Deep-learning-based removal of autofluorescence and fluorescence quantification in plant-colonizing bacteria in vivo.

Fluorescence microscopy is common in bacteria-plant interaction studies. However, strong autofluorescence from plant tissues impedes in vivo studies on endophytes tagged with fluorescent proteins. To solve this problem, we developed a deep-learning-based approach to eliminate plant autofluorescence from fluorescence microscopy images, tested for the model endophyte Azoarcus olearius BH72 colonizing Oryza sativa roots. Micrographs from three channels (tdTomato for gene expression, green fluorescent protein (GFP) and AutoFluorescence (AF)) were processed by a neural network based approach, generating images that simulate the background autofluorescence in the tdTomato channel. After subtracting the model-generated signals from each pixel in the genuine channel, the autofluorescence in the tdTomato channel was greatly reduced or even removed. The deep-learning-based approach can be applied for fluorescence detection and quantification, exemplified by a weakly expressed, a cell-density modulated and a nitrogen-fixation gene in A. olearius. A transcriptional nifH::tdTomato fusion demonstrated stronger induction of nif genes inside roots than outside, suggesting extension of the rhizosphere effect for diazotrophs into the endorhizosphere. The pre-trained convolutional neural network model is easily applied to process other images of the same plant tissues with the same settings. This study showed the high potential of deep-learning-based approaches in image processing. With proper training data and strategies, autofluorescence in other tissues or materials can be removed for broad applications.

Aromatoleum gen. nov., a novel genus accommodating the phylogenetic lineage including Azoarcus evansii and related species, and proposal of Aromatoleum aromaticum sp. nov., Aromatoleum petrolei sp. nov., Aromatoleum bremense sp. nov., Aromatoleum toluolicum sp. nov. and Aromatoleum diolicum sp. nov.

Comparative 16S rRNA gene sequence analysis and major physiological differences indicate two distinct sublineages within the genus Azoarcus: the Azoarcus evansii lineage, comprising Azoarcusevansii (type strain KB740T=DSM 6898T=CIP 109473T=NBRC 107771T), Azoarcusbuckelii (type strain U120T=DSM 14744T=LMG 26916T), Azoarcusanaerobius (type strain LuFRes1T=DSM 12081T=LMG 30943T), Azoarcustolulyticus (type strain Tol-4T=ATCC 51758T=CIP 109470T), Azoarcustoluvorans (type strain Td21T=ATCC 700604T=DSM 15124T) and Azoarcustoluclasticus (type strain MF63T=ATCC 700605T), and the Azoarcus indigens lineage, comprising Azoarcusindigens (type strain VB32T=ATCC 51398T=LMG 9092T), Azoarcus communis (type strain SWub3T=ATCC 51397T=LMG 9095T) and Azoarcusolearius (type strain DQS-4T=BCRC 80407T=KCTC 23918T=LMG 26893T). Az. evansii lineage members have remarkable anaerobic degradation capacities encompassing a multitude of alkylbenzenes, aromatic compounds and monoterpenes, often involving novel biochemical reactions. In contrast, Az. indigens lineage members are diazotrophic endophytes lacking these catabolic capacities. It is proposed that species of the Az. evansii lineage should be classified in a novel genus, Aromatoleum gen. nov. Finally, based on the literature and new growth, DNA-DNA hybridization and proteomic data, the following five new species are proposed: Aromatoleum aromaticum sp. nov. (type strain EbN1T=DSM 19018T=LMG 30748T and strain pCyN1=DSM 19016=LMG 31004), Aromatoleum petrolei sp. nov. (type strain ToN1T=DSM 19019T=LMG 30746T), Aromatoleumbremense sp. nov. (type strain PbN1T=DSM 19017T=LMG 31005T), Aromatoleum toluolicum sp. nov. (type strain TT=DSM 19020T=LMG 30751T) and Aromatoleum diolicum sp. nov. (type strain 22LinT=DSM 15408T=LMG 30750T).

Bradyrhizobium ripae sp. nov., a nitrogen-fixing symbiont isolated from nodules of wild legumes in Namibia.

Root-nodule bacteria were isolated from wild legumes growing in the Kavango region, Namibia. Using a polyphasic approach, four strains belonging to the genus Bradyrhizobium (WR4T, WR87, T10 and T12) were further characterized to clarify the taxonomic status of this group. On the basis of 16S rRNA gene sequences, the four strains showed highest similarity to Bradyrhizobium elkanii USDA 76T (99.9 %), Bradyrhizobium pachyrhizi PAC48T (identical) and to Bradyrhizobiumbrasilense UFLA03-321T (identical). Multilocus sequence analysis of concatenated glnII-recA-gyrB-dnaK-rpoB sequences and comparison of the intergenic transcribed spacer (ITS) sequences confirmed that the novel group belongs to a distinct lineage of the genus Bradyrhizobium, with <96.7 % (MLSA) and 97.25 % (ITS) nucleotide identity with B. elkanii USDA 76T. Results from the sequence-based analysis were validated by DNA-DNA hybridization experiments and suggested a novel species. Several phenotypic features including carbon compound utilization and growth characteristics supported the phylogenetic data, thus it is concluded that the strains represent a novel species, for which the name Bradyrhizobium ripae sp. nov. is proposed, with type strain WR4T [LMG 30283, DSM 105795, NTCCM 0019 (Windhoek)].

The Regulatory Network Controlling Ethanol-Induced Expression of Alcohol Dehydrogenase in the Endophyte Azoarcus sp. Strain BH72.

The habitat of the nitrogen-fixing endophyte Azoarcus sp. strain BH72 is grass roots grown under waterlogged conditions that produce, under these conditions, ethanol. Strain BH72 is well equipped to metabolize ethanol, with eight alcohol dehydrogenases (ADHs), of which ExaA2 and ExaA3 are the most relevant ones. exaA2 and exaA3 cluster and are surrounded by genes encoding two-component regulatory systems (TCSs) termed ExaS-ExaR and ElmS-GacA. Functional genomic analyses revealed that i) expression of the corresponding genes was induced by ethanol, ii) the genes were also expressed in the rhizoplane or even inside of rice roots, iii) both TCSs were indispensable for growth on ethanol, and iv) they were important for competitiveness during rice root colonization. Both TCSs form a hierarchically organized ethanol-responsive signal transduction cascade with ExaS-ExaR as the highest level, essential for effective expression of the ethanol oxidation system based on ExaA2. Transcript and expression levels of exaA3 increased in tcs deletion mutants, suggesting no direct influence of both TCSs on its ethanol-induced expression. In conclusion, this underscores the importance of ethanol for the endophytic lifestyle of Azoarcus sp. strain BH72 and indicates a tight regulation of the ethanol oxidation system during root colonization.

Global expression analysis of the response to microaerobiosis reveals an important cue for endophytic establishment of Azoarcus sp. BH72.

The endophyte Azoarcus sp. BH72, fixing nitrogen microaerobically, encounters low O2 tensions in flooded roots. Therefore, its transcriptome upon shift to microaerobiosis was analyzed using oligonucleotide microarrays. A total of 8.7% of the protein-coding genes were significantly modulated. Aerobic conditions induced expression of genes involved in oxidative stress protection, while under microaerobiosis, 233 genes were upregulated, encoding hypothetical proteins, transcriptional regulators, and proteins involved in energy metabolism, among them a cbb3 -type terminal oxidase contributing to but not essential for N2 fixation. A newly established sensitive transcriptional reporter system using tdTomato allowed to visualize even relatively low bacterial gene expression in association with roots. Beyond metabolic changes, low oxygen concentrations seemed to prime transcription for plant colonization: Several genes known to be required for endophytic rice interaction were induced, and novel bacterial colonization factors were identified, such as azo1653. The cargo of the type V autotransporter Azo1653 had similarities to the attachment factor pertactin. Although for short term swarming-dependent colonization, it conferred a competitive disadvantage, it contributed to endophytic long-term establishment inside roots. Proteins sharing such opposing roles in the colonization process appear to occur more generally, as we demonstrated a very similar phenotype for another attachment protein, Azo1684. This suggests distinct cellular strategies for endophyte establishment.

Bradyrhizobium namibiense sp. nov., a symbiotic nitrogen-fixing bacterium from root nodules of Lablab purpureus, hyacinth bean, in Namibia.

Four strains of symbiotic bacteria from root nodules of hyacinth bean (Lablab purpureus (L.) Sweet) from Namibia were previously identified as a novel group within the genus Bradyrhizobium. To confirm their taxonomic status, these strains were further characterized by taking a polyphasic approach. The type strain possessed 16S rRNA gene sequences identical to Bradyrhizobium paxllaeri LMTR 21T and Bradyrhizobiumicense LMTR 13T, the full-length sequences were identical to those retrieved from SAMN05230119 and SAMN05230120, respectively. However, the intergenic spacer sequences of the novel group showed identities of less than 93.1 % to described Bradyrhizobium species and were placed in a well-supported separate lineage in the phylogenetic tree. Phylogenetic analyses of six concatenated housekeeping genes, recA, glnII, gyrB, dnaK, atpD and rpoB, corroborated that the novel strains belonged to a lineage distinct from named species of the genus Bradyrhizobium, with highest sequence identities to Bradyrhizobiumjicamae and B. paxllaeri (below 93 %). The species status was validated by results of DNA-DNA hybridization and average nucleotide identity values of genome sequences. The combination of phenotypic characteristics from several tests, including carbon source utilization and antibiotic resistance, could be used to differentiate representative strains from recognized species of the genus Bradyrhizobium. Phylogenetic analysis of nodC and nifH genes placed the novel strains in a group with B. paxllaeri and B.lablabi. Novel strain 5-10T induces effective nodules on Lablab purpureus, Vigna subterranea, Vigna unguiculata and Arachis hypogaea. Based on our results, we conclude that our strains represent a novel species for which the name Bradyrhizobium namibiense sp. nov. is proposed, with type strain 5-10T[LMG 28789, DSM 100300, NTCCM0017 (Windhoek)].

Bradyrhizobium vignae sp. nov., a nitrogen-fixing symbiont isolated from effective nodules of Vigna and Arachis.

Twenty one strains of symbiotic bacteria from root nodules of local races of cowpea (Vigna unguiculata), Bambara groundnut (Vigna subterranea) and peanuts (Arachis hypogaea) grown on subsistence farmers' fields in the Kavango region of Namibia, were previously characterized as a novel group within the genus Bradyrhizobium. To verify their taxonomic position, the strains were further analysed using a polyphasic approach. 16S rRNA gene sequences were most similar to Bradyrhizobium manausense BR 3351T, with Bradyrhizobium ganzhouense RITF806T being the most closely related type strain in the phylogenetic analysis, and Bradyrhizobium yuanmingense CCBAU 10071T in the ITS sequence analysis. Phylogenetic analysis of concatenated glnII-recA-rpoB-dnaK placed the strains in a highly supported lineage distinct from species of the genus Bradyrhizobium with validly published names; they were most closely related to Bradyrhizobium subterraneum 58 2-1T. The status of the species was validated by results of DNA-DNA hybridization. The combination of phenotypic characteristics from several tests, including carbon source utilization and antibiotic resistance, could be used to differentiate representative strains of species of the genus Bradyrhizobium with validly published names. Novel strain 7-2T induced effective nodules on Vigna subterranea, Vigna unguiculata, Arachis hypogaea and on Lablab purpureus. The DNA G+C content of strain 7-2T was 65.4 mol% (Tm). Based on the data presented, we conclude that these strains represent a novel species for which the name Bradyrhizobium vignae sp. nov. is proposed, with strain 7-2T [LMG 28791T, DSMZ 100297T, NTCCM0018T (Windhoek)] as the type strain.

Rice responds to endophytic colonization which is independent of the common symbiotic signaling pathway.

As molecular interactions of plants with N2 -fixing endophytes are largely uncharacterized, we investigated whether the common signaling pathway (CSP) shared by root nodule symbioses (RNS) and arbuscular mycorrhizal (AM) symbioses may have been recruited for the endophytic Azoarcus sp.-rice (Oryza sativa) interaction, and combined this investigation with global approaches to characterize rice root responses to endophytic colonization. Putative homologs of genes required for the CSP were analyzed for their putative role in endophytic colonization. Proteomic and suppressive subtractive hybridization (SSH) approaches were also applied, and a comparison of defense-related processes was carried out by setting up a pathosystem for flooded roots with Xanthomonas oryzae pv. oryzae strain PXO99 (Xoo). All tested genes were expressed in rice roots seedlings but not induced upon Azoarcus sp. inoculation, and the oscyclops and oscastor mutants were not impaired in endophytic colonization. Global approaches highlighted changes in rice metabolic activity and Ca(2+) -dependent signaling in roots colonized by endophytes, including some stress proteins. Marker genes for defense responses were induced to a lesser extent by the endophytes than by the pathogen, indicating a more compatible interaction. Our results thus suggest that rice roots respond to endophytic colonization by inducing metabolic shifts and signaling events, for which the CSP is not essential.

Bradyrhizobium subterraneum sp. nov., a symbiotic nitrogen-fixing bacterium from root nodules of groundnuts.

Seven strains of symbiotic bacteria from root nodules of local races of Bambara groundnut (Vigna subterranea) and peanuts (Arachis hypogaea) grown on subsistence farmers' fields in the Kavango region, Namibia, were previously characterized and identified as a novel group within the genus Bradyrhizobium. To corroborate their taxonomic status, these strains were further characterized using a polyphasic approach. All strains possessed identical 16S rRNA gene sequences with Bradyrhizobium yuanmingense CCBAU 10071T being the most closely related type strain in the 16S rRNA gene phylogenetic analysis, and Bradyrhizobium daqingense CCBAU 15774T in the ITS sequence analysis. Phylogenetic analysis of concatenated glnII-recA-rpoB-dnaK placed the strains in a highly supported lineage distinct from named species of the genus Bradyrhizobium, most closely related to Bradyrhizobium yuanmingense CCBAU 10071T. The species status was validated by results of DNA­DNA hybridization. Phylogenetic analysis of nifH genes placed the novel strains in a group with nifH of 'Bradyrhizobium arachidis' CCBAU 051107 that also nodulates peanuts. The combination of phenotypic characteristics from several tests including carbon source utilization and antibiotic resistance could be used to differentiate representative strains from recognized species of the genus Bradyrhizobium. Novel strain 58 2-1T induced effective nodules on V. subterranea, Vigna unguiculata and A. hypogaea, and some strains on Lablab purpureus. Based on the data presented, we conclude that our strains represent a novel species for which the name Bradyrhizobium subterraneum sp. nov. is proposed, with 58 2-1T [ = DSM 100298T = LMG 28792T = NTCCM0016T (Windhoek)] as the type strain. The DNA G+C content of strain 58 2-1T was 64.7 mol% (T m).

Bradyrhizobium kavangense sp. nov., a symbiotic nitrogen-fixing bacterium from root nodules of traditional Namibian pulses.

Eight strains of symbiotic bacteria from root nodules of local races of cowpea (Vigna unguiculata) and Bambara groundnut (Vigna subterranea) grown on subsistence farmers' fields in the Kavango region, Namibia, were previously characterized and identified as a novel group within the genus Bradyrhizobium. To clarify their taxonomic status, these strains were further characterized using a polyphasic approach. In phylogenetic analysis of the 16S rRNA gene sequence the novel group was most closely related to Bradyrhizobium iriomotense EK05T and Bradyrhizobium ingae BR 10250T, and to 'Bradyrhizobium arachidis' CCBAU 051107 in the ITS sequence analysis. Phylogenetic analysis of concatenated glnII-recA-rpoB-dnaK sequences placed the strains in a lineage distinct from named species of the genus Bradyrhizobium. The species status was validated by results of DNA-DNA hybridization. Phylogenetic analysis of nifH and nodC genes placed the novel strains in a group with 'B. arachidis' CCBAU 051107. The combination of phenotypic characteristics from several tests including carbon source utilization and antibiotic resistance could be used to differentiate representative strains from recognized species of the genus Bradyrhizobium. Novel strain 14-3T induces effective nodules on Vigna subterranea, Vigna unguiculata, Arachis hypogaea and Lablab purpureus. Based on the data presented, it is concluded that the strains represent a novel species of the genus Bradyrhizobium, for which the name Bradyrhizobium kavangense sp. nov. is proposed. The type strain is 14-3T [ = DSM 100299T = LMG 28790T = NTCCM 0012T (Windhoek)]. The DNA G+C content of strain 14-3T is 63.8 mol% (Tm).

Rhizobia Indigenous to the Okavango Region in Sub-Saharan Africa: Diversity, Adaptations, and Host Specificity.

The rhizobial community indigenous to the Okavango region has not yet been characterized. The isolation of indigenous rhizobia can provide a basis for the formulation of a rhizobial inoculant. Moreover, their identification and characterization contribute to the general understanding of species distribution and ecology. Isolates were obtained from nodules of local varieties of the pulses cowpea, Bambara groundnut, peanut, hyacinth bean, and common bean. Ninety-one of them were identified by BOX repetitive element PCR (BOX-PCR) and sequence analyses of the 16S-23S rRNA internally transcribed spacer (ITS) and the recA, glnII, rpoB, and nifH genes. A striking geographical distribution was observed. Bradyrhizobium pachyrhizi dominated at sampling sites in Angola which were characterized by acid soils and a semihumid climate. Isolates from the semiarid sampling sites in Namibia were more diverse, with most of them being related to Bradyrhizobium yuanmingense and Bradyrhizobium daqingense. Host plant specificity was observed only for hyacinth bean, which was nodulated by rhizobia presumably representing yet-undescribed species. Furthermore, the isolates were characterized with respect to their adaptation to high temperatures, drought, and local host plants. The adaptation experiments revealed that the Namibian isolates shared an exceptionally high temperature tolerance, but none of the isolates showed considerable adaptation to drought. Moreover, the isolates' performance on different local hosts showed variable results, with most Namibian isolates inducing better nodulation on peanut and hyacinth bean than the Angolan strains. The local predominance of distinct genotypes implies that indigenous strains may exhibit a better performance in inoculant formulations.

A novel regulatory role of the Rnf complex of Azoarcus sp. strain BH72.

The superfamily of P(II) proteins contains the most widely distributed signalling proteins in nature. Remarkable is the variety of targets whose activity is affected by protein-protein interactions. Here we identified as novel partner for interaction with GlnK an Rnf complex, known to couple the energy of ion transport to reduce ferredoxins. The endophytic diazotrophic betaproteobacterium Azoarcus sp. strain BH72 harbours two rnf-like clusters in the genome, of which only the rnf1 cluster was induced under conditions of N(2) fixation under control of the transcriptional activator NifA. Rapid inactivation ('DraT-independent switch off') of nitrogenase activity upon ammonium upshift was dependent on the Rnf1 complex. Membrane sequestration of GlnK in steady-state N-surplus conditions occurred in its unmodified form, signalling N-surplus, and was dependent on presence of the Rnf1 complex, suggesting physical interaction. In vitro binding studies by Far-Western analysis indicated interactions of RnfC1 with specifically GlnK but not with GlnB. As ammonium upshift led to decreased activity of the Rnf1 complex in membranes, it might be inactivated by GlnK binding, leading to an interruption of electron flow to nitrogenase and thus a rapid, DraT-independent nitrogenase switch off. Our data imply a hitherto unknown interaction partner for a P(II)-like protein and an additional process under its control.

Exploring the benefits of inoculated cowpeas under different climatic conditions in Namibia.

Cowpeas (Vigna uniculata L. Walp) are grown by many smallholder farmers in sub-Saharan Africa for food and their ability to fix nitrogen even under stress. Their performance depends on the indigenous rhizobial strains that live in symbiotic association with the roots; it can be enhanced if the seeds are inoculated with more effective ones. Data of the effectiveness of the technique under a variety of climatic conditions is rare. Here, we thus use a model to upscale two field experiments conducted in Namibia to include different climate change scenarios. The simulations show that non-inoculated cowpeas have mean yields of 0.5 t/ha and inoculated cowpeas 1 t/ha. If climatic conditions are favorable (cool and wet), estimated yield differences increase to over 1 t/ha. In dry years (< 200 mm), the average yield difference is only 0.1 t/ha. In the far future (2080-2100), instances of dry and hot years will increase. Using inoculated cowpea seeds instead of non-inoculated ones thus does not benefit farmers as much then as in the near future (2030-2050). In conclusion, using cowpea seeds inoculated with an efficient rhizobial strain can significantly increase yields under varying climatic conditions, but yield advantages decrease markedly in very dry and hot years.

Towards inoculant development for Bambara groundnut (Vigna subterranean (L.) Verdc) pulse crop production in Namibia.

Introduction: The globally expanding population, together with climate change, poses a risk to the availability of food for humankind. Bambara groundnut (BGN) (Vigna subterranea (L.) Verdc) is a neglected, relatively drought-tolerant native legume of Sub-Saharan Africa that has the potential to become a successful food crop because of its nutritional quality and climate-smart features. Nitrogen fixation from root nodule symbiosis with climate-adapted rhizobial symbionts can contribute nitrogen and organic material in nutrient-poor soil and improve yields. However, high soil temperature and drought often reduce the abundance of native rhizobia in such soil. Therefore, the formulation of climate-smart biofertilizers has the potential to improve the farming of BGN at a low cost in a sustainable manner. Method: The effect of seven Bradyrhizobium spp. strains native to Namibia, including B. vignae and B. subterraneum, were tested on three Namibian BGN varieties (red, brown, cream) in greenhouse pot experiments in Namibia, using soil from the target region of Kavango. Each variety was treated with a mixed inoculant consisting of seven preselected strains ("MK") as well as with one promising single inoculant strain. Results: The results revealed that in all three varieties, the two inoculants (mixed or single) outperformed the non-inoculated cultivars in terms of shoot dry weight by up to 70%; the mixed inoculant treatment performed significantly better (p < 0.05) in all cases compared to the single inoculant used. To test whether the inoculant strains were established in root nodules, they were identified by sequence analysis. In many cases, the indigenous strains of Kavango soil outcompeted the inoculant strains of the mix for nodule occupancy, depending on the BGN variety. As a further preselection, each of the individual strains of the mix was used to inoculate the three varieties under sterile conditions in a phytotron. The agronomic trait and root nodulation response of the host plant inoculations strongly differed with the BGN variety. Even competitiveness in nodule occupancy without involving any indigenous strains from soil differed and depended strictly on the variety. Discussion: Severe differences in symbiont-plant interactions appear to occur in BGN depending on the plant variety, demanding for coupling of breeding efforts with selecting efficient inoculant strains.

Flagella mediate endophytic competence rather than act as MAMPS in rice-Azoarcus sp. strain BH72 interactions.

Azoarcus sp. strain BH72 is an endophytic betaproteobacterium able to colonize rice roots without induction of visible disease symptoms. BH72 possesses one polar flagellum. The genome harbors three copies of putative fliC genes, generally encoding the major structural protein flagellin. It is not clear whether, in endophytic interactions, flagella mediate endophytic competence or act as MAMPs (microbe-associated molecular patterns) inducing plant defense responses. Therefore, possible functions of the three FliC proteins were investigated. Only fliC3 was found to be highly expressed in pure culture and in association with rice roots and to be required for bacterial motility, suggesting that it encodes the major flagellin. Endophytic colonization of rice roots was significantly reduced in the in-frame deletion mutant, while the establishment of microcolonies on the root surface was not affected. Moreover, an elicitation of defense responses related to FliC3 was not observed. In conclusion, our data support the hypothesis that FliC3 does not play a major role as a MAMP but is required for endophytic colonization in the Azoarcus-rice interaction, most likely for spreading inside the plant.

Transcriptomic analysis of responses to exudates reveal genes required for rhizosphere competence of the endophyte Azoarcus sp. strain BH72.

Endophytic colonization is a very complex process which is not yet completely understood. Molecules exuded by the plants may act as signals which influence the ability of the microbe to colonize the host or survive in the rhizosphere. Here we used the whole genome microarray approach to investigate the response of the diazotrophic model endophyte, Azoarcus sp. strain BH72, to exudates of O. sativa cv. Nipponbare in order to identify differentially regulated genes. On exposure to exudates, an overall expression of 4.4% of the 3992 protein coding genes of Azoarcus sp. strain BH72 was altered, out of which 2.4% was upregulated and 2.0% was downregulated. Genes with modulated expression included a few whose involvement in plant-microbe interaction had already been established, whereas a large fraction comprised of genes encoding proteins with putative or unknown functions. Mutational analysis of several differentially regulated genes like those encoding a minor pilin PilX, signal transduction proteins containing GGDEF domains and a serine-threonine kinase as a putative component of the type IV secretion system (T6SS), revealed their role in host colonization. Our data suggest that strain BH72 may be primed for the endophytic lifestyle by exudates, as the expression of bacterial genes relevant for endophytic colonization of roots is induced by root exudates.

amoA-based consensus phylogeny of ammonia-oxidizing archaea and deep sequencing of amoA genes from soils of four different geographic regions.

Ammonia-oxidizing archaea (AOA) play an important role in nitrification and many studies exploit their amoA genes as marker for their diversity and abundance. We present an archaeal amoA consensus phylogeny based on all publicly available sequences (status June 2010) and provide evidence for the diversification of AOA into four previously recognized clusters and one newly identified major cluster. These clusters, for which we suggest a new nomenclature, harboured 83 AOA species-level OTU (using an inferred species threshold of 85% amoA identity). 454 pyrosequencing of amoA amplicons from 16 soils sampled in Austria, Costa Rica, Greenland and Namibia revealed that only 2% of retrieved sequences had no database representative on the species-level and represented 30-37 additional species-level OTUs. With the exception of an acidic soil from which mostly amoA amplicons of the Nitrosotalea cluster were retrieved, all soils were dominated by amoA amplicons from the Nitrososphaera cluster (also called group I.1b), indicating that the previously reported AOA from the Nitrosopumilus cluster (also called group I.1a) are absent or represent minor populations in soils. AOA richness estimates on the species level ranged from 8-83 co-existing AOAs per soil. Presence/absence of amoA OTUs (97% identity level) correlated with geographic location, indicating that besides contemporary environmental conditions also dispersal limitation across different continents and/or historical environmental conditions might influence AOA biogeography in soils.

Elucidation of Essential Genes and Mutant Fitness during Adaptation toward Nitrogen Fixation Conditions in the Endophyte Azoarcus olearius BH72 Revealed by Tn-Seq.

Azoarcus olearius BH72 is a diazotrophic model endophyte that contributes fixed nitrogen to its host plant, Kallar grass, and expresses nitrogenase genes endophytically. Despite extensive studies on biological nitrogen fixation (BNF) of diazotrophic endophytes, little is known about global genetic players involved in survival under respective physiological conditions. Here, we report a global genomic screen for putatively essential genes of A. olearius employing Tn5 transposon mutagenesis with a modified transposon combined with high-throughput sequencing (Tn-Seq). A large Tn5 master library of ~6 × 105 insertion mutants of strain BH72 was obtained. Next-generation sequencing identified 183,437 unique insertion sites into the 4,376,040-bp genome, displaying one insertion every 24 bp on average. Applying stringent criteria, we describe 616 genes as putatively essential for growth on rich medium. COG (Clusters of Orthologous Groups) assignment of the 564 identified protein-coding genes revealed enrichment of genes related to core cellular functions and cell viability. To mimic gradual adaptations toward BNF conditions, the Tn5 mutant library was grown aerobically in synthetic medium or microaerobically on either combined or atmospheric nitrogen. Enrichment and depletion analysis of Tn5 mutants not only demonstrated the role of BNF- and metabolism-related proteins but also revealed that, strikingly, many genes relevant for plant-microbe interactions decrease bacterial competitiveness in pure culture, such type IV pilus- and bacterial envelope-associated genes. IMPORTANCE A constantly growing world population and the daunting challenge of climate change demand new strategies in agricultural crop production. Intensive usage of chemical fertilizers, overloading the world's fields with organic input, threaten terrestrial and marine ecosystems as well as human health. Long overlooked, the beneficial interaction of endophytic bacteria and grasses has attracted ever-growing interest in research in the last decade. Capable of biological nitrogen fixation, diazotrophic endophytes not only provide a valuable source of combined nitrogen but also are known for diverse plant growth-promoting effects, thereby contributing to plant productivity. Elucidation of an essential gene set for a prominent model endophyte such as A. olearius BH72 provides us with powerful insights into its basic lifestyle. Knowledge about genes detrimental or advantageous under defined physiological conditions may point out a way of manipulating key steps in the bacterium's lifestyle and plant interaction toward a more sustainable agriculture.

Exploring the function of alcohol dehydrogenases during the endophytic life of Azoarcus Sp. strain BH72.

The endophytic bacterium Azoarcus sp. strain BH72 is capable of colonizing the interior of rice roots, where it finds suitable physicochemical properties for multiplying and fixing nitrogen. Because these properties are poorly understood, a microtiter-plate-based screening of a transcriptional gfp (green fluorescent protein) fusion library of Azoarcus sp. grown under different conditions was performed. Monitoring of the GFP activity allowed the identification of a gene highly expressed in medium supplemented with ethanol. Sequence analysis revealed that this gene encodes a pyrrolo-quinoline quinone-dependent alcohol dehydrogenase (ADH). Inspection of the complete genome sequence of the Azoarcus sp. strain BH72 identified seven additional genes encoding putative ADH, indicating that BH72 is well equipped to survive in different environmental conditions offering various alcohols as carbon source. Analyses of these eight putative ADH showed that expression of three was induced by ethanol, of which two were also expressed inside rice roots. The fact that waterlogged plants such as rice accumulate ethanol suggests that ethanol occurs in sufficiently high concentration within the root to induce expression of bacterial ADH. Disruption of these two ADH evoked a reduced competitiveness to the wild type in colonizing rice roots internally. Thus, it is likely that ethanol is an important carbon source for the endophytic life of Azoarcus sp.

RNA-Seq Provides New Insights into the Gene Expression Changes in Azoarcus olearius BH72 under Nitrogen-Deficient and Replete Conditions beyond the Nitrogen Fixation Process.

Azoarcus olearius BH72 is an endophyte capable of biological nitrogen fixation (BNF) and of supplying nitrogen to its host plant. Our previous microarray approach provided insights into the transcriptome of strain BH72 under N2-fixation in comparison to ammonium-grown conditions, which already indicated the induction of genes not related to the BNF process. Due to the known limitations of the technique, we might have missed additional differentially expressed genes (DEGs). Thus, we used directional RNA-Seq to better comprehend the transcriptional landscape under these growth conditions. RNA-Seq detected almost 24% of the annotated genes to be regulated, twice the amount identified by microarray. In addition to confirming entire regulated operons containing known DEGs, the new approach detected the induction of genes involved in carbon metabolism and flagellar and twitching motility. This may support N2-fixation by increasing energy production and by finding suitable microaerobic niches. On the other hand, energy expenditures were reduced by suppressing translation and vitamin biosynthesis. Nonetheless, strain BH72 does not appear to be content with N2-fixation but is primed for alternative economic N-sources, such as nitrate, urea or amino acids; a strong gene induction of machineries for their uptake and assimilation was detected. RNA-Seq has thus provided a better understanding of a lifestyle under limiting nitrogen sources by elucidating hitherto unknown regulated processes.