MPrESS: An R-Package for Accurately Predicting Power for Comparisons of 16S rRNA Microbiome Taxa Distributions including Simulation by Dirichlet Mixture Modeling.

Deep sequencing has revealed that the 16S rRNA gene composition of the human microbiome can vary between populations. However, when existing data are insufficient to address the desired study questions due to limited sample sizes, Dirichlet mixture modeling (DMM) can simulate 16S rRNA gene predictions from experimental microbiome data. We examined the extent to which simulated 16S rRNA gene microbiome data can accurately reflect the diversity within that identified from experimental data and calculate the power. Even when experimental and simulated datasets differed by less than 10%, simulation by DMM consistently overestimates power, except when using only highly discriminating taxa. Admixtures of DMM with experimental data performed poorly compared to pure simulation and did not show the same correlation with experimental data p-value and power values. While multiple replications of random sampling remain the favored method of determining the power, when the estimated sample size required to achieve a certain power exceeds the sample number, then simulated samples based on DMM can be used. We introduce an R-Package, MPrESS, to assist in power calculation and sample size estimation for a 16S rRNA gene microbiome dataset to detect a difference between populations. MPrESS can be downloaded from GitHub.

Escherichia coli ST131 Associated with Increased Mortality in Bloodstream Infections from Urinary Tract Source.

Escherichia coli sequence type 131 (ST131) is a globally dominant multidrug-resistant clone, although its clinical impact on patients with bloodstream infection (BSI) is incompletely understood. This study aims to further define the risk factors, clinical outcomes, and bacterial genetics associated with ST131 BSI. A prospectively enrolled cohort study of adult inpatients with E. coli BSI was conducted from 2002 to 2015. Whole-genome sequencing was performed with the E. coli isolates. Of the 227 patients with E. coli BSI in this study, 88 (39%) were infected with ST131. Patients with E. coli ST131 BSI and those with non-ST131 BSI did not differ with respect to in-hospital mortality (17/82 [20%] versus 26/145 [18%]; P = 0.73). However, in patients with BSI from a urinary tract source, ST131 was associated with a numerically higher in-hospital mortality than patients with non-ST131 BSI (8/42 [19%] versus 4/63 [6%]; P = 0.06) and increased mortality in an adjusted analysis (odds ratio of 5.85; 95% confidence interval of 1.44 to 29.49; P = 0.02). Genomic analyses showed that ST131 isolates primarily had an H4:O25 serotype, had a higher number of prophages, and were associated with 11 flexible genomic islands as well as virulence genes involved in adhesion (papA, kpsM, yfcV, and iha), iron acquisition (iucC and iutA), and toxin production (usp and sat). In patients with E. coli BSI from a urinary tract source, ST131 was associated with increased mortality in an adjusted analysis and contained a distinct repertoire of genes influencing pathogenesis. These genes could contribute to the higher mortality observed in patients with ST131 BSI.

The evolutionary history of cribellate orb-weaver capture thread spidroins.

BACKGROUND: Spiders have evolved two types of sticky capture threads: one with wet adhesive spun by ecribellate orb-weavers and another with dry adhesive spun by cribellate spiders. The evolutionary history of cribellate capture threads is especially poorly understood. Here, we use genomic approaches to catalog the spider-specific silk gene family (spidroins) for the cribellate orb-weaver Uloborus diversus. RESULTS: We show that the cribellar spidroin, which forms the puffy fibrils of cribellate threads, has three distinct repeat units, one of which is conserved across cribellate taxa separated by ~ 250 Mya. We also propose candidates for a new silk type, paracribellar spidroins, which connect the puffy fibrils to pseudoflagelliform support lines. Moreover, we describe the complete repeat architecture for the pseudoflagelliform spidroin (Pflag), which contributes to extensibility of pseudoflagelliform axial fibers. CONCLUSIONS: Our finding that Pflag is closely related to Flag, supports homology of the support lines of cribellate and ecribellate capture threads. It further suggests an evolutionary phase following gene duplication, in which both Flag and Pflag were incorporated into the axial lines, with subsequent loss of Flag in uloborids, and increase in expression of Flag in ecribellate orb-weavers, explaining the distinct mechanical properties of the axial lines of these two groups.

Gene expression profiling reveals candidate genes for defining spider silk gland types.

Molecular studies of the secretory glands involved in spider silk production have revealed candidate genes for silk synthesis and a complicated history of spider silk gene evolution. However, differential gene expression profiles of the multiple silk gland types within an individual orb-web weaving spider are lacking. Each of these gland types produces a functionally distinct silk type. Comparison of gene expression among spider silk gland types would provide insight into the genes that define silk glands generally from non-silk gland tissues, and the genes that define silk glands from each other. Here, we perform 3' tag digital gene expression profiling of the seven silk gland types of the silver garden orb weaver Argiope argentata. Five of these gland types produce silks that are non-adhesive fibers, one silk includes both fibers and glue-like adhesives, and one silk is exclusively glue-like. We identify 1275 highly expressed, significantly upregulated, and tissue specific silk gland specific transcripts (SSTs). These SSTs include seven types of spider silk protein encoding genes known as spidroin genes. We find that the fiber-producing major ampullate and minor ampullate silk glands have more similar expression profiles than any other pair of glands. We also find that a subset of the SSTs is enriched for transmembrane transport and oxidoreductases, and that these transcripts highlight differences and similarities among the major ampullate, minor ampullate, and aggregate silk glands. Furthermore, we show that the wet glue-producing aggregate glands have the most unique SSTs, but still share some SSTs with fiber producing glands. Aciniform glands were the only gland type to share a majority of SSTs with other silk gland types, supporting previous hypotheses that duplication of aciniform glands and subsequent divergence of the duplicates gave rise to the multiple silk gland types within an individual spider.

AbGRI4, a novel antibiotic resistance island in multiply antibiotic-resistant Acinetobacter baumannii clinical isolates.

OBJECTIVES: To investigate the genomic context of a novel resistance island (RI) in multiply antibiotic-resistant Acinetobacter baumannii clinical isolates and global isolates. METHODS: Using a combination of long and short reads generated from the Oxford Nanopore and Illumina platforms, contiguous chromosomes and plasmid sequences were determined. BLAST-based analysis was used to identify the RI insertion target. RESULTS: Genomes of four multiply antibiotic-resistant A. baumannii clinical strains, from a US hospital system, belonging to prevalent MLST ST2 (Pasteur scheme) and ST281 (Oxford scheme) clade F isolates were sequenced to completion. A class 1 integron carrying aadB (tobramycin resistance) and aadA2 (streptomycin/spectinomycin resistance) was identified. The class 1 integron was 6.8 kb, bounded by IS26 at both ends, and embedded in a new target location between an α/ß-hydrolase and a reductase. Due to its novel insertion site and unique RI composition, we suggest naming this novel RI AbGRI4. Molecular analysis of global A. baumannii isolates identified multiple AbGRI4 RI variants in non-ST2 clonal lineages, including variations in the resistance gene cassettes, integron backbone and insertion breakpoints at the hydrolase gene. CONCLUSIONS: A novel RI insertion target harbouring a class 1 integron was identified in a subgroup of ST2/ST281 clinical isolates. Variants of the RI suggested evolution and horizontal transfer of the RI across clonal lineages. Long- and short-read hybrid assembly technology completely resolved the genomic context of IS-bounded RIs, which was not possible using short reads alone.

Bacteriophage targeting of gut bacterium attenuates alcoholic liver disease.

Chronic liver disease due to alcohol-use disorder contributes markedly to the global burden of disease and mortality1-3. Alcoholic hepatitis is a severe and life-threatening form of alcohol-associated liver disease. The gut microbiota promotes ethanol-induced liver disease in mice4, but little is known about the microbial factors that are responsible for this process. Here we identify cytolysin-a two-subunit exotoxin that is secreted by Enterococcus faecalis5,6-as a cause of hepatocyte death and liver injury. Compared with non-alcoholic individuals or patients with alcohol-use disorder, patients with alcoholic hepatitis have increased faecal numbers of E. faecalis. The presence of cytolysin-positive (cytolytic) E. faecalis correlated with the severity of liver disease and with mortality in patients with alcoholic hepatitis. Using humanized mice that were colonized with bacteria from the faeces of patients with alcoholic hepatitis, we investigated the therapeutic effects of bacteriophages that target cytolytic E. faecalis. We found that these bacteriophages decrease cytolysin in the liver and abolish ethanol-induced liver disease in humanized mice. Our findings link cytolytic E. faecalis with more severe clinical outcomes and increased mortality in patients with alcoholic hepatitis. We show that bacteriophages can specifically target cytolytic E. faecalis, which provides a method for precisely editing the intestinal microbiota. A clinical trial with a larger cohort is required to validate the relevance of our findings in humans, and to test whether this therapeutic approach is effective for patients with alcoholic hepatitis.

Spidroins and Silk Fibers of Aquatic Spiders.

Spiders are commonly found in terrestrial environments and many rely heavily on their silks for fitness related tasks such as reproduction and dispersal. Although rare, a few species occupy aquatic or semi-aquatic habitats and for them, silk-related specializations are also essential to survive in aquatic environments. Most spider silks studied to date are from cob-web and orb-web weaving species, leaving the silks from many other terrestrial spiders as well as water-associated spiders largely undescribed. Here, we characterize silks from three Dictynoidea species: the aquatic spiders Argyroneta aquatica and Desis marina as well as the terrestrial Badumna longinqua. From silk gland RNA-Seq libraries, we report a total of 47 different homologs of the spidroin (spider fibroin) gene family. Some of these 47 spidroins correspond to known spidroin types (aciniform, ampullate, cribellar, pyriform, and tubuliform), while other spidroins represent novel branches of the spidroin gene family. We also report a hydrophobic amino acid motif (GV) that, to date, is found only in the spidroins of aquatic and semi-aquatic spiders. Comparison of spider silk sequences to the silks from other water-associated arthropods, shows that there is a diversity of strategies to function in aquatic environments.

Stop paying through the nose: student and trainee medical conferences offer better value for money than professional alternatives.

BACKGROUND: Scientific conferences in the UK are attended by practising doctors and medical students for sharing research, networking and professional development. Student/trainee conferences are typically cheaper than professional conferences, but as they are not acknowledged in national scoring systems for medical and surgical training applications, they may have worse attendance than otherwise possible. We questioned whether student/trainee conferences are of a similar scientific quality to professional conferences, while being considerably cheaper. METHODS: In this cross-sectional database review, 162 conferences were identified through a systematic search of two conference databases by three independent researchers. χ2 tests were used to compare scientific quality between student/trainee and professional conferences and the likelihood of offering different types of discounts. Independent t-tests were employed to determine cost differences between the two categories of conferences. RESULTS: Our data revealed that there was no significant difference between student/trainee and professional conferences likelihood of declaring information on their abstract review processes (p=0.105). There was no difference in speaker seniority, determined by the tool the authors developed (p=0.172). Student/trainee conferences were significantly more likely to offer workshops (p<0.0005) and were cheaper than professional conferences (p<0.0005). CONCLUSION: Our results show that student/trainee conferences offer a similar level of scientific quality to professional medical conferences in the UK at a fraction of the cost, which should be reflected within the national scoring systems.

CD28 null CD4 T-cell expansions in autoimmune disease suggest a link with cytomegalovirus infection.

Immunosenescence is thought to contribute to the increase of autoimmune diseases in older people. Immunosenescence is often associated with the presence of an expanded population of CD4 T cells lacking expression of CD28 (CD28 null). These highly cytotoxic CD4 T cells were isolated from disease-affected tissues in patients with rheumatoid arthritis, systemic lupus erythematosus, multiple sclerosis, or other chronic inflammatory diseases and their numbers appeared to be linked to disease severity. However, we recently demonstrated that the common herpes virus, cytomegalovirus (CMV), not ageing, is the major driver of this subset of cytotoxic T cells. In this review, we discuss how CMV might potentiate and exacerbate autoimmune disease through the expansion of CD28 null CD4 T cells.

Large-scale comparative analysis of microbial pan-genomes using PanOCT.

SUMMARY: The JCVI pan-genome pipeline is a collection of programs to run PanOCT and tools that support and extend the capabilities of PanOCT. PanOCT (pan-genome ortholog clustering tool) is a tool for pan-genome analysis of closely related prokaryotic species or strains. The JCVI Pan-Genome Pipeline wrapper invokes command-line utilities that prepare input genomes, invoke third-party tools such as NCBI Blast+, run PanOCT, generate a consensus pan-genome, annotate features of the pan-genome, detect sets of genes of interest such as antimicrobial resistance (AMR) genes and generate figures, tables and html pages to visualize the results. The pipeline can run in a hierarchical mode, lowering the RAM and compute resources used. AVAILABILITY AND IMPLEMENTATION: Source code, demo data, and detailed documentation are freely available at https://github.com/JCVenterInstitute/PanGenomePipeline.

Enterobacterhormaechei subsp. hoffmannii subsp. nov., Enterobacter hormaechei subsp. xiangfangensis comb. nov., Enterobacter roggenkampii sp. nov., and Enterobacter muelleri is a later heterotypic synonym of Enterobacter asburiae based on computational analysis of sequenced Enterobacter genomes.

Background: The predominant species in clinical Enterobacter isolates is E. hormaechei. Many articles, clinicians, and GenBank submissions misname these strains as E. cloacae. The lack of sequenced type strains or named species/subspecies for some clades in the E. cloacae complex complicate the issue. Methods: The genomes of the type strains for Enterobacter hormaechei subsp.  oharae, E.  hormaechei subsp.  steigerwaltii, and E. xiangfangensis, and two strains from Hoffmann clusters III and IV of the E. cloacae complex were sequenced. These genomes, the E.  hormaechei subsp.  hormaechei type strain, and other available Enterobacter type strains were analysed in conjunction with all extant Enterobacter genomes in NCBI's RefSeq using Average Nucleotide Identity (ANI). Results: There were five recognizable subspecies of E. hormaechei: E. hormaechei subsp. hoffmannii subsp. nov., E. hormaechei subsp. xiangfangensis comb. nov., and the three previously known subspecies. One of the strains sequenced from the E. cloacae complex was not a novel E. hormaechei subspecies but rather a member of a clade of a novel species: E. roggenkampii sp. nov.. E. muelleri was determined to be a later heterotypic synonym of E. asburiae which should take precedence. Conclusion: The phylogeny of the Enterobacter genus, particularly the cloacae complex, was re-evaluated based on the type strain genome sequences and all other available Enterobacter genomes in RefSeq.

Silk genes and silk gene expression in the spider Tengella perfuga (Zoropsidae), including a potential cribellar spidroin (CrSp).

Most spiders spin multiple types of silk, including silks for reproduction, prey capture, and draglines. Spiders are a megadiverse group and the majority of spider silks remain uncharacterized. For example, nothing is known about the silk molecules of Tengella perfuga, a spider that spins sheet webs lined with cribellar silk. Cribellar silk is a type of adhesive capture thread composed of numerous fibrils that originate from a specialized plate-like spinning organ called the cribellum. The predominant components of spider silks are spidroins, members of a protein family synthesized in silk glands. Here, we use silk gland RNA-Seq and cDNA libraries to infer T. perfuga silks at the protein level. We show that T. perfuga spiders express 13 silk transcripts representing at least five categories of spider silk proteins (spidroins). One category is a candidate for cribellar silk and is thus named cribellar spidroin (CrSp). Studies of ontogenetic changes in web construction and spigot morphology in T. perfuga have documented that after sexual maturation, T. perfuga females continue to make capture webs but males halt web maintenance and cease spinning cribellar silk. Consistent with these observations, our candidate CrSp was expressed only in females. The other four spidroin categories correspond to paralogs of aciniform, ampullate, pyriform, and tubuliform spidroins. These spidroins are associated with egg sac and web construction. Except for the tubuliform spidroin, the spidroins from T. perfuga contain novel combinations of amino acid sequence motifs that have not been observed before in these spidroin types. Characterization of T. perfuga silk genes, particularly CrSp, expand the diversity of the spidroin family and inspire new structure/function hypotheses.

Spatial and Environmental Variation of the Human Hair Microbiota.

The skin is a complex living ecosystem harboring diverse microbial communities. Its highly variable properties and influence of intrinsic and extrinsic factors creates unique microenvironments where niche-specific microbes thrive. As part of the skin, hair supports its own microbial habitat that is also intra and inter-personal variable. This little explored substrate has significant potential in forensics microbiome research due to the unique signatures that are available on an individual. To further investigate this, we explored the hair microbiota from scalp and pubic regions in healthy adults to investigate how the hair shaft microenvironment varies microbially. Our results suggest that there are distinct differences between the microbial communities identified on hair shafts originating from different parts of the body. The taxonomic composition of the communities from different hair sources are most reminiscent of those identified from their associated cutaneous region. We further demonstrate that the hair microbiota varies by geographical origin and has the potential to be used to predict the source location of the hair.

PanACEA: a bioinformatics tool for the exploration and visualization of bacterial pan-chromosomes.

BACKGROUND: Bacterial pan-genomes, comprised of conserved and variable genes across multiple sequenced bacterial genomes, allow for identification of genomic regions that are phylogenetically discriminating or functionally important. Pan-genomes consist of large amounts of data, which can restrict researchers ability to locate and analyze these regions. Multiple software packages are available to visualize pan-genomes, but currently their ability to address these concerns are limited by using only pre-computed data sets, prioritizing core over variable gene clusters, or by not accounting for pan-chromosome positioning in the viewer. RESULTS: We introduce PanACEA (Pan-genome Atlas with Chromosome Explorer and Analyzer), which utilizes locally-computed interactive web-pages to view ordered pan-genome data. It consists of multi-tiered, hierarchical display pages that extend from pan-chromosomes to both core and variable regions to single genes. Regions and genes are functionally annotated to allow for rapid searching and visual identification of regions of interest with the option that user-supplied genomic phylogenies and metadata can be incorporated. PanACEA's memory and time requirements are within the capacities of standard laptops. The capability of PanACEA as a research tool is demonstrated by highlighting a variable region important in differentiating strains of Enterobacter hormaechei. CONCLUSIONS: PanACEA can rapidly translate the results of pan-chromosome programs into an intuitive and interactive visual representation. It will empower researchers to visually explore and identify regions of the pan-chromosome that are most biologically interesting, and to obtain publication quality images of these regions.

GGRaSP: a R-package for selecting representative genomes using Gaussian mixture models.

Motivation: The vast number of available sequenced bacterial genomes occasionally exceeds the facilities of comparative genomic methods or is dominated by a single outbreak strain, and thus a diverse and representative subset is required. Generation of the reduced subset currently requires a priori supervised clustering and sequence-only selection of medoid genomic sequences, independent of any additional genome metrics or strain attributes. Results: The Gaussian Genome Representative Selector with Prioritization (GGRaSP) R-package described below generates a reduced subset of genomes that prioritizes maintaining genomes of interest to the user as well as minimizing the loss of genetic variation. The package also allows for unsupervised clustering by modeling the genomic relationships using a Gaussian mixture model to select an appropriate cluster threshold. We demonstrate the capabilities of GGRaSP by generating a reduced list of 315 genomes from a genomic dataset of 4600 Escherichia coli genomes, prioritizing selection by type strain and by genome completeness. Availability and implementaion: GGRaSP is available at https://github.com/JCVenterInstitute/ggrasp/. Supplementary information: Supplementary data are available at Bioinformatics online.

Genomic perspectives of spider silk genes through target capture sequencing: Conservation of stabilization mechanisms and homology-based structural models of spidroin terminal regions.

A powerful system for studying protein aggregation, particularly rapid self-assembly, is spider silk. Spider silks are proteinaceous and silk proteins are synthesized and stored within silk glands as liquid dope. As needed, liquid dope is near-instantaneously transformed into solid fibers or viscous adhesives. The dominant constituents of silks are spidroins (spider fibroins) and their terminal domains are vital for the tight control of silk self-assembly. To better understand spidroin termini, we used target capture and deep sequencing to identify spidroin gene sequences from six species representing the araneoid families of Araneidae, Nephilidae, and Theridiidae. We obtained 145 terminal regions, of which 103 are newly annotated here, as well as novel variants within nine diverse spidroin types. Our comparative analyses demonstrated the conservation of acidic, basic, and cysteine amino acid residues across spidroin types that had been proposed to be important for monomer stability, dimer formation, and self-assembly from a limited sampling of spidroins. Computational, protein homology modeling revealed areas of spidroin terminal regions that are highly conserved in three-dimensions despite sequence divergence across spidroin types. Analyses of our dense sampling of terminal regions suggest that most spidroins share stabilization mechanisms, dimer formation, and tertiary structure, despite producing functionally distinct materials.

Evolutionary shifts in gene expression decoupled from gene duplication across functionally distinct spider silk glands.

Spider silk synthesis is an emerging model for the evolution of tissue-specific gene expression and the role of gene duplication in functional novelty, but its potential has not been fully realized. Accordingly, we quantified transcript (mRNA) abundance in seven silk gland types and three non-silk gland tissues for three cobweb-weaving spider species. Evolutionary analyses based on expression levels of thousands of homologous transcripts and phylogenetic reconstruction of 605 gene families demonstrated conservation of expression for each gland type among species. Despite serial homology of all silk glands, the expression profiles of the glue-forming aggregate glands were divergent from fiber-forming glands. Also surprising was our finding that shifts in gene expression among silk gland types were not necessarily coupled with gene duplication, even though silk-specific genes belong to multi-paralog gene families. Our results challenge widely accepted models of tissue specialization and significantly advance efforts to replicate silk-based high-performance biomaterials.

Integrating the microbiome as a resource in the forensics toolkit.

The introduction of DNA fingerprinting to forensic science rapidly expanded the available evidence that could be garnered from a crime scene and used in court cases. Next generation sequencing technologies increased available genetic data that could be used as evidence by orders of magnitude, and as such, significant additional genetic information is now available for use in forensic science. This includes DNA from the bacteria that live in and on humans, known as the human microbiome. Next generation sequencing of the human microbiome demonstrates that its bacterial DNA can be used to uniquely identify an individual, provide information about their life and behavioral patterns, determine the body site where a sample came from, and estimate postmortem intervals. Bacterial samples from the environment and objects can also be leveraged to address similar questions about the individual(s) who interacted with them. However, the applications of this new field in forensic sciences raises concerns on current methods used in sample processing, including sample collection, storage, and the statistical power of published studies. These areas of human microbiome research need to be fully addressed before microbiome data can become a regularly incorporated evidence type and routine procedure of the forensic toolkit. Here, we summarize information on the current status of microbiome research as applies to the forensic field, the mathematical models used to make predictions, and the possible legal and practical difficulties that can limit the application of microbiomes in forensic science.

Silk gene expression of theridiid spiders: implications for male-specific silk use.

Spiders (order Araneae) rely on their silks for essential tasks, such as dispersal, prey capture, and reproduction. Spider silks are largely composed of spidroins, members of a protein family that are synthesized in silk glands. As needed, silk stored in silk glands is extruded through spigots on the spinnerets. Nearly all studies of spider silks have been conducted on females; thus, little is known about male silk biology. To shed light on silk use by males, we compared silk gene expression profiles of mature males to those of females from three cob-web weaving species (Theridiidae). We de novo assembled species-specific male transcriptomes from Latrodectus hesperus, Latrodectus geometricus, and Steatoda grossa followed by differential gene expression analyses. Consistent with their complement of silk spigots, male theridiid spiders express appreciable amounts of aciniform, major ampullate, minor ampullate, and pyriform spidroin genes but not tubuliform spidroin genes. The relative expression levels of particular spidroin genes varied between sexes and species. Because mature males desert their prey-capture webs and become cursorial in their search for mates, we anticipated that major ampullate (dragline) spidroin genes would be the silk genes most highly expressed by males. Indeed, major ampullate spidroin genes had the highest expression in S. grossa males. However, minor ampullate spidroin genes were the most highly expressed spidroin genes in L. geometricus and L. hesperus males. Our expression profiling results suggest species-specific adaptive divergence of silk use by male theridiids.

Duplication and concerted evolution of MiSp-encoding genes underlie the material properties of minor ampullate silks of cobweb weaving spiders.

BACKGROUND: Orb-web weaving spiders and their relatives use multiple types of task-specific silks. The majority of spider silk studies have focused on the ultra-tough dragline silk synthesized in major ampullate glands, but other silk types have impressive material properties. For instance, minor ampullate silks of orb-web weaving spiders are as tough as draglines, due to their higher extensibility despite lower strength. Differences in material properties between silk types result from differences in their component proteins, particularly members of the spidroin (spider fibroin) gene family. However, the extent to which variation in material properties within a single silk type can be explained by variation in spidroin sequences is unknown. Here, we compare the minor ampullate spidroins (MiSp) of orb-weavers and cobweb weavers. Orb-web weavers use minor ampullate silk to form the auxiliary spiral of the orb-web while cobweb weavers use it to wrap prey, suggesting that selection pressures on minor ampullate spidroins (MiSp) may differ between the two groups. RESULTS: We report complete or nearly complete MiSp sequences from five cobweb weaving spider species and measure material properties of minor ampullate silks in a subset of these species. We also compare MiSp sequences and silk properties of our cobweb weavers to published data for orb-web weavers. We demonstrate that all our cobweb weavers possess multiple MiSp loci and that one locus is more highly expressed in at least two species. We also find that the proportion of ß-spiral-forming amino acid motifs in MiSp positively correlates with minor ampullate silk extensibility across orb-web and cobweb weavers. CONCLUSIONS: MiSp sequences vary dramatically within and among spider species, and have likely been subject to multiple rounds of gene duplication and concerted evolution, which have contributed to the diverse material properties of minor ampullate silks. Our sequences also provide templates for recombinant silk proteins with tailored properties.