Archives of Microbiology

Four rhizobial strains isolated from root nodules of tropical leguminous plants in Costa Rica were characterized. Strain LEGMi-135b was isolated from Lonchocarpus felipei root nodules, LEGMi-12c from Cojoba arborea, LEGMi-198b from Calliandra haematocephala and Rhizobium altiplani LEGMi-166a from Mimosa pigra. Phylogenetic analyses based on the 16S rRNA gene and four concatenated housekeeping genes (recA, atpD, GATase and gyrB) placed these strains within the genera Martinezella (LEGMi-12c, LEGMi-135b, and LEGMi-198b) and Rhizobium (LEGMi-166a). Genome-based analyses showed that strain LEGMi-135b exhibited an average nucleotide identity (ANI) value of 94% and a digital DNA-DNA hybridization (dDDH) value of 53.2% compared with its closest related species, Martinezella hainanensis CCBAU 57015T, supporting its classification as a distinct species. LEGMI-12c exhibited ANI and dDDH values of 98% and 87%, respectively, compared with Martinezella hainanensis CCBAU 57015T. LEGMi-198b showed the highest similarity to Rhizobium cerradonense CNPSo 3464, with ANI and dDDH values of 98% and 81.4%, respectively; however, this taxon has not been validly published. LEGMi-166a exhibited ANI and dDDH values of 100% and 99.3%, respectively, compared with Rhizobium altiplani BR 10423, confirming its assignment to this species. Additionally, differentiation of strains LEGMi-135b and LEGMi-198b from their closest phylogenetic relatives was supported by phenotypic, physiological, and biochemical analyses. Based on the combined genomic, phenotypic, and biochemical evidence, we propose the establishment of the novel rhizobial species Martinezella legmii sp. nov., with strain LEGMi-135b designated as the type strain (LEGMi-135b), and Martinezella ivanii sp. nov., with strain LEGMi-198b designated as the type strain (LEGMi-198b). This study expands our understanding of rhizobial diversity associated with nitrogen-fixing symbioses, particularly among tropical leguminous plants from Costa Rica

Pectobacterium comprises Gram-negative, pectinolytic plant pathogens that cause soft rot and blackleg diseases on numerous economically important crops. During sampling in 2018 in taro (Colocasia esculenta) fields in Hawaii, two bacterial strains PL152T and PL155 were isolated from taro. Peliminary molecular characterization placed both strains in the genus Pectobacterium. However, their taxanomic postions could not be resoved at the species level. A polyphasic taxonomic approach incorporating phenotypic characterization, 16S rRNA gene sequence analysis, multilocus sequence analysis (MLSA), whole genome based phylogeny, average nucleotide identity (ANI), and digital DNA-DNA hybridization (dDDH) was therefore employed to determine their taxonomic status. Phylogenetic and phylogenomic analyses consistently placed PL152 and PL155 together in a distinct and well supported lineage within the genus Pectobacterium, separate from currently recognized species. The two strains exhibited high genomic relatedness to each other, whereas the average nucleotide identity (ANI) and digital DNA-DNA hybridization (dDDH) values, were lower than the accepted species delineation thresholds of 95% and 70%, respectively, supported the delineation of a novel bacterial species. The genomic characteristic of PL152 included a genome size of 5.02 Mb and a DNA G+C content of 52%. Collectively, based on a polyphasic approach including phylogenetic, phlyogenomic, biochemical, and phenotypic evidences supports the classification of strains PL152 and PL155 as representatives of a novel species within the genus Pectobacterium, for which the name Pectobacterium pacificum sp. nov. is proposed. Strain PL152 (ICMP 2436274, LMG 32536) is designated as the type strain.

Prevotella melaninogenica is a Gram-negative bacterium that has been implicated in numerous disorders such as chronic obstructive pulmonary disease (COPD) and cystic fibrosis, and despite its abundance in the human microbiome, there is still limited knowledge on the fundamental physiology describing Prevotella melaninogenica. To properly advance Prevotella biology, it is critical to report and document such characterizations of Prevotella; therefore, the purpose of this study is to examine and define the phenotypic and growth characteristics of P. melaninogenica ATCC 25845. In this study, we found that Prevotella melaninogenica ATCC 25845 is a Gram-negative bacterium that is predominantly short and medium rod-shaped, with occasional filamentous rods. SEM imaging revealed that this filamentation may serve as division centers, points from which multiple daughter cells form. We also describe the antibiotic profile, which showed susceptibility to four classes and resistance to six classes of antibiotics. Carbon source growth kinetic characterization revealed a preference for carbohydrate sources.

Although therapeutic advancements have dramatically improved outcomes for cancer patients in recent decades, advanced cancer remains a significant challenge. There is still an urgent need for new and more effective strategies that minimize toxicity and improve survival outcomes in cancer patients. Natural products from microorganisms are a rich and promising source of bioactive compounds with substantial therapeutic potential in cancer treatment. Recently, B. proteolyticus strain OSUB18, isolated from switchgrass, has been demonstrated to promote plant growth and regulate reactive oxygen species (ROS) production. Here, we investigated the anticancer potential of this plant-associated microbe and demonstrated that its crude extracts exhibited broad-spectrum cytostatic activity across diverse solid tumor cell lines. Importantly, OSUB18 extracts displayed limited cytotoxicity toward normal human mammary epithelial cells and mouse fibroblasts. This potentially selective cytostatic activity was retained within a water soluble, heat-labile, and protease-resistant fraction. The extracts primarily suppressed cell proliferation, accompanied by increased lipid peroxidation, with preliminary evidence suggesting the potential involvement of ferroptosis. These findings suggest that the bioactive component may be a selective cytostatic agent with multifaceted mechanisms of action.

Anaerobic gut fungi (AGF) are an integral part of the herbivore gut microbiome, primary decomposers of lignocellulose, and play a crucial role in establishing foundational links of syntrophic associations. The syntrophy with methanogens appears beneficial for mitigating feedback inhibition and results in the augmentation of AGF activity. However, the thermodynamic advantages of the presence of methanogens to AGF remain poorly understood. This study explores the preferred pathways adopted by Cyllamyces sp. and Methanobrevibacter olleyae, using 13C-glucose as a substrate and spiked with malate and pyruvate in different experimental set-ups. The results indicated reduced biomass formation by Cyllamyces sp. in monoxenic culture with Methanobrevibacter olleyae compared to axenic AGF culture supplemented with glucose. Additionally, metabolic analyses revealed the pyruvate:ferredoxin oxidoreductase (PFO) as a key contributor to pyruvate metabolism. Malate supplementation notably enhanced the growth of Cyllamyces sp. and M. olleyae, whereas pyruvate supplementation enhanced the growth of Cyllamyces sp. only in axenic culture. This investigation provides insights into the preferred metabolic pathways operating in the hydrogenosomes of axenic anaerobic gut fungi (AGF) cultures and monoxenic AGF-methanogen co-cultures.

The human gut is home to more than 100 trillion bacteria, where various environmental factors, such as nutrients and gases, exist and influence them. The levels of fermentation gases in the gut, including CO2, fluctuate depending on the host's diet. However, how CO2, an environmental factor, affects the physiological states of gut anaerobes remains poorly understood, but CO2 is often utilized in their cultivation. Akkermansia muciniphila (A. muciniphila) is widely studied as a beneficial human gut microbe. However, the effects of CO2 on the physiological state of A. muciniphila are not fully understood. In this study, we focused on CO2 as an environmental factor and aimed to clarify how CO2 affects the physiological state of A. muciniphila. Culture experiments showed that compared with the absence of CO2, the presence of CO2 significantly increased the growth of A. muciniphila. The results of the metabolomic analysis revealed distinct metabolic profiles in the presence or absence of CO2. The tracing analysis using stable 13C-labelled CO2 demonstrated that CO2 was incorporated into various metabolites in the TCA cycle, the de novo purine/pyrimidine synthesis pathways, and other pathways. In addition, the activity of pyruvate carboxylase, which catalyses an anaplerotic reaction involving CO2 fixation to supply oxaloacetate (OAA) to the TCA cycle, was significantly higher in A. muciniphila cells grown in the presence of CO2. Moreover, supplementation with OAA increased the growth of A. muciniphila in a dose-dependent manner, mirroring the effects of CO2 supplementation. These findings suggest that A. muciniphila utilizes CO2 for an anaplerotic reaction in the TCA cycle and other pathways involving CO2 incorporation, facilitating metabolic flux to promote its robust growth.

Microalgae are promising biological platforms for sustainable energy production because they can convert photosynthetic activity into electricity in biophotovoltaic (BPV) systems. Identifying strains that grow well at elevated temperatures and under reduced irradiance is an important step toward BPV applications in seasonally variable environments. Microalgae were isolated from Seondong Lake (Seoul, Republic of Korea) during three sampling periods and screened for growth at different temperatures. Among 45 isolates, KHUC-11 showed the highest weighted growth index based on growth rates at 25, 30, and 35 under dark conditions and was selected for further characterization. Phylogenetic reconstruction of the 45S rRNA gene cluster, internal transcribed spacer 2 (ITS2) secondary-structure comparison, and morphological characterization supported the description of KHUC-11 as Desmodesmus ecotolerans sp. nov. D. ecotolerans produced more biomass than Chlamydomonas reinhardtii CC-125 under both moderate- and low-light conditions. Under low light, its final dry biomass concentrations were 1.81 and 1.58 times those of CC-125 under mixotrophic and photoautotrophic conditions, respectively. Despite its smaller cell size, D. ecotolerans showed higher photosynthetic performance than CC-125, and screen-printed electrode (SPE)-based electrochemical assays demonstrated enhanced anodic current generation and CV-derived estimated power output. Taken together, these findings identify D. ecotolerans as a novel climate-resilient Desmodesmus species with bioelectrochemical properties that support further evaluation for BPV applications.

Acinetobacter sp. strain Tol 5 is an attractive microorganism because of its ability to utilize various aromatic compounds, including toluene. However, because Tol 5 is a non-model microorganism, the utilization of carbon sources in central carbon metabolism remains unclear. In this study, the activity of the central carbon metabolism was characterized using 13C-metabolic flux analysis (13C-MFA). Tol 5 was cultured in synthetic medium using ethanol or acetate as the sole carbon source. In addition, because toluene is degraded into pyruvate and acetyl-CoA via the toluene dioxygenase pathway, this situation was mimicked by culturing with ethanol and lactate as co-substrates. The 13C-MFA confirmed flux through the glyoxylate shunt, which bypasses part of the TCA cycle when either ethanol or acetate was used as the sole carbon source. In ethanol, the flux ratio of isocitrate dehydrogenase at the isocitrate branch point decreased markedly. Unlike acetate, the conversion of ethanol to acetyl-CoA is accompanied by NADH production. Therefore, the observed change in this flux ratio was influenced by the excess NADH generated. Under ethanol-lactate conditions, Tol 5 simultaneously utilized ethanol and lactate. The ethanol consumption rate decreased to less than half of that observed when ethanol was the sole carbon source. Most of the lactate uptake was converted to pyruvate and metabolized via the TCA cycle. The glyoxylate shunt flux was lower than that in ethanol. These metabolic characteristics provide valuable insights into the use of Tol 5 as a next-generation microbial cell factory.

Background: Cyanobacteria are the major phototrophs in many endolithic ecosystems, yet their genomic diversity and metabolic adaptations remain poorly characterized. Here, we investigated cyanobacterial diversity in endolithic dolomite rocks from the Piora Valley in the southern Swiss Alps using genome-resolved metagenomics. Results: We reconstructed 138 medium-quality metagenome-assembled genomes (MAGs) from four neighboring endolithic rock communities, including 15 cyanobacterial MAGs distributed across six order-level lineages. The cyanobacterial communities showed limited taxonomic overlap among rocks, and most recovered MAGs represented potentially novel species. Only one Gloeobacter lineage occurred in all sampled rocks, which we propose as Gloeobacter dolomitensis sp. nov., representing the first Gloeobacter genome recovered from an endolithic environment. Comparative genomic analyses identified a cynS gene encoding cyanase unique to G. dolomitensis among the Gloeobacter. Phylogenetic analyses support its horizontal acquisition from Pseudomonadota. Interestingly, the genome of G. dolomitensis lacks a canonical CynABD cyanate transporter and instead contains a distinct ATP-synthase binding cassette (ABC) transporter adjacent to cynS. Although the available genomic and structural evidence does not establish cyanate transport, its genomic association with cynS suggests a potentially non-canonical mechanism. We further show that horizontal gene transfer was unevenly distributed among cyanobacterial lineages, with Acaryochloridales acting predominantly as donors, Chroococcidiopsidales as recipients, and Gloeobacterales showing a comparatively high frequency of interphylum gene exchange. Conclusions: The Piora Valley dolomite environment represents an underexplored reservoir of cyanobacterial diversity, including basal and previously undescribed lineages. The discovery of G. dolomitensis and its unusual cyanate-associated genomic architecture expands our understanding of Gloeobacterales diversity and highlights the potential importance of horizontal gene acquisition and metabolic innovation in endolithic microbial communities.

Extremophilic fungi are a relatively unexplored source of stress-resilient microbes for sustainable agriculture. Here, we describe Talaromyces pseudofuniculosus FAR, isolated from the highly acidic site of Piccolo Inferno in southern Italy and identified by ITS and {beta}-tubulin (BenA) sequencing. Despite its origin from an extreme environment, T. pseudofuniculosus FRA remained fully functional under non-stress conditions and displayed tolerance to salinity and heavy metals while maintaining key plant growth-promoting traits, including phosphate solubilization, auxin-related compound production and hydrolytic activities. In soybean (Glycine max L.), the live fungus promoted plant growth under both non-stress conditions and agriculturally relevant salinity and copper stress, and successfully colonized the roots. A cell-free extracellular extract (exCFS) promoted early seedling development in vitro, but its effects were not sustained in pot experiments. GC-MS analysis provided a qualitative characterization of the extracellular metabolite profile and revealed condition-dependent differences between control, NaCl and CuSO4; growth conditions, indicating environment-dependent modulation of fungal metabolism. Overall, these findings highlight the physiological and metabolic plasticity of T. pseudofuniculosus FRA and support its potential as a fungal biostimulant for saline- and metal-impacted agricultural soils.

BackgroundMethicillin-resistant Staphylococcus aureus (MRSA) is one the most common cause of post-surgical infections in Malawi and poses a significant public health challenge due to resistance to commonly used antibiotics. We isolated and evaluated the stability and lytic activity of a MRSA-specific bacteriophage (Staphylophage) from Mzuzu Central hospital (MZCH) sewage against MRSA strains isolated from surgical wards. MethodsA prospective in vitro experimental study was conducted using 56 pus swabs collected from post-surgical patients at Mzuzu Central Hospital (MZCH), Malawi. Standard microbiological methods and VITEK analysis were used for bacterial isolation, identification and antimicrobial susceptibility testing of Staphylococcus aureus and MRSA according to the 2024 EUCAST guidelines. A lytic Staphylophage was isolated and purified from sewage wastewater using filtration, enrichment, and double-layer agar techniques. The purified Staphylophage was evaluated for its lytic activity through host range testing and stability under ultraviolet (UV) exposure (up to 60 minutes), different pH levels (2-12), and temperatures (4{degrees}C, -20{degrees}C, and -80{degrees}C). ResultsOut of 56 bacterial isolates, 18 (32.1%) were S. aureus, of which 11 (61.1%) were MRSA. The isolated Staphylophage showed lytic activity against 87% of Staphylococcus species tested. The phage remained stable at temperatures between -80{degrees}C and 4{degrees}C and across a pH range of 2- 12. It also retained viability after UV exposure for up to 15 minutes, although a reduction in phage titre was observed after 30 minutes. ConclusionsA lytic Staphylophage isolated from hospital sewage at MZCH demonstrated activity against Staphylococcus species, including MRSA strains from post-surgical wound infections. These findings illustrate the feasibility of generating candidate phages for alternative therapeutic approaches in a central hospital setting in Malawi.

A subgroup of membrane-anchored bacterial Alkaline Phosphatase superfamily members transfers phospholipid headgroups from phospholipids to diverse acceptor molecules. As part of type II and hybrid type I/II cellulose biosynthesis gene clusters BcsG transfers phosphorylethanolamine pEtN from phospholipid to the emerging 1,4 {beta}-D-glucan chain synthesized by the BcsABC cellulose biosynthesis nanomachine. BcsG homologs throughout the phylogenetic tree possess a uniquely structured tripartite linker that connects the fifth transmembrane domain with the periplasmic catalytic domain. A N-terminal unstructured sequence of highly variable length and biased amino acid composition, the N-linker, is followed by an -helix and an unstructured sequence of high similarity and constant length leading into the first {beta}-strand of the catalytic domain. With BcsG from Salmonella typhimurium synthesizing pEtN cellulose as a model, linkers were shown to be promiscuous and linker length positively correlated with transfer efficiency of the pEtN headgroup and protein stability. As the donor substrate, the phosphatidylethanolamine content of the membranes affects pEtN transfer efficiency. With Alkaline Phosphatase superfamily members to be promiscuous enzymes the catalytic domain of OpgE transfers pEtN to osmoregulated periplasmic glucan. BcsG-OpgE hybrid proteins cause colony morphology alterations thus potentially transferring pEtN to the BcsA synthesized glucan chain. With BcsG with shortest linkers not associated with a cellulose biosynthesis gene cluster, multiple hypotheses can be proposed that select for different length of the N-linker in BcsG homologs located within a cellulose biosynthesis gene cluster in other Pseudomonadati.

Abstract The Bacillus cereus group is a complex of closely related species, which vary in terms of their geographic spread, industrial utility, and pathogenic potential. In addition to encompassing well-known species represented by thousands of genomes (e.g., bioterrorism agent B. anthracis, foodborne pathogen B. cereus, biopesticide B. thuringiensis), the B. cereus group houses "rare" species discovered/first sequenced over a decade ago. Until recently, four rare species (B. manliponensis, B. bingmayongensis, B. gaemokensis, "B. clarus") were each represented by a single unique genome; as such, virtually nothing is known about these species beyond their type strains. We hypothesized that additional representatives of rare B. cereus group species may already be present in public databases as yet-unassembled metagenomes. To this end, we used species-specific markers to identify 7 metagenomic samples in NCBI's Sequence Read Archive, which harbored B. manliponensis, B. bingmayongensis, or B. gaemokensis. Metagenomic assembly and binning yielded an isolate-quality B. manliponensis metagenome-assembled genome (MAG) from avocado orchard soil in Israel, plus a partial B. bingmayongensis MAG from commercial spinach spiked with Shiga toxin-producing Escherichia coli. For rare species represented by >1 unique genome, we compared our novel MAGs to all available genomes and identified strain- and lineage-specific genomic elements (e.g., sphingomyelinase C and hemolysin BL homologs, variably present in B. manliponensis and B. bingmayongensis, respectively). Altogether, our study indicates that some rare B. cereus group species may be more common and geographically widespread than previously thought, and highlights the utility of culture-independent methods for recovering rare/under-isolated taxa. Impact Statement Individual B. cereus group strains vary in terms of their pathogenic potential and industrial utility. While some strains cause illness in humans and/or animals (e.g., anthrax, emetic intoxication, diarrheal toxicoinfection, non-gastrointestinal illnesses), other strains have important biotechnological applications (e.g., biocontrol, plant growth promotion, production of antimicrobials and other natural products). Because many virulence- and biotechnological-related phenotypes are strain-dependent (rather than species-dependent), phenotypes associated with an individual strain (e.g., a species type strain) cannot be extrapolated to an entire B. cereus group species. Within this context, rare B. cereus group species represent a major gap in our understanding of B. cereus group evolution, ecology, and pathogenicity, as until 2021, each rare species was represented solely by its type strain genome. Here, we aggregate all publicly available genomes/MAGs of rare B. cereus group species and supplement them with their first novel representatives since 2024. Our efforts bring the total number of unique B. manliponensis and B. bingmayongensis genomes/MAGs to >2, allowing us to conduct the first-ever phylogeographic survey and strain-level assessment of functional potential for each species. Overall, our study represents a critical first step toward understanding rare B. cereus group species beyond their type strains.

Microorganisms inhabiting geothermal springs and volcanic systems experience fluctuating temperatures that can periodically exceed their upper thermal limits, but the demographic consequences of such exposure remain poorly understood. Here, we investigated demographic responses of the thermophilic archaeon Sulfolobus acidocaldarius to an extreme temperature (94.1{degrees}C) under two regimes: sustained exposure varying in duration, and episodic exposure interspersed with recovery at a permissive temperature (75{degrees}C). Under sustained exposure, populations showed no detectable loss of viability after 15 min but declined thereafter, decreasing by approximately five orders of magnitude after 120 min. Under episodic exposure, populations remained viable across nine exposure-recovery cycles but declined in density with successive cycles. Similar responses were observed for three strains, including a DNA mismatch repair knockout ({Delta}nucS), indicating that mismatch repair deficiency did not affect viability or recovery. Together, these results demonstrate that S. acidocaldarius can withstand brief and repeated exposure to near-boiling temperatures, with mortality determined primarily by cumulative exposure duration rather than a fixed thermal threshold.

Carbohydrate inducible promoters on expression plasmids are widely used for biotechnology and basic science research. However, using carbohydrates for inducers has drawbacks including influencing bacterial metabolism, the inducer being used as a carbon source by the bacteria, high background expression, and only a subset of cells experiencing promoter activation. Furthermore, some bacteria including the pathogen Acinetobacter baumannii currently lack good carbohydrate-inducible promoters. To address this issue, a tetracycline-inducible promoter, PtetB, isolated from A. baumannii was cloned and evaluated in a number of important bacteria: Acinetobacter baumannii, Escherichia coli, Klebsiella pneumoniae, Pseudomonas aeruginosa, Pseudomonas fluorescens, and Serratia marcescens. A highly sensitive luxCDABE reporter was used to evaluate promoter activity. S. marcescens strains with elevated and reduced cyclic-AMP levels were used to evaluate the impact of cAMP on PtetB function. The PtetB promoter was induced over 800-fold in A. baumannii and 10-750-fold inducible in the other tested species. Background expression was lower from PtetB in the pseudomonads than previously described carbohydrate inducible promoters. Data indicated that the PtetB promoter system may be a useful tool for future basic science research with the tested bacteria.

Stratified benthic deposits offer crucial modern analogs for Precambrian biogeochemical dynamics, yet whether the vertical organization of deep sedimentary columns reflects depositional history, present-day geochemical conditions, or both remains poorly understood, particularly under extreme oligotrophy. Here, we present a depth-resolved exploratory survey of a continuous 34-cm sediment core partitioned into 11 discrete horizons (L1 to L11) from Poza Roja 2, an alkaline, sub-hypersaline pond in the Cuatro Cienegas Basin, Mexico. Integrating physical stratigraphy, organic carbon and nutrient stoichiometry, and 16S rRNA gene metabarcoding, we evaluated down-core microbial community structure alongside acute phosphorus scarcity. Molar C:Po and N:Po ratios exceeded canonical Redfield proportions (106:16:1) in every layer, by up to 48.5-fold and 87.8-fold, respectively, both peaking in layer L4. Unconstrained ordination (PCoA) and hierarchical clustering grouped the layers into three compositional zones: upper (L2-L4), intermediate (L5-L6), and basal (L8-L11), with L1 and L7 occupying transitional positions. Archaea accounted for 31.0% of prokaryotic reads, ranging from 8.3% (L5) to 43.7% (L9), and were dominated by SM1-50 (Thermoplasmatota) and Halobacteriota. The phototrophic heliobacterium Heliorestis peaked in the intermediate zone (47.6% of bacterial reads in L6). Constrained Redundancy Analysis models lacked statistical significance (P >= 0.12), highlighting the exploratory nature of a single core (n = 11). This pilot characterization provides a baseline for understanding deep biogenic archives under severe nutrient limitation.

Marine microbial communities vary seasonally and spatially, shaped by physiochemical gradients and biotic interactions. Spatiotemporal variability of seawater microbiomes in tropical coastal regions is comparatively understudied, especially in Southeast Asia with high pressures from development and anthropogenic activities. Here, we examined the microspatial and temporal variability of bacterioplankton in the Singapore Strait, which is a tropical system in Southeast Asia influenced by monsoons. By applying total community amplicon sequencing to three particle size fractions reflecting microspatial habitats of seawater (0.22-1 {micro}m ''free-living'', 1-10 {micro}m ''large cells and small aggregates'' and 10-150 {micro}m ''particle-associated''), we show that the seawater microbial community was structured by their microspatial habitats, monsoon seasons and physiochemical variables. Monsoon-driven alternation of water masses originating from the South China Sea to the East and from off the Sumatran coast to the West resulted in a distinct community during the Southwest monsoon across all three size fractions. Total DNA (biomass) was lowest in the 10-150 {micro}m fraction, but these particle-associated sequences had the highest diversity and were surprisingly enriched with picocyanobacterial and some pathogenic taxa, potentially reflecting aggregate formation and/or host-derived particle association. This fraction also exhibited greater temporal variability than the two smaller fractions, which could be related to a more heterogenous environment in particle microhabitats. Overall, this study demonstrated compositional variability in the bacterioplankton across space and time in an equatorial region influenced by monsoons.

Mangrove reforestation is expanding across the Caribbean, but restoration success is still judged almost exclusively by tree survival and canopy cover. Whether the sediment microbiome, which governs carbon burial and nutrient cycling, follows the recovery of the vegetation remains largely unknown. We combined 16S rRNA gene metabarcoding (V4, Illumina MiSeq) with CHNS elemental analysis, pH and salinity measurements to compare sediment prokaryotic communities along 0-100 cm profiles in a reference, a reforested (Rhizophora mangle plantings since 2019) and an altered mangrove within the Las Dunas de Las Calderas Natural Monument, Dominican Republic (39 cores pooled into 28 depth-resolved composite samples). Reference sediments contained 2.5-5.5-fold more organic carbon (median 7.5% vs 3.0% in altered and 1.3% in reforested sediments), had higher C/N ratios and were less alkaline (pH 8.2 vs 8.8). Reference communities were more diverse and even, and differed in composition from both disturbed stands (pairwise PERMANOVA, R^2 ~ 0.09, adjusted P = 0.0015), whereas reforested and altered communities were statistically indistinguishable (R^2 = 0.04, P = 0.25). Disturbed sediments were dominated by Cyanobacteriota (~41-45% of reads), which tracked high pH, whereas reference sediments were enriched in Chloroflexota, Thermodesulfobacteriota and Thermoproteota, lineages linked to anaerobic degradation of complex organic matter and sulfate reduction. Depth structured communities by separating surface from subsurface layers, and sediment organic carbon and nitrogen were the only significant constraints in distance-based redundancy analysis. These results indicate that, within a few years of the first plantings (2019), reforestation had not restored the carbon-rich, anaerobic sediment habitat of the reference forest. We propose sediment organic carbon and microbial community structure as complementary indicators for mangrove restoration monitoring in the Caribbean.

Stemflow transports water and biological material from forest canopies to the stem base, yet fungal studies of this flux have largely relied on microscopy of conidia. We used ITS2 metabarcoding to characterize fungal environmental DNA (eDNA) in stemflow from 15 trees - five each of American beech (Fagus grandifolia), sugar maple (Acer saccharum), and tulip poplar (Liriodendron tulipifera) in a closed-canopy forest in northeastern Ohio, USA. Stemflow was sampled during eight rain events in the leafless season (November 2024-May 2025), along with simulated bark and gutter washes to assess near-collar and collector signals. Natural stemflow communities were compositionally distinct from both controls and occupied greater ordination space. Ascomycota dominated across all samples, with Parafenestella, Cytospora, Microcera, Nectria, and Aureobasidium among the most abundant resolved genera and 13 of the 25 most abundant taxa differed significantly among host species. Woody-plant-associated endophytes, canker fungi, and saprobes dominated ecological guild composition. Storm event explained 28.6% of taxonomic variation, host tree species 24.1%, and their interaction 10.0%. Guild composition was more strongly structured by host species (35.4%) than storm event (20.4%). Lichenized fungi and lichen-associated fungi were detected. Thus, leafless-season stemflow integrates broad bark-associated fungal eDNA, including recurrent lichen-derived material, beyond the morphologically distinctive conidia captured by traditional methods. Stemflow eDNA therefore offers a minimally invasive means of monitoring canopy fungal diversity and the strong host and temporal filtering of DNA exported from woody surfaces.

Ugba, a traditional alkaline-fermented condiment produced from African oil bean seeds (Pentaclethra macrophylla Benth.), remains poorly characterised at genome resolution. This study characterised a single spontaneous ugba fermentation using genome-resolved shotgun metagenomics across Early (0-24 h), Mid (48-96 h), and Late (120-144 h) composite stages. Thirty metagenome-assembled genomes (MAGs) were recovered and grouped according to their temporal abundance patterns. The Early-stage retained MAG population was dominated by Bacteroidota-associated MAGs, which exhibited the highest carbohydrate-active enzyme density and broad glycoside hydrolase repertoires, consistent with substantial genome-encoded potential for utilisation of the plant-derived seed matrix. The Mid stage showed marked restructuring of MAG composition, while Core and Late-associated MAGs increased towards fermentation maturation. Functional differentiation was also evident among Corynebacterium MAGs: C. nuruki encoded a complete urease system, whereas C. phoceense_A encoded glutamate dehydrogenase and nitrate-reduction genes. These stage-associated changes coincided with progressive alkalinisation from pH 7.15 to 8.01. Together, the results show that temporal restructuring of reconstructed microbial populations was accompanied by differentiation in genome-encoded functional potential. This study provides a genome-resolved characterisation of microbial composition and functional potential during spontaneous ugba fermentation and expands current understanding of the microbial organisation of this traditional African fermented food.