Complete-genome comparison of six linuron-degrading Variovorax strains (Belgium, Denmark, Japan) showed that near-identical IS1071-flanked composite-transposon catabolic modules sit on different plasmids in each strain, including a novel tRNA-carrying megaplasmid family, a ~560 kb trb-containing trfA-lacking plasmid, and broad-host-range IncP-1delta and PromA plasmids, with 99%-identical IncP-1delta plasmids shared across continents (Öztürk et al. 2020). How this system assembled and spread remains unexplained: the molecular mechanism of IS1071-mediated array formation and inter-replicon movement is unproven (transposition versus translocatable-unit-like circles versus homologous recombination); the transferability and host range of the novel plasmid groups are unknown; the basis for clade-1 Variovorax's predisposition to recruit and express xenobiotic catabolic genes is unresolved; and the origin and long-distance transmission routes of the limited worldwide catabolic reservoir are undetermined.

Background

Linuron is a phenylurea herbicide (3-(3,4-dichlorophenyl)-1-methoxy-1-methylurea) used worldwide since the 1960s. Its complete microbial mineralization requires a three-step pathway: an amide hydrolase (LibA or HylA) cleaves the urea bond to release 3,4-dichloroaniline (DCA), the dca gene cluster oxidizes DCA, and the ccd (chlorocatechol degradation) gene cluster channels the resulting chlorocatechol into the 3-oxoadipate pathway and central metabolism. Variovorax is a genus of beta-proteobacteria common in soils, and linuron mineralization is a specialization of one lineage within it, called clade 1 (the other major lineage of the genus being clade 2). Yet the six known linuron-degrading strains, isolated in Belgium, Denmark, and Japan, do not descend from a single ancestor that already carried the pathway, so the trait must have been assembled repeatedly by horizontal gene transfer.

Öztürk et al. (2020) sequenced the complete genomes of the six strains and found that each carries two to six extrachromosomal replicons—plasmids and, in two strains, chromids—whereas none of the non-degrading Variovorax isolates carries plasmids. The catabolic clusters (hylA or libA, dca, ccd) are nearly identical and syntenic across strains but reside on different plasmids in each strain, and most are bordered by IS1071, an insertion sequence of the Tn3 family (a class II transposon) first described flanking chlorobenzoate catabolic genes on plasmid pBRC60 of Comamonas testosteroni (Fulthorpe and Wyndham 1992). Two copies of the same insertion sequence flanking a DNA segment form a composite transposon, here often stacked into multimeric arrays in which one IS1071 copy is shared between neighboring composite transposons. Two observations argue against these arrays having arisen by ordinary transposition: transposition of a class II composite transposon should generate short direct repeats (duplicated host DNA) at the target site, and these were observed at only one locus; and direct experimental transposition of an IS1071-flanked composite transposon has never been demonstrated in any organism. The authors therefore weighed three candidate mechanisms: genuine transposition; a translocatable-unit mechanism, described for the unrelated IS26 element, in which a circle of one IS plus adjacent cargo inserts next to another identical IS copy (Harmer, Moran and Hall 2014), which would build exactly the observed directly oriented multimeric arrays but has so far been shown only for IS6-family elements; and homologous recombination between IS1071 copies.

The plasmid vehicles are themselves largely uncharacterized. Each strain except one carries at least one plasmid of a known promiscuous family: IncP-1 plasmids (a classic incompatibility group of self-transmissible broad-host-range plasmids, here the IncP-1delta subgroup) or PromA plasmids (another broad-host-range family previously considered mostly cryptic). Strikingly, the ~71 kb IncP-1delta plasmids of the Belgian, Danish, and Japanese strains are 99% identical over their entire length and all carry libA at the same insertion hot spot between trfA (the plasmid replication-initiator gene) and the plasmid origin of replication, evidence of worldwide sharing of essentially a single plasmid. Beyond these known families, the strains carry a family of ~800 kb megaplasmids that are 99% identical to one another, encode a concentrated array of tRNA genes covering all proteinogenic amino acids, contain conjugative-transfer (tra/trb) gene homologs but no identifiable relaxase (the enzyme that nicks the transfer origin to initiate conjugative DNA transfer), and carry most of the catabolic cargo; and a ~560 kb plasmid (pSRS16-2) with IncP-1-like trb transfer genes but no trfA, proposed as the first member of a novel plasmid group. Whether these replicons can actually transfer, and to which hosts, is unknown.

The trait's distribution finally poses two ecological puzzles. Only clade-1 Variovorax repeatedly recruit and express these foreign catabolic genes and are overrepresented among degraders of other anthropogenic compounds, for no identified genomic or physiological reason. And the worldwide pool of catabolic modules is strikingly small, the same few IS1071-flanked loci and near-identical IncP-1delta plasmids recurring on different continents, but whether similar plasmids arose independently at different locations or the same broad-host-range plasmids were transferred across large geographic distances is undetermined. The authors' own explicit closing recommendation is sequence analysis of further genomes within clade 1 and comparison with clade-2 genome sequences, which might unravel the mechanisms of catabolic-gene recruitment; the Discussion elsewhere leaves unresolved whether the new megaplasmids are transferable (no putative relaxase was identified) and notes that movement of the composite transposons as discrete units between replicons has never been shown for IS1071-flanked elements.

Problem Statement

Determine how the linuron catabolic pathway was assembled and disseminated in Variovorax. The comparative genomics of the six linuron-degrading strains (Öztürk et al. 2020) leaves four interlocking questions open toward that account: (1) the molecular mechanism by which IS1071 generates the observed composite-transposon arrays and moves catabolic cargo between replicons, that is, whether the arrays form by direct transposition (which should leave direct repeats at target sites), by a translocatable-unit-like mechanism as described for IS26, or by homologous recombination between IS1071 copies; (2) the transferability and host range of the newly identified tRNA-carrying megaplasmid family and of the novel conjugative plasmid groups such as the ~560 kb trb-containing, trfA-lacking plasmids; (3) the genomic or physiological basis for the predisposition of Variovorax clade-1 members, relative to clade 2, to recruit and express xenobiotic-degrading functions; and (4) how the limited worldwide reservoir of catabolic loci arose, that is, whether the nearly identical IncP-1delta plasmids and composite transposons found at geographically distant sites reflect independent evolution of similar plasmids at different locations or long-distance transfer of the same broad-host-range plasmids. These strands share one objective, an account of the assembly and spread of the pathway.

References

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