Biology subjects

Koga, R.

Publications and source records attributed to Koga, R..

15 recordsLinked to original sources

bioRxiv2026

Global transcriptional rewiring provides recurrent evolutionary routes to insect-Escherichia coli mutualism

Microbial symbionts often evolve specialized functions that compensate for host nutritional deficiencies, yet the evolutionary routes by which such mutualisms arise remain poorly understood. Here, using an experimental symbiosis between the stinkbug Plautia stali and Escherichia coli, we investigated the early evolution of insect-bacterium mutualism. Among 144 independently evolved bacterial lineages, six acquired the ability to improve host performance. Four lineages carried independent disruptive mutations in cyaA, disabling carbon catabolite repression and thereby increasing the essential amino acid tryptophan. The remaining two lineages evolved through nonsynonymous mutations in rpoB or rpoD, encoding core components of the transcriptional machinery. Transcriptomic analyses revealed extensive regulatory rewiring in both mutants, including marked upregulation of biosynthetic pathways for branched-chain essential amino acids. Our results identify global transcriptional regulatory systems as major evolutionary targets for the emergence of mutualism and demonstrate how large-effect regulatory mutations can rapidly generate host-beneficial traits. (146 < 150 words)

evolutionary biology↗

bioRxiv2026

Adaptive laboratory evolution of a minimal cell to low temperature

Temperature is a fundamental constraint on life. Most organisms adapt to temperature change through complex regulatory and stress-response systems. Whether such adaptation is possible in a genomically minimal cell remains unclear. Here, adaptive laboratory evolution was performed on the minimal genome bacterium JCVI-syn3B, shifting growth from 37{degrees}C to 25{degrees}C, a temperature at which the original strain cannot grow. After 40 serial passages, the evolved strains exhibited robust growth at 25{degrees}C. Multi-omics analyses revealed the adaptation mechanisms including increased mRNA turnover, enhanced DNA unwinding and stabilization of replication intermediates, elevated glycerolipid synthesis, and upregulation of division proteins associated with Z-ring assembly. Whole-genome transplantation was performed to distinguish genetic or non-genetic contributions, demonstrating that the cold-adapted phenotype was largely genetically encoded. These results indicated that even a minimal cell retains substantial evolvability and highlight its potential as a tractable research platform for cellular and evolutionary biology.

evolutionary biology↗

bioRxiv2026

Symbiotic Escherichia coli strains can better colonize host stinkbugs and outcompete natural symbiotic bacteria, but confer less fitness benefits

The stinkbug Plautia stali harbors essential gut symbiotic bacteria of the genus Pantoea, whose natural strains differ in cultivability and host benefits. Using this system, we evaluated how laboratory-evolved and genetically-engineered symbiotic Escherichia coli strains compete against native Pantoea symbionts and how they influence host fitness. In single infection assays, the native uncultivable symbiont Sym A conferred the highest host performance, whereas the evolved (CmL05G13) and artificial ({Delta}cyaA) symbiotic E. coli strains supported host survival at levels comparable to cultivable Pantoea symbionts (Sym C-F). In competitive co-infection assays, the symbiotic E. coli strains generally showed unexpectedly strong colonization ability. CmL05G13 outcompeted all the cultivable symbionts Sym C-F and even displaced the native uncultivable symbiont Sym A, whereas {Delta}cyaA and the nonsymbiotic control E. coli {Delta}intS were dominated by Sym A at the adult stage. Despite their superior infection competitiveness, the symbiotic E. coli strains provided limited reproductive benefits, behaving as "cheater-like" associates. They were able to invade and dominate the symbiotic organ but failed to match the fitness contributions of native symbionts. These results demonstrate that the experimentally evolved E. coli can rapidly acquire strong colonization ability surpassing that of the natural symbionts that have coevolved with P. stali in nature. At the same time, the mismatch between infection success and host fitness benefits highlights potential evolutionary conflicts and provides an experimental model for studying the dynamics of cheating, mutualism, and symbiont replacement in vertically transmitted symbioses. IMPORTANCEUnderstanding how novel symbionts invade and displace long-term mutualists is central to the evolution of symbiosis. This study demonstrates that Escherichia coli, originally a nonsymbiotic bacterium, can rapidly evolve potent colonization ability and even outcompete native Pantoea symbionts of the stinkbug Plautia stali. Meanwhile, these competitive E. coli strains confer markedly lower reproductive benefits compared with the native symbionts that have developed intimate mutualistic association with host P. stali over evolutionary time, revealing a striking decoupling between infection success and host fitness. This finding highlights the potential for cheater-like microbes to invade vertically transmitted symbioses and destabilize coevolved partnerships. By combining experimental evolution, controlled co-infections, and quantitative analyses, the P. stali-E. coli experimental symbiotic system provides a powerful model for studying the mechanisms and evolutionary dynamics of mutualism, cheating, and symbiont replacement.

microbiology↗

bioRxiv2024

Tryptophanase disruption underlies the evolution of insect-bacterium mutualism

Animal-microbe symbioses are omnipresent, where both partners often gain benefits as mutualists. How such mutualism has evolved between originally unrelated organisms is of interest. Here we report that, using an experimental symbiotic system between the stinkbug Plautia stali and the model bacterium Escherichia coli, disruption of a single bacterial gene tnaA encoding tryptophanase makes E. coli mutualistic to P. stali. Survey of natural bacterial mutualists across wild populations of P. stali and other stinkbug species uncovered that their Pantoea-allied symbionts consistently lack tnaA gene. Some Pantoea species like P. ananatis retain tnaA gene and cannot establish symbiosis with P. stali, but tnaA-disrupted P. ananatis partially restored the symbiotic capability. When a natural Pantoea mutualist of P. stali was transformed with a functional tna operon, its symbiotic capability reduced significantly. Our finding suggests that tryptophanase disruption may have facilitated the evolution of gut bacterial mutualists in insects.

evolutionary biology↗

bioRxiv2024

Frequent and asymmetric cell division in endosymbiotic bacteria of cockroaches

Many insects are obligatorily associated with and dependent on specific microbial species as essential mutualistic partners. In the host insects, such microbial mutualists are usually maintained in specialized cells or organs, called bacteriocytes or symbiotic organs. Hence, potentially exponential microbial growth cannot be realized but must be strongly constrained by spatial and resource limitations within the host cells or tissues. How such endosymbiotic bacteria grow, divide and proliferate is important for understanding the interactions and dynamics underpinning intimate host-microbe symbiotic associations. Here we report that Blattabacterium, the ancient and essential endosymbiont of cockroaches, exhibits unexpectedly high rates of cell division (20-58%) and, in addition, the cell division is asymmetric (average asymmetry index > 1.5) when isolated from the German cockroach Blattella germanica. The asymmetric division of endosymbiont cells at high frequencies was observed irrespective of host tissues (fat bodies vs. ovaries) or developmental stages (adults vs. nymphs vs. embryos) of B. germanica, and also observed in several different cockroach species. By contrast, such asymmetric and frequent cell division was observed neither in Buchnera, the obligatory bacterial endosymbiont of aphids, nor in Pantoea, the obligatory bacterial gut symbiont of stinkbugs. Comparative genomics of cell division-related genes uncovered that the Blattabacterium genome lacks the Min system genes that determine the cell division plane, which may be relevant to the asymmetric cell division. These observations combined with comparative symbiont genomics provide insight into what processes and regulations may underpin the growth, division and proliferation of such bacterial mutualists continuously constrained under within-host conditions. IMPORTANCEDiverse insects are dependent on specific bacterial mutualists for their survival and reproduction. Due to the long-lasting coevolutionary history, such symbiotic bacteria tend to exhibit degenerative genomes and suffer uncultivability. Because of their microbiological fastidiousness, the cell division patterns of such uncultivable symbiotic bacteria have been poorly described. Here, using fine microscopic and quantitative morphometric approaches, we report that, although bacterial cell division usually proceeds through symmetric binary fission, Blattabacterium, the ancient and essential endosymbiont of cockroaches, exhibits frequent and asymmetric cell division. Such peculiar cell division patterns were not observed with other uncultivable essential symbiotic bacteria of aphids and stinkbugs. Gene repertoire analysis revealed that the molecular machineries for regulating the bacterial cell division plane are lost in the Blattabacterium genome, suggesting the possibility that the general trend toward the reductive genome evolution of symbiotic bacteria may underpin their bizarre cytological/morphological traits.

microbiology↗

bioRxiv2024

Host range of naturally and artificially evolved symbiotic bacteria for a specific host insect

Diverse insects are intimately associated with specific symbiotic bacteria, where host and symbiont are integrated into an almost inseparable biological entity. These symbiotic bacteria usually exhibit host specificity, uncultivability, reduced genome size, and other peculiar traits relevant to their symbiotic lifestyle. How host-symbiont specificity is established at the very beginning of symbiosis is of interest but poorly understood. To gain insight into the evolutionary issue, we adopted an experimental approach using the recently developed evolutionary model of symbiosis between the stinkbug Plautia stali and Escherichia coli. Based on the laboratory evolution of P. stali-E. coli mutualism, we selected {Delta}cyaA mutant of E. coli as an artificial symbiont of P. stali that has established mutualism by a single mutation. In addition, we selected a natural cultivable symbiont of P. stali of relatively recent evolutionary origin. These artificial and natural symbiotic bacteria of P. stali were experimentally inoculated to symbiont-deprived newborn nymphs of diverse stinkbug species. Strikingly, the mutualistic E. coli was unable to establish infection and support growth and survival of all the stinkbug species except for P. stali, uncovering that host specificity can be established at a very early stage of symbiotic evolution. Meanwhile, the natural symbiont was able to establish infection and support growth and survival of several stinkbug species in addition to P. stali, unveiling that a broader host range of the symbiont has evolved in nature. Based on these findings, we discuss what factors are relevant to the establishment of host specificity in the evolution of symbiosis. IMPORTANCEHow does host-symbiont specificity emerge at the very beginning of symbiosis? This question is difficult to address, because it is generally difficult to directly observe the onset of symbiosis. However, recent development of experimental evolutionary approaches to symbiosis has brought about a breakthrough. Here we tackled this evolutionary issue using a symbiotic Escherichia coli created in laboratory and a natural Pantoea symbiont, which are both mutualistic to the stinkbug Plautia stali. We experimentally replaced essential symbiotic bacteria of diverse stinkbugs by the artificial and natural symbionts of P. stali, and evaluated whether the symbiotic bacteria evolved for a specific host can establish infection and support growth and survival of heterospecific hosts. Strikingly, the artificial symbiont showed strict host specificity to P. stali whereas the natural symbiont was capable of symbiosis with diverse stinkbugs, which provide insight into how host-symbiont specificity can be established at early evolutionary stages of symbiosis.

microbiology↗

bioRxiv2024

Defensive fungal symbiosis on insect hindlegs

Tympanal organs as "insect ears" have evolved repeatedly. Dinidorid stinkbugs were reported to possess a conspicuous tympanal organ on females hindlegs. Here we report an unexpected discovery that the stinkbugs "tympanal organ" is actually a novel symbiotic organ. The stinkbugs "tympanum" is not membranous but a porous cuticle, where each pore connects to glandular secretory cells. In reproductive females, the hindleg organ is covered with fungal hyphae growing out of the pores. Upon oviposition, the females skillfully transfer the fungi from the organ to the eggs. The eggs are quickly covered with hyphae and physically protected against wasp parasitism. The fungi are mostly benign Cordycipitaceae entomopathogens and show considerable diversity among insect individuals and populations, indicating environmental acquisition of specific fungal associates. These results uncover a novel external fungal symbiosis in which hosts elaborate morphological, physiological and behavioral specializations underpin the selective recruitment of benign entomopathogens for a defensive purpose.

microbiology↗

bioRxiv2023

Structural Characterization of TRAF6 N-terminal for Therapeutic Uses

Tumor Necrosis Factor Receptor Associated Factors (TRAFs) are a protein family with a wide variety of roles and binding partners. Among them, TRAF6, a ubiquitin ligase, possesses unique receptor binding specificity and shows diverse functions in immune system regulation, cellular signaling, central nervous system (CNS), and tumor formation. TRAF6 consists of an N-terminal Really Interesting New Gene (RING) domain, multiple zinc fingers, and a C-terminal TRAF domain. RING domain and zinc fingers mediate the activation of nuclear factor kappa B (NF-{kappa}B), which has essential roles in the regulation of inflammatory responses, proliferation, differentiation, migration, cell adhesion, and apoptosis. Therefore, it has been found that TRAF6 is overexpressed in various types of cancer including pancreatic, liver, lung, head and neck, breast, colorectal cancers, and melanoma along with inflammatory, autoimmune and neurodegenerative disorders. Furthermore, TRAF6 is an important therapeutic target for numerous disorders and structural studies of this protein are crucial for the development of next-generation therapeutics. Here, we present a TRAF6 N-terminal structure determined at the Turkish Light Source "Turkish DeLight" to 2.6 [A] resolution at cryogenic temperature. This structure offers insight into the domain organization and zinc-binding, which are critical for protein function. Since the RING domain and the zinc fingers are key targets for TRAF6 therapeutics, structural insights are crucial for future research.

molecular biology↗

bioRxiv2022

Single mutation makes Escherichia coli an insect mutualist

We report an experimental system in which Escherichia coli evolves into an insect mutualist. When the essential gut symbiont of the stinkbug Plautia stali was replaced by E. coli, a few survivor insects exhibited specific localization and vertical transmission of E. coli. Through trans-generational maintenance with P. stali, several hyper-mutating E. coli lines independently evolved hosts high adult emergence and improved body color. Such "mutualistic" E. coli lines exhibited independent mutations disrupting the carbon catabolite repression (CCR) global transcriptional regulator. Each of the mutations reproduced the mutualistic phenotypes when introduced into wild-type E. coli, confirming that the single CCR mutations instantly make E. coli an insect mutualist. Our discovery uncovers that evolution of elaborate mutualism can proceed more easily and rapidly than conventionally envisaged.

evolutionary biology↗

bioRxiv2021

Exploration of novel αβ-protein folds through de novo design

Most naturally occurring protein folds have likely been discovered1-3. The question is whether natural evolution has exhaustively sampled almost all possible protein folds4, or whether a large fraction of the possible folds remains unexplored5-7. To address this question, we introduce a set of rules for {beta}-sheet topology to predict novel folds, and carry out the systematic de novo protein design for the novel folds predicted by the rules. The rules predicted eight novel {beta}-folds with a four-stranded {beta}-sheet, including a knot-forming one. We designed proteins for all the predicted {beta}-folds and found that all the designs are monomeric with high thermal stability and fold into the structures close to the design models, demonstrating the ability of the set of rules to predict novel {beta}-folds. The rules also predicted about twelve thousand novel {beta}-folds with five- to eight-stranded {beta}-sheets; the number is far exceeding the number of {beta}-folds observed so far. This result suggests that the enormous number of {beta}-folds are possible but have not emerged or become extinct due to evolutionary bias. The predicted novel folds should open up the possibility of designing functional proteins of our interests.

biophysics↗

bioRxiv2021

Design of complicated all-α protein structures

A wide range of de novo protein structure designs have been achieved, but the complexity of naturally occurring protein structures is still far beyond these designs. To expand the diversity and complexity of de novo designed protein structures, we sought to develop a method for designing "difficult-to-describe"-helical protein structures composed of irregularly aligned -helices like globins. Backbone structure libraries consisting of a myriad of -helical structures with 5- or 6-helices were generated by combining 18 helix-loop-helix motifs and canonical -helices, and five distinct topologies were selected for de novo design. The designs were found to be monomeric with high thermal stability in solution and fold into the target topologies with atomic accuracy. This study demonstrated that complicated -helical proteins are created using typical building blocks. The method we developed would enable us to explore the universe of protein structures for designing novel functional proteins.

biophysics↗

bioRxiv2021

Structural insight into host plasma membrane association and assembly of HIV-1 Matrix protein

HIV-1 continues to be a global health concern since AIDS was first recognized by the World Health Organization (WHO). It is estimated that there were 38 million people infected with HIV-1 and 1.5 million deaths in 2019 alone. A better understanding of the details of the HIV late-stage life cycle, involving Pr55Gag attachment to the membrane for the further oligomerization to release virion, will provide us new avenues for potential treatment. Inositol hexakisphosphate (IP6) is an abundant endogenous cyclitol molecule and its binding was linked to the oligomerization of Pr55Gag via the MA domain. However, the binding site of IP6 on MA was unknown and the structural details of this interaction were missing. Here, we present three high-resolution crystal structures of the MA domain in complex with IP6 molecules to reveal its binding mode. Additionally, extensive Differential Scanning Fluorimetry analysis combined with cryo- and ambient-temperature X-ray crystallography and computational biology identify the key residues that participate in IP6 binding. Our data provide novel insights about the multilayered HIV-1 virion assembly process that involves the interplay of IP6 with PIP2, a phosphoinositide essential for the membrane binding of Pr55Gag. IP6 and PIP2 have neighboring alternate binding sites within the same highly basic region (residues 18-33). This indicates that IP6 and PIP2 bindings are not mutually exclusive and may play a key role in coordinating virion particles membrane localization. Based on our three different IP6-MA complex crystal structures, we propose a new model that involves the IP6 coordination of the oligomerization of outer MA and inner CA domain 2D layers during assembly and budding.

microbiology↗

bioRxiv2019

Serial femtosecond X-ray diffraction of HIV-1 Gag MA-IP6 microcrystals at ambient temperature

The Human immunodeficiency virus-1 (HIV-1) matrix (MA) domain is involved in the highly regulated assembly process of the virus particles that occur at the host cells plasma membrane. High-resolution structures of the MA domain determined using cryo X- ray crystallography have provided initial insights into the possible steps in the viral assembly process. However, these structural studies have relied on large and frozen crystals in order to reduce radiation damage caused by the intense X-rays. Here, we report the first XFEL study of the HIV-1 MA domains interaction with inositol hexaphosphate (IP6), a phospholipid headgroup mimic. We also describe the purification, characterization and microcrystallization of two MA crystal forms obtained in the presence of IP6. In addition, we describe the capabilities of serial femtosecond X-ray crystallography (SFX) using X-ray free-electron laser (XFEL) to elucidate the diffraction data of MA-IP6 complex microcrystals in liquid suspension at ambient temperature. Two different microcrystal forms of MA-IP6 complex both diffracted to beyond 3.5 [A] resolution, demonstrating the feasibility of using SFX to study the complexes of MA domain of HIV-1 Gag polyprotein with IP6 at near-physiological temperatures. Further optimization of the experimental and data analysis procedures will lead to better understanding of the MA domain of HIV-1 Gag and IP6 interaction at high resolution and provide basis for optimization of the lead compounds for efficient inhibition of the Gag protein recruitment to the plasma membrane prior to virion formation.

biophysics↗

bioRxiv2017

Dramatic differences in gut bacterial densities help to explain the relationship between diet and habitat in rainforest ants

Abundance is a key parameter in microbial ecology, and important to estimates of potential metabolite flux, impacts of dispersal, and sensitivity of samples to technical biases such as laboratory contamination. However, modern amplicon-based sequencing techniques by themselves typically provide no information about the absolute abundance of microbes. Here, we use fluorescence microscopy and quantitative PCR as independent estimates of microbial abundance to test the hypothesis that microbial symbionts have enabled ants to dominate tropical rainforest canopies by facilitating herbivorous diets, and compare these methods to microbial diversity profiles from 16S rRNA amplicon sequencing. Through a systematic survey of ants from a lowland tropical forest, we show that the density of gut microbiota varies across several orders of magnitude among ant lineages, with median individuals from many genera only marginally above detection limits. Supporting the hypothesis that microbial symbiosis is important to dominance in the canopy, we find that the abundance of gut bacteria is positively correlated with stable isotope proxies of herbivory among canopy-dwelling ants, but not among ground-dwelling ants. Notably, these broad findings are much more evident in the quantitative data than in the 16S rRNA sequencing data. Our results help to resolve a longstanding question in tropical rainforest ecology, and have broad implications for the interpretation of sequence-based surveys of microbial diversity.

ecology↗

bioRxiv2016

The structured diversity of specialized gut symbionts of the New World army ants

Symbiotic bacteria play important roles in the biology of their arthropod hosts. Yet the microbiota of many diverse and influential groups remain understudied, resulting in a paucity of information on the fidelities and histories of these associations. Motivated by prior findings from a smaller scale, 16S rRNA-based study, we conducted a broad phylogenetic and geographical survey of microbial communities in the ecologically dominant New World army ants (Formicidae: Dorylinae). Amplicon sequencing of the 16S rRNA gene across 28 species spanning the five New World genera showed that the microbial communities of army ants consist of very few common and abundant bacterial species. The two most abundant microbes, referred to as Unclassified Firmicutes and Unclassified Entomoplasmatales, appear to be specialized army ant associates that dominate microbial communities in the gut lumen of three host genera, Eciton, Labidus and Nomamyrmex. Both are present in other army ant genera, including those from the Old World, suggesting that army ant symbioses date back to the Cretaceous. Extensive sequencing of bacterial protein-coding genes revealed multiple strains of these symbionts co-existing within colonies, but seldom within the same individual ant. Bacterial strains formed multiple host species-specific lineages on phylogenies, which often grouped strains from distant geographic locations. These patterns deviate from those seen in other social insects, and raise intriguing questions about the influence of army ant colony swarm-founding and within-colony genetic diversity on strain co-existence, and the effects of hosting a diverse suite of symbiont strains on colony ecology.

evolutionary biology↗