Sympatric speciation in a bacterial endosymbiont results in two genomes with the functionality of one.

Mutualisms that become evolutionarily stable give rise to organismal interdependencies. Some insects have developed intracellular associations with communities of bacteria, where the interdependencies are manifest in patterns of complementary gene loss and retention among members of the symbiosis. Here, using comparative genomics and microscopy, we show that a three-member symbiotic community has become a four-way assemblage through a novel bacterial lineage-splitting event. In some but not all cicada species of the genus Tettigades, the endosymbiont Candidatus Hodgkinia cicadicola has split into two new cytologically distinct but metabolically interdependent species. Although these new bacterial genomes are partitioned into discrete cell types, the intergenome patterns of gene loss and retention are almost perfectly complementary. These results defy easy classification: they show genomic patterns consistent with those observed after both speciation and whole-genome duplication. We suggest that our results highlight the potential power of nonadaptive forces in shaping organismal complexity.

Patterns of bacterial motility in microfluidics-confining environments.

Understanding the motility behavior of bacteria in confining microenvironments, in which they search for available physical space and move in response to stimuli, is important for environmental, food industry, and biomedical applications. We studied the motility of five bacterial species with various sizes and flagellar architectures (Vibrio natriegens, Magnetococcus marinus, Pseudomonas putida, Vibrio fischeri, and Escherichia coli) in microfluidic environments presenting various levels of confinement and geometrical complexity, in the absence of external flow and concentration gradients. When the confinement is moderate, such as in quasi-open spaces with only one limiting wall, and in wide channels, the motility behavior of bacteria with complex flagellar architectures approximately follows the hydrodynamics-based predictions developed for simple monotrichous bacteria. Specifically, V. natriegens and V. fischeri moved parallel to the wall and P. putida and E. coli presented a stable movement parallel to the wall but with incidental wall escape events, while M. marinus exhibited frequent flipping between wall accumulator and wall escaper regimes. Conversely, in tighter confining environments, the motility is governed by the steric interactions between bacteria and the surrounding walls. In mesoscale regions, where the impacts of hydrodynamics and steric interactions overlap, these mechanisms can either push bacteria in the same directions in linear channels, leading to smooth bacterial movement, or they could be oppositional (e.g., in mesoscale-sized meandered channels), leading to chaotic movement and subsequent bacterial trapping. The study provides a methodological template for the design of microfluidic devices for single-cell genomic screening, bacterial entrapment for diagnostics, or biocomputation.

General Stress Signaling in the Alphaproteobacteria.

The Alphaproteobacteria uniquely integrate features of two-component signal transduction and alternative σ factor regulation to control transcription of genes that ensure growth and survival across a range of stress conditions. Research over the past decade has led to the discovery of the key molecular players of this general stress response (GSR) system, including the sigma factor σ(EcfG), its anti-σ factor NepR, and the anti-anti-σ factor PhyR. The central molecular event of GSR activation entails aspartyl phosphorylation of PhyR, which promotes its binding to NepR and thereby releases σ(EcfG) to associate with RNAP and direct transcription. Recent studies are providing a new understanding of complex, multilayered sensory networks that activate and repress this central protein partner switch. This review synthesizes our structural and functional understanding of the core GSR regulatory proteins and highlights emerging data that are defining the systems that regulate GSR transcription in a variety of species.

When bacteria meet mitochondria: The strange case of the tick symbiont Midichloria mitochondrii†.

Mitochondria are key eukaryotic organelles that perform several essential functions. Not surprisingly, many intracellular bacteria directly or indirectly target mitochondria, interfering with innate immunity, energy production or apoptosis, to make the host cell a more hospitable niche for bacterial replication. The alphaproteobacterium Midichloria mitochondrii has taken mitochondrial targeting to another level by physically colonising mitochondria, as shown by transmission electron micrographs of bacteria residing in the mitochondrial intermembrane space. This unique localization provokes a number of questions around the mechanisms allowing, and reasons driving intramitochondrial tropism. We suggest possible scenarios that could lead to this peculiar localization and hypothesize potential costs and benefits of mitochondrial colonisation for the bacterium and its host.

An aryl-homoserine lactone quorum-sensing signal produced by a dimorphic prosthecate bacterium.

Many species of Proteobacteria produce acyl-homoserine lactone (AHL) compounds as quorum-sensing (QS) signals for cell density-dependent gene regulation. Most known AHL synthases, LuxI-type enzymes, produce fatty AHLs, and the fatty acid moiety is derived from an acyl-acyl carrier protein (ACP) intermediate in fatty acid biosynthesis. Recently, a class of LuxI homologs has been shown to use CoA-linked aromatic or amino acid substrates for AHL synthesis. By using an informatics approach, we found the CoA class of LuxI homologs exists primarily in α-Proteobacteria. The genome of Prosthecomicrobium hirschii, a dimorphic prosthecate bacterium, possesses a luxI-like AHL synthase gene that we predicted to encode a CoA-utilizing enzyme. We show the P. hirschii LuxI homolog catalyzes synthesis of phenylacetyl-homoserine lactone (PA-HSL). Our experiments show P. hirschii obtains phenylacetate from its environment and uses a CoA ligase to produce the phenylacetyl-CoA substrate for the LuxI homolog. By using an AHL degrading enzyme, we showed that PA-HSL controls aggregation, biofilm formation, and pigment production in P. hirschii These findings advance a limited understanding of the CoA-dependent AHL synthases. We describe how to identify putative members of the class, we describe a signal synthesized by using an environmental aromatic acid, and we identify phenotypes controlled by the aryl-HSL.

Parasitic 'Candidatus Aquarickettsia rohweri' is a marker of disease susceptibility in Acropora cervicornis but is lost during thermal stress.

Holobiont phenotype results from a combination of host and symbiont genotypes as well as from prevailing environmental conditions that alter the relationships among symbiotic members. Corals exemplify this concept, where shifts in the algal symbiont community can lead to some corals becoming more or less thermally tolerant. Despite linkage between coral bleaching and disease, the roles of symbiotic bacteria in holobiont resistance and susceptibility to disease remains less well understood. This study thus characterizes the microbiome of disease-resistant and -susceptible Acropora cervicornis coral genotypes (hereafter referred to simply as 'genotypes') before and after high temperature-mediated bleaching. We found that the intracellular bacterial parasite 'Ca. Aquarickettsia rohweri' was strikingly abundant in disease-susceptible genotypes. Disease-resistant genotypes, however, had notably more diverse and even communities, with correspondingly low abundances of 'Ca. Aquarickettsia'. Bleaching caused a dramatic reduction of 'Ca. Aquarickettsia' within disease-susceptible corals and led to an increase in bacterial community dispersion, as well as the proliferation of opportunists. Our data support the hypothesis that 'Ca. Aquarickettsia' species increase coral disease risk through two mechanisms: (i) the creation of host nutritional deficiencies leading to a compromised host-symbiont state and (ii) the opening of niche space for potential pathogens during thermal stress.

Perception and Homeostatic Control of Iron in the Rhizobia and Related Bacteria.

Iron is an essential nutrient, but it can also be toxic. Therefore, iron homeostasis must be strictly regulated. Transcriptional control of iron-dependent gene expression in the rhizobia and other taxa of the Alphaproteobacteria is fundamentally different from the Fur paradigm in Escherichia coli and other model systems. Rather than sense iron directly, the rhizobia employ the iron response regulator (Irr) to monitor and respond to the status of an iron-dependent process, namely, heme biosynthesis. This novel control mechanism allows iron homeostasis to be integrated with other cellular processes, and it permits differential control of iron regulon genes in a manner not readily achieved by Fur. Moreover, studies of Irr have defined a role for heme in conditional protein stability that has been subsequently described in eukaryotes. Finally, Irr-mediated control of iron metabolism may reflect a cellular strategy that accommodates a greater reliance on manganese.

Multifunctional Periphytic Biofilms: Polyethylene Degradation and Cd2+ and Pb2+ Bioremediation under High Methane Scenario.

Priority pollutants such as polyethylene (PE) microplastic, lead (Pb2+), and cadmium (Cd2+) have attracted the interest of environmentalists due to their ubiquitous nature and toxicity to all forms of life. In this study, periphytic biofilms (epiphyton and epixylon) were used to bioremediate heavy metals (HMs) and to biodegrade PE under high (120,000 ppm) methane (CH4) doses. Both periphytic biofilms were actively involved in methane oxidation, HMs accumulation and PE degradation. Epiphyton and epixylon both completely removed Pb2+ and Cd2+ at concentrations of 2 mg L-1 and 50 mg L-1, respectively, but only partially removed these HMs at a relatively higher concentration (100 mg L-1). Treatment containing 12% 13CH4 proved to be most effective for biodegradation of PE. A synergistic effect of HMs and PE drastically changed microbial biota and methanotrophic communities. High-throughput 16S rRNA gene sequencing revealed that Cyanobacteria was the most abundant class, followed by Gammaproteobacteria and Alphaproteobacteria in all high-methane-dose treatments. DNA stable-isotope probing was used to label 13C in a methanotrophic community. A biomarker for methane-oxidizing bacteria, pmoA gene sequence of a 13C-labeled fraction, revealed that Methylobacter was most abundant in all high-methane-dose treatments compared to near atmospheric methane (NAM) treatment, followed by Methylococcus. Methylomonas, Methylocystis, Methylosinus, and Methylocella were also found to be increased by high doses of methane compared to NAM treatment. Overall, Cd+2 had a more determinantal effect on methanotrophic activity than Pb2+. Epiphyton proved to be more effective than epixylon in HMs removal and PE biodegradation. The findings proved that both epiphyton and epixylon can be used to bioremediate HMs and biodegrade PE as an efficient ecofriendly technique under high methane concentrations.

The impact of DNA methylation in Alphaproteobacteria.

Alphaproteobacteria include bacteria with very different modes of life, from free-living to host-associated and pathogenic bacteria. Their genomes vary in size and organization from single circular chromosomes to multipartite genomes and are often methylated by one or more adenine or cytosine methyltransferases (MTases). These include MTases that are part of restriction/modification systems and so-called orphan MTases. The development of novel technologies accelerated the analysis of methylomes and revealed the existence of epigenetic patterns in several Alphaproteobacteria. This review describes the known functions of DNA methylation in Alphaproteobacteria and also discusses its potential drawbacks through the accidental deamination of methylated cytosines. Particular emphasis is given to the strong connection between the cell cycle-regulated orphan MTase CcrM and the complex network that controls gene expression and cell cycle progression in Alphaproteobacteria.

Pathogen infections and primary biliary cholangitis.

Primary biliary cholangitis (PBC) is a multi-factorial disease caused by the interaction of both genetic predisposition and environmental triggers. Bacterial infection has been investigated most intensively, both epidemiologically and experimentally, as a prime environmental aetiology in PBC. The association of recurrent history of urinary tract infection (UTI) with PBC has been frequently confirmed by several large-scale, case-control studies, despite variation in geographic area or case-finding methods. Escherichia coli is a predominant pathogen in most cases with UTI. Animal studies and molecular mimicry analysis between the human and E. coli E2 subunit of the 2-oxo-acid dehydrogenase complexes demonstrated that E. coli infection is a key factor in breaking immunological tolerance against the mitochondria, resulting in the production of anti-mitochondrial autoantibodies (AMA), the disease-specific autoantibodies of PBC. Novosphingobium aromaticivorans, a ubiquitous xenobiotic-metabolizing bacterium, is another candidate which may be involved in the aetiology of PBC. Meanwhile, improved environmental hygiene and increased prevalence of PBC, especially in males, may argue against the aetiological role of bacterial infection in PBC. Multiple mechanisms can result in the loss of tolerance to mitochondrial autoantigens in PBC; nonetheless, bacterial infection is probably one of the dominant pathways, especially in female patients. Notably, there is a rising prevalence of male patients with PBC. With increasing exposure to environmental xenobiotics in both genders, studies directed towards identifying the environmental culprit with systematically designed case-control studies are much needed to further determine the environmental factors and role of bacterial infections in PBC.

The evolution of abdominal microbiomes in fungus-growing ants.

The attine ants are a monophyletic lineage that switched to fungus farming ca. 55-60 MYA. They have become a model for the study of complex symbioses after additional fungal and bacterial symbionts were discovered, but their abdominal endosymbiotic bacteria remain largely unknown. Here, we present a comparative microbiome analysis of endosymbiotic bacteria spanning the entire phylogenetic tree. We show that, across 17 representative sympatric species from eight genera sampled in Panama, abdominal microbiomes are dominated by Mollicutes, α- and γ-Proteobacteria, and Actinobacteria. Bacterial abundances increase from basal to crown branches in the phylogeny reflecting a shift towards putative specialized and abundant abdominal microbiota after the ants domesticated gongylidia-bearing cultivars, but before the origin of industrial-scale farming based on leaf-cutting herbivory. This transition coincided with the ancestral single colonization event of Central/North America ca. 20 MYA, documented in a recent phylogenomic study showing that almost the entire crown group of the higher attine ants, including the leaf-cutting ants, evolved there and not in South America. Several bacterial species are located in gut tissues or abdominal organs of the evolutionarily derived, but not the basal attine ants. The composition of abdominal microbiomes appears to be affected by the presence/absence of defensive antibiotic-producing actinobacterial biofilms on the worker ants' cuticle, but the significance of this association remains unclear. The patterns of diversity, abundance and sensitivity of the abdominal microbiomes that we obtained explore novel territory in the comparative analysis of attine fungus farming symbioses and raise new questions for further in-depth research.

ACC deaminase plays a major role in Pseudomonas fluorescens YsS6 ability to promote the nodulation of Alpha- and Betaproteobacteria rhizobial strains.

Ethylene acts as a major regulator of the nodulation process of leguminous plants. Several rhizobial strains possess the ability to modulate plant ethylene levels through the expression of the enzyme 1-aminocyclopropane-1-carboxylate (ACC) deaminase; however, rhizobia present low enzymatic activities. One possible alternative to this problem resides on the use of free-living bacteria, such as Pseudomonas, presenting high levels of ACC deaminase activity that may be used as adjuvants in the nodulation process by decreasing inhibitory ethylene levels. Nevertheless, not much is understood about the specific role of ACC deaminase in the possible role of free-living bacteria as nodulation adjuvants. Therefore, this work aims to study the effect of ACC deaminase in the plant growth-promoting bacterium, Pseudomonas fluorescens YsS6, ability to facilitate alpha- and beta-rhizobia nodulation. The ACC deaminase-producing P. fluorescens YsS6 and its ACC deaminase mutant were used in co-inoculation assays to evaluate their impact in the nodulation process of alpha- (Rhizobium tropici CIAT899) and beta-rhizobia (Cupriavidus taiwanensis STM894) representatives, in Phaseolus vulgaris and Mimosa pudica plants, respectively. The results obtained indicate that the wild-type P. fluorescens YsS6, but not its mutant defective in ACC deaminase production, increase the nodulation abilities of both alpha- and beta-rhizobia, resulting in an increased leguminous plant growth. Moreover, this is the first report of the positive effect of free-living bacteria in the nodulation process of beta-rhizobia. The modulation of inhibitory ethylene levels by free-living ACC deaminase-producing bacteria plays an important role in facilitating the nodulation process of alpha- and beta-rhizobia.

Diverse cellular colonizing endophytic bacteria in field shoots and in vitro cultured papaya with physiological and functional implications.

The study was envisaged to assess the extent of normally uncultivable endophytic bacteria in field papaya plants and in vitro established cultures adopting cultivation vs molecular analysis and microscopy. Surface-sterilized axillary shoot-buds of papaya 'Arka Surya' revealed high bacterial diversity as per 16S rRNA metagene amplicon sequencing (6 phyla, 10 classes, 21 families) with an abundance of Pseudomonas (Gammaproteobacteria), which also formed a common contaminant for in vitro cultured field explants. Molecular analysis of seedling shoot-tip-derived healthy proliferating cultures of three genotypes ('Arka Surya', 'Arka Prabhath', 'Red Lady') with regular monthly subculturing also displayed high bacterial diversity (11-16 phyla, >25 classes, >50 families, >200 genera) about 12-18 months after initial establishment. 'Arka Surya' and 'Red Lady' cultures bore predominantly Actinobacteria (75-78%) while 'Arka Prabhath' showed largely Alphaproteobacteria corroborating the slowly activated Methylobacterium sp. Bright-field direct microscopy on tissue sections and tissue homogenate and epi-fluorescence microscopy employing bacterial DNA probe SYTO-9 revealed abundant intracellular bacteria embracing the next-generation sequencing elucidated high taxonomic diversity. Phylogenetic investigation of communities by reconstruction of unobserved states- PICRUSt- functional annotation suggested significant operational roles for the bacterial-biome. Metabolism, environmental information processing, and genetic information processing constituted major Kyoto Encyclopedia of Genes and Genomes KEGG attributes. Papaya stocks occasionally displayed bacterial growth on culture medium arising from the activation of originally uncultivable organisms to cultivation. The organisms included Bacillus (35%), Methylobacterium (15%), Pseudomonas (10%) and seven other genera (40%). This study reveals a hidden world of diverse and abundant conventionally uncultivable cellular-colonizing endophytic bacteria in field shoots and micropropagating papaya stocks with high genotypic similarity and silent participation in various plant processes/pathways.

Jiella endophytica sp. nov., a novel endophytic bacterium isolated from root of Ficus microcarpa Linn. f.

A Gram-negative, aerobic, short rodshaped, asporogenous bacterium, designated CBS5Q-3T, was isolated from a surface-sterilised root of Ficus microcarpa Linn. f. collected from Guangxi, China and investigated by a polyphasic approach to determine its taxonomic position. Strain CBS5Q-3T was found to grow optimally with 2% (w/v) NaCl at 30 °C, pH 7.0-8.0. Substrate mycelia and aerial mycelia were not formed, and no diffusible pigments were observed on the media tested. Phylogenetic analysis based on 16S rRNA gene sequences showed that strain CBS5Q-3T is closely related to species of genus Jiella and shares high 16S rRNA gene sequence similarity of 98.1% with Jiella aquimaris JCM 30119T. The average nucleotide identity and in silico DNA-DNA hybridization values between strain CBS5Q-3T and J. aquimaris JCM 30119T were 82.8% and 26.0%, respectively. The DNA G + C content of strain CBS5Q-3T was determined to be 66.5 mol %. The cell wall peptidoglycan was found to contain meso-diaminopimelic acid and ubiquinone Q-10 identified as the respiratory lipoquinone. The polar lipids were found to be comprised of diphosphatidylglycerol, phosphatidylglycerol, phosphatidylcholine, phosphatidylmonomethylethanolamine, phosphatidylethanolamine and three unidentified aminolipids, while the major fatty acids were identified as C18:1ω7c and cyclo-C19:0ω8c. On the basis of phylogenetic, chemotaxonomic and phenotypic data, strain CBS5Q-3T can be concluded to represent a novel species of the genus Jiella, for which the name Jiella endophytica sp. nov. is proposed. The type strain is CBS5Q-3T (= JCM 33167T = CGMCC 1.13863T).

Complete Genome Sequence of Qipengyuania sediminis CGMCC 1.12928T, Shed Light on Its Role in Matter-Cycle and Cold Adaption Mechanism of the Genus Qipengyuania.

Qipengyuania sediminis CGMCC 1.12928T, a family member of Erythrobacteraceae, the class of Alphaproteobacteria, was isolated from a borehole sediment sample collected from Qiangtang Basin in Qinghai-Tibetan Plateau, the largest permafrost in China. Understanding bacterial molecular feature may shed light on the ecological strategy in the extreme environment. Here we describe the complete genome sequence and annotation of strain CGMCC 1.12928T, including the complete genome sequence and annotation. The genome of strain CGMCC 1.12928T consist of a single-circular chromosome, comprises 2,416,000 bp with an average G + C content of 66.7 mol%, and contains 2414 genes; including 2367 CDSs, 44 tRNA genes, as well as one operon of 16S-23S-5S rRNA genes. Genomic properties indicated that strain CGMCC 1.12928T has a relatively smaller genome size and higher G + C content within the family Erythrobacteraceae. In addition, genomic analysis revealed its genome contains multiple function genes responsible for nitrogen, sulfur and phosphorus cycles and explained the cold adaption mechanism. Thus, this strain plays an active role in the biogeochemical cycle in cold niche. The whole-genome of this isolate will widen our understanding of the ecological role of the genus Qipengyuania in permafrost.

Investigation of the thermophilic mechanism in the genus Porphyrobacter by comparative genomic analysis.

BACKGROUND: Type strains of the genus Porphyrobacter belonging to the family Erythrobacteraceae and the class Alphaproteobacteria have been isolated from various environments, such as swimming pools, lake water and hot springs. P. cryptus DSM 12079T and P. tepidarius DSM 10594T out of all Erythrobacteraceae type strains, are two type strains that have been isolated from geothermal environments. Next-generation sequencing (NGS) technology offers a convenient approach for detecting situational types based on protein sequence differences between thermophiles and mesophiles; amino acid substitutions can lead to protein structural changes, improving the thermal stabilities of proteins. Comparative genomic studies have revealed that different thermal types exist in different taxa, and few studies have been focused on the class Alphaproteobacteria, especially the family Erythrobacteraceae. In this study, eight genomes of Porphyrobacter strains were compared to elucidate how Porphyrobacter thermophiles developed mechanisms to adapt to thermal environments. RESULTS: P. cryptus DSM 12079T grew optimally at 50 °C, which was higher than the optimal growth temperature of other Porphyrobacter type strains. Phylogenomic analysis of the genus Porphyrobacter revealed that P. cryptus DSM 12079T formed a distinct and independent clade. Comparative genomic studies uncovered that 1405 single-copy genes were shared by Porphyrobacter type strains. Alignments of single-copy proteins showed that various types of amino acid substitutions existed between P. cryptus DSM 12079T and the other Porphyrobacter strains. The primary substitution types were changes from glycine/serine to alanine. CONCLUSIONS: P. cryptus DSM 12079T was the sole thermophile within the genus Porphyrobacter. Phylogenomic analysis and amino acid frequencies indicated that amino acid substitutions might play an important role in the thermophily of P. cryptus DSM 12079T. Bioinformatic analysis revealed that major amino acid substitutional types, such as changes from glycine/serine to alanine, increase the frequency of α-helices in proteins, promoting protein thermostability in P. cryptus DSM 12079T. Hence, comparative genomic analysis broadens our understanding of thermophilic mechanisms in the genus Porphyrobacter and may provide a useful insight in the design of thermophilic enzymes for agricultural, industrial and medical applications.

Dynamics of the peptidoglycan biosynthetic machinery in the stalked budding bacterium Hyphomonas neptunium.

Most commonly studied bacteria grow symmetrically and divide by binary fission, generating two siblings of equal morphology. An exception to this rule are budding bacteria, in which new offspring emerges de novo from a morphologically invariant mother cell. Although this mode of proliferation is widespread in diverse bacterial lineages, the underlying mechanisms are still incompletely understood. Here, we report the first molecular-level analysis of growth and morphogenesis in the stalked budding alphaproteobacterium Hyphomonas neptunium. Peptidoglycan labeling shows that, in this species, buds originate from a stalk-like extension of the mother cell whose terminal segment is gradually remodeled into a new cell compartment. As a first step toward identifying the machinery mediating the budding process, we performed comprehensive mutational and localization studies of predicted peptidoglycan biosynthetic proteins in H. neptunium. These analyses identify factors that localize to distinct zones of dispersed and zonal growth, and they suggest a critical role of the MreB-controlled elongasome in cell morphogenesis. Collectively, our work shows that the mechanism of growth in H. neptunium is distinct from that in related, polarly growing members of the order Rhizobiales, setting the stage for in-depth analyses of the molecular principles regulating the fascinating developmental cycle of this species.

Genome sequence, metabolic properties and cyanobacterial attachment of Porphyrobacter sp. HT-58-2 isolated from a filamentous cyanobacterium-microbial consortium.

Tolyporphins are structurally diverse tetrapyrrole macrocycles produced by the cyanobacterial culture HT-58-2. Although tolyporphins were discovered over 25 years ago, little was known about the microbiology of the culture. The studies reported herein expand the description of the community of predominantly alphaproteobacteria associated with the filamentous HT-58-2 cyanobacterium and isolate a dominant bacterium, Porphyrobacter sp. HT-58-2, for which the complete genome is established and growth properties are examined. Fluorescence in situ hybridization (FISH) analysis of the cyanobacterium-microbial community with a probe targeting the 16S rRNA of Porphyrobacter sp. HT-58-2 showed fluorescence emanating from the cyanobacterial sheath. Although genes for the biosynthesis of bacteriochlorophyll a (BChl a) are present in the Porphyrobacter sp. HT-58-2 genome, the pigment was not detected under the conditions examined, implying the absence of phototrophic growth. Comparative analysis of four Porphyrobacter spp. genomes from worldwide collection sites showed significant collinear gene blocks, with two inversions and three deletion regions. Taken together, the results enrich our understanding of the HT-58-2 cyanobacterium-microbial culture.

Is there foul play in the leaf pocket? The metagenome of floating fern Azolla reveals endophytes that do not fix N2 but may denitrify.

Dinitrogen fixation by Nostoc azollae residing in specialized leaf pockets supports prolific growth of the floating fern Azolla filiculoides. To evaluate contributions by further microorganisms, the A. filiculoides microbiome and nitrogen metabolism in bacteria persistently associated with Azolla ferns were characterized. A metagenomic approach was taken complemented by detection of N2 O released and nitrogen isotope determinations of fern biomass. Ribosomal RNA genes in sequenced DNA of natural ferns, their enriched leaf pockets and water filtrate from the surrounding ditch established that bacteria of A. filiculoides differed entirely from surrounding water and revealed species of the order Rhizobiales. Analyses of seven cultivated Azolla species confirmed persistent association with Rhizobiales. Two distinct nearly full-length Rhizobiales genomes were identified in leaf-pocket-enriched samples from ditch grown A. filiculoides. Their annotation revealed genes for denitrification but not N2 -fixation. 15 N2 incorporation was active in ferns with N. azollae but not in ferns without. N2 O was not detectably released from surface-sterilized ferns with the Rhizobiales. N2 -fixing N. azollae, we conclude, dominated the microbiome of Azolla ferns. The persistent but less abundant heterotrophic Rhizobiales bacteria possibly contributed to lowering O2 levels in leaf pockets but did not release detectable amounts of the strong greenhouse gas N2 O.