ABSTRACT
This study reports draft genomes of 30 bacteria representative of the plant food system microbiota and isolated from different sources in Italy and France. Individual genomes were reconstructed using PacBIO DNA sequencing: taxonomic classification and distribution of genes involved in microbe-environment interactions are reported to facilitate strains' characterization and utilization.
KEYWORDS: food system microbiota, rhizobia, bacilli, whole-genome sequencing, PacBio HiFi
ANNOUNCEMENT
The microbial communities thriving in association with plants, collectively referred to as the plant microbiota, have long been identified as a resource to reduce the environmental footprint of plant food systems (1). Within the framework of the H2020 Innovation Action “CIRCLES”, we report the sequencing and preliminary characterization of 30 bacterial genomes representative of taxa enriched in the plant food system microbiota and part of collections held at a commercial company in Italy (MS Biotech) and at the Agroecology Department of the French National Research Institute for Agriculture, Food and Environment Bourgogne Franche-Comté (INRAE-BFC).
Cultures of Rhizobuim sp., Bradyrhizobium sp., and Rhodococcus sp. strains were obtained from frozen glycerol stock cultures by inoculating a 10 microliter loop of frozen crystals in liquid broth (TY) (10 g/L tryptone, 5 g/L yeast extract, and 0.06 M CaCl2) incubated at 28°C until late exponential phase (from 2 to 5 days). For the other strains described in this study, 50 microliters of glycerol stocks was transferred into 100 mL of Nutrient Broth and incubated at 30°C until exponential phase was reached (~24 hours). Genomic DNA was extracted using the Qiagen Blood & Cell Culture DNA Kit (Cat. No. 19060) and high molecular weight genomic was subsequently purified from lysate using the Qiagen Genomic-tip 500/G columns (Cat. No. 10262) according to manufacturer's instructions. Genomic DNA was quantified by a Qubit fluorometric assay, and its size was estimated by Agilent Tape Station analysis (using the genomic DNA Kit) following the manufacturer's protocol. Sequencing libraries were prepared using the SMRTbell Express Template Prep Kit 2.0 (PacBio, Menlo Park, USA) following manufacturer's recommendations for an insert size target of 10 Kb. Sequencing was performed using the “HiFi sequencing” using the platform PacBio Sequel II (PacBio, Menlo Park, USA) at Genotoul-GeT-PlaGe (Toulouse, France). PacBio HiFi data in fastq format were assembled, including overlap alignment and correction, using hifiasm (version 0.19.5-r587). The final graph assembly was converted to fasta format using gfatools (0.4-r214-dirty). Assemblies were interrogated for quality and contamination using quast (v5.2.0) (2) and Bloobtools (version 1.1.1) (3), respectively. Assembly completeness was assessed using BUSCO (version 5.4.3) (4) with bacteria_odb10 models. Genome annotation was performed using bakta (1.8.1) (5). Genome taxonomic classification was performed using GTDB-tk (version 2.3.2) (6). Orthology, comparative, and phylogenetic analyses were performed using OrthoFinder (version 2.5.5) (7), and bcgTree (version 1.2.0) (8) was used for tree's construction. Annotated genomes were mined using tools in Galaxi Europe (9) and the RAST server (10). We used iTOL (version 6) (11) to visualize and annotate a phylogenetic tree; unless otherwise specified, default parameters were used for the computational tools utilized. Sequencing and assembly information is available in data set “CIRCLES_WP3 S1” accessible at https://zenodo.org/records/14356128 .
As a proof of concept, we mined the annotation of individual genomes for key traits in microbe-environment interactions, including genes putatively implicated in (a) nodulation and nitrogen fixation in rhizobia (12), (b) nitrous oxide reduction (13), (c) the production of phytohormones (14), and (d) induced system resistance (15) as summarized in Table 1. To facilitate sample retrieving, we constructed a phylogenetic tree highlighting genomes' relatedness with reference strains (Fig. 1).
TABLE 1.
Taxonomic affiliation, prediction, functional genes, accession numbers of genomes, and isolation source of the 30 individual bacterial isolates described in this study
| Isolate ID and taxonomya | nif | nod | nosZ | cheA | YsnE | trpA,B | men | bgl | Accession | Source & cultivationb |
|---|---|---|---|---|---|---|---|---|---|---|
| CIRCLES_WGS1 | S,U | - | - | Y | + | + | + | + | SAMEA115779467 | Spinach soil, Italy. |
| Rhodococcus qingshengii | Minimal salt medium containing metamitron 100 mg/L as sole source of carbon and nitrogen (16) | |||||||||
| CIRCLES_WGS2 | U,Z | - | - | Y,C,X | + | + | + | + | SAMEA115779468 | |
| Rhodococcus qingshengii | ||||||||||
| CIRCLES_WGS3 | U | - | - | Y | + | + | + | + | SAMEA115779469 | |
| Rhodococcus qingshengii | ||||||||||
| CIRCLES_WGS4 | U,T,B,A,D,K,E,X,N,H | + | + | A,W,Y,D,B,R,C,X | + | + | + | + | SAMEA115779470 | Trigonella foenum graecum isolate, France. |
| Sinorhizobium medicae | Reference isolation protocol (17) | |||||||||
| CIRCLES_WGS6 | T,B,A,D,K,E,X,N,U,S,H | + | + | A,W,Y,D,B,R | + | + | + | + | SAMEA115779471 | Medicago sativa isolate, France. |
| Sinorhizobium meliloti | Reference isolation protocol (17) | |||||||||
| CIRCLES_WGS7 | T,B,A,D,K,E,X,N,U,S,H | + | + | A,W,Y,D,B,R,C,X | + | + | + | + | SAMEA115779472 | |
| Sinorhizobium meliloti | ||||||||||
| CIRCLES_WGS8 | T,B,A,D,K,E,X,N,U,S,H | + | - | A,W,Y,D,B,R | + | + | + | + | SAMEA115779473 | Trigonella foenum graecum isolate, France. |
| Sinorhizobium meliloti | Reference isolation protocol (17) | |||||||||
| CIRCLES_WGS9 | D,K,E,H,N,X,S,T,B,Q,W,A,U | + | - | A,W,Y,B,R,C,X | + | + | + | + | SAMEA115779474 | Soybean nodule isolate, France. |
| Bradyrhizobium japonicum | Reference isolation protocol (17) | |||||||||
| CIRCLES_WGS10 | D,K,E,H,N,X,S,T,B,Q,W,A,U | + | - | A,W,Y,B,R,C,X | + | + | + | + | SAMEA115779475 | |
| Bradyrhizobium japonicum | ||||||||||
| CIRCLES_WGS11 | D,K,E,H,N,X,S,T,B,Q,W,A,U | + | - | A,W,Y,B.R | + | + | + | + | SAMEA115779476 | |
| Bradyrhizobium diazoefficiens | ||||||||||
| CIRCLES_WGS12 | D,K,E,H,N,X,S,T,B,Q,W,A,U | + | - | W,Y,B.R | + | + | + | + | SAMEA115779477 | |
| Bradyrhizobium sp011516635 | ||||||||||
| CIRCLES_WGS14 | D,K,E,H,N,X,S,T,B,Q,W,A,U | + | - | W,Y,B.R | + | + | + | + | SAMEA115779478 | |
| Bradyrhizobium diazoefficiens | ||||||||||
| CIRCLES_WGS15 | D,K,E,H,N,X,S,T,B,Q,W,A,U,Z | + | + | W,Y,B,R | + | + | + | + | SAMEA115779479 | |
| Bradyrhizobium diazoefficiens | ||||||||||
| CIRCLES_WGS16 | S,U | - | - | A,W,Y,D,B,R,C,X | + | + | + | - | SAMEA115779480 | Sandy soil, Italy. |
| Cytobacillus firmus | Nutrient agar plus cycloheximide, 30°C | |||||||||
| CIRCLES_WGS17 | S,U | - | - | A,W,Y,D,B,R,C,X | + | + | + | + | SAMEA115779481 | Bean leaf, Italy. |
| Bacillus safensis | Nutrient agar plus cycloheximide, 30°C | |||||||||
| CIRCLES_WGS18 | F,M,Z,W,V,S,U,X,N,E,Y,T,K,D,H,Q,B,A,L | - | - | A,W,Y,B,R, | + | + | - | - | SAMEA115779482 | Wheat rhizosphere, Italy. |
| Azotobacter salinestris | Enrichment culture in nitrogen-free mannitol medium, 25°C (18) | |||||||||
| CIRCLES_WGS19 | S,U | - | - | A,W,Y,D,B,R | - | + | + | + | SAMEA115779483 | Wheat rhizosphere, Italy. |
| Priestia endophytica | Nutrient agar plus cycloheximide, 30°C | |||||||||
| CIRCLES_WGS20 | S,U | - | - | A,W,Y,D,B,R,C,X | + | + | + | + | SAMEA115779484 | Sandy soil, Italy. Peptone-meat extract- KNO3 medium, 45°C (19) |
| Bacillus licheniformis | ||||||||||
| CIRCLES_WGS21 | U | - | - | A,W,Y,D,B,R,C,X | + | + | - | - | SAMEA115779485 | Succulent root, Italy. |
| Paenibacillus zeisoli | Nutrient agar plus cycloheximide, 30°C | |||||||||
| CIRCLES_WGS22 | S,U | - | - | W,Y,B,R | - | + | - | + | SAMEA115779486 | Pomegranate roots, Italy |
| Pseudomonas chlororaphis | Triptic soy agar, 30°C | |||||||||
| CIRCLES_WGS23 | S,U | - | - | A,W,Y,D,B,R | + | + | + | + | SAMEA115779487 | Popillia japonica, Italy. |
| Priestia megaterium | Nutrient agar plus cycloheximide, 30°C | |||||||||
| CIRCLES_WGS24 | S,U | - | - | A,W,Y,B,R, | - | + | + | + | SAMEA115779488 | Vine leaf treated with copper, Italy. |
| Bacillus bombysepticus | Nutrient agar plus cycloheximide, 30°C | |||||||||
| CIRCLES_WGS25 | S,U | - | - | A,W,Y,D,B,R | + | + | + | - | SAMEA115779489 | Sandy soil, Italy. |
| Bacillus thuringiensis | Nutrient agar plus cycloheximide, 30°C | |||||||||
| CIRCLES_WGS27 | U,S,X,N,E,K,D,H,B, | - | - | A,W,Y,D,R,C,X | - | + | + | + | SAMEA115779490 | Aloe leaf compost, Italy. |
| Paenibacillus polymyxa | Nutrient agar plus cycloheximide, 30°C | |||||||||
| CIRCLES_WGS28 | U | - | - | A,W,Y,D,B,R,C,X | - | + | + | - | SAMEA115779491 | Nematode eggs, Italy. |
| Lysinibacillus xylanilyticus | Nutrient agar plus cycloheximide, 30°C | |||||||||
| CIRCLES_WGS29 | U | - | - | A,W,Y,D,B,R,C,X | + | + | - | + | SAMEA115779492 | Underwood, Italy. |
| Bacillus simplex | Nutrient agar plus cycloheximide, 30°C | |||||||||
| CIRCLES_WGS30 | S | - | - | A,W,Y,D,B,R,C,X | + | + | + | + | SAMEA115779493 | Cabbage leaf, Italy. |
| Bacillus velezensis | Nutrient agar plus cycloheximide, 30°C | |||||||||
| CIRCLES_WGS31 | S | - | - | A,W,Y,D,B,R,C,X | + | + | + | + | SAMEA115779494 | Copper-based product MSBiotech, Italy. |
| Bacillus velezensis | Nutrient agar plus cycloheximide, 30°C | |||||||||
| CIRCLES_WGS32 | S | - | - | A,W,Y,D,B,R,C,X | + | + | + | + | SAMEA115779495 | Sandy soil, Italy. |
| Bacillus halotolerans | Nutrient agar plus cycloheximide, 45°C | |||||||||
| CIRCLES_WGS33 | S | - | - | A,W,Y,D,B,R,C,X | + | + | + | + | SAMEA115779496 | Blended aloe leaf, Italy. |
| Bacillus velezensis | Nutrient agar plus cycloheximide, 30°C |
Strain taxonomy reflects the lowest and unique rank as defined by GTDBTK (v2.3.2).
Fig 1.
Whole-genome phylogenetic tree of individual genomes (ANI cutoff, 95%) constructed incorporating additional reference sequences, depicted with genus and species name for each clade. The tree was constructed with 100 bootstrap iterations; the color code depicts strain’s taxonomic affiliation at phylum level. The outer shapes indicate strain’s availability in either MS Biotech or INRAE collection.
ACKNOWLEDGMENTS
This work was supported by the Horizon 2020 Framework Program Innovation Action “CIRCLES” (European Commission grant agreement 818290) awarded to the University of Dundee, INRAE, MSBiotech, and the University of Bologna. C.A. is supported by a UKRI BBSRC eastBIO PhD studentship (project 2734186). We thank Matteo Pollini and Michela Matassoni (OROGEL, Italy) for having provided the spinach soil samples for the enrichment cultures. We thank Genotoul-GeT-PlaGe staff (Toulouse, France) for their support for the full sequencing of the genomes of the microbial cultures.
L.P. and M.D-L., bacterial isolation; M.D-L., sample preparation; P.T., raw sequencing data analysis, assembly, annotation, and phylogenetic tree construction; L.P., annotated data analysis; C.A., figure preparation; C.S., data archiving and preparation for dissemination; L.P. and D.B., draft writing; M.D-L., P.T., C.A., C.S., S.R-A., A.H., F.M-L., and J.A., reviewing and editing; L.P., A.H., F.M-L., and D.B., conceptualization; D.B., funding acquisition.
Contributor Information
Davide Bulgarelli, Email: d.bulgarelli@dundee.ac.uk.
Julia A. Maresca, SUNY College of Environmental Science and Forestry, Syracuse, New York, USA
DATA AVAILABILITY
The genomic sequences reported in this study are deposited in the European Nucleotide Archive (ENA), project PRJEB75963. Accession numbers for the individual genomes are provided in Table 1. To acquire isolates, or for questions or suggestions, please contact Laura Pietrantonio at MSBiotech or Alain Hartmann and Marion Devers-Lamrani at the Agroecology department of INRAE. The codes to reproduce data analyses are available at https://github.com/bartongroup/PT_HiFi_rhizobial_assemblies/.
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Associated Data
This section collects any data citations, data availability statements, or supplementary materials included in this article.
Data Availability Statement
The genomic sequences reported in this study are deposited in the European Nucleotide Archive (ENA), project PRJEB75963. Accession numbers for the individual genomes are provided in Table 1. To acquire isolates, or for questions or suggestions, please contact Laura Pietrantonio at MSBiotech or Alain Hartmann and Marion Devers-Lamrani at the Agroecology department of INRAE. The codes to reproduce data analyses are available at https://github.com/bartongroup/PT_HiFi_rhizobial_assemblies/.