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Background The Anaerolineae lineage of Chloroflexi had been identified as one of the core microbial populations in anaerobic digesters; however, the ecological role of the Anaerolineae remains uncertain due to the scarcity of isolates and annotated genome sequences. Our previous metatranscriptional analysis revealed this prevalent population that showed minimum involvement in the main pathways of cellulose hydrolysis and subsequent methanogenesis in the thermophilic cellulose fermentative consortium (TCF). Results In further pursuit, five high-quality curated draft genomes (>98 % completeness) of this population, including two affiliated with the inaccessible lineage of SBR1031, were retrieved by sequence-based multi-dimensional coverage binning. Comparative genomic analyses revealed versatile genetic capabilities for carbohydrate-based fermentative lifestyle including key genes catalyzing cellulose hydrolysis in Anaerolinea phylotypes. However, the low transcriptional activities of carbohydrate-active genes (CAGs) excluded cellulolytic capability as the selective advantage for their prevalence in the community. Instead, a substantially active type VI pili (Tfp) assembly was observed. Expression of the tight adherence protein on the Tfp indicated its function for cellular attachment which was further testified to be more likely related to cell aggregation other than cellulose surface adhesion. Meanwhile, this Tfp structure was found not contributing to syntrophic methanogenesis. Members of the SBR1031 encoded key genes for acetogenic dehydrogenation that may allow ethanol to be used as a carbon source. Conclusion The common prevalence of Anaerolineae in anaerobic digesters should be originated from advantageous cellular adhesiveness enabled by Tfp assembly other than its potential as cellulose degrader or anaerobic syntrophs.

The adaptation of the phylum Chloroflexota to various geochemical conditions is thought to have originated in primitive microbial ecosystems, involving hydrogenotrophic energy conservation under ferruginous anoxia. Oligotrophic deep waters displaying anoxic ferruginous conditions, such as those of Lake Towuti, and their sediments may thus constitute a preferential ecological niche for investigating metabolic versatility in modern Chloroflexota. Combining pore water geochemistry, cell counts, sulfate reduction rates, and 16S rRNA genes with in-depth analysis of metagenome-assembled genomes, we show that Chloroflexota benefit from cross-feeding on metabolites derived from canonical respiration chains and fermentation. Detailing their genetic contents, we provide molecular evidence that Anaerolineae have metabolic potential to use unconventional electron acceptors, different cytochromes, and multiple redox metalloproteins to cope with oxygen fluctuations, and thereby effectively colonizing the ferruginous sediment-water interface. In sediments, Dehalococcoidia evolved to be acetogens, scavenging fatty acids, haloacids, and aromatic acids, apparently bypassing specific steps in carbon assimilation pathways to perform energy-conserving secondary fermentations combined with CO2 fixation via the Wood–Ljungdahl pathway. Our study highlights the partitioning of Chloroflexota populations according to alternative electron acceptors and donors available at the sediment-water interface and below. Chloroflexota would have developed analogous primeval features due to oxygen fluctuations in ancient ferruginous ecosystems.

In the metabolic pathway of chlorophylls (Chls), an enzyme called STAY-GREEN or SGR catalyzes the removal of the central magnesium ion of Chls and their derivatives to their corresponding free bases, including pheophytins. The substrate specificity of SGR has been investigated through in vitro reactions using Chl-related molecules. However, information about the biochemical properties and reaction mechanisms of SGR and its substrate specificity remains elusive. In this study, we synthesized various Chl derivatives and investigated their in vitro dechelations using an SGR enzyme. Chl-a derivatives with the C3-vinyl group on the A-ring, which is commonly found as a substituent in natural substrates, and their analogs with ethyl, hydroxymethyl, formyl, and styryl groups at the C3-position were prepared as substrates. In vitro dechelatase reactions of these substrates were performed using an SGR enzyme derived from an Anaerolineae bacterium, allowing us to investigate their specificity. Reactivity was reduced for substrates with an electron-withdrawing formyl or sterically demanding styryl group at the C3-position. Furthermore, the Chl derivative with the C8-styryl group on the B-ring was less reactive for SGR dechelation than the C3-styryl substrate. These results indicate that the SGR enzyme recognizes substituents on the B-ring of substrates more than those on the A-ring.

In this study, we report for the first time an exploration of the physicochemical characteristics and the prokaryotic diversity of three different types of microbial mats from karstic habitats located in Sisal, Progreso and Ría Lagartos, in the Yucatan Peninsula, Mexico. Our results showed that lift-off mats were found in the lower salinity (2.2%) area (Sisal), while flat and pustular mats were detected in hypersaline (6–9%) sites (Progreso and Ría Lagartos). Notably, some of these microbial mat structures were in close proximity to mangrove forest ecosystems with both degraded and restored regimes. XRD analysis revealed different mineral compositions of the mats; however, aragonite, calcite, and halite were commonly found in all samples studied. High-throughput sequencing of the 16S rRNA gene identified differences in microbial communities across the different mat types, and statistical analyses revealed that salinity, redox potential, and temperature were significant factors in explaining the variance of the prokaryotic assemblages. Microbial groups identified in this study include those known to be important in the biogeochemical cycling of key elements, such as carbon, nitrogen, and sulfur. Interestingly, the community composition of flat and pustular mats from Progreso was similar, with Bacteroidia, Anaerolineae, and Phycisphaerae being the most abundant microbial groups in flat mats; and Bacteroidia, Anaerolineae, and Alphaproteobacteria dominating pustular mats. By contrast, flat mats from Ría Lagartos were dominated by Halobacteria, Cyanobacteria and Bacteroidota, while Bacteroidia, Gammaproteobacteria and Cyanobacteria dominated lift-off mats from Sisal. This work contributes to understanding the distribution, physicochemical characteristics and microbial diversity of coastal microbial mats, providing valuable new insights into microbial mats that develop in karstic ecosystems. This information is relevant to ongoing and future efforts to manage and preserve coastal ecosystems in the Yucatan Peninsula.

Understanding how long-term agricultural practices affect soil bacteriome is essential for sustainable land management. In the Guadalquivir Marshes of southwestern Spain, which encompass both Doñana National Park and one of Europe’s most productive rice cultivation areas, decades of rice farming have transformed natural wetlands into artificial agroecosystems. Although bacterial degradation in cultivated soils has been previously suggested, comparative analyses between rice paddies and adjacent natural wetlands remain scarce. Here, we characterized the soil bacteriome across a cultivation gradient by comparing undisturbed natural marshes, within Doñana National Park, with rice fields cultivated for 25 years (Cantarita) and 80 years (Mínima 2). Using full 16S rRNA gene via long-read metabarcoding and standardized soil physicochemical assays, we analysed taxonomic composition, environmental associations, and predicted functional profiles. Our results reveal a progressive restructuring of bacterial communities with increased cultivation time, notably a significant enrichment of Chloroflexota (especially Anaerolineae ) and a decline in Actinomycetota and Planctomycetota in paddy soils. Functional predictions indicated a higher potential for denitrification in cultivated soils—likely involving Chloroflexota taxa—compared to more diverse nitrogen pathways in natural sites. These shifts were strongly associated with changes in pH, electrical conductivity, calcium carbonate, and nitrate levels. Remarkably, most bacterial differences were already evident within the first 25 years of cultivation, underscoring the rapid ecological impact of intensive rice cultivation. Notably, we identified specific bacterial groups ( Anaerolineae and Nocardioides in paddy soils; Euzebya , Rubrobacter , and Planctomycetota in natural wetlands), whose enrichment was associated with soil type. This approach highlights the value of integrating bacterial-based assessments into sustainable wetland management strategies.

The phylum Chloroflexota (formerly Chloroflexi) encompasses metabolically diverse bacteria that often have high prevalence in terrestrial and aquatic habitats, some even with biotechnological application. However, there is substantial disagreement in public databases which lineage should be considered a member of the phylum and at what taxonomic level. Here, we addressed these issues through extensive phylogenomic analyses. The analyses were based on a collection of >5000 Chloroflexota genomes and metagenome-assembled genomes (MAGs) from public databases, novel environmental sites, as well as newly generated MAGs from publicly available sequence reads via an improved binning approach incorporating covariance information. Based on calculated relative evolutionary divergence, we propose that Candidatus Dormibacterota should be listed as a class (i.e., Ca. Dormibacteria) within Chloroflexota together with the classes Anaerolineae, Chloroflexia, Dehalococcoidia, Ktedonobacteria, Ca. Limnocylindria, Thermomicrobia, and two other classes containing only uncultured members. All other Chloroflexota lineages previously listed at the class rank appear to be rather orders or families in the Anaerolineae and Dehalococcoidia, which contain the vast majority of genomes and exhibited the strongest phylogenetic radiation within the phylum. Furthermore, the study suggests that a common ecophysiological capability of members of the phylum is to successfully cope with low energy fluxes.

Branched glycerol dialkyl glycerol tetraethers (brGDGTs) are promising molecular biomarkers widely applied in paleoenvironmental reconstructions, including temperature and pH. However, knowledge of the microorganisms responsible for brGDGT production in marine environments remains limited, which constrains the further development and application of brGDGT-based proxies for reconstructing past marine conditions. In this study, both ‘living’ intact polar lipid-derived brGDGTs (IPL-brGDGTs) and ‘fossil’ core brGDGTs (CL-brGDGTs), together with bacterial community compositions, were analysed in multiple sediment cores collected along a nearshore-to-offshore transect in the East China Sea (ECS). The potential correlations between brGDGT distributions and bacterial community compositions at varying sediment depths across an environmental gradient were also explored. Results revealed that IPL-brGDGTs were predominantly biosynthesised in situ, whereas CL-brGDGTs reflected a mixture of marine autochthonous production and terrestrial inputs. Potential brGDGT-producing bacteria in nearshore environments were primarily composed of chemolithoautotrophic taxa (e.g., Gammaproteobacteria and Dehalococcoidia) and chemoheterotrophic taxa (e.g., Alphaproteobacteria, Bacilli, and Actinobacteria). In contrast, offshore regions were dominated by chemoheterotrophic hypoxic bacteria (e.g., Anaerolineae and Phycisphaerae) and facultatively anaerobic chemolithoautotrophic bacteria (e.g., Gammaproteobacteria and Desulfobacteria). A significant difference in bacterial community composition and IPL-brGDGT distribution was observed at a depth of 17 cm, likely due to physical disturbance in near-surface sediments, such as wave action, tidal forces, and storm events. Variance partitioning analysis (VPA) revealed that the bacterial community composition alone accounted for 14.1% of the variation in IPL-brGDGTs and 6.5% in CL-brGDGTs, further suggesting that the distribution of brGDGTs is primarily influenced by the composition of the bacterial community in the nearshore-to-offshore sedimentary ecosystems of the ECS. These findings regarding the potential biosynthesis of brGDGTs in coastal habitats advance our understanding of the microbial mechanisms that regulate brGDGT distribution in marine ecosystems. Moreover, they emphasise the importance of considering physical disturbance effects when interpreting sedimentary brGDGT records for paleoenvironmental reconstructions in marginal seas, such as the ECS.

Given that coastal salt marshes are recognized as biogeochemical hotspots, it is fundamentally important to understand the functional role of the microbiome in this ecosystem. In particular, Anaerolineae are abundant members of the salt marsh rhizosphere and have been identified as core microbes, suggesting they play an important functional role. Yet, little is known about the metabolic pathways encoded and expressed in this abundant salt marsh clade. Using an ‘omics-based approach, we determined that Anaerolineae are capable of oxidizing a range of carbon compounds, including simple sugars to complex carbon compounds, while also encoding fermentation and carbon fixation. Surprisingly, Anaerolineae encoded and transcribed genes involved in aerobic respiration, which was unexpected given the reduced nature of the salt marsh rhizosphere. Finally, the majority of Anaerolineae appear to be involved in secondary metabolite production, suggesting that this group represents an unexplored reservoir of novel and important secondary metabolites.

Hydrothermal vents, widely occurring along middle-ocean ridges and volcanic arcs, have been well-studied in vent-associated microbiology, mineralogy, and geochemistry. However, there are rarely investigations regarding the detailed microbial community in the hydrothermal vent-influenced sediment. To explore hydrothermal activities on microbial diversity at the Carlsberg Ridge in the northwestern Indian Ocean, four sediment cores were sampled from the near-vent fields to distant vent sedimentary fields in the Tianxiu hydrothermal field, and the microbial community compositions were analyzed. The sediment microorganisms closest to the hydrothermal vent were primarily composed of Acidimicrobiia, Gammaproteobacteria, Anaerolineae, and Planctomycetes. The microbial communities at the depth containing extensive signals of hydrothermal activity consisted mainly of Dehalococcoidia, Aerophoria, Anaerolineae, and Gammaproteobacteria. No significant differences in microbial composition were observed between the two weak hydrothermal sediment cores, primarily composed of Nitrososphaeria, Gammaproteobacteria, Alphaproteobacteria, and Acidimicrobiia. Moreover, heterogeneous selection substantially impacted the bacterial community assembly in near-vent sediments other than stochasticity. Multivariate statistical analysis identified that environmental fluctuations accounted for 55.59% of the community variation, with hydrothermal inputs (such as Fe, Pb, Cu, and Zn) being the primary factors shaping the construction of hydrothermal sediment microbial communities. These results enhance understanding of the response of deep-sea sediments to hydrothermal activity.

The deep marine subsurface is one of the largest unexplored biospheres on Earth and is widely inhabited by members of the phylum Chloroflexi . In this report, we investigated genomes of single cells obtained from deep-sea sediments of the Peruvian Margin, which are enriched in such Chloroflexi . 16S rRNA gene sequence analysis placed two of these single-cell-derived genomes (DscP3 and Dsc4) in a clade of subphylum I Chloroflexi which were previously recovered from deep-sea sediment in the Okinawa Trough and a third (DscP2-2) as a member of the previously reported DscP2 population from Peruvian Margin site 1230. The presence of genes encoding enzymes of a complete Wood-Ljungdahl pathway, glycolysis/gluconeogenesis, a Rhodobacter nitrogen fixation (Rnf) complex, glyosyltransferases, and formate dehydrogenases in the single-cell genomes of DscP3 and Dsc4 and the presence of an NADH-dependent reduced ferredoxin:NADP oxidoreductase (Nfn) and Rnf in the genome of DscP2-2 imply a homoacetogenic lifestyle of these abundant marine Chloroflexi . We also report here the first complete pathway for anaerobic benzoate oxidation to acetyl coenzyme A (CoA) in the phylum Chloroflexi (DscP3 and Dsc4), including a class I benzoyl-CoA reductase. Of remarkable evolutionary significance, we discovered a gene encoding a formate dehydrogenase (FdnI) with reciprocal closest identity to the formate dehydrogenase-like protein (complex iron-sulfur molybdoenzyme [CISM], DET0187) of terrestrial Dehalococcoides/Dehalogenimonas spp. This formate dehydrogenase-like protein has been shown to lack formate dehydrogenase activity in Dehalococcoides/Dehalogenimonas spp. and is instead hypothesized to couple HupL hydrogenase to a reductive dehalogenase in the catabolic reductive dehalogenation pathway. This finding of a close functional homologue provides an important missing link for understanding the origin and the metabolic core of terrestrial Dehalococcoides/Dehalogenimonas spp. and of reductive dehalogenation, as well as the biology of abundant deep-sea Chloroflexi . IMPORTANCE The deep marine subsurface is one of the largest unexplored biospheres on Earth and is widely inhabited by members of the phylum Chloroflexi . In this report, we investigated genomes of single cells obtained from deep-sea sediments and provide evidence for a homacetogenic lifestyle of these abundant marine Chloroflexi . Moreover, genome signature and key metabolic genes indicate an evolutionary relationship between these deep-sea sediment microbes and terrestrial, reductively dehalogenating Dehalococcoides . The deep marine subsurface is one of the largest unexplored biospheres on Earth and is widely inhabited by members of the phylum Chloroflexi . In this report, we investigated genomes of single cells obtained from deep-sea sediments and provide evidence for a homacetogenic lifestyle of these abundant marine Chloroflexi . Moreover, genome signature and key metabolic genes indicate an evolutionary relationship between these deep-sea sediment microbes and terrestrial, reductively dehalogenating Dehalococcoides .