The cell envelope of Thermotogae suggests a mechanism for outer membrane biogenesis.

The presence of a cell membrane is one of the major structural components defining life. Recent phylogenomic analyses have supported the hypothesis that the last universal common ancestor (LUCA) was likely a diderm. Yet, the mechanisms that guided outer membrane (OM) biogenesis remain unknown. Thermotogae is an early-branching phylum with a unique OM, the toga. Here, we use cryo-electron tomography to characterize the in situ cell envelope architecture of Thermotoga maritima and show that the toga is made of extended sheaths of ß-barrel trimers supporting small (~200 nm) membrane patches. Lipidomic analyses identified the same major lipid species in the inner membrane (IM) and toga, including the rare to bacteria membrane-spanning ether-bound diabolic acids (DAs). Proteomic analyses revealed that the toga was composed of multiple SLH-domain containing Ompα and novel ß-barrel proteins, and homology searches detected variable conservations of these proteins across the phylum. These results highlight that, in contrast to the SlpA/OmpM superfamily of proteins, Thermotoga possess a highly diverse bipartite OM-tethering system. We discuss the implications of our findings with respect to other early-branching phyla and propose that a toga-like intermediate may have facilitated monoderm-to-diderm cell envelope transitions.

Thermophilic Dehalococcoidia with unusual traits shed light on an unexpected past.

Although the phylum Chloroflexota is ubiquitous, its biology and evolution are poorly understood due to limited cultivability. Here, we isolated two motile, thermophilic bacteria from hot spring sediments belonging to the genus Tepidiforma and class Dehalococcoidia within the phylum Chloroflexota. A combination of cryo-electron tomography, exometabolomics, and cultivation experiments using stable isotopes of carbon revealed three unusual traits: flagellar motility, a peptidoglycan-containing cell envelope, and heterotrophic activity on aromatics and plant-associated compounds. Outside of this genus, flagellar motility has not been observed in Chloroflexota, and peptidoglycan-containing cell envelopes have not been described in Dehalococcoidia. Although these traits are unusual among cultivated Chloroflexota and Dehalococcoidia, ancestral character state reconstructions showed flagellar motility and peptidoglycan-containing cell envelopes were ancestral within the Dehalococcoidia, and subsequently lost prior to a major adaptive radiation of Dehalococcoidia into marine environments. However, despite the predominantly vertical evolutionary histories of flagellar motility and peptidoglycan biosynthesis, the evolution of enzymes for degradation of aromatics and plant-associated compounds was predominantly horizontal and complex. Together, the presence of these unusual traits in Dehalococcoidia and their evolutionary histories raise new questions about the timing and selective forces driving their successful niche expansion into global oceans.

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

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 to 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. IMPORTANCEDehalococcoidia 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 three-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.

Structural, Metabolic and Evolutionary Comparison of Bacterial Endospore and Exospore Formation.

Sporulation is a specialized developmental program employed by a diverse set of bacteria which culminates in the formation of dormant cells displaying increased resilience to stressors. This represents a major survival strategy for bacteria facing harsh environmental conditions, including nutrient limitation, heat, desiccation, and exposure to antimicrobial compounds. Through dispersal to new environments via biotic or abiotic factors, sporulation provides a means for disseminating genetic material and promotes encounters with preferable environments thus promoting environmental selection. Several types of bacterial sporulation have been characterized, each involving numerous morphological changes regulated and performed by non-homologous pathways. Despite their likely independent evolutionary origins, all known modes of sporulation are typically triggered by limited nutrients and require extensive membrane and peptidoglycan remodeling. While distinct modes of sporulation have been observed in diverse species, two major types are at the forefront of understanding the role of sporulation in human health, and microbial population dynamics and survival. Here, we outline endospore and exospore formation by members of the phyla Firmicutes and Actinobacteria, respectively. Using recent advances in molecular and structural biology, we point to the regulatory, genetic, and morphological differences unique to endo- and exospore formation, discuss shared characteristics that contribute to the enhanced environmental survival of spores and, finally, cover the evolutionary aspects of sporulation that contribute to bacterial species diversification.

No endospore formation confirmed in members of the phylum Proteobacteria.

Endospore formation is used by members of the phylum Firmicutes to withstand extreme environmental conditions. Several recent studies have proposed endospore formation in species outside of Firmicutes, particularly in Rhodobacter johrii and Serratia marcescens, members of the phylum Proteobacteria. Here, we aimed to investigate endospore formation in these two species by using advanced imaging and analytical approaches. Examination of the phase-bright structures observed in R. johrii and S. marcescens using cryo-electron tomography failed to identify endospores or stages of endospore formation. We determined that the phase-bright objects in R. johrii cells were triacylglycerol storage granules and those in S. marcescens were aggregates of cellular debris. In addition, R. johrii and S. marcescens containing phase-bright objects do not possess phenotypic and genetic features of endospores, including enhanced resistance to heat, presence of dipicolinic acid, or the presence of many of the genes associated with endospore formation. Our results support the hypothesis that endospore formation is restricted to the phylum Firmicutes.Importance: Bacterial endospore formation is an important process that allows the formation of dormant life forms called spores. As such, organisms able to sporulate can survive harsh environmental conditions for hundreds of years. Here, we follow up on previous claims that two members of Proteobacteria, Serratia marcescens and Rhodobacter johrii, are able to form spores. We conclude that those claims were incorrect and show that the putative spores in R. johrii and S. marcescens are storage granules and cellular debris, respectively. This study concludes that endospore formation is still unique to the phylum Firmicutes.

Genomics and metatranscriptomics of biogeochemical cycling and degradation of lignin-derived aromatic compounds in thermal swamp sediment.

Thermal swamps are unique ecosystems where geothermally warmed waters mix with decomposing woody biomass, hosting novel biogeochemical-cycling and lignin-degrading microbial consortia. Assembly of shotgun metagenome libraries resolved 351 distinct genomes from hot-spring (30-45 °C) and mesophilic (17 °C) sediments. Annotation of 39 refined draft genomes revealed metabolism consistent with oligotrophy, including pathways for degradation of aromatic compounds, such as syringate, vanillate, p-hydroxybenzoate, and phenol. Thermotolerant Burkholderiales, including Rubrivivax ssp., were implicated in diverse biogeochemical and aromatic transformations, highlighting their broad metabolic capacity. Lignin catabolism was further investigated using metatranscriptomics of sediment incubated with milled or Kraft lignin at 45 °C. Aromatic compounds were depleted from lignin-amended sediment over 148 h. The metatranscriptomic data revealed upregulation of des/lig genes predicted to specify the catabolism of syringate, vanillate, and phenolic oligomers in the sphingomonads Altererythrobacter ssp. and Novosphingobium ssp., as well as in the Burkholderiales genus, Rubrivivax. This study demonstrates how temperature structures biogeochemical cycling populations in a unique ecosystem, and combines community-level metagenomics with targeted metatranscriptomics to identify pathways with potential for bio-refinement of lignin-derived aromatic compounds. In addition, the diverse aromatic catabolic pathways of Altererythrobacter ssp. may serve as a source of thermotolerant enzymes for lignin valorization.

Steryl Ester Formation and Accumulation in Steroid-Degrading Bacteria.

Steryl esters (SEs) are important storage compounds in many eukaryotes and are often prominent components of intracellular lipid droplets. Here, we demonstrate that selected Actino- and Proteobacteria growing on sterols are also able to synthesize SEs and to sequester them in cytoplasmic lipid droplets. We found cholesteryl ester (CE) formation in members of the actinobacterial genera Rhodococcus, Mycobacterium, and Amycolatopsis, as well as several members of the proteobacterial Cellvibrionales order. CEs maximally accumulated under nitrogen-limiting conditions, suggesting that steryl ester formation plays a crucial role for storing excess energy and carbon under adverse conditions. Rhodococcus jostii RHA1 was able to synthesize phytosteryl and cholesteryl esters, the latter reaching up to 7% of its cellular dry weight and 69% of its lipid droplets. Purified lipid droplets from RHA1 contained CEs, free cholesterol, and triacylglycerols. In addition, we found formation of CEs in Mycobacterium tuberculosis when it was grown with cholesterol plus an additional fatty acid substrate. This study provides a basis for the application of bacterial whole-cell systems in the biotechnological production of SEs for use in functional foods and cosmetics.IMPORTANCE Oleaginous bacteria exhibit great potential for the production of high-value neutral lipids, such as triacylglycerols and wax esters. This study describes the formation of steryl esters (SEs) as neutral lipid storage compounds in sterol-degrading oleaginous bacteria, providing a basis for biotechnological production of SEs using bacterial systems with potential applications in the functional food, nutraceutical, and cosmetic industries. We found cholesteryl ester (CE) formation in several sterol-degrading Actino- and Proteobacteria under nitrogen-limiting conditions, suggesting an important role of this process in storing energy and carbon under adverse conditions. In addition, Mycobacterium tuberculosis grown on cholesterol accumulated CEs in the presence of an additional fatty acid substrate.

Criibacterium bergeronii gen. nov., sp. nov., a new member of the family Peptostreptococcaceae, isolated from human clinical samples.

A rod-shaped, motile anaerobic bacterium, designated CCRI-22567T, was isolated from a vaginal sample of a woman diagnosed with bacterial vaginosis and subjected to a polyphasic taxonomic study. The novel strain was capable of growth at 30-42 °C (optimum, 42 °C), at pH 5.5-8.5 (optimum, pH 7.0-7.5) and in the presence of 0-1.5 % (w/v) NaCl (optimally at 0.5 % NaCl). The phylogenetic trees based on 16S rRNA gene sequences showed that strain CCRI-22567T forms a distinct evolutionary lineage independent of other taxa in the family Peptostreptococcaceae. Strain CCRI-22567T exhibited 90.1 % 16S rRNA gene sequence similarity to Peptoanaerobacter stomatis ACC19aT and 89.7 % to Eubacterium yurii subsp. schtitka ATCC 43716. The three closest organisms with an available whole genome were compared to strain CCRI-22567T for genomic relatedness assessment. The genomic average nucleotide identities (OrthoANIu) obtained with Peptoanaerobacter stomatis ACC19aT, Eubacterium yurii subsp. margaretiae ATCC 43715 and Filifactor alocis ATCC 35896T were 71.8, 70.3 and 69.6 %, respectively. Strain CCRI-22567T contained C18 : 1 ω9c and C18 : 1 ω9c DMA as the major fatty acids. The DNA G+C content of strain CCRI-22567T based on its genome sequence was 33.8 mol%. On the basis of the phylogenetic, chemotaxonomic and other phenotypic properties, strain CCRI-22567T is considered to represent a new genus and species within the family Peptostreptococcaceae, for which the name Criibacterium bergeronii gen. nov., sp. nov., is proposed. The type strain of Criibacterium bergeronii is CCRI-22567T (=LMG 31278T=DSM 107614T=CCUG 72594T).