Plant roots affect free-living diazotroph communities in temperate grassland soils despite decades of fertilization.

Fixation of atmospheric N2 by free-living diazotrophs accounts for an important proportion of nitrogen naturally introduced to temperate grasslands. The effect of plants or fertilization on the general microbial community has been extensively studied, yet an understanding of the potential combinatorial effects on the community structure and activity of free-living diazotrophs is lacking. In this study we provide a multilevel assessment of the single and interactive effects of different long-term fertilization treatments, plant species and vicinity to roots on the free-living diazotroph community in relation to the general microbial community in grassland soils. We sequenced the dinitrogenase reductase (nifH) and the 16S rRNA genes of bulk soil and root-associated compartments (rhizosphere soil, rhizoplane and root) of two grass species (Arrhenatherum elatius and Anthoxanthum odoratum) and two herb species (Galium album and Plantago lanceolata) growing in Austrian grassland soils treated with different fertilizers (N, P, NPK) since 1960. Overall, fertilization has the strongest effect on the diazotroph and general microbial community structure, however with vicinity to the root, the plant effect increases. Despite the long-term fertilization, plants strongly influence the diazotroph communities emphasizing the complexity of soil microbial communities' responses to changing nutrient conditions in temperate grasslands.

Global abundance patterns, diversity, and ecology of Patescibacteria in wastewater treatment plants.

BACKGROUND: Microorganisms are responsible for nutrient removal and resource recovery in wastewater treatment plants (WWTPs), and their diversity is often studied by 16S rRNA gene amplicon sequencing. However, this approach underestimates the abundance and diversity of Patescibacteria due to the low coverage of commonly used PCR primers for this highly divergent bacterial phylum. Therefore, our current understanding of the global diversity, distribution, and ecological role of Patescibacteria in WWTPs is very incomplete. This is particularly relevant as Patescibacteria are considered to be associated with microbial host cells and can therefore influence the abundance and temporal variability of other microbial groups that are important for WWTP functioning. RESULTS: Here, we evaluated the in silico coverage of widely used 16S rRNA gene-targeted primer pairs and redesigned a primer pair targeting the V4 region of bacterial and archaeal 16S rRNA genes to expand its coverage for Patescibacteria. We then experimentally evaluated and compared the performance of the original and modified V4-targeted primers on 565 WWTP samples from the MiDAS global sample collection. Using the modified primer pair, the percentage of ASVs classified as Patescibacteria increased from 5.9 to 23.8%, and the number of detected patescibacterial genera increased from 560 to 1576, while the detected diversity of the remaining microbial community remained similar. Due to this significantly improved coverage of Patescibacteria, we identified 23 core genera of Patescibacteria in WWTPs and described the global distribution pattern of these unusual microbes in these systems. Finally, correlation network analysis revealed potential host organisms that might be associated with Patescibacteria in WWTPs. Interestingly, strong indications were found for an association between Patescibacteria of the Saccharimonadia and globally abundant polyphosphate-accumulating organisms of the genus Ca. Phosphoribacter. CONCLUSIONS: Our study (i) provides an improved 16S rRNA gene V4 region-targeted amplicon primer pair inclusive of Patescibacteria with little impact on the detection of other taxa, (ii) reveals the diversity and distribution patterns of Patescibacteria in WWTPs on a global scale, and (iii) provides new insights into the ecological role and potential hosts of Patescibacteria in WWTPs. Video Abstract.

Future of the Search for Life: Workshop Report.

The 2-week, virtual Future of the Search for Life science and engineering workshop brought together more than 100 scientists, engineers, and technologists in March and April 2022 to provide their expert opinion on the interconnections between life-detection science and technology. Participants identified the advances in measurement and sampling technologies they believed to be necessary to perform in situ searches for life elsewhere in our Solar System, 20 years or more in the future. Among suggested measurements for these searches, those pertaining to three potential indicators of life termed "dynamic disequilibrium," "catalysis," and "informational polymers" were identified as particularly promising avenues for further exploration. For these three indicators, small breakout groups of participants identified measurement needs and knowledge gaps, along with corresponding constraints on sample handling (acquisition and processing) approaches for a variety of environments on Enceladus, Europa, Mars, and Titan. Despite the diversity of these environments, sample processing approaches all tend to be more complex than those that have been implemented on missions or envisioned for mission concepts to date. The approaches considered by workshop breakout groups progress from nondestructive to destructive measurement techniques, and most involve the need for fluid (especially liquid) sample processing. Sample processing needs were identified as technology gaps. These gaps include technology and associated sampling strategies that allow the preservation of the thermal, mechanical, and chemical integrity of the samples upon acquisition; and to optimize the sample information obtained by operating suites of instruments on common samples. Crucially, the interplay between science-driven life-detection strategies and their technological implementation highlights the need for an unprecedented level of payload integration and extensive collaboration between scientists and engineers, starting from concept formulation through mission deployment of life-detection instruments and sample processing systems.

Identification of inulin-responsive bacteria in the gut microbiota via multi-modal activity-based sorting.

Prebiotics are defined as non-digestible dietary components that promote the growth of beneficial gut microorganisms. In many cases, however, this capability is not systematically evaluated. Here, we develop a methodology for determining prebiotic-responsive bacteria using the popular dietary supplement inulin. We first identify microbes with a capacity to bind inulin using mesoporous silica nanoparticles functionalized with inulin. 16S rRNA gene amplicon sequencing of sorted cells revealed that the ability to bind inulin was widespread in the microbiota. We further evaluate which taxa are metabolically stimulated by inulin and find that diverse taxa from the phyla Firmicutes and Actinobacteria respond to inulin, and several isolates of these taxa can degrade inulin. Incubation with another prebiotic, xylooligosaccharides (XOS), in contrast, shows a more robust bifidogenic effect. Interestingly, the Coriobacteriia Eggerthella lenta and Gordonibacter urolithinfaciens are indirectly stimulated by the inulin degradation process, expanding our knowledge of inulin-responsive bacteria.

Ecophysiology and interactions of a taurine-respiring bacterium in the mouse gut.

Taurine-respiring gut bacteria produce H2S with ambivalent impact on host health. We report the isolation and ecophysiological characterization of a taurine-respiring mouse gut bacterium. Taurinivorans muris strain LT0009 represents a new widespread species that differs from the human gut sulfidogen Bilophila wadsworthia in its sulfur metabolism pathways and host distribution. T. muris specializes in taurine respiration in vivo, seemingly unaffected by mouse diet and genotype, but is dependent on other bacteria for release of taurine from bile acids. Colonization of T. muris in gnotobiotic mice increased deconjugation of taurine-conjugated bile acids and transcriptional activity of a sulfur metabolism gene-encoding prophage in other commensals, and slightly decreased the abundance of Salmonella enterica, which showed reduced expression of galactonate catabolism genes. Re-analysis of metagenome data from a previous study further suggested that T. muris can contribute to protection against pathogens by the commensal mouse gut microbiota. Together, we show the realized physiological niche of a key murine gut sulfidogen and its interactions with selected gut microbiota members.

Cultivation and genomic characterization of novel and ubiquitous marine nitrite-oxidizing bacteria from the Nitrospirales.

Nitrospirales, including the genus Nitrospira, are environmentally widespread chemolithoautotrophic nitrite-oxidizing bacteria. These mostly uncultured microorganisms gain energy through nitrite oxidation, fix CO2, and thus play vital roles in nitrogen and carbon cycling. Over the last decade, our understanding of their physiology has advanced through several new discoveries, such as alternative energy metabolisms and complete ammonia oxidizers (comammox Nitrospira). These findings mainly resulted from studies of terrestrial species, whereas less attention has been given to marine Nitrospirales. In this study, we cultured three new marine Nitrospirales enrichments and one isolate. Three of these four NOB represent new Nitrospira species while the fourth represents a novel genus. This fourth organism, tentatively named "Ca. Nitronereus thalassa", represents the first cultured member of a Nitrospirales lineage that encompasses both free-living and sponge-associated nitrite oxidizers, is highly abundant in the environment, and shows distinct habitat distribution patterns compared to the marine Nitrospira species. Partially explaining this, "Ca. Nitronereus thalassa" harbors a unique combination of genes involved in carbon fixation and respiration, suggesting differential adaptations to fluctuating oxygen concentrations. Furthermore, "Ca. Nitronereus thalassa" appears to have a more narrow substrate range compared to many other marine nitrite oxidizers, as it lacks the genomic potential to utilize formate, cyanate, and urea. Lastly, we show that the presumed marine Nitrospirales lineages are not restricted to oceanic and saline environments, as previously assumed.

Taurine as a key intermediate for host-symbiont interaction in the tropical sponge Ianthella basta.

Marine sponges are critical components of marine benthic fauna assemblages, where their filter-feeding and reef-building capabilities provide bentho-pelagic coupling and crucial habitat. As potentially the oldest representation of a metazoan-microbe symbiosis, they also harbor dense, diverse, and species-specific communities of microbes, which are increasingly recognized for their contributions to dissolved organic matter (DOM) processing. Recent omics-based studies of marine sponge microbiomes have proposed numerous pathways of dissolved metabolite exchange between the host and symbionts within the context of the surrounding environment, but few studies have sought to experimentally interrogate these pathways. By using a combination of metaproteogenomics and laboratory incubations coupled with isotope-based functional assays, we showed that the dominant gammaproteobacterial symbiont, 'Candidatus Taurinisymbion ianthellae', residing in the marine sponge, Ianthella basta, expresses a pathway for the import and dissimilation of taurine, a ubiquitously occurring sulfonate metabolite in marine sponges. 'Candidatus Taurinisymbion ianthellae' incorporates taurine-derived carbon and nitrogen while, at the same time, oxidizing the dissimilated sulfite into sulfate for export. Furthermore, we found that taurine-derived ammonia is exported by the symbiont for immediate oxidation by the dominant ammonia-oxidizing thaumarchaeal symbiont, 'Candidatus Nitrosospongia ianthellae'. Metaproteogenomic analyses also suggest that 'Candidatus Taurinisymbion ianthellae' imports DMSP and possesses both pathways for DMSP demethylation and cleavage, enabling it to use this compound as a carbon and sulfur source for biomass, as well as for energy conservation. These results highlight the important role of biogenic sulfur compounds in the interplay between Ianthella basta and its microbial symbionts.

Rapid nitrification involving comammox and canonical Nitrospira at extreme pH in saline-alkaline lakes.

Nitrite-oxidizing bacteria (NOB) catalyse the second nitrification step and are the main biological source of nitrate. The most diverse and widespread NOB genus is Nitrospira, which also contains complete ammonia oxidizers (comammox) that oxidize ammonia to nitrate. To date, little is known about the occurrence and biology of comammox and canonical nitrite oxidizing Nitrospira in extremely alkaline environments. Here, we studied the seasonal distribution and diversity, and the effect of short-term pH changes on comammox and canonical Nitrospira in sediments of two saline, highly alkaline lakes. We identified diverse canonical and comammox Nitrospira clade A-like phylotypes as the only detectable NOB during more than a year, suggesting their major importance for nitrification in these habitats. Gross nitrification rates measured in microcosm incubations were highest at pH 10 and considerably faster than reported for other natural, aquatic environments. Nitrification could be attributed to canonical and comammox Nitrospira and to Nitrososphaerales ammonia-oxidizing archaea. Furthermore, our data suggested that comammox Nitrospira contributed to ammonia oxidation at an extremely alkaline pH of 11. These results identify saline, highly alkaline lake sediments as environments of uniquely strong nitrification with novel comammox Nitrospira as key microbial players.

Metabolic reconstruction of the near complete microbiome of the model sponge Ianthella basta.

Many marine sponges host highly diverse microbiomes that contribute to various aspects of host health. Although the putative function of individual groups of sponge symbionts has been increasingly described, the extreme diversity has generally precluded in-depth characterization of entire microbiomes, including identification of syntrophic partnerships. The Indo-Pacific sponge Ianthella basta is emerging as a model organism for symbiosis research, hosting only three dominant symbionts: a Thaumarchaeotum, a Gammaproteobacterium, and an Alphaproteobacterium and a range of other low abundance or transitory taxa. Here, we retrieved metagenome assembled genomes (MAGs) representing >90% of I. basta's microbial community, facilitating the metabolic reconstruction of the sponge's near complete microbiome. Through this analysis, we identified metabolic complementarity between microbes, including vitamin sharing, described the importance of low abundance symbionts, and characterized a novel microbe-host attachment mechanism in the Alphaproteobacterium. We further identified putative viral sequences, highlighting the role viruses can play in maintaining symbioses in I. basta through the horizontal transfer of eukaryotic-like proteins, and complemented this data with metaproteomics to identify active metabolic pathways in bacteria, archaea, and viruses. This data provide the framework to adopt I. basta as a model organism for studying host-microbe interactions and provide a basis for in-depth physiological experiments.

Nutrient niche specificity for glycosaminoglycans is reflected in polysaccharide utilization locus architecture of gut Bacteroides species.

Introduction: Glycosaminoglycans (GAGs) present in the mucosal layer can be used as nutrients by certain intestinal bacteria, particularly members of the Bacteroides. GAG abundances are altered in some diseases such as inflammatory bowel diseases, which may affect microbial composition and activity, and it is therefore important to understand GAG utilization by members of the gut microbiota. Methods: We used growth assays, transcriptomics, and comparative genomics to evaluate chondroitin sulfate (CS) and hyaluronan (HA) degradation ability by multiple gut Bacteroides species. Results and discussion: We found that not all Bacteroides species able to degrade CS could also degrade HA, despite having lyases which act on both compounds. We propose that in the model organism Bacteroides thetaiotaomicron, the lyase BT_3328 in combination with surface binding proteins BT_3329 and BT_3330 and potentially BT_4411 are involved in HA breakdown. Furthermore, degradation of both compounds provides public goods for other Bacteroides, including non-degraders, suggesting that cooperative degradation as well as cross-feeding may be widespread in the mucosal glycan utilization clade.

Atypical enteropathogenic E. coli are associated with disease activity in ulcerative colitis.

With increasing urbanization and industrialization, the prevalence of inflammatory bowel diseases (IBDs) has steadily been rising over the past two decades. IBD involves flares of gastrointestinal (GI) inflammation accompanied by microbiota perturbations. However, microbial mechanisms that trigger such flares remain elusive. Here, we analyzed the association of the emerging pathogen atypical enteropathogenic E. coli (aEPEC) with IBD disease activity. The presence of diarrheagenic E. coli was assessed in stool samples from 630 IBD patients and 234 age- and sex-matched controls without GI symptoms. Microbiota was analyzed with 16S ribosomal RNA gene amplicon sequencing, and 57 clinical aEPEC isolates were subjected to whole-genome sequencing and in vitro pathogenicity experiments including biofilm formation, epithelial barrier function and the ability to induce pro-inflammatory signaling. The presence of aEPEC correlated with laboratory, clinical and endoscopic disease activity in ulcerative colitis (UC), as well as microbiota dysbiosis. In vitro, aEPEC strains induce epithelial p21-activated kinases, disrupt the epithelial barrier and display potent biofilm formation. The effector proteins espV and espG2 distinguish aEPEC cultured from UC and Crohn's disease patients, respectively. EspV-positive aEPEC harbor more virulence factors and have a higher pro-inflammatory potential, which is counteracted by 5-ASA. aEPEC may tip a fragile immune-microbiota homeostasis and thereby contribute to flares in UC. aEPEC isolates from UC patients display properties to disrupt the epithelial barrier and to induce pro-inflammatory signaling in vitro.

Discovery of a new genus of anaerobic ammonium oxidizing bacteria with a mechanism for oxygen tolerance.

In the past 20 years, there has been a major stride in understanding the core mechanism of anaerobic ammonium-oxidizing (anammox) bacteria, but there are still several discussion points on their survival strategies. Here, we discovered a new genus of anammox bacteria in a full-scale wastewater-treating biofilm system, tentatively named "Candidatus Loosdrechtia aerotolerans". Next to genes of all core anammox metabolisms, it encoded and transcribed genes involved in the dissimilatory nitrate reduction to ammonium (DNRA), which coupled to oxidation of small organic acids, could be used to replenish ammonium and sustain their metabolism. Surprisingly, it uniquely harbored a new ferredoxin-dependent nitrate reductase, which has not yet been found in any other anammox genome and might confer a selective advantage to it in nitrate assimilation. Similar to many other microorganisms, superoxide dismutase and catalase related to oxidative stress resistance were encoded and transcribed by "Ca. Loosdrechtia aerotolerans". Interestingly, bilirubin oxidase (BOD), likely involved in oxygen resistance of anammox bacteria under fluctuating oxygen concentrations, was identified in "Ca. Loosdrechtia aerotolerans" and four Ca. Brocadia genomes, and its activity was demonstrated using purified heterologously expressed proteins. A following survey of oxygen-active proteins in anammox bacteria revealed the presence of other previously undetected oxygen defense systems. The novel cbb3-type cytochrome c oxidase and bifunctional catalase-peroxidase may confer a selective advantage to Ca. Kuenenia and Ca. Scalindua that face frequent changes in oxygen concentrations. The discovery of this new genus significantly broadens our understanding of the ecophysiology of anammox bacteria. Furthermore, the diverse oxygen tolerance strategies employed by distinct anammox bacteria advance our understanding of their niche adaptability and provide valuable insight for the operation of anammox-based wastewater treatment systems.

Ecological Processes Shaping Microbiomes of Extremely Low Birthweight Infants.

The human microbiome has been implicated in affecting health outcomes in premature infants, but the ecological processes governing early life microbiome assembly remain poorly understood. Here, we investigated microbial community assembly and dynamics in extremely low birth weight infants (ELBWI) over the first 2 weeks of life. We profiled the gut, oral cavity and skin microbiomes over time using 16S rRNA gene amplicon sequencing and evaluated the ecological forces shaping these microbiomes. Though microbiomes at all three body sites were characterized by compositional instability over time and had low body-site specificity (PERMANOVA, r 2 = 0.09, p = 0.001), they could nonetheless be clustered into four discrete community states. Despite the volatility of these communities, deterministic assembly processes were detectable in this period of initial microbial colonization. To further explore these deterministic dynamics, we developed a probabilistic approach in which we modeled microbiome state transitions in each ELBWI as a Markov process, or a "memoryless" shift, from one community state to another. This analysis revealed that microbiomes from different body sites had distinctive dynamics as well as characteristic equilibrium frequencies. Time-resolved microbiome sampling of premature infants may help to refine and inform clinical practices. Additionally, this work provides an analysis framework for microbial community dynamics based on Markov modeling that can facilitate new insights, not only into neonatal microbiomes but also other human-associated or environmental microbiomes.

Ammonia-oxidizing archaea possess a wide range of cellular ammonia affinities.

Nitrification, the oxidation of ammonia to nitrate, is an essential process in the biogeochemical nitrogen cycle. The first step of nitrification, ammonia oxidation, is performed by three, often co-occurring guilds of chemolithoautotrophs: ammonia-oxidizing bacteria (AOB), archaea (AOA), and complete ammonia oxidizers (comammox). Substrate kinetics are considered to be a major niche-differentiating factor between these guilds, but few AOA strains have been kinetically characterized. Here, the ammonia oxidation kinetic properties of 12 AOA representing all major cultivated phylogenetic lineages were determined using microrespirometry. Members of the genus Nitrosocosmicus have the lowest affinity for both ammonia and total ammonium of any characterized AOA, and these values are similar to previously determined ammonia and total ammonium affinities of AOB. This contrasts previous assumptions that all AOA possess much higher substrate affinities than their comammox or AOB counterparts. The substrate affinity of ammonia oxidizers correlated with their cell surface area to volume ratios. In addition, kinetic measurements across a range of pH values supports the hypothesis that-like for AOB-ammonia and not ammonium is the substrate for the ammonia monooxygenase enzyme of AOA and comammox. Together, these data will facilitate predictions and interpretation of ammonia oxidizer community structures and provide a robust basis for establishing testable hypotheses on competition between AOB, AOA, and comammox.

Activities and metabolic versatility of distinct anammox bacteria in a full-scale wastewater treatment system.

Anaerobic ammonium oxidation (anammox) is a key N2-producing process in the global nitrogen cycle. Major progress in understanding the core mechanism of anammox bacteria has been made, but our knowledge of the survival strategies of anammox bacteria in complex ecosystems, such as full-scale wastewater treatment plants (WWTPs), remains limited. Here, by combining metagenomics with in situ metatranscriptomics, complex anammox-driven nitrogen cycles in an anoxic tank and a granular activated carbon (GAC) biofilm module of a full-scale WWTP treating landfill leachate were constructed. Four distinct anammox metagenome-assembled genomes (MAGs), representing a new genus named Ca. Loosdrechtii, a new species in Ca. Kuenenia, a new species in Ca. Brocadia, and a new strain in "Ca. Kuenenia stuttgartiensis", were simultaneously retrieved from the GAC biofilm. Metabolic reconstruction revealed that all anammox organisms highly expressed the core metabolic enzymes and showed a high metabolic versatility. Pathways for dissimilatory nitrate reduction to ammonium (DNRA) coupled to volatile fatty acids (VFAs) oxidation likely assist anammox bacteria to survive unfavorable conditions and facilitate switches between lifestyles in oxygen fluctuating environments. The new Ca. Kuenenia species dominated the anammox community of the GAC biofilm, specifically may be enhanced by the uniquely encoded flexible ammonium and iron acquisition strategies. The new Ca. Brocadia species likely has an extensive niche distribution that is simultaneously established in the anoxic tank and the GAC biofilm, the two distinct niches. The highly diverse and impressive metabolic versatility of anammox bacteria revealed in this study advance our understanding of the survival and application of anammox bacteria in the full-scale wastewater treatment system.

Gilbert's Syndrome and the Gut Microbiota - Insights From the Case-Control BILIHEALTH Study.

The heme catabolite bilirubin has anti-inflammatory, anti-oxidative and anti-mutagenic effects and its relation to colorectal cancer (CRC) risk is currently under evaluation. Although the main metabolic steps of bilirubin metabolism, including the formation of stercobilin and urobilin, take place in the human gastrointestinal tract, potential interactions with the human gut microbiota are unexplored. This study investigated, whether gut microbiota composition is altered in Gilbert's Syndrome (GS), a mild form of chronically elevated serum unconjugated bilirubin (UCB) compared to matched controls. Potential differences in the incidence of CRC-associated bacterial species in GS were also assessed. To this end, a secondary investigation of the BILIHEALTH study was performed, assessing 45 adults with elevated UCB levels (GS) against 45 age- and sex-matched controls (C). Fecal microbiota analysis was performed using 16S rRNA gene sequencing. No association between mildly increased UCB and the composition of the gut microbiota in this healthy cohort was found. The alpha and beta diversity did not differ between C and GS and both groups showed a typical representation of the known dominant phyla. Furthermore, no difference in abundance of Firmicutes and Proteobacteria, which have been associated with the mucosa of CRC patients were observed between the groups. A sequence related to the Christensenella minuta strain YIT 12065 was identified with a weak association value of 0.521 as an indicator species in the GS group. This strain has been previously associated with a lower body mass index, which is typical for the GS phenotype. Overall, sex was the only driver for an identifiable difference in the study groups, as demonstrated by a greater bacterial diversity in women. After adjusting for confounding factors and multiple testing, we can conclude that the GS phenotype does not affect the composition of the human gut microbiota in this generally healthy study group.

Novel Alcaligenes ammonioxydans sp. nov. from wastewater treatment sludge oxidizes ammonia to N2 with a previously unknown pathway.

Heterotrophic nitrifiers are able to oxidize and remove ammonia from nitrogen-rich wastewaters but the genetic elements of heterotrophic ammonia oxidation are poorly understood. Here, we isolated and identified a novel heterotrophic nitrifier, Alcaligenes ammonioxydans sp. nov. strain HO-1, oxidizing ammonia to hydroxylamine and ending in the production of N2 gas. Genome analysis revealed that strain HO-1 encoded a complete denitrification pathway but lacks any genes coding for homologous to known ammonia monooxygenases or hydroxylamine oxidoreductases. Our results demonstrated strain HO-1 denitrified nitrite (not nitrate) to N2 and N2 O at anaerobic and aerobic conditions respectively. Further experiments demonstrated that inhibition of aerobic denitrification did not stop ammonia oxidation and N2 production. A gene cluster (dnfT1RT2ABCD) was cloned from strain HO-1 and enabled E. coli accumulated hydroxylamine. Sub-cloning showed that genetic cluster dnfAB or dnfABC already enabled E. coli cells to produce hydroxylamine and further to 15 N2 from (15 NH4 )2 SO4 . Transcriptome analysis revealed these three genes dnfA, dnfB and dnfC were significantly upregulated in response to ammonia stimulation. Taken together, we concluded that strain HO-1 has a novel dnf genetic cluster for ammonia oxidation and this dnf genetic cluster encoded a previously unknown pathway of direct ammonia oxidation (Dirammox) to N2 .

Ecological memory of recurrent drought modifies soil processes via changes in soil microbial community.

Climate change is altering the frequency and severity of drought events. Recent evidence indicates that drought may produce legacy effects on soil microbial communities. However, it is unclear whether precedent drought events lead to ecological memory formation, i.e., the capacity of past events to influence current ecosystem response trajectories. Here, we utilize a long-term field experiment in a mountain grassland in central Austria with an experimental layout comparing 10 years of recurrent drought events to a single drought event and ambient conditions. We show that recurrent droughts increase the dissimilarity of microbial communities compared to control and single drought events, and enhance soil multifunctionality during drought (calculated via measurements of potential enzymatic activities, soil nutrients, microbial biomass stoichiometry and belowground net primary productivity). Our results indicate that soil microbial community composition changes in concert with its functioning, with consequences for soil processes. The formation of ecological memory in soil under recurrent drought may enhance the resilience of ecosystem functioning against future drought events.