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Frankia strains induce the formation of nitrogen-fixing nodules on roots of actinorhizal plants. Phylogenetically, Frankia strains can be grouped in four clusters. The earliest divergent cluster, cluster-2, has a particularly wide host range. The analysis of cluster-2 strains has been hampered by the fact that with two exceptions, they could never be cultured. In this study, 12 Frankia-enriched metagenomes of Frankia cluster-2 strains or strain assemblages were sequenced based on seven inoculum sources. Sequences obtained via DNA isolated from whole nodules were compared with those of DNA isolated from fractionated preparations enhanced in the Frankia symbiotic structures. The results show that cluster-2 inocula represent groups of strains, and that strains not represented in symbiotic structures, that is, unable to performsymbiotic nitrogen fixation, may still be able to colonize nodules. Transposase gene abundance was compared in the different Frankia-enriched metagenomes with the result that NorthAmerican strains contain more transposase genes than Eurasian strains. An analysis of the evolution and distribution of the host plants indicated that bursts of transposition may have coincided with niche competition with other cluster-2 Frankia strains. The first genome of an inoculum from the Southern Hemisphere, obtained from nodules of Coriaria papuana in Papua NewGuinea, represents a novel species, postulated as Candidatus Frankiameridionalis. All Frankia-enrichedmetagenomes obtained in this study contained homologs of the canonical nod genes nodABC; the North American genomes also contained the sulfotransferase gene nodH, while the genome from the Southern Hemisphere only contained nodC and a truncated copy of nodB.

Background: Insertion sequences (ISs) are approximately 1 kbp long jumping genes found in prokaryotes. ISs encode the protein Transposase, which facilitates the excision and reinsertion of ISs in genomes, making these sequences a type of class I (cut-and-paste) Mobile Genetic Elements. ISs are proposed to be involved in the reductive evolution of symbiotic prokaryotes. Our previous sequencing of the genome of the cyanobacterium 'Nostoc azollae' 0708, living in a tight perpetual symbiotic association with a plant (the water fern Azolla), revealed the presence of an eroding genome, with a high number of insertion sequences (ISs) together with an unprecedented large proportion of pseudogenes. To investigate the role of ISs in the reductive evolution of 'Nostoc azollae' 0708, and potentially in the formation of pseudogenes, a bioinformatic investigation of the IS identities and positions in 47 cyanobacterial genomes was conducted. To widen the scope, the IS contents were analysed qualitatively and quantitatively in 20 other genomes representing both free-living and symbiotic bacteria. Results: Insertion Sequences were not randomly distributed in the bacterial genomes and were found to transpose short distances from their original location (local hopping) and pseudogenes were enriched in the vicinity of IS elements. In general, symbiotic organisms showed higher densities of IS elements and pseudogenes than non-symbiotic bacteria. A total of 1108 distinct repeated sequences over 500 bp were identified in the 67 genomes investigated. In the genome of 'Nostoc azollae' 0708, IS elements were apparent at 970 locations (14.3%), with 428 being full-length. Morphologically complex cyanobacteria with large genomes showed higher frequencies of IS elements, irrespective of life style. Conclusions: The apparent co-location of IS elements and pseudogenes found in prokaryotic genomes implies earlier IS transpositions into genes. As transpositions tend to be local rather than genome wi, AuthorCount:4

Background: An ancient cyanobacterial incorporation into a eukaryotic organism led to the evolution of plastids (chloroplasts) and subsequently to the origin of the plant kingdom. The underlying mechanism and the identities of the partners in this monophyletic event remain elusive. Methodology/Principal Findings: To shed light on this evolutionary process, we sequenced the genome of a cyanobacterium residing extracellularly in an endosymbiosis with a plant, the water-fern Azolla filiculoides Lam. This symbiosis was selected as it has characters which make it unique among extant cyanobacterial plant symbioses: the cyanobacterium lacks autonomous growth and is vertically transmitted between plant generations. Our results reveal features of evolutionary significance. The genome is in an eroding state, evidenced by a large proportion of pseudogenes (31.2%) and a high frequency of transposable elements (,600) scattered throughout the genome. Pseudogenization is found in genes such as the replication initiator dnaA and DNA repair genes, considered essential to free-living cyanobacteria. For some functional categories of genes pseudogenes are more prevalent than functional genes. Loss of function is apparent even within the ‘core’ gene categories of bacteria, such as genes involved in glycolysis and nutrient uptake. In contrast, serving as a critical source of nitrogen for the host, genes related to metabolic processes such as cell differentiation and nitrogen-fixation are well preserved. Conclusions/Significance: This is the first finding of genome degradation in a plant symbiont and phenotypically complex cyanobacterium and one of only a few extracellular endosymbionts described showing signs of reductive genome evolution. Our findings suggest an ongoing selective streamlining of this cyanobacterial genome which has resulted in an organism devoted to nitrogen fixation and devoid of autonomous growth. The cyanobacterial symbiont of Azolla can thus be considered at the ini

Insertion sequences (ISs) are mobile genetic elements found in almost all prokaryotic genomes. They consist of a gene encoding a transposase, surrounded by inverted repeats. The transposase has the ability to excise the IS and insert it elsewhere in the genome, a process referred to as transposition. ISs have high copy numbers in prokaryotes inhabiting “extreme” environments, and it is proposed that their activity facilitates adaptation to environmental changes and subsequent adaptive evolution. The initial step in the transposition of an IS is the transcription of the open reading frame encoding the transposase. In an effort to evaluate the presence, activity and role of ISs in microbes of a temperate water body offering steep changes in salinity and nutrient conditions, the metatranscriptomes and metagenomes of ten water samples from the brackish water Baltic Sea were examined. ISs in the limnic Lake Torne Träsk, the marine waters off the Swedish west coast and off the coast of California were included to get perspective. The results reveal that insertion sequences make up a considerably higher fraction of the metatranscriptomes of brackish waters (0.3-1.8%) than of marine waters (0.0005-0.2%), and that the IS fraction of the metatranscriptome is commonly double that of the IS fraction of the metagenome. From these data it is concluded that ISs occupy a significant part of Baltic Sea bacterial transcription activity, in line with their proposed function as facilitators of adaptive change to changing and stressful environments.