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The mutualistic association between leguminous plants and endosymbiotic rhizobial bacteria is a paradigmatic example of a symbiosis driven by metabolic exchanges. Here, we report the reconstruction and modelling of a genome-scale metabolic network of Medicago truncatula (plant) nodulated by Sinorhizobium meliloti (bacterium). The reconstructed nodule tissue contains five spatially distinct developmental zones and encompasses the metabolism of both the plant and the bacterium. Flux balance analysis (FBA) suggests that the metabolic costs associated with symbiotic nitrogen fixation are primarily related to supporting nitrogenase activity, and increasing N 2 -fixation efficiency is associated with diminishing returns in terms of plant growth. Our analyses support that differentiating bacteroids have access to sugars as major carbon sources, ammonium is the main nitrogen export product of N 2 -fixing bacteria, and N 2 fixation depends on proton transfer from the plant cytoplasm to the bacteria through acidification of the peribacteroid space. We expect that our model, called ‘Virtual Nodule Environment’ (ViNE), will contribute to a better understanding of the functioning of legume nodules, and may guide experimental studies and engineering of symbiotic nitrogen fixation. The association between leguminous plants and rhizobial bacteria is a paradigmatic example of a symbiosis driven by metabolic exchanges. Here, diCenzo et al. report the reconstruction and modelling of a genome-scale metabolic network of the plant Medicago truncatula nodulated by the bacterium Sinorhizobium meliloti .

Dickeya solani causes soft rot and blackleg mainly on potato crops. High pathogenicity of this species results from efficient production of plant cell wall-degrading enzymes, especially pectate lyases, potent root colonization, and fast vascular movement. Despite genomic homogeneity, variations in virulence-related phenotypes suggest differences in the gene expression patterns between diverse strains. Therefore, the methylomes and transcriptomes of two strains (virulent IFB0099 and low virulent IFB0223), differing in tissue maceration capacity and virulence factors production, have been studied. Methylation analysis revealed no significant differences. However, the analysis of transcriptomes, studied under both non-induced and induced by polygalacturonic acid conditions (in order to mimic diverse stages of plant infection process), unveiled higher expression of pectate lyases ( pelD , pelE , pelL ), pectin esterase ( pemA ), proteases ( prtE , prtD ) and Vfm-associated quorum-sensing genes ( vfmC , vfmD , vfmE ) in IFB0099 strain compared to IFB0223. Additionally, the genes related to the secretion system II (T2SS) ( gspJ , nipE ) displayed higher induction of expression in IFB0099. Furthermore, IFB0099 showed more elevated expression of genes involved in flagella formation, which coincides with enhanced motility and pathogenicity of this strain compared to IFB0223. To sum up, differential expression analysis of genes important for the virulence of D. solani indicated candidate genes, which might be crucial for the pathogenicity of this species.

Bacterial genome evolution is characterized by gains, losses, and rearrangements of functional genetic segments. The extent to which large-scale genomic alterations influence genotype-phenotype relationships has not been investigated in a high-throughput manner. In the symbiotic soil bacterium Sinorhizobium meliloti, the genome is composed of a chromosome and two large extrachromosomal replicons (pSymA and pSymB, which together constitute 45% of the genome). Massively parallel transposon insertion sequencing (Tn-seq) was employed to evaluate the contributions of chromosomal genes to growth fitness in both the presence and absence of these extrachromosomal replicons. Ten percent of chromosomal genes from diverse functional categories are shown to genetically interact with pSymA and pSymB. These results demonstrate the pervasive robustness provided by the extrachromosomal replicons, which is further supported by constraint-based metabolic modeling. A comprehensive picture of core S. meliloti metabolism was generated through a Tn-seq-guided in silico metabolic network reconstruction, producing a core network encompassing 726 genes. This integrated approach facilitated functional assignments for previously uncharacterized genes, while also revealing that Tn-seq alone missed over a quarter of wild-type metabolism. This work highlights the many functional dependencies and epistatic relationships that may arise between bacterial replicons and across a genome, while also demonstrating how Tn-seq and metabolic modeling can be used together to yield insights not obtainable by either method alone.

Rhizobia are bacteria that can form symbiotic associations with plants of the Fabaceae family, during which they reduce atmospheric di-nitrogen to ammonia. The symbiosis between rhizobia and leguminous plants is a fundamental contributor to nitrogen cycling in natural and agricultural ecosystems. Rhizobial microsymbionts are a major reason why legumes can colonize marginal lands and nitrogen-deficient soils. Several leguminous species have been found in metal-contaminated areas, and they often harbor metal-tolerant rhizobia. In recent years, there have been numerous efforts and discoveries related to the genetic determinants of metal resistance by rhizobia, and on the effectiveness of such rhizobia to increase the metal tolerance of host plants. Here, we review the main findings on the metal resistance of rhizobia: the physiological role, evolution, and genetic determinants, and the potential to use native and genetically-manipulated rhizobia as inoculants for legumes in phytoremediation practices.

Zophobas morio (=Zophobas atratus) and Tenebrio molitor are darkling beetles with industrial importance due to their use as feeder insects and their apparent ability to biodegrade plastics. High quality genome assemblies were recently reported for both species. Here, we report additional independent Z. morio and T. molitor genome assemblies generated from Nanopore and Illumina data. Following scaffolding against the published genomes, haploid assemblies of 462 Mb (scaffold N90 of 16.8 Mb) and 258 Mb (scaffold N90 of 5.9 Mb) were produced for Z. morio and T. molitor, respectively. Gene prediction led to the prediction of 28,544 and 19,830 genes for Z. morio and T. molitor, respectively. Benchmarking Universal Single Copy Orthologs (BUSCO) analyses suggested that both assemblies have a high level of completeness; 91.5 and 89.0% of the BUSCO endopterygota marker genes were complete in the Z. morio assembly and proteome, respectively, while 99.1 and 92.8% were complete in the T. molitor assembly and proteome, respectively. Phylogenomic analyses of four genera from the family Tenebrionidae yielded phylogenies consistent with those previously constructed based on mitochondrial genomes. Synteny analyses revealed large stretches of macrosynteny across the family Tenebrionidae, as well as numerous within-chromosome rearrangements. Finally, orthogroup analysis identified ∼28,000 gene families across the family Tenebrionidae, of which 8,185 were identified in all five of the analyzed species, and 10,837 were conserved between Z. morio and T. molitor. We expect that the availability of multiple whole genome sequences for Z. morio and T. molitor will facilitate population genetics studies to identify genetic variation associated with industrially relevant phenotypes.

Many bacteria carry two or more chromosome-like replicons. This occurs in pathogens such as Vibrio cholerea and Brucella abortis as well as in many N2-fixing plant symbionts including all isolates of the alfalfa root-nodule bacteria Sinorhizobium meliloti. Understanding the evolution and role of this multipartite genome organization will provide significant insight into these important organisms; yet this knowledge remains incomplete, in part, because technical challenges of large-scale genome manipulations have limited experimental analyses. The distinct evolutionary histories and characteristics of the three replicons that constitute the S. meliloti genome (the chromosome (3.65 Mb), pSymA megaplasmid (1.35 Mb), and pSymB chromid (1.68 Mb)) makes this a good model to examine this topic. We transferred essential genes from pSymB into the chromosome, and constructed strains that lack pSymB as well as both pSymA and pSymB. This is the largest reduction (45.4%, 3.04 megabases, 2866 genes) of a prokaryotic genome to date and the first removal of an essential chromid. Strikingly, strains lacking pSymA and pSymB (ΔpSymAB) lost the ability to utilize 55 of 74 carbon sources and various sources of nitrogen, phosphorous and sulfur, yet the ΔpSymAB strain grew well in minimal salts media and in sterile soil. This suggests that the core chromosome is sufficient for growth in a bulk soil environment and that the pSymA and pSymB replicons carry genes with more specialized functions such as growth in the rhizosphere and interaction with the plant. These experimental data support a generalized evolutionary model, in which non-chromosomal replicons primarily carry genes with more specialized functions. These large secondary replicons increase the organism's niche range, which offsets their metabolic burden on the cell (e.g. pSymA). Subsequent co-evolution with the chromosome then leads to the formation of a chromid through the acquisition of functions core to all niches (e.g. pSymB).

The genome of about 10% of bacterial species is divided among two or more large chromosome-sized replicons. The contribution of each replicon to the microbial life cycle (for example, environmental adaptations and/or niche switching) remains unclear. Here we report a genome-scale metabolic model of the legume symbiont Sinorhizobium meliloti that is integrated with carbon utilization data for 1,500 genes with 192 carbon substrates. Growth of S. meliloti is modelled in three ecological niches (bulk soil, rhizosphere and nodule) with a focus on the role of each of its three replicons. We observe clear metabolic differences during growth in the tested ecological niches and an overall reprogramming following niche switching. In silico examination of the inferred fitness of gene deletion mutants suggests that secondary replicons evolved to fulfil a specialized function, particularly host-associated niche adaptation. Thus, genes on secondary replicons might potentially be manipulated to promote or suppress host interactions for biotechnological purposes. The genome of some bacteria consists of two or more chromosomes or replicons. Here, diCenzo et al . integrate genome-scale metabolic modelling and growth data from a collection of mutants of the plant symbiont Sinorhizobium meliloti to estimate the fitness contribution of each replicon in three environments.

Background Symbiotic nitrogen-fixation between bacteria called rhizobia and leguminous plants is a critical aspect of sustainable agriculture. Complex, two-way communication governs the invasion of plant roots and the formation of nodules in which the rhizobia reduce N 2 to bioavailable ammonia. Research has uncovered many of the genes required for the symbiosis; however, engineering the symbiosis to function with alternative hosts such as cereal crops necessitates the establishment of a core set of symbiotic players. Results We examined the symbiotic relevance of the genes on the 1.68 Mb pSymB chromid of the model rhizobium Sinorhizobium meliloti . By employing a strain in which pSymB was removed, we used a gain-of-function approach to assess a select group of known symbiotic regions totalling 261 kb (15.5%) of pSymB. This gene set enabled symbiotic N 2 -fixation with alfalfa with a high degree of plant genotype-dependent variation in which nodules often senesced prematurely. We demonstrate that additional regions lacking canonical symbiosis genes are important for the efficient formation of symbiosis with the plant host. These regions appear to contain auxiliary symbiotic loci whose genes encode products with quasi-essential functions for the symbiosis and that are redundant in nature. We further established a 673-kb pSymB genome that engages consistently in N 2 -fixation with alfalfa with 45% efficiency. Conclusions The reduction of the pSymB genome showcases the complexity and nuance of its involvement in the N 2 -fixing symbiosis.

To combat the alarming global increase in superbugs amid the simultaneous scarcity of new drugs, we can create synergistic combinations of currently available antibiotics or chimeric molecules with dual activities, to minimize resistance. Here we show that older anti-folate drugs synergize with specific cell wall biosynthesis inhibitors to kill the priority pathogen, Pseudomonas aeruginosa . Anti-folate drugs caused a dose-dependent loss of rod cell shape followed by explosive lysis, and synergized with β-lactams that target D,D-carboxypeptidases required to tailor the cell wall. Anti-folates impaired cell wall recycling and subsequent downstream expression of the chromosomally encoded β-lactamase, AmpC, which normally destroys β-lactam antibiotics. Building on the anti-folate-like scaffold of a metallo-β-lactamase inhibitor, we created a new molecule, MLLB-2201, that potentiates β-lactams and anti-folates and restores meropenem activity against metallo-β-lactamase-expressing Escherichia coli . These strategies are useful ways to tackle the ongoing rise in dangerous bacterial pathogens.

Nitrogen-fixing root nodule symbioses between rhizobia and legume plants provide more than half of the combined nitrogen incorporated annually into terrestrial ecosystems, rendering plant growth independent of environmentally unfriendly chemical fertilizers. The success of symbiosis depends primarily on the capacity of rhizobia to establish competitive populations in soil and rhizosphere environments. The rhizosphere and rhizoplane are nutrient-rich but selective environments for the root microbiome. Here, we deciphered a posttranscriptional network regulated by the homologous trans -small RNAs (sRNAs) AbcR1 and AbcR2, which rewire the metabolism of the nitrogen-fixing α-rhizobium Sinorhizobium meliloti during preinfection stages of symbiosis with its legume host alfalfa. The LysR-type regulator LsrB, which transduces the cell redox state, is indispensable for AbcR1 expression in actively dividing bacteria, whereas the stress-induced transcription of AbcR2 depends on the alternative σ factor RpoH1. MS2 affinity purification coupled with RNA sequencing unveiled exceptionally large and overlapping AbcR1/2 mRNA interactomes, jointly representing ⁓6% of the S. meliloti protein-coding genes. Most mRNAs encode transport/metabolic proteins whose translation is silenced by base pairing to two distinct anti-Shine Dalgarno motifs that function independently in both sRNAs. A metabolic model-aided analysis of the targetomes predicted changes in AbcR1/2 expression driven by shifts in carbon/nitrogen sources, which were confirmed experimentally. Low AbcR1/2 levels in some defined media anticipated overexpression growth phenotypes linked to the silencing of specific mRNAs. As a proof of principle, we confirmed AbcR1/2-mediated downregulation of the l -amino acid AapQ permease. AbcR1/2 interactomes are well represented in rhizosphere-related S. meliloti transcriptomic signatures. Remarkably, a lack of AbcR1 specifically compromised the ability of S. meliloti to colonize the root rhizoplane. The AbcR1 regulon likely ranks the utilization of available substrates to optimize metabolism, thus conferring on S. meliloti an advantage for efficient rhizosphere/rhizoplane colonization. AbcR1 regulation is predicted to be conserved in related α-rhizobia, which opens unprecedented possibilities for engineering highly competitive biofertilizers. IMPORTANCE Nitrogen-fixing root nodule symbioses between rhizobia and legume plants provide more than half of the combined nitrogen incorporated annually into terrestrial ecosystems, rendering plant growth independent of environmentally unfriendly chemical fertilizers. The success of symbiosis depends primarily on the capacity of rhizobia to establish competitive populations in soil and rhizosphere environments. Here, we provide insights into the regulation and architecture of an extensive RNA posttranscriptional network that fine-tunes the metabolism of the alfalfa symbiont S. meliloti , thereby enhancing the ability of this beneficial bacterium to colonize nutrient-rich but extremely selective niches, such as the rhizosphere of its host plant. This pervasive RNA regulation of metabolism is a major adaptive mechanism, predicted to operate in diverse rhizobial species. Because RNA regulation relies on modifiable base-pairing interactions, our findings open unexplored avenues for engineering the legumes rhizobiome within sustainable agricultural practices.