A Cristae-Like Microcompartment in Desulfobacterota

Abstract

Some Alphaproteobacteria contain intracytoplasmic membranes (ICMs) and proteins homologous to those responsible for the mitochondrial cristae, an observation which has given rise to the hypothesis that the Alphaproteobacteria endosymbiont had already evolved cristae-like structures and functions. However, our knowledge of microbial fine structure is still limited, leaving open the possibility of structurally homologous ICMs outside the Alphaproteobacteria. Here, we report on the detailed characterization of lamellar cristae-like ICMs in environmental sulfate-reducing Desulfobacterota that form syntrophic partnerships with anaerobic methane-oxidizing (ANME) archaea. These structures are junction-bound to the cytoplasmic membrane and resemble the form seen in the lamellar cristae of opisthokont mitochondria. Extending these observations, we also characterized similar structures in Desulfovibrio carbinolicus, a close relative of the magnetotactic D. magneticus, which does not contain magnetosomes. Despite a remarkable structural similarity, the key proteins involved in cristae formation have not yet been identified in Desulfobacterota, suggesting that an analogous, but not a homologous, protein organization system developed during the evolution of some members of Desulfobacterota. IMPORTANCE Working with anaerobic consortia of methane oxidizing ANME archaea and their sulfate-reducing bacterial partners recovered from deep sea sediments and with the related sulfate-reducing bacterial isolate D. carbinolicus, we discovered that their intracytoplasmic membranes (ICMs) appear remarkably similar to lamellar cristae. Three-dimensional electron microscopy allowed for the novel analysis of the nanoscale attachment of ICMs to the cytoplasmic membrane, and these ICMs are structurally nearly identical to the crista junction architecture seen in metazoan mitochondria. However, the core junction-forming proteins must be different. The outer membrane vesicles were observed to bud from syntrophic Desulfobacterota, and darkly stained granules were prominent in both Desulfobacterota and D. carbinolicus. These findings expand the taxonomic breadth of ICM-producing microorganisms and add to our understanding of three-dimensional microbial fine structure in environmental microorganisms.

Keywords: Desulfobacterota; anaerobic oxidation of methane; compartmentalization; electron microscope tomography; intracytoplasmic membrane; outer membrane vesicles; pera; pera junction; sulfate reduction.

FIG 1

Application of DAB to seep derived microbial consortia to investigate the positioning of transition metal-based redox activity. (A) Panel and inset: ANME-SRB consortia showing the strong staining of bacterial ICMs by DAB. (B) ANME-SRB consortia showing the staining of bacterial ICMs with the inset showing (left to right) the stain intensity profile of the archaeal membrane (A), the intercellular location of the putative heme proteins that are involved in electron transfer (I), and the bacterial membranes (B). The x axis is measured in μm. Cells with morphological similarity to ANME-2b (which contain polyphosphate-like bodies) are stained by DAB on the cell exterior, indicating redox activity outside the cell (Panels A, B, C, and E). Other cells which experienced the same reaction conditions but do not appear to be ANME-2b (and are likely 2c) did not show staining between cells (Panels D and F). Panels A, B, and C are from the 5133 methane seep sample, and panels D, E, and F are from the 3730 methane seep sample.

FIG 2

The T3B1 morphotype has short, stubby pera. (A) A 1.6 nm thick slice through the middle of a tomographic volume of a T3B1 ANME-SRB consortium. The ANME archaea were spatially separated from their partner SRB cells, as noted by the demarcating line. The SRB clustered in aggregates. Uniformly appearing pera are clearly seen by their dark membrane staining inside the profiles of 3 whole and 6 partially visualized SRB. Dark granules are prominent in the SRB. An example of a pera with a pera junction is boxed. Scale = 500 nm. (B) A “top” view of the surface-rendered volume shown in panel A after the segmentation of the cell (outer) membranes (transparent maroon color), dark granules (slate gray color), and pera (goldenrod color). (C) The pera are arranged mostly around the cell periphery and in rows. The boxed region of 11 pera is the most prominent example. (D) A side view of the pera in the boxed region, showing uniformity when viewed from the top and also from the side. (E) The cytoplasmic membrane, viewed from the outside, showing the 11 pera junctions of the pera in the boxed region. The pera junction openings (dark) are also strikingly uniform in size. The boxed region is shown in panel F. (F) In the left panel, with the cytoplasmic membrane made transparent, the placement of the pera junctions along the height of their respective pera is clear. Even though the pera junctions tend toward the middle of the pera, there is sufficient variation to conclude that the pera junction placement is not uniform. Rather, these junctions can appear anywhere along the pera height. The right panel shows the perpendicular view of one of the middle pera to show that a pera junction is a short, tube-like structure that connects the pera interior across the cytoplasmic membrane (translucent maroon) to the periplasmic space. This view emphasizes why pera, Latin for pouch, appropriately describes this structure. (G) Even though the preponderance of pera is arrayed along the periphery of the cell, there are examples of pera that appear to be in the middle of the cell. However, because the slice thickness only captures less than half of the cell, they may be closer to the “top” or “bottom” of the cell. Scale = 500 nm.

FIG 3

The T3B2 SRB morphotype has narrower, longer pera, compared with the T3B1 morphotype. (A) A 1.6 nm thick slice through the middle of a tomographic volume of an ANME-SRB consortium, showing 2 SRB cells. The pera lengths are noticeably less uniform than their T3B1 counterparts. Five open pera junctions are seen (arrowheads). The other pera junctions are closed, as seen by a cap across their entrance. As with the T3B1, the dark granules are prominent. Scale = 500 nm. (B) A “top” view of the surface-rendered volume after the segmentation of the cell (outer) membranes (transparent maroon) and pera (various shades of brown). Whereas the short, stubby pera found in the T3B1 cells were invariably straight, these pera are curvier. (C) Perpendicular view showing the difference in sizes of these pera.

FIG 4

The T2B SRB morphotype has even longer and curvier pera, compared with the T3B2 morphotype, and it closely resembles the typical mitochondrial architecture. (A) A 1.6 nm thick slice through the middle of a tomographic volume of an ANME-SRB consortium, showing an SRB cell. No dark granule is seen. This cell could easily be mistaken for a mitochondrion because of the shape, size, and distribution of the pera, which resemble cristae. Although not common, there is even branching of the pera (arrow), reminiscent of branched cristae. Moreover, the pera junctions (example is boxed) look identical to crista junctions. Scale = 500 nm. (B) A “top” view of the surface-rendered volume after the segmentation of the cell membrane (transparent maroon) and pera (various shades of brown). These pera look like lamellar mitochondrial cristae. (C) Perpendicular view showing that these pera are larger than those in the T3B1 and T3B2 morphotypes. (D) An oblique view of the branched pera to better see that the branches are not connected by tubes but are rather joined throughout. (E) Oblique view of a pera (chocolate), emphasizing the pera junction (sandy brown) shape. This pera has 2 pera junctions (arrowhead), differing from the T3B1 pera, which have at the most only 1 pera junction each (Fig. 2F), and is more similar to mitochondrial lamellar cristae, which often have more than 1 crista junction.

FIG 5

The DC morphotype has narrow, straight pera. (A) A 1.6 nm thick slice through the middle of a tomographic volume of a DC1 cell from a late exponential culture which had been transferred to grow on EtOH from MeOH. The contrast is reversed so that the pera membrane is light and the interior is dark. This reversal makes it harder to identify the pera, so each pera is marked by a white arrowhead. As with the T3B1 and T3B2 SRB, the dark granules are prominent. The inset shows another slice through the same volume that is closer to the section surface, showing pera arranged at the cell periphery and in a row, resembling the predominant arrangement of the T3B1 pera. These pera are numbered from 1 to 9 to help mark each. Scale = 250 nm. (B) A “top” view of the 9 surface-rendered pera shown in the inset after the segmentation of the cell membrane (transparent maroon) and pera (goldenrod). The regular array of pera was reminiscent of the T3B1 morphotype, except that the T3B1 pera were shorter and stubbier, and these pera were narrower and longer. (C) Perpendicular view showing that these pera are short. (D) A top view of all of the pera in this cell. There appear to be 2 classes of pera in the DC1 cells. One class (goldenrod) is T3B1-like and is arranged in an array but is narrower. The other class is comprised of pera of divergent orientation, but not random orientation, of different sizes (shades of brown).These pera are straight, unlike the curvier pera of the T2B morphotype. (E) Perpendicular view reinforcing the impression that the second class of pera is not randomly oriented but tends to be within 30 degrees of vertical. (F) A 1.6 nm thick slice through the middle of a tomographic volume of a DC2 cell from an early stationary culture which had been transferred to grow on EtOH from MeOH. Each pera is marked by a white arrowhead. The dark granules are also prominent with the DC2 cells. (G) A side view of all of the pera in this cell. (H) A perpendicular view, reinforcing the observation that many of the pera are positioned along the periphery of the cell. (I) A 1.6 nm thick slice through the middle of a tomographic volume of a DC3 cell grown in culture in the exponential-phase on EtOH from a culture grown on EtOH. Each pera is marked by a white arrowhead. As with the DC1 and the DC2 cells, the dark granules are also prominent with the DC3 cells. (J) A side view of all of the pera in this cell. (K) A perpendicular view, showing two interesting features. One, there tend to be separate aggregations of pera, with a few on the left to more than 10 on the right. Two, because we can track most of the pera completely through the volume, those pera in the middle of the cell cannot have pera junctions. (L) Side-by-side comparison of pera length from pera chosen near the median length for each type. Images were taken from slices of the volumes. From left to right: T3B1, T3B2, T2B, DC1, and DC3. An arrowhead points to each pera. DC2 (not shown) was similar to DC1. Scale = 50 nm. (M) Side-by-side comparison of the segmented pera with sizes chosen near the median for each type. Surface-rendered pera volumes are shown. Scale = 200 nm.

FIG 6

Measurements of pera and pera junction features. (A) Oblique view of a pera (chocolate) connected to the inner boundary membrane (translucent maroon), showing how the width, length, and height measurements were made. The black double arrow corresponds to the pera width. The blue double arrow corresponds to the pera length. The red double arrow corresponds to the pera height. The white double arrow corresponds to the pera junction (sandy brown) width. (B) The pera of the T3B1 morphotype have considerably greater maximum widths than do the pera of the T3B2 or T2B morphotypes, reenforcing the impression that these are stubbier pouches (*, P < 0.01; n = 40; from 10 cells each for T2B, T3B1, T3B2, DC1, DC2, and DC3).The DC1, DC2, and DC3 pera all have similar maximum widths that are no different from T3B2 or T2B. The maximum widths for 5 out of the 6 morphotypes are remarkably uniform. (C) The maximum pera lengths are strikingly different for T3B1, T3B2, and T2B, with the T3B1 being the shortest, reinforcing the impression that the T3B1 pera are short and stubby. Among the DC morphotypes, the DC3 pera are significantly shorter (*, P < 0.01; n = 20; 10 cells for each SRB type). (D) The maximum pera height was also different between the morphotypes, with the T3B1 being shorter than the T3B2 or T2B. Note that it was not possible to accurately measure the maximum height for the T2B pera because most extended beyond the section thickness of 300 nm used for the tomographic analyses. Clearly, though, this morphotype had the tallest pera (Fig. 5M). Among the DC samples, DC3 had pera shorter than those of DC1 or DC2 (*, P < 0.01; n = 20; 10 cells for each SRB type). (E) The pera membrane density is highest for the T2B morphotype (*, P < 0.01; n = 10 cells for each SRB type). (F) The pera membrane surface area divided by the cytoplasmic membrane surface area was greatest for the T2B morphotype, which was the only morphotype with a mean (and median) ratio that was greater than 1 (*, P < 0.01; n = 10 cells for each SRB type). (G) Individually, the T3B1 and DC3 pera had the least membrane surface area. Note that the y scale is different for the DC samples because their pera are generally smaller than those of T2B, T3B1, and T3B2. (*, P < 0.01; n = 20; 10 cells for each SRB type). (H) The pera junction width is greater for T3B1 than for the other morphotypes (*, P < 0.01; n = 20; 10 cells for each SRB type). (I) The Pera junction opening (cross-sectional) area at the pera junction entrance. The T3B1 and T3B2 cells have larger openings. This is the case for the T3B1 cells because they have larger circular openings and for the T3B2 because they have elongated (oblong) openings (*, P < 0.01; n = 20; 10 cells for each SRB type). (J) The pera junction density is greatest for the T3B1 pera, consistent with the observation of more pera, hence more pera junctions, per cellular volume in this morphotype, compared with the other morphotypes (*, P < 0.01; n = 10 cells for each SRB type).

FIG 7

Dense granules, present in both methane seep and cultured bacteria, differ in volume, density, and size between the 6 classes of SRB. (A) An example of a T2B SRB containing a few large, dense granules that appear similar to granules prevalent in T3B1, T3B2, and DC1-3 SRB. An arrowhead points to a large granule. Scale = 200 nm. (B) The granule volume density is much lower in T2B SRB compared with T3B1, T3B2, and DC1-3 SRB (P < 0.01; n = 10 cells). (C) The granule volume is bigger for T3B2 SRB than for T2B or T3B1 SRB, and the DC3 granule volume is bigger than the volume of either DC1 or DC2 (P < 0.05; n = 50 granules).

FIG 8

OMVs bud from the outer (cell) membrane of T2B, T3B1, and T3B2 SRB. (A) A T2B SRB showing 2 buds (arrows) and a nearby cluster of spherical OMVs (boxed). Scale = 200 nm. (B) A view from the top, angled to look through the cell membrane made translucent so as to see the nearby OMVs, three of which are still attached to the outer membrane (arrows). All of the pera are shown to demonstrate that they are positioned in a subvolume that is separated from the portion of the outer membrane where the OMV budding is observed. (C) A side view of the adjacent OMVs against the backdrop of the opaque outer membrane, providing another perspective of the distribution of the OMVs. (D) Another side view, giving a good perspective of how close these OMVs are to the outer membrane after budding off. The large pera typical of T2B SRB provide a structural landmark reference to the size of the OMVs. (E) Even though the vast majority of the OMVs are spherical, lamellar-type OMVs are also observed. An arrow points to a lamellar-type OMV budding from the cell membrane of a T3B2 SRB. Scale = 200 nm. (F) A side view, looking through the translucent outer membrane, partly cut away, at the lamellar OMV (circled), with the budding portion indicated by an arrow. The smallish pera are shown for perspective. (G) A rotated side view, showing how the lamellar OMV parallels the outer membrane. An arrow points to the attached portion of the OMV. (H) Large lamellar OMVs (arrows) are also observed adjacent to the SRB outer membranes. Part of a T3B1 SRB is shown. Scale = 200 nm. (I) The lamellar OMV marked with an asterisk in panels H and I is attached to the outer membrane with a tubular bud (arrow). A side view with the outer membrane partially cut away to show that this lamellar OMV is larger than the pera. 3 are shown. (J) A rotated side view shows how this lamellar OMV is closely apposed to the outer membrane. (K) Measurements of the diameter of the spherical-type OMVs, with examples shown in panel A. The diameters were relatively uniform and did not vary between T2B, T3B1, or T3B2 (n = 50).