3.5 Genes for a novel opine system in pRi1855
The pRi1855 plasmid has several regions with genes of unknown function.
It shares a large region of about 65 kbp (area from 88-153 kbp on the
map of figure 2) with the other Ri plasmids. This region contains genes
putatively involved in sugar transport, glycerol metabolism and encodes
several transcription regulators and two chemoreceptors (Moriguchi et
al. 2001). Besides, the pRi1855 plasmid has several unique areas with
genes that are found in none of the other types of Ri and Ti plasmids
described. These include transposable elements (fig.2) and two larger
areas of about 20 kbp (area from 20-40 kbp on the map adjacent to the
right border of the TL-region in fig.2) and about 24 kbp (area from
64-88 kbp on the map adjacent to the agropine catabolic genes). The
latter area contains mainly metabolic genes and may have been introduced
into pRi1855 by transposition as it is surrounded by IS5-like insertion
sequences. It may have originated from the chromosome of anotherRhizobium species, as a very similar stretch of DNA was detected
in the recently sequenced chromosome of Rhizobium lusitanumstrain 629 (supplementary figure S6). The 20 kbp segment adjacent to the
right border of the TL-region may be involved in the transport and
catabolism of a new opine. In this area we identified all the three
characteristic genes that together code for a putative flavin-containing
opine dehydrogenase (supplementary figures S7-S9). Flavin-containing
opine dehydrogenases such as octopine, nopaline and succinamopine
dehydrogenase consist of three subunits OdhABC that are encoded bynoxABC/ooxABC -like genes arranged in tandem in the genome
(Watanabe et al. 2015). The three genes in pRi1855 (F3X89_28345,
F3X89_28350, F3X89_28355) encode closely related proteins in which the
characteristic binding sites for the FAD and FMN co-factors and the Fe-S
cluster have fully been conserved (supplementary figures S7-S9). These
three genes are surrounded on both sides by genes for a transport system
and a LysR-type regulator. In Ti and Ri plasmids genes encoding an opine
dehydrogenase are often accompanied by genes encoding the permease
required for uptake of specific opine into the bacterial cell. Besides
in the vicinity often genes are present encoding enzymes involved in the
catabolism of the specific products released by the opine dehydrogenase.
In this area of pRi1855 genes encoding such metabolic proteins are also
present including genes encoding a putative saccharopine dehydrogenase
and a putative aminoadipate semialdehyde dehydrogenase, which may form
part of a catabolic pathway of the amino acid lysine (de Mello Serrano
et al. 2012). A gene for an AsnC/Lrp regulator is located at the end of
this DNA segment. The Lrp family of transcriptional regulators is known
to control amino acid metabolism in bacteria (Brinkman et al. 2003).
If these genes are involved in the catabolism of an opine, a gene for an
unknown opine synthase should be present in the T-region of pRi1855.
Genes for agrocinopine synthase are located at the extreme left end of
the T-region in Ti and Ri plasmids, while genes for nopaline synthase,
octopine synthase, and succinamopine synthase are located at the extreme
right end of the T-region in Ti plasmids. In cucumopine, mikimopine, and
mannopine Ri plasmids the genes for cucumopine, mikimopine, and
mannopine synthesis are likewise located immediately next to the right
border repeat. We find at the very right end of the TL-region of pRi1855
two related genes (fig. 3): orf15/rolD and orf16 , which
share 55% identity (fig. 4, table 1). These genes are not present in
the T-regions of any of the other types of Ri plasmids (fig. 3). Theorf15 has been calledrolD ; the encoded RolD protein has weak sequence homology with
ornithine cyclodeaminases and indeed can convert ornithine into proline
(Trovato et al. 2001). The role of rolD in hairy root formation
is marginal, but the gene can influence plant development by its
metabolic activity (Trovato et al. 2018). Using BLASTP with the proteins
encoded by orf15 and orf16 as a query we picked up the
succinamopine synthases encoded by the T-region of chrysopine pTiChry5
(Shao et al. 2018) and the agropine pTiBo542 (Oger et al. 2001) as the
most related proteins. The proteins encoded by orf15 andorf16 share 44-47% identity with the two succinamopine
synthases, which themselves share 93% identity (table 1).
All these proteins (encoded byorf15/rolD, orf16, susL ) are evolutionary related to
ornithine cyclodeaminases (encoded
by ocd genes). They share, for instance, about 19-21% identity
with the ornithine cyclodeaminase encoded by the nopaline Ti plasmid.
That ornithine cyclodeaminase
(ocd ) genes can evolve novel biochemical functions during
evolution is known for some time. For instance its function has been
reported to evolve into an alanine dehydrogenase in Archaeoglobus
fulgidus and into a tauropine dehydrogenase in Halichondria
japonica (Sharma et al. 2013;
Watanabe et al. 2014). Apparently, it can evolve also in an opine
(succinamopine) synthase. Therefore, it would seem possible that either
or both of the ocd -like genes at the right end of pRi1855
(orf15, orf16 ) similarly have evolved a novel opine synthase
function, producing an unknown opine that can be degraded by the
putative opine dehydrogenase encoded in the area with genes of unknown
function located adjacent to the right border of the TL-region.
Discussion
The second complete genomic sequence of a Rhizobium rhizogenesstrain and the first of a virulent strain enabled us to make a
comparison with the sequence of the previously sequenced biocontrol
strain K84. This revealed a high conservation of the primary chromosome,
but showed large differences in the secondary megacircle, the chromid.
It has been described that chromids have a plasmid-like RepABC
replication system, but have a similar GC content as the primary
chromosome and this is also the case in LBA9402. It has been proposed
that chromids are plasmids that evolve into secondary chromosomes and
over time exchange genes with the primary chromosome (Slater et al.
2009; Harrison et al. 2010). We found that the chromid of strain LBA9402
was much smaller than that of strain K84 due to the absence of a segment
of 724 kbp that may have been deleted in LBA9402 or inserted in K84.
Also we found that this insertion/deletion was accompanied by a large
inversion of a segment of 1.8 Mbp. The presence of such complex
rearrangements is in line with their plasmid descent and the genes which
they carry being mostly non-essential.
Our genomic sequence includes the complete sequence of the agropine
pRi1855 plasmid. Over the years sequences have already been published
dealing with specific parts of the closely related agropine Ri plasmid
pRiA4 and earlier this year a draft of the completed pRiA4 sequence was
published (Thompson et al. 2020). When compared our pRi1855 sequence is
indeed closely related, but still differs at numerous areas, both by
base substitutions and small insertions/deletions.
Hairy roots formed by agropine strains contain agropine, agropinic acid,
mannopinic acid and mannopine (Petit et al. 1983). The agropine Ri
plasmid, however, enables host strains to degrade agropine, but not the
other mannityl opines. R. rhizogenes strains such as A4 , but not
1855 or HRI, contain a second, catabolic plasmid with genes for
catabolism of the other three mannityl opines (Petit et al. 1983). We
have now identified the genes for agropine catabolism in pRi1855
adjacent to the right border of the TR-region. The region embraces anagcA gene for the delactonase converting agropine into mannopine,mocC for oxidizing mannopine, and mocD and mocEtogether determining the enzymes that can release the amino acid and a
phosphorylated sugar from the conjugate. This pathway would allow the
bacterium to degrade both agropine and mannopine. However, it is known
that the bacteria carrying pRi1855 cannot degrade mannopine. This may be
because mannopine cannot induce the catabolic genes or because the
bacterium cannot import mannopine in the cell. Indeed the pRi1855
plasmid contains genes for an agropine permease, but not for a mannopine
transport system.
Agrobacteria induce neoplasias in which opines are formed that serve as
a nutritional source of the bacteria. All the different types of Ti and
Ri plasmids described sofar have a gene coding for an opine synthase at
the very right end of the T-region. Our sequence now shows that also the
agropine Ri plasmid has one larger orf (orf16 ) at the very
right end of the TL-region, which shares 55% identity with the
neighboring rolD gene. This gene encodes a protein that is
evolutionary related to ornithine cyclodeaminase (ocd) and which still
has ornithine cyclodeaminase activity (Trovato et al. 2001). Here we
discovered that both rolD and orf16 have also significant
identity of 44-47% with the susL genes encoding succinamopine
synthase in the agropine and chrysopine Ti plasmids.
Ornithine
cyclodeaminase encoding (ocd ) genes have been reported to have
evolved into genes encoding new enzymatic activities such as alanine
dehydrogenase activity in Archaeoglobus fulgidus and tauropine
dehydrogenase activity in Halichondria japonica (Sharma et al.
2013; Watanabe et al. 2014). We hypothesize that an ocd -like gene
may have evolved in the agropine Ri plasmid into a gene for a new opine
synthase. Opine catabolic genes are often located close to the synthase
gene, but on the other side of the right border, an arrangement seen in
many different Ti and Ri plasmids. Three of the genes located here
together indeed have the signature of the trios of genes that are known
to encode the octopine and nopaline dehydrogenase (Watanabe et al.
2015), and thus may encode the opine dehydrogenase needed for catabolism
of the novel unknown opine.
In view of its frequent application, the available sequence will
facilitate the use of R. rhizogenes and especially LBA9402 in
both the laboratory and for biotechnological purposes.