Genome sequence of the enterobacterial phytopathogen Erwinia carotovora subsp. atroseptica and characterization of virulence factors

Abstract

The bacterial family Enterobacteriaceae is notable for its well studied human pathogens, including Salmonella, Yersinia, Shigella, and Escherichia spp. However, it also contains several plant pathogens. We report the genome sequence of a plant pathogenic enterobacterium, Erwinia carotovora subsp. atroseptica (Eca) strain SCRI1043, the causative agent of soft rot and blackleg potato diseases. Approximately 33% of Eca genes are not shared with sequenced enterobacterial human pathogens, including some predicted to facilitate unexpected metabolic traits, such as nitrogen fixation and opine catabolism. This proportion of genes also contains an overrepresentation of pathogenicity determinants, including possible horizontally acquired gene clusters for putative type IV secretion and polyketide phytotoxin synthesis. To investigate whether these gene clusters play a role in the disease process, an arrayed set of insertional mutants was generated, and mutations were identified. Plant bioassays showed that these mutants were significantly reduced in virulence, demonstrating both the presence of novel pathogenicity determinants in Eca, and the impact of functional genomics in expanding our understanding of phytopathogenicity in the Enterobacteriaceae.

Fig. 1.

Comparison of the Eca genome sequence with other bacterial genomes. Inner to outer tracks show the locations of RBHs found by reciprocal fasta of Eca CDSs against those from 32 bacterial genomes (circular plot): Gram-positive (gray); Shewanella oneidensis (ochre); nonenteric animal pathogens (green); plant-associated bacteria (brown); nonenteric plant pathogens (red); and enterobacteria (blue) (Table 2). The locations of CDSs on the Eca genome colored by functional class (see legend to Fig. 5). Two tracks indicating HAIs listed in Table 1. Shown are islands with evidence of recent acquisition (red bars) and possible islands based on reciprocal FASTA analysis (green bars). A plot of G+C skew (red) and percent G+C content (blue).

Fig. 2.

Ratio of observed to expected CDSs by functional class and bacterial group distribution. The log₂(C_func/C_all) ratio of CDSs from Eca strain SCRI1043 shared with no other species (i, dark blue), nonenteric only (ii, cyan) enteric only (iii, yellow), or enteric and nonenteric species (iv, red), by functional class.

Fig. 3.

Screen shot adapted from Artemis Comparison Tool (www.sanger.ac.uk/Software/ACT). (A–C) RBHs between the SPI-7-like regions of S. enterica serovar Typhi (A) and Eca SCRI1043 (B), and the genomic region containing the cfa gene cluster of P. syringae pv. tomato (C). The locations of the cfa gene cluster and the viaB operon are indicated. Shaded lines between the genomes represent RBHs.

Fig. 4.

Mutations in putative type IV secretion and polyketide phytotoxin synthesis genes affect disease development. Lesion development on potato plant stem over a 14-day period after inoculation of Eca strain SCRI1043 wild-type and mutant strains at 10⁴ cells per inoculation site. (A) Wild type. (B) Mutant Eca1043 virB4::Tn5. (C) Mutant Eca1043 cfa6::Tn5. (D) Bar graph showing length of rot (millimeters) for wild-type and mutants Eca1043virB4::Tn5, Eca1043cfa6::Tn5, and Eca1043cfa7::Tn5. LSD = LSD of means across all days (value 4.82).