Interstrain transfer of the large pathogenicity island (PAPI-1) of Pseudomonas aeruginosa

Abstract

The large Pseudomonas aeruginosa pathogenicity island PAPI-1 of strain PA14 is a cluster of 108 genes that encode a number of virulence features. We demonstrate that, in a subpopulation of cells, PAPI-1 can exist in an extrachromosomal circular form after precise excision from its integration site within the 3' terminus of the tRNA(Lys) gene. Circular PAPI-1 can reintegrate into either of the two tRNA(Lys) genes, including the one that was used for integration of small pathogenicity island PAPI-2 in strain PA14. The excision requires PAPI-1-encoded integrase, a member of the tyrosine recombinase family. PAPI-1 Soj contains the conserved domains of proteins that are related to chromosome and plasmid partition. soj plays a role in maintaining PAPI-1 and mutations in soj result in the loss of PAPI-1 from P. aeruginosa. We further demonstrate that, during coculture, the PAPI-1-containing strains are able to transfer it into P. aeruginosa recipient strains that do not harbor this island naturally. After transfer, PAPI-1 integrates into either of the two tRNA(Lys) genes. PAPI-1 encompasses many features of mobile elements, including mobilization and maintenance modules. Together with the virulence determinants, PAPI-1 plays an important role in the evolution of P. aeruginosa, by expanding its natural habitat from soil and water to animal and human infections.

Fig. 1.

Detection of integrated and circular PAPI-1 in P. aeruginosa. (A) Model for PAPI-1 excision from the chromosome. Open arrowheads indicate the primers used for detection of integrated and circular PAPI-1 in various P. aeruginosa strains. The genes are not drawn to scale. (B). I, II, III, and IV are PCR amplifications with primer pairs 4542F + intF, sojR + 4541F, intF+sojR, and 4542F + 4541F, respectively.

Fig. 2.

Detection of the composite island adjacent to PA0976.1 in strain PA14. (A) Schematic diagram shows the gene organization between PA0976 and PA0988 after integration of PAPI-1 at PA0976.1. Open arrows indicate the primers used to amplify the junctions between chromosome and PAPI-1 after PAPI-1 integrates into the PA0976.1. (B) PCR amplifications with primer pairs 976F + PAPI-1R and sojR167 + PAPI-2R.

Fig. 3.

Transcriptional analysis of soj and quantification of PA14 cells carrying circular PAPI-1. (A) Chromosomal locations of various soj promoter-containing fragments. The red arrows indicate the orientation of the fragments relative to the lacZ reporter gene. Open arrows indicate the primers used to amplify various soj promoter-containing fragments. Primer pairs P1F + R, P2F + R, P3F + R, and P4F + R were used to amplify DNA fragments sojP1, sojP2, sojP3, and sojP4, respectively. (B) Determination of β-galactosidase at midexponential (open bars) and early stationary phases (filled bars) in strains PA14lacZ (vector), PA14sojP1lacZ (sojP1), PA14sojP2lacZ (sojP2), PA14sojP3lacZ (sojP3), and PA14sojP4lacZ (sojP4). (C) Quantification of the proportion of cells that carry circular PAPI-1 by flow cytometry. Vector is the strain PA14Δint soj::gfp transformed with empty vector pPSV35. pint is the strain PA14Δint soj::gfp transformed with cloned int on pPSV35. The percentage of GFP-positive cells (0.16%) is the average of three independent measurements.