Agrobacterium radiobacter is the only known non-phytopathogenic species inAgrobacterium genus. In this study, the whole-genome sequence ofA. radiobacter type strain DSM 30147^T was described and compared to the other availableAgrobacterium genomes. This bacterium has a genome size of 7,122,065 bp distributed in 612 contigs, including 6,834 protein-coding genes and 41 RNA genes. It harbors a circular chromosome and a linear chromosome but not a tumor-inducing (Ti) plasmid. To the best of our knowledge, this is the first report of a genome from theA. radiobacter species. In addition, an emended description ofA. radiobacter is described. This study reveals information that enhances the current understanding of its non-phytopathogenicity and its phylogenetic position withinAgrobacterium genus.

Agrobacterium radiobacter DSM 30147^T (= ATCC 19358^T) was first isolated from saprobic soil in 1902 asBacillus radiobacter [1] and obtained its current name untilAgrobacterium genus established by Conn in 1942 [2]. Based on phytopathogenic properties, Conn dividedAgrobacterium into 3 species,A. radiobacter, A. tumefaciens andA. rhizogenes [2]. Subsequently,A. rubi, A. vitis andA. larrymoorei were also identified within theAgrobacterium genus [3–6]. Recently,A. rhizogenes was transferred toRhizobium genus, asRhizobium rhizogenes, based on multilocus sequence analysis (MLSA) using several housekeeping genes (rrs, atpD andrecA) [7,8]. In addition, Younget al. proposed thatA. radiobacter should have priority overA. tumefaciens, andA. tumefaciens may not officially represent a species [8,9]. Thus, currently, the genusAgrobacterium contains four validly named species,A. radiobacter, A. vitis, A. rubi andA. larrymoorei [7–9].

A taxonomic classification that relies on the phytopathogenic phenotypes may not accurately reflect the actual phylogenetic relationships of strains withinAgrobacterium [10]. Accordingly, an alternative classification method was applied which divided mostAgrobacterium strains into 3 biovariants (Biovars I, II and III) [10]. Among the 3 biovariants, Biovar I is the most complex group and includes several members (genomovars), designated as genomovar G1 through G9 and G13 [8,11]. At present, two strains in Biovar I have been completely sequenced:Agrobacterium sp. H13-3 (G1) andA. tumefaciens C58 (G8). The genome sequencing revealed that these strains contained two chromosomes and different numbers of plasmids.A. radiobacter DSM 30147^T also belongs to Biovar I (it is classified as a member of genomovar G4), which indicates its close relationship toA. tumefaciens C58 andAgrobacterium sp. H13-3 [12].

Most strains in the genusAgrobacterium are phytopathogens and induce crown gall tumors or hairy root diseases in their host plants [2]. However,A. radiobacter is an exception because it does not have the tumor-inducing (Ti) plasmid that contributes to the pathogenicity [13–16].A. radiobacter members have been widely found in soil, in the rhizosphere of plants and in clinical specimens [17]. A strain ofA. radiobacter was reported to enhance soil arsenic phytoremediation, indicating a potential application in bioremediation [18]. However, some members have been identified as opportunistic human pathogens [19]. So far, a total of 11Agrobacterium genomes (3 finished and 8 draft genomes, listed in Table1) have been sequenced but no genome ofA. radiobacter has been reported. Considering its essential biological feature and important phylogenetic position in the genusAgrobacterium, we present the genome sequence ofA. radiobacter DSM 30147^T, the first sequenced strain in this species.

The descriptions ofA. radiobacter have been reported in 1902 [1], 1942 [2], 1980 [21] and 1993 [22]. After that, fatty acids and utilization of more carbon and nitrogen sources have been tested and showed that the major fatty acids (> 5%) are 16:0, 19:0 cycloω8c, summed feature 2 (one or more of 12:0 aldehyde, iso-16:1 I and 14:0 3-OH) and summed feature 8 (18:1ω7c and/or 18:1ω6c) [23]. The strain can utilize adonitol, D-fructose, D-galactose, D-mannitol, lactose and raffinose as sole carbon sources and L-ornithine, L-proline and L-serine as sole nitrogen sources [23]. Citrate utilization, nitrate reduction and urease are all positive [23]. In this study, we performed more physiological/biochemical analysis and present the emended description ofA. radiobacter.

Genome sequences and 16S rRNA genes were used for phylogenetic analysis. In view of the close evolutionary relationship and the inconsistent phylogeny betweenAgrobacterium andRhizobium [12], we pre-analyzed all sequenced strains in these two genera and found that two “Rhizobium” members were very closely related to the 12Agrobacterium members (including strain DSM 30147^T). Thus, all of the 12Agrobacterium members with sequenced genomes, twoRhizobium strains [R. lupini HPC(L) andRhizobium sp. PDO1-076] (Table1) and an out-group strainR. rhizogenes K84 [7,8], were included in the phylogenetic analysis. A comparison of the 15 genomes revealed a total of 370 proteins that were shared across these genomes. A rooted neighbor-jointing (NJ) phylogenetic tree was constructed based on the shared amino acid sequences. As shown in Figure1a,A. radiobacter DSM 30147^T was in the same cluster as the Biovar I membersAgrobacterium sp. H13-3 (G1) andA. tumefaciens C58 (G8), and showed the closest relationship withA. tumefaciens str. Cherry 2E-2-2. A NJ phylogenetic tree was also constructed based on the 16S rRNA genes (Figure1b). When comparing the trees generated by the core protein sequences with those generated by 16S rRNA gene sequences, small topological differences in topology were found between them. In comparison to the tree generated using the 370 conserved proteins, some strains could not be distinguished with a high degree of clarity using the 16S rRNA genes. Therefore, phylogenomic analysis was considered a more robust approach than that using the 16S rRNA genes to infer the phylogeny, especially for closely related strains [21,25,26].

Phylogenetic trees highlighting the relationships amongA. radiobacter DSM 30147^T and other closely related sequenced strains. (a) A tree was built based on 370 conserved proteins shared among the 15 genomes (12Agrobacterium strains, 2Rhizobium strains very closely related toAgrobacterium and one out-group strain,R. rhizogenes K84); (b) A tree inferred from the 16S rRNA genes of the same strains. The phylogenies were inferred by MEGA 5.05 using the neighbor-joining algorithm [20,24], and 1,000 bootstrap repetitions were computed to estimate the reliability of the branching order. The genome accession numbers of the strains used in the phylogenetic reconstructions:A. albertimagni AOL15, ALJF00000000;Rhizobium sp. PDO1-076, AHZC00000000;A. vitis S4,A. radiobacter, ASXY01000000; GCA_000016285;Agrobacterium sp. H13-3, GCA_000192635;Agrobacterium sp. 10MFCol1.1, ARLJ00000000;A. tumefaciens 5A, AGVZ00000000;A. tumefaciens F2, AFSD00000000;A. tumefaciens C58, GCA_000092025;Agrobacterium sp. ATCC 31749, AECL00000000;R. lupini HPC(L), AMQQ00000000;A. tumefaciens str. Cherry 2E-2-2, APCC00000000;Agrobacterium sp. 224MFTsu3.1, ARQL00000000;A. tumefaciens CCNWGS0286, AGSM00000000 andR. rhizogenes K84 GCA_000016265.

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Strain DSM 30147^T is rod-shaped (0.6–0.8 × 1.5–1.8 µm) (Figure2). The enzyme activities and carbon sources utilization of strain DSM 30147^T were tested using API ZYM, API 20 NE and API ID 32 GN systems and the results are shown in Table2 and in the emended description ofA. radiobacter.

A transmission micrograph ofA. radiobacter DSM 30147^T, using 200 kV transmission electron microscopy FEI Tecnai G² 20 TWIN (USA). The scale bar represents 1 µm.

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Genome project history

To make a comprehensive genomic comparison for theAgrobacterium genomes, the whole genome sequence ofA. radiobacter DSM 30147^T was determined. This draft genome sequence has been deposited at DDBJ/EMBL/GenBank under accession number ASXY00000000. The version described in this study is the first version, ASXY01000000. The project information is summarized in Table3.

Growth condition and DNA isolation

A. radiobacter DSM 30147^T was grown aerobically in LB medium [38] at 28 °C for 24 h. The DNA was extracted, concentrated and purified using the QiAamp kit according to the manufacturer’s instruction (Qiagen, Germany).

Genome sequencing and assembly

Illumina Hiseq2000 with the Paired-End library strategy (300 bp insert size) was used to determine the whole-genome sequence ofA. radiobacter DSM 30147^T and obtained a total of 15,140,909 reads (1.41 Gb data). The detailed methods of library construction and sequencing can be found at Illumina’s official website [39]. Using SOAPdenovo v1.05 [40], these reads were assembled into 612 contigs (> 200 bp) with a genome size of 7,122,065 bp and an average coverage of 196.3 ×.

Genome annotation

The draft genome ofA. radiobacter DSM 30147^T was annotated using the National Center for Biotechnology Information (NCBI) Prokaryotic Genome Annotation Pipeline (PGAP) [41], which combines the gene caller GeneMarkS⁺ [42] with the similarity-based gene detection approach. Protein function classification was performed by searching all the predicted coding sequences of strain DSM 30147^T against the Clusters of Orthologous Groups (COGs) protein database [43] using Blastp algorithm withE-value cutoff 1-e¹⁰.

The whole genome ofA. radiobacter DSM 30147^T is 7,122,065 bp in length, with an average GC content of 59.9%, and distributed in 612 contigs. Compared to the complete reference genomeA. tumefaciens C58 [44] (also belonging to Biovar I, Figure1), the whole genome of strain DSM 30147^T could clearly be divided into 2 replicons, a circular chromosome and a linear chromosome (Figure3). In accordance with its non-phytopathogenicity phenotype, strain DSM 30147^T did not contain a Ti plasmid. Of the 6,894 genes predicted, 6,853 were protein-coding genes (CDSs), and 41 RNA genes. A total of 5,320 CDSs (77.85%) were assigned with putative functions, and the remaining proteins were annotated as the hypothetical proteins. The genome properties and statistics are summarized in Table4 and Figure3. The distribution of the genes into COG functional categories is shown in Table5.

The circular representation of theA. radiobacter DSM 30147^T circular chromosome (left) and linear chromosome (right). From outside to center, ring 1, 4 show protein-coding genes colored by COG categories on forward/reverse strand; ring 2, 3 denote genes on forward/reverse strand; ring 5 shows G+C% content plot, and the innermost ring shows GC skew.

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Strain DSM 30147^T has the largest genome size of the 12Agrobacterium strains sequenced to date and is larger than the 2 very closely relatedRhizobium strain genomes as well (Table1). OrthoMCL [45] was used to perform orthologs clustering analysis for the 14 genomes (Table1). The results indicate thatA. radiobacter DSM 30147^T shares 1,636 genes with the other 13 strains and contains 548 strain-specific genes (Table1), which potentially encode products that contribute to species-specific features differentiatingA. radiobacter from otherAgrobacterium species [46]. In addition, on average, only 31% core genes were shared among the 14 genomes, which reveals a high-degree of diversity withinAgrobacterium genus.

Emended description ofAgrobacterium radiobacter (Beijerinck and van Delden 1902) Conn 1942 (Approved Lists 1980) emend. Sawadaet al. 1993

This emended description is based on that given by Beijerinck and van Delden 1902, Conn 1942 (Approved Lists 1980) and Sawada et al. 1993 with the following changes. Positive results are observed for acid phosphatase, α-glucosidase, alkaline phosphatase, arginine dihydrolase, β-glucosidase, citrate utilization, esterase (C4), leucine arylamidase, N-acetyl-β-glucosaminidase, naphthol-AS-BI-phosphohydrolase, nitrate reduction, urease and valine arylamidase, but negative results for α-galactosidase, α-mannosidase, β-fucosidase, β-galactosidase, β-glucuronidase, chymotrypsin, cystine arylamidase, esterase lipase (C8), lipase (C14) and trypsin. Arabinose, D-glucose, D-melibiose, D-ribose, D-sorbitol, gluconates, histidine, 4-hydroxybenzoate, 3-hydroxybutyrate, inositol, 2-ketogluconate, L-alanine, L-fucose, L-lactate, L-rhamnose, malate, maltose, mannose, N-acetyl glucosamine, propionate, salicin, sodium acetate and sucrose source while cannot assimilate adipate, caprate, 3-hydroxy-benzoate, itaconic acid, glycogen, 5-ketogluconate, phenylacetate, potassium, sodium malonate, suberate and valerate are utilized as the sole carbon sources. L-ornithine, L-proline and L-serine are utilized as nitrogen sources. The major fatty acids (> 5%) are 16:0, 19:0 cycloω8c, summed feature 2 (one or more of 12:0 aldehyde, iso-16:1 I and 14:0 3-OH) and summed feature 8 (18:1ω7c and/or 18:1ω6c). The members of this species are nonphytopathogenic, but in individual cases, some members of this species are detected as possible human pathogens.

This work was supported by the National Natural Science Foundation of China (31010103903 and J1103510). The authors thank the German Collection of Microorganisms and Cell Cultures (DSMZ) for providingA. radiobacter DSM 30147^T cultures.

Authors and Affiliations

1. State Key Laboratory of Agricultural Microbiology, College of Life Sciences and Technology, Huazhong Agricultural University, Wuhan, 430070, P. R. China

   Linshuang Zhang, Xiangyang Li, Feng Zhang & Gejiao Wang

Corresponding author

Correspondence toGejiao Wang.

Authors contributed equally.

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