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 ΔstaB 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 (ΔstaA and ΔmltA) 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.

Evolution recovers the fitness of Acinetobacter baylyi strains with large deletions through mutations in deletion-specific targets and global post-transcriptional regulators.

Organelles and endosymbionts have naturally evolved dramatically reduced genome sizes compared to their free-living ancestors. Synthetic biologists have purposefully engineered streamlined microbial genomes to create more efficient cellular chassis and define the minimal components of cellular life. During natural or engineered genome streamlining, deletion of many non-essential genes in combination often reduces bacterial fitness for idiosyncratic or unknown reasons. We investigated how and to what extent laboratory evolution could overcome these defects in six variants of the transposon-free Acinetobacter baylyi strain ADP1-ISx that each had a deletion of a different 22- to 42-kilobase region and two strains with larger deletions of 70 and 293 kilobases. We evolved replicate populations of ADP1-ISx and each deletion strain for ~300 generations in a chemically defined minimal medium or a complex medium and sequenced the genomes of endpoint clonal isolates. Fitness increased in all cases that were examined except for two ancestors that each failed to improve in one of the two environments. Mutations affecting nine protein-coding genes and two small RNAs were significantly associated with one of the two environments or with certain deletion ancestors. The global post-transcriptional regulators rnd (ribonuclease D), csrA (RNA-binding carbon storage regulator), and hfq (RNA-binding protein and chaperone) were frequently mutated across all strains, though the incidence and effects of these mutations on gene function and bacterial fitness varied with the ancestral deletion and evolution environment. Mutations in this regulatory network likely compensate for how an earlier deletion of a transposon in the ADP1-ISx ancestor of all the deletion strains restored csrA function. More generally, our results demonstrate that fitness lost during genome streamlining can usually be regained rapidly through laboratory evolution and that recovery tends to occur through a combination of deletion-specific compensation and global regulatory adjustments.

CryptKeeper: a negative design tool for reducing unintentional gene expression in bacteria.

Foundational techniques in molecular biology-such as cloning genes, tagging biomolecules for purification or identification, and overexpressing recombinant proteins-rely on introducing non-native or synthetic DNA sequences into organisms. These sequences may be recognized by the transcription and translation machinery in their new context in unintended ways. The cryptic gene expression that sometimes results has been shown to produce genetic instability and mask experimental signals. Computational tools have been developed to predict individual types of gene expression elements, but it can be difficult for researchers to contextualize their collective output. Here, we introduce CryptKeeper, a software pipeline that visualizes predictions of bacterial gene expression signals and estimates the translational burden possible from a DNA sequence. We investigate several published examples where cryptic gene expression in E. coli interfered with experiments. CryptKeeper accurately postdicts unwanted gene expression from both eukaryotic virus infectious clones and individual proteins that led to genetic instability. It also identifies off-target gene expression elements that resulted in truncations that confounded protein purification. Incorporating negative design using CryptKeeper into reverse genetics and synthetic biology workflows can help to mitigate cloning challenges and avoid unexplained failures and complications that arise from unintentional gene expression.

Pathogen contingency loci and the evolution of host specificity: Simple sequence repeats mediate Bartonella adaptation to a wild rodent host.

Parasites, including pathogens, can adapt to better exploit their hosts on many scales, ranging from within an infection of a single individual to series of infections spanning multiple host species. However, little is known about how the genomes of parasites in natural communities evolve when they face diverse hosts. We investigated how Bartonella bacteria that circulate in rodent communities in the dunes of the Negev Desert in Israel adapt to different species of rodent hosts. We propagated 15 Bartonella populations through infections of either a single host species (Gerbillus andersoni or Gerbillus pyramidum) or alternating between the two. After 20 rodent passages, strains with de novo mutations replaced the ancestor in most populations. Mutations in two mononucleotide simple sequence repeats (SSRs) that caused frameshifts in the same adhesin gene dominated the evolutionary dynamics. They appeared exclusively in populations that encountered G. andersoni and altered the dynamics of infections of this host. Similar SSRs in other genes are conserved and exhibit ON/OFF variation in Bartonella isolates from the Negev Desert dunes. Our results suggest that SSR-based contingency loci could be important not only for rapidly and reversibly generating antigenic variation to escape immune responses but that they may also mediate the evolution of host specificity.

One-step genome engineering in bee gut bacterial symbionts.

Mechanistic understanding of interactions in many host-microbe systems, including the honey bee microbiome, is limited by a lack of easy-to-use genome engineering approaches. To this end, we demonstrate a one-step genome engineering approach for making gene deletions and insertions in the chromosomes of honey bee gut bacterial symbionts. Electroporation of linear or non-replicating plasmid DNA containing an antibiotic resistance cassette flanked by regions with homology to a symbiont genome reliably results in chromosomal integration. This lightweight approach does not require expressing any exogenous recombination machinery. The high concentrations of large DNAs with long homology regions needed to make the process efficient can be readily produced using modern DNA synthesis and assembly methods. We use this approach to knock out genes, including genes involved in biofilm formation, and insert fluorescent protein genes into the chromosome of the betaproteobacterial bee gut symbiont Snodgrassella alvi. We are also able to engineer the genomes of multiple strains of S. alvi and another species, Snodgrassella communis, which is found in the bumble bee gut microbiome. Finally, we use the same method to engineer the chromosome of another bee symbiont, Bartonella apis, which is an alphaproteobacterium. As expected, gene knockout in S. alvi using this approach is recA-dependent, suggesting that this straightforward procedure can be applied to other microbes that lack convenient genome engineering methods. IMPORTANCE: Honey bees are ecologically and economically important crop pollinators with bacterial gut symbionts that influence their health. Microbiome-based strategies for studying or improving bee health have utilized wild-type or plasmid-engineered bacteria. We demonstrate that a straightforward, single-step method can be used to insert cassettes and replace genes in the chromosomes of multiple bee gut bacteria. This method can be used for investigating the mechanisms of host-microbe interactions in the bee gut community and stably engineering symbionts that benefit pollinator health.

Measuring the burden of hundreds of BioBricks defines an evolutionary limit on constructability in synthetic biology.

Engineered DNA will slow the growth of a host cell if it redirects limiting resources or otherwise interferes with homeostasis. Escape mutants that alleviate this burden can rapidly evolve and take over cell populations, making genetic engineering less reliable and predictable. Synthetic biologists often use genetic parts encoded on plasmids, but their burden is rarely characterized. We measured how 301 BioBrick plasmids affected Escherichia coli growth and found that 59 (19.6%) were burdensome, primarily because they depleted the limited gene expression resources of host cells. Overall, no BioBricks reduced the growth rate of E. coli by >45%, which agreed with a population genetic model that predicts such plasmids should be unclonable. We made this model available online for education ( https://barricklab.org/burden-model ) and added our burden measurements to the iGEM Registry. Our results establish a fundamental limit on what DNA constructs and genetic modifications can be successfully engineered into cells.

Promoter recruitment drives the emergence of proto-genes in a long-term evolution experiment with Escherichia coli.

The phenomenon of de novo gene birth-the emergence of genes from non-genic sequences-has received considerable attention due to the widespread occurrence of genes that are unique to particular species or genomes. Most instances of de novo gene birth have been recognized through comparative analyses of genome sequences in eukaryotes, despite the abundance of novel, lineage-specific genes in bacteria and the relative ease with which bacteria can be studied in an experimental context. Here, we explore the genetic record of the Escherichia coli long-term evolution experiment (LTEE) for changes indicative of "proto-genic" phases of new gene birth in which non-genic sequences evolve stable transcription and/or translation. Over the time span of the LTEE, non-genic regions are frequently transcribed, translated and differentially expressed, with levels of transcription across low-expressed regions increasing in later generations of the experiment. Proto-genes formed downstream of new mutations result either from insertion element activity or chromosomal translocations that fused preexisting regulatory sequences to regions that were not expressed in the LTEE ancestor. Additionally, we identified instances of proto-gene emergence in which a previously unexpressed sequence was transcribed after formation of an upstream promoter, although such cases were rare compared to those caused by recruitment of preexisting promoters. Tracing the origin of the causative mutations, we discovered that most occurred early in the history of the LTEE, often within the first 20,000 generations, and became fixed soon after emergence. Our findings show that proto-genes emerge frequently within evolving populations, can persist stably, and can serve as potential substrates for new gene formation.

Identifying widespread and recurrent variants of genetic parts to improve annotation of engineered DNA sequences.

Engineered plasmids have been workhorses of recombinant DNA technology for nearly half a century. Plasmids are used to clone DNA sequences encoding new genetic parts and to reprogram cells by combining these parts in new ways. Historically, many genetic parts on plasmids were copied and reused without routinely checking their DNA sequences. With the widespread use of high-throughput DNA sequencing technologies, we now know that plasmids often contain variants of common genetic parts that differ slightly from their canonical sequences. Because the exact provenance of a genetic part on a particular plasmid is usually unknown, it is difficult to determine whether these differences arose due to mutations during plasmid construction and propagation or due to intentional editing by researchers. In either case, it is important to understand how the sequence changes alter the properties of the genetic part. We analyzed the sequences of over 50,000 engineered plasmids using depositor metadata and a metric inspired by the natural language processing field. We detected 217 uncatalogued genetic part variants that were especially widespread or were likely the result of convergent evolution or engineering. Several of these uncatalogued variants are known mutants of plasmid origins of replication or antibiotic resistance genes that are missing from current annotation databases. However, most are uncharacterized, and 3/5 of the plasmids we analyzed contained at least one of the uncatalogued variants. Our results include a list of genetic parts to prioritize for refining engineered plasmid annotation pipelines, highlight widespread variants of parts that warrant further investigation to see whether they have altered characteristics, and suggest cases where unintentional evolution of plasmid parts may be affecting the reliability and reproducibility of science.

Measuring the burden of hundreds of BioBricks defines an evolutionary limit on constructability in synthetic biology.

Engineered DNA will slow the growth of a host cell if it redirects limiting resources or otherwise interferes with homeostasis. Populations of engineered cells can rapidly become dominated by "escape mutants" that evolve to alleviate this burden by inactivating the intended function. Synthetic biologists working with bacteria rely on genetic parts and devices encoded on plasmids, but the burden of different engineered DNA sequences is rarely characterized. We measured how 301 BioBricks on high-copy plasmids affected the growth rate of Escherichia coli. Of these, 59 (19.6%) negatively impacted growth. The burden imposed by engineered DNA is commonly associated with diverting ribosomes or other gene expression factors away from producing endogenous genes that are essential for cellular replication. In line with this expectation, BioBricks exhibiting burden were more likely to contain highly active constitutive promoters and strong ribosome binding sites. By monitoring how much each BioBrick reduced expression of a chromosomal GFP reporter, we found that the burden of most, but not all, BioBricks could be wholly explained by diversion of gene expression resources. Overall, no BioBricks reduced the growth rate of E. coli by >45%, which agreed with a population genetic model that predicts such plasmids should be "unclonable" because escape mutants will take over during growth of a bacterial colony or small laboratory culture from a transformed cell. We made this model available as an interactive web tool for synthetic biology education and added our burden measurements to the iGEM Registry descriptions of each BioBrick.

Promoter capture drives the emergence of proto-genes in Escherichia coli.

The phenomenon of de novo gene birth-the emergence of genes from non-genic sequences-has received considerable attention due to the widespread occurrence of genes that are unique to particular species or genomes. Most instances of de novo gene birth have been recognized through comparative analyses of genome sequences in eukaryotes, despite the abundance of novel, lineage-specific genes in bacteria and the relative ease with which bacteria can be studied in an experimental context. Here, we explore the genetic record of the Escherichia coli Long-Term Evolution Experiment (LTEE) for changes indicative of "proto-genic" phases of new gene birth in which non-genic sequences evolve stable transcription and/or translation. Over the time-span of the LTEE, non-genic regions are frequently transcribed, translated and differentially expressed, thereby serving as raw material for new gene emergence. Most proto-genes result either from insertion element activity or chromosomal translocations that fused pre-existing regulatory sequences to regions that were not expressed in the LTEE ancestor. Additionally, we identified instances of proto-gene emergence in which a previously unexpressed sequence was transcribed after formation of an upstream promoter. Tracing the origin of the causative mutations, we discovered that most occurred early in the history of the LTEE, often within the first 20,000 generations, and became fixed soon after emergence. Our findings show that proto-genes emerge frequently within evolving populations, persist stably, and can serve as potential substrates for new gene formation.

No assembly required: Time for stronger, simpler publishing standards for DNA sequences.

Uniformly accessible DNA sequences are needed to improve experimental reproducibility and automation. Rather than descriptions of how engineered DNA is assembled, publishers should require complete and empirically validated sequences.

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. alvi's 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.

Bartonella infections are prevalent in rodents despite efficient immune responses.

BACKGROUND: Pathogens face strong selection from host immune responses, yet many host populations support pervasive pathogen populations. We investigated this puzzle in a model system of Bartonella and rodents from Israel's northwestern Negev Desert. We chose to study this system because, in this region, 75-100% of rodents are infected with Bartonella at any given time, despite an efficient immunological response. In this region, Bartonella species circulate in three rodent species, and we tested the hypothesis that at least one of these hosts exhibits a waning immune response to Bartonella, which allows reinfections. METHODS: We inoculated captive animals of all three rodent species with the same Bartonella strain, and we quantified the bacterial dynamics and Bartonella-specific immunoglobulin G antibody kinetics over a period of 139 days after the primary inoculation, and then for 60 days following reinoculation with the same strain. RESULTS: Contrary to our hypothesis, we found a strong, long-lasting immunoglobulin G antibody response, with protective immunological memory in all three rodent species. That response prevented reinfection upon exposure of the rodents to the same Bartonella strain. CONCLUSIONS: This study constitutes an initial step toward understanding how the interplay between traits of Bartonella and their hosts influences the epidemiological dynamics of these pathogens in nature.

Daily Transfers, Archiving Populations, and Measuring Fitness in the Long-Term Evolution Experiment with Escherichia coli.

The Long-Term Evolution Experiment (LTEE) has followed twelve populations of Escherichia coli as they have adapted to a simple laboratory environment for more than 35 years and 77,000 bacterial generations. The setup and procedures used in the LTEE epitomize reliable and reproducible methods for studying microbial evolution. In this protocol, we first describe how the LTEE populations are transferred to fresh medium and cultured each day. Then, we describe how the LTEE populations are regularly checked for possible signs of contamination and archived to provide a permanent frozen "fossil record" for later study. Multiple safeguards included in these procedures are designed to prevent contamination, detect various problems when they occur, and recover from disruptions without appreciably setting back the progress of the experiment. One way that the overall tempo and character of evolutionary changes are monitored in the LTEE is by measuring the competitive fitness of populations and strains from the experiment. We describe how co-culture competition assays are conducted and provide both a spreadsheet and an R package (fitnessR) for calculating relative fitness from the results. Over the course of the LTEE, the behaviors of some populations have changed in interesting ways, and new technologies like whole-genome sequencing have provided additional avenues for investigating how the populations have evolved. We end by discussing how the original LTEE procedures have been updated to accommodate or take advantage of these changes. This protocol will be useful for researchers who use the LTEE as a model system for studying connections between evolution and genetics, molecular biology, systems biology, and ecology. More broadly, the LTEE provides a tried-and-true template for those who are beginning their own evolution experiments with new microbes, environments, and questions.

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 microbial ecology and host-associated microbes, including bacteria that are a key constituent of the gut microbiome of a model insect species.

Identifying widespread and recurrent variants of genetic parts to improve annotation of engineered DNA sequences.

Engineered plasmids have been workhorses of recombinant DNA technology for nearly half a century. Plasmids are used to clone DNA sequences encoding new genetic parts and to reprogram cells by combining these parts in new ways. Historically, many genetic parts on plasmids were copied and reused without routinely checking their DNA sequences. With the widespread use of high-throughput DNA sequencing technologies, we now know that plasmids often contain variants of common genetic parts that differ slightly from their canonical sequences. Because the exact provenance of a genetic part on a particular plasmid is usually unknown, it is difficult to determine whether these differences arose due to mutations during plasmid construction and propagation or due to intentional editing by researchers. In either case, it is important to understand how the sequence changes alter the properties of the genetic part. We analyzed the sequences of over 50,000 engineered plasmids using depositor metadata and a metric inspired by the natural language processing field. We detected 217 uncatalogued genetic part variants that were especially widespread or were likely the result of convergent evolution or engineering. Several of these uncatalogued variants are known mutants of plasmid origins of replication or antibiotic resistance genes that are missing from current annotation databases. However, most are uncharacterized, and 3/5 of the plasmids we analyzed contained at least one of the uncatalogued variants. Our results include a list of genetic parts to prioritize for refining engineered plasmid annotation pipelines, highlight widespread variants of parts that warrant further investigation to see whether they have altered characteristics, and suggest cases where unintentional evolution of plasmid parts may be affecting the reliability and reproducibility of science.

Addressing the challenges of symbiont-mediated RNAi in aphids.

Because aphids are global agricultural pests and models for bacterial endosymbiosis, there is a need for reliable methods to study and control their gene function. However, current methods available for aphid gene knockout and knockdown of gene expression are often unreliable and time consuming. Techniques like CRISPR-Cas genome editing can take several months to achieve a single gene knockout because they rely on aphids going through a cycle of sexual reproduction, and aphids often lack strong, consistent levels of knockdown when fed or injected with molecules that induce an RNA interference (RNAi) response. In the hopes of addressing these challenges, we attempted to adapt a new method called symbiont-mediated RNAi (smRNAi) for use in aphids. smRNAi involves engineering a bacterial symbiont of the insect to continuously supply double-stranded RNA (dsRNA) inside the insect body. This approach has been successful in thrips, kissing bugs, and honeybees. We engineered the laboratory Escherichia coli strain HT115 and the native aphid symbiont Serratia symbiotica CWBI-2.3T to produce dsRNA inside the gut of the pea aphid (Acyrthosiphon pisum) targeting salivary effector protein (C002) or ecdysone receptor genes. For C002 assays, we also tested co-knockdown with an aphid nuclease (Nuc1) to reduce RNA degradation. However, we found that smRNAi was not a reliable method for aphid gene knockdown under our conditions. We were unable to consistently achieve the expected phenotypic changes with either target. However, we did see indications that elements of the RNAi pathway were modestly upregulated, and expression of some targeted genes appeared to be somewhat reduced in some trials. We conclude with a discussion of the possible avenues through which smRNAi, and aphid RNAi in general, could be improved in the future.

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. IMPORTANCE: To 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.

Electron Tomography and Machine Learning for Understanding the Highly Ordered Structure of Leafhopper Brochosomes.

Insects known as leafhoppers (Hemiptera: Cicadellidae) produce hierarchically structured nanoparticles known as brochosomes that are exuded and applied to the insect cuticle, thereby providing camouflage and anti-wetting properties to aid insect survival. Although the physical properties of brochosomes are thought to depend on the leafhopper species, the structure-function relationships governing brochosome behavior are not fully understood. Brochosomes have complex hierarchical structures and morphological heterogeneity across species, due to which a multimodal characterization approach is required to effectively elucidate their nanoscale structure and properties. In this work, we study the structural and mechanical properties of brochosomes using a combination of atomic force microscopy (AFM), electron microscopy (EM), electron tomography, and machine learning (ML)-based quantification of large and complex scanning electron microscopy (SEM) image data sets. This suite of techniques allows for the characterization of internal and external brochosome structures, and ML-based image analysis methods of large data sets reveal correlations in the structure across several leafhopper species. Our results show that brochosomes are relatively rigid hollow spheres with characteristic dimensions and morphologies that depend on leafhopper species. Nanomechanical mapping AFM is used to determine a characteristic compression modulus for brochosomes on the order of 1-3 GPa, which is consistent with crystalline proteins. Overall, this work provides an improved understanding of the structural and mechanical properties of leafhopper brochosomes using a new set of ML-based image classification tools that can be broadly applied to nanostructured biological materials.