Dimorphism in methane seep-dwelling ecotypes of the largest known bacteria

Abstract

We present evidence for a dimorphic life cycle in the vacuolate sulfide-oxidizing bacteria that appears to involve the attachment of a spherical Thiomargarita-like cell to the exteriors of invertebrate integuments and other benthic substrates at methane seeps. The attached cell elongates to produce a stalk-like form before budding off spherical daughter cells resembling free-living Thiomargarita that are abundant in surrounding sulfidic seep sediments. The relationship between the attached parent cell and free-living daughter cell is reminiscent of the dimorphic life modes of the prosthecate Alphaproteobacteria, but on a grand scale, with individual elongate cells reaching nearly a millimeter in length. Abundant growth of attached Thiomargarita-like bacteria on the integuments of gastropods and other seep fauna provides not only a novel ecological niche for these giant bacteria, but also for animals that may benefit from epibiont colonization.

Figure 1

Vacuolate sulfide-oxidizing bacteria are abundant on a variety of substrates along the Costa Rica Margin including (a) free-living Thiomargarita-like cells and large Beggiatoa-like filaments inhabiting sulfidic sediments; (b) slightly elongate Thiomargarita-like morphotypes attached to authigenic carbonates; and (c) elongate Thiomargarita-like cells attached to Bathymodiolus mussel byssal threads. Scale bars represent 500 μm.

Figure 2

(a–c) The shells of living gastropods (Provanna laevis) from the Costa Rica margin and the Cascadia margin (Hydrate Ridge) are colonized by attached, elongate Thiomargarita-like cells. Scale bars represent ∼1 mm.

Figure 3

An apparent reproductive developmental cycle in attached sulfur bacteria (a) appears to begin with the attachment of a spherical cell to a solid substrate (arrows 1), followed by elongation to a stalked form (arrows 2), initial bud formation (arrows 3) and finally budding (arrows 4) to produce a spherical daughter cell (arrows 5). (b, c) Attached sulfur bacteria on a gastropod shell. (d, e) Detail of attached cell and intracellular sulfur globules. (f) Bacteria attached to mussel byssal threads. (g) Budded Thiomargarita-like cell was observed to undergo complete detachment from the mother cell between 12 and 20 h after collection. After bud detachment, the elongate mother cell (a4 → 3) can continue to produce additional buds. Scale bar in b=350 μm; c=1 mm; d=200 μm; f=300 μm.

Figure 4

Attached sulfur bacteria. (a, b) Densely packed Thiomargarita-like cells on gastropod shell co-occur with attached filamentous bacteria. Scale bar in a=30 μm; b=20 μm. (c) Particularly during early stages of attachment and elongation they have rounded bases where they are attached to benthic substrates. As the cell elongates, the base of the cells becomes tapered. In some specimens, a small appendage formed by extrusion of the cell ultrastructure is present. (d) Transmission electron micrograph of vacuolated Thiomargarita-like cells (t) attached to plaque (p) and mucous occupied by smaller filamentous bacteria. This material surrounds the Bathymodiolus mussel byssus. Scale bar=2 μm.

Figure 5

Phylogenetic tree of gammaproteobacterial 16S rRNA gene sequences showing sequences of different morphotypes from Costa Rica (COS) and Namibia (NAM) in gray and their affiliation to previously published Thiomargarita spp. These sequences are shown within the context of the broader clade of conspicuous sulfide-oxidizing Gammaproteobacteria. Of each newly sequenced cell the morphology and diameter is indicated. In attached forms, the widest diameter of the attached basal body is reported. The scale bar represents ∼10% sequence divergence.

Figure 6

The gastropod Provanna laevis burrows into sulfidic mud in a collection container, (a) first digging a burrow, (b) then occupying the burrow with its carapace oriented downward, which exposes the population of attached sulfur bacteria to sulfidic conditions. (c) The head of the gastropod remains partially exposed above the sediment/water interface.