Ultrastructure of Organohalide-Respiring Dehalococcoidia Revealed by Cryo-Electron Tomography

Abstract

Dehalococcoides mccartyi ( Dhc ) and Dehalogenimonas spp. ( Dhgm ) are members of the class Dehalococcoidia , phylum Chloroflexi, characterized by streamlined genomes and a strict requirement for organohalogens as electron acceptors. Here, we used cryo-electron tomography to reveal morphological and ultrastructural features of Dhc strain BAV1 and ‘ Candidatus Dehalogenimonas etheniformans’ strain GP cells at unprecedented resolution. Dhc cells were irregularly shaped discs (890 ± 110 nm long, 630 ± 110 nm wide and 130 ± 15 nm thick) with curved and straight sides that intersected at acute angles, whereas Dhgm cells appeared as slightly flattened cocci (760 ± 85 nm). The cell envelopes were composed of a cytoplasmic membrane (CM), a paracrystalline surface layer (S-layer) with hexagonal symmetry and ∼22 nm spacing between repeating units, and a layer of unknown composition separating the CM and the S-layer. Cell surface appendages were only detected in Dhc cells, whereas both cell types had bundled cytoskeletal filaments. Repetitive globular structures, ∼5 nm in diameter and ∼9 nm apart, were observed associated with the outer leaflet of the CM. We hypothesized that those represent organohalide respiration (OHR) complexes and estimated ∼30,000 copies per cell. In Dhgm cultures, extracellular lipid vesicles (20 - 110 nm in diameter) decorated with putative OHR complexes but lacking an S-layer were observed. The new findings expand our understanding of the unique cellular ultrastructure and biology of organohalide-respiring Dehalococcoidia . Importance: Dehalococcoidia respire organohalogen compounds and play relevant roles in bioremediation of groundwater, sediments and soils impacted with toxic chlorinated pollutants. Using advanced imaging tools, we have obtained 3-dimensional images at macromolecular resolution of whole Dehalococcoidia cells revealing their unique structural components. Our data detail the overall cellular shape, cell envelope architecture, cytoskeletal filaments, the likely localization of enzymatic complexes involved in reductive dehalogenation, and the structure of extracellular vesicles. The new findings expand our understanding of the cell structure-function relationship in Dehalococcoidia with implications for Dehalococcoidia biology and bioremediation.

Thermophilic Dehalococcoidia with unusual traits shed light on an unexpected past

“…2E). This is similar to some cultivated Dehalococcoidaceae species 43 , but unlike other Chloroflexota where hexagonal S-layers only cover cell apices 38 .…”

“…Cryo-ET also revealed other structural features that are distinct from known organohalide-respiring Dehalococcoidia. Members of the order Dehalococcoidales range in cell shape from irregular discs (Dehalococcoides mccartyi 42,43 ) to irregular or slightly flattened cocci (Dehalogenimonas spp. 6,43,44 ).…”

“…The non-thermophilic Dehalococcoidia genera Dehalococcoides and Dehalogenimonas have either been reported to lack peptidoglycan in their cell envelopes 42,43,54 , or no data are available regarding envelope architecture 6,44,45 . The apparent lack of peptidoglycan was previously also supported by the limited available genome data, as evidence for peptidoglycan biosynthesis has largely been lacking from environmental genomes 1,35,55 .…”

“…Both novel Tepidiforma species were only capable of growth under fully aerobic conditions and showed significantly reduced growth at lower oxygen concentrations. Although the class Dehalococcoidia was initially considered to be strictly anaerobic, obligately using halogenated organic compounds for growth 12,35,43 , diverse ecological roles in anoxic and oxic settings have been proposed based on genome data 1,8,12,26,30,55 . As aerobes, these taxa do not encode a Wood-Ljungdahl pathway, like many anaerobic marine Dehalococcoidia 1,29 and lack robustly identified homologs for use of terminal electron acceptors other than oxygen (Table S6 and S7).…”

“…Physiologies within the Dehalococcoidia, both known and predicted, differ markedly. Though cultivated members of the class are best known for reductive dehalogenation 6,[42][43][44][45][46] or consortial degradation of recalcitrant organic carbon, and syntrophic interactions have been suggested based on environmental genomes 12,31,47 . For example, some marine Dehalococcoidia that encode genes involved in the degradation of aromatic and plant-derived organic compounds have been predicted to partially degrade terrestrial recalcitrant organic carbon for utilization by the rest of the community 14,32,55,57,58 .…”