Publications by authors named "George C diCenzo"

32 Publications

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

G3 (Bethesda) 2021 Sep;11(9)

Department of Biology, Queen's University, Kingston, ON K7L 3N6, Canada.

In order to survive subzero temperatures, some plants undergo cold acclimation (CA) where low, nonfreezing temperatures, and/or shortened day lengths allow cold-hardening and survival during subsequent freeze events. Central to this response is the plasma membrane (PM), where low temperature is perceived and cellular homeostasis must be preserved by maintaining membrane integrity. Here, we present the first PM proteome of cold-acclimated Brachypodium distachyon, a model species for the study of monocot crops. A time-course experiment investigated CA-induced changes in the proteome following two-phase partitioning PM enrichment and label-free quantification by nano-liquid chromatography-mass spectrophotometry. Two days of CA 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 CA 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 CA response elicited over several days. A meta-analysis revealed that the identified PM 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 CA may prime plants for other abiotic and biotic stresses. The PM 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.
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http://dx.doi.org/10.1093/g3journal/jkab198DOI Listing
September 2021

Contain Diverse flg22 Epitopes That Elicit Varying Immune Responses in .

Mol Plant Microbe Interact 2021 May 7;34(5):504-510. Epub 2021 Apr 7.

Department of Biology, Queen's University, Kingston ON, K7L 3N6, Canada.

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 , using a dataset of 2,470 flg22 sequences. To predict which species encode highly immunogenic flg22 epitopes, we queried a custom motif ([ST]xx[DN][DN]xAGxxI) in the flg22 sequences, followed by sequence conservation analysis and protein structural modeling. These data led us to hypothesize that most flg22 epitopes of the γ- and β- are highly immunogenic, whereas most flg22 epitopes of the α-, δ-, and ε- 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 . The flg22 peptides of γ- and β- triggered strong oxidative bursts, whereas peptides from the ε-, δ-, and α- triggered moderate, weak, or no response, respectively. These data suggest flg22 immunogenicity is not highly conserved across the phylum . We postulate that sequence divergence of each taxonomic class was present prior to the evolution of FLS2, and that the ligand specificity of FLS2 was driven by the flg22 epitopes of the γ- and β-, a monophyletic group containing many common phytopathogens.[Formula: see text] Copyright © 2021 The Author(s). This is an open access article distributed under the CC BY 4.0 International license.
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http://dx.doi.org/10.1094/MPMI-11-20-0314-SCDOI Listing
May 2021

Minimal gene set from () pSymA required for efficient symbiosis with .

Proc Natl Acad Sci U S A 2021 01;118(2)

Department of Biology, McMaster University, Hamilton, ON, Canada L8S 4K1

Reduction of N gas to ammonia in legume root nodules is a key component of sustainable agricultural systems. Root nodules are the result of a symbiosis between leguminous plants and bacteria called rhizobia. Both symbiotic partners play active roles in establishing successful symbiosis and nitrogen fixation: while root nodule development is mostly controlled by the plant, the rhizobia induce nodule formation, invade, and perform N fixation once inside the plant cells. Many bacterial genes involved in the rhizobia-legume symbiosis are known, and there is much interest in engineering the symbiosis to include major nonlegume crops such as corn, wheat, and rice. We sought to identify and combine a minimal bacterial gene complement necessary and sufficient for symbiosis. We analyzed a model rhizobium, () , using a background strain in which the 1.35-Mb symbiotic megaplasmid pSymA was removed. Three regions representing 162 kb of pSymA were sufficient to recover a complete N-fixing symbiosis with alfalfa, and a targeted assembly of this gene complement achieved high levels of symbiotic N fixation. The resulting gene set contained just 58 of 1,290 pSymA protein-coding genes. To generate a platform for future synthetic manipulation, the minimal symbiotic genes were reorganized into three discrete , , and modules. These constructs will facilitate directed studies toward expanding the symbiosis to other plant partners. They also enable forward-type approaches to identifying genetic components that may not be essential for symbiosis, but which modulate the rhizobium's competitiveness for nodulation and the effectiveness of particular rhizobia-plant symbioses.
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http://dx.doi.org/10.1073/pnas.2018015118DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7814474PMC
January 2021

Symbiotic and Nonsymbiotic Members of the Genus Ensifer (syn. Sinorhizobium) Are Separated into Two Clades Based on Comparative Genomics and High-Throughput Phenotyping.

Genome Biol Evol 2020 12;12(12):2521-2534

Department of Biology, University of Florence, Sesto Fiorentino, Italy.

Rhizobium-legume symbioses serve as paradigmatic examples 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 interkingdom mutualisms more generally, we performed extensive phenotypic, genomic, and phylogenetic analyses of the genus Ensifer. The data suggest that SNF likely emerged several times within the genus Ensifer 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 "nonsymbiotic" 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 nonsymbiotic clade. Initial characterization of a subset of ten Ensifer strains identified several putative phenotypic differences between the clades. Tested strains of the nonsymbiotic 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, the tested 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.
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http://dx.doi.org/10.1093/gbe/evaa221DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7719227PMC
December 2020

Tn-Core: Functionally Interpreting Transposon-Sequencing Data with Metabolic Network Analysis.

Methods Mol Biol 2021 ;2189:199-215

Department of Biology, University of Florence, Sesto Fiorentino, FI, Italy.

Transposon-sequencing (Tn-seq) is a powerful tool facilitating the genome-scale identification of genes required for bacterial growth or survival in an environment of interest. However, Tn-seq suffers from two primary drawbacks: (1) genetic interactions masking phenotypes thereby resulting in important cellular functions remaining undiscovered and (2) a difficulty in easily going from a list of essential genes to a functional understanding of cell physiology. Tn-Core is a computational toolbox to help overcome these limitations through combining the output of Tn-seq studies with in silico genome-scale metabolic networks. In this chapter, we outline how to use Tn-Core to contextualize Tn-seq data (and optionally RNA-seq data) with metabolic models to: (1) generate a complete view of essential metabolism, (2) prepare context-specific metabolic models for further computational analyses, and (3) refine genome-scale metabolic models. All functions of Tn-Core are provided for download from a freely available repository ( github.com/diCenzo-GC/Tn-Core ), and a web-app requiring limited computational experience is also available ( combo.dbe.unifi.it /tncore).
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http://dx.doi.org/10.1007/978-1-0716-0822-7_15DOI Listing
March 2021

Rhizobium indicum sp. nov., isolated from root nodules of pea (Pisum sativum) cultivated in the Indian trans-Himalayas.

Syst Appl Microbiol 2020 Sep 30;43(5):126127. Epub 2020 Jul 30.

National Centre for Microbial Resource, National Centre for Cell Science, Pune, Maharashtra 411007, India.

Three strains of rhizobia isolated from effective root nodules of pea (Pisum sativum L.) collected from the Indian trans-Himalayas were characterized using 16S rRNA, atpD and recA genes. Phylogeny of the 16S rRNA genes revealed that the newly isolated strains were members of the genus Rhizobium with ≥99.9% sequence similarity to the members within the "Rhizobium leguminosarum" group. Phylogenetic analyses based on the concatenated sequences of atpD and recA gene, and 92 core genes extracted from the genome sequences indicated that strains JKLM 12A2 and JKLM 13E are grouped as a separate clade closely related to R. laguerreae FB206. In contrast, the strain JKLM 19E was placed with "R. hidalgonense" FH14. Whole-genome average nucleotide identity (ANI) values were 97.6% within strains JKLM 12A2 and JKLM 13E, and less than 94% with closely related species. The digital DNA-DNA hybridization (dDDH) values were 81.45 within the two strains and less than 54.8% to closely related species. The major cellular fatty acids were C in summed feature 8, C/C in summed feature 2, and C. The DNA G+C content of JKLM 12A2 and JKLM 13E was 60.8mol%. The data on genomic, chemotaxonomic, and phenotypic characteristics indicates that the strains JKLM 12A2 and JKLM 13E represent a novel species, Rhizobium indicum sp. nov. The type strain is JKLM 12A2 (= MCC 3961=KACC 21380=JCM 33658). However, the strain JKLM 19E represents a member of "R. hidalgonense" and the symbiovar viciae.
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http://dx.doi.org/10.1016/j.syapm.2020.126127DOI Listing
September 2020

Genome-scale metabolic reconstruction of the symbiosis between a leguminous plant and a nitrogen-fixing bacterium.

Nat Commun 2020 05 22;11(1):2574. Epub 2020 May 22.

Department of Biology, University of Florence, Sesto Fiorentino, Italy.

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-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-fixing bacteria, and N 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.
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http://dx.doi.org/10.1038/s41467-020-16484-2DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7244743PMC
May 2020

Chromids Aid Genome Expansion and Functional Diversification in the Family Burkholderiaceae.

Mol Biol Evol 2019 03;36(3):562-574

Department of Biology, University of Florence, Sesto Fiorentino, Florence, Italy.

Multipartite genomes, containing at least two large replicons, are found in diverse bacteria; however, the advantage of this genome structure remains incompletely understood. Here, we perform comparative genomics of hundreds of finished β-proteobacterial genomes to gain insights into the role and emergence of multipartite genomes. Almost 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. 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 interreplicon 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 indicated a clear functional separation of the replicons. Chromids appeared biased to environmental adaptation, as the functional categories enriched on chromids were also overrepresented on the chromosomes of the environmental genera (Paraburkholderia and Cupriavidus) compared with the pathogenic genera (Burkholderia and 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 replicons enriched in genes associated with adaptation to novel environments.
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http://dx.doi.org/10.1093/molbev/msy248DOI Listing
March 2019

Tn-Core: A Toolbox for Integrating Tn-seq Gene Essentiality Data and Constraint-Based Metabolic Modeling.

ACS Synth Biol 2019 01 27;8(1):158-169. Epub 2018 Dec 27.

Department of Biology , University of Florence , Sesto Fiorentino , Florence , 50019 , Italy.

The design of synthetic cells requires a detailed understanding of the relevance of genes and gene networks underlying complex cellular phenotypes. Transposon-sequencing (Tn-seq) and constraint-based metabolic modeling can be used to probe the core genetic and metabolic networks underlying a biological process. Integrating these highly complementary experimental and in silico approaches has the potential to yield a highly comprehensive understanding of the core networks of a cell. Specifically, it can facilitate the interpretation of Tn-seq data sets and identify gaps in the data that could hinder the engineering of the cellular system, while also providing refined models for the accurate predictions of cellular metabolism. Here, we present Tn-Core, the first easy-to-use computational pipeline specifically designed for integrating Tn-seq data with metabolic modeling, prepared for use by both experimental and computational biologists. Tn-Core is a MATLAB toolbox that contains several custom functions, and it is built upon existing functions within the COBRA Toolbox and the TIGER Toolbox. Tn-Core takes as input a genome-scale metabolic model, Tn-seq data, and optionally RNA-seq data, and returns: (i) a context-specific core metabolic model; (ii) an evaluation of redundancies within core metabolic pathways, and optionally (iii) a refined genome-scale metabolic model. A simple, user-friendly workflow, requiring limited knowledge of metabolic modeling, is provided that allows users to run the analyses and export the data as easy-to-explore files of value to both experimental and computational biologists. We demonstrate the utility of Tn-Core using Sinorhizobium meliloti, Pseudomonas aeruginosa, and Rhodobacter sphaeroides genome-scale metabolic reconstructions as case studies.
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http://dx.doi.org/10.1021/acssynbio.8b00432DOI Listing
January 2019

Harnessing Rhizobia to Improve Heavy-Metal Phytoremediation by Legumes.

Genes (Basel) 2018 Nov 8;9(11). Epub 2018 Nov 8.

Department of Biology, University of Florence, Via Madonna del Piano 6, 50019 Sesto Fiorentino, Italy.

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.
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http://dx.doi.org/10.3390/genes9110542DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6266702PMC
November 2018

Creation and Characterization of a Genomically Hybrid Strain in the Nitrogen-Fixing Symbiotic Bacterium Sinorhizobium meliloti.

ACS Synth Biol 2018 10 3;7(10):2365-2378. Epub 2018 Oct 3.

Department of Biology , University of Florence , 50019 Sesto Fiorentino , Italy.

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. meliloti 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.
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http://dx.doi.org/10.1021/acssynbio.8b00158DOI Listing
October 2018

Multidisciplinary approaches for studying rhizobium-legume symbioses.

Can J Microbiol 2019 Jan 11;65(1):1-33. Epub 2018 Sep 11.

a Department of Biology, University of Florence, Sesto Fiorentino, FI 50019, Italy.

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.
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http://dx.doi.org/10.1139/cjm-2018-0377DOI Listing
January 2019

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

Genes (Basel) 2018 Jul 27;9(8). Epub 2018 Jul 27.

Laboratory of Environmental Pollution Analysis, Faculty of Biology, University of Warsaw, Miecznikowa 1, 02-096 Warsaw, Poland.

() 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 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 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 sp. M14 in arsenic removal performed in environmental settings.
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http://dx.doi.org/10.3390/genes9080379DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6115938PMC
July 2018

Pulse-Modulated Radio-Frequency Alternating-Current-Driven Atmospheric-Pressure Glow Discharge for Continuous-Flow Synthesis of Silver Nanoparticles and Evaluation of Their Cytotoxicity toward Human Melanoma Cells.

Nanomaterials (Basel) 2018 Jun 2;8(6). Epub 2018 Jun 2.

Department of Analytical Chemistry and Chemical Metallurgy, Faculty of Chemistry, Wroclaw University of Science and Technology, Wybrzeze St. Wyspianskiego 27, 50-370 Wroclaw, Poland.

An innovative and environmentally friendly method for the synthesis of size-controlled silver nanoparticles (AgNPs) is presented. Pectin-stabilized AgNPs were synthesized in a plasma-reaction system in which pulse-modulated radio-frequency atmospheric-pressure glow discharge (pm-rf-APGD) was operated in contact with a flowing liquid electrode. The use of pm-rf-APGD allows for better control of the size of AgNPs and their stability and monodispersity. AgNPs synthesized under defined operating conditions exhibited average sizes of 41.62 ± 12.08 nm and 10.38 ± 4.56 nm, as determined by dynamic light scattering and transmission electron microscopy (TEM), respectively. Energy-dispersive X-ray spectroscopy (EDS) confirmed that the nanoparticles were composed of metallic Ag. Furthermore, the ξ-potential of the AgNPs was shown to be -43.11 ± 0.96 mV, which will facilitate their application in biological systems. Between 70% and 90% of the cancerous cells of the human melanoma Hs 294T cell line underwent necrosis following treatment with the synthesized AgNPs. Furthermore, optical emission spectrometry (OES) identified reactive species, such as NO, NH, N₂, O, and H, as pm-rf-APGD produced compounds that may be involved in the reduction of the Ag(I) ions.
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http://dx.doi.org/10.3390/nano8060398DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6027456PMC
June 2018

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

PLoS Genet 2018 04 19;14(4):e1007357. Epub 2018 Apr 19.

Department of Microbiology and Molecular Biology, Brigham Young University, Provo, UT, United States of America.

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.
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http://dx.doi.org/10.1371/journal.pgen.1007357DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5929573PMC
April 2018

Inter-replicon Gene Flow Contributes to Transcriptional Integration in the Multipartite Genome.

G3 (Bethesda) 2018 05 4;8(5):1711-1720. Epub 2018 May 4.

Department of Biology, McMaster University, Hamilton, Ontario, Canada L8S 4K1

Integration of newly acquired genes into existing regulatory networks is necessary for successful horizontal gene transfer (HGT). Ten percent of bacterial species contain at least two DNA replicons over 300 kilobases in size, with the secondary replicons derived predominately through HGT. The genome is split between a 3.7 Mb chromosome, a 1.7 Mb chromid consisting largely of genes acquired through ancient HGT, and a 1.4 Mb megaplasmid consisting primarily of recently acquired genes. Here, RNA-sequencing is used to examine the transcriptional consequences of massive, synthetic genome reduction produced through the removal of the megaplasmid and/or the chromid. Removal of the pSymA megaplasmid influenced the transcription of only six genes. In contrast, removal of the chromid influenced expression of ∼8% of chromosomal genes and ∼4% of megaplasmid genes. This was mediated in part by the loss of the ETR DNA region whose presence on pSymB is due to a translocation from the chromosome. No obvious functional bias among the up-regulated genes was detected, although genes with putative homologs on the chromid were enriched. Down-regulated genes were enriched in motility and sensory transduction pathways. Four transcripts were examined further, and in each case the transcriptional change could be traced to loss of specific pSymB regions. In particularly, a chromosomal transporter was induced due to deletion of likely mediated through 3-hydroxybutyrate accumulation. These data provide new insights into the evolution of the multipartite bacterial genome, and more generally into the integration of horizontally acquired genes into the transcriptome.
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http://dx.doi.org/10.1534/g3.117.300405DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5940162PMC
May 2018

Techniques for Large-Scale Bacterial Genome Manipulation and Characterization of the Mutants with Respect to In Silico Metabolic Reconstructions.

Methods Mol Biol 2018 ;1716:291-314

Department of Biology, McMaster University, Hamilton, ON, Canada.

The rate at which all genes within a bacterial genome can be identified far exceeds the ability to characterize these genes. To assist in associating genes with cellular functions, a large-scale bacterial genome deletion approach can be employed to rapidly screen tens to thousands of genes for desired phenotypes. Here, we provide a detailed protocol for the generation of deletions of large segments of bacterial genomes that relies on the activity of a site-specific recombinase. In this procedure, two recombinase recognition target sequences are introduced into known positions of a bacterial genome through single cross-over plasmid integration. Subsequent expression of the site-specific recombinase mediates recombination between the two target sequences, resulting in the excision of the intervening region and its loss from the genome. We further illustrate how this deletion system can be readily adapted to function as a large-scale in vivo cloning procedure, in which the region excised from the genome is captured as a replicative plasmid. We next provide a procedure for the metabolic analysis of bacterial large-scale genome deletion mutants using the Biolog Phenotype MicroArray™ system. Finally, a pipeline is described, and a sample Matlab script is provided, for the integration of the obtained data with a draft metabolic reconstruction for the refinement of the reactions and gene-protein-reaction relationships in a metabolic reconstruction.
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http://dx.doi.org/10.1007/978-1-4939-7528-0_13DOI Listing
July 2018

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

Front Microbiol 2017 9;8:2207. Epub 2017 Nov 9.

Department of Biology, University of Florence, Florence, Italy.

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.
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http://dx.doi.org/10.3389/fmicb.2017.02207DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5684177PMC
November 2017

Succinate Transport Is Not Essential for Symbiotic Nitrogen Fixation by Sinorhizobium meliloti or Rhizobium leguminosarum.

Appl Environ Microbiol 2018 Jan 15;84(1). Epub 2017 Dec 15.

Department of Biology, McMaster University, Hamilton, Ontario, Canada

Symbiotic nitrogen fixation (SNF) is an energetically expensive process performed by bacteria during endosymbiotic relationships with plants. The bacteria require the plant to provide a carbon source for the generation of reductant to power SNF. While C-dicarboxylates (succinate, fumarate, and malate) appear to be the primary, if not sole, carbon source provided to the bacteria, the contribution of each C-dicarboxylate is not known. We address this issue using genetic and systems-level analyses. Expression of a malate-specific transporter (MaeP) in Rm1021 mutants unable to transport C-dicarboxylates resulted in malate import rates of up to 30% that of the wild type. This was sufficient to support SNF with , with acetylene reduction rates of up to 50% those of plants inoculated with wild-type bv. viciae 3841 mutants unable to transport C-dicarboxylates but expressing the transporter had strong symbiotic properties, with plants inoculated with these strains appearing similar to plants inoculated with wild-type This was despite malate transport rates by the mutant bacteroids being 10% those of the wild type. An RNA-sequencing analysis of the combined - nodule transcriptome was performed to identify systems-level adaptations in response to the inability of the bacteria to import succinate or fumarate. Few transcriptional changes, with no obvious pattern, were detected. Overall, these data illustrated that succinate and fumarate are not essential for SNF and that, at least in specific symbioses, l-malate is likely the primary C-dicarboxylate provided to the bacterium. Symbiotic nitrogen fixation (SNF) is an economically and ecologically important biological process that allows plants to grow in nitrogen-poor soils without the need to apply nitrogen-based fertilizers. Much research has been dedicated to this topic to understand this process and to eventually manipulate it for agricultural gains. The work presented in this article provides new insights into the metabolic integration of the plant and bacterial partners. It is shown that malate is the only carbon source that needs to be available to the bacterium to support SNF and that, at least in some symbioses, malate, and not other C-dicarboxylates, is likely the primary carbon provided to the bacterium. This work extends our knowledge of the minimal metabolic capabilities the bacterium requires to successfully perform SNF and may be useful in further studies aiming to optimize this process through synthetic biology approaches. The work describes an engineering approach to investigate a metabolic process that occurs between a eukaryotic host and its prokaryotic endosymbiont.
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http://dx.doi.org/10.1128/AEM.01561-17DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5734035PMC
January 2018

A Key Regulator of the Glycolytic and Gluconeogenic Central Metabolic Pathways in .

Genetics 2017 11 29;207(3):961-974. Epub 2017 Aug 29.

Department of Biology, McMaster University, Hamilton, Ontario L8S 4K1, Canada

The order Rhizobiales contains numerous agriculturally, biotechnologically, and medically important bacteria, including the rhizobia, and the genera , , and , among others. These organisms tend to be metabolically versatile, but there has been relatively little investigation into the regulation of their central carbon metabolic pathways. Here, RNA-sequencing and promoter fusion data are presented to show that the PckR protein is a key regulator of central carbon metabolism in ; during growth with gluconeogenic substrates, PckR represses expression of the complete Entner-Doudoroff glycolytic pathway and induces expression of the and gluconeogenic genes. Electrophoretic mobility shift assays indicate that PckR binds an imperfect palindromic sequence that overlaps the promoter or transcriptional start site in the negatively regulated promoters, or is present in tandem upstream the promoter motifs in the positively regulated promoters. Genetic and electrophoretic mobility shift assay experiments suggest that elevated concentrations of a PckR effector ligand results in the dissociation of PckR from its target binding site, and evidence is presented that suggests phosphoenolpyruvate may function as the effector. Characterization of missense alleles identified three conserved residues important for increasing the affinity of PckR for its cognate effector molecule. Bioinformatics analyses illustrates that PckR is limited to a narrow phylogenetic range consisting of the , , , and families. These data provide novel insights into the regulation of the core carbon metabolic pathways of this pertinent group of α-proteobacteria.
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http://dx.doi.org/10.1534/genetics.117.300212DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5676232PMC
November 2017

The Divided Bacterial Genome: Structure, Function, and Evolution.

Microbiol Mol Biol Rev 2017 09 9;81(3). Epub 2017 Aug 9.

Department of Biology, McMaster University, Hamilton, Ontario, Canada

Approximately 10% of bacterial genomes are split between two or more large DNA fragments, a genome architecture referred to as a multipartite genome. This multipartite organization is found in many important organisms, including plant symbionts, such as the nitrogen-fixing rhizobia, and plant, animal, and human pathogens, including the genera , , and . The availability of many complete bacterial genome sequences means that we can now examine on a broad scale the characteristics of the different types of DNA molecules in a genome. Recent work has begun to shed light on the unique properties of each class of replicon, the unique functional role of chromosomal and nonchromosomal DNA molecules, and how the exploitation of novel niches may have driven the evolution of the multipartite genome. The aims of this review are to (i) outline the literature regarding bacterial genomes that are divided into multiple fragments, (ii) provide a meta-analysis of completed bacterial genomes from 1,708 species as a way of reviewing the abundant information present in these genome sequences, and (iii) provide an encompassing model to explain the evolution and function of the multipartite genome structure. This review covers, among other topics, salient genome terminology; mechanisms of multipartite genome formation; the phylogenetic distribution of multipartite genomes; how each part of a genome differs with respect to genomic signatures, genetic variability, and gene functional annotation; how each DNA molecule may interact; as well as the costs and benefits of this genome structure.
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http://dx.doi.org/10.1128/MMBR.00019-17DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5584315PMC
September 2017

PhoU Allows Rapid Adaptation to High Phosphate Concentrations by Modulating PstSCAB Transport Rate in Sinorhizobium meliloti.

J Bacteriol 2017 09 22;199(18). Epub 2017 Aug 22.

Department of Biology, McMaster University, Hamilton, Ontario, Canada

Maintenance of cellular phosphate homeostasis is essential for cellular life. The PhoU protein has emerged as a key regulator of this process in bacteria, and it is suggested to modulate phosphate import by PstSCAB and control activation of the phosphate limitation response by the PhoR-PhoB two-component system. However, a proper understanding of PhoU has remained elusive due to numerous complications of mutating , including loss of viability and the genetic instability of the mutants. Here, we developed two sets of strains of that overcame these limitations and allowed a more detailed and comprehensive analysis of the biological and molecular activities of PhoU. The data showed that cannot be deleted in the presence of phosphate unless PstSCAB is inactivated also. However, deletions were readily recovered in phosphate-free media, and characterization of these mutants revealed that addition of phosphate to the environment resulted in toxic levels of PstSCAB-mediated phosphate accumulation. Phosphate uptake experiments indicated that PhoU significantly decreased the PstSCAB transport rate specifically in phosphate-replete cells but not in phosphate-starved cells and that PhoU could rapidly respond to elevated environmental phosphate concentrations and decrease the PstSCAB transport rate. Site-directed mutagenesis results suggested that the ability of PhoU to respond to phosphate levels was independent of the conformation of the PstSCAB transporter. Additionally, PhoU-PhoU and PhoU-PhoR interactions were detected using a bacterial two-hybrid screen. We propose that PhoU modulates PstSCAB and PhoR-PhoB in response to local, internal fluctuations in phosphate concentrations resulting from PstSCAB-mediated phosphate import. Correct maintenance of cellular phosphate homeostasis is critical in all kingdoms of life and in bacteria involves the PhoU protein. This work provides novel insights into the role of the PhoU protein, which plays a key role in rapid adaptation to elevated phosphate concentrations. It is shown that PhoU rapidly responds to elevated phosphate levels by significantly decreasing the phosphate transport of PstSCAB, thereby preventing phosphate toxicity and cell death. Additionally, a new model for phosphate sensing in bacterial species which involves the PhoR-PhoB two-component system is presented. This work provides new insights into the bacterial response to changing environmental conditions and into regulation of the phosphate limitation response that influences numerous bacterial processes, including antibiotic production and virulence.
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http://dx.doi.org/10.1128/JB.00143-17DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5573078PMC
September 2017

Heterologous Complementation Reveals a Specialized Activity for BacA in the Medicago-Sinorhizobium meliloti Symbiosis.

Mol Plant Microbe Interact 2017 04 10;30(4):312-324. Epub 2017 Apr 10.

Department of Biology, McMaster University, 1280 Main St. W., Hamilton, Ontario L8S 4K1, Canada.

The bacterium Sinorhizobium meliloti Rm2011 forms N-fixing root nodules on alfalfa and other leguminous plants. The pSymB chromid contains a 110-kb region (the ETR region) showing high synteny to a chromosomally located region in Sinorhizobium fredii NGR234 and related rhizobia. We recently introduced the ETR region from S. fredii NGR234 into the S. meliloti chromosome. Here, we report that, unexpectedly, the S. fredii NGR234 ETR region did not complement deletion of the S. meliloti ETR region in symbiosis with Medicago sativa. This phenotype was due to the bacA gene of NGR234 not being functionally interchangeable with the S. meliloti bacA gene during M. sativa symbiosis. Further analysis revealed that, whereas bacA genes from S. fredii or Rhizobium leguminosarum bv. viciae 3841 failed to complement the Fix phenotype of a S. meliloti bacA mutant with M. sativa, they allowed for further developmental progression prior to a loss of viability. In contrast, with Melilotus alba, bacA from S. fredii and R. leguminosarum supported N fixation by a S. meliloti bacA mutant. Additionally, the S. meliloti bacA gene can support N fixation of a R. leguminosarum bacA mutant during symbiosis with Pisum sativum. A phylogeny of BacA proteins illustrated that S. meliloti BacA has rapidly diverged from most rhizobia and has converged toward the sequence of pathogenic genera Brucella and Escherichia. These data suggest that the S. meliloti BacA has evolved toward a specific interaction with Medicago and highlights the limitations of using a single model system for the study of complex biological topics.
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http://dx.doi.org/10.1094/MPMI-02-17-0030-RDOI Listing
April 2017

A putative 3-hydroxyisobutyryl-CoA hydrolase is required for efficient symbiotic nitrogen fixation in Sinorhizobium meliloti and Sinorhizobium fredii NGR234.

Environ Microbiol 2017 01 12;19(1):218-236. Epub 2016 Dec 12.

Department of Biology, McMaster University, 1280 Main St. W., Hamilton, Ontario, Canada, L8S 4K1.

We report that the smb20752 gene of the alfalfa symbiont Sinorhizobium meliloti is a novel symbiotic gene required for full N -fixation. Deletion of smb20752 resulted in lower nitrogenase activity and smaller nodules without impacting overall nodule morphology. Orthologs of smb20752 were present in all alpha and beta rhizobia, including the ngr_b20860 gene of Sinorhizobium fredii NGR234. A ngr_b20860 mutant formed Fix determinate nodules that developed normally to a late stage of the symbiosis on the host plants Macroptilium atropurpureum and Vigna unguiculata. However an early symbiotic defect was evident during symbiosis with Leucaena leucocephala, producing Fix indeterminate nodules. The smb20752 and ngr_b20860 genes encode putative 3-hydroxyisobutyryl-CoA (HIB-CoA) hydrolases. HIB-CoA hydrolases are required for l-valine catabolism and appear to prevent the accumulation of toxic metabolic intermediates, particularly methacrylyl-CoA. Evidence presented here and elsewhere (Curson et al., , PLoS ONE 9:e97660) demonstrated that Smb20752 and NGR_b20860 can also prevent metabolic toxicity, are required for l-valine metabolism, and play an undefined role in 3-hydroxybutyrate catabolism. We present evidence that the symbiotic defect of the HIB-CoA hydrolase mutants is independent of the inability to catabolize l-valine and suggest it relates to the toxicity resulting from metabolism of other compounds possibly related to 3-hydroxybutyric acid.
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http://dx.doi.org/10.1111/1462-2920.13570DOI Listing
January 2017

Loss of malic enzymes leads to metabolic imbalance and altered levels of trehalose and putrescine in the bacterium Sinorhizobium meliloti.

BMC Microbiol 2016 07 26;16(1):163. Epub 2016 Jul 26.

Department of Biology, McMaster University, 1280 Main St. West, Hamilton, ON, L8S 4K1, Canada.

Background: Malic enzymes decarboxylate the tricarboxylic acid (TCA) cycle intermediate malate to the glycolytic end-product pyruvate and are well positioned to regulate metabolic flux in central carbon metabolism. Despite the wide distribution of these enzymes, their biological roles are unclear in part because the reaction catalyzed by these enzymes can be by-passed by other pathways. The N2-fixing alfalfa symbiont Sinorhizobium meliloti contains both a NAD(P)-malic enzyme (DME) and a separate NADP-malic enzyme (TME) and to help understand the role of these enzymes, we investigated growth, metabolomic, and transcriptional consequences resulting from loss of these enzymes in free-living cells.

Results: Loss of DME, TME, or both enzymes had no effect on growth with the glycolytic substrate, glucose. In contrast, the dme mutants, but not tme, grew slowly on the gluconeogenic substrate succinate and this slow growth was further reduced upon the addition of glucose. The dme mutant strains incubated with succinate accumulated trehalose and hexose sugar phosphates, secreted malate, and relative to wild-type, these cells had moderately increased transcription of genes involved in gluconeogenesis and pathways that divert metabolites away from the TCA cycle. While tme mutant cells grew at the same rate as wild-type on succinate, they accumulated the compatible solute putrescine.

Conclusions: NAD(P)-malic enzyme (DME) of S. meliloti is required for efficient metabolism of succinate via the TCA cycle. In dme mutants utilizing succinate, malate accumulates and is excreted and these cells appear to increase metabolite flow via gluconeogenesis with a resulting increase in the levels of hexose-6-phosphates and trehalose. For cells utilizing succinate, TME activity alone appeared to be insufficient to produce the levels of pyruvate required for efficient TCA cycle metabolism. Putrescine was found to accumulate in tme cells growing with succinate, and whether this is related to altered levels of NADPH requires further investigation.
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http://dx.doi.org/10.1186/s12866-016-0780-xDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4960864PMC
July 2016

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

Nat Commun 2016 07 22;7:12219. Epub 2016 Jul 22.

Department of Biology, University of Florence, 50019 Sesto Fiorentino, Italy.

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.
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http://dx.doi.org/10.1038/ncomms12219DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4961836PMC
July 2016

L-Hydroxyproline and d-Proline Catabolism in Sinorhizobium meliloti.

J Bacteriol 2016 Feb 1;198(7):1171-81. Epub 2016 Feb 1.

Department of Biology, McMaster University, Hamilton, Ontario, Canada

Unlabelled: Sinorhizobium meliloti forms N2-fixing root nodules on alfalfa, and as a free-living bacterium, it can grow on a very broad range of substrates, including l-proline and several related compounds, such as proline betaine, trans-4-hydroxy-l-proline (trans-4-l-Hyp), and cis-4-hydroxy-d-proline (cis-4-d-Hyp). Fourteen hyp genes are induced upon growth of S. meliloti on trans-4-l-Hyp, and of those, hypMNPQ encodes an ABC-type trans-4-l-Hyp transporter and hypRE encodes an epimerase that converts trans-4-l-Hyp to cis-4-d-Hyp in the bacterial cytoplasm. Here, we present evidence that the HypO, HypD, and HypH proteins catalyze the remaining steps in which cis-4-d-Hyp is converted to α-ketoglutarate. The HypO protein functions as a d-amino acid dehydrogenase, converting cis-4-d-Hyp to Δ(1)-pyrroline-4-hydroxy-2-carboxylate, which is deaminated by HypD to α-ketoglutarate semialdehyde and then converted to α-ketoglutarate by HypH. The crystal structure of HypD revealed it to be a member of the N-acetylneuraminate lyase subfamily of the (α/β)8 protein family and is consistent with the known enzymatic mechanism for other members of the group. It was also shown that S. meliloti can catabolize d-proline as both a carbon and a nitrogen source, that d-proline can complement l-proline auxotrophy, and that the catabolism of d-proline is dependent on the hyp cluster. Transport of d-proline involves the HypMNPQ transporter, following which d-proline is converted to Δ(1)-pyrroline-2-carboxylate (P2C) largely via HypO. The P2C is converted to l-proline through the NADPH-dependent reduction of P2C by the previously uncharacterized HypS protein. Thus, overall, we have now completed detailed genetic and/or biochemical characterization of 9 of the 14 hyp genes.

Importance: Hydroxyproline is abundant in proteins in animal and plant tissues and serves as a carbon and a nitrogen source for bacteria in diverse environments, including the rhizosphere, compost, and the mammalian gut. While the main biochemical features of bacterial hydroxyproline catabolism were elucidated in the 1960s, the genetic and molecular details have only recently been determined. Elucidating the genetics of hydroxyproline catabolism will aid in the annotation of these genes in other genomes and metagenomic libraries. This will facilitate an improved understanding of the importance of this pathway and may assist in determining the prevalence of hydroxyproline in a particular environment.
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http://dx.doi.org/10.1128/JB.00961-15DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4800863PMC
February 2016

Genomic resources for identification of the minimal N2 -fixing symbiotic genome.

Environ Microbiol 2016 09 16;18(8):2534-47. Epub 2016 Feb 16.

Department of Biology, McMaster University, 1280 Main St. W., Hamilton, Ontario, Canada, L8S 4K1.

The lack of an appropriate genomic platform has precluded the use of gain-of-function approaches to study the rhizobium-legume symbiosis, preventing the establishment of the genes necessary and sufficient for symbiotic nitrogen fixation (SNF) and potentially hindering synthetic biology approaches aimed at engineering this process. Here, we describe the development of an appropriate system by reverse engineering Sinorhizobium meliloti. Using a novel in vivo cloning procedure, the engA-tRNA-rmlC (ETR) region, essential for cell viability and symbiosis, was transferred from Sinorhizobium fredii to the ancestral location on the S. meliloti chromosome, rendering the ETR region on pSymB redundant. A derivative of this strain lacking both the large symbiotic replicons (pSymA and pSymB) was constructed. Transfer of pSymA and pSymB back into this strain restored symbiotic capabilities with alfalfa. To delineate the location of the single-copy genes essential for SNF on these replicons, we screened a S. meliloti deletion library, representing > 95% of the 2900 genes of the symbiotic replicons, for their phenotypes with alfalfa. Only four loci, accounting for < 12% of pSymA and pSymB, were essential for SNF. These regions will serve as our preliminary target of the minimal set of horizontally acquired genes necessary and sufficient for SNF.
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http://dx.doi.org/10.1111/1462-2920.13221DOI Listing
September 2016

Proline auxotrophy in Sinorhizobium meliloti results in a plant-specific symbiotic phenotype.

Microbiology (Reading) 2015 Dec 21;161(12):2341-51. Epub 2015 Sep 21.

Department of Biology, McMaster University, 1280 Main Street West, Hamilton, Ontario L8S 4K1, Canada.

In order to effectively manipulate rhizobium-legume symbioses for our benefit, it is crucial to first gain a complete understanding of the underlying genetics and metabolism. Studies with rhizobium auxotrophs have provided insight into the requirement for amino acid biosynthesis during the symbiosis; however, a paucity of available L-proline auxotrophs has limited our understanding of the role of L-proline biosynthesis. Here, we examined the symbiotic phenotypes of a recently described Sinorhizobium meliloti L-proline auxotroph. Proline auxotrophy was observed to result in a host-plant-specific phenotype. The S. meliloti auxotroph displayed reduced symbiotic capability with alfalfa (Medicago sativa) due to a decrease in nodule mass formed and therefore a reduction in nitrogen fixed per plant. However, the proline auxotroph formed nodules on white sweet clover (Melilotus alba) that failed to fix nitrogen. The rate of white sweet clover nodulation by the auxotroph was slightly delayed, but the final number of nodules per plant was not impacted. Examination of white sweet clover nodules by confocal microscopy and transmission electron microscopy revealed the presence of the S. meliloti proline auxotroph cells within the host legume cells, but few differentiated bacteroids were identified compared with the bacteroid-filled plant cells of WT nodules. Overall, these results indicated that L-proline biosynthesis is a general requirement for a fully effective nitrogen-fixing symbiosis, likely due to a transient requirement during bacteroid differentiation.
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http://dx.doi.org/10.1099/mic.0.000182DOI Listing
December 2015

Genetic redundancy is prevalent within the 6.7 Mb Sinorhizobium meliloti genome.

Mol Genet Genomics 2015 Aug 1;290(4):1345-56. Epub 2015 Feb 1.

Department of Biology, McMaster University, 1280 Main St. W., Hamilton, ON, L8S 4K1, Canada.

Biological pathways are frequently identified via a genetic loss-of-function approach. While this approach has proven to be powerful, it is imperfect as illustrated by well-studied pathways continuing to have missing steps. One potential limiting factor is the masking of phenotypes through genetic redundancy. The prevalence of genetic redundancy in bacterial species has received little attention, although isolated examples of functionally redundant gene pairs exist. Here, we made use of a strain of Sinorhizobium meliloti whose genome was reduced by 45 % through the complete removal of a megaplasmid and a chromid (3 Mb of the 6.7 Mb genome was removed) to begin quantifying the level of genetic redundancy within a large bacterial genome. A mutagenesis of the strain with the reduced genome identified a set of transposon insertions precluding growth of this strain on minimal medium. Transfer of these mutations to the wild-type background revealed that 10-15 % of these chromosomal mutations were located within duplicated genes, as they did not prevent growth of cells with the full genome. The functionally redundant genes were involved in a variety of metabolic pathways, including central carbon metabolism, transport, and amino acid biosynthesis. These results indicate that genetic redundancy may be prevalent within large bacterial genomes. Failing to account for redundantly encoded functions in loss-of-function studies will impair our understanding of a broad range of biological processes and limit our ability to use synthetic biology in the construction of designer cell factories.
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http://dx.doi.org/10.1007/s00438-015-0998-6DOI Listing
August 2015
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