George C. diCenzo

Metabolic modelling reveals the specialization of secondary replicons for niche adaptation in Sinorhizobium meliloti

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.

Robustness encoded across essential and accessory replicons of the ecologically versatile bacterium Sinorhizobium meliloti

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.

Tn-Core: context-specific reconstruction of core metabolic models using Tn-seq data

Motivation Tn-seq (transposon mutagenesis and sequencing) and constraint-based metabolic modelling represent highly complementary approaches. They can be used to probe the core genetic and metabolic networks underlying a biological process, revealing invaluable information for synthetic biology engineering of microbial cell factories. However, while algorithms exist for integration of –omics data sets with metabolic models, no method has been explicitly developed for integration of Tn-seq data with metabolic reconstructions. Results We report the development of Tn-Core, a Matlab toolbox designed to generate gene-centric, context-specific core reconstructions consistent with experimental Tn-seq data. Extensions of this algorithm allow: i) the generation of context-specific functional models through integration of both Tn-seq and RNA-seq data; ii) to visualize redundancy in core metabolic processes; and iii) to assist in curation of de novo draft metabolic models. The utility of Tn-Core is demonstrated primarily using a Sinorhizobium meliloti model as a case study. Availability and implementation The software can be downloaded from https://github.com/diCenzo-GC/Tn-Core. All results presented in this work have been obtained with Tn-Core v. 1.0. Contact georgecolin.dicenzo@unifi.it, marco.fondi@unifi.it Supplementary information Supplementary data are available at Bioinformatics online.

Trade, Diplomacy, and Warfare: The Quest for Elite Rhizobia Inoculant Strains

Rhizobia form symbiotic nitrogen-fixing nodules on leguminous plants, which provides an important source of fixed nitrogen input into the soil ecosystem. The improvement of symbiotic nitrogen fixation is one of the main challenges facing agriculture research. Doing so will reduce the usage of chemical nitrogen fertilizer, contributing to the development of sustainable agriculture practices to deal with the increasing global human population. Sociomicrobiological studies of rhizobia have become a model for the study of the evolution of mutualistic interactions. The exploitation of the wide range of social interactions rhizobia establish among themselves, with the soil and root microbiota, and with the host plant, could constitute a great advantage in the development of a new generation of highly effective rhizobia inoculants. Here, we provide a brief overview of the current knowledge on three main aspects of rhizobia interaction: trade of fixed nitrogen with the plant; diplomacy in terms of communication and possible synergistic effects; and warfare, as antagonism and plant control over symbiosis. Then, we propose new areas of investigation and the selection of strains based on the combination of the genetic determinants for the relevant rhizobia symbiotic behavioral phenotypes.

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 β-proteobacterial genomes to study the role and emergence of multipartite genomes. Nearly all essential secondary replicons (chromids) of the β-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 replicon’s 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.

Genomic and Biotechnological Characterization of the Heavy-Metal Resistant, Arsenic-Oxidizing Bacterium Ensifer sp. M14

Ensifer (Sinorhizobium) sp. M14 is an efficient arsenic-oxidizing bacterium (AOB) that displays high resistance to numerous metals and various stressors. Here, we report the draft genome sequence and genome-guided characterization of Ensifer sp. M14, and we describe a pilot-scale installation applying the M14 strain for remediation of arsenic-contaminated waters. The M14 genome contains 6874 protein coding sequences, including hundreds not found in related strains. Nearly all unique genes that are associated with metal resistance and arsenic oxidation are localized within the pSinA and pSinB megaplasmids. Comparative genomics revealed that multiple copies of high-affinity phosphate transport systems are common in AOBs, possibly as an As-resistance mechanism. Genome and antibiotic sensitivity analyses further suggested that the use of Ensifer sp. M14 in biotechnology does not pose serious biosafety risks. Therefore, a novel two-stage installation for remediation of arsenic-contaminated waters was developed. It consists of a microbiological module, where M14 oxidizes As(III) to As(V) ion, followed by an adsorption module for As(V) removal using granulated bog iron ores. During a 40-day pilot-scale test in an abandoned gold mine in Zloty Stok (Poland), water leaving the microbiological module generally contained trace amounts of As(III), and dramatic decreases in total arsenic concentrations were observed after passage through the adsorption module. These results demonstrate the usefulness of Ensifer sp. M14 in arsenic removal performed in environmental settings.

Multi-disciplinary approaches for studying rhizobium - legume symbioses

The rhizobium-legume symbiosis is a major source of fixed nitrogen (ammonia) in the biosphere. The potential for this process to increase agricultural yield while reducing the reliance on nitrogen-based fertilizers has generated interest in understanding and manipulating this process. For decades, rhizobium research has benefited from the use of leading techniques from a very broad set of fields, including population genetics, molecular genetics, genomics, and systems biology. In this review, we summarize many of the research strategies that have been employed in the study of rhizobia and the unique knowledge gained from these diverse tools, with a focus on genome- and systems-level approaches. We then describe ongoing synthetic biology approaches aimed at improving existing symbioses or engineering completely new symbiotic interactions. The review concludes with our perspective of the future directions and challenges of the field, with an emphasis on how the application of a multidisciplinary approach and the development of new methods will be necessary to ensure successful biotechnological manipulation of the symbiosis.

Creation and 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, harbor a multipartite genome composed of 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. We term this strain as cis-hybrid, since it is produced by genomic material coming from the same species pangenome. In particular, we moved the secondary replicon pSymA (accounting for nearly 20% of total genome content) from a donor S.xa0meliloti strain to an acceptor strain. The cis-hybrid strain was screened for a panel of complex phenotypes (carbon/nitrogen utilization phenotypes, intra- and extracellular metabolomes, symbiosis, and various microbiological tests). Additionally, metabolic network reconstruction and constraint-based modeling 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.

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.