Genomic species are ecological species as revealed by comparative genomics in Agrobacterium tumefaciens

Abstract

The definition of bacterial species is based on genomic similarities, giving rise to the operational concept of genomic species, but the reasons of the occurrence of differentiated genomic species remain largely unknown. We used the Agrobacterium tumefaciens species complex and particularly the genomic species presently called genomovar G8, which includes the sequenced strain C58, to test the hypothesis of genomic species having specific ecological adaptations possibly involved in the speciation process. We analyzed the gene repertoire specific to G8 to identify potential adaptive genes. By hybridizing 25 strains of A. tumefaciens on DNA microarrays spanning the C58 genome, we highlighted the presence and absence of genes homologous to C58 in the taxon. We found 196 genes specific to genomovar G8 that were mostly clustered into seven genomic islands on the C58 genome-one on the circular chromosome and six on the linear chromosome-suggesting higher plasticity and a major adaptive role of the latter. Clusters encoded putative functional units, four of which had been verified experimentally. The combination of G8-specific functions defines a hypothetical species primary niche for G8 related to commensal interaction with a host plant. This supports that the G8 ancestor was able to exploit a new ecological niche, maybe initiating ecological isolation and thus speciation. Searching genomic data for synapomorphic traits is a powerful way to describe bacterial species. This procedure allowed us to find such phenotypic traits specific to genomovar G8 and thus propose a Latin binomial, Agrobacterium fabrum, for this bona fide genomic species.

FIG. 1.—

Presence and estimated similarity of C58 CDS homologs in other genomovar G8 members. Percentage of WES of C58 CDS homologs were plotted against their coordinates on the four C58 replicons. Dot colors indicate the presence (green) or absence (red) of C58 CDS homologs. Diamonds indicate actual similarity values of sequenced PCR products.

FIG. 2.—

Clustering of Agrobacterium tumefaciens strains based on absence/presence of C58 CDS homologs. Neighbor-joining trees were constructed using the paralinear distances of Lake (1994) calculated from the presence/absence of C58 CDSs in other strains. With the reference genome being C58, this strain and its nearly identical relative DC07-004 were excluded from the analysis.

FIG. 3.—

Ubiquity in the Agrobacterium tumefaciens species complex of C58 CDSs according to their localization on C58 chromosomes. Tracks are numbered from inner to outer track (circular chromosome) or top to bottom track (linear chromosome). tRNA and rRNA genes are represented track 1 and 2, respectively (pink). CDSs are represented according to their levels of ubiquity: track 3, specific to C58 (black); track 4, sporadic in G8 (yellow); track 5, strictly specific to G8, and track 6, specific to G8 with a loose criterion (red); track 7, sporadic in At (purple); track 8, specific to At (blue); track 9, “At-Al core-genome” (green). Boxes indicate G8-specific (SpG8) gene clusters.

FIG. 4.—

Codon usage signatures of SpG8 genes. First factorial plan in the correspondence analysis of C58 CDSs according to their codon usage. First and second axes explain 4.5% and 2.2% of total variance, respectively. Grey dots represent CDSs, ellipses represent the inertia of ubiquity classes, and boxes represent barycenters of SpG8 loci named as detailed in table 2 and supplementary table S6 (Supplementary Material online) (and 0 for interspersed SpG8 CDSs). Codon usage of ubiquity classes were found to gradually vary from core to sporadic genes revealing, in turn, that SpG8 clusters can be distinguished by this criterion from core-like ones to sporadic-like ones. Interestingly, SpG8-1a, SpG8-4, and SpG8-7a—which are shared by the most closely related non-G8 strain G6-NCPPB 925 (table 2)—displayed a core-like codon usage.

FIG. 5.—

Experimental evidences of G8-specific phenotypes determined by SpG8 loci. (A) Ferulic acid degradation by A. tumefaciens strains determined by HPLC and UV spectrum at 320 nm. mAU, milli absorbance units. All genomovar G8 members were able to catabolize all ferulic acid in 12 h, contrary to non-G8 strains lacking SpG8-1b. (B) Curdlan production revealed by red dye on Congo red medium. C58: Agrobacterium tumefaciens wild-type strain C58 (red colonies), C58ΔSpG8-2a: SpG8-2a-deleted mutant (white colonies).

FIG. 6.—

Putative ferulic acid catabolism pathway encoded by SpG8-1b. (A) SpG8-1 CDSs organization in C58 (top): subregions SpG8-1a and SpG8-1b are colored in purple and red, respectively. Presence in other Agrobacterium tumefaciens strains (bottom): presence, black; absence, white. (B) Reconstructed ferulic acid catabolism pathway encoded by SpG8-1b according to similarities to sequences in databases and associated literature: Fcs, feruloyl-CoA synthetase (Overhage et al. 1999; Plaggenborg et al. 2003); Ech, enoyl-CoA hydratase (Pelletier and Harwood 1998); Fcd, feruloyl-CoA dehydratase; LigM, tetrahydrofolate-dependent vanillate O-demethylase (Nishikawa et al. 1998); MetF, methylenetetrahydrofolate reductase (Nishikawa et al. 1998). (C) Putative transcriptional regulation of SpG8-1b genes inferred from sequence similarities in databases: VanR, vanillate catabolism repressor (Morawski et al. 2000); FerR, ferulate catabolism regulator (Breese and Fuchs 1998; Calisti et al. 2008).

FIG. 7.—

Hypothetical integrated functioning of SpG8 genes allowing G8 member adaptation to their specific ecological niche. EPS, exopolysaccharide; CrdS, curdlan synthase; McsS, mechano-sensitive channel; NodVW, two-component system sensor kinase and response regulator; BraCDEFG, branched-chain amino acid transporter; RbsABC, ribose transporter; TetA, tetracycline extrusion pump; MatE, multidrug transporter; FecBCD, iron-siderophore transporter.