Genome sequences of three agrobacterium biovars help elucidate the evolution of multichromosome genomes in bacteria

Abstract

The family Rhizobiaceae contains plant-associated bacteria with critical roles in ecology and agriculture. Within this family, many Rhizobium and Sinorhizobium strains are nitrogen-fixing plant mutualists, while many strains designated as Agrobacterium are plant pathogens. These contrasting lifestyles are primarily dependent on the transmissible plasmids each strain harbors. Members of the Rhizobiaceae also have diverse genome architectures that include single chromosomes, multiple chromosomes, and plasmids of various sizes. Agrobacterium strains have been divided into three biovars, based on physiological and biochemical properties. The genome of a biovar I strain, A. tumefaciens C58, has been previously sequenced. In this study, the genomes of the biovar II strain A. radiobacter K84, a commercially available biological control strain that inhibits certain pathogenic agrobacteria, and the biovar III strain A. vitis S4, a narrow-host-range strain that infects grapes and invokes a hypersensitive response on nonhost plants, were fully sequenced and annotated. Comparison with other sequenced members of the Alphaproteobacteria provides new data on the evolution of multipartite bacterial genomes. Primary chromosomes show extensive conservation of both gene content and order. In contrast, secondary chromosomes share smaller percentages of genes, and conserved gene order is restricted to short blocks. We propose that secondary chromosomes originated from an ancestral plasmid to which genes have been transferred from a progenitor primary chromosome. Similar patterns are observed in select Beta- and Gammaproteobacteria species. Together, these results define the evolution of chromosome architecture and gene content among the Rhizobiaceae and support a generalized mechanism for second-chromosome formation among bacteria.

FIG. 1.

Phylogenetic tree relating 19 genomes in the Rhizobiales. The tree was inferred from 119,758 aligned protein positions from 507 genes located strictly on the primary chromosome in each genome. Bootstrap support was 100% for all nodes except that linking Bradyrhizobium and Nitrobacter genomes, which was 98 out of 100. Bar, number of amino acid substitutions per site.

FIG. 2.

Phylogenetic analysis of RepC proteins among the Rhizobiaceae. The organism name is followed by the NCBI accession number. Red indicates membership in the Rhizobiales, purple in the Sphingomonadales, blue in the Rhodospirillales, green in the Rhodobacterales, and orange in the Caulobacterales. Arad, A. radiobacter; Avi, A. vitis; Atu, A. tumefaciens; A., Agrobacterium; S., Sinorhizobium; R. etli, Rhizobium etli; R. leguminosarum, Rhizobium leguminosarum; R. sphaeroides, Rhodabacter sphaeroides.

FIG. 3.

Gene conservation among replicons of the Rhizobiales. Graphic depicts ortholog gene alignments shown from the outer circle and moving inward as follows (GenBank accession numbers are in parentheses): Sinorhizobium meliloti 1021 (NC_003047.1), Rhizobium leguminosarum biovar viciae 3841 (NC_008380.1), Rhizobium etli CFN42 (NC_007761.1), K84, S4, C58, Ochrobactrum anthropi ATCC 49188 (NC_009668.1), and Brucella suis 1330 (NC_004310.3). Top, the alignment is anchored by C58 chromosome I; bottom, the alignment is anchored by C58 chromosome II. The anchor replicons themselves are represented by the circles bordered by scales with marks every one-eighth of their total size. Each gene is colored according to its replicon of origin: blue for the primary chromosome, green for secondary chromosomes (including the K84 2.65-Mbp replicon), and orange for plasmids. Note that in all circles except the anchor, the location of a gene in the figure is not tied to physical position in that genome. At higher resolution (http://agro.vbi.vt.edu/public), it is possible to see that many genes in the nonanchor circles occur consecutively in their respective replicons, thus representing syntenic blocks or clusters. The positions of clusters that occur in C58 are listed in Tables S9 and S13 to S15 in the supplemental material and are indicated in the figure by the outermost-arc sections colored black. Each such arc is labeled as Sx-y, where x is the number of the table in the supplemental material and y is the order of the cluster in the table. The alignment in the top panel is predominantly blue, suggesting the high degree of conservation among Rhizobiales primary chromosomes. The alignment in the bottom panel is a mixture of blue, green, and orange, suggesting the mosaic nature of chromosome II and hinting at the various genomic transfers hypothesized to have taken place, as explained in the text.

FIG. 4.

Reconstruction of the origin of secondary chromosomes and related large replicons within the Rhizobiales through transfers of gene clusters from the primordial chromosome to what originally was a repABC-type plasmid (called here the ITR plasmid). LGT, lateral gene transfer.

FIG. 5.

Key gene clusters present on ITR plasmid progenitor of chromosome II and related large replicons during evolution of Rhizobiales. C58 is the reference, and its genes are represented as arrows consistent with the strand they are found on in the deposited genome sequence. Genes for the other genomes were aligned with the C58 genes and are represented with circles or squares. Circles/squares are connected with lines when corresponding genes are consecutive. A black or gray circle means that the gene represented is in a secondary chromosome or plasmid; a black or gray square means that the gene represented is in the primary chromosome. A black circle or square means that the alignment to the C58 ortholog covered 80% or more of both genes; a gray circle or square means the alignment covered less than 80%. Gene numbering is shown for C58 (Agro C58), S4 (Agro vitis S4), K84 (Agro K84), R. etli CFN42, R. leguminosarum biovar viciae 3841 (R.leg), S. meliloti 1021 (S.meli), B. suis 1330, and O. anthropi ATCC49188.