Cyclitol metabolism is a central feature of Burkholderia leaf symbionts.

The symbioses between plants of the Rubiaceae and Primulaceae families with Burkholderia bacteria represent unique and intimate plant-bacterial relationships. Many of these interactions have been identified through PCR-dependent typing methods, but there is little information available about their functional and ecological roles. We assembled 17 new endophyte genomes representing endophytes from 13 plant species, including those of two previously unknown associations. Genomes of leaf endophytes belonging to Burkholderia s.l. show extensive signs of genome reduction, albeit to varying degrees. Except for one endophyte, none of the bacterial symbionts could be isolated on standard microbiological media. Despite their taxonomic diversity, all endophyte genomes contained gene clusters linked to the production of specialized metabolites, including genes linked to cyclitol sugar analog metabolism and in one instance non-ribosomal peptide synthesis. These genes and gene clusters are unique within Burkholderia s.l. and are likely horizontally acquired. We propose that the acquisition of secondary metabolite gene clusters through horizontal gene transfer is a prerequisite for the evolution of a stable association between these endophytes and their hosts.

Whole-Genome Sequencing of Bacterial Endophytes From Fresh and Preserved Plant Specimens.

Many plants harbor symbiotic bacteria in their leaves, sometimes within structures visible with the naked eye. These bacteria play critical roles for host development and defense, but are often not amenable to culture. Gaining insight into the functions of these obligate endophytic bacteria hinges on culture-independent omics approaches, which have seen tremendous development in recent years. We describe in this chapter a set of protocols for the extraction and bioinformatic analysis of bacterial genomic DNA from leaf samples of various origins, including fresh, silica-preserved, or herbarium specimens.

Patterns of transmission and horizontal gene transfer in the Dioscorea sansibarensis leaf symbiosis revealed by whole-genome sequencing.

Leaves of the wild yam species Dioscorea sansibarensis display prominent forerunner or "drip" tips filled with extracellular bacteria of the species Orrella dioscoreae.1 This species of yam is native to Madagascar and tropical Africa and reproduces mainly asexually through aerial bulbils and underground tubers, which also contain a small population of O. dioscoreae.2,3 Despite apparent vertical transmission, the genome of O. dioscoreae does not show any of the hallmarks of genome erosion often found in hereditary symbionts (e.g., small genome size and accumulation of pseudogenes).4-6 We investigated here the range and distribution of leaf symbiosis between D. sansibarensis and O. dioscoreae using preserved leaf samples from herbarium collections that were originally collected from various locations in Africa. We recovered DNA from the extracellular symbiont in all samples, showing that the symbiosis is widespread throughout continental Africa and Madagascar. Despite the degraded nature of this DNA, we constructed 17 symbiont genomes using de novo methods without relying on a reference. Phylogenetic and genomic analyses revealed that horizontal transmission of symbionts and horizontal gene transfer have shaped the evolution of the symbiont. These mechanisms could help explain lack of signs of reductive genome evolution despite an obligate host-associated lifestyle. Furthermore, phylogenetic analysis of D. sansibarensis based on plastid genomes revealed a strong geographical clustering of samples and provided evidence that the symbiosis originated at least 13 mya, earlier than previously estimated.3.

Adaptations and evolution of a heritable leaf nodule symbiosis between Dioscorea sansibarensis and Orrella dioscoreae.

Various plant species establish intimate symbioses with bacteria within their aerial organs. The bacteria are contained within nodules or glands often present in distinctive patterns on the leaves in what is commonly referred to as leaf nodule symbiosis. We describe here a highly specific symbiosis between a wild yam species from Madagascar, Dioscorea sansibarensis and bacteria of the species Orrella dioscoreae. Using whole-genome sequencing of plastids and bacteria from wild-collected samples, we show phylogenetic patterns consistent with a dominant vertical mode of transmission of the symbionts. Unique so far among leaf nodule symbioses, the bacteria can be cultured and are amenable to comparative transcriptomics, revealing a potential role in complementing the host's arsenal of secondary metabolites. We propose a recent establishment of a vertical mode of transmission in this symbiosis which, together with a large effective population size explains the cultivability and apparent lack of genome reductive evolution in O. dioscoreae. We leverage these unique features to reveal pathways and functions under positive selection in these specialized endophytes, highlighting the candidate mechanisms enabling a permanent association in the phyllosphere.

Patterns of Nucleotide Deletion and Insertion Inferred from Bacterial Pseudogenes.

Pseudogenes are a paradigm of neutral evolution and their study has the potential to reveal intrinsic mutational biases. However, this potential is mitigated by the fact that pseudogenes are quickly purged from bacterial genomes. Here, we assembled a large set of pseudogenes from genomes experiencing reductive evolution as well as functional references for which we could establish reliable phylogenetic relationships. Using this unique dataset, we identified 857 independent insertion and deletion mutations and discover a pervasive bias towards deletions, but not insertions, with sizes multiples of 3 nt. We further show that selective constraints for the preservation of gene frame are unlikely to account for the observed mutational bias and propose that a mechanistic bias in alternative end-joining repair, a recombination-independent double strand break DNA repair mechanism, is responsible for the accumulation of 3n deletions.

Nutrient removal from horticultural wastewater by benthic filamentous algae Klebsormidium sp., Stigeoclonium spp. and their communities: From laboratory flask to outdoor Algal Turf Scrubber (ATS).

Benthic filamentous algae have evident advantages in wastewater treatment over unicellular microalgae, including the ease in harvesting and resistance to predation. To assess the potentials of benthic filamentous algae in treating horticultural wastewater under natural conditions in Belgium, three strains and their mixture with naturally wastewater-borne microalgae were cultivated in 250 ml Erlenmeyer flasks in laboratory as well as in 1 m(2) scale outdoor Algal Turf Scrubber (ATS) with different flow rates. Stigeoclonium competed well with the natural wastewater-borne microalgae and contributed to most of the biomass production both in Erlenmeyer flasks and outdoor ATS at flow rates of 2-6 L min(-1) (water velocity 3-9 cm s(-1)), while Klebsormidium was not suitable for growing in horticultural wastewater under the tested conditions. Flow rate had great effects on biomass production and nitrogen removal, while phosphorus removal was less influenced by flow rate due to other mechanisms than assimilation by algae.