Comparative Genomics of Novel Agrobacterium G3 Strains Isolated From the International Space Station and Description of Agrobacterium tomkonis sp. nov.

Nitin K Singh,Joseph Nesme,Florent Lassalle,Christopher E Mason,Xavier Nesme,Kasthuri Venkateswaran,Céline Lavire,Ludovic Vial

doi:10.3389/fmicb.2021.765943

Abstract

Strains of Agrobacterium genomospecies 3 (i.e., genomovar G3 of the Agrobacterium tumefaciens species complex) have been previously isolated from diverse environments, including in association with plant roots, with algae, as part of a lignocellulose degrading community, from a hospital environment, as a human opportunistic pathogen, or as reported in this study, from a surface within the International Space Station. Polyphasic taxonomic methods revealed the relationship of Agrobacterium G3 strains to other Agrobacterium spp., which supports the description of a novel species. The G3 strains tested (n = 9) were phenotypically distinguishable among the strains from other genomospecies of the genus Agrobacterium. Phylogenetic analyses of the 16S rRNA gene, gyrB gene, multi-locus sequence analysis, and 1,089-gene core-genome gene concatenate concur that tested G3 strains belong to the Agrobacterium genus and they form a clade distinct from other validly described Agrobacterium species. The distinctiveness of this clade was confirmed by average nucleotide identity (ANI) and in silico digital DNA–DNA hybridization (dDDH) comparisons between the G3 tested strains and all known Agrobacterium species type strains, since obtained values were considerably below the 95% (ANI) and 70% (dDDH) thresholds used for the species delineation. According to the core-genome phylogeny and ANI comparisons, the closest relatives of G3 strains were Agrobacterium sp. strains UGM030330-04 and K599, members of a novel genomospecies we propose to call genomovar G21. Using this polyphasic approach, we characterized the phenotypic and genotypic synapomorphies of Agrobacterium G3, showing it is a bona fide bacterial species, well separated from previously named Agrobacterium species or other recognized genomic species. We thus propose the name Agrobacterium tomkonis for this species previously referred to as Agrobacterium genomospecies 3. The type strain of A. tomkonis is IIF1SW-B1T (= LMG 32164 = NRRL B-65602). Comparative genomic analysis show A. tomkonis strains have species-specific genes associated with secretion of secondary metabolites, including an exopolysaccharide and putative adhesins and resistance to copper. A. tomkonis specific gene functions notably relate to surface adhesion and could be involved to colonize nutrient-poor and harsh habitats. The A. tomkonis strains from the ISS showed presence of a 40-kbp plasmid and several other potential mobile genetic elements detected that could also be part of conjugative elements or integrated prophages.

Highlights

Agrobacterium species are firstly known for their pathogenic potential on plant, causing either crown gall or hairy root disease, depending on the presence of a tumor-inducing (Ti) or rootinducing (Ri) megaplasmid in their genome; most strains lack such plasmids and are not pathogenic
The three strains collected from ISS belonging to Agrobacterium G3 along with six other G3 strains were subjected to a polyphasic characterization to determine the variable, conserved or distinctive traits of this genomic species
We found 556 genes present in the three ISS strains; we identified these genes based on their presence in all ISS strains and none of the 17 genomes in the background clade comprising the other A. tomkonis strains, all strains of Agrobacterium arsenijevicii, Agrobacterium nepotum, Agrobacterium genomospecies G1, G5, G13, and G2 (Supplementary Data online

Summary

Introduction

Agrobacterium species are firstly known for their pathogenic potential on plant, causing either crown gall or hairy root disease, depending on the presence of a tumor-inducing (Ti) or rootinducing (Ri) megaplasmid in their genome; most strains lack such plasmids and are not pathogenic. Further refinement of the taxonomy of Agrobacterium biovar 1, known as the Agrobacterium tumefaciens species complex, was conducted based on whole-genome information using DDH, leading to the classification of strains into species-level units called genomic species or genomovars (Popoff et al, 1984) This classification framework was later validated and enriched by studies using amplified fragment-length polymorphism (AFLP), marker gene phylogeny, multi-locus sequence analysis (MLSA), comparative genome hybridization (CGH) and core-genome phylogeny (Portier et al, 2006; Costechareyre et al, 2010; Lassalle et al, 2011, 2017; Mousavi et al, 2015), as recently reviewed by Flores-Felix et al (2020). Nine out of 15 described genomic species within the Agrobacterium biovar 1 are not yet formally named: G3 (this study), G5, G6, G7 (“Agrobacterium deltaense”), G8 (“Agrobacterium fabrum”), G13, G15 (“Agrobacterium viscosum”), G19 and G20 (Mafakheri et al, 2019)

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