Domestication and evolutionary histories of specialized gut symbionts across cephalotine ants.

The evolution of animals and their gut symbionts is a complex phenomenon, obscured by lability and diversity. In social organisms, transmission of symbionts among relatives may yield systems with more stable associations. Here, we study the history of a social insect symbiosis involving cephalotine ants and their extracellular gut bacteria, which come predominantly from host-specialized lineages. We perform multi-locus phylogenetics for symbionts from nine bacterial orders, and map prior amplicon sequence data to lineage-assigned symbiont genomes, studying distributions of rigorously defined symbionts across 20 host species. Based on monophyly and additional hypothesis testing, we estimate that these specialized gut bacteria belong to 18 distinct lineages, of which 15 have been successfully isolated and cultured. Several symbiont lineages showed evidence for domestication events that occurred later in cephalotine evolutionary history, and only one lineage was ubiquitously detected in all 20 host species and 48 colonies sampled with amplicon 16S rRNA sequencing. We found evidence for phylogenetically constrained distributions in four symbionts, suggesting historical or genetic impacts on community composition. Two lineages showed evidence for frequent intra-lineage co-infections, highlighting the potential for niche divergence after initial domestication. Nearly all symbionts showed evidence for occasional host switching, but four may, more often, co-diversify with their hosts. Through our further assessment of symbiont localization and genomic functional profiles, we demonstrate distinct niches for symbionts with shared evolutionary histories, prompting further questions on the forces underlying the evolution of hosts and their gut microbiomes.

Complete genome sequences of nine Rhodococcus equi phages.

We report genomes of nine phages isolated from Actinobacteria Rhodococcus equi NRRL B-16538. Six of these phages belong to actinobacteriophage cluster CR, which otherwise contains Gordonia phages; two form the CF cluster; and one is a singleton. Genome lengths are 62,017-80,980 bp with 63.9%-67.3% GC content.

Physiological and evolutionary contexts of a new symbiotic species from the nitrogen-recycling gut community of turtle ants.

While genome sequencing has expanded our knowledge of symbiosis, role assignment within multi-species microbiomes remains challenging due to genomic redundancy and the uncertainties of in vivo impacts. We address such questions, here, for a specialized nitrogen (N) recycling microbiome of turtle ants, describing a new genus and species of gut symbiont-Ischyrobacter davidsoniae (Betaproteobacteria: Burkholderiales: Alcaligenaceae)-and its in vivo physiological context. A re-analysis of amplicon sequencing data, with precisely assigned Ischyrobacter reads, revealed a seemingly ubiquitous distribution across the turtle ant genus Cephalotes, suggesting ≥50 million years since domestication. Through new genome sequencing, we also show that divergent I. davidsoniae lineages are conserved in their uricolytic and urea-generating capacities. With phylogenetically refined definitions of Ischyrobacter and separately domesticated Burkholderiales symbionts, our FISH microscopy revealed a distinct niche for I. davidsoniae, with dense populations at the anterior ileum. Being positioned at the site of host N-waste delivery, in vivo metatranscriptomics and metabolomics further implicate I. davidsoniae within a symbiont-autonomous N-recycling pathway. While encoding much of this pathway, I. davidsoniae expressed only a subset of the requisite steps in mature adult workers, including the penultimate step deriving urea from allantoate. The remaining steps were expressed by other specialized gut symbionts. Collectively, this assemblage converts inosine, made from midgut symbionts, into urea and ammonia in the hindgut. With urea supporting host amino acid budgets and cuticle synthesis, and with the ancient nature of other active N-recyclers discovered here, I. davidsoniae emerges as a central player in a conserved and impactful, multipartite symbiosis.

Turtle ants harbor metabolically versatile microbiomes with conserved functions across development and phylogeny.

Gut bacterial symbionts can support animal nutrition by facilitating digestion and providing valuable metabolites. However, changes in symbiotic roles between immature and adult stages are not well documented, especially in ants. Here, we explored the metabolic capabilities of microbiomes sampled from herbivorous turtle ant (Cephalotes sp.) larvae and adult workers through (meta)genomic screening and in vitro metabolic assays. We reveal that larval guts harbor bacterial symbionts with impressive metabolic capabilities, including catabolism of plant and fungal recalcitrant dietary fibers and energy-generating fermentation. Additionally, several members of the specialized adult gut microbiome, sampled downstream of an anatomical barrier that dams large food particles, show a conserved potential to depolymerize many dietary fibers. Symbionts from both life stages have the genomic capacity to recycle nitrogen and synthesize amino acids and B-vitamins. With help of their gut symbionts, including several bacteria likely acquired from the environment, turtle ant larvae may aid colony digestion and contribute to colony-wide nitrogen, B-vitamin and energy budgets. In addition, the conserved nature of the digestive capacities among adult-associated symbionts suggests that nutritional ecology of turtle ant colonies has long been shaped by specialized, behaviorally-transferred gut bacteria with over 45 million years of residency.

Gut bacteria are essential for normal cuticle development in herbivorous turtle ants.

Across the evolutionary history of insects, the shift from nitrogen-rich carnivore/omnivore diets to nitrogen-poor herbivorous diets was made possible through symbiosis with microbes. The herbivorous turtle ants Cephalotes possess a conserved gut microbiome which enriches the nutrient composition by recycling nitrogen-rich metabolic waste to increase the production of amino acids. This enrichment is assumed to benefit the host, but we do not know to what extent. To gain insights into nitrogen assimilation in the ant cuticle we use gut bacterial manipulation, 15N isotopic enrichment, isotope-ratio mass spectrometry, and 15N nuclear magnetic resonance spectroscopy to demonstrate that gut bacteria contribute to the formation of proteins, catecholamine cross-linkers, and chitin in the cuticle. This study identifies the cuticular components which are nitrogen-enriched by gut bacteria, highlighting the role of symbionts in insect evolution, and provides a framework for understanding the nitrogen flow from nutrients through bacteria into the insect cuticle.

Parallel Evolution of Host-Attachment Proteins in Phage PP01 Populations Adapting to Escherichia coli O157:H7.

The emergence of antibiotic resistance has sparked interest in phage therapy, which uses virulent phages as antibacterial agents. Bacteriophage PP01 has been studied for potential bio-control of Escherichia coli O157:H7, its natural host, but in the laboratory, PP01 can be inefficient at killing this bacterium. Thus, the goal of this study was to improve the therapeutic potential of PP01 through short-term experimental evolution. Four replicate populations of PP01 were serially passaged 21 times on non-evolving E. coli O157:H7 with the prediction that the evolved phage populations would adsorb faster and more efficiently kill the host bacteria. Dead-cell adsorption assays and in vitro killing assays confirmed that evolved viruses improved their adsorption ability on E. coli O157:H7, and adapted to kill host bacteria faster than the wildtype ancestor. Sequencing of candidate tail-fiber genes revealed that the phage populations evolved in parallel; the lineages shared two point mutations in gp38 that encodes a host recognition protein, and surprisingly shared a ~600 bp deletion in gp37 that encodes the distal tail fibers. In contrast, no mutations were observed in the gp12 gene encoding PP01’s short tail fibers. We discuss the functional role of the observed mutations, including the possible adaptive role of the evolved deletions. This study demonstrates how experimental evolution can be used to select for viral traits that improve phage attack of an important bacterial pathogen, and that the molecular targets of selection include loci contributing to cell attachment and phage virulence.

Author Correction: Herbivorous turtle ants obtain essential nutrients from a conserved nitrogen-recycling gut microbiome.

The originally published version of the Supplementary Information file associated with this Article contained an error in Supplementary Figure 3. Panel b was inadvertently replaced with a duplicate of panel a. The error has now been fixed and the corrected version of the Supplementary Information PDF is available to download from the HTML version of the Article.

Herbivorous turtle ants obtain essential nutrients from a conserved nitrogen-recycling gut microbiome.

Nitrogen acquisition is a major challenge for herbivorous animals, and the repeated origins of herbivory across the ants have raised expectations that nutritional symbionts have shaped their diversification. Direct evidence for N provisioning by internally housed symbionts is rare in animals; among the ants, it has been documented for just one lineage. In this study we dissect functional contributions by bacteria from a conserved, multi-partite gut symbiosis in herbivorous Cephalotes ants through in vivo experiments, metagenomics, and in vitro assays. Gut bacteria recycle urea, and likely uric acid, using recycled N to synthesize essential amino acids that are acquired by hosts in substantial quantities. Specialized core symbionts of 17 studied Cephalotes species encode the pathways directing these activities, and several recycle N in vitro. These findings point to a highly efficient N economy, and a nutritional mutualism preserved for millions of years through the derived behaviors and gut anatomy of Cephalotes ants.

The U.S. Culture Collection Network Responding to the Requirements of the Nagoya Protocol on Access and Benefit Sharing.

The U.S. Culture Collection Network held a meeting to share information about how culture collections are responding to the requirements of the recently enacted Nagoya Protocol on Access to Genetic Resources and the Fair and Equitable Sharing of Benefits Arising from their Utilization to the Convention on Biological Diversity (CBD). The meeting included representatives of many culture collections and other biological collections, the U.S. Department of State, U.S. Department of Agriculture, Secretariat of the CBD, interested scientific societies, and collection groups, including Scientific Collections International and the Global Genome Biodiversity Network. The participants learned about the policies of the United States and other countries regarding access to genetic resources, the definition of genetic resources, and the status of historical materials and genetic sequence information. Key topics included what constitutes access and how the CBD Access and Benefit-Sharing Clearing-House can help guide researchers through the process of obtaining Prior Informed Consent on Mutually Agreed Terms. U.S. scientists and their international collaborators are required to follow the regulations of other countries when working with microbes originally isolated outside the United States, and the local regulations required by the Nagoya Protocol vary by the country of origin of the genetic resource. Managers of diverse living collections in the United States described their holdings and their efforts to provide access to genetic resources. This meeting laid the foundation for cooperation in establishing a set of standard operating procedures for U.S. and international culture collections in response to the Nagoya Protocol.

Complete Genome Sequences of 38 Gordonia sp. Bacteriophages.

We report here the genome sequences of 38 newly isolated bacteriophages using Gordonia terrae 3612 (ATCC 25594) and Gordonia neofelifaecis NRRL59395 as bacterial hosts. All of the phages are double-stranded DNA (dsDNA) tail phages with siphoviral morphologies, with genome sizes ranging from 17,118 bp to 93,843 bp and spanning considerable nucleotide sequence diversity.

Saccharedens versatilis gen. nov., sp. nov., a sugar-degrading member of the Burkholderiales isolated from Cephalotes rohweri ant guts.

Cephalotes 'turtle' ants host a core group of gut-associated symbionts, but their potential contributions to ant nutrition and disease resistance remain uncharacterized in vitro. To gain a better understanding of the metabolic capability of core symbionts belonging to the Burkholderiales, we cultivated and characterized strain CAG32T from the guts of Cephalotes rohweri ants. Strain CAG32T was rod-shaped, Gram-stain-negative, motile and formed pale-white colonies on trypticase soy agar. Optimum growth occurred under an atmosphere of 20 % O2 supplemented with 1 % CO2. Strain CAG32T grew under NaCl concentrations of 0-2.0 %, temperatures of 23-47 °C and pH values of 4.0-8.0, and was capable of producing n-butyric acid and degrading carbohydrates for growth. The G+C content of the genomic DNA was 59.2±0.6 mol% and the major fatty acids were C16 : 0, C16 : 1ω7c/C16 : 1ω6c, C17 : 0 cylcopropane, C12 : 0 and C14 : 0 3-OH/C16 : 1 iso I. The only respiratory quinone detected was ubiquinone-8 (Q-8) and the major polar lipids were phosphatidylethanolamine, phosphatidylglycerol and diphosphatidylglycerol. Based on phylogenetic analysis of the 16S rRNA gene sequence, strain CAG32T shared 96.9 % nucleotide similarity with its closest cultivated neighbours Bordetella petrii Se-1111RT and Bordetella bronchiseptica ATCC 19395T. This, combined with differences in the phenotypic and biochemical profile from neighbouring strains, warrants the classification of strain CAG32T as representing a novel species of a new genus within the Burkholderiales family Alcaligenaceae. The name Saccharedens versatilis gen. nov., sp. nov. is proposed. The type strain of Saccharedens versatilis is CAG32T (=NCIMB 15010T=DSM 100909T).

Cephaloticoccus gen. nov., a new genus of 'Verrucomicrobia' containing two novel species isolated from Cephalotes ant guts.

Two novel members of the bacterial phylum 'Verrucomicrobia', strains CAG34T and CV41T, were isolated from the guts of Cephalotes rohweri and Cephalotes varians ants, respectively. Strains CAG34T and CV41T were coccoid, Gram-stain-negative, non-motile, and formed cream-coloured colonies on trypticase soy agar. Optimum growth occurred under an atmosphere of 12-20 % O2 and 1 % CO2 for both strains, although strain CV41T could not grow without supplemental CO2. Growth was possible under NaCl concentrations of 0.5-1.5 % (w/v) and temperatures of 23-37 °C for both strains, and pH values of 6.9-7.7 for strain CAG34T and 6.9-7.3 for strain CV41T. The G+C content of the genomic DNA was 60.7 mol% for strain CAG34T and 60.5 mol% for strain CV41T. The major fatty acids for both strains were anteiso-C15 : 0, iso-C14 : 0, C16 : 0, and C16 : 1ω5c. Based on the phylogenetic analysis of 16S rRNA gene sequences, the closest cultivated relative for both strains was the type strain of Opitutus terrae (91.8 % similarity). Hence, strains CAG34T and CV41T are considered to represent a new genus within the 'Verrucomicrobia' family Opitutaceae, for which we propose the name Cephaloticoccus gen. nov. Given that strains CAG34T and CV41T share 97.7 % 16S rRNA gene sequence similarity with each other and are physiologically distinct, we propose to classify the isolates as representing two novel species, Cephaloticoccus primus sp. nov. for strain CAG34T (=NCIMB 15004T =ATCC TSD-38T) and Cephaloticoccus capnophilus sp. nov. for strain CV41T (=NCIMB 15005T =ATCC TSD-39T =DSM 100879T).

Phage selection restores antibiotic sensitivity in MDR Pseudomonas aeruginosa.

Increasing prevalence and severity of multi-drug-resistant (MDR) bacterial infections has necessitated novel antibacterial strategies. Ideally, new approaches would target bacterial pathogens while exerting selection for reduced pathogenesis when these bacteria inevitably evolve resistance to therapeutic intervention. As an example of such a management strategy, we isolated a lytic bacteriophage, OMKO1, (family Myoviridae) of Pseudomonas aeruginosa that utilizes the outer membrane porin M (OprM) of the multidrug efflux systems MexAB and MexXY as a receptor-binding site. Results show that phage selection produces an evolutionary trade-off in MDR P. aeruginosa, whereby the evolution of bacterial resistance to phage attack changes the efflux pump mechanism, causing increased sensitivity to drugs from several antibiotic classes. Although modern phage therapy is still in its infancy, we conclude that phages, such as OMKO1, represent a new approach to phage therapy where bacteriophages exert selection for MDR bacteria to become increasingly sensitive to traditional antibiotics. This approach, using phages as targeted antibacterials, could extend the lifetime of our current antibiotics and potentially reduce the incidence of antibiotic resistant infections.

Complete Draft Genome Sequence of Escherichia coli JF733.

ITALIC! Escherichia coliJF733 is a strain with a long history in research on membrane proteins and processes. However, tracing back the strain development raises some questions concerning the correct genotype of JF733. Here, we present the complete draft genome of ITALIC! E. coliJF733 in order to resolve any remaining uncertainties.

Ventosimonas gracilis gen. nov., sp. nov., a member of the Gammaproteobacteria isolated from Cephalotes varians ant guts representing a new family, Ventosimonadaceae fam. nov., within the order 'Pseudomonadales'.

Cephalotes 'turtle' ants are known to harbor a core group of gut symbionts, including members belonging to the Gammaproteobacteria. Here, we describe the cultivation and characterization of strain CV58T, a novel member of the Gammaproteobacteria order Pseudomonadales isolated from the guts of the ant Cephalotes varians. Strain CV58T was rod-shaped, Gram-stain-negative, non-motile and formed pale-yellow colonies on trypticase soy agar. Optimum growth occurred under an atmosphere of 4-20 % (v/v) O2. Growth was possible for strain CV58Tat NaCl concentrations of 0-1.5 % (w/v), temperatures of 23-40 °C, and pH values of 5.5-8.5. The G+C content of the genomic DNA was 54.9 mol% and the major fatty acids were C18 : 1ω7c, C16 : 0, C16 : 1ω7c/C16 : 1ω6c, C12 : 0 and C12 : 03OH. The only respiratory quinone detected was ubiquinone-9 (Q-9) and the major polar lipids were phosphatidylethanolamine, phosphatidylglycerol and diphosphatidylglycerol. Based on phylogenetic analysis of the 16S rRNA gene sequence, strain CV58T shared an 88.3 % nucleotide identity with its closest cultivated neighbor, Pseudomonas putida R43. We believe that this, combined with the housekeeping gene phylogeny, differences in phenotypic characteristics and cellular fatty acid compositions of other cultivated members indicates that strain CV58T represents a novel species occupying a novel genus and family within the order Pseudomonadales. Thus, we propose the name Ventosimonadaceae fam nov., followed by Ventosimonas gracilis gen. nov., sp. nov., to classify strain CV58T (=NCIMB 15011T =DSM 100910T).

The U.S. Culture Collection Network Lays the Foundation for Progress in Preservation of Valuable Microbial Resources.

The U.S. Culture Collection Network was formed in 2012 by a group of culture collection scientists and stakeholders in order to continue the progress established previously through efforts of an ad hoc group. The network is supported by a Research Coordination Network grant from the U.S. National Science Foundation (NSF) and has the goals of promoting interaction among collections, encouraging the adoption of best practices, and protecting endangered or orphaned collections. After prior meetings to discuss best practices, shared data, and synergy with genome programs, the network held a meeting at the U.S. Department of Agriculture (USDA)-Agricultural Research Service (ARS) National Center for Genetic Resources Preservation (NCGRP) in Fort Collins, Colorado in October 2015 specifically to discuss collections that are vulnerable because of changes in funding programs, or are at risk of loss because of retirement or lack of funding. The meeting allowed collection curators who had already backed up their resources at the USDA NCGRP to visit the site, and brought collection owners, managers, and stakeholders together. Eight formal collections have established off-site backups with the USDA-ARS, ensuring that key material will be preserved for future research. All of the collections with backup at the NCGRP are public distributing collections including U.S. NSF-supported genetic stock centers, USDA-ARS collections, and university-supported collections. Facing the retirement of several pioneering researchers, the community discussed the value of preserving personal research collections and agreed that a mechanism to preserve these valuable collections was essential to any future national culture collection system. Additional input from curators of plant and animal collections emphasized that collections of every kind face similar challenges in developing long-range plans for sustainability.

The United States Culture Collection Network (USCCN): Enhancing Microbial Genomics Research through Living Microbe Culture Collections.

The mission of the United States Culture Collection Network (USCCN; http://usccn.org) is "to facilitate the safe and responsible utilization of microbial resources for research, education, industry, medicine, and agriculture for the betterment of human kind." Microbial culture collections are a key component of life science research, biotechnology, and emerging global biobased economies. Representatives and users of several microbial culture collections from the United States and Europe gathered at the University of California, Davis, to discuss how collections of microorganisms can better serve users and stakeholders and to showcase existing resources available in public culture collections.