Fatal affairs - conjugational transfer of a dinoflagellate-killing plasmid between marine Rhodobacterales

Abstract

The roseobacter group of marine bacteria is characterized by a mosaic distribution of ecologically important phenotypes. These are often encoded on mobile extrachromosomal replicons. So far, conjugation had only been experimentally proven between the two model organisms Phaeobacter inhibens and Dinoroseobacter shibae. Here, we show that two large natural RepABC-type plasmids from D. shibae can be transferred into representatives of all known major Rhodobacterales lineages. Complete genome sequencing of the newly established Phaeobacter inhibens transconjugants confirmed their genomic integrity. The conjugated plasmids were stably maintained as single copy number replicons in the genuine as well as the new host. Co-cultivation of Phaeobacter inhibens and the transconjugants with the dinoflagellate Prorocentrum minimum demonstrated that Phaeobacter inhibens is a probiotic strain that improves the yield and stability of the dinoflagellate culture. The transconjugant carrying the 191 kb plasmid, but not the 126 kb sister plasmid, killed the dinoflagellate in co-culture.

Keywords: Roseobacter; bacteria–algae interaction; conjugation; horizontal gene transfer.

Fig. 1.

Conjugational transfer of D. shibae 126 and 191 kb plasmids within the roseobacter group. Phylogenomic tree of Rhodobacterales clades 1 to 10 (as defined by Bartling et al. [30]); the branches for the clades hve been collapsed and the complete tree is shown in Fig. S1. Naturally occurring homologues of the 126 and 191 kb D. shibae plasmids are indicated in blue beside the respective leaf. Successful experimental conjugation of either plasmid is indicated in red. Phaeobacter and Dinoroseobacter are according to their natural pigmentation highlighted in brown and pink, respectively. Branch lengths show substitutions per site. Bootstrap values are shown near the branch nodes.

Fig. 2.

Genome organization and ECR copy number of D. shibae and Phaeobacter inhibens strains. (a) Genome organization of Phaeobacter inhibens and D. shibae strains used for comparative analyses. Host cell and replicon affiliation is colour-coded in brown and pink for Phaeobacter and Dinoroseobacter , respectively. X indicates absence of the respective replicon. Chr, chromosome. ECRs are indicated by their size in kb. (b) The mean coverage was calculated via mapping of Illumina sequences on the different replicons. The coverage range of each replicon is illustrated in violin plots on the left-hand side and the chromosomal median indicated by a solid line. The 191 kb killer plasmid and the 126 kb sister plasmid are highlighted in red and orange, respectively.

Fig. 3.

Co-cultivation of Prorocentrum minimum and Phaeobacter inhibens . Axenic cultures of Prorocentrum minimum were inoculated with Phaeobacter inhibens control strains ( Phaeobacter inhibens Δ65kb and Phaeobacter inhibens Δ65kbΔ262kb) or transconjugants carrying either the 191 kb killer plasmid of D. shibae ( Phaeobacter inhibens Δ65kbΔ262kb+191kb) or the 126 kb sister plasmid of D. shibae ( Phaeobacter inhibens Δ65kbΔ262kb+126kb). Cultures of Prorocentrum minimum in vitamin B₁₂-replete and -limited medium (L1+B₁₂, L1−B₁₂) served as positive and negative controls, respectively. Mean values and standard deviations of eight replicates are shown. Control experiments without bacteria are shown in blue, co-cultivation experiments with Phaeobacter reference strains and transconjugants are indicated in green and red/orange, respectively. d, Days. A.U.: arbitrary units