Genomic analysis of the Phalaenopsis pathogen Dickeya sp. PA1, representing the emerging species Dickeya fangzhongdai

Abstract

Background: Dickeya sp. strain PA1 is the causal agent of bacterial soft rot in Phalaenopsis, an important indoor orchid in China. PA1 and a few other strains were grouped into a novel species, Dickeya fangzhongdai, and only the orchid-associated strains have been shown to cause soft rot symptoms.

Methods: We constructed the complete PA1 genome sequence and used comparative genomics to explore the differences in genomic features between D. fangzhongdai and other Dickeya species.

Results: PA1 has a 4,979,223-bp circular genome with 4269 predicted protein-coding genes. D. fangzhongdai was phylogenetically similar to Dickeya solani and Dickeya dadantii. The type I to type VI secretion systems (T1SS-T6SS), except for the stt-type T2SS, were identified in D. fangzhongdai. The three phylogenetically similar species varied significantly in terms of their T5SSs and T6SSs, as did the different D. fangzhongdai strains. Genomic island (GI) prediction and synteny analysis (compared to D. fangzhongdai strains) of PA1 also indicated the presence of T5SSs and T6SSs in strain-specific regions. Two typical CRISPR arrays were identified in D. fangzhongdai and in most other Dickeya species, except for D. solani. CRISPR-1 was present in all of these Dickeya species, while the presence of CRISPR-2 varied due to species differentiation. A large polyketide/nonribosomal peptide (PK/NRP) cluster, similar to the zeamine biosynthetic gene cluster in Dickeya zeae rice strains, was discovered in D. fangzhongdai and D. solani. The D. fangzhongdai and D. solani strains might recently have acquired this gene cluster by horizontal gene transfer (HGT).

Conclusions: Orchid-associated strains are the typical members of D. fangzhongdai. Genomic analysis of PA1 suggested that this strain presents the genomic characteristics of this novel species. Considering the absence of the stt-type T2SS, the presence of CRISPR loci and the zeamine biosynthetic gene cluster, D. fangzhongdai is likely a transitional form between D. dadantii and D. solani. This is supported by the later acquisition of the zeamine cluster and the loss of CRISPR arrays by D. solani. Comparisons of phylogenetic positions and virulence determinants could be helpful for the effective quarantine and control of this emerging species.

Keywords: CRISPR; Comparative genomics; Dickeya; Novel species; PKs/NRPs; Secretion systems.

Fig. 1

Circular visualization of the complete genome of D. fangzhongdai PA1. Circles from outside to inside indicate predicted genes in the positive strand, predicted genes in the negative strand, ncRNA (black indicates tRNA, red indicates rRNA), G + C content and GC skew value (GC skew = (G-C)/(G + C); purple indicates > 0, orange indicates < 0)

Fig. 2

Phylogenetic characterization of D. fangzhongdai PA1. a Phylogenetic analysis of Dickeya strains from different species based on concatenated sequences of the genes dnaX, recA, dnaN, fusA, gapA, purA, rplB, rpoS and gyrA. Confidence values on the branches were obtained with Mega 5.1, bootstrapped at 1000 replicates. Twenty-four Dickeya strains, including strain PA1, were used for phylogenetic analyses. b ANI analysis based on the complete or draft genomes of Dickeya strains

Fig. 3

Linearity analysis between D. fangzhongdai PA1 and other Dickeya strains based on whole-genome sequences. The strains used have complete genome sequences, and linearity analysis was performed based on a nucleic acid sequence BLAST. Red indicates homologous regions present in the same orientation; blue indicates homologous regions present in inverted orientation

Fig. 4

Genomic organization of the flagellar-type T3SS in Dickeya strains. The flagellar-type T3SS in D. fangzhongdai PA1 is at locus B6N31_13995–B6N31_14255. Variable region 1 is located between the loci of fliA and fliC; variable region 2 extends from the locus of flhB to the end of the fli and che clusters. Variable region 2 in D. zeae MS1 is at locus J417_RS0103870–J417_RS0104170. flagellar protein and flagellin; transcription factor; flagellar hook-associated protein; flagellar ring protein; flagellar motor switch protein; ATP synthase; flagellar basal-body protein; methyltransferase; oxidoreductase; fatty acid synthase; transketolase; acyl carrier protein; maltose O-acetyltransferase; aminotransferase; carbamoyl-phosphate synthase; transposase; chemotaxis protein; chemotaxis family TCS; flagellar motor protein; integrase

Fig. 5

Genomic organization of the T5SS in Dickeya strains. The cdi1 and cdi2 genes in D. fangzhongdai PA1 are at loci B6N31_12005 and B6N31_11515, respectively. Homologous gene domains are shown in the same color. The N-terminal and C-terminal toxin domains are shown individually, and diagonal lines indicate divergent domains

Fig. 6

Genomic organization of the T6SS in Dickeya strains. The three hcp genes in D. fangzhongdai PA1 are at loci B6N31_00250, B6N31_08590, B6N31_07000. Homologous gene domains are presented in the same color and diagonal lines indicate divergent domains

Fig. 7

Genomic organization of CRISPRs in Dickeya strains. Homologous gene domains are shown in the same color. The toxin PIN gene in D. fangzhongdai PA1 is at locus B6N31_0200. The Ail/Lom family gene in D. zeae MS1 is at locus J417_RS0102360

Fig. 8

Genomic organization of the homologous zeamine biosynthetic gene clusters in Dickeya strains. Genes zmsO and zmsP–zmN in D. fangzhongdai PA1 are at loci B6N31_07205 and B6N31_07230–B6N31_07310, respectively. Genes aligned with a shadow were homologous, and the numbers indicate the percentages of identity of each protein compared with homologous proteins in D. fangzhongdai PA1. peroxidase; ABC transporter; secretion protein HlyD; membrane protein; polyketide synthase; polyunsaturated fatty acid synthase; thioester reductase; hydrolase; nonribosomal peptide synthase; phosphopantetheinyl transferase; hypothetical protein. Circles = the domain predicted in PK or NRP genes. KS = keto reductase domain; AT = acyl transferase domain; KR = keto reductase domain; DH = dehydratase domain; NAD = NAD-binding domain; A = AMP-binding domain; C = condensation domain; E = epimerization domain. The PK domains include KS, AT, KR and DH, and the NRP domains include A, C and E