Gene fragmentation in bacterial draft genomes: extent, consequences and mitigation

<p/> <p>Background</p> <p>Ongoing technological advances in genome sequencing are allowing bacterial genomes to be sequenced at ever-lower cost. However, nearly all of these new techniques concomitantly decrease genome quality, primarily due to the inability of their relatively short read lengths to bridge certain genomic regions, e.g., those containing repeats. Fragmentation of predicted open reading frames (ORFs) is one possible consequence of this decreased quality. In this study we quantify ORF fragmentation in draft microbial genomes and its effect on annotation efficacy, and we propose a solution to ameliorate this problem.</p> <p>Results</p> <p>A survey of draft-quality genomes in GenBank revealed that fragmented ORFs comprised > 80% of the predicted ORFs in some genomes, and that increased fragmentation correlated with decreased genome assembly quality. In a more thorough analysis of 25 <it>Streptomyces </it>genomes, fragmentation was especially enriched in some protein classes with repeating, multi-modular structures such as polyketide synthases, non-ribosomal peptide synthetases and serine/threonine kinases. Overall, increased genome fragmentation correlated with increased false-negative Pfam and COG annotation rates and increased false-positive KEGG annotation rates. The false-positive KEGG annotation rate could be ameliorated by linking fragmented ORFs using their orthologs in related genomes. Whereas this strategy successfully linked up to 46% of the total ORF fragments in some genomes, its sensitivity appeared to depend heavily on the depth of sampling of a particular taxon's variable genome.</p> <p>Conclusions</p> <p>Draft microbial genomes contain many ORF fragments. Where these correspond to the same gene they have particular potential to confound comparative gene content analyses. Given our findings, and the rapid increase in the number of microbial draft quality genomes, we suggest that accounting for gene fragmentation and its associated biases is important when designing comparative genomic projects.</p

Parasites may help stabilize cooperative relationships

Background: The persistence of cooperative relationships is an evolutionary paradox; selection should favor those individuals that exploit their partners (cheating), resulting in the breakdown of cooperation over evolutionary time. Our current understanding of the evolutionary stability of mutualisms (cooperation between species) is strongly shaped by the view that they are often maintained by partners having mechanisms to avoid or retaliate against exploitation by cheaters. In contrast, we empirically and theoretically examine how additional symbionts, specifically specialized parasites, potentially influence the stability of bipartite mutualistic associations. In our empirical work we focus on the obligate mutualism between fungus-growing ants and the fungi they cultivate for food. This mutualism is exploited by specialized microfungal parasites (genus Escovopsis) that infect the ant's fungal gardens. Using sub-colonies of fungus-growing ants, we investigate the interactions between the fungus garden parasite and cooperative and experimentally-enforced uncooperative ("cheating") pairs of ants and fungi. To further examine if parasites have the potential to help stabilize some mutualisms we conduct Iterative Prisoner's Dilemma (IPD) simulations, a common framework for predicting the outcomes of cooperative/non-cooperative interactions, which incorporate parasitism as an additional factor. Results: In our empirical work employing sub-colonies of fungus-growing ants, we found that Escovopsis-infected sub-colonies composed of cheating populations of ants or fungi lost significantly more garden biomass than sub-colonies subjected to infection or cheating (ants or fungi) alone. Since the loss of fungus garden compromises the fitness of both mutualists, our findings suggest that the potential benefit received by the ants or fungi for cheating is outweighed by the increased concomitant cost of parasitism engendered by non-cooperation (cheating). IPD simulations support our empirical results by confirming that a purely cooperative strategy, which is unsuccessful in the classic IPD model, becomes stable when parasites are included. Conclusion: Here we suggest, and provide evidence for, parasitism being an external force that has the potential to help stabilize cooperation by aligning the selfish interests of cooperative partners in opposition to a common enemy. Specifically, our empirical results and IPD simulations suggest that when two mutualists share a common enemy selection can favor cooperation over cheating, which may help explain the evolutionary stability of some mutualisms

Symbiont Interactions in a Tripartite Mutualism: Exploring the Presence and Impact of Antagonism between Two Fungus-Growing Ant Mutualists

Mutualistic associations are shaped by the interplay of cooperation and conflict among the partners involved, and it is becoming increasingly clear that within many mutualisms multiple partners simultaneously engage in beneficial interactions. Consequently, a more complete understanding of the dynamics within multipartite mutualism communities is essential for understanding the origin, specificity, and stability of mutualisms. Fungus-growing ants cultivate fungi for food and maintain antibiotic-producing Pseudonocardia actinobacteria on their cuticle that help defend the cultivar fungus from specialized parasites. Within both ant-fungus and ant-bacterium mutualisms, mixing of genetically distinct strains can lead to antagonistic interactions (i.e., competitive conflict), which may prevent the ants from rearing multiple strains of either of the mutualistic symbionts within individual colonies. The success of different ant-cultivar-bacterium combinations could ultimately be governed by antagonistic interactions between the two mutualists, either as inhibition of the cultivar by Pseudonocardia or vice versa. Here we explore cultivar-Pseudonocardia antagonism by evaluating in vitro interactions between strains of the two mutualists, and find frequent antagonistic interactions both from cultivars towards Pseudonocardia and vice versa. To test whether such in vitro antagonistic interactions affect ant colonies in vivo, we performed sub-colony experiments using species of Acromyrmex leaf-cutting ants. We created novel ant-fungus-bacterium pairings in which there was antagonism from one, both, or neither of the ants' microbial mutualists, and evaluated the effect of directional antagonism on cultivar biomass and Pseudonocardia abundance on the cuticle of workers within sub-colonies. Despite the presence of frequent in vitro growth suppression between cultivars and Pseudonocardia, antagonism from Pseudonocardia towards the cultivar did not reduce sub-colony fungus garden biomass, nor did cultivar antagonism towards Pseudonocardia reduce bacteria abundance on the cuticle of sub-colony workers. Our findings suggest that inter-mutualist antagonism does not limit what combinations of cultivar and Pseudonocardia strains Acromyrmex fungus-growing ants can maintain within nests

A community of ants, fungi, and bacteria: A multilateral approach to studying symbiosis

The ancient and highly evolved mutualism between fungus-growing ants and their fungi is a textbook example of symbiosis. The ants carefully tend the fungus, which serves as their main food source, and traditionally are believed to be so successful at fungal cultivation that they are able to maintain the fungus free of microbial pathogens. This assumption is surprising in light of theories on the evolution of parasitism, especially for those species of ants that have been clonally propagating their cultivars for millions of years. Recent work has established that, as theoretically predicted, the gardens of fungus-growing ants are host to a specialized, virulent, and highly evolved fungal pathogen in the genus Escovopsis. In addition, the ants have evolved a mutualistic association with filamentous bacteria (actinomycetes) that produce antibiotics that suppress the growth of Escovopsis. Thus, the attine symbiosis appears to be a coevolutionary "arms race" between the garden parasite Escovopsis on the one hand and the ant-fungus-actinomycete tripartite mutualism on the other. These recent findings indicate that microbes may be key components in the regulation of other symbiotic associations between higher organisms

Complex host-pathogen coevolution in the Apterostigma fungus-growing ant-microbe symbiosis

BACKGROUND: The fungus-growing ant-microbe symbiosis consists of coevolving microbial mutualists and pathogens. The diverse fungal lineages that these ants cultivate are attacked by parasitic microfungi of the genus Escovopsis. Previous molecular analyses have demonstrated strong phylogenetic congruence between the ants, the ants-cultivated fungi and the garden pathogen Escovopsis at ancient phylogenetic levels, suggesting coevolution of these symbionts. However, few studies have explored cophylogenetic patterns between these symbionts at the recent phylogenetic levels necessary to address whether these parasites are occasionally switching to novel hosts or whether they are diversifying with their hosts as a consequence of long-term host fidelity. RESULTS: Here, a more extensive phylogenetic analysis of Escovopsis lineages infecting the gardens of Apterostigma ants demonstrates that these pathogens display patterns of phylogenetic congruence with their fungal hosts. Particular clades of Escovopsis track particular clades of cultivated fungi, and closely-related Escovopsis generally infect closely-related hosts. Discordance between host and parasite phylogenies, however, provides the first evidence for occasional host-switches or acquisitions of novel infections from the environment. CONCLUSION: The fungus-growing ant-microbe association has a complex coevolutionary history. Though there is clear evidence of host-specificity on the part of diverse Escovopsis lineages, these pathogens have switched occasionally to novel host fungi. Such switching is likely to have profound effects on how these host and parasites adapt to one another over evolutionary time scales and may impact how disease spreads over ecological time scales

Design and Development of a Myoelectric Transradial Prosthesis

The loss of a limb is a life-changing event and reality for 441,000 transradial amputees in the United States. Limb loss can have substantial physical, social, psychological, and economic consequences. A prototype prosthesis was created that has sophisticated hand functionality, an adjustable and comfortable socket, and a lightweight yet durable design utilizing 3D printing, all available at a reasonable price point. The prosthesis integrated force sensors, servo motors, and a myoelectric means of control so the user may perform activities of daily living. The overall outcome was a prosthesis that met its design requirements, offering increased usability, functionality, and availability

Chemical Analyses of Wasp-Associated Streptomyces Bacteria Reveal a Prolific Potential for Natural Products Discovery

Identifying new sources for small molecule discovery is necessary to help mitigate the continuous emergence of antibiotic-resistance in pathogenic microbes. Recent studies indicate that one potentially rich source of novel natural products is Actinobacterial symbionts associated with social and solitary Hymenoptera. Here we test this possibility by examining two species of solitary mud dauber wasps, Sceliphron caementarium and Chalybion californicum. We performed enrichment isolations from 33 wasps and obtained more than 200 isolates of Streptomyces Actinobacteria. Chemical analyses of 15 of these isolates identified 11 distinct and structurally diverse secondary metabolites, including a novel polyunsaturated and polyoxygenated macrocyclic lactam, which we name sceliphrolactam. By pairing the 15 Streptomyces strains against a collection of fungi and bacteria, we document their antifungal and antibacterial activity. The prevalence and anti-microbial properties of Actinobacteria associated with these two solitary wasp species suggest the potential role of these Streptomyces as antibiotic-producing symbionts, potentially helping defend their wasp hosts from pathogenic microbes. Finding phylogenetically diverse and chemically prolific Actinobacteria from solitary wasps suggests that insect-associated Actinobacteria can provide a valuable source of novel natural products of pharmaceutical interest

The role of symbiont genetic distance and potential adaptability in host preference towards Pseudonocardia symbionts in <i>Acromyrmex</i> leaf-cutting ants

Fungus-growing ants display symbiont preference in behavioral assays, both towards the fungus they cultivate for food and Actinobacteria they maintain on their cuticle for antibiotic production against parasites. These Actinobacteria, genus Pseudonocardia Henssen (Pseudonocardiacea: Actinomycetales), help defend the ants' fungal mutualist from specialized parasites. In Acromyrmex Mayr (Hymenoptera: Formicidae) leaf-cutting ants, individual colonies maintain either a single or a few strains of Pseudonocardia, and the symbiont is primarily vertically transmitted between generations by colony-founding queens. A recent report found that Acromyrmex workers are able to differentiate between their native Pseudonocardia strain and non-native strains isolated from sympatric or allopatric Acromyrmex species, and show preference for their native strain. Here we explore worker preference when presented with two non-native strains, elucidating the role of genetic distance on preference between strains and Pseudonocardia origin. Our findings suggest that ants tend to prefer bacteria more closely related to their native bacterium and that genetic similarity is probably more important than whether symbionts are ant-associated or free-living. Preliminary findings suggest that when continued exposure to a novel Pseudonocardia strain occurs, ant symbiont preference is potentially adaptable, with colonies apparently being able to alter symbiont preference over time. These findings are discussed in relation to the role of adaptive recognition, potential ecological flexibility in symbiont preference, and more broadly, in relation to self versus non-self recognition

Preliminary in vitro insights into the use of natural fungal pathogens of leaf-cutting ants as biocontrol agents

Leaf-cutting ants are one of the main herbivores of the Neotropics, where they represent an important agricultural pest. These ants are particularly difficult to control because of the complex network of microbial symbionts. Leaf-cutting ants have traditionally been controlled through pesticide application, but there is a need for alternative, more environmentally friendly, control methods such as biological control. Potential promising biocontrol candidates include the microfungi Escovopsis spp. (anamorphic Hypocreales), which are specialized pathogens of the fungi the ants cultivate for food. These pathogens are suppressed through ant behaviors and ant-associated antibiotic-producing Actinobacteria. In order to be an effective biocontrol agent, Escovopsis has to overcome these defenses. Here, we evaluate, using microbial in vitro assays, whether defenses in the ant-cultivated fungus strain (Leucoagaricus sp.) and Actinobacteria from the ant pest Acromyrmex lundii have the potential to limit the use of Escovopsis in biocontrol. We also explore, for the first time, possible synergistic biocontrol between Escovopsis and the entomopathogenic fungus Lecanicillium lecanii. All strains of Escovopsis proved to overgrow A. lundii cultivar in less than 7 days, with the Escovopsis strain isolated from a different leaf-cutting ant species being the most efficient. Escovopsis challenged with a Streptomyces strain isolated from A. lundii did not exhibit significant growth inhibition. Both results are encouraging for the use of Escovopsis as a biocontrol agent. Although we found that L. lecanii can suppress the growth of the cultivar, it also had a negative impact on Escovopsis, making the success of simultaneous use of these two fungi for biocontrol of A. lundii questionable.Fil: Folgarait, Patricia Julia. Universidad Nacional de Quilmes; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; ArgentinaFil: Gorosito, Norma Beatriz. Universidad Nacional de Quilmes; ArgentinaFil: Poulsen, Michael. University of Wisconsin; Estados UnidosFil: Currie, Cameron R.. University of Wisconsin; Estados Unido

Antibiotic and Antimalarial Quinones from Fungus-Growing Ant-Associated Pseudonocardia sp.

Three new members of the angucycline class of antibiotics, pseudonocardones A–C (1–3), along with the known antibiotics 6-deoxy-8-O-methylrabelomycin (4) and X-14881 E (5) have been isolated from the culture of a Pseudonocardia strain associated with the fungus-growing ant Apterostigma dentigerum. Compounds 4 and 5 showed antibiotic activity against Bacillus subtilis 3610 and liver-stage Plasmodium berghei, while 1–3 were inactive or only weakly active in a variety of biological assays. Compound 5 also showed moderate cytotoxicity against HepG2 cells