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).

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).

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).

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.

Genomic and physiological characterization of the Verrucomicrobia isolate Geminisphaera colitermitum gen. nov., sp. nov., reveals microaerophily and nitrogen fixation genes.

Previously we reported the cultivation of novel verrucomicrobia, including strain TAV2 (93% 16S rRNA gene identity to its nearest cultivated representative, Opitutus terreae PB90-1) from the gut of the termite Reticulitermes flavipes. To gain better insight into the Verrucomicrobia as a whole and understand the role of verrucomicrobia within the termite gut ecosystem, we analyzed a draft genome and undertook a physiological characterization of TAV2. Strain TAV2 is an autochthonous member of the R. flavipes gut microbiota and groups phylogenetically among diverse Verrucomicrobia from R. flavipes and other termites that are represented by 16S rRNA gene sequences alone. TAV2 is a microaerophile, possessing a high-affinity cbb(3)-type terminal oxidase-encoding gene and exhibiting an optimum growth rate between 2 and 8% (vol/vol) oxygen. It has the genetic potential to degrade cellulose, an important function within termite guts, but its in vitro substrate utilization spectrum was limited to starch and a few mono- and disaccharides. Growth occurred on nitrogen-free medium, and genomic screening revealed genes for dinitrogenases, heretofore detected in only a few members of the Verrucomicrobia. This represents the first (i) characterization of a verrucomicrobial species from the termite gut, (ii) report of nif and anf genes in a nonacidophilic verrucomicrobial species, and (iii) description of a microaerophilic genotype and phenotype in this phylum of bacteria. The genetic and physiological distinctiveness of TAV2 supports its recognition as the type strain of a new genus and species, for which the name Geminisphaera colitermitum gen. nov., sp. nov., is proposed.

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.

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.

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.

Development of an ecophysiological model for Diplosphaera colotermitum TAV2, a termite hindgut Verrucomicrobium.

Termite hindguts are populated by a dense and diverse community of microbial symbionts working in concert to transform lignocellulosic plant material and derived residues into acetate, to recycle and fix nitrogen, and to remove oxygen. Although much has been learned about the breadth of microbial diversity in the hindgut, the ecophysiological roles of its members is less understood. In this study, we present new information about the ecophysiology of microorganism Diplosphaera colotermitum strain TAV2, an autochthonous member of the Reticulitermes flavipes gut community. An integrated high-throughput approach was used to determine the transcriptomic and proteomic profiles of cells grown under hypoxia (2% O2) or atmospheric (20% O2) concentrations of oxygen. Our results revealed that genes and proteins associated with energy production and utilization, carbohydrate transport and metabolism, nitrogen fixation, and replication and recombination were upregulated under 2% O2. The metabolic map developed for TAV2 indicates that this microorganism may be involved in biological nitrogen fixation, amino-acid production, hemicellulose degradation and consumption of O2 in the termite hindgut. Variation of O2 concentration explained 55.9% of the variance in proteomic profiles, suggesting an adaptive evolution of TAV2 to the hypoxic periphery of the hindgut. Our findings advance the current understanding of microaerophilic microorganisms in the termite gut and expand our understanding of the ecological roles for members of the phylum Verrucomicrobia.

Identifying proteins that can form tyrosine-cysteine crosslinks.

Protein cofactors represent a unique class of redox active posttranslational protein modifications formed in or by metalloproteins. Once formed, protein cofactors provide a one-electron oxidant, which is tethered to the protein backbone. Twenty-five proteins are known to contain protein cofactors, but this number is likely limited by the use of crystallography as the identification technique. In order to address this limitation, a search of all reported protein structures for chemical environments conducive to forming a protein cofactor through tyrosine and cysteine side chain crosslinking yielded three hundred candidate proteins. Using hydrogen bonding and metal center proximity, the three hundred proteins were narrowed to four highly viable candidates. An orphan metalloprotein (BF4112) was examined to validate this methodology, which identifies proteins capable of crosslinking tyrosine and cysteine sidechains. A tyrosine-cysteine crosslink was formed in BF4112 using copper-dioxygen chemistry, as in galactose oxidase. Liquid chromatography-MALDI mass spectrometry and optical spectroscopy confirmed tyrosine-cysteine crosslink formation in BF4112. This finding demonstrates the efficacy of these predictive methods and the minimal constraints, provided by the BF4112 protein structure, in tyrosine-cysteine crosslink formation. This search method, when coupled with physiological evidence for crosslink formation and function as a cofactor, could identify additional protein-derived cofactors.

Cluster K mycobacteriophages: insights into the evolutionary origins of mycobacteriophage TM4.

Five newly isolated mycobacteriophages--Angelica, CrimD, Adephagia, Anaya, and Pixie--have similar genomic architectures to mycobacteriophage TM4, a previously characterized phage that is widely used in mycobacterial genetics. The nucleotide sequence similarities warrant grouping these into Cluster K, with subdivision into three subclusters: K1, K2, and K3. Although the overall genome architectures of these phages are similar, TM4 appears to have lost at least two segments of its genome, a central region containing the integration apparatus, and a segment at the right end. This suggests that TM4 is a recent derivative of a temperate parent, resolving a long-standing conundrum about its biology, in that it was reportedly recovered from a lysogenic strain of Mycobacterium avium, but it is not capable of forming lysogens in any mycobacterial host. Like TM4, all of the Cluster K phages infect both fast- and slow-growing mycobacteria, and all of them--with the exception of TM4--form stable lysogens in both Mycobacterium smegmatis and Mycobacterium tuberculosis; immunity assays show that all five of these phages share the same immune specificity. TM4 infects these lysogens suggesting that it was either derived from a heteroimmune temperate parent or that it has acquired a virulent phenotype. We have also characterized a widely-used conditionally replicating derivative of TM4 and identified mutations conferring the temperature-sensitive phenotype. All of the Cluster K phages contain a series of well conserved 13 bp repeats associated with the translation initiation sites of a subset of the genes; approximately one half of these contain an additional sequence feature composed of imperfectly conserved 17 bp inverted repeats separated by a variable spacer. The K1 phages integrate into the host tmRNA and the Cluster K phages represent potential new tools for the genetics of M. tuberculosis and related species.

Stenoxybacter acetivorans gen. nov., sp. nov., an acetate-oxidizing obligate microaerophile among diverse O2-consuming bacteria from termite guts.

In termite hindguts, fermentative production of acetate--a major carbon and energy source for the insect--depends on efficient removal of inwardly diffusing oxygen by microbes residing on and near the hindgut wall. However, little is known about the identity of these organisms or about the substrate(s) used to support their respiratory activity. A cultivation-based approach was used to isolate O(2)-consuming organisms from hindguts of Reticulitermes flavipes. A consistently greater (albeit not statistically significant) number of colonies developed under hypoxia (2% [vol/vol] O(2)) than under air, and the increase coincided with the appearance of morphologically distinct colonies of a novel, rod-shaped, obligately microaerophilic beta-proteobacterium that was <95% similar (based on the 16S rRNA gene sequence) to its closest known relative (Eikenella corrodens). Nearly identical organisms (and/or their 16S rRNA genes) were obtained from geographically separated and genetically distinct populations of Reticulitermes. PCR-based procedures implied that the novel isolates were autochthonous to the hindgut of R. flavipes and comprised ca. 2 to 7% of the hindgut prokaryote community. Representative strain TAM-DN1 utilized acetate and a limited range of other organic and amino acids as energy sources and possessed catalase and superoxide dismutase. On solid medium, the optimal O(2) concentration for growth was about 2%, and no growth occurred with O(2) concentrations above 4% or under anoxia. However, cells in liquid medium could grow with higher O(2) concentrations (up to 16%), but only after proportionately extended lag phases. The genetic and physiological distinctiveness of TAM-DN1 and related strains supports their recognition as a new genus and species, for which the name Stenoxybacter acetivorans gen. nov., sp. nov. is proposed.

Physiological ecology of Stenoxybacter acetivorans, an obligate microaerophile in termite guts.

Stenoxybacter acetivorans is a newly described, obligately microaerophilic beta-proteobacterium that is abundant in the acetate-rich hindgut of Reticulitermes. Here we tested the hypotheses that cells are located in the hypoxic, peripheral region of Reticulitermes flavipes hindguts and use acetate to fuel their O(2)-consuming respiratory activity in situ. Physical fractionation of R. flavipes guts, followed by limited-cycle PCR with S. acetivorans-specific 16S rRNA gene primers, indicated that cells of this organism were indeed located primarily among the microbiota colonizing the hindgut wall. Likewise, reverse transcriptase PCR of hindgut RNA revealed S. acetivorans-specific transcripts for acetate-activating enzymes that were also found in cell extracts (acetate kinase and phosphotransacetylase), as well as transcripts of ccoN, which encodes the O(2)-reducing subunit of high-affinity cbb(3)-type cytochrome oxidases. However, S. acetivorans strains did not possess typical enzymes of the glyoxylate cycle (isocitrate lyase and malate synthase A), suggesting that they may use an alternate pathway to replenish tricarboxylic acid cycle intermediates or they obtain such compounds (or their precursors) in situ. Respirometric measurements indicated that much of the O(2) consumption by R. flavipes worker larvae was attributable to their guts, and the potential contribution of S. acetivorans to O(2) consumption by extracted guts was about 0.2%, a value similar to that obtained for other hindgut bacteria examined. Similar measurements obtained with guts of larvae prefed diets to disrupt major members of the hindgut microbiota implied that most of the O(2) consumption observed with extracted guts was attributable to protozoans, a group of microbes long thought to be "strict anaerobes."