Molecular evolution of pathogenicity-island genes in Pseudomonas viridiflava

Abstract

The bacterial pathogen Pseudomonas viridiflava possesses two pathogenicity islands (PAIs) that share many gene homologs, but are structurally and phenotypically differentiated (T-PAI and S-PAI). These PAIs are paralogous, but only one is present in each isolate. While this dual presence/absence polymorphism has been shown to be maintained by balancing selection, little is known about the molecular evolution of individual genes on the PAIs. Here we investigate genetic variation of 12 PAI gene loci (7 on T-PAI and 5 on S-PAI) in 96 worldwide isolates of P. viridiflava. These genes include avirulence genes (hopPsyA and avrE), their putative chaperones (shcA and avrF), and genes encoding the type III outer proteins (hrpA, hrpZ, and hrpW). Average nucleotide diversities in these genes (pi = 0.004-0.020) were close to those in the genetic background. Large numbers of recombination events were found within PAIs and a sign of positive selection was detected in avrE. These results suggest that the PAI genes are evolving relatively freely from each other on the PAIs, rather than as a single unit under balancing selection. Evolutionarily stable PAIs may be preferable in this species because preexisting genetic variation enables P. viridiflava to respond rapidly to natural selection.

Figure 1.—

Schematics for locations of the PAI genes investigated in this study. T-PAI is located in region 1 (left) and S-PAI is located in region 2 (right). Physical distance of the two regions is unknown in P. viridiflava. [AT], [AS], and [BS] represent [clade, PAI-type] of the isolates. In all the samples, the presence of T-PAI in region 1 was perfectly associated with the absence of S-PAI in region 2, and vice versa. Arrowheads represent locations where at least one recombination event was detected by Hudson and Kaplan's R_m (1985), and the number below the arrowhead represents R_m found within or between loci.

Figure 2.—

Genealogical relationships of hopPsyA and shcA (A) and a part of hrpK (B). The neighbor-joining trees were constructed on the basis of the nucleotide variations in the third codon position in the combined coding sequences of hopPsyA + schA (1704 bp) and the partial sequences of hrpK (175 bp). The sequences in P. syringae were obtained from either Charity et al. (2003) or Deng et al. (2003). Bootstrap probabilities (%) with 1000 replicates, which were >80%, were represented on or below the major branch. The partial hrpK sequences in P. viridiflava were only available in LP23.1a and PNA3.3a (Araki et al. 2006).

Figure 3.—

Genealogical relationships of five shared genes between T-PAI and S-PAI. The neighbor-joining trees were constructed on the basis of the nucleotide variations in the third codon position in the combined coding sequences of the five genes (aveE, avrF, hrpA, hrpZ, and hrpW, 6720 bp comparable in total) after the Jukes–Cantor correction (Kumar et al. 2001). Bootstrap probabilities (percentage) with 1000 replicates were represented on or below the major branches.

Figure 4.—

Window plot analysis of nucleotide divergence between [AS] and [BS] in avrE. The genetic divergences in replacement sites (solid line) and in synonymous sites (dashed line) are plotted with relative position of the start position of the coding region in avrE. The window size and step size were 100 and 25 bp, respectively. An asterisk represents a midpoint of the windows that showed statistical significance (P < 0.05) by both Fu and Li's D* and F* in [BS] with a sequence from [AS] as an outgroup. D* = −2.35 and F* = −2.46 in a window of 392–503 (bp from the start codon), D* = −2.35 and F* = −2.46 in a window of 2493–2592, and D* = −2.41 and F* = −2.77 in a window of 2718–2817.