Genome comparison of the epiphytic bacteria Erwinia billingiae and E. tasmaniensis with the pear pathogen E. pyrifoliae

Abstract

Background: The genus Erwinia includes plant-associated pathogenic and non-pathogenic Enterobacteria. Important pathogens such as Erwinia amylovora, the causative agent of fire blight and E. pyrifoliae causing bacterial shoot blight of pear in Asia belong to this genus. The species E. tasmaniensis and E. billingiae are epiphytic bacteria and may represent antagonists for biocontrol of fire blight. The presence of genes that are putatively involved in virulence in E. amylovora and E. pyrifoliae is of special interest for these species in consequence.

Results: Here we provide the complete genome sequences of the pathogenic E. pyrifoliae strain Ep1/96 with a size of 4.1 Mb and of the non-pathogenic species E. billingiae strain Eb661 with a size of 5.4 Mb, de novo determined by conventional Sanger sequencing and next generation sequencing techniques. Genome comparison reveals large inversions resulting from homologous recombination events. Furthermore, comparison of deduced proteins highlights a relation of E. billingiae strain Eb661 to E. tasmaniensis strain Et1/99 and a distance to E. pyrifoliae for the overall gene content as well as for the presence of encoded proteins representing virulence factors for the pathogenic species. Pathogenicity of E. pyrifoliae is supposed to have evolved by accumulation of potential virulence factors. E. pyrifoliae carries factors for type III secretion and cell invasion. Other genes described as virulence factors for E. amylovora are involved in the production of exopolysaccharides, the utilization of plant metabolites such as sorbitol and sucrose. Some virulence-associated genes of the pathogenic species are present in E. tasmaniensis but mostly absent in E. billingiae.

Conclusion: The data of the genome analyses correspond to the pathogenic lifestyle of E. pyrifoliae and underlines the epiphytic localization of E. tasmaniensis and E. billingiae as a saprophyte.

Figure 1

Venn diagram for the deduced proteins of E. pyrifoliae strain Ep1/96, E. tasmaniensis strain Et1/99 and E. billingiae strain Eb661. Values were calculated by BLASTCLUST using an identity of >60%, an alignment length of >70% and an e-value of 1e-6 as cut-off. The number of proteins per chromosome is given. The number of clusters (#) is given in brackets representing the non-redundant protein-coding genes per intersection. The overlapping sections indicate shared numbers of proteins. The total number of all deduced proteins of these three species is 11,659.

Figure 2

Chromosome organization of strain Eb661 vs. Et1/99 vs. Ep1/96. Chromosomes were compared using ACT. Red lines connect homologous regions present in the same orientation while the blue lines connect regions of inverted orientation. Black bars symbolize chromosomes. Localization of the rRNA operons is indicated by white (16S-23S-5 S rRNA organization) and yellow bars (16S-23S-5S-5 S rRNA organization). Translocated and inverted regions are highlighted in grey and the positions on the chromosomes are provided. The estimated size of these regions, which were modulated by several re-arrangements, is noted in megabases.

Figure 3

Alignment of genes involved in thiamine biosynthesis (thiOSGF), a toxin-antitoxin system (stbDE) and a choline transporter (betT) for E. tasmaniensis, and E. pyrifoliae. The maps show corresponding parts from the chromosome of E. tasmaniensis strain Et1/99 and from plasmid pEP36 of E. pyrifoliae strain Ep1/96. BlastP results (percentage identity) are shown for related genes (grey boxes). Labels indicating the locus tag number are given within the grey boxes, if no gene name is assigned. White boxes indicate genes without similarity. The proteins for thiamine biosynthesis (thi) and the flanking regions are highly related and the genes are in conserved order. The stbD and stbE genes of pEp36 encode for a toxin-antitoxin system. A comparison for genes in this region was done before for the E. amylovora plasmid pEA29, the E. pyrifoliae plasmid pEP36 and plasmid pEJ30 from the Japanese E. pyrifoliae strain Ejp557 [89]. Locus tags of the genes are abbreviated for this overview.

Figure 4

Alignment of the sucrose and sorbitol operons from E. amylovora, E. pyrifoliae and E. tasmaniensis with Mauve v.2.3.0. The similarity profile of the alignment in the progressive mode is shown in red [55]. The height of the bars corresponds to the average level of conservation in the sequence. White boxes indicate genes. The alignment of the sucrose operons (A-D) comprise nucleotide sequences of E. amylovora strain CFBP 1430 (A; Acc. No. AJ250722, positions 230 6950), E. pyrifoliae strain Ep1/96 (B; Acc. No. FP236842, positions 2206738- 2213359) and E. tasmaniensis strain Et1/99 (C; Acc. No. CU468135, positions 2119695- 2126241). The genes scrA and scrB are duplicated in the genome of E. tasmaniensis Et1/99 (D; positions 1064052- 1070604). There is a continuous homology in the sucrose operons shown in the similarity profiles. Alignment of the sorbitol operons (E-G) comprises nucleotide sequences of E. amylovora strain Ea7/74 (E; Acc. No. Y14603, positions 276- 4479), E. pyrifoliae strain Ep1/96 (F; Acc. No. FP236842, positions 668798- 672810) and E. billingiae strain Eb661 (G; Acc. No. FP236843, positions 2907872- 2911878).

Figure 5

Analysis of NRPSs for the strains Ep1/96 (position 1,277,774- 1,298,860), Et1/99 (pos. 3,243,033- 3,253,088) and Eb661 (pos. complement 749,230- 753,177). Locus tag and size are given in brackets. NRPSs were identified by BLASTP and domains were assigned according to Pfam. Predicted amino acids to be synthesized are noted, if a prediction was available. Abbreviations of amino acids: Ala, Alanine; Gln, Glutamine; Leu, Leucine, Phe, Phenylalanine; Ser, Serine; Thr, Threonine.