3.2 Comparison to the sequence of R. rhizogenes strain K84
The only other complete genome of R. rhizogenes obtained thus far is that of the avirulent biocontrol strain K84 (Slater et al.2009). This latter strain does not contain an Ri plasmid, but the two LBA9402 megacircles could be aligned to those of K84 using MUMmer (NUCmer) (Kurtz et al. 2004). As can be seen in supplementary figure S2 the largest contig of LBA9402 is largely collinear with the primary chromosome of K84 (96% of the sequence can be aligned to that of K84 at >95% sequence identity). It contains the genes for replication such as for a DnaA replication protein, repair and DNA recombination, for cell division, for transcription and translation including the 3 rRNA clusters of the bacterium and the 53 tRNA genes. We annotated an extra tRNA in one of the unique regions of the LBA9402 primary chromosome. The chromosome contains a putative genomic island of about 100 kbp with a set of genes for conjugative DNA transfer encoding not only a Type4 secretion system for mating pair formation (from position 3,324,071-3,314,501), but also the enzymes necessary for DNA transfer and replication (from position 3,302,269-3,307,564). The genomic island contains a gene for a putative integrase and is surrounded by a direct repeat of 15 bp, which may be (the remains of) two att -sites. Larger differences between the chromosomes of LBA9402 and K84 are mainly due to presence/absence of other mobile elements. For example, various proteins encoded in the unique segment of DNA from position 745,195-784,185 in LBA9402, have homology to phage proteins (Rhizobium phage vB_RleM_PPF1 and other tailed phages) as revealed by the phage search tool PHASTER (Arndt et al. 2016) and thus seems due to the insertion of a prophage (supplementary figure S3).
The second-largest replicons are less similar (supplementary figure S2, fig. 1), but still 85% of the LBA9402 sequence aligns to that of K84 (and 64% of K84 aligns to LBA9402). The LBA9402 sequence is smaller, mainly due to a large approximately 724 kb deletion, which seems accompanied by a large 1.8 Mbp inversion (fig. 1). The large deletion did not affect one functional category of genes in particular as can be seen in supplementary figure 1, demonstrating that there are no major differences in the categories of genes present according to eggNOG-mapper analysis.
This secondary megacircle has a plasmid-like RepABC replication system, but has a similar GC content as the primary chromosome. Such secondary megacircles are considered (developing) secondary chromosomes that over evolutionary time exchange genes with the primary chromosome and have been coined ‘chromids’ (Slater et al. 2009; Harrison et al. 2010). The chromid of LBA9402 contains many metabolic genes, but also genes for the production of cell wall polysaccharides and fimbriae/pili. We found in the chromid a set of genes homologous to the erythritol region inSinorhizobium meliloti and Rhizobium leguminosarum(supplementary figure S4) including a transport operon with geneseryEFG , a catabolic operon with genes eryABCD and adeoR -type regulator (also called eryR ) followed by genes called eryH and eryI (Yost et al. 2006; Geddes and Oresnik 2012; Barbier et al. 2014). The ability to catabolize erythritol is one of the key characteristics distinguishing biotype 1 and biotype 2 agrobacteria (Kerr and Panagopoulos 1977). The presence of erythritol catabolic genes was thus expected, but it was remarkable that they were present on the more dynamic chromid instead of the chromosome. Finally the 252 kbp circle represents the agropine pRi1855 plasmid, which is very different from the large nopaline catabolic plasmid carried by strain K84, and will be described below.