Purifying Selection and Molecular Adaptation in the Genome of Verminephrobacter, the Heritable Symbiotic Bacteria of Earthworms

While genomic erosion is common among intracellular symbionts, patterns of genome evolution in heritable extracellular endosymbionts remain elusive. We study vertically transmitted extracellular endosymbionts (Verminephrobacter, Betaproteobacteria) that form a beneficial, species-specific, and evolutionarily old (60–130 Myr) association with earthworms. We assembled a draft genome of Verminephrobacter aporrectodeae and compared it with the genomes of Verminephrobacter eiseniae and two nonsymbiotic close relatives (Acidovorax). Similar to V. eiseniae, the V. aporrectodeae genome was not markedly reduced in size and showed no A–T bias. We characterized the strength of purifying selection (ω = dN/dS) and codon usage bias in 876 orthologous genes. Symbiont genomes exhibited strong purifying selection (ω = 0.09 ± 0.07), although transition to symbiosis entailed relaxation of purifying selection as evidenced by 50% higher ω values and less codon usage bias in symbiont compared with reference genomes. Relaxation was not evenly distributed among functional gene categories but was overrepresented in genes involved in signal transduction and cell envelope biogenesis. The same gene categories also harbored instances of positive selection in the Verminephrobacter clade. In total, positive selection was detected in 89 genes, including also genes involved in DNA metabolism, tRNA modification, and TonB-dependent iron uptake, potentially highlighting functions important in symbiosis. Our results suggest that the transition to symbiosis was accompanied by molecular adaptation, while purifying selection was only moderately relaxed, despite the evolutionary age and stability of the host association. We hypothesize that biparental transmission of symbionts and rare genetic mixing during transmission can prevent genome erosion in heritable symbionts

Ubiquitous Presence and Novel Diversity of Anaerobic Alkane Degraders in Cold Marine Sediments

Alkanes are major constituents of crude oil and are released to the marine environment by natural seepage and from anthropogenic sources. Due to their chemical inertness, their removal from anoxic marine sediments is primarily controlled by the activity of anaerobic alkane-degrading microorganisms. To facilitate comprehensive cultivation-independent surveys of the diversity and distribution of anaerobic alkane degraders, we designed novel PCR primers that cover all known diversity of the 1-methylalkyl succinate synthase gene (masD/assA), which catalyzes the initial activation of alkanes. We studied masD/assA gene diversity in pristine and seepage-impacted Danish coastal sediments, as well as in sediments and alkane-degrading enrichment cultures from the Middle Valley (MV) hydrothermal vent system in the Pacific Northwest. MasD/assA genes were ubiquitously present, and the primers captured the diversity of both known and previously undiscovered masD/assA gene diversity. Seepage sediments were dominated by a single masD/assA gene cluster, which is presumably indicative of a substrate-adapted community, while pristine sediments harbored a diverse range of masD/assA phylotypes including those present in seepage sediments. This rare biosphere of anaerobic alkane degraders will likely increase in abundance in the event of seepage or accidental oil spillage. Nanomolar concentrations of short-chain alkanes (SCA) were detected in pristine and seepage sediments. Interestingly, anaerobic alkane degraders closely related to strain BuS5, the only SCA degrader in pure culture, were found in mesophilic MV enrichments, but not in cold sediments from Danish waters. We propose that the new masD/assA gene lineages in these sediments represent novel phylotypes that are either fueled by naturally occurring low levels of SCA or that metabolize medium- to long-chain alkanes. Our study highlights that masD/assA genes are a relevant diagnostic marker to identify seepage and microseepage, e.g., during prospecting for oil and gas, and may act as an indicator of anthropogenic oil spills in marine sediments

Single-Cell Genomics Reveals a Diverse Metabolic Potential of Uncultivated Desulfatiglans-Related Deltaproteobacteria Widely Distributed in Marine Sediment

Desulfatiglans-related organisms comprise one of the most abundant deltaproteobacterial lineages in marine sediments where they occur throughout the sediment column in a gradient of increasing sulfate and organic carbon limitation with depth. Characterized Desulfatiglans isolates are dissimilatory sulfate reducers able to grow by degrading aromatic hydrocarbons. The ecophysiology of environmental Desulfatiglans-populations is poorly understood, however, possibly utilization of aromatic compounds may explain their predominance in marine subsurface sediments. We sequenced and analyzed seven Desulfatiglans-related single-cell genomes (SAGs) from Aarhus Bay sediments to characterize their metabolic potential with regard to aromatic compound degradation and energy metabolism. The average genome assembly size was 1.3 Mbp and completeness estimates ranged between 20 and 50%. Five of the SAGs (group 1) originated from the sulfate-rich surface part of the sediment while two (group 2) originated from sulfate-depleted subsurface sediment. Based on 16S rRNA gene amplicon sequencing group 2 SAGs represent the more frequent types of Deaufatig/ans-populations in Aarhus Bay sediments. Genes indicative of aromatic compound degradation could be identified in both groups, but the two groups were metabolically distinct with regard to energy conservation. Group 1 SAGs carry a full set of genes for dissimilatory sulfate reduction, whereas the group 2 SAGs lacked any genetic evidence for sulfate reduction. The latter may be due to incompleteness of the SAGs, but as alternative energy metabolisms group 2 SAGs carry the genetic potential for growth by acetogenesis and fermentation. Group 1 SAGs encoded reductive dehalogenase genes, allowing them to access organohalides and possibly conserve energy by their reduction. Both groups possess sulfatases unlike their cultured relatives allowing them to utilize sulfate esters as source of organic carbon and sulfate. In conclusion, the uncultivated marine Desulfatiglans populations are metabolically diverse, likely reflecting different strategies for coping with energy and sulfate limitation in the subsurface seabed

Microbial community assembly and evolution in subseafloor sediment

Bacterial and archaeal communities inhabiting the subsurface seabed live under strong energy limitation and have growth rates that are orders of magnitude slower than laboratory-grown cultures. It is not understood how subsurface microbial communities are assembled and whether populations undergo adaptive evolution or accumulate mutations as a result of impaired DNA repair under such energy-limited conditions. Here we use amplicon sequencing to explore changes of microbial communities during burial and isolation from the surface to the > 5,000-y-old subsurface of marine sediment and identify a small core set of mostly uncultured bacteria and archaea that is present throughout the sediment column. These persisting populations constitute a small fraction of the entire community at the surface but become predominant in the subsurface. We followed patterns of genome diversity with depth in four dominant lineages of the persisting populations by mapping metagenomic sequence reads onto single-cell genomes. Nucleotide sequence diversity was uniformly low and did not change with age and depth of the sediment. Likewise, therewas no detectable change inmutation rates and efficacy of selection. Our results indicate that subsurface microbial communities predominantly assemble by selective survival of taxa able to persist under extreme energy limitation

Marine Deep Biosphere Microbial Communities Assemble in Near-Surface Sediments in Aarhus Bay

Analyses of microbial diversity in marine sediments have identified a core set of taxa unique to the marine deep biosphere. Previous studies have suggested that these specialized communities are shaped by processes in the surface seabed, in particular that their assembly is associated with the transition from the bioturbated upper zone to the nonbioturbated zone below. To test this hypothesis, we performed a fine-scale analysis of the distribution and activity of microbial populations within the upper 50 cm of sediment from Aarhus Bay (Denmark). Sequencing and qPCR were combined to determine the depth distributions of bacterial and archaeal taxa (16S rRNA genes) and sulfate-reducing microorganisms (SRM) (dsrB gene). Mapping of radionuclides throughout the sediment revealed a region of intense bioturbation at 0–6 cm depth. The transition from bioturbated sediment to the subsurface below (7 cm depth) was marked by a shift from dominant surface populations to common deep biosphere taxa (e.g., Chloroflexi and Atribacteria). Changes in community composition occurred in parallel to drops in microbial activity and abundance caused by reduced energy availability below the mixed sediment surface. These results offer direct evidence for the hypothesis that deep subsurface microbial communities present in Aarhus Bay mainly assemble already centimeters below the sediment surface, below the bioturbation zone

Sulfate Transporters in Dissimilatory Sulfate Reducing Microorganisms: A Comparative Genomics Analysis

The first step in the sulfate reduction pathway is the transport of sulfate across the cell membrane. This uptake has a major effect on sulfate reduction rates. Much of the information available on sulfate transport was obtained by studies on assimilatory sulfate reduction, where sulfate transporters were identified among several types of protein families. Despite our growing knowledge on the physiology of dissimilatory sulfate-reducing microorganisms (SRM) there are no studies identifying the proteins involved in sulfate uptake in members of this ecologically important group of anaerobes. We surveyed the complete genomes of 44 sulfate-reducing bacteria and archaea across six phyla and identified putative sulfate transporter encoding genes from four out of the five surveyed protein families based on homology. We did not find evidence that ABC-type transporters (SulT) are involved in the uptake of sulfate in SRM. We speculate that members of the CysP sulfate transporters could play a key role in the uptake of sulfate in thermophilic SRM. Putative CysZ-type sulfate transporters were present in all genomes examined suggesting that this overlooked group of sulfate transporters might play a role in sulfate transport in dissimilatory sulfate reducers alongside SulP. Our in silico analysis highlights several targets for further molecular studies in order to understand this key step in the metabolism of SRMs

A Novel Extracellular Gut Symbiont in the Marine Worm Priapulus caudatus (Priapulida) Reveals an Alphaproteobacterial Symbiont Clade of the Ecdysozoa

Priapulus caudatus (phylum Priapulida) is a benthic marine predatory worm with a cosmopolitan distribution. In its digestive tract we detected symbiotic bacteria that were consistently present in specimens collected over eight years from three sites at the Swedish west coast. Based on their 16S rRNA gene sequence, these symbionts comprise a novel genus of the order Rickettsiales (Alphaproteobacteria). Electron microscopy and fluorescence in situ hybridization (FISH) identified them as extracellular, elongate bacteria closely associated with the microvilli, for which we propose the name ‘Candidatus Tenuibacter priapulorum’. Within Rickettsiales, they form a phylogenetically well-defined, family-level clade with uncultured symbionts of marine, terrestrial, and freshwater arthropods. Cand. Tenuibacter priapulorum expands the host range of this candidate family from Arthropoda to the entire Ecdysozoa, which may indicate an evolutionary adaptation of this bacterial group to the microvilli-lined guts of the Ecdysozoa

Concurrent Methane Production and Oxidation in Surface Sediment from Aarhus Bay, Denmark

Marine surface sediments, which are replete with sulfate, are typically considered to be devoid of endogenous methanogenesis. Yet, methanogenic archaea are present in those sediments, suggesting a potential for methanogenesis. We used an isotope dilution method based on sediment bag incubation and spiking with 13C-CH4 to quantify CH4 turnover rates in sediment from Aarhus Bay, Denmark. In two independent experiments, highest CH4 production and oxidation rates (>200 pmol cm-3 d-1) were found in the top 0–2 cm, below which rates dropped below 100 pmol cm-3 d-1 in all other segments down to 16 cm. This drop in overall methane turnover with depth was accompanied by decreasing rates of organic matter mineralization with depth. Molecular analyses based on quantitative PCR and MiSeq sequencing of archaeal 16S rRNA genes showed that the abundance of methanogenic archaea also peaked in the top 0–2 cm segment. Based on the community profiling, hydrogenotrophic and methylotrophic methanogens dominated among the methanogenic archaea in general, suggesting that methanogenesis in surface sediment could be driven by both CO2 reduction and fermentation of methylated compounds. Our results show the existence of elevated methanogenic activity and a dynamic recycling of CH4 at low concentration in sulfate-rich marine surface sediment. Considering the common environmental conditions found in other coastal systems, we speculate that such a cryptic methane cycling can be ubiquitous