig. S1A for purification details). C) and D) TEM analysis of OMVs purified from 4 mM and 7 mM sulfide cultures in panel A corresponding to the green and black traces in panel B.

Figure 6.

OMVs and S(0) share multiple characteristic. Characteristic Raman scattering (A) or IR absorption (B) spectra for purified OMVs (black), biogenic S(0) (red), and PBS (blue). Scattering (A) or absorption (B) wavenumbers are indicated by vertical dashed lines for sulfur (orange), polysaccharide (pink), and protein (black). (C) Chromatograms of elemental sulfur standards (0–0.3 mM S(0), black traces) and OMV’s purified from three independent 7 mM sulfide Cba. tepidum cultures (orange traces). The standards indicate the peak from OMV samples co-migrates with authentic S(0) and the replicates are within the range of detection.

Figure 7.

Summary of the proposed OMV S(0) transport mechanism. A) Cba. tepidum produces both OMVs and polysulfides during growth that contribute to the growth of extracellular S(0) globules. B) Enlarged view of the periplasm and OM with increased detail. Sulfide oxidation may induce unfolded protein stress that leads to OMV production. The dashed arrows for sulfide and polysulfide indicate possible routes to enter and exit either OMVs or S(0) globules. SQR = sulfide:quinone oxidoreductase (CT0117 and CT1087), Psr = putative polysulfide oxidoreductase complex (CT0496-CT0494). C) Enlarged view of a Cba. tepidum OMV with selected cargo molecules detected by proteomics analyses denoted at the right. Created in BioRender https://BioRender.com/bzod9yf