Genome Content and Phylogenomics Reveal both Ancestral and Lateral Evolutionary Pathways in Plant-Pathogenic Streptomyces Species

Abstract

Streptomyces spp. are highly differentiated actinomycetes with large, linear chromosomes that encode an arsenal of biologically active molecules and catabolic enzymes. Members of this genus are well equipped for life in nutrient-limited environments and are common soil saprophytes. Out of the hundreds of species in the genus Streptomyces, a small group has evolved the ability to infect plants. The recent availability of Streptomyces genome sequences, including four genomes of pathogenic species, provided an opportunity to characterize the gene content specific to these pathogens and to study phylogenetic relationships among them. Genome sequencing, comparative genomics, and phylogenetic analysis enabled us to discriminate pathogenic from saprophytic Streptomyces strains; moreover, we calculated that the pathogen-specific genome contains 4,662 orthologs. Phylogenetic reconstruction suggested that Streptomyces scabies and S. ipomoeae share an ancestor but that their biosynthetic clusters encoding the required virulence factor thaxtomin have diverged. In contrast, S. turgidiscabies and S. acidiscabies, two relatively unrelated pathogens, possess highly similar thaxtomin biosynthesis clusters, which suggests that the acquisition of these genes was through lateral gene transfer.

FIG 1

Gene content dendrogram. Shown are phylogenetic relationships of 14 streptomycete strains based on gene content. The dendrogram was constructed by UPGMA clustering based on similarities in gene content for strains (cophenetic correlation coefficient of 0.95). Horizontal bars represent dissimilarities, measured by the Jaccard coefficient, which range from 0 to 1. Pathogenic Streptomyces spp. are highlighted in red. The gene content of Saccharopolyspora erythraea strain NRRL2338 was used as an outgroup.

FIG 2

Distribution of pathogen-specific genes. The Venn diagram shows the distribution of the pathogen-specific ortholog groups among the four pathogenic Streptomyces spp. The numbers of ortholog groups not specific to the pathogens (i.e., present in the indicated pathogen[s] and one or more saprophytic species) are shown in parentheses.

FIG 3

Phylogenetic relationships and synteny analysis of the tomA clusters in plant-pathogenic Streptomyces spp. On the left is the topology of the tree constructed from the concatenated nucleotide sequences of genes in the 15-kb tomA cluster. The Clavibacter michiganensis subsp. michiganensis tomA cluster was used as the outgroup. Labels in the genes indicate the names of the genes or the locus tags for each species. Orange, tomA (tomatinase) and blgA (beta-glucosidase); green, tetR (transcriptional regulator); red, ABC transporter system; blue (from left to right), beta-glucosidase and glycosyl hydrolase. Gray open reading frames 1374 and 1375 in tomA cluster 1 encode a transcriptional regulator and a putative cutinase, respectively. The gray arrow in tomA cluster 2 represents a hypothetical protein. The gray arrow in the tomA cluster in Clavibacter michiganensis subsp. michiganensis represents a transcriptional regulator. Pink areas designate syntenic gene regions, and only these regions were used in the phylogenetic analysis.

FIG 4

Consensus supertree inferred from common genes. The 1,000 individual gene trees are summarized in this consensus supertree. The percentages of genes that support the branches of the tree are indicated in red. The horizontal bar at the top represents substitutions per nucleotide site. Pathogenic Streptomyces strains are highlighted in red. Sequences of Saccharopolyspora erythraea NRRL2338 were used as outgroups in the analyses. Five well-supported clades, designated STRA, STRA1, STRB, STRB1, and STRC, are indicated.

FIG 5

Gene content analysis of the thaxtomin biosynthetic cluster. The thaxtomin biosynthetic operon of each pathogen is depicted. Orthologs of the txtD and txtE genes are found in S. lavendulae and Streptomyces sp. Mg1. The GC content is depicted at the top of each cluster (upward peaks in gold indicate above-average GC content, and downward peaks in purple designate below-average GC content). S. scabies and S. acidiscabies clusters are identical and depicted only once. Genes in blue are predicted insertion sequences; truncated coding regions that might represent pseudogenes are in gray.

FIG 6

Comparison of topologies derived from the common gene phylogenic tree (core tree) and the gene content (Gcont) tree with the thaxtomin tree (Txt tree). The top diagram shows the general structure of the thaxtomin biosynthesis cluster. Depicted within the txtA and txtB genes are the relative positions of the encoded AMP-binding adenylation (A), methyltransferase (M), peptidyl carrier protein (P), and condensation (C) domains. Asterisks denote the entire txt genes or domain regions of txtA and txtB used to construct the trees. The txtC sequence was not included in the analysis because it is absent in S. ipomoeae.

FIG 7

Architecture of domains in the NRPSs TxtA and TxtB. NRPSs contain four conserved domains: adenylation (AMP), methyltransferase (MET), peptidyl carrier protein (PP), and condensation (COND). In the cases of TxtA (top) and TxtB (bottom), the MET domain is embedded within the C-terminal region of the AMP domain. TxtA of all 4 Streptomyces plant pathogens and TxtB of S. ipomoeae contain the MET12 (methyltransferase type 12) domain, while TxtB in S. scabies, S. acidiscabies, and S. turgidiscabies contains the methyltransferase type 11 domain. Alignments and conserved residues are shown in blue.