Biology subjects

diCenzo, G. C.

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

At least 19 recordsLinked to original sources

bioRxiv2026

Evolution and remodeling of acceptor specificity in the NodZ α-1,6-fucosyltransferase

Glycosyltransferases have broad diversity in their acceptor substrates, yet the molecular and evolutionary basis of this diversity remains poorly understood. NodZ is an -1,6-fucosyltransferase that fucosylates Nod factors, lipochitooligosaccharide molecules involved in the establishment of rhizobium-legume symbioses. While NodZ is the only known bacterial member of the GT23 family of -1,6-fucosyltransferases, the determinants of its acceptor selectivity and the evolutionary origins thereof are unclear. Here, a phylogenetic analysis of 332 NodZ-like proteins revealed a distinct rhizobial clade and significantly greater structural diversity in the N-terminus than in the C-terminus. Kinetic analysis of a Mesorhizobium japonicum NodZ demonstrated selectivity for longer chitooligosaccharide acceptors (comprised of four to five GlcNAc units), while alanine scanning revealed W139 as a primary determinant of this chain length preference. We propose that W139 forms an aromatic platform that engages distal GlcNAc units present only in longer acceptors, coupling productive binding to acceptor chain length. We then used the NodZ phylogeny to reconstruct ancestral NodZ homolog sequences, which indicated that W139 emerged at the base of the rhizobial clade, and thus may represent a key evolutionary step towards canonical NodZ function. Additionally, the most recent resurrected ancestor was capable of fucosylating chitooligosaccharides with lower efficiency than the extant NodZ, suggesting that efficiency towards chitooligosaccharides was improved during the evolution of this family. Results described here demonstrate that evolutionary diversification of the N-terminal region can remodel acceptor specificity while preserving the conserved catalytic architecture of GT23 fucosyltransferases, providing a framework for understanding and engineering fucosyltransferase specificity.

biochemistry↗

bioRxiv2026

Sinorhizobium meliloti possesses a complete Embden-Meyerhoff-Parnas pathway that is indispensable for symbiotic nitrogen fixation

Rhizobia are an agronomically valuable group of bacteria capable of entering into endosymbiotic relationships with leguminous plants, during which they fix atmospheric nitrogen using energy derived from the metabolism of plant-provided dicarboxylic acids. It is generally assumed that the gluconeogenic catabolism of dicarboxylic acids proceeds via the Embden-Meyerhoff-Parnas pathway in rhizobia. However, rhizobia are classically thought to lack the phosphofructokinase enzyme required for conversion of fructose-1,6-bisphosphate to fructore-6-phosphate as part of this pathway. Here, we demonstrate that a model rhizobium, Sinorhizobium meliloti, encodes a phosphofructokinase, completing the Embden-Meyerhoff-Parnas pathway of this organism. Biochemical characterization of the S. meliloti phosphofructokinase demonstrates that it can catalyze the reversible phosphorylation of fructose-6-phosphate under in vitro conditions in a pyrophosphate-dependent, rather than ATP-dependent, manner. We further show that S. meliloti also encodes a distinct fructose-1,6-bisphosphatase that can phenotypically complement the loss of the phosphofructokinase enzyme. Loss of both enzymes results in a block of the gluconeogenic pathway in S. meliloti and results in S. meliloti being unable to fix nitrogen in symbiosis with alfalfa (Medicago sativa). Phylogenetic analyses and complementation studies demonstrate that PPi-dependent phosphofructokinases are broadly distributed across the phylum Pseudomonadota (syn. Proteobacteria), including most rhizobial species of the class Alphaproteobacteria, suggesting both that PPi-dependent phosphofructokinases are likely more broadly distributed than is generally recognized, and that the catabolism of dicarboxylic acids in most rhizobia proceeds via a PPi-dependent phosphofructokinase. SIGNIFIGANCECentral carbon metabolism is an important biochemical network that bridges the gap between substrate catabolism and biosynthetic reactions in all living organisms. However, much of what we know about metabolism comes from the study of a few model organisms such as the bacterium Escherichia coli. Here, we identified the enzyme catalyzing a key step of central carbon metabolism in rhizobia (nitrogen-fixing bacterial symbionts of legumes), which until now had remained undetected. We show that this enzyme is dependent on pyrophosphate, which is different than the situation in E. coli, helping to explain why previous studies failed to identify this enzyme in rhizobia and highlighting the limitations associated with generalizing our understanding of metabolism from a limited subset of organisms.

microbiology↗

bioRxiv2026

Isolation of rhizobia from Ontario soils that are effective at fixing nitrogen with common bean (Phaseolus vulgaris)

Common bean (Phaseolus vulgaris) is an important crop in Canada and globally. Like other legumes, common bean (Phaseolus vulgaris) establishes symbiotic interactions with nitrogen fixing bacteria called rhizobia. However, nitrogen fixation by rhizobia in association with common bean is often suboptimal, constraining its productivity and necessitating the application of nitrogen fertilizer. To support the development of high-performing, locally adapted rhizobial inoculants for Ontario common bean growers, we isolated 216 common bean-nodulating rhizobia from southern Ontario soils using a nodule trapping approach with four common bean cultivars. Whole genome sequencing followed by phylogenomic analyses of the 216 rhizobial isolates revealed substantial diversity, assigning them to 11 Rhizobium species, including two novel species. Nearly all isolates belong to the symbiovar phaseoli, spanning the nodC {gamma}-a, {gamma}-b, and alleles, with four isolates belonging to the symbiovar gallica. Soil origin had a significant impact on the species-level community composition recovered during the nodule trapping experiments, indicative of biogeographical structuring of common bean-nodulating rhizobia across southern Ontario. In contrast, host trapping cultivar had only a minor influence of the recovered Rhizobium population diversity. Greenhouse assays demonstrated that one of the novel Rhizobium species exhibited the highest average symbiotic effectiveness, although high-quality isolates were found across multiple species. Together, these results revealed a diverse and genomically variable Rhizobium community capable of forming effective symbioses with common bean in southern Ontario soils. Importantly, our genome-sequenced Rhizobium collection will serve as a valuable resource for identifying competitive and high-quality strains for the development of inoculants tailored to Ontario common bean production. IMPORTANCECommon bean is a globally important food crop, yet its productivity is often limited by suboptimal nitrogen fixation, forcing growers to rely on synthetic fertilizers. Consequently, identifying high-performing, locally adapted inoculant strains is essential for reducing dependence on synthetic nitrogen fertilizers and improving the sustainability of temperate agroecosystems. Our study provides a genome-sequenced collection of common bean-nodulating Rhizobium from southern Ontario, revealing substantial species and genomic diversity across sampling locations. Greenhouse studies allowed us to identify multiple isolates, including isolates from a novel Rhizobium species, that consistently fix nitrogen with, and enhance the growth of, common bean plants. Our findings highlight strong biogeographical structuring of rhizobial communities and demonstrate that Ontario soils already harbour strains with high symbiotic potential. In addition, our Rhizobium collection represents a foundational resource to support future inoculant development and enables future work on the ecology, evolution, and applied optimization of legume-rhizobium symbioses.

microbiology↗

bioRxiv2026

Host cohort has a larger impact on the gut microbiomes of mealworms and superworms than does the ingestion of polyethylene or polystyrene

Beetles (order Coleoptera) are a diverse insect group whose associated microbial communities remain understudied despite their ecological, commercial, and biotechnological importance. Larvae of the darkling beetle species Tenebrio molitor (mealworms) and Zophobas atratus (superworms) have gained attention for their reported ability to ingest and possibly degrade plastics such as polyethylene and polystyrene. This degradation is believed to be mediated by enzymes from their gut microbiota, yet the underlying microbial mechanisms remain unclear. Using deep shotgun metagenomics, we generated comprehensive gut metagenomes for mealworms and superworms, recovering 53 and 100 high-quality metagenome-assembled genomes (MAGs), respectively. We found that the gut microbiomes of both insect species are dominated by bacteria from the phyla Bacillota and Pseudomonadota, with superworms trending towards greater bacterial diversity than mealworms. Comparing gut bacterial communities of insects fed polyethylene or polystyrene versus controls revealed no clear plastic ingestion effects beyond those attributable to starvation. On the other hand, insect cohort contributed substantially to variation in bacterial community composition, explaining 23% and 33% of the total variation in mealworms and superworms, respectively. Finally, we computationally identified numerous secreted proteins with sequence similarity to enzymes previously implicated in polyethylene and polystyrene degradation, and we provide support for dye decolorizing peroxidase (DyP)-type peroxidases as being the enzyme class mostly likely to initiate plastic oxidation in the digestive tracts of mealworms and superworms. Overall, this study provides high-quality metagenomes and MAGs for mealworms and superworms, revealing substantial under-explored microbial biodiversity, and highlighting DyP-type peroxidases as promising targets for future plastic biodegradation studies.

microbiology↗

bioRxiv2025

Updated taxonomy of the family Rhizobiaceae with proposals for 10 novel genera and 35 novel combinations

The family Rhizobiaceae of the class Alphaproteobacteria is highly diverse and currently consists of at least 276 validly published or proposed species across 38 genera. Despite several recent studies proposing revisions to the family Rhizobiaceae, anomalies and inconsistencies in the taxonomy of this family remain. Here, we revisit the taxonomy of the family Rhizobiaceae with a focus on the genus Rhizobium. First, we generated whole genome sequences for 12 Rhizobium type strains that previously lacked publicly available genome sequences. We then applied an established phylogenomic framework to reappraise the taxonomic classification of 242 Rhizobiaceae type strains. Our data suggest that Rhizobium aegyptiacum is a later heterotypic synonym of Rhizobium aethiopicum, and they contradict a recent suggestion that Rhizobium azibense and Rhizobium gallicum are synonymous. In addition, we propose the formation of ten new genera (Allohoeflea gen. nov., Arminia gen. nov., Fluviimicrobium gen. nov., Gillisella gen. nov., Limnomicrobium gen. nov., Martinezia gen. nov., Neohoeflea gen. nov., Parahoeflea gen. nov., Velazquezia gen. nov., and Yannia gen. nov.) and 35 novel combinations to fix paraphyletic genera or account for monophyletic type strains that are clearly distinguishable based on core-proteome average amino acid identity (cpAAI) comparisons. Lastly, our data suggest that the type strain of Rhizobium arsenicireducens may have been lost, and that either a neotype should be designated or the taxonomic status of this species should be revised.

microbiology↗

bioRxiv2025

Corylus avellana disease management: using metagenomics to illuminate the rhizosphere microbiome of Corylus

The European hazelnut, Corylus avellana, is one of the most economically important tree nut crops globally. The biotrophic ascomycete pathogen Anisogramma anomala, found naturally associated with wild C. americana, continues to pose a significant threat to European hazelnut production across North America. Here, metagenomics was used to examine the taxonomic and functional features of the rhizosphere microbial communities of hazelnut trees differing in their levels of resistance to A. anomala: highly tolerant Corylus americana, and resistant and susceptible Corylus avellana. No statistically significant differences in microbial alpha diversity or beta diversity were noted between the three rhizosphere groups. Compared to bulk soil, all three rhizosphere groups were enriched for the fungal phylum Basidiomycota and bacterial phylum "Candidatus Rokubacteriota". At the genus level, the bacterial genera Actinospica, Occallatibacter, and "Candidatus Sulfotelmatobacter" were under-represented, while the genus Rhizobacter was over-represented, in the resistant and susceptible C. avellana rhizosphere samples compared to the bulk soil. A total of 45 dereplicated, high-quality metagenome-assembled genomes (MAGs) were generated, corresponding to 41 bacteria and 4 archaea. Many of the MAGs carried multiple biosynthetic gene clusters, including MAGs corresponding to the genera Lysobacter and Actinospica. Overall, the low differentiation of the rhizosphere microbiomes suggest that differences in A. anomala disease expression are likely not associated with differences in the rhizosphere microbiome. Nevertheless, the results shed new light on the rhizosphere communities of two species of hazelnut, and woody perennials more broadly, and identify potential avenues for future research into the development of microbial inoculants for Corylus spp..

microbiology↗

bioRxiv2025

Structure-based phylogenetic analysis reveals multiple events of convergent evolution of cysteine-rich antimicrobial peptides in legume-rhizobium symbiosis

Nitrogen is essential for plant growth, yet its availability often limits agricultural productivity. Some legumes have evolved a unique ability to form symbiotic relationships with nitrogen-fixing soil bacteria called rhizobia, enabling them to thrive in nitrogen-deficient soils. In five legume clades, an exploitive strategy has evolved in which rhizobia undergo Terminal Bacteroid Differentiation (TBD), where the bacteria become larger, polyploid, and have a permeabilized membrane. Terminally differentiated bacteria are associated with higher N2-fixation and, thus, a higher return on investment to the plant. In several members of the IRLC (Inverted Repeat-Lacking Clade) and the Dalbergioid clades of legumes, this differentiation process is triggered by a set of apparently unrelated plant antimicrobial peptides with membrane-damaging activity, known as Nodule-specific Cysteine-Rich (NCR) peptides. However, whether NCR peptides are also implicated in symbiotic TBD in other legume clades and whether they are evolutionarily related remains unknown. Here, to address the molecular identity of NCR peptides and their evolution in different legume clades, we performed inter- and intra-clade comparisons of NCR peptides in representative species of four TBD-inducing legume clades. First, we collected genomic and proteomic data of species for which NCR peptides are known (1523 NCR peptides). We then used sequence similarity-based clustering to regroup the NCR peptides, resulting in over 400 different NCR clusters, each clade-specific. We obtained Hidden Markov Models for each cluster and used them to predict NCR peptides in 21 legume genomes (6 clades), including newly generated deep-sequenced root and nodule RNA-seq data of Indigofera argentea (Indigoferoid clade) and newly assembled high-quality transcriptomes of Lupinus luteus and Lupinus mariae-josephae (Genistoid clade), using tailored gene prediction pipeline and transcriptome matching. This resulted in 3710 NCR peptides in species that induce TBD. To date, the rapid diversification of NCR peptides that reduces the sequence similarities has masked the origin of NCR peptide evolution. We obtained high-confidence structural models for one sequence of each cluster. We performed structure-based clustering and phylogenetics, which resulted in 23 superclusters (14 inter-clade and nine clade-specific) that we represent in a structural distance-based tree. Our study revealed that the evolution of NCR peptides is a mix of divergent and convergent processes within each clade. We further chose nine independently evolved NCR peptides to test in vitro whether they are functional analogs in symbiosis. Graphical abstract O_FIG O_LINKSMALLFIG WIDTH=145 HEIGHT=200 SRC="FIGDIR/small/675119v1_ufig1.gif" ALT="Figure 1"> View larger version (50K): org.highwire.dtl.DTLVardef@8d1698org.highwire.dtl.DTLVardef@c65b98org.highwire.dtl.DTLVardef@a75994org.highwire.dtl.DTLVardef@ea1a73_HPS_FORMAT_FIGEXP M_FIG Overview of the experimental and computational workflow for NCR peptide detection, characterization, and structural analysis. Nodule and root samples from Indigofera argentea (8 weeks post-inoculation) were collected and subjected to RNA extraction, library preparation, and Illumina PE150 sequencing. Raw RNA-seq reads from two Lupinus species were also included (Lupinus luteus and Lupinus mariae-josephae). Bacteroid differentiation of I. argentea was assessed by flow cytometry and confocal microscopy. Transcriptomes were assembled de novo and analyzed for differential gene expression between root and nodule tissues. NCR peptides were identified from them and other legume genomes and transcriptomes using the SPADA pipeline and HMM profiles from NCR clusters of the known NCR peptides. The putative NCR peptides were filtered based on conserved cysteine motifs, length, and nodule expression to build an exhaustive NCR peptide database. 3D structural predictions of NCR clusters were performed using AlphaFold2 (pLDDT >70), followed by structural clustering (Foldseek) and phylogenetic analysis (Foldtree). Functional validation involved flow cytometry and antimicrobial assays (against Eschericha coli, Sinorhizobium meliloti, and Bacillus subtilis), enabling structural and evolutionary characterization of NCR peptides. The green box at the top represents the experimental analysis, the blue box represents the sequence-based computational pipeline, the red box represents the structure-based computational pipeline, and the grey box at the bottom left represents the functional validation and interpretation of the results. C_FIG

evolutionary biology↗

bioRxiv2025

Variations in carbapenem resistance associated with the VIM-1 metallo-β-lactamase across the Enterobacterales

The VIM-1 metallo-{beta}-lactamase enzyme, encoded as a cassette within class 1 integrons, is found in Gram-negative clinical isolates worldwide and has been linked to outbreaks of bacterial pathogens in nosocomial settings. Six vim-1+ clinical isolates, from the genera Escherichia, Klebsiella, and Enterobacter, were obtained from Kingston, Ontario, Canada. Whole genome sequencing revealed that vim-1 was plasmid-borne in all strains and situated as the first gene in In916 or In110 integrons. Analysis of related plasmids suggested that these vim-1-containing plasmids are globally disseminated and have spread via horizontal gene transfer and autochthonous vertical spread within Ontario. Interestingly, the minimum inhibitory concentrations of ertapenem and meropenem, two clinically relevant carbapenem antibiotics, against these six isolates varied more than tenfold, suggesting the effects of VIM-1 are dependent on the genomic content of the host microbe. To further study the genomic content dependency of VIM-1, we introduced vim-1 into three common Enterobacterales laboratory strains. Although introduction of vim-1 into Escherichia coli DH5 resulted in little resistance to ertapenem or meropenem, multiple rounds of adaptive laboratory evolution allowed us to identify variants with extremely high levels of resistance to both carbapenems. DNA sequencing revealed that the increase in carbapenem resistance was due to a combination of increased vim-1 gene dosage and epistatic interactions with mutations of ompC that likely would have decreased outer membrane permeability to these antibiotics. Together, these results provide additional support for the role of gene epistasis is modulating the antimicrobial resistance phenotypes of acquired resistance genes, as well as previous results suggesting that the presence of a {beta}-lactamase gene is insufficient to confer strong resistance to carbapenems without being paired with reduced outer membrane permeability.

microbiology↗

bioRxiv2025

Characterization of bacterial fucokinase/GDP-fucose pyrophosphorylase (FKP) enzymes supports the evolution of interdomain communication and modularity in the FKP family

L-fucokinase (FUK) and GDP-fucose pyrophosphorylase (GFPP) salvage free L-fucose and synthesize the valuable nucleotide-sugar GDP-L-fucose (GDP-Fuc). Some organisms express these enzymes as one bifunctional polypeptide called L-fucokinase/GDP-fucose pyrophosphorylase (FKP), which has attracted attention for use in the chemoenzymatic synthesis of GDP-Fuc. Despite the documented use of the FKP from Bacteroides fragilis (BfFKP), the evolutionary origins of these enzymes and their relationships to monofunctional FUKs and GFPPs are poorly understood. We hypothesized that biochemical characterization of these proteins coupled with an evolutionary analysis would uncover the natural diversity of FKPs, facilitating the discovery of new biocatalysts. Phylogenetic and sequence similarity network (SSN) analyses distinguished FKPs from FUKs and GFPPs, suggesting that FKPs originate from one ancestral fusion event between these domains. To evaluate how environmental factors might select for functional diversity within the FKP family, we recombinantly expressed and purified a putative FKP from the thermophilic bacterium Thermophagus xiamenensis (TxFKP). This enzyme exhibited in vitro kinase and pyrophosphorylase activities and demonstrated subtle kinetic differences compared to BfFKP. While alanine scanning mutational analysis of the TxFKP FUK and GFPP domains supported the role of conserved residues that TxFKP uses to coordinate substrate binding and catalysis, other mutations in the TxFKP GFPP domain influenced kinase activity differentially for the substrates L-fucose and D-arabinose, showing an unprecedented role for the GFPP domain in FUK substrate specificity. Finally, thermal shift profiles of TxFKP and BfFKP were biphasic and provided new insights into how these enzymes have evolved to respond to different sugar substrates.

biochemistry↗

bioRxiv2025

Re-evaluation of the nodulation capacity of Sphingomonas sediminicola DSM 18106T indicates that this strain is not capable of inducing root nodule formation on Pisum sativum (pea)

Rhizobia are soil-dwelling proteobacteria that can enter into symbiotic nitrogen-fixing relationships with compatible leguminous plants. Taxonomically, rhizobia are divided into alpha-rhizobia, which belong to the class Alpharoteobacteria, and beta-rhizobia, which belong to the class Betaproteobacteria. To date, all bona fide alpha-rhizobia belong to the order Hyphomicrobiales. However, a recent study suggested that Sphingomonas sediminicola DSM 18106T is also a rhizobium and is capable of nodulating pea plants (Pisum sativum), which would expand the known taxonomic distribution of alpha-rhizobia to include the order Sphingomonadales. Here, we attempted to replicate the results of that previous study. Resequencing and computational analysis of the genome of S. sediminicola DSM 18106T failed to identify genes encoding proteins involved in legume nodulation or nitrogen fixation. In addition, experimental plant assays indicated that S. sediminicola DSM 18106T is unable to nodulate the two cultivars of pea tested in our study, unlike the rhizobium Rhizobium johnstonii 3841T. Taken together, and in contrast to the previous study, these results suggest that S. sediminicola DSM 18106T is not capable of inducing root nodule formation on pea, meaning that the taxonomic distribution of all known alpha-rhizobia remains limited to the class Hyphomicrobiales.

microbiology↗

bioRxiv2024

BEVA2.0: Modular Assembly of Golden Gate-Compatible Vectors with Expanded Utility for Genetic Engineering

This expansion for the modular vector assembly platform BEVA (Bacterial Expression Vector Archive) introduces 11 new BEVA parts including two new cloning site variants, two new antibiotic resistance modules, three new origins of replication, and four new accessary modules. As a result, the modular system is now doubled in size and expanded in its capacity to produce diverse replicating plasmids. Furthermore, it is now amenable to genetic engineering methods involving genome-manipulation of target strains through deletions or integrations. In addition to introducing the new modules, we provide several BEVA-derived Golden Gate cloning plasmids that are used to validate parts and that may be useful for genetic engineering of proteobacteria and other bacteria. We also introduce new parts to allow compatibility with the CIDAR MoClo parts libraries.

synthetic biology↗

bioRxiv2024

Taxonomic distribution of SbmA/BacA and BacA-like antimicrobial peptide transporters suggests independent recruitment and convergent evolution in host-microbe interactions

Small, antimicrobial peptides are often produced by eukaryotes to control bacterial populations in both pathogenic and mutualistic symbioses. These include proline-rich mammalian immune peptides and cysteine-rich peptides produced by legume plants in symbiosis with rhizobia. The fitness of the bacterial partner is dependent upon their ability to persist in the presence of these antimicrobial peptides. In the case of Escherichia coli and Mycobacterium tuberculosis pathogens and nitrogen-fixing legume symbionts (rhizobia), the ability to survive exposure to these peptides depends on peptide transporters called SbmA (also known as BacA) or BclA (for BacA-like). However, how broadly these transporters are distributed amongst bacteria, and their evolutionary history, is poorly understood. Here, we used hidden Markov models, phylogenetic analysis, and sequence similarity networks to examine the distribution of SbmA/BacA and BclA proteins across a representative set of 1,255 species from across the domain Bacteria. We identified a total of 71 and 177 SbmA/BacA and BclA proteins, respectively. Phylogenetic and sequence similarity analyses suggest that these protein families likely did not evolve from a common ancestor and that their functional similarity is instead a result of convergent evolution. In vitro sensitivity assays using the legume peptide NCR247 and several of the newly-identified BclA proteins confirmed that transport of antimicrobial peptides is a common feature of this protein family. Analysis of the taxonomic distribution of these proteins showed that SbmA/BacA orthologs were encoded only by species in the phylum Pseudomonadota and that they were primarily identified in just two orders: Hyphomicrobiales (class Alphaproteobacteria) and Enterobacterales (class Gammaproteobacteria). BclA orthologs were somewhat more broadly distributed and were found in clusters across four phyla. These included several orders of the phyla Pseudomonadota and Cyanobacteriota, as well as the order Mycobacteriales (phylum Actinomycetota) and the class Negativicutes (phylum Bacillota). Notably, many of the clades enriched for species encoding BacA or BclA orthologs also include many species known to interact with eukaryotic hosts in mutualistic or pathogenic interactions. Collectively, these observations suggest that SbmA/BacA and BclA proteins have been repeatedly co-opted to facilitate both mutualistic and pathogenic associations with eukaryotic hosts by allowing bacteria to cope with host-encoded antimicrobial peptides.

microbiology↗

bioRxiv2024

Comparative genomic analysis of Bradyrhizobium strains with natural variability in the efficiency of nitrogen fixation, competitiveness, and adaptation to stressful edaphoclimatic conditions

Bradyrhizobium is known for its ability to fix atmospheric nitrogen in symbiosis with agronomically important crops. This study focused on two groups of strains, each containing eight putative natural variants of B. japonicum SEMIA 586 (=CNPSo 17) or B. diazoefficiens SEMIA 566 (=CNPSo 10), previously used as commercial inoculants for soybean crops in Brazil. We aimed to detect genetic variations that might be related to biological nitrogen fixation, competitiveness for nodule occupancy, and adaptation to the stressful conditions of the Brazilian Cerrado soils. High-quality genome assemblies were produced for all strains and used for comparative genomic analyses. The core genome phylogeny revealed that strains of each group are closely related, confirmed by high average nucleotide identity (ANI) values. However, variants accumulated divergences resulting from horizontal gene transfer (HGT), genomic rearrangements, and nucleotide polymorphisms. The B. japonicum group presented a larger pangenome and a higher number of nucleotide polymorphisms than the B. diazoefficiens group, probably due to its longer adaptation time to the Cerrado soil. Interestingly, five strains of the B. japonicum group carry two plasmids. The genetic variability found in both groups is discussed in light of the observed differences in their nitrogen fixation capacity, competitiveness for nodule occupancy, and environmental adaptation. SIGNIFICANCEThe two main reference strains for soybean inoculation in Brazil, B. japonicum CPAC 15 (=SEMIA 5079) and B. diazoefficiens CPAC 7 (=SEMIA 5080), have been considered highly competitive and highly efficient in nitrogen fixation, respectively. In this study, we obtained and analyzed the genomes of the parental and variant strains. We detected two plasmids in five strains and several genetic differences that might be related to adaptation to the stressful conditions of the soils of the Brazilian Cerrado biome. We also detected genetic variations in specific regions that may impact symbiotic nitrogen fixation. Our analysis contributes to new insights into evolution of Bradyrhizobium, and some of the identified differences may be applied as genetic markers to assist strain selection programs.

microbiology↗

bioRxiv2023

Identifying functional multi-host shuttle plasmids to advance synthetic biology applications in Mesorhizobium and Bradyrhizobium

Ammonia availability has a crucial role in agriculture as it ensures healthy plant growth and increased crop yields. Since diazotrophs are the only organisms capable of reducing dinitrogen to ammonium, they have a great ecological importance and potential to mitigate the environmental and economic costs of synthetic fertilizer use. Rhizobia are especially valuable being that they can engage in nitrogen-fixing symbiotic relationships with legumes, and they demonstrate great diversity and plasticity in genomic and phenotypic traits. However, few rhizobial species have improved genetic tractability for synthetic biology applications. This study established a basic genetic toolbox with antibiotic resistance markers, multi-host shuttle plasmids and a streamlined protocol for biparental conjugation with Mesorhizobium and Bradyrhizobium species. We identified two repABC origins of replication from Sinorhizobium meliloti (pSymB) and Rhizobium etli (p42d) that were stable across all three strains of interest. Furthermore, the NZP2235 genome was sequenced and phylogenetic analysis determined its reclassification to Mesorhizobium huakuii. These tools will enable the use of plasmid-based strategies for more advanced genetic engineering projects and ultimately contribute towards the development of more sustainable agriculture practices by means of novel nitrogen-fixing organelles, elite bioinoculants or symbiotic association with non-legumes. GRAPHICAL ABSTRACT O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=94 SRC="FIGDIR/small/572671v1_ufig1.gif" ALT="Figure 1"> View larger version (43K): org.highwire.dtl.DTLVardef@70e377org.highwire.dtl.DTLVardef@165b06corg.highwire.dtl.DTLVardef@12196a9org.highwire.dtl.DTLVardef@2a33e0_HPS_FORMAT_FIGEXP M_FIG C_FIG

synthetic biology↗

bioRxiv2023

Metabolic connections between folate and peptidoglycan pathways in Pseudomonas aeruginosa inform rational design of a dual-action inhibitor

Peptidoglycan is an important bacterial macromolecule that confers cell shape and structural integrity, and a key antibiotic target. The synthesis and turnover of peptidoglycan are carefully coordinated with other cellular processes and pathways. Although there are established connections between peptidoglycan and DNA replication or outer membrane biosynthesis, connections between peptidoglycan and folate metabolism are comparatively unexplored. Folate is an essential cofactor for bacterial growth and required for the synthesis of many important metabolites. Here we show that inhibition of folate synthesis in the important Gram-negative pathogen Pseudomonas aeruginosa has downstream effects on peptidoglycan metabolism and integrity. Folate inhibitors reduced expression of the AmpC {beta}-lactamase through perturbation of peptidoglycan recycling, potentiating the activity of {beta}-lactams normally cleaved by that resistance enzyme. Folate inhibitors also synergized with fosfomycin, which inhibits MurA - the first committed step in peptidoglycan synthesis - resulting in dose-dependent formation of round cells that underwent explosive lysis.The insights from this work were used to design a dual-active inhibitor that overcomes NDM-1-mediated meropenem resistance and synergizes with the folate inhibitor, trimethoprim. This work shows that folate and peptidoglycan metabolism are intimately connected and offers new opportunities to exploit this relationship in strategies to overcome antibiotic resistance in Gram-negative pathogens.

microbiology↗

bioRxiv2023

Refining the taxonomy of the order Hyphomicrobiales (Rhizobiales) based on whole genome comparisons of over 130 genus type strains

The alphaproteobacterial order Hyphomicrobiales consists of 38 families comprising 155 validly published genera as of June 2023. The order Hyphomicrobiales was first described in 1957 and underwent important revisions in 2020. However, several inconsistencies in the taxonomy of this order remain, and there is a need for a consistent framework for defining families within the order. We propose a common genome-based framework for defining families within the order Hyphomicrobiales, suggesting that families represent monophyletic groups in core-genome phylogenies that share pairwise average amino acid identity values above [~]75% when calculated from a core set of 59 proteins. Applying this framework, we propose the formation of four new families and to reassign the genera Salaquimonas, Rhodoblastus, and Rhodoligotrophos into Salaquimonadaceae fam. nov., Rhodoblastaceae fam. nov., and Rhodoligotrophaceae fam. nov., respectively, and the genera Albibacter, Chenggangzhangella, Hansschlegelia, and Methylopila into Methylopilaceae fam. nov.. We further propose to unify the families Bartonellaceae, Brucellaceae, Phyllobacteriaceae, and Notoacmeibacteraceae as Bartonellaceae; the families Segnochrobactraceae and Pseudoxanthobacteraceae as Segnochrobactraceae; the families Lichenihabitantaceae and Lichenibacteriaceae as Lichenihabitantaceae; and the families Breoghaniaceae and Stappiaceae as Stappiaceae. Lastly, we propose to reassign several genera to existing families. Specifically, we propose to reassign the genus Pseudohoeflea to the family Rhizobiaceae; the genera Oricola, Roseitalea, and Oceaniradius to the family Ahrensiaceae; the genus Limoniibacter to the emended family Bartonellaceae; the genus Faunimonas to the family Afifellaceae; and the genus Pseudochelatococcus to the family Chelatococcaceae. Our data also support the recent proposal to reassign the genus Prosthecomicrobium to the family Kaistiaceae.

microbiology↗

bioRxiv2023

Complete genome sequences of the species type strains Sinorhizobium garamanticum LMG 24692T and Sinorhizobium numidicum LMG 27395T and CIP 109850T

The genus Sinorhizobium comprises rhizobia that fix nitrogen in symbiosis with legumes. To support taxonomic studies of this genus and of rhizobia more broadly, we report complete genome sequences and annotations for the species type strains Sinorhizobium garamanticum LMG 24692T and Sinorhizobium numidicum LMG 27395T and CIP 109850T. Average nucleotide identity and core-genome phylogenetic analyses confirm that S. garamanticum and S. numidicum represent distinct species.

microbiology↗

bioRxiv2023

Genomics of the "tumorigenes" clade of the family Rhizobiaceae and description of Rhizobium rhododendri sp. nov.

Tumorigenic members of the family Rhizobiaceae, known as agrobacteria, are responsible for crown and cane gall diseases of various agricultural crops worldwide. Tumorigenic agrobacteria are commonly found in the genera Agrobacterium, Allorhizobium, and Rhizobium. In this study, we analyzed a distinct "tumorigenes" clade of the genus Rhizobium, which includes the tumorigenic species Rhizobium tumorigenes, as well as strains causing crown gall disease on rhododendron. Here, high quality, closed genomes of representatives of the "tumorigenes" clade were generated, followed by comparative genomic and phylogenomic analyses. Additionally, phenotypic characteristics of representatives of the "tumorigenes" clade were analyzed. Our results showed that the tumorigenic strains isolated from rhododendron represent a novel species of the genus Rhizobium for which the name Rhizobium rhododendri sp. nov. is proposed. This species also includes additional strains originating from blueberry and Himalayan blackberry in USA, whose genome sequences were retrieved from GenBank. Both R. tumorigenes and R. rhododendri contain multipartite genomes, including a chromosome, putative chromids, and megaplasmids. Synteny and phylogenetic analyses indicated that a large putative chromid of R. rhododendri resulted from the cointegration of an ancestral megaplasmid and two putative chromids, following its divergence from R. tumorigenes. Moreover, gene clusters specific for both species of the "tumorigenes" clade were identified, and their biological functions and roles in ecological diversification of R. rhododendri and R. tumorigenes were predicted and discussed.

microbiology↗