Comparative phylogenomics and phylotranscriptomics provide insights into the genetic complexity of nitrogen-fixing root-nodule symbiosis.

Plant root-nodule symbiosis (RNS) with mutualistic nitrogen-fixing bacteria is restricted to a single clade of angiosperms, the Nitrogen-Fixing Nodulation Clade (NFNC), and is best understood in the legume family. Nodulating species share many commonalities, explained either by divergence from a common ancestor over 100 million years ago or by convergence following independent origins over that same time period. Regardless, comparative analyses of diverse nodulation syndromes can provide insights into constraints on nodulation-what must be acquired or cannot be lost for a functional symbiosis-and the latitude for variation in the symbiosis. However, much remains to be learned about nodulation, especially outside of legumes. Here, we employed a large-scale phylogenomic analysis across 88 species, complemented by 151 RNA-seq libraries, to elucidate the evolution of RNS. Our phylogenomic analyses further emphasize the uniqueness of the transcription factor NIN as a master regulator of nodulation and identify key mutations that affect its function across the NFNC. Comparative transcriptomic assessment revealed nodule-specific upregulated genes across diverse nodulating plants, while also identifying nodule-specific and nitrogen-response genes. Approximately 70% of symbiosis-related genes are highly conserved in the four representative species, whereas defense-related and host-range restriction genes tend to be lineage specific. Our study also identified over 900 000 conserved non-coding elements (CNEs), over 300 000 of which are unique to sampled NFNC species. NFNC-specific CNEs are enriched with the active H3K9ac mark and are correlated with accessible chromatin regions, thus representing a pool of candidate regulatory elements for genes involved in RNS. Collectively, our results provide novel insights into the evolution of nodulation and lay a foundation for engineering of RNS traits in agriculturally important crops.

Identification and evolution of nsLTPs in the root nodule nitrogen fixation clade and molecular response of Frankia to AgLTP24.

Non-specific lipid transfer proteins (nsLTPs) are antimicrobial peptides, involved in several plant biological processes including root nodule nitrogen fixation (RNF). Nodulating plants belonging to the RNF clade establish symbiosis with the nitrogen-fixing bacteria rhizobia (legumes symbiosis model) and Frankia (actinorhizal symbiosis model) leading to root nodule formation. nsLTPs are involved in processes active in early step of symbiosis and functional nodule in both models. In legumes, nsLTPs have been shown to regulate symbiont entry, promote root cortex infection, membrane biosynthesis, and improve symbiosis efficiency. More recently, a nsLTP, AgLTP24 has been described in the context of actinorhizal symbiosis between Alnus glutinosa and Frankia alni ACN14a. AgLTP24 is secreted at an early step of symbiosis on the deformed root hairs and targets the symbiont in the nitrogen-fixing vesicles in functional nodules. nsLTPs are involved in RNF, but their functions and evolutionary history are still largely unknown. Numerous putative nsLTPs were found up-regulated in functional nodules compared to non-infected roots in different lineages within the RNF clade. Here, results highlight that nodulating plants that are co-evolving with their nitrogen-fixing symbionts appear to have independently specialized nsLTPs for this interaction, suggesting a possible convergence of function, which opens perspectives to investigate nsLTPs functions in RNF.

Exploring the Phytobeneficial and Biocontrol Capacities of Endophytic Bacteria Isolated from Hybrid Vanilla Pods.

In this study, 58 endophytic bacterial strains were isolated from pods of two hybrid vanilla plants from Madagascar, Manitra ampotony and Tsy taitra. They were genetically characterized and divided into four distinct phylotypes. Three were associated to genus Bacillus species, and the fourth to the genus Curtobacterium. A selection of twelve strains corresponding to the identified genetic diversity were tested in vitro for four phytobeneficial capacities: phosphate solubilisation, free nitrogen fixation, and phytohormone and siderophore production. They were also evaluated in vitro for their ability to biocontrol the growth of the vanilla pathogenic fungi, Fusarium oxysporum f. sp. radicis vanillae and Cholletotrichum orchidophilum. Three bacteria of phylotype 4, m62a, m64 and m65, showed a high nitrogen fixation capacity in vitro, similar to the Pseudomonas florescens F113 bacterium used as a control (phospate solubilizing efficiency respectively 0.50 ± 0.07, 0.43 ± 0.07 and 0.40 ± 0.06 against 0.48 ± 0.03). Strain t2 related to B. subtilis showed a higher siderophore production than F113 (respectively 1.40 ± 0.1 AU and 1.2 ± 0.1 AU). The strain m72, associated with phylotype 2, showed the highest rate of production of Indole-3-acetic acid (IAA) in vitro. Bacteria belonging to the pylotype 4 showed the best capacity to inhibit fungal growth, especially the strains m62b m64 and t24, which also induced a significant zone of inhibition, suggesting that they may be good candidates for controlling fungal diseases of vanilla. This competence was highlighted with spectral imaging showing the production of lipopeptides (Iturin A2 and A3, C16 and C15-Fengycin A and C14 and C15-Surfactin) by the bacterial strains m65 confronted with the pathogenic fungi of vanilla.

Genomic Insights of Alnus-Infective Frankia Strains Reveal Unique Genetic Features and New Evidence on Their Host-Restricted Lifestyle.

The present study aimed to use comparative genomics to explore the relationships between Frankia and actinorhizal plants using a data set made of 33 Frankia genomes. The determinants of host specificity were first explored for "Alnus-infective strains" (i.e., Frankia strains belonging to Cluster Ia). Several genes were specifically found in these strains, including an agmatine deiminase which could possibly be involved in various functions as access to nitrogen sources, nodule organogenesis or plant defense. Within "Alnus-infective strains", Sp+ Frankia genomes were compared to Sp- genomes in order to elucidate the narrower host specificity of Sp+ strains (i.e., Sp+ strains being capable of in planta sporulation, unlike Sp- strains). A total of 88 protein families were lost in the Sp+ genomes. The lost genes were related to saprophytic life (transcriptional factors, transmembrane and secreted proteins), reinforcing the proposed status of Sp+ as obligatory symbiont. The Sp+ genomes were also characterized by a loss of genetic and functional paralogs, highlighting a reduction in functional redundancy (e.g., hup genes) or a possible loss of function related to a saprophytic lifestyle (e.g., genes involved in gas vesicle formation or recycling of nutrients).

A Nonspecific Lipid Transfer Protein with Potential Functions in Infection and Nodulation.

The response of Alnus glutinosa to Frankia alni ACN14a is driven by several sequential physiological events from calcium spiking and root-hair deformation to the development of the nodule. Early stages of actinorhizal symbiosis were monitored at the transcriptional level to observe plant host responses to Frankia alni. Forty-two genes were significantly upregulated in inoculated compared with noninoculated roots. Most of these genes encode proteins involved in biological processes induced during microbial infection, such as oxidative stress or response to stimuli, but a large number of them are not differentially modulated or downregulated later in the process of nodulation. In contrast, several of them remained upregulated in mature nodules, and this included the gene most upregulated, which encodes a nonspecific lipid transfer protein (nsLTP). Classified as an antimicrobial peptide, this nsLTP was immunolocalized on the deformed root-hair surfaces that are points of contact for Frankia spp. during infection. Later in nodules, it binds to the surface of F. alni ACN14a vesicles, which are the specialized cells for nitrogen fixation. This nsLTP, named AgLTP24, was biologically produced in a heterologous host and purified for assay on F. alni ACN14a to identify physiological effects. Thus, the activation of the plant immunity response occurs upon first contact, while the recognition of F. alni ACN14a genes switches off part of the defense system during nodulation. AgLTP24 constitutes a part of the defense system that is maintained all along the symbiosis, with potential functions such as the formation of infection threads or nodule primordia to the control of F. alni proliferation. [Formula: see text] Copyright © 2022 The Author(s). This is an open access article distributed under the CC BY-NC-ND 4.0 International license.

Implications of carbon catabolite repression for plant-microbe interactions.

Carbon catabolite repression (CCR) plays a key role in many physiological and adaptive responses in a broad range of microorganisms that are commonly associated with eukaryotic hosts. When a mixture of different carbon sources is available, CCR, a global regulatory mechanism, inhibits the expression and activity of cellular processes associated with utilization of secondary carbon sources in the presence of the preferred carbon source. CCR is known to be executed by completely different mechanisms in different bacteria, yeast, and fungi. In addition to regulating catabolic genes, CCR also appears to play a key role in the expression of genes involved in plant-microbe interactions. Here, we present a detailed overview of CCR mechanisms in various bacteria. We highlight the role of CCR in beneficial as well as deleterious plant-microbe interactions based on the available literature. In addition, we explore the global distribution of known regulatory mechanisms within bacterial genomes retrieved from public repositories and within metatranscriptomes obtained from different plant rhizospheres. By integrating the available literature and performing targeted meta-analyses, we argue that CCR-regulated substrate use preferences of microorganisms should be considered an important trait involved in prevailing plant-microbe interactions.

Feedback Regulation of N Fixation in Frankia-Alnus Symbiosis Through Amino Acids Profiling in Field and Greenhouse Nodules.

Symbiosis established between actinorhizal plants and Frankia spp., which are nitrogen-fixing actinobacteria, promotes nodule organogenesis, the site of metabolic exchange. The present study aimed to identify amino acid markers involved in Frankia-Alnus interactions by comparing nodules and associated roots from field and greenhouse samples. Our results revealed a high level of citrulline in all samples, followed by arginine (Arg), aspartate (Asp), glutamate (Glu), γ-amino-n-butyric acid (GABA), and alanine (Ala). Interestingly, the field metabolome approach highlighted more contrasted amino acid patterns between nodules and roots compared with greenhouse samples. Indeed, 12 amino acids had a mean relative abundance significantly different between field nodule and root samples, against only four amino acids in greenhouse samples, underlining the importance of developing "ecometabolome" approaches. In order to monitor the effects on Frankia cells (respiration and nitrogen fixation activities) of amino acid with an abundance pattern evocative of a role in symbiosis, in-vitro assays were performed by supplementing them in nitrogen-free cultures. Amino acids had three types of effects: i) those used by Frankia as nitrogen source (Glu, Gln, Asp), ii) amino acids stimulating both nitrogen fixation and respiration (e.g., Cit, GABA, Ala, valine, Asn), and iii) amino acids triggering a toxic effect (Arg, histidine). In this paper, a N-metabolic model was proposed to discuss how the host plant and bacteria modulate amino acids contents in nodules, leading to a fine regulation sustaining high bacterial nitrogen fixation.

Comparative genomics and proteogenomics highlight key molecular players involved in Frankia sporulation.

Sporulation is a microbial adaptive strategy to resist inhospitable conditions for vegetative growth and to disperse to colonise more favourable environments. This microbial trait is widespread in Actinobacteria. Among them, Frankia strains are able to differentiate sporangia in pure culture, while others can sporulate even when in symbiosis with sporulation occurring within host cells. The molecular determinants controlling Frankia sporulation have not been yet described. In order to highlight, for the first time, the molecular players potentially involved in Frankia sporulation, we conducted (i) a comparison of protein contents between Frankia spores and hyphae and (ii) a comparative genomic analysis of Frankia proteomes with sporulating and non-sporulating Actinobacteria. Among the main results, glycogen-metabolism related proteins, as well as oxidative stress response and protease-like proteins were overdetected in hyphae, recalling lytic processes that allow Streptomyces cells to erect sporogenic hyphae. Several genes encoding transcriptional regulators, including GntR-like, appeared up-regulated in spores, as well as tyrosinase, suggesting their potential role in mature spore metabolism. Finally, our results highlighted new proteins potentially involved in Frankia sporulation, including a pyrophosphate-energized proton pump and YaaT, described as involved in the phosphorelay allowing sporulation in Bacillus subtilis, leading us to discuss the role of a phosphorelay in Frankia sporulation.

Molecular response to nitrogen starvation by Frankia alni ACN14a revealed by transcriptomics and functional analysis with a fosmid library in Escherichia coli.

The transcriptome of Frankia alni strain ACN14a was compared between in vitro ammonium-replete (N-replete) and ammonium-free dinitrogen-fixing (N-fixing) conditions using DNA arrays. A Welch-test (p < 0.05) revealed significant upregulation of 252 genes under N-fixing vs. N-replete (fold-change (FC) ≥ 2), as well as significant downregulation of 48 other genes (FC ≤ 0.5). Interestingly, there were 104 Frankia genes upregulated in vitro that were also significantly upregulated in symbiosis with Alnus glutinosa, while the other 148 genes were not, showing that the physiology of in vitro fixation is markedly different from that under symbiotic conditions. In particular,in vitro fixing cells were seen to upregulate genes identified as coding for a nitrite reductase, and amidases that were not upregulated in symbiosis. Confirmatory assays for nitrite reductase showed that Frankia indeed reduced nitrite and used it as a nitrogen source. An Escherichia coli fosmid clone carrying the nirB region was able to grow better in the presence of 5 mM nitrite than without it, confirming the function of the genome region. The physiological pattern that emerges shows that Frankia undergoes nitrogen starvation that induces a molecular response different from that seen in symbiosis.

Physiological effects of major up-regulated Alnus glutinosa peptides on Frankia sp. ACN14a.

Alnus glutinosa has been shown previously to synthesize, in response to nodulation by Frankia sp. ACN14a, an array of peptides called Alnus symbiotic up-regulated peptides (ASUPs). In a previous study one peptide (Ag5) was shown to bind to Frankia nitrogen-fixing vesicles and to modify their porosity. Here we analyse four other ASUPs, alongside Ag5, to determine whether they have different physiological effects on in vitro grown Frankia sp. ACN14a. The five studied peptides were shown to have different effects on nitrogen fixation, respiration, growth, the release of ions and amino acids, as well as on cell clumping and cell lysis. The mRNA abundance for all five peptides was quantified in symbiotic nodules and one (Ag11) was found to be more abundant in the meristem part of the nodule. These findings point to some peptides having complementary effects on Frankia cells.

The Absence of Pupylation (Prokaryotic Ubiquitin-Like Protein Modification) Affects Morphological and Physiological Differentiation in Streptomyces coelicolor.

UNLABELLED: Protein turnover is essential in all living organisms for the maintenance of normal cell physiology. In eukaryotes, most cellular protein turnover involves the ubiquitin-proteasome pathway, in which proteins tagged with ubiquitin are targeted to the proteasome for degradation. In contrast, most bacteria lack a proteasome but harbor proteases for protein turnover. However, some actinobacteria, such as mycobacteria, possess a proteasome in addition to these proteases. A prokaryotic ubiquitination-like tagging process in mycobacteria was described and was named pupylation: proteins are tagged with Pup (prokaryotic ubiquitin-like protein) and directed to the proteasome for degradation. We report pupylation in another actinobacterium, Streptomyces coelicolor. Both the morphology and life cycle of Streptomyces species are complex (formation of a substrate and aerial mycelium followed by sporulation), and these bacteria are prolific producers of secondary metabolites with important medicinal and agricultural applications. The genes encoding the pupylation system in S. coelicolor are expressed at various stages of development. We demonstrated that pupylation targets numerous proteins and identified 20 of them. Furthermore, we established that abolition of pupylation has substantial effects on morphological and metabolic differentiation and on resistance to oxidative stress. In contrast, in most cases, a proteasome-deficient mutant showed only modest perturbations under the same conditions. Thus, the phenotype of the pup mutant does not appear to be due solely to defective proteasomal degradation. Presumably, pupylation has roles in addition to directing proteins to the proteasome. IMPORTANCE: Streptomyces spp. are filamentous and sporulating actinobacteria, remarkable for their morphological and metabolic differentiation. They produce numerous bioactive compounds, including antifungal, antibiotic, and antitumor compounds. There is therefore considerable interest in understanding the mechanisms by which Streptomyces species regulate their complex physiology and production of bioactive compounds. We studied the role in Streptomyces of pupylation, a posttranslational modification that tags proteins that are then directed to the proteasome for degradation. We demonstrated that the absence of pupylation had large effects on morphological differentiation, antibiotic production, and resistance to oxidative stress in S. coelicolor. The phenotypes of pupylation and proteasome-defective mutants differed and suggest that pupylation acts in a proteasome-independent manner in addition to its role in proteasomal degradation.

Alnus peptides modify membrane porosity and induce the release of nitrogen-rich metabolites from nitrogen-fixing Frankia.

Actinorhizal plant growth in pioneer ecosystems depends on the symbiosis with the nitrogen-fixing actinobacterium Frankia cells that are housed in special root organs called nodules. Nitrogen fixation occurs in differentiated Frankia cells known as vesicles. Vesicles lack a pathway for assimilating ammonia beyond the glutamine stage and are supposed to transfer reduced nitrogen to the plant host cells. However, a mechanism for the transfer of nitrogen-fixation products to the plant cells remains elusive. Here, new elements for this metabolic exchange are described. We show that Alnus glutinosa nodules express defensin-like peptides, and one of these, Ag5, was found to target Frankia vesicles. In vitro and in vivo analyses showed that Ag5 induces drastic physiological changes in Frankia, including an increased permeability of vesicle membranes. A significant release of nitrogen-containing metabolites, mainly glutamine and glutamate, was found in N2-fixing cultures treated with Ag5. This work demonstrates that the Ag5 peptide is central for Frankia physiology in nodules and uncovers a novel cellular function for this large and widespread defensin peptide family.

Replication fork reversal after replication-transcription collision.

Replication fork arrest is a recognized source of genetic instability, and transcription is one of the most prominent causes of replication impediment. We analyze here the requirement for recombination proteins in Escherichia coli when replication-transcription head-on collisions are induced at a specific site by the inversion of a highly expressed ribosomal operon (rrn). RecBC is the only recombination protein required for cell viability under these conditions of increased replication-transcription collisions. In its absence, fork breakage occurs at the site of collision, and the resulting linear DNA is not repaired and is slowly degraded by the RecJ exonuclease. Lethal fork breakage is also observed in cells that lack RecA and RecD, i.e. when both homologous recombination and the potent exonuclease V activity of the RecBCD complex are inactivated, with a slow degradation of the resulting linear DNA by the combined action of the RecBC helicase and the RecJ exonuclease. The sizes of the major linear fragments indicate that DNA degradation is slowed down by the encounter with another rrn operon. The amount of linear DNA decreases nearly two-fold when the Holliday junction resolvase RuvABC is inactivated in recB, as well as in recA recD mutants, indicating that part of the linear DNA is formed by resolution of a Holliday junction. Our results suggest that replication fork reversal occurs after replication-transcription head-on collision, and we propose that it promotes the action of the accessory replicative helicases that dislodge the obstacle.

The helicases DinG, Rep and UvrD cooperate to promote replication across transcription units in vivo.

How living cells deal with head-on collisions of the replication and transcription complexes has been debated for a long time. Even in the widely studied model bacteria Escherichia coli, the enzymes that take care of such collisions are still unknown. We report here that in vivo, the DinG, Rep and UvrD helicases are essential for efficient replication across highly transcribed regions. We show that when rRNA operons (rrn) are inverted to face replication, the viability of the dinG mutant is affected and over-expression of RNase H rescues the growth defect, showing that DinG acts in vivo to remove R-loops. In addition, DinG, Rep and UvrD exert a common function, which requires the presence of two of these three helicases. After replication blockage by an inverted rrn, Rep in conjunction with DinG or UvrD removes RNA polymerase, a task that is fulfilled in its absence by the SOS-induced DinG and UvrD helicases. Finally, Rep and UvrD also act at inverted sequences other than rrn, and promote replication through highly transcribed regions in wild-type E. coli.

Recombination proteins and rescue of arrested replication forks.

Recombination proteins play crucial roles in the rescue of inactivated replication forks in Escherichia coli. The enzymes that catalyze the repair of DNA double-strand breaks by a classical strand-exchange reaction (RecBCD, RecA) act in two well-characterized fork repair pathways. They repair the DNA double-strand end made when a replication fork runs into a single-strand interruption. They reset the DNA double-strand end generated by replication fork reversal when a component of the replication machinery is inactivated. In addition, recombination proteins also act at replication forks in ways other than the classical strand-exchange reaction. For example, the RuvAB enzyme that catalyzes Holliday junction branch-migration during homologous recombination is also able to catalyze replication fork reversal in certain replication mutants, i.e. to convert certain blocked replication forks into Holliday junctions. Finally, some of the actions of recombination proteins after replication impairment are still unclear, as for example in UV-irradiated cells, where RecFOR and RecA catalyze gap repair but also participate, in a yet undefined way, in "replisome reactivation".

Development of metagenomic DNA shuffling for the construction of a xenobiotic gene.

We describe a metagenomic DNA shuffling process by combining protein engineering process mutation generator and the high potential diversity of metagenomic DNA derived from the environment. Numerous previous shuffling processes attempted to recombine more or less related parental sequences. At the same time, metagenomic approaches unveiled a huge diversity of DNA sequences and genomes, which have not yet been identified to date. In this study, we attempted to combine these two approaches in order to regenerate a novel gene. Here, we present the possibility that DNA fragments from an entire microbial community (metagenome) might be available for the creation of novel genes capable of degrading pollutants. Metagenomic DNA extracted from non-polluted soil was shuffled in vitro to recreate the linA gene responsible for the first steps of lindane degradation. In this work, 74% of the ORF came from separate subsets of the metagenomic pool from a lindane-free and linA-free soil. Our results demonstrate that microbial community genetic diversity can serve as a source for novel gene construction during in vitro manipulation. This in vitro gene construction might also simulate the mosaic nature of novel genes. This demonstration might lead to other attempts to mimic bacterial adaptation and to construct degradative genes for novel compounds not yet released into the environment.

Plasmid-encoded gamma-hexachlorocyclohexane degradation genes and insertion sequences in Sphingobium francense (ex-Sphingomonas paucimobilis Sp+).

The lin genes encode the gamma-hexachlorocyclohexane (gamma-HCH or lindane) catabolic pathway in lindane-degrading strains. The location and stability of these genes have been explored in the lindane-degrading Sphingobium francense strain Sp+, and in two non-lindane-degrading mutants (Sp1- and Sp2-). The lin genes, linA, linB, linE and linX were localized by hybridization on three of the six plasmids of the S. francense strain Sp+ showing dispersal within the genome. The linC gene was detected by PCR, but was not detected by hybridization on any of the plasmids. The hybridization of the linA and linX genes was negative with the two non-lindane-degrading mutants S. francense strains, Sp1- and Sp2-. The dynamic of this genome associated with gene loss and acquisition, and plasmid rearrangement was explored by a search for associated insertion sequences. A new insertion sequence, ISSppa4, belonging to the IS21 family was detected and compared with IS6100 and ISsp1. Insertion sequence localization was explored on different hybridization patterns (plasmid, total genome) with the lindane-degrading Sp+ strain and the two non-degrading derivatives (Sp1-, Sp2-). Insertion sequence movement and plasmid rearrangement could explain the emergence of the non-lindane-degrading mutants.