The Sixth Element: a 102-kb RepABC Plasmid of Xenologous Origin Modulates Chromosomal Gene Expression in Dinoroseobacter shibae

Abstract

The model organism Dinoroseobacter shibae and many other marine Rhodobacterales (Roseobacteraceae, Alphaproteobacteria) are characterized by a multipartite genome organization. Here, we show that the original isolate (Dshi-6) contained six extrachromosomal replicons (ECRs), whereas the strain deposited at the DSMZ (Dshi-5) lacked a 102-kb plasmid. To determine the role of the sixth plasmid, we investigated the genomic and physiological differences between the two strains. Therefore, both genomes were (re)sequenced, and gene expression, growth, and substrate utilization were examined. For comparison, we included additional plasmid-cured strains in the analysis. In the Dshi-6 population, the conjugative 102-kb RepABC-9 plasmid was present in only about 50% of the cells, irrespective of its experimentally validated stability. In the presence of the sixth plasmid, copy number changes of other ECRs, in particular, a decrease of the 86-kb plasmid, were observed. The most conspicuous finding was the strong influence of plasmids on chromosomal gene expression, especially the repression of the CtrA regulon and the activation of the denitrification gene cluster. Expression is inversely controlled by either the presence of the 102-kb plasmid or the absence of the 86-kb plasmid. We identified regulatory genes on both plasmids, i.e., a sigma 70 factor and a quorum sensing synthase, that might be responsible for these major changes. The tremendous effects that were probably even underestimated challenge the current understanding of the relevance of volatile plasmids not only for the original host but also for new recipients after conjugation. IMPORTANCE Plasmids are small DNA molecules that replicate independently of the bacterial chromosome. The common view of the role of plasmids is dominated by the accumulation of resistance genes, which is responsible for the antibiotic crisis in health care and livestock breeding. Beyond rapid adaptations to a changing environment, no general relevance for the host cell's regulome was attributed to these volatile ECRs. The current study shows for the model organism D. shibae that its chromosomal gene expression is strongly influenced by two plasmids. We provide evidence that the gain or loss of plasmids not only results in minor alterations of the genetic repertoire but also can have tremendous effects on bacterial physiology. The central role of some plasmids in the regulatory network of the host could also explain their persistence despite fitness costs, which has been described as the "plasmid paradox."

Keywords: CtrA regulon; Roseobacteraceae; denitrification; heavy metal resistance; plasmid stability; transcriptomics.

FIG 1

The 102-kb plasmid of D. shibae. (A) Pulsed-field gel electrophoresis of strain Dshi-6. (B) Circular visualization of the 102-kb plasmid, with major gene groups indicated. The plasmid was oriented based on its RepABC-9_II-type replication system. The outer to inner rings show (i) the scale of plasmid size in kilobases; (ii) the locations of gene groups on the plus and minus strands, where groups were identified based on automatic and manual annotations; (iii) gene expression via bar plots of the log reads per kilobase per million mapped reads (RPKM) with a scale of 5 to 19; and (iv) GC skew. (C) Classification of chromids and plasmids based on relative synonymous codon usage (RSCU) analysis. (D) Synteny plot of the 102-kb plasmid from D. shibae and the 86-kb plasmid from Roseobacter litoralis. Mob, virD-type mobilization genes. (E) RSCU analysis of Dshi-6 and R. litoralis. (F) Coverage of all replicons in Dshi-5 and Dshi-6. Coverage is shown relative to the chromosome with 1 equivalent per cell.

FIG 2

Chromosomal gene expression and phenotypic microarray of Dshi-6 and Dshi-5 reflect the regulatory role of the 102-kb plasmid in D. shibae. (A) Differential gene expression of Dshi-6 with the respective location on the chromosome. Strongly regulated clusters are color-coded and labeled. (B) Categories of up- and downregulated genes in Dshi-6. (C) Differential conversion of metabolites. AUC, area under the curve.

FIG 3

Gene expression of D. shibae strains with four and six ECRs (Δ86kb, Δ191kb, and Dshi-6) in comparison with the model organism DSM 16493 (Dshi-5) harboring five ECRs. (A) Venn diagram representing the overlap of significantly (P ≤ 0.05) regulated chromosomal genes in three strains compared to the reference strain. (B) Correlation of log₂ fold change (FC) values of all genes in response to the loss of the 86-kb plasmid or the “gain” of the 102-kb plasmid compared to the reference strain. (C) Heatmap of differentially expressed genes on the D. shibae chromosome. Each gene is significantly expressed in at least one of the four strains. Regulators include the partner-switching system rsbV-rsbW (Dshi_0072-Dshi_0073); Dshi_1507; a stress response regulator, lexA (Dshi_1803); two divL genes (Dshi_3433-Dshi_3346); and gafA (Dshi_1584) with its adjacent gene Dshi_1585.