Biology subjects

diCenzo, G. C.

Publications and source records attributed to diCenzo, G. C..

33 records · Page 2Linked to original sources

bioRxiv2022

Whole genome assemblies of Zophobas morio and Tenebrio molitor

Zophobas morio (=Zophobas atratus) and Tenebrio molitor are darkling beetles with industrial importance due to their use as feeder insects, their potential for use in aquafeed and human food products, and their apparent ability to biodegrade various plastic polymers. We report draft genome assemblies for Z. morio and T. molitor generated from Nanopore and Illumina data. Following scaffolding against 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. BUSCO analyses suggested 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.

genomics↗

bioRxiv2022

Whole genome sequencing of three mesorhizobia isolated from northern Canada to identify genomic adaptations promoting nodulation in cold climates

The N2-fixing symbiosis between rhizobia and legumes is negatively impacted by numerous stresses, including low temperatures. To identify genomic features and biochemical pathways of rhizobia that could foster improved symbiotic function under low temperatures, we isolated and characterized three Mesorhizobium strains from legume nodules collected at two distant northern Canadian sites. Whereas the classical determinants of nodulation and nitrogen fixation are located on the chromosome of most mesorhizobia, whole genome sequencing revealed that these genes are on a large symbiotic megaplasmid in all three of the newly isolated strains. A pangenome-wide association study identified 25 genes putatively associated with mesorhizobia isolated from arctic or subarctic environments, with the genomic location of many of these genes implying a relationship with legume symbiosis. Phylogenetic and sequence analyses of the common nodulation genes revealed alleles that are highly conserved amongst mesorhizobia isolated from northern climates but uncommon in mesorhizobia isolated from similar plant hosts in other climatic regions, suggesting potential functional adaptive differences and the horizontal transfer of these alleles between northern rhizobia. We speculate that nod sequence divergence was driven by climatic factors, and that the encoded proteins may be particularly stable and/or active at low temperatures.

microbiology↗

bioRxiv2022

Brachypodium antifreeze protein gene products inhibit ice recrystallization, attenuate ice nucleation, and reduce immune response

Antifreeze proteins (AFPs) from the model crop, Brachypodium distachyon, allow freeze survival and attenuate pathogen-mediated ice nucleation. Intriguingly, each Brachypodium AFP gene encodes two proteins, an autonomous AFP and a leucine-rich repeat (LRR). We present structural models suggesting that ice-binding motifs on the ~13 kDa AFPs can "spoil" nucleating arrays on the ~120 kDa bacterial ice nucleating proteins that form ice at high sub-zero temperatures, consistent with decreases in ice nucleating activity by lysates from wildtype compared to transgenic Brachypodium lines. Strikingly, the expression of Brachypodium LRRs in transgenic Arabidopsis inhibited an immune response to pathogen flagella peptides (flg22), with structural modelling suggesting this was due to affinity of the LRR domains to flg22. Thus, these genes play distinctive roles in connecting freeze survival and anti-pathogenic systems via their encoded proteins ability to adsorb to ice as well as attenuating bacterial ice nucleation and the host immune response.

plant biology↗

bioRxiv2022

Cold-inducible promoter-driven knockdown of Brachypodium antifreeze proteins confers freeze sensitivity

The model forage crop, Brachypodium distachyon, has a family of ice recrystallization inhibition (BdIRI) genes, which encode antifreeze proteins that function by adsorbing to ice crystals and inhibiting their growth. The genes were previously targeted for knockdown using a constitutive CaMV 35S promoter and the resulting transgenic Brachypodium showed reduced antifreeze activity and a greater susceptibility to freezing. However, the transgenic plants also showed developmental defects with shortened stem lengths and were almost completely sterile, raising the possibility that their reduced freeze tolerance could be attributed to developmental deficits. A cold-induced promoter from rice (prOsMYB1R35) has now been substituted for the constitutive promoter to generate temporal miRNA-mediated Brachypodium antifreeze protein knockdowns. Although transgenic lines showed no apparent pleiotropic developmental defects, they demonstrated reduced antifreeze activity as assessed by assays for ice-recrystallization inhibition, thermal hysteresis, electrolyte leakage, leaf infrared thermography, and leaf damage after infection with an ice nucleating phytopathogen. Strikingly, the number of cold-acclimated transgenic plants that survived freezing at -8 {degrees}C was reduced by half or killed entirely, depending on the line, compared to cold-acclimated wild type plants. Although these proteins have been studied for almost 60 years, this is the first unequivocal demonstration in any organism of the utility of antifreeze protein function and their contribution to freeze protection, independent of obvious developmental defects. These proteins are thus of potential interest in a wide range of biotechnological applications from accessible cryopreservation, to frozen product additives, to the engineering of transgenic crops with enhanced freezing tolerance.

plant biology↗

bioRxiv2022

Reference nodule transcriptomes for Melilotus officinalis and Medicago sativa cv. Algonquin

Host/symbiont compatibility is a hallmark of the symbiotic nitrogen-fixing interaction between rhizobia and legumes, mediated in part by plant produced nodule-specific cysteine-rich (NCR) peptides and the bacterial BacA membrane protein that can act as a NCR peptide transporter. In addition, the genetic and metabolic properties supporting symbiotic nitrogen fixation often differ between compatible partners, including those sharing a common partner, highlighting the need for multiple study systems. Here, we report high quality nodule transcriptome assemblies for Medicago sativa cv. Algonquin and Melilotus officinalis, two legumes able to form compatible symbioses with Sinorhizobium meliloti. The compressed M. sativa and M. officinalis assemblies consisted of 79,978 and 64,593 contigs, respectively, of which 33,341 and 28,278 were assigned putative annotations, respectively. As expected, the two transcriptomes showed broad similarity at a global level. We were particularly interested in the NCR peptide profiles of these plants, as these peptides drive bacterial differentiation during the symbiosis. A total of 412 and 308 NCR peptides were predicted from the M. sativa and M. officinalis transcriptomes, respectively, with approximately 9% of the transcriptome of both species consisting of NCR transcripts. Notably, transcripts encoding highly-cationic NCR peptides (isoelectric point > 9.5), which are known to have antimicrobial properties, were ~2-fold more abundant in M. sativa than in M. officinalis, and ~27-fold more abundant when considering only NCR peptides in the six-cysteine class. We hypothesize that the difference in abundance of highly-cationic NCR peptides explains our previous observation that some rhizobial bacA alleles which can support symbiosis with M. officinalis are unable to support symbiosis with M. sativa.

plant biology↗

bioRxiv2021

Taxonomy of Rhizobiaceae revisited: proposal of a new framework for genus delimitation

The alphaproteobacterial family Rhizobiaceae is highly diverse, with 168 species with validly published names classified into 17 genera with validly published names. Most named genera in this family are delineated based on genomic relatedness and phylogenetic relationships, but some historically named genera show inconsistent distribution and phylogenetic breadth. Most problematic is Rhizobium, which is notorious for being highly paraphyletic, as most newly described species in the family being assigned to this genus without consideration for their proximity to existing genera, or the need to create novel genera. In addition, many Rhizobiaceae genera lack synapomorphic traits that would give them biological and ecological significance. We propose a common framework for genus delimitation within the family Rhizobiaceae. We propose that genera in this family should be defined as monophyletic groups in a core-genome gene phylogeny, that are separated from related species using a pairwise core-proteome average amino acid identity (cpAAI) threshold of approximately 86%. We further propose that the presence of additional genomic or phenotypic evidence can justify the division of species into separate genera even if they all share greater than 86% cpAAI. Applying this framework, we propose to reclassify Rhizobium rhizosphaerae and Rhizobium oryzae into the new genus Xaviernesmea gen. nov. Data is also provided to support the recently proposed genus "Peteryoungia", and the reclassifications of Rhizobium yantingense as Endobacterium yantingense comb. nov., Rhizobium petrolearium as Neorhizobium petrolearium comb. nov., Rhizobium arenae as Pararhizobium arenae comb. nov., Rhizobium tarimense as Pseudorhizobium tarimense comb. nov., and Rhizobium azooxidefex as Mycoplana azooxidifex comb. nov. Lastly, we present arguments that the unification of the genera Ensifer and Sinorhizobium in Opinion 84 of the Judicial Commission is no longer justified by current genomic and phenotypic data. We thus argue that the genus Sinorhizobium is not illegitimate and now encompasses 17 species.

microbiology↗

bioRxiv2021

The Brachypodium distachyon cold-acclimated plasma membrane proteome is primed for stress resistance

In order to survive sub-zero temperatures, some plants undergo cold acclimation where low, non-freezing temperatures and/or shortened day lengths allow cold hardening and survival during subsequent freeze events. Central to this response is the plasma membrane, where low-temperature is perceived and cellular homeostasis must be preserved by maintaining membrane integrity. Here, we present the first plasma membrane proteome of cold-acclimated Brachypodium distachyon, a model species for the study of monocot crops. A time course experiment investigated cold acclimation-induced changes in the proteome following two-phase partitioning plasma membrane enrichment and label-free quantification by nano-liquid chromatography mass spectrophotometry. Two days of cold acclimation were sufficient for membrane protection as well as an initial increase in sugar levels, and coincided with a significant change in the abundance of 154 proteins. Prolonged cold acclimation resulted in further increases in soluble sugars and abundance changes in more than 680 proteins, suggesting both a necessary early response to low-temperature treatment, as well as a sustained cold acclimation response elicited over several days. A meta-analysis revealed that the identified plasma membrane proteins have known roles in low-temperature tolerance, metabolism, transport, and pathogen defense as well as drought, osmotic stress and salt resistance suggesting crosstalk between stress responses, such that cold acclimation may prime plants for other abiotic and biotic stresses. The plasma membrane proteins identified here present keys to an understanding of cold tolerance in monocot crops and the hope of addressing economic losses associated with modern climate-mediated increases in frost events.

plant biology↗

bioRxiv2021

DNA methylation patterns in bacteria of the genus Ensifer during free living growth and during nitrogen-fixing symbiosis with Medicago spp.

Methylation of specific DNA sequences is ubiquitous in bacteria and has known roles in immunity and regulation of cellular processes, such as the cell cycle. Using single-molecule real-time sequencing, six genome-wide methylated motifs were identified across four Ensifer strains, five of which were strain-specific. Only the GANTC motif, recognized by the cell cycle-regulated CcrM methyltransferase, was methylated in all strains. In actively dividing cells, methylation of GANTC motifs increased progressively from the ori to ter regions in each replicon, in agreement with a cell cycle-dependent regulation of CcrM. In contrast, there was near full genome-wide GANTC methylation in the early stage of symbiotic differentiation. This was followed by a moderate decrease in the overall extent methylation and a progressive decrease in chromosomal GANTC methylation from the ori to ter regions in later stages of differentiation. We interpret these observations as evidence of dysregulated and constitutive CcrM activity during terminal differentiation, and we hypothesize that it is a driving factor for endoreduplication of terminally differentiated bacteroids.

microbiology↗

bioRxiv2020

Proteobacteria encode diverse flg22 peptides that elicit varying immune responses in Arabidopsis thaliana

Bacterial flagellin protein is a potent microbe-associated molecular pattern. Immune responses are triggered by a 22 amino acid epitope derived from flagellin, known as flg22, upon detection by the pattern recognition receptor FLAGELLIN-SENSING2 (FLS2) in multiple plant species. However, increasing evidence suggests that flg22 epitopes of several bacterial species are not universally immunogenic to plants. We investigated whether flg22 immunogenicity systematically differs between classes of the phylum Proteobacteria, using a dataset of 2,470 flg22 sequences. To predict which species encode highly immunogenic flg22 epitopes, we queried a custom motif (11[ST]xx[DN][DN]xAGxxI21) in the flg22 sequences, followed by sequence conservation analysis and protein structural modelling. These data led us to hypothesize that most flg22 epitopes of the {gamma}- and {beta}-Proteobacteria are highly immunogenic, whereas most flg22 epitopes of the -, {delta}-, and {varepsilon}-Proteobacteria are weakly to moderately immunogenic. To test this hypothesis, we generated synthetic peptides representative of the flg22 epitopes of each proteobacterial class, and we monitored their ability to elicit an immune response in Arabidopsis thaliana. Flg22 peptides of the {gamma}- and {beta}-Proteobacteria triggered strong oxidative bursts, whereas peptides from the {varepsilon}-, {delta}-, and -Proteobacteria triggered moderate, weak, or no response, respectively. These data suggest flg22 immunogenicity is not highly conserved across the phylum Proteobacteria. We postulate that sequence divergence of each taxonomic class was present prior to the evolution of FLS2, and that the ligand specificity of A. thaliana FLS2 was driven by the flg22 epitopes of the {gamma}- and {beta}-proteobacteria, a monophyletic group containing many common phytopathogens.

plant biology↗

bioRxiv2020

Dissecting transcriptomic signatures of genotype x genotype interactions during the initiation of plant-rhizobium symbiosis

Rhizobia are ecologically important, facultative plant symbiotic microbes. In nature there exists large variability in the association of rhizobial strains and host plant of the same species. Here, we evaluated whether plant and rhizobial genotypes influence the initial transcriptional response of rhizobium following perception of host plant. RNA-sequencing of the model rhizobium Sinorhizobium meliloti exposed to root exudates or luteolin was performed in a combination of three S. meliloti strains and three Medicago sativa varieties. The response to root exudates involved hundreds of changes in the rhizobium transcriptome. Of the differentially expressed genes, expression of 35% were influenced by strain genotype, 16% by the plant genotype, and 29% by strain x host plant genotype interactions. We also examined the response of a hybrid S. meliloti strain, in which the symbiotic megaplasmid (~ 20% of the genome) was mobilized between two of the above-mentioned strains. Dozens of genes resulted up-regulated in the hybrid strain, indicative of nonadditive variation in the transcriptome. In conclusion, this study demonstrated that transcriptional responses of rhizobia upon perception of legumes is influenced by the genotypes of both symbiotic partners, and their interaction, suggesting a wide genetic spectrum of partner choice selection in plant-rhizobium symbiosis.

microbiology↗

bioRxiv2020

Symbiotic and non-symbiotic members of the genus Ensifer (syn. Sinorhizobium) are separated into two clades based on comparative genomics and high-throughput phenotyping

Rhizobium - legume symbioses serve as a paradigmatic example for the study of mutualism evolution. The genus Ensifer (syn. Sinorhizobium) contains diverse plant-associated bacteria, a subset of which can fix nitrogen in symbiosis with legumes. To gain insights into the evolution of symbiotic nitrogen fixation (SNF), and inter-kingdom mutualisms more generally, we performed extensive phenotypic, genomic, and phylogenetic analyses of the genus Ensifer. The data suggest that SNF emerged several times within the genus Ensifer, likely through independent horizontal gene transfer events. Yet, the majority (105 of 106) of the Ensifer strains with the nodABC and nifHDK nodulation and nitrogen fixation genes were found within a single, monophyletic clade. Comparative genomics highlighted several differences between the "symbiotic" and "non-symbiotic" clades, including divergences in their pangenome content. Additionally, strains of the symbiotic clade carried 325 fewer genes, on average, and appeared to have fewer rRNA operons than strains of the non-symbiotic clade. Characterizing a subset of ten Ensifer strains identified several phenotypic differences between the clades. Strains of the non-symbiotic clade could catabolize 25% more carbon sources, on average, than strains of the symbiotic clade, and they were better able to grow in LB medium and tolerate alkaline conditions. On the other hand, strains of the symbiotic clade were better able to tolerate heat stress and acidic conditions. We suggest that these data support the division of the genus Ensifer into two main subgroups, as well as the hypothesis that pre-existing genetic features are required to facilitate the evolution of SNF in bacteria.

microbiology↗

bioRxiv2019

A Virtual Nodule Environment (ViNE) for modelling the inter-kingdom metabolic integration during symbiotic nitrogen fixation

Biological associations are often premised upon metabolic cross-talk between the organisms, with the N2-fixing endosymbiotic relationship between rhizobia and leguminous plants being a prime example. Here, we report the in silico reconstruction of a metabolic network of a Medicago truncatula plant nodulated by the bacterium Sinorhizobium meliloti. The nodule tissue of the model contains five spatially distinct developmental zones and encompasses the metabolism of both the plant and the bacterium. Flux balance analysis (FBA) suggested that the majority of the metabolic costs associated with symbiotic nitrogen fixation are directly related to supporting nitrogenase activity, while a minority is related to the formation and maintenance of nodule and bacteroid tissue. Interestingly, FBA simulations suggested there was a non-linear relationship between the rate of N2-fixation per gram of nodule and the rate of plant growth; increasing the N2-fixation efficiency was associated with diminishing returns in terms of plant growth. Evaluating the metabolic exchange between the symbiotic partners provided support for: i) differentiating bacteroids having access to sugars (e.g., sucrose) as a major carbon source, ii) ammonium being the major nitrogen export product of N2-fixing bacteria, and iii) N2-fixation being dependent on the transfer of protons from the plant cytoplasm to the bacteria through acidification of the peribacteroid space. Our simulations further suggested that the use of C4-dicarboxylates by N2-fixing bacteroids may be, in part, a consequence of the low concentration of free oxygen in the nodule limiting the activity of the plant mitochondria. These results demonstrate the power of this integrated model to advance our understanding of the functioning of legume nodules, and its potential for hypothesis generation to guide experimental studies and engineering of symbiotic nitrogen fixation.

systems biology↗

bioRxiv2018

Emergence and features of the multipartite genome structure of the family Burkholderiaceae revealed through comparative and evolutionary genomics

The multipartite genome structure is found in a diverse group of important symbiotic and pathogenic bacteria; however, the advantage of this genome structure remains incompletely understood. Here, we perform comparative genomics of hundreds of finished {beta}-proteobacterial genomes to study the role and emergence of multipartite genomes. Nearly all essential secondary replicons (chromids) of the {beta}-proteobacteria are found in the family Burkholderiaceae. These replicons arose from just two plasmid acquisition events, and they were likely stabilized early in their evolution by the presence of core genes, at least some of which were likely acquired through an inter-replicon translocation event. On average, Burkholderiaceae genera with multipartite genomes had a larger total genome size, but smaller chromosome, than genera without secondary replicons. Pangenome-level functional enrichment analyses suggested that inter-replicon functional biases are partially driven by the enrichment of secondary replicons in the accessory pangenome fraction. Nevertheless, the small overlap in orthologous groups present in each replicons pangenome indicates a clear functional separation of the replicons. Chromids appeared biased to environmental adaptation, as the functional categories enriched on chromids were also over-represented on the chromosomes of the environmental genera (Paraburkholderia, Cupriavidus) compared to the pathogenic genera (Burkholderia, Ralstonia). Using ancestral state reconstruction, it was predicted that the rate of accumulation of modern-day genes by chromids was more rapid than the rate of gene accumulation by the chromosomes. Overall, the data are consistent with a model where the primary advantage of secondary replicons is in facilitating increased rates of gene acquisition through horizontal gene transfer, consequently resulting in a replicon enriched in genes associated with adaptation to novel environments.

genomics↗

bioRxiv2018

Creation and multi-omics characterization of a genomically hybrid strain in the nitrogen-fixing symbiotic bacterium Sinorhizobium meliloti

Many bacteria, often associated with eukaryotic hosts and of relevance for biotechnological applications, harbour a multipartite genome composed by more than one replicon. Biotechnologically relevant phenotypes are often encoded by genes residing on the secondary replicons. A synthetic biology approach to developing enhanced strains for biotechnological purposes could therefore involve merging pieces or entire replicons from multiple strains into a single genome. Here we report the creation of a genomic hybrid strain in a model multipartite genome species, the plant-symbiotic bacterium Sinorhizobium meliloti. In particular, we moved the secondary replicon pSymA (accounting for nearly 20% of total genome content) from a donor S. meliloti strain to an acceptor strain. The cis-hybrid strain was screened for a panel of complex phenotypes (carbon/nitrogen utilization phenotypes, intra- and extra-cellular metabolomes, symbiosis, and various microbiological tests). Additionally, metabolic network reconstruction and constraint-based modelling were employed for in silico prediction of metabolic flux reorganization. Phenotypes of the cis-hybrid strain were in good agreement with those of both parental strains. Interestingly, the symbiotic phenotype showed a marked cultivar-specific improvement with the cis-hybrid strains compared to both parental strains. These results provide a proof-of-principle for the feasibility of genome-wide replicon-based remodelling of bacterial strains for improved biotechnological applications in precision agriculture.

synthetic biology↗

bioRxiv2017

Robustness encoded across essential and accessory replicons in an ecologically versatile bacterium

Bacterial genome evolution is characterized by gains, losses, and rearrangements of functional genetic segments. The extent to which genotype-phenotype relationships are influenced by large-scale genomic alterations 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 contributions of chromosomal genes to 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 modelling. 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 misses over a quarter of wild type metabolism. This work highlights the strong functional dependencies and epistatic relationships that may arise between bacterial replicons and across a genome, while also demonstrating how Tn-seq and metabolic modelling can be used together to yield insights not obtainable by either method alone.

systems biology↗