Search

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., (© 2022 Applied Microbiology International and John Wiley & Sons Ltd.)

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., (© 2023. The Author(s), under exclusive license to Springer Science+Business Media, LLC, part of Springer Nature.)

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 ., Competing Interests: Declaration of interests The authors declare no competing interests., (Copyright © 2021 Elsevier Inc. All rights reserved.)

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.

Lytic polysaccharide monooxygenases (LPMOs) are copper-dependent enzymes that oxidatively cleave many of nature's most recalcitrant polysaccharides by acting on the C1- and/or C4-carbon of the glycosidic bond. Here, the results of an extensive mutagenesis study on three LPMO representatives, Phanerochaete chrysosporium LPMO9D (C1-oxidizer), Neurospora crassa LPMO9C (C4), and Hypocrea jecorina LPMO9A (C1/C4), are reported. Using a previously published indicator diagram, the authors demonstrate that several structural determinants of LPMOs play an important role in their oxidative regioselectivity. N-glycan removal and alterations of the aromatic residues on the substrate-binding surface are shown to alter C1/C4-oxidation ratios. Removing the carbohydrate binding module (CBM) is found not to alter the regioselectivity of HjLPMO9A, although the effect of mutational changes is shown to increase in a CBM-free context. The accessibility to the solvent-exposed axial position of the copper-site reveales not to be a major regioselectivity indicator, at least not in PcLPMO9D. Interestingly, a HjLPMO9A variant lacking two surface exposed aromatic residues combines decreased binding capacity with a 22% increase in synergetic efficiency. Similarly to recent LPMO10 findings, our results suggest a complex matrix of surface-interactions that enables LPMO9s not only to bind their substrate, but also to accurately direct their oxidative force., (© 2018 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

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.

Lytic polysaccharide monooxygenases (LPMOs) have changed our understanding of lignocellulosic degradation dramatically over the last years. These metalloproteins catalyze oxidative cleavage of recalcitrant polysaccharides and can act on the C1 and/or C4 position of glycosidic bonds. Structural data have led to several hypotheses, but we are still a long way from reaching complete understanding of the factors that determine their divergent regioselectivity. Site-directed mutagenesis enables the investigation of structure-function relationship in enzymes and will be of major importance in unraveling this intriguing matter. In this context, it is crucial to have an enzyme assay or screening approach with a direct correlation with the desired functionality. LPMOs render this search extra challenging due to their insoluble substrates, complex pattern of reaction products and lack of synthetic standards of most oxidized products. Here, we describe a regioselectivity indicator diagram based on the time-course of only 2 HPAEC-PAD signals. The diagram was successfully used to confirm the hypothesis that aromatic surface residues influence the C1/C4 oxidation ratio in Hypocrea jecorina LPMO9A. Consequently, the diagram should become a valuable tool in the search towards better understanding and engineering of regioselectivity in LPMOs.

Lytic polysaccharide monooxygenases (LPMOs) are crucial components of cellulase mixtures but their stability has not yet been studied in detail, let alone been engineered for industrial applications. In this work, we have evaluated the importance of disulfide bridges for the thermodynamic stability of Streptomyces coelicolor LPMO10C. Interestingly, this enzyme was found to retain 34% of its activity after 2-h incubation at 80°C while its apparent melting temperature (Tm) is only 51°C. When its three disulfide bridges were broken, however, irreversible unfolding occurred and no residual activity could be detected after a similar heat treatment. Based on these findings, additional disulfide bridges were introduced, as predicted by computational tools (MOdelling of DIsulfide bridges in Proteins (MODiP) and Disulfide by Design (DbD)) and using the most flexible positions in the structure as target sites. Four out of 16 variants displayed an improvement in Tm, ranging from 2 to 9°C. Combining the positive mutations yielded additional improvements (up to 19°C) but aberrant unfolding patterns became apparent in some cases, resulting in a diminished capacity for heat resistance. Nonetheless, the best variant, a combination of A143C-P183C and S73C-A115C, displayed a 12°C increase in Tm and was able to retain and was able to retain no less than 60% of its activity after heat treatment., (© The Author 2017. Published by Oxford University Press. All rights reserved. For Permissions, please e-mail: journals.permissions@oup.com.)

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., (Copyright © 2016 Elsevier Ltd. All rights reserved.)

The auxiliary activity family 9 (AA9, formerly GH61) harbors a recently discovered group of oxidative enzymes that boost cellulose degradation. Indeed, these lytic polysaccharide monooxygenases (LPMOs) are able to disrupt the crystalline structure of cellulose, thereby facilitating the work of hydrolytic enzymes involved in biomass degradation. Since these enzymes require an N-terminal histidine residue for activity, their recombinant production as secreted protein is not straightforward. We here report the expression optimization of Trichoderma reesei Cel61A (TrCel61A) in the host Pichia pastoris. The use of the native TrCel61A secretion signal instead of the alpha-mating factor from Saccharomyces cerevisiae was found to be crucial, not only to obtain high protein yields (>400 mg/L during fermentation) but also to enable the correct processing of the N-terminus. Furthermore, the LPMO activity of the enzyme is demonstrated here for the first time, based on its degradation profile of a cellulosic substrate.

(3-Pyrrolin-1-yl)- and (2-oxopyrrolidin-1-yl)quinolines were prepared via cyclization of diallylaminoquinolines and 4-chloro-N-quinolinylbutanamides, respectively, as novel synthetic intermediates en route to N-functionalized 3-, 5-, 6- and 8-aminoquinolines with potential biological activity. (3-Pyrrolin-1-yl)quinolines were subjected to bromination reactions, and the reactivity of (2-oxopyrrolidin-1-yl)quinolines toward lithium aluminum hydride and methyllithium was assessed, providing an entry into a broad range of novel functionalized (pyrrolidin-1-yl)- and (hydroxyalkylamino)quinolines. Antiplasmodial evaluation of these novel quinolines and their functionalized derivatives revealed moderate micromolar potency against a chloroquine-sensitive strain of the malaria parasite Plasmodium falciparum, and the two most potent compounds also showed micromolar activity against a chloroquine-resistant strain of P. falciparum. Antifungal assessment of (hydroxyalkylamino)quinolines revealed three compounds with promising MIC values against Rhodotorula bogoriensis and one compound with potent activity against Aspergillus flavus., (Copyright © 2014 Elsevier Masson SAS. All rights reserved.)