Biology subjects

Nishikawa, Y.

Publications and source records attributed to Nishikawa, Y..

At least 19 recordsLinked to original sources

bioRxiv2026

Extensive membrane lipid remodeling at the host-parasite interface of Toxoplasma gondii

Phosphoinositides regulate diverse membrane trafficking and signaling events in eukaryotic cells, yet their transbilayer distribution remains largely unexplored, particularly at host-parasite membrane interfaces. Here, we determined the membrane leaflet localization of phosphatidylinositol 4-phosphate (PtdIns(4)P) and phosphatidylinositol 4,5-bisphosphate (PtdIns(4,5)P2) in Toxoplasma gondii using quick-freezing freeze-fracture replica labeling combined with phosphoinositide-specific probes. Both phosphoinositides were predominantly localized to the cytoplasmic leaflet of the parasite plasma membrane, whereas only weak labeling was detected in the inner membrane complex. PtdIns(4)P, but not PtdIns(4,5)P2, was additionally detected on intracellular vesicles, while mature micronemes lacked detectable labeling for either phosphoinositide. Surprisingly, both phosphoinositides exhibited nearly symmetric distributions between the two leaflets of the parasitophorous vacuole membrane (PVM), in striking contrast to their asymmetric localization in the parasite plasma membrane. Together with our previous demonstration that phosphatidylserine and phosphatidylethanolamine also lose their transbilayer asymmetry in the PVM, these findings indicate that the PVM undergoes global membrane lipid remodeling that extends from major phospholipids to phosphoinositides. We propose that this extensive lipid reorganization represents a defining structural feature of the apicomplexan PVM and provides a new framework for understanding membrane identity at the host-parasite interface.

cell biology↗

bioRxiv2026

Parasitophorous vacuole membranes of Toxoplasma gondii and Plasmodium falciparum lack the lipid asymmetry characteristic of host cell plasma membranes

Apicomplexan parasites, including Toxoplasma gondii and Plasmodium falciparum, reside within a specialized compartment known as the parasitophorous vacuole (PV) during their intracellular life cycle. The PV membrane (PVM), which derives from the host plasma membrane upon invasion, serves as a selective barrier that permits nutrient acquisition while shielding the parasite from host defense mechanisms. Although the protein composition of the PVM has been studied extensively, its lipid organization remains poorly understood. Using the quick-freeze, freeze-fracture replica labeling (QF-FRL) method, we quantitatively analyzed the transbilayer distribution of phosphatidylserine (PtdSer), phosphatidylethanolamine (PtdEtn), and GM3 ganglioside in the PVM of T. gondii and P. falciparum. Unlike host cell plasma membranes, where these lipids exhibit strict asymmetry--PtdSer and PtdEtn confined to the cytoplasmic leaflet and GM3 to the exoplasmic leaflet--we found that all three lipids were symmetrically distributed across both leaflets of the PVM. This striking loss of lipid asymmetry suggests that the PVM undergoes profound remodeling during infection. The presence of PtdSer and PtdEtn in the luminal leaflet may facilitate the binding of perforin-like proteins (PLP1s) during egress. These findings reveal a unique feature of the PVM that redefines our understanding of host-parasite membrane biology.

cell biology↗

bioRxiv2026

Single-cell genomics reveals opportunistic Enterobacterales carrying putative cationic antimicrobial peptide resistance genes in red crown rot-affected soybean rhizoplanes

1.Structured Abstract1.1 AbstractSoybean red crown rot, caused by the soil-borne fungus Calonectria ilicicola, causes substantial yield losses, but the response of the root-associated bacterial microbiome remains poorly understood. Here, we combined 16S rRNA gene sequencing, shotgun metagenomics, and single-cell genomics to characterize bacterial communities in soybean root-associated soils. 16S rRNA gene sequencing showed that diseased plants had rhizosphere and, more strikingly, rhizoplane microbiomes distinct from those of healthy plants, often with increased Enterobacterales. Shotgun metagenomics further revealed enrichment of genes associated with antibiotic resistance, particularly cationic antimicrobial peptide resistance, in diseased rhizoplane samples. Single-cell genomics recovered seven nonredundant Enterobacterales genomes and showed that plant pathogenicity-related genes were broadly distributed across these lineages. In contrast, dlt genes, which are associated with cationic antimicrobial peptide resistance, were detected only in the Enterobacterales lineages enriched in diseased rhizoplane soils. These results support a model in which soybean red crown rot is accompanied by microbiome restructuring and opportunistic enrichment of specific Enterobacterales lineages carrying putative cationic antimicrobial peptide resistance genes. More broadly, this study highlights the value of strain-resolved single-cell genomics for linking disease-associated community shifts to specific bacterial traits. 1.2 ImportanceUnderstanding crop disease requires resolving not only the primary pathogen but also the root-associated bacteria that respond to infection. Here, we used 16S rRNA gene sequencing, shotgun metagenomics, and single-cell genomics to examine the soybean rhizoplane microbiome under red crown rot. Diseased plants showed reproducible shifts in bacterial composition, including frequent enrichment of Enterobacterales and antimicrobial resistance-related functions. Strain-resolved genomes further revealed that the Enterobacterales lineages enriched in diseased rhizoplane soils specifically carried putative dlt-mediated resistance to cationic antimicrobial peptides, whereas general pathogenicity-related genes were broadly shared. These findings suggest that host defense-associated selection, rather than pathogenicity genes alone, may help shape disease-associated root microbiomes. This study demonstrates how single-cell genomics can uncover strain-level traits hidden within bulk community data and thereby clarify plant-pathogen-microbiome interactions.

microbiology↗

bioRxiv2025

Longitudinal analysis of antibody titers after primary and booster mRNA COVID-19 vaccination can identify individuals at risk for breakthrough infection

A key issue in the post-COVID-19 era is the ongoing administration of COVID-19 vaccines. Repeated vaccination is essential for preparing against currently circulating and newly emerging SARS-CoV-2 variants while enabling people to continue with daily life. Optimizing vaccination strategies is crucial to efficiently manage medical resources and establish an effective vaccination framework. Therefore, it is important to quantitatively understand vaccine-induced immunity dynamics and to be able to identify poor responders with lower sustained antibody titers as potential priorities for revaccination. We investigated longitudinal antibody titer data in a cohort of 2,526 people in Fukushima, Japan, from April 2021 to November 2022 for whom basic demographic and health information was available. Using mathematical modeling and machine learning, we stratified the time-course patterns of antibody titers after 2 primary doses and 1 booster dose of mRNA COVID-19 vaccines. We identified 3 notable populations, which we refer to as the durable, the vulnerable, and the rapid-decliner populations, approximately half of which remained in the same population after the booster dose. Notably, the rapid-decliner population experienced earlier infections than the others. Furthermore, when comparing IgG(S) titers, IgA(S) titers, and T-spot counts between participants who experienced breakthrough infections after booster vaccination and those who did not, we found that IgA(S) titers were significantly lower in breakthrough infected participants during the early stage after booster vaccination. Our computational approach is adaptable to various types of vaccinations. This flexibility can inform policy decisions on vaccine distribution to enhance immunity both in future pandemics and in the post-COVID-19 era.

immunology↗

bioRxiv2025

Pediococcus pentosaceus decreases α-synuclein accumulation and alleviates parkinsonism in Caenorhabditis elegans and Oryzias latipes

In Parkinsons disease (PD), -synuclein (Syn) accumulation drives neuropathological progression, establishing it as a potential therapeutic target. Lactic acid bacteria (LAB) provide various health benefits to the host and are expected to offer protective effects on neurological functions through the brain-gut connection, as indicated by animal studies. However, the protective effects of LAB against Syn are not well understood. We investigated whether LAB feeding could reduce Syn accumulation and improve mobility in transgenic Caenorhabditis elegans, an invertebrate model organism that expresses human Syn in muscle. Among the nine screened strains, Pediococcus pentosaceus suppressed Syn accumulation and the decrease in bending counts, a locomotion index in nematodes. Additionally, feeding P. pentosaceus to Oryzias latipes (medaka), a vertebrate model organism, alleviated PD-like behavioral defects induced by 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine. P. pentosaseus acts as a probiotic against PD, and PD model worms and medaka can be used to screen effective LAB for prevention.

microbiology↗

bioRxiv2024

Conserved interhemispheric morphogenesis in amniotes preceded the evolution of the corpus callosum

The corpus callosum (CC) is the large axon bundle connecting the telencephalic hemispheres. The CC is formed exclusively in placental mammals, and the lack of comparable structures in other amniotes obscures the evolutionary origin of the CC. We here demonstrate that interhemispheric remodeling, a prior developmental step for CC formation, is highly conserved in non-mammalian amniotes, such as reptiles and birds. In these animal groups, the spatiotemporal dynamics of interhemispheric remodeling are tightly connected with distinct commissural formations. We observed a high degree of similarity between the mammalian CC and reptilian rostral pallial commissure, (RPC) and significant modifications in the avian pallial projection. Furthermore, we determined that Satb2 plays crucial roles in interhemispheric remodeling, which is associated with proper formation of both the CC and RPC in mice and geckoes, via the use of CRISPR-mediated gene-targeting. Our findings suggest that developmental mechanisms for midline remodeling were already present in the common ancestor of amniotes, which contributed to the evolution of eutherian-specific CC formation.

developmental biology↗

bioRxiv2024

Contrasting defense strategies of oligotrophs and copiotrophs revealed by single-cell-resolved virus-host pairing of freshwater bacteria

The ecological importance of virus-host interactions is unclear due to the limited ability of metagenomics to resolve virus-host pairs and the infection state of individual cells. We addressed these problems using single-cell genomics combined with published metagenomic data on lake bacterioplankton. We obtained 862 medium- to high-quality single-cell amplified genomes (SAGs) from two water layers and two seasons in Lake Biwa, Japan. We assembled 176 viral (dsDNA phage) contigs in the SAGs, and identified novel virus-host pairs including the discovery of viruses infecting CL500-11, the dominant bacterioplankton lineage in deep freshwater lakes worldwide. A virus was detected in 133 (15.4%) SAGs through read mapping analysis. The viral detection rate showed little variation among samples (12.1-18.1%) but significant variation in host taxonomy (4.2-65.3%), with copiotrophs showing higher values than oligotrophs. The high infection rates of copiotrophs were achieved by collective infection by diverse viruses, suggesting weak density-dependent virus-host selections, presumably because of their non-persistent interactions with viruses due to their fluctuating abundance. In contrast, the low infection rates of oligotrophs supported the idea that their co-dominance with viruses is achieved by genomic microdiversification that diversifies the virus-host specificity, sustained by their large population size and persistent density-dependent fluctuating selection. Overall, we demonstrated that virus-host interactions are highly diverse within and between host lineages, which was overlooked by metagenomics analysis, as exemplified by the CL500-11 virus, which showed extremely high read coverages in cellular and virion metagenomes, but infected < 1% of host cells. Significance statementVirus-host interactions are among the most significant driving forces of microbial biogeochemical cycles and genomic diversification. Unlike experimental conditions, bacterial cells in the natural environment are not uniformly infected by a single virus, but interact with diverse viruses under heterogeneous eco-physiological and genetic conditions. The specificity and heterogeneity of infection are the keys to understanding complex virus-host interactions and the mechanisms behind their co-existence. However, these interactions remain unclear due to the limitations of conventional metagenomic approaches. We addressed this issue by detecting viral signals from single-cell-amplified genomes of lake bacterial communities. The results revealed novel virus-host pairs and their infection rates, suggesting that viral defense strategies differ among host lineages, reflecting their ecological characteristics.

ecology↗

bioRxiv2024

Efficacy and mechanism of actions of cipargamin as an antibabesial drug candidate

Babesiosis is a disease brought on by intraerythrocytic parasites of the genus Babesia. Current chemotherapies are accompanied by side effects and parasite relapse. Therefore, it is crucial to develop highly effective drugs against Babesia. Cipargamin (CIP) has shown inhibition against apicomplexan parasites, mainly Plasmodium and Toxoplasma. This study evaluated the growth-inhibiting properties of CIP against Babesia spp. and investigated the mechanism of CIP on B. gibsoni. The half inhibitory concentration (IC50) values of CIP against the in vitro growth of B. bovis and B. gibsoni were 20.2 {+/-} 1.4 nM and 69.4 {+/-} 2.2 nM, respectively. CIP significantly inhibited the growth of B. microti and B. rodhaini in vivo. Resistance was conferred by L921V and L921I mutations in BgATP4, which reduced the sensitivity to CIP by 6.1- and 12.8-fold. The inhibitory potency of CIP against BgATP4-associated ATPase activity was moderately reduced in mutant strains, with a 1.3-fold and 2.4-fold decrease in BgATP4L921V and BgATP4L921I, respectively, compared to that of BgATP4WT. An in silico investigation revealed reductions in affinity for CIP binding to BgATP4L921V and BgATP4L921I compared to BgATP4WT. Resistant strains showed no significant cross-resistance to atovaquone (ATO) or tafenoquine succinate (TQ), with less than a onefold change in IC50 values. Combining CIP with TQ effectively eliminated B. microti infection in SCID mice with no relapse, and parasite DNA was not detected by qPCR within 90 days post-infection. Our findings reveal the efficacy of CIP as an anti-babesial agent, its limitations as a monotherapy due to resistance development, and the potential of combination therapy with TQ to overcome said resistance and achieve complete parasite clearance.

microbiology↗

bioRxiv2024

Large-scale single-virus genomics uncovers hidden diversity of river water viruses and diversified gene profiles

Environmental viruses (primarily bacteriophages) are widely recognized as playing an important role in ecosystem homeostasis through the infection of host cells. However, the majority of environmental viruses are still unknown as their mosaic structure and frequent mutations in their sequences hinder genome construction in current metagenomics. To enable the large-scale acquisition of environmental viral genomes, we developed a new single-viral genome sequencing platform with microfluidic-generated gel beads. Amplification of individual DNA viral genomes in mass-produced gel beads allows high-throughput genome sequencing compared to conventional single-virus genomics. The sequencing analysis of river water samples yielded 1431 diverse viral single-amplified genomes, while viral metagenomics recovered 100 viral metagenome-assembled genomes at the comparable sequence depth. The 99.5% of viral single-amplified genomes were determined novel at the species level, most of which could not be recovered by a metagenomic assembly. The large-scale acquisition of diverse viral genomes identified protein clusters commonly detected in different viral strains, allowing the gene transfer to be tracked. Moreover, comparative genomics within the same viral species revealed that the profiles of various methyltransferase subtypes were diverse, suggesting an enhanced escape from host bacterial internal defense mechanisms. Our use of gel bead-based single-virus genomics will contribute to exploring the nature of viruses by accelerating the accumulation of draft genomes of environmental DNA viruses.

genomics↗

bioRxiv2024

Effect of translation enhancing nascent SKIK peptide on the arrest peptides containing consecutive Proline

Ribosome arrest peptides (RAPs) such as SecM arrest peptide (SecM AP) and WPPP with consecutive Pro residues, are known to induce translational stalling in Escherichia coli. We demonstrate that the translation enhancing SKIK peptide tag, which consisted of four amino acid residues Ser-Lys-Ile-Lys, effectively alleviate translational arrest caused by WPPP. Moreover, the proximity between SKIK and WPPP significantly influences the extent of this alleviation, observed in both PURE cell-free protein synthesis and in vivo protein production systems, resulting in a substantial increase in the yield of proteins containing such RAPs. Furthermore, we unveil that nascent SKIK peptide tag and translation elongation factor P (EF-P) which alleviate ribosome stalling in consecutive-Pro rich protein, synergistically promote translation. A kinetic analysis based on the generation of super folder green fluorescent protein under in vitro translation reaction reveals that the ribosome turnover is enhanced by more than 10-fold when the SKIK peptide tag is positioned immediately upstream of the SecM AP sequence. Our findings provide valuable insights into optimizing protein production processes, which are essential for advancing synthetic biology applications. O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=98 SRC="FIGDIR/small/582505v2_ufig1.gif" ALT="Figure 1"> View larger version (13K): org.highwire.dtl.DTLVardef@c97150org.highwire.dtl.DTLVardef@12e3fb1org.highwire.dtl.DTLVardef@157af4forg.highwire.dtl.DTLVardef@1b67da9_HPS_FORMAT_FIGEXP M_FIG C_FIG

synthetic biology↗

bioRxiv2023

Characterization of carotenoid-producing Muricauda sp. strains isolated from the reef-building coral Galaxea fascicularis and subtropical seawater

Bacterial symbionts in corals and coral-associated zooxanthellae are attracting increasing attention as potential probiotics. Some members of the family Flavobacteriaceae are zooxanthellae-associated bacteria, which are known to protect zooxanthellae from stresses such as heat and light by producing carotenoids that reduce reactive oxygen species production. This study characterized two Flavobacteriaceae bacteria, Muricauda sp. strains ORYM1 (NBRC115792) and ORYM2 (NBRC115793), isolated from the reef-building coral Galaxea fascicularis and its surrounding seawater in Okinawa, Japan, respectively. The Muricauda sp. strain ORYM2 was isolated from subtropical seawater, and carotenoid production was assessed using ORYM2 as well as ORYM1, which was previously isolated from G. fascicularis. De novo genome sequencing revealed that both strains contain complete sets of zeaxanthin biosynthesis genes, similar to those found in other Muricauda spp. Thin-layer chromatography and high-performance liquid chromatography analyses demonstrated that ORYM1 and ORYM2 produce several carotenoids. The bacterial strains and carotenoids identified in this study provide insights into the biological roles of zooxanthellae-associated bacteria in protecting zooxanthellae and reef-building corals from environmental stresses. Statements and declarationsCompeting interests: The authors declare no competing interests.

microbiology↗

bioRxiv2023

Wistar rats raised in an affectionate environment display lifesaving-like behaviors while distinguishing life from death

It is generally believed that humans are the only species that values life. However, it is not well understood whether animals have a nature that values life. In this study, we attempted to determine whether male Wistar rats have this nature. Normally-reared rats did not show lifesaving-like actions towards anesthesia-induced comatose rats, although they seemed to distinguish life from death. Considering the possibility that different rearing conditions may foster a life-valuing nature, male Wistar rat pups were reared in several ways: normal rearing, loving rearing (LR; rats reared as if they were cute pets), rearing in an enriched environment, reared with gentle stroking of the back, and normal rearing of offspring of rats raised under LR conditions. When placed in an anxiety-producing environment, only LR rats escaped into the hands of the person who reared them, indicating attachment. Only the LR rats displayed lifesaving-like actions towards unknown comatose rats or drowning pups. LR rats also stopped attacks by biting ICR mice that were attacking C57BL/6 mice. Thus, rearing in an affectionate environment may foster a life-valuing nature, even in rats, suggesting that the valuing of life may be neither innate nor human-specific.

animal behavior and cognition↗

bioRxiv2022

Nascent MSKIK peptide prevents or releases translation arrest in Escherichia coli

The insertion of the sequence encoding SKIK peptide adjacent to the M start codon of a difficult-to-express protein enhances protein production in Escherichia coli. In this report, we show that the increased production of the SKIK-tagged protein is not due to codon usage of the SKIK sequence. In addition, insertion of SKX, KKX, and AKX (X = G, L, H, Y, E, and F) at the N-terminus increased protein production. Furthermore, insertion of MSKIK just before the SecM arrest peptide (FSTPVWISQAQGIRAGP), which causes translational stalling on mRNA, greatly increased the production of the protein containing the SecM arrest peptide in the E. coli reconstituted cell-free protein synthesis system (PURE system). A similar phenomenon was observed for CmlA leader whose arrest is induced by chloramphenicol. These results suggest that the nascent MSKIK peptide prevents or releases translational stalling immediately following its generation during the translation process.

biochemistry↗

bioRxiv2022

Male-killing-associated bacteriophage WO identified from comparisons of Wolbachia endosymbionts of Homona magnanima

The origin and mechanism of male-killing, an advantageous strategy employed by maternally transmitted symbionts such as Wolbachia, remain unclear. We compared genomes of four Wolbachia strains derived from Homona magnanima, a male-killing strain wHm-t (1.5 Mb), and three non-male-killing strains, wHm-a (1.1 Mb), wHm-b (1.3 Mb), and wHm-c (1.4 Mb). A wHm-t-specific 76-kbp prophage region harboured two tandemly arrayed WO-mediated killing (wmk) gene homologs (wmk-1/wmk-2 and wmk-3/wmk-4). Of these, wmk-1 or wmk-3 killed almost all Drosophila melanogaster individuals when transgenically overexpressed. Dual expression of wmk-3 and wmk-4 killed all males and rescued females. We propose a novel hypothesis wherein horizontally transmitted proto-Wolbachia with a single wmk killed both sexes, and tandem duplication of wmk allowed an evolutionary transition to a vertically transmitted symbiont, causing male-killing. Our study highlights the bacteriophage as a critical driver of the evolution of male-killing and argues for a conserved male-killing mechanism in diverse insects.

evolutionary biology↗

bioRxiv2022

Targeted single-cell genomics reveals novel host adaptation strategies of the symbiotic bacteria Endozoicomonas in Acropora tenuis coral

Endozoicomonas bacteria symbiose with various marine organisms and are known to be beneficial for coral health. However, genome analysis of coral-associated Endozoicomonas has been limited owing to the difficulty in cultivation and metagenomic approach by contamination of host-derived sequences. In this study, we applied a novel single-cell genomics technique using droplet microfluidics to obtain single-cell amplified genome (SAGs) for coral-associated Endozoicomonas spp. genome. We obtained seven novel Endozoicomonas genomes from Acropora tenuis coral. These genomes revealed that Endozoicomonas bacteria played host-associated functions in host corals and had undergone independent host-adaptive evolution in different clades. These adaptive evolutions were mediated by host-derived eukaryotic-like genes, some of which were speculated to influence host immune mechanisms. These genes are speculated to enhance coral tolerance to environmental stresses. This study suggests the possibility of host adaptation of Endozoicomonas spp. in symbiosis with corals and their contribution to coral bleaching tolerance.

microbiology↗

bioRxiv2022

Revealing within-species diversity in uncultured human gut bacteria with single-cell long-read sequencing

Bacterial genome structure changes dynamically, and structural variants can change bacterial phenotype; However, obtaining the complete genome and analyzing genome structure of uncultured bacteria has been challenging. We aimed to develop a single-cell amplified genome long-read assembly (scALA) workflow to construct circular single-cell amplified genomes (cSAGs) from long-read single-cell sequencing data of targeted uncultured bacteria. In particular, scALA generated cSAGs from nanopore long-read sequencing data of SAGs by producing contiguous sequences with repeated bias reduction and assembly processes. From 12 human fecal samples, scALA generated 16 cSAGs of three specifically targeted bacterial species, Anaerostipes hadrus, Agathobacter rectalis, and Ruminococcus gnavus. A. hadrus cSAGs exhibited large, ten kbp-long, phage insertions, saccharide metabolic capacity, and frequent genomic recombination with related strains from cohabitant hosts. Noteworthy, cSAGs constructed using this method could expand bacterial genome databases and our understanding of within-species diversities in uncultured bacteria.

microbiology↗

bioRxiv2021

Multi-omics analysis reveals cross-organism interactions in coral holobiont

Corals create an ecosystem, called a holobiont, with intracellular algae (zooxanthellae) and resident bacteria. Zooxanthellae and some bacteria play major roles in the physiological properties of the coral host. However, because of the difficulties in experimental verification of cross-organism interactions, the mechanisms underpinning these interactions are largely unknown. To address this, we here generated and then analyzed multi-omics datasets for corals, zooxanthellae, and bacteria collected at Okinawa, Japan, from November 2014 to September 2016. Using cross-organism co-expression analysis, we successfully characterized the host-alga relationship in the coral holobiont. Specifically, we observed that the coral host dominates the zooxanthellae. The multi-omics analysis also suggested that infection with coral-associated bacteria Endozoicomonas likely involves coral-like ephrin ligands, triggering an immune response of the coral host. This study highlights the potential of the multi-omics approach to elucidate coral-microbe interactions.

systems biology↗

bioRxiv2021

Single-cell metabolite detection and genomics reveals uncultivated talented producer

The production of bioactive metabolites is increasingly recognized as an important function of host-associated bacteria. An example is defensive symbiosis that might account for much of the chemical richness of marine invertebrates including sponges (Porifera), one of the oldest metazoans. However, as most complex microbiomes remain largely uncultivated and lack reference genomes, unequivocally linking metabolic functions to a cellular source is a challenge. Here we report an analysis pipeline of microfluidic encapsulation, Raman microscopy, and integrated digital genomics (MERMAID) for an efficient identification of uncultivated producers. We applied this method to the chemically rich bacteriosponge Theonella swinhoei, previously shown to contain Entotheonella symbionts providing most of its bioactive substances except for the antifungal aurantosides that lacked biosynthetic gene candidates in the metagenome. Raman-guided single-bacterial analysis and sequencing revealed a cryptic, distinct multiproducer, Candidatus Poriflexus aureus from a new Chloroflexi lineage. Its exceptionally large genome contains numerous biosynthetic loci and suggested an even higher chemical richness of this sponge than previously appreciated. This study highlights the importance of complementary technologies to uncover microbiome functions, reveals remarkable parallels between distantly related symbionts of the same host, and adds functional support for diverse chemically prolific lineages being present in microbial dark matter. Significance StatementThe production of bioactive metabolites is increasingly recognized as an important function of host-associated bacteria. However, the acquisition of integrated genomic and metabolic data from uncultivated environmental bacteria is still challenging. In this work, we explored the combination of Raman microscopy and single-cell sequencing to localize chemical features to a specific bacterium in an uncultivated microbiome, and we specified the bacteria in the uncultured lineage as a producer of aurantoside, an antifungal natural product, from a chemically and microbially complex sponge. This study offers a new methodology as well as insights into chemical functions of uncultivated life.

microbiology↗