Biology subjects

Moran, N. A.

Publications and source records attributed to Moran, N. A..

At least 19 recordsLinked to original sources

bioRxiv2026

Tailocin as an alternative weapon to type VI secretion system in bee gut symbionts

Antagonistic interactions often shape microbial communities, as co-residents compete for resources. One system of interbacterial antagonism involves tailocins: phage tail-like particles produced by some bacteria to kill close relatives. Their roles in natural communities are poorly studied. Here, we identified putative tailocin loci ubiquitously present in genomes of Snodgrassella species, core members of the bee gut microbiota. We purified tailocins from culture supernatants of different Snodgrassella strains and found that these inhibited growth of multiple Snodgrassella strains but not of strains of other species that cooccur in bee guts. Proteomic analyses corroborated the production of tailocins, and cryogenic electron microscopy imaging revealed their morphology and killing activity via membrane disruption. We generated single- and double-gene knockout mutants of a predator Snodgrassella strain that naturally possesses both tailocin and a type VI secretion system (T6SS) that was previously shown to function in intraspecies competition in bee guts. These mutants were used in tests for antagonism against a prey Snodgrassella strain in vivo. Co-inoculations within gnotobiotic honeybees including visualization within guts showed that both tailocin and T6SS are important for Snodgrassella in intraspecies competition. Each of these two antagonism mechanisms inhibits the establishment of susceptible strains within the ileum. Outcomes depend on the timing of colonization by competing strains, with both tailocin and T6SS giving a stronger advantage when the producing strain colonizes first. Thus, tailocin production can underlie priority effects that have been widely observed in the bee gut community and other host-associated bacterial communities.

microbiology↗

bioRxiv2025

Colibactin produced by a honeybee symbiont defends against pathogens and shapes the gut community

Colibactin is a bacterial genotoxin that is linked to colorectal cancer and implicated in interbacterial competition; however, its role in natural communities remains unknown. Frischella perrara, a symbiont living only in honeybee guts, produces colibactin and causes DNA damage. Here, we found that F. perrara mono-colonization reduces bee lifespan but increases survivorship following challenge with an opportunistic pathogen. F. perrara reduces pathogen loads by causing colibactin-dependent DNA damage and prophage induction. clbS, a gene conferring protection from colibactin, is ubiquitous among bacteria restricted to bee guts but is absent from opportunistic colonizers including bee pathogens. Our findings provide evidence that colibactin functions in interbacterial competition, exerts selective pressure on community members, and confers protection against pathogens, while permitting co-evolved gut symbionts to persist.

microbiology↗

bioRxiv2025

A secreted endosymbiont protein essential for colonizing host cells

ABSTRACTIntracellular bacterial symbioses have arisen myriad times in eukaryotes, with dozens known from insects alone1,2. Beginning with Buchnera, the obligate endosymbiont of aphids, genomes of endosymbionts have illuminated their evolutionary origins and metabolic contributions to hosts3,4. However, the mechanisms by which nonculturable endosymbionts enter host cells and suppress cellular immune processes have remained unknown. We show that an uncharacterized Buchnera protein, here designated SyeA, was present in the Buchnera ancestor, is secreted and homologous to secreted effectors of bacterial pathogens and is essential for Buchnera transmission. Buchnera is transmitted via expulsion from specialized maternal cells and uptake by embryos5. Using immunofluorescence microscopy, we found that SyeA levels peak upon colonization, accompanied by actin accumulation at the entry site. SyeA localizes outside the host-derived membrane and actin layer surrounding each Buchnera cell and colocalizes in host cytoplasm with Rho1, which regulates actin polymerization. syeA knockdown disrupts colonization and embryonic development and elevates lysosomal activity, leading to Buchnera destruction6. Our findings provide rare insight into how an anciently associated, mutualistic endosymbiont achieves its intracellular existence. SyeA is a vestige of pathogenic origins followed by evolution of increased host control and erosion of the original, more complex pathogenicity machinery.

microbiology↗

bioRxiv2025

Gut microbiota of Brazilian Melipona stingless bees: dominant members and their localization in different gut regions

The gut microbiome of eusocial corbiculate bees, which include honeybees, bumblebees, and stingless bees, consists of anciently associated, host-specific bacteria that are vital for bee health. Two symbionts, Snodgrassella and Gilliamella, are ubiquitous in honeybees and bumblebees. However, their presence varies in the stingless bee clade (Meliponini), a group with pantropical distribution. They are absent or rare in the diverse genus Melipona, indicating a shift in microbiota composition in this lineage. To identify the main members of the Melipona microbiota, we combined newly collected and published data from field-collected individuals of several species. Additionally, we identified the localization of the dominant microbiota members within the gut regions of Melipona quadrifasciata anthidioides. The dominant microbiota of Melipona species includes members of the genera Bifidobacterium, Lactobacillus, Apilactobacillus, Floricoccus, and Bombella. Among these, Apilactobacillus and Bombella dominate in the crop, whereas Apilactobacillus and other members of the Lactobacillaceae dominate the ventriculus. The ileum lacks Snodgrassella or Gilliamella but contains a putative new symbiont close to Floricoccus, as well as strains of Bifidobacterium, Lactobacillaceae (including Apilactobacillus), and Bombella. The rectum is dominated by Bifidobacterium and Lactobacillus. In summary, the Melipona microbiota is compositionally distinct but shows spatial organization paralleling that of other eusocial corbiculate bees.

microbiology↗

bioRxiv2025

Effects of priority on strain-level composition of the honey bee gut community

Host-associated microbiomes are complex communities shaped by interactions between members. The Type VI Secretion System (T6SS), among other bacterial weapons, allows gram-negative bacteria to deliver toxic effectors into competitors. In this study, we investigated the impact of differential colonization timing on the competitive advantage associated with T6SS possession using Snodgrassella alvi, a core symbiont of the honey bee gut microbiome. Following a timeline based on the natural establishment window of the gut microbiome, we sequentially inoculated newly emerged bees with fluorescently labeled strains that differed in presence of the T6SS-1. When inoculated simultaneously, the T6SS-1-possessing strain (wkB2) consistently excluded the T6SS-1-lacking strain (wkB332); however, when given a five-day advantage, the second strain was consistently excluded regardless of strain identities. With a one-day advantage, the effect of priority was weakened, but wkB332 was able to persist following introduction of wkB2. Utilizing a wkB2 T6SS-1 knockout strain, we repeated our 24-hour priority experiments and found that the T6SS-1 contributes to invasion outcomes along with other mechanisms of competition. Through fluorescent microscopy, we explored how coexisting strains in these experimental scenarios organize spatially within the bee ileum. Our results demonstrate that colonization timing can have lasting consequences for strain composition of the established microbiome. These findings illustrate the influence of stochastic processes in microbial community assembly and emphasize that differences in colonization timing may alter competitive outcomes between taxa, impacting taxon coexistence. ImportanceThe bacterial gut communities of honey bees possess considerable strain-level diversity between hives, between individual bees and within individual bees. However, the factors underlying strain coexistence are unclear. Here, we provide support for timing of colonization, or priority effects, as one factor driving this strain level diversity. Our results show that priority inoculation can prevent colonization by subsequent competing bacterial strains and mitigate advantages conferred through bacterial weaponry. Further, a brief window of priority can facilitate the coexistence of strongly and weakly competitive strains within single bees. These results add to our understanding of the impacts of priority effects in host-associated microbial communities and provide support for the development of future probiotic strategies aimed directing microbiome structure to benefit honey bee health.

ecology↗

bioRxiv2024

Virulence-linked adhesin drives mutualist colonization of the bee gut via biofilm formation

Bacterial biofilms are stable multicellular structures that can enable long term host association. Yet, the role of biofilms in supporting gut mutualism is still not fully understood. Here, we investigate Snodgrassella alvi, a beneficial bacterial symbiont of honey bees, and find that biofilm formation is required for its colonization of the bee gut. We constructed fifteen S. alvi mutants containing knockouts of genes known to promote colonization with putative roles in biofilm formation. Genes required for colonization included staA and staB, encoding trimeric autotransporter adhesins (TAAs) and mltA, encoding a lytic transglycosylase. Intriguingly, TAAs are considered virulence factors in pathogens but support mutualism by the symbiont S. alvi. In vitro, biofilm formation was reduced in {Delta}staB cells and abolished in the other two mutants. Loss of staA also reduced auto-aggregation and cell-cell connections. Based on structural predictions, StaA/B are massive (>300 nm) TAAs with many repeats in their stalk regions. Further, we find that StaA/B are conserved across Snodgrassella species, suggesting that StaA/B-dependent colonization is characteristic of this symbiont lineage. Finally, staA deletion increases sensitivity to bactericidal antimicrobials, suggesting that the biofilm indirectly buffers against antibiotic stress. In all, the inability of two biofilm-deficient strains ({Delta}staA and {Delta}mltA) to effectively mono-colonize bees indicates that S. alvi biofilm formation is required for colonization of the bee gut. We envision the bee gut system as a genetically tractable model for studying the physical basis of biofilm-mutualist-gut interactions.

microbiology↗

bioRxiv2024

The gut microbiome of honey-producing wasps has converged on that of social bees

Honey-feeding social bees, including honey bees, bumbles bees, and stingless bees, possess distinctive gut bacterial communities that provide benefits to hosts, such as defense against pathogens and parasites. Members of these communities are transmitted through social interactions within colonies. The Mexican honey wasp (Brachygastra mellifica) represents an independent origin of honey-storing within a group of social Hymenoptera. Honey wasps feed on and store honey, but, unlike bees, they prey on other insects as a protein source, and do not consume pollen. We surveyed the gut bacterial communities of Mexican honey wasps across sites within Texas using 16S rRNA profiling, and we estimated bacterial titer per bee using qPCR. For comparison, we also surveyed non-honey feeding wasps from six families, collected in the same region. We found that honey wasp communities are dominated by characteristic bacterial species. In contrast, other wasps had lower absolute titers and more variable communities, dominated by environmental bacteria. Honey wasps from all sampled nests contained strains of Bifidobacterium and Bombilactobacillus that were closely related to symbionts of bumble bees and other bees, suggesting acquisition via host-switching. Some individuals also harbored a close relative of Candidatus Schmidhempelia bombi (Orbaceae), an uncultured bumble bee symbiont, again suggesting host-switching. The most prevalent species was an uncultured Lactobacillus that potentially represents an independent acquisition of environmental Lactobacillus. The transition to honey feeding, combined with a highly social life history, appears to have facilitated the establishment of a bacterial community with similarities to those of social bees. IMPORTANCEHoney-feeding social insects such as honey bees and bumble bees have conserved gut bacterial communities that are transmitted among nestmates. These bacteria benefit hosts by providing defense against pathogens, and potentially by contributing to pollen digestion. The bacterial communities of wasps are less studied. Whereas most wasps are carnivorous and consume nectar, honey wasps (Brachygastra spp.) store and eat honey. Here, we address the consequences of this dietary shift for the gut community. Using field collections of Mexican honey wasps and other co-occurring wasps, we found that honey wasps have distinctive gut bacterial communities. These include several bacteria most closely related to bacteria in bumble bees, suggesting their acquisition via host-switching. Solitary wasps and social wasps that do not make honey have smaller gut communities dominated by environmental bacteria, suggesting that honey feeding has shaped the gut bacterial communities of honey wasps.

microbiology↗

bioRxiv2024

Glyphosate Effects on Growth and Biofilm Formation in Bee Gut Symbionts and Diverse Associated Bacteria

Biofilm formation is a common adaptation enabling bacteria to thrive in various environments and to withstand external pressures. In the context of host-microbe interactions, biofilms play vital roles in establishing microbiomes associated with animals and plants and are used by opportunistic microbes to facilitate proliferation within hosts. Investigating biofilm dynamics, composition, and responses to environmental stressors is crucial for understanding microbial community assembly and biofilm regulation in health and disease. In this study, we explore the independent gut colonization and in vitro biofilm formation abilities of core members of the honey bee (Apis mellifera) gut microbiota. Additionally, we assess the impact of glyphosate, a widely used herbicide with antimicrobial properties, and a glyphosate-based formulation on growth and biofilm formation in bee gut symbionts as well as in other biofilm-forming bacteria associated with diverse animals and plants. Our results demonstrate that several strains of core bee gut bacterial species can independently colonize the bee gut, which probably depends on their ability to form biofilms. Furthermore, glyphosate exposure has varying effects on bacterial growth and biofilm formation. These findings imply specific impacts of environmental stressors on microbial biofilms with both ecological and host health-related implications. Importance Biofilms are essential for microbial communities to establish and thrive in diverse environments. In the honey bee gut, the core microbiota member Snodgrassella alvi forms biofilms, potentially aiding the establishment of other members and promoting interactions with the host. In this study, we show that specific strains of other core members, including Bifidobacterium, Bombilactobacillus, Gilliamella, and Lactobacillus, also form biofilms. We then examine the impact of glyphosate, a widely used herbicide that disrupts the bee microbiota, on their growth and biofilm formation. Our findings demonstrate diverse effects of glyphosate on biofilm formation, ranging from inhibition to enhancement, reflecting observations in other beneficial or pathogenic bacteria associated with animals and plants. Thus, glyphosate exposure may influence bacterial growth and biofilm formation, potentially shaping microbial establishment on host surfaces and impacting health outcomes.

microbiology↗

bioRxiv2023

Intracellular defensive symbiont is culturable and capable of transovarial, vertical transmission

Insects frequently form heritable associations with beneficial bacteria that are vertically transmitted from parent to offspring. Long term vertical transmission has repeatedly resulted in genome reduction and gene loss rendering many such bacteria incapable of independent culture. Among aphids, heritable endosymbionts often provide a wide range of context-specific benefits to their hosts. Although these associations have large impacts on host phenotypes, experimental approaches are often limited by an inability to independently cultivate these microbes. Here, we report the axenic culture of Candidatus Fukatsuia symbiotica strain WIR, a heritable bacterial endosymbiont of the pea aphid, Acyrthosiphon pisum. Whole genome sequencing revealed similar genomic features and high sequence similarity to previously described strains, suggesting the cultivation techniques used here may be applicable to Ca. F. symbiotica strains from distantly related aphids. Microinjection of the isolated strain into uninfected aphids revealed that it can reinfect developing embryos, and is maintained in subsequent generations via transovarial maternal transmission. Artificially infected aphids exhibit similar phenotypic and life history traits compared to native infections, including protective effects against an entomopathogenic Fusarium species. Overall, our results show that Ca. F. symbiotica may be a useful tool for experimentally probing the molecular mechanisms underlying heritable symbioses and antifungal defense in the pea aphid system. IMPORTANCEDiverse eukaryotic organisms form stable, symbiotic relationships with bacteria that provide benefits to their hosts. While these associations are often biologically important, they can be difficult to probe experimentally, because intimately host-associated bacteria are difficult to access within host tissues, and most cannot be cultured. This is especially true of the intracellular, maternally inherited bacteria associated with many insects, including aphids. Here, we demonstrate that a pea aphid-associated strain of the heritable endosymbiont, Candidatus Fukatsuia symbiotica, can be grown outside of its host using standard microbiology techniques, and can readily re-establish infection that is maintained across host generations. These artificial infections recapitulate the effects of native infections making this host-symbiont pair a useful experimental system. Using this system, we demonstrate that Ca. F. symbiotica infection reduces host fitness under benign conditions, but protects against a previously unreported fungal pathogen.

microbiology↗

bioRxiv2023

Single-step genome engineering in the bee gut symbiont Snodgrassella alvi

Honey bees are economically relevant pollinators experiencing population declines due to a number of threats. As in humans, the health of bees is influenced by their microbiome. The bacterium Snodgrassella alvi is a key member of the bee gut microbiome and has a role in excluding pathogens. Despite this importance, there are not currently any easy-to-use methods for modifying the S. alvi chromosome to study its genetics. To solve this problem, we developed a one-step procedure that uses electroporation and homologous recombination, which we term SnODIFY (Snodgrassella-specific One-step gene Deletion or Insertion to alter FunctionalitY). We used SnODIFY to create seven single-gene knockout mutants and recovered mutants for all constructs tested. Nearly all transformants had the designed genome modifications, indicating that SnODIFY is highly accurate. Mutant phenotypes were validated through knockout of Type 4 pilus genes, which led to reduced biofilm formation. We also used SnODIFY to insert heterologous sequences into the genome by integrating fluorescent protein-coding genes. Finally, we confirmed that genome modification is dependent on S. alvis endogenous RecA protein. Because it does not require expression of exogenous recombination machinery, SnODIFY is a straightforward, accurate, and lightweight method for genome editing in S. alvi. This workflow can be used to study the functions of S. alvi genes and to engineer this symbiont for applications including protection of honey bee health.

microbiology↗

bioRxiv2023

Phylloxera and aphids show distinct features of genome evolution despite similar reproductive modes

Genomes of aphids (family Aphididae) show several unusual evolutionary patterns. In particular, within the XO sex determination system of aphids, the X chromosome exhibits a lower rate of interchromosomal rearrangements, fewer highly expressed genes, and faster evolution at nonsynonymous sites compared to the autosomes. In contrast, other hemipteran lineages have similar rates of interchromosomal rearrangement for autosomes and X chromosomes. One possible explanation for these differences is the aphids life cycle of cyclical parthenogenesis, where multiple asexual generations alternate with one sexual generation. If true, we should see similar features in the genomes of Phylloxeridae, an outgroup of aphids which also undergoes cyclical parthenogenesis. To investigate this, we generated a chromosome-level assembly for the grape phylloxera, an agriculturally important species of Phylloxeridae, and identified its single X chromosome. We then performed synteny analysis using the phylloxerid genome and 30 high-quality genomes of aphids and other hemipteran species. Unexpectedly, we found that the phylloxera does not share aphids patterns of chromosome evolution. By estimating interchromosomal rearrangement rates on an absolute time scale, we found that rates are elevated for aphid autosomes compared to their X chromosomes, but this pattern does not extend to the phylloxera branch. Potentially, the conservation of X chromosome gene content is due to selection on XO males that appear in the sexual generation. We also examined gene duplication patterns across Hemiptera and uncovered horizontal gene transfer events contributing to phylloxera evolution.

evolutionary biology↗

bioRxiv2023

The Pathfinder plasmid toolkit for genetically engineering newly isolated bacteria enables the study of Drosophila-colonizing Orbaceae

Toolkits of plasmids and genetic parts streamline the process of assembling DNA constructs and engineering microbes. Many of these kits were designed with specific industrial or laboratory microbes in mind. For researchers interested in non-model microbial systems, it is often unclear which tools and techniques will function in newly isolated strains. To address this challenge, we designed the Pathfinder toolkit for quickly determining the compatibility of a bacterium with different plasmid components. Pathfinder plasmids combine three different broad-host-range origins of replication with multiple antibiotic resistance cassettes and reporters, so that sets of parts can be rapidly screened through multiplex conjugation. We first tested these plasmids in Escherichia coli, a strain of Sodalis praecaptivus that colonizes insects, and a Rosenbergiella isolate from leafhoppers. Then, we used the Pathfinder plasmids to engineer previously unstudied bacteria from the family Orbaceae that were isolated from several fly species. Engineered Orbaceae strains were able to colonize Drosophila melanogaster and could be visualized in fly guts. Orbaceae are common and abundant in the guts of wild-caught flies but have not been included in laboratory studies of how the Drosophila microbiome affects fly health. Thus, this work provides foundational genetic tools for studying new host-associated microbes, including bacteria that are a key constituent of the gut microbiome of a model insect species. IMPORTANCETo fully understand how microbes have evolved to interact with their environments, one must be able to modify their genomes. However, it can be difficult and laborious to discover which genetic tools and approaches work for a new isolate. Bacteria from the recently described Orbaceae family are common in the microbiomes of insects. We developed the Pathfinder plasmid toolkit for testing the compatibility of different genetic parts with newly cultured bacteria. We demonstrate its utility by engineering Orbaceae strains isolated from flies to express fluorescent proteins and characterizing how they colonize the Drosophila melanogaster gut. Orbaceae are widespread in Drosophila in the wild but have not been included in laboratory studies examining how the gut microbiome affects fly nutrition, health, and longevity. Our work establishes a path for genetic studies aimed at understanding and altering interactions between these and other newly isolated bacteria and their hosts.

microbiology↗

bioRxiv2022

The genomic basis of evolutionary novelties in a leafhopper

Evolutionary innovations generate phenotypic and species diversity. Elucidating the genomic processes underlying such innovations is central to understanding biodiversity. In this study, we addressed the genomic basis of evolutionary novelties in the Glassy-Winged Sharpshooter (Homalodisca vitripennis, GWSS), an agricultural pest. Prominent evolutionary innovations in leafhoppers include brochosomes, proteinaceous structures that are excreted and used to coat the body, and obligate symbiotic associations with two bacterial types that reside within cytoplasm of distinctive cell types. Using PacBio long-read sequencing and Dovetail Omni-C technology, we generated a chromosome-level genome assembly for the GWSS, then validated the assembly using flow cytometry and karyotyping. Additional transcriptomic and proteomic data were used to identify novel genes that underlie brochosome production. We found that brochosome-associated genes include novel gene families that have diversified through tandem duplications. We also identified the locations of genes involved in interactions with bacterial symbionts. Ancestors of the GWSS acquired bacterial genes through horizontal gene transfer (HGT), and these genes appear to contribute to symbiont support. Using a phylogenomics approach, we inferred HGT sources and timing. We found that some HGT events date to the common ancestor of the hemipteran suborder Auchenorrhyncha, representing some of the oldest known examples of HGT in animals. Overall, we show that evolutionary novelties in leafhoppers are generated by the combination of acquiring novel genes, produced both de novo and through tandem duplication, acquiring new symbiotic associations that enable use of novel diets and niches, and recruiting foreign genes to support symbionts and enhance herbivory.

genomics↗

bioRxiv2022

Microbiome assembly and maintenance across the lifespan of bumble bee workers

How a hosts microbiome changes over its lifespan can influence development and aging. As these temporal patterns have only been described in detail for humans and a handful of other hosts, an important next step is to compare microbiome dynamics across a broader array of host-microbe symbioses, and to investigate how and why they vary. Here we characterize the temporal dynamics and stability of the bumblebee worker gut microbiome. Bumblebees are a useful symbiosis model given their relatively well-understood life history and simple, host-specific gut bacterial communities. Furthermore, microbial dynamics may influence bumblebee health and pollination services. We combined high-temporal-resolution sampling with 16S rRNA gene sequencing, quantitative PCR, and shotgun metagenomics to characterize gut microbiomes over the adult lifespan of Bombus impatiens workers. To understand how hosts may control (or lose control of) the gut microbiome as they age, we also sequenced hindgut transcriptomes. We found that, at the community level, microbiome assembly is highly predictable and similar to patterns of primary succession observed in the human gut. At the same time, partitioning of strain-level bacterial variants among colonies suggests stochastic colonization events similar to those observed in flies and nematodes. We also find strong differences in temporal dynamics among symbiont species, suggesting ecological differences among microbiome members in colonization and persistence. Finally, we show that both the gut microbiome and host transcriptome--including expression of key immunity genes--stabilize, as opposed to senesce, with age. We suggest that in highly social groups such as bumblebees, maintenance of both microbiomes and immunity contribute to the inclusive fitness of workers, and thus remain under selection even in old age. Our findings provide a foundation for exploring the mechanisms and functional outcomes of bee microbiome succession, and for comparative analyses with other host-microbe symbioses.

microbiology↗

bioRxiv2021

Global composition of the bacteriophage community in honeybees

The microbial communities in animal digestive systems are critical to host development and health. These assemblages of primarily viruses, bacteria, and fungi stimulate the immune system during development, synthesize important chemical compounds like hormones, aid in digestion, competitively exclude pathogens, etc. The bacteriophages in animal microbiomes are harder to characterize than the bacterial or fungal components of the microbiome and thus we know comparatively little about the temporal and spatial dynamics of bacteriophage communities in animal digestive systems. Recently, the bacteriophages of the honeybee gut were characterized in two European bee populations. Most of the bacteriophages described in these two reports were novel, encoded many metabolic genes in their genomes, and had a community structure that suggests coevolution with their bacterial hosts. To describe the conservation of bacteriophages in bees and begin to understand their role in the bee microbiome, we sequenced the virome of Apis mellifera from Austin, Texas and compared bacteriophage composition between three locations around the world. We found that the majority of bacteriophages from Austin are novel, sharing no sequence similarity to anything in public repositories. However, many bacteriophages are shared among the three bee viromes, indicating specialization of bacteriophages in the bee gut. Our study along with the two previous bee virome studies shows that the bee gut bacteriophage community is simple compared to that of many animals, consisting of several hundred types of bacteriophages that primarily infect four of the dominant bacterial phylotypes in the bee gut.

microbiology↗

bioRxiv2021

Isolation of the Buchnera aphidicola flagellum basal body from the Buchnera membrane

Buchnera aphidicola is an intracellular bacterial symbiont of aphids and maintains a small genome of only 600 kbps. Buchnera is thought to maintain only genes relevant to the symbiosis with its aphid host. Curiously, the Buchnera genome contains gene clusters coding for flagellum basal body structural proteins and for flagellum type III export machinery. These structures have been shown to be highly expressed and present in large numbers on Buchnera cells. No recognizable pathogenicity factors or secreted proteins have been identified in the Buchnera genome, and the relevance of this protein complex to the symbiosis is unknown. Here, we show isolation of Buchnera flagella from the cellular membrane of Buchnera, confirming the enrichment of flagellum proteins relative to other proteins in the Buchnera proteome. This will facilitate studies of the structure and function of the Buchnera flagellum structure, and its role in this model symbiosis.

biochemistry↗

bioRxiv2020

Engineering a culturable Serratia symbiotica strain for aphid paratransgenesis

Aphids are global agricultural pests and important models for bacterial symbiosis. To date, none of the native symbionts of aphids have been genetically manipulated, which limits our understanding of how they interact with their hosts. Serratia symbiotica CWBI-2.3T is a culturable, gut-associated bacterium isolated from the black bean aphid. Closely related Serratia symbiotica strains are facultative aphid endosymbionts that are vertically transmitted from mother to offspring during embryogenesis. We demonstrate that CWBI-2.3T can be genetically engineered using a variety of techniques, plasmids, and gene expression parts. Then, we use fluorescent protein expression to track the dynamics with which CWBI-2.3T colonizes the guts of multiple aphid species, and we measure how this bacterium affects aphid fitness. Finally, we show that we can induce heterologous gene expression from engineered CWBI-2.3T in living aphids. These results inform the development of CWBI-2.3T for aphid paratransgenesis, which could be used to study aphid biology and enable future agricultural technologies. IMPORTANCEInsects have remarkably diverse and integral roles in global ecosystems. Many harbor symbiotic bacteria, but very few of these bacteria have been genetically engineered. Aphids are major agricultural pests and an important model system for the study of symbiosis. This work describes methods for engineering a culturable aphid symbiont, Serratia symbiotica CWBI-2.3T. These approaches and genetic tools could be used in the future to implement new paradigms for the biological study and control of aphids.

synthetic biology↗

bioRxiv2020

Vertical transmission at the pathogen-symbiont interface: Serratia symbiotica and aphids

Many insects possess beneficial bacterial symbionts that occupy specialized host cells and are maternally transmitted. As a consequence of their host-restricted lifestyle, these symbionts often possess reduced genomes and cannot be cultured outside hosts, limiting their study. The bacterial species Serratia symbiotica was originally described by noncultured strains that live as mutualistic symbionts of aphids and are vertically transmitted through transovarial endocytosis within the mothers body. More recently, culturable strains of S. symbiotica were discovered that retain a larger set of ancestral Serratia genes, are gut pathogens in aphid hosts, and are principally transmitted via a fecal-oral route. We find that these culturable strains, when injected into pea aphids, replicate in the hemolymph and are pathogenic. Unexpectedly, they are also capable of maternal transmission via transovarial endocytosis: using GFP-tagged strains, we observe that pathogenic S. symbiotica, but not Escherichia coli, are endocytosed into early embryos. Furthermore, pathogenic S. symbiotica strains are compartmentalized into specialized aphid cells in a similar fashion to mutualistic S. symbiotica strains during later stages of embryonic development. Thus, cultured, pathogenic strains of S. symbiotica have the latent capacity to transition to lifestyles as mutualistic symbionts of aphid hosts. This capacity is blocked by pathogenicity: their hosts die before infected progeny are born. To transition into stably inherited symbionts, culturable S. symbiotica strains may need to adapt to regulate their titer, limit their pathogenicity, and/or provide benefits to aphids that outweigh their cost. ImportanceInsects have evolved various mechanisms to reliably transmit their beneficial bacterial symbionts to the next generation. Sap-sucking insects, including aphids, transmit symbionts by endocytosis of the symbiont into cells of the early embryo within the mothers body. Experimental studies of this process are hampered by the inability to culture or genetically manipulate host-restricted, symbiotic bacteria. Serratia symbiotica is a bacterial species that includes strains ranging from obligate, heritable symbionts to culturable gut pathogens. We demonstrate that culturable S. symbiotica strains, that are aphid gut pathogens, can be maternally transmitted by endocytosis. Cultured S. symbiotica therefore possess a latent capacity for evolving a host-restricted lifestyle and can be used to understand the transition from pathogenicity to beneficial symbiosis.

microbiology↗