RcgA and RcgR, Two Novel Proteins Involved in the Conjugative Transfer of Rhizobial Plasmids

Abstract

Rhizobia are Gram-negative bacteria that are able to establish a nitrogen-fixing symbiotic interaction with leguminous plants. Rhizobia genomes usually harbor several plasmids which can be transferred to other organisms by conjugation. Two main mechanisms of the regulation of rhizobial plasmid transfer have been described: quorum sensing (QS) and the rctA/rctB system. Nevertheless, new genes and molecules that modulate conjugative transfer have recently been described, demonstrating that new actors can tightly regulate the process. In this work, by means of bioinformatics tools and molecular biology approaches, two hypothetical genes are identified as playing key roles in conjugative transfer. These genes are located between conjugative genes of plasmid pRfaLPU83a from Rhizobium favelukesii LPU83, a plasmid that shows a conjugative transfer behavior depending on the genomic background. One of the two mentioned genes, rcgA, is essential for conjugation, while the other, rcgR, acts as an inhibitor of the process. In addition to introducing this new regulatory system, we show evidence of the functions of these genes in different genomic backgrounds and confirm that homologous proteins from non-closely related organisms have the same functions. These findings set up the basis for a new regulatory circuit of the conjugative transfer of plasmids. IMPORTANCE Extrachromosomal DNA elements, such as plasmids, allow for the adaptation of bacteria to new environments by conferring new determinants. Via conjugation, plasmids can be transferred between members of the same bacterial species, different species, or even to organisms belonging to a different kingdom. Knowledge about the regulatory systems of plasmid conjugative transfer is key in understanding the dynamics of their dissemination in the environment. As the increasing availability of genomes raises the number of predicted proteins with unknown functions, deeper experimental procedures help to elucidate the roles of these determinants. In this work, two uncharacterized proteins that constitute a new regulatory circuit with a key role in the conjugative transfer of rhizobial plasmids were discovered.

Keywords: Rhizobia; Rhizobium; conjugation; gene regulation; plasmid.

FIG 1

Phylogenetic analysis of LPU83a_0146 and LPU83a_0148. (A) Phylogenetic tree based on LPU83a_0146. (B) Phylogenetic tree based on LPU83a_0148. (C) Genomic organization of conjugative genes in plasmids from groups X and Y. In pLPU83a, genes hyp1 and hyp2 correspond to LPU83a_0146 and LPU83a_0148, respectively. The accession numbers of each protein are listed in Table S2. Bootstrap values higher than 50 are shown on the branches. Proteins encoded in plasmids are annotated as the name of the plasmid. Black arrows highlight the hypothetical proteins of pLPU83a and the homologs found in pLPU88a and Shinella sp. DD12. The * symbol indicates that a sequence is annotated as a scaffold, meaning that it is not possible to determine whether it belongs to a plasmid.

FIG 2

Conjugative transfer frequency of mutants in LPU83a_0145, LPU83a_0146, LPU83a_0148, and the complemented strains. (A) Evaluation of CT frequencies of plasmid pLPU83a-13 and the LPU83a_0145, LPU83a_0146, and LPU83a_0148 mutants of this plasmid. (B) Evaluation of the CT frequencies of plasmid pLPU83a-13 and the LPU83a_0146 and LPU83a_0148 mutants containing the empty vector pBBR1MCS-5 or a vector carrying an entire copy of the deleted gene. (C) CT frequencies of the rcgR mutant in the presence of homologous genes encoded by S. meliloti LPU88 and Shinella sp. DD12. (D) CT frequencies of the rcgA mutant in the presence of homologous genes encoded by S. meliloti LPU88 and Shinella sp. DD12. n.d: not detected. A statistical comparison was performed via Dunnett’s multiple comparison test in relation to the wild-type values. ns: not significant. *, P = 0.0134; **, P = 0.0024; ***, P = 0.0004; ****, P < 0.0001.

FIG 3

Conjugative transfer frequencies of the plasmid pLPU83a and the rcgR and rcgA derivative mutants from different genomic backgrounds. (A) CT frequencies from the S. meliloti 20MP6 genomic background. (B) CT frequencies from the A. tumefaciens UBAPF2 genomic background. (C) CT frequencies from the S. meliloti LPU88 genomic background. (D) CT frequencies from the Shinella sp. DD12 genomic background. n.d: not detected. A statistical comparison was performed via Dunnett’s multiple comparison test in relation to the wild-type values. ns: not significant. *, P = 0.0435; **, P < 0.0001.

FIG 4

Evaluation of traR transcription through fluorescence quantification. (A) Evaluation of the traR transcription from the R. favelukesii LPU83 genomic background. (B) Evaluation of the traR transcription from the A. tumefaciens UBAPF2 genomic background. Each strain carries a GFP protein encoded downstream of traR, so the GFP transcription depends on the traR promoter activity. A statistical comparison was performed via Dunnett’s multiple comparison test in relation to the wild-type values. ns: not significant. ****, P  < 0.0001.