R391: a conjugative integrating mosaic comprised of phage, plasmid, and transposon elements

Abstract

The conjugative, chromosomally integrating element R391 is the archetype of the IncJ class of mobile genetic elements. Originally found in a South African Providencia rettgeri strain, R391 carries antibiotic and mercury resistance traits, as well as genes involved in mutagenic DNA repair. While initially described as a plasmid, R391 has subsequently been shown to be integrated into the bacterial chromosome, employing a phage-like integration mechanism closely related to that of the SXT element from Vibrio cholerae O139. Analysis of the complete 89-kb nucleotide sequence of R391 has revealed a mosaic structure consisting of elements originating in bacteriophages and plasmids and of transposable elements. A total of 96 open reading frames were identified; of these, 30 could not be assigned a function. Sequence similarity suggests a relationship of large sections of R391 to sequences from Salmonella, in particular those corresponding to the putative conjugative transfer proteins, which are related to the IncHI1 plasmid R27. A composite transposon carrying the kanamycin resistance gene and a novel insertion element were identified. Challenging the previous assumption that IncJ elements are plasmids, no plasmid replicon was identified on R391, suggesting that they cannot replicate autonomously.

FIG. 1.

Linear map of R391 showing all ORFs and their orientations in the chromosomally integrated state, from the left junction attL to the right junction attR. The boxes above and below the axis represent ORFs in the forward and reverse frames, respectively. ORF numbers, names, and sequence features are indicated; the scale bar is given in kilobases. See also Table 1.

FIG. 2.

G+C plot of the DNA sequence of R391. Regions indicated are the transfer regions TRA I to IV (I to IV), the kanamycin resistance transposon (K), the IS element (IS), the putative restriction enzyme methylase subunit ORF 27 (R), and the mercury resistance operon (M). Asterisks indicate putative transposase genes.

FIG. 3.

Bootstrap neighbor-joining tree of integrases from R391 and SXT, including genomic islands (SGI1, clc, and HiGI), pathogenicity islands (PAI, SHI, SAI, LEE, and HPI), phages (Gifsy1, CP-933C, φ80, lambda, Fels-2, and P4), and uncharacterized putative integrases. Host organisms, accession numbers of protein sequences, and bootstrap values (1,000 replicates) are indicated.

FIG. 4.

Relationship between the putative transfer genes of R391 and homologs present in related plasmids and genomic islands. The names of the R391 transfer genes and transfer regions are indicated. Homologous proteins are depicted by arrows with the same shading and connected by dotted lines. Clustered genes are shown as linked. Note that the structural organization and gene order in the different plasmids and genomic islands differ from those of R391. Both traI and orf34 have no homologs with significant similarity in plasmids except R27. orf34 is a homolog of traG on R27 (based on IncP nomenclature); however, in R391, ORF 83 has been designated traG, in accordance with the IncF nomenclature.