Comparative genomic analysis of the pPT23A plasmid family of Pseudomonas syringae

Abstract

Members of the pPT23A plasmid family of Pseudomonas syringae play an important role in the interaction of this bacterial pathogen with host plants. Complete sequence analysis of several pPT23A family plasmids (PFPs) has provided a glimpse of the gene content and virulence function of these plasmids. We constructed a macroarray containing 161 genes to estimate and compare the gene contents of 23 newly analyzed and eight known PFPs from 12 pathovars of P. syringae, which belong to four genomospecies. Hybridization results revealed that PFPs could be distinguished by the type IV secretion system (T4SS) encoded and separated into four groups. Twelve PFPs along with pPSR1 from P. syringae pv. syringae, pPh1448B from P. syringae pv. phaseolicola, and pPMA4326A from P. syringae pv. maculicola encoded a type IVA T4SS (VirB-VirD4 conjugative system), whereas 10 PFPs along with pDC3000A and pDC3000B from P. syringae pv. tomato encoded a type IVB T4SS (tra system). Two plasmids encoded both T4SSs, whereas six other plasmids carried none or only a few genes of either the type IVA or type IVB secretion system. Most PFPs hybridized to more than one putative type III secretion system effector gene and to a variety of additional genes encoding known P. syringae virulence factors. The overall gene contents of individual PFPs were more similar among plasmids within each of the four groups based on T4SS genes; however, a number of genes, encoding plasmid-specific functions or hypothetical proteins, were shared among plasmids from different T4SS groups. The only gene shared by all PFPs in this study was the repA gene, which encoded sequences with 87 to 99% amino acid identityamong 25 sequences examined. We proposed a model to illustrate the evolution and gene acquisition of the pPT23A plasmid family. To our knowledge, this is the first such attempt to conduct a global genetic analysis of this important plasmid family.

FIG. 1.

Representative plasmid macroarray hybridization images for PFPs containing the type IVB (A) and type IVA (B) secretion system genes. A total of 164 genes were printed in duplicate on a 7.5- by 11.5-cm membrane. The position of each gene on the array was labeled as a combination of P (plate), C (column), and R (row). (A) Array hybridized with pPSM8810 from P. syringae pv. maculicola strain 88-10; (B) array hybridized with pPSS4918 from P. syringae pv. syringae strain 4918. Arrowheads, spots containing the blank and chromosomal genes used as negative controls.

FIG. 2.

Phylogenetic tree constructed by using the neighbor-joining method indicating relationships among RepA proteins encoded by the pPT23A plasmid family. The GenBank accession numbers for RepA protein sequences encoded by plasmids pDC3000A, pDC3000B, pPMA4326A, pPMA4326B, pPT23A, pPSR1, and pFKN areAE016855, AE016854, AY603979, AY603980, AJ224509, AY342395, and AF359557, respectively. The RepA sequences encoded by pPh1448A and pPh1448B were obtained from the TIGR website (www.tigr.org). The scale is shown in number of substitutions per site. The genomospecies that the P. syringae pathovar host of each plasmid belongs to is indicated as a superscript.

FIG. 3.

(A) Schematic map for the type IVA secretion genes from plant-related bacteria. The type IVA secretion system is normally encoded by 11 virB genes and the virD4 gene, as represented by the A. tumefaciens pTi VirB-VirD4 reference system. Structural variations and gene rearrangements occur in different plasmids and/or genomes. Genes with similar functions were drawn with similar colors. Genes without color have no known functions. Solid lines indicate genes that are physically unlinked. A superscript “a” indicates that two or more virB6 genes with ISs were found either upstream or downstream of the operon. The GenBank accession numbers for plasmids or genomes are as follows: X. campestris pv. campestris ATCC 33913 genome (XCC) and plasmid pXcB, NC_003902 and NC_005240; X. axonopodis pv. citri 306 genome (XAC) and plasmid pXAC64, NC_003919 and NC_003922; Erwinia amylovora EA110 plasmid pEU30, NC_005247; Erwinia carotovora subsp. atroseptica SCRI1043 genome (ECA), BX950851; P. syringae pv. maculicola 4326 plasmid pPMA4326A, NC_005918; P. syringae pv. syringae A2 plasmid pPSR1, NC_005205; A. tumefaciens strain C58 plasmid pAT_washington, NC_003306; A. tumefaciens plasmid pTi_Washington, Sakura, NC_003308, NC_002147, and NC_002377; Agrobacterium rhizogenes plasmid pRi1724, NC_002575. (B) Structural conservation of type IVB secretion genes. Representatives of the type IVB subfamilies are the R64 and ColIb-P9 (IncI) tra systems. Solid lines indicate genes that are physically unlinked. *, essential genes for the type IVB secretion system (13). 1, pR64 and ColIb-P9; 2, pCTX-M3; 3, pEL60; 4, pDC3000A; 5, pDC3000B. The GenBank accession numbers for plasmids are as follows: P. syringae pv. tomato DC3000 plasmids pDC3000A and pDC3000B: NC_004633 and NC_004632; Citrobacter freundii plasmid pCTX-M3, NC_004464; E. amylovora plasmid pEL60, NC_005246; Salmonella enterica serovar Typhimurium plasmid pR64, NC_005014; ColIb-P9, NC_002122.