Last week of annotation by our students (see list of annotation topics). Let’s see what they’re up to. This is a snapshot of an unfinished annotation.
In which of the three replicons are the core genes? And first, are they chromosomes or plasmids? GC content, GC skew, and codon usage all indicate that the first replicon is a chromosome, the second a chromid (or megaplasmid or secondary chromosome), and the third a plasmid. So far, no more than 5 genes out of 206 are found on the chromid or the plasmid, and all the others on the chromosome, confirming its chromosome status. Next, to check the replication partitionning machinery.
Ralstonia 705 has a Nitrogen fixating system, but it cannot do symbiosis with plants, since they lack the genes for symbiosis, such as the Nod factors. It can use nitrate respiration, with a denitrification process, although it lacks Nitrous oxide reductase. Relative to closely related strains, such as Burkholderia vietnamensis, which is N2 fixing, gene content and order is well conserved (see Fig. 1), and is on the chrosome as in R705, whereas relative to also closely related symbiotic bacteria gene order is not conserved, and the genes are on a plasmid.
In Ralstonia 743, we found almost an entire phage, in one block in the genome, and the integration regions AttL and AttR. It’s on the chromosome, whereas there are no plasmids found in the other replicons. There are two other regions with phage sequences, but not a complete phage. We also found 5 genomic islands on the chromosome, and 2 on the chromid (chromosome 2), which include many heavy metal transporters. In one of the islands, an interesting resistance to polymyxin, an antibiotic, was also found.
Both Ralstonia have many transporters, generally the same ones. There are notably many heavy metal transporters, which are often conserved in other bacteria and organized in operons. For example, Co, Ni, Ar and Hg all have such conserved operons.
Concerning the degration of aromatic compounds, the 705 strain has one operon of the aerobic degradation of benzoyl-CoA pathway almost complete, and there are two such operons in 743. BoxE is missing in all operons, but PcaF is always present and seems to have the same Beta-ketoadipyl-CoA thiolase function. Thus both strains can apparently do complete aerobic degradation of benzoyl-CoA. For the degradation of toluene, 743 has the complete pathway whereas 705 misses the end of the pathway, and is bracketed by two transposases which are absent from 743. This raises the question of how the difference appeared, especially since at least one other Ralstonia strain also has the partial operon (Müller et al 2003).
Staying on aromatic compounds, we looked for transporters. 743 has a chlorobenzene transporter, whereas we didn’t find any in 705; this transporter seems to also be able to transport styrene, based on Blast hits and its gene neighborhood on the chromosome; we checked for this styrene transport based on the KEGG pathways mapped to Ralstonia genes. We also found a benzoate transporter in both strains, with an interesting conserved neighborhood (Fig. 2) containing benzoate-CoA ligase and sub-units of an ABC transporter, as well as a transporter for 4-hydrobenzoate in a different chromosomal location. Overall, this partially explains how these strains degrade aromatic compounds in the environment.
Still on aromatics, 743 seems to have many operons for aromatic degradation, mostly through benzoate metabolism. There is notably a complete Tod operon for toluene degradation (Fig. 3), on the chromid. Benzoate is converted to catechol though the BenABCD operon, which is entirely present. We also found a large PAAX operon present in all Ralstonia, but without clearly described function; of note, the same gene names are annotated in E. coli, but with very limited sequence conservation, so function may or may not be conserved. PAAX neighbors Box on the chromid. 743 also seems to degrade phtalates and protocatechuates.
Finally, in 705, most of these pathways are conserved. But there is a gene missing for the degradation of toluene (Fig. 4), which is consistent with preliminary unpublished experimental results. There are mcb genes to degrade chlorobenzene. There is a locus with all genes to degrade aminophenol which is inside a mobile CLC element.
Genomic islands of 705: the CLC island is obviously there, but seems misassembled, because a small insertion surrounded by duplicate sequences which should be in the island is in the “unplaced” contigs. There is a large deletion (≈10kb) relative to the Knackmussi CLC island. We found another genomic island, which apparently belongs to the Tn4371 family; the integration and conjugal transfer are well conserved, whereas the rest of the island is not (Fig. 5). The non conserved regions seem to contain arsenic resistance and a multidrug efflux transporter. There are clearly more genomic islands, but without clear homology to known elements in the ICEberg database. This illustrates how genomic islands are still poorly known in general. We also found two rather well conserved Mu-like phages; one has most of the structural proteins, so is probably quite young, whereas the other is more degraded. There are also more phage remains which are difficult to identify further. They are all on chromosome 1, there are no phages detected on the other replicons. Althoug we didn’t find any specific resistance genes inside the phages, we did find a multidrug resistance and a Vibrio cholera toxin just neighboring phages.
And now for something completely different: comparing different assemblies of 705 and 743, to improve the assembly. Thus we improved the alignment between the strains. Improving the assembly of the chromid was easier based on a published strain (H16) than based on our two strains. Interestingly, the differences between our two strains have odd GC content, a direction which is being pursued.
Also in comparisons, the phylogeny of Ralstonia seems different between the 16S phylogeny and that based on core genes (unique copy, present in all 47 species of Burkhonderiaceae compared). We also did a phylogeny based on the ICE elements, one based on 23S, and 6 randomly selected genes. This work is ongoing.
So, an emerging picture of dynamic evolution of Ralstonia with three replicons carrying heavy metal resistance and aromatic compound transport and degradation, for a species found in contaminated environments.