AbstractExtracellular DNA is a major macromolecule in global element cycles, and is a particularly crucial phosphorus, nitrogen and carbon source for microorganisms in the seafloor. Nevertheless, the identities, ecophysiology and genetic features of DNA-foraging microorganisms in marine sediments are largely unknown. Here, we combined microcosm experiments, DNA stable isotope probing (SIP), single-cell SIP using nano-scale secondary isotope mass spectrometry (NanoSIMS) and genome-centric metagenomics to study microbial catabolism of DNA and its subcomponents in marine sediments. 13C-DNA added to sediment microcosms was largely degraded within 10 d and mineralized to 13CO2. SIP probing of DNA revealed diverse ‘Candidatus Izemoplasma’, Lutibacter, Shewanella and Fusibacteraceae incorporated DNA-derived 13C-carbon. NanoSIMS confirmed incorporation of 13C into individual bacterial cells of Fusibacteraceae sorted from microcosms. Genomes of the 13C-labelled taxa all encoded enzymatic repertoires for catabolism of DNA or subcomponents of DNA. Comparative genomics indicated that diverse ‘Candidatus Izemoplasmatales’ (former Tenericutes) are exceptional because they encode multiple (up to five) predicted extracellular nucleases and are probably specialized DNA-degraders. Analyses of additional sediment metagenomes revealed extracellular nuclease genes are prevalent among Bacteroidota at diverse sites. Together, our results reveal the identities and functional properties of microorganisms that may contribute to the key ecosystem function of degrading and recycling DNA in the seabed.
The class
Deltaproteobacteria
comprises an ecologically and metabolically diverse group of bacteria best known for dissimilatory sulphate reduction and predatory behaviour. Although this lineage is the fourth described class of the phylum
Proteobacteria
, it rarely affiliates with other proteobacterial classes and is frequently not recovered as a monophyletic unit in phylogenetic analyses. Indeed, one branch of the class
Deltaproteobacteria
encompassing Bdellovibrio-like predators was recently reclassified into a separate proteobacterial class, the
Oligoflexia
. Here we systematically explore the phylogeny of taxa currently assigned to these classes using 120 conserved single-copy marker genes as well as rRNA genes. The overwhelming majority of markers reject the inclusion of the classes
Deltaproteobacteria
and
Oligoflexia
in the phylum
Proteobacteria
. Instead, the great majority of currently recognized members of the class
Deltaproteobacteria
are better classified into four novel phylum-level lineages. We propose the names Desulfobacterota phyl. nov. and Myxococcota phyl. nov. for two of these phyla, based on the oldest validly published names in each lineage, and retain the placeholder name SAR324 for the third phylum pending formal description of type material. Members of the class
Oligoflexia
represent a separate phylum for which we propose the name Bdellovibrionota phyl. nov. based on priority in the literature and general recognition of the genus Bdellovibrio. Desulfobacterota phyl. nov. includes the taxa previously classified in the phylum
Thermodesulfobacteria
, and these reclassifications imply that the ability of sulphate reduction was vertically inherited in the
Thermodesulfobacteria
rather than laterally acquired as previously inferred. Our analysis also indicates the independent acquisition of predatory behaviour in the phyla Myxococcota and Bdellovibrionota, which is consistent with their distinct modes of action. This work represents a stable reclassification of one of the most taxonomically challenging areas of the bacterial tree and provides a robust framework for future ecological and systematic studies.
For
Thaumarchaeota
, the ratio of their glycerol dialkyl glycerol tetraether (GDGT) lipids depends on growth temperature, a premise that forms the basis of the widely applied TEX
86
paleotemperature proxy. A thorough understanding of which GDGTs are produced by which
Thaumarchaeota
and what the effect of temperature is on their GDGT composition is essential for constraining the TEX
86
proxy. “
Ca
. Nitrosotenuis uzonensis” is a moderately thermophilic thaumarchaeote enriched from a thermal spring, setting it apart in its environmental niche from the other marine mesophilic members of its order. Indeed, we found that the GDGT composition of “
Ca
. Nitrosotenuis uzonensis” cultures was distinct from those of other members of its order and was more similar to those of other thermophilic, terrestrial
Thaumarchaeota
. This suggests that while phylogeny has a strong influence on GDGT distribution, the environmental niche that a thaumarchaeote inhabits also shapes its GDGT composition.
ABSTRACT
Nitrification is a key process of the biogeochemical nitrogen cycle and of biological wastewater treatment. The second step, nitrite oxidation to nitrate, is catalyzed by phylogenetically diverse, chemolithoautotrophic nitrite-oxidizing bacteria (NOB). Uncultured NOB from the genus “
Candidatus
Nitrotoga” are widespread in natural and engineered ecosystems. Knowledge about their biology is sparse, because no genomic information and no pure “
Ca
. Nitrotoga” culture was available. Here we obtained the first “
Ca
. Nitrotoga” isolate from activated sludge. This organism, “
Candidatus
Nitrotoga fabula,” prefers higher temperatures (>20°C; optimum, 24 to 28°C) than previous “
Ca
. Nitrotoga” enrichments, which were described as cold-adapted NOB. “
Ca
. Nitrotoga fabula” also showed an unusually high tolerance to nitrite (activity at 30 mM NO
2
−
) and nitrate (up to 25 mM NO
3
−
). Nitrite oxidation followed Michaelis-Menten kinetics, with an apparent
K
m
(
K
m
(app)
) of ~89 µM nitrite and a
V
max
of ~28 µmol of nitrite per mg of protein per h. Key metabolic pathways of “
Ca
. Nitrotoga fabula” were reconstructed from the closed genome. “
Ca
. Nitrotoga fabula” possesses a new type of periplasmic nitrite oxidoreductase belonging to a lineage of mostly uncharacterized proteins. This novel enzyme indicates (i) separate evolution of nitrite oxidation in “
Ca
. Nitrotoga” and other NOB, (ii) the possible existence of phylogenetically diverse, unrecognized NOB, and (iii) together with new metagenomic data, the potential existence of nitrite-oxidizing archaea. For carbon fixation, “
Ca
. Nitrotoga fabula” uses the Calvin-Benson-Bassham cycle. It also carries genes encoding complete pathways for hydrogen and sulfite oxidation, suggesting that alternative energy metabolisms enable “
Ca
. Nitrotoga fabula” to survive nitrite depletion and colonize new niches.
IMPORTANCE
Nitrite-oxidizing bacteria (NOB) are major players in the biogeochemical nitrogen cycle and critical for wastewater treatment. However, most NOB remain uncultured, and their biology is poorly understood. Here, we obtained the first isolate from the environmentally widespread NOB genus “
Candidatus
Nitrotoga” and performed a detailed physiological and genomic characterization of this organism (“
Candidatus
Nitrotoga fabula”). Differences between key phenotypic properties of “
Ca
. Nitrotoga fabula” and those of previously enriched “
Ca
. Nitrotoga” members reveal an unexpectedly broad range of physiological adaptations in this genus. Moreover, genes encoding components of energy metabolisms outside nitrification suggest that “
Ca
. Nitrotoga” are ecologically more flexible than previously anticipated. The identification of a novel nitrite-oxidizing enzyme in “
Ca
. Nitrotoga fabula” expands our picture of the evolutionary history of nitrification and might lead to discoveries of novel nitrite oxidizers. Altogether, this study provides urgently needed insights into the biology of understudied but environmentally and biotechnologically important microorganisms.
AbstractAmmonia-oxidizing archaea (AOA) within the phylumThaumarchaeaare the only known aerobic ammonia oxidizers in geothermal environments. Although molecular data indicate the presence of phylogenetically diverse AOA from theNitrosocaldusclade, group 1.1b and group 1.1aThaumarchaeain terrestrial high-temperature habitats, only one enrichment culture of an AOA thriving above 50 °C has been reported and functionally analyzed. In this study, we physiologically and genomically characterized a novelThaumarchaeonfrom the deep-branchingNitrosocaldaceaefamily of which we have obtained a high (∼85 %) enrichment from biofilm of an Icelandic hot spring (73 °C). This AOA, which we provisionally refer to as “CandidatusNitrosocaldus islandicus”, is an obligately thermophilic, aerobic chemolithoautotrophic ammonia oxidizer, which stoichiometrically converts ammonia to nitrite at temperatures between 50 °C and 70 °C.Ca.N. islandicus encodes the expected repertoire of enzymes proposed to be required for archaeal ammonia oxidation, but unexpectedly lacks anirKgene and also possesses no identifiable other enzyme for nitric oxide (NO) generation. Nevertheless, ammonia oxidation by this AOA appears to be NO-dependent asCa.N. islandicus is, like all other tested AOA, inhibited by the addition of an NO scavenger. Furthermore, comparative genomics revealed thatCa.N. islandicus has the potential for aromatic amino acid fermentation as its genome encodes an indolepyruvate oxidoreductase(iorAB)as well as a type 3b hydrogenase, which are not present in any other sequenced AOA. A further surprising genomic feature of this thermophilic ammonia oxidizer is the absence of DNA polymerase D genes - one of the predominant replicative DNA polymerases in all other ammonia-oxidizingThaumarchaea.Collectively, our findings suggest that metabolic versatility and DNA replication might differ substantially between obligately thermophilic and other AOA.
AbstractThaumarchaeota have been detected in several industrial and municipal wastewater treatment plants (WWTPs), despite the fact that ammonia-oxidizing archaea (AOA) are thought to be adapted to low ammonia environments. However, the activity, physiology and metabolism of WWTP-associated AOA remain poorly understood. We report the cultivation and complete genome sequence of Candidatus Nitrosocosmicus exaquare, a novel AOA representative from a municipal WWTP in Guelph, Ontario (Canada). In enrichment culture, Ca. N. exaquare oxidizes ammonia to nitrite stoichiometrically, is mesophilic, and tolerates at least 15 mm of ammonium chloride or sodium nitrite. Microautoradiography (MAR) for enrichment cultures demonstrates that Ca. N. exaquare assimilates bicarbonate in association with ammonia oxidation. However, despite using inorganic carbon, the ammonia-oxidizing activity of Ca. N. exaquare is greatly stimulated in enrichment culture by the addition of organic compounds, especially malate and succinate. Ca. N. exaquare cells are coccoid with a diameter of ~1–2 μm. Phylogenetically, Ca. N. exaquare belongs to the Nitrososphaera sister cluster within the Group I.1b Thaumarchaeota, a lineage which includes most other reported AOA sequences from municipal and industrial WWTPs. The 2.99 Mbp genome of Ca. N. exaquare encodes pathways for ammonia oxidation, bicarbonate fixation, and urea transport and breakdown. In addition, this genome encodes several key genes for dealing with oxidative stress, including peroxidase and catalase. Incubations of WWTP biofilm demonstrate partial inhibition of ammonia-oxidizing activity by 2-phenyl-4,4,5,5-tetramethylimidazoline-1-oxyl 3-oxide (PTIO), suggesting that Ca. N. exaquare-like AOA may contribute to nitrification in situ. However, CARD-FISH-MAR showed no incorporation of bicarbonate by detected Thaumarchaeaota, suggesting that detected AOA may incorporate non-bicarbonate carbon sources or rely on an alternative and yet unknown metabolism.