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BMC Genomics

, 19:889 | Cite as

Comparative genomics of 84 Pectobacterium genomes reveals the variations related to a pathogenic lifestyle

  • Xiaoying Li
  • Yali Ma
  • Shuqing Liang
  • Yu Tian
  • Sanjun Yin
  • Sisi Xie
  • Hua Xie
Open Access
Research article
  • 39 Downloads
Part of the following topical collections:
  1. Comparative and evolutionary genomics

Abstract

Background

Pectobacterium spp. are necrotrophic bacterial plant pathogens of the family Pectobacteriaceae, responsible for a wide spectrum of diseases of important crops and ornamental plants including soft rot, blackleg, and stem wilt. P. carotovorum is a genetically heterogeneous species consisting of three valid subspecies, P. carotovorum subsp. brasiliense (Pcb), P. carotovorum subsp. carotovorum (Pcc), and P. carotovorum subsp. odoriferum (Pco).

Results

Thirty-two P. carotovorum strains had their whole genomes sequenced, including the first complete genome of Pco and another circular genome of Pcb, as well as the high-coverage genome sequences for 30 additional strains covering Pcc, Pcb, and Pco. In combination with 52 other publicly available genome sequences, the comparative genomics study of P. carotovorum and other four closely related species P. polaris, P. parmentieri, P. atrosepticum, and Candidatus P. maceratum was conducted focusing on CRISPR-Cas defense systems and pathogenicity determinants. Our analysis identified two CRISPR-Cas types (I-F and I-E) in Pectobacterium, as well as another I-C type in Dickeya that is not found in Pectobacterium. The core pathogenicity factors (e.g., plant cell wall-degrading enzymes) were highly conserved, whereas some factors (e.g., flagellin, siderophores, polysaccharides, protein secretion systems, and regulatory factors) were varied among these species and/or subspecies. Notably, a novel type of T6SS as well as the sorbitol metabolizing srl operon was identified to be specific to Pco in Pectobacterium.

Conclusions

This study not only advances the available knowledge about the genetic differentiation of individual subspecies of P. carotovorum, but also delineates the general genetic features of P. carotovorum by comparison with its four closely related species, thereby substantially enriching the extent of information now available for functional genomic investigations about Pectobacterium.

Keywords

Pectobacterium carotovorum Comparative genomics Genetic variation Pathogenicity determinants CRISPR-Cas 

Abbreviations

BLAT

BLAST-like alignment tool

COG

Clustering of orthologous groups

CRISPR-Cas

Clustered regularly interspaced palindromic repeats/CRISPR-associated proteins

ECA

Enterobacterial common antigen

IS

Identity scores

LPS

Lipopolysaccharide

Nip

Necrosis-inducing protein

Pap

Pyelonephritis-associated pili

Pca

P. carotovorum subsp. actinidiae

Pcb

P. carotovorum subsp. brasiliense

Pcc

P. carotovorum subsp. carotovorum

Pco

P. carotovorum subsp. odoriferum

PCWDEs

Plant cell wall-degrading enzymes

PGAAP

Prokaryotic Genome Annotation Pipeline

SNPs

Single nucleotide polymorphisms

Svx

A homologue of an avirulence protein of Xanthomonas

TNSS

Protein secretion system

Background

Pectobacterium spp. are necrotrophic bacterial plant pathogens of the family Pectobacteriaceae, responsible for a wide spectrum of diseases (including soft rot, blackleg, and stem wilt) of important crops and ornamental plants worldwide [1]. At the time of writing, the genus has ten species (P. carotovorum, P. wasabiae, P. betavasculorum, P. cacticida, P. aroidearum, P. peruviense, P. atrosepticum, P. parmentieri, P. polaris, and Candidatus P. maceratum), of which P. carotovorum, P. atrosepticum, and P. parmentieri are three soft rot species that cause the most severe economic losses [2, 3]. In contrast to P. atrosepticum and P. parmentieri, the causal agent of potato rots, P. carotovorum exhibits a wide host range and high heterogeneity, and is further divided into three valid subspecies: P. carotovorum subsp. brasiliense (Pcb), P. carotovorum subsp. carotovorum (Pcc), P. carotovorum subsp. odoriferum (Pco), as well as a the newly proposed but as yet not validly published subspecies P. carotovorum subsp. actinidiae (Pca) [4, 5, 6]. In addition, some strains previously known as P. carotovorum has been separated from P. carotovorum recently and have been proposed as four new species: P. aroidearum, P. peruviense, P. polaris and Candidatus P. maceratum [7, 8, 9, 10].

Pectobacterium spp. possess a great number of pathogenicity determinants that contribute to the ability to cause maceration-associated diseases [4]. Most of them are secreted and injected into host cells through one or a combination of the six known types of bacterial protein secretion systems: T1SS-T6SS [11]. Recent studies on CRISPR-Cas systems, which function as a type of microbial immune system, may also influence Pectobacterium pathogenicity [12]. We focus our studies on genome-level variation in known virulence factors such as Plant Cell Wall Degrading Enzymes (PCWDEs), secretion systems, flagellin, siderophores, and polysaccharides, as well as CRISPR-Cas systems, in genus Pectobacterium, and especially in the heterogeneous species P. carotovorum in order to improve our understanding of the genetic basis for pathogenicity in general.

Genome sequencing has provided new insights into the lifestyle of Pectobacterium. P. atrosepticum SCRI1043 (accession: BX950851) was the first soft rot pathogen sequenced in 2004 [11]. By July 2018, many genome data have been released successively of which 16 more Pectobacterium complete genome sequences have become publicly available: P. atrosepticum 21A (accession: CP009125) [13], P. atrosepticum JG10–08 (accession: CP007744), P. wasabiae CFBP3304 (accession: CP015750), P. aroidearum PC1 (accession: CP001657), P. parmentieri WPP163 (accession: CP001790), P. parmentieri SCC3193 (accession: CP003415) [14], P. parmentieri RNS08.42.1A (accession: CP015749) [3], P. carotovorum 3–2 (accession: CP024842) and PCC21 (accession: CP003776) [15], Candidatus P. maceratum SCC1 (accession: CP021894) [16], P. polaris NIBIO1392 (accession: CP017482) and NIBIO1006 (accession: CP017481) [9], Pcb SX309 (accession: CP020350) and BZA12 (accession: CP024780) [17], as well as two new sequenced genomes Pcb BC1 (CP009769), Pco BCS7 (accession: CP009678) in this study (Additional file 1: Table S1). Previous genomic studies have compared Pectobacterium with other genera [18, 19, 20], and we are aware of two studies that examined Pectobacterium species/subspecies differences among P. atrosepticum, Pcc, and Pcb [21], and another comparing P. parmentieri and P. atrosepticum [22]. However, no reports have as yet compared the genomes of all of the subspecies of P. carotovorum.

The present study provides an important resource for functional genomics studies of the heterogeneous P. carotovorum. A total of 32 P. carotovorum strains including 14 Pcb, 5 Pcc, and 13 Pco strains were collected from 3 economically important Chinese vegetables (Chinese cabbage, Bok choy, and celery) (Additional file 1: Table S1 and Additional file 2: Figure S1), and these were characterized by conducting full genome sequencing and extensive phenotypic analyses. In addition, we conducted comparative genomics based on the 32 newly sequenced sequences and 52 additional genomes from P. carotovorum, P. polaris, Candidatus P. maceratum, P. atrosepticum, and P. parmentieri available from GenBank with three complete Dickeya genomes (D. zeae Ech586, D. paradisiaca Ech703, and D. dadantii 3937) as the reference. Our results not only represent progress in the knowledge regarding the genetic differentiation of individual subspecies of P. carotovorum, but also delineate the general genetic features of P. carotovorum by comparison with that of other four closely related species in following aspects: phylogenetic analysis, general genomic properties, genome similarity, pan-genome, CRISPR-Cas systems, pathogenicity-related genes, and TNSSs. The results showed the srl operon/T6SSc is apparently specific to Pco subspecies, and we therefore further studied the distribution and the similarity of srl operon/T6SSc in the Pectobacterium genus and even in the Enterobacterales order by using an expanding genome data set. This study represents a major step towards filling in our understanding about genomic differentiation in pathogenicity-related genes and pathways of the economically important Pectobacterium soft rot pathogens.

Result

Phenotypic and genomic features of 32 Chinese Pectobacterium carotovorum isolates

Thirty-two Chinese soft rot isolates including 14 Pcb, 5 Pcc, and 13 Pco strains were collected from 3 economically important Chinese vegetables (Chinese cabbage, bok choy, and celery) afflicted with bacterial soft rot disease (Additional file 2: Figure S1). All isolates tested caused soft rot lesions at the locations that were inoculated onto Chinese cabbage petioles (Additional file 1: Table S1). The extent of the decay caused by the Pcb strains (mean diameter of decay in Chinese cabbage = 3.29 cm) was, on average, less substantial than the decay caused by the Pcc or Pco strains (mean diameter of decay = 3.62 and 3.72 cm, respectively); however, consider that the Pcb strains exhibited a very wide range of values Pcb (0.24~4.06 cm), Pcc (range 3.13~3.83 cm), and Pco (range 1.19~4.20 cm). All tested strains were able to grow at 37 °C and were positive for utilization of cellobiose, sucrose, α-D-glucose, D-fructose, D-mannitol, and glycerol. They were unable to utilize propionic acid, L-alanine, D-maltose, D-serine, tween 40, and L-histidine. However, all tested Pco strains produced acid from D-sorbitol, palatinose, α-methylglucoside, and utilized D-arabitol whereas the Pcc and Pcb strains could not (Additional file 1: Table S1-S2).

Subsequently, to examine their genomic variation that may underly phenotypic differences, we sequenced the 32 P. carotovorum isolates to an average depth of 100-fold coverage. The genomes of both Pcb BC1 and Pco BCS7 (Fig. 1a and b) consist of a single circular chromosome of 4,920,350 bp and 4,933,575 bp, GC content of 51.84 and 51.75%, 4868 and 3855 predicted CDS sequences, respectively, and both possessing 7 rRNA operons, and 77 tRNA genes. The sizes of the draft genomes for 30 additional P. carotovorum strains ranged from 4,710,507 to 5,144,582 bp with GC% content ranging from 50.97 to 52.18% (Additional file 1: Table S3). No apparent autonomous plasmids were identified among these strains. We found that several genomic features that distinguish these Chinese Pco strains from others: the genomes of these Pco strains are the largest in size, have the lowest GC content, and have the least number of both tandem repeats and minisatellite DNA sequences compared with these Chinese Pcc and Pcb strains (Fig. 1c, d, and e).
Fig. 1

Genomic features of the 32 newly sequenced Pectobacterium carotovorum strains. a Circular chromosome map of Pcb strain BC1. b Pco strain BCS7. The distribution of genes is shown on the two outer rings. The next circle indicates the GC-skew values (pink: the GC-skew values > the average; black: the GC-skew values < the average) and the central circle shows the GC content (salmon: the GC content> the average; black: the GC content < the average). Two-dimensional plots showing relationships between genome size and GC content (c), minisatellite DNA count (d), and TR count (e) for the 32 newly sequenced P. carotovorum strains. The salmon dots represent the 14 Pcb strains, the pink squares represent the 5 Pcc strains, and the light green triangles represent the 13 Pco strains

Comparative genomics analysis of Pectobacterium carotovorum and other closely related species

To enrich the extent of information available for functional genomic investigations about P. carotovorum, we conducted comparative genomics analyses of individual subspecies of P. carotovorum and compared P. carotovorum genetic features with those of P. atrosepticum, P. parmentieri, P. polaris, Candidatus P. maceratum. A total of 84 Pectobacterium genomes including the 32 newly sequenced P. carotovorum genomes and the other available 52 Pectobacterium genomes downloaded from GenBank in July 2018 were used in this analysis (Additional file 1: Table S3).

Genomics features for distinguishing closely related species

Generally, we found three major genomic features of these 84 Pectobacterium genomes seemed to have regularity with their classification of species and subspecies, including GC content and the numbers of both long terminal repeats and minisatellite DNA sequences (Additional file 1: Table S4). The differing GC content of closely related species has been considered as a useful means of discrimination for a long time [23]. At the subspecies level, Pco and Pca had lower GC content (51.27–51.70%) than the other two subspecies (Pcc and Pcb) (51.84–52.18%; except Pcc Y16 (50.97%) and Pcb CFIA1033 (51.25%)). At the species level, P. carotovorum had a similar GC content with P. polaris and Candidatus P. maceratum, which was higher than P. atrosepticum (50.87–51.15%), whereas P. parmentieri (50.37–50.59%) had the lowest GC content among these four species. Overall, these Pectobacterium spp. had a lower GC content (50.37–52.18%) in comparison with the three Dickeya strains (53.63–56.29%). There were also fewer long terminal repeats in P. carotovorum, P. polaris, and Candidatus P. maceratum (56–122; except Pcc WPP14 (134), Pcb PBR1692T (137), and Pca ICMP 19971 (126)) than in the other two species P. atrosepticum and P. parmentieri (125–157). For minisatellite DNA sequences, Pca had the lowest number (84–88) among these subspecies and species, and the number of Pco (115–129; except NCPPB3841 (143)) was less than that of Pcc/Pcb (131–172) at the subspecies level. In addition, P. parmentieri had fewer such sequences (94–100) than P. atrosepticum (124–141), which was far fewer than that in Candidatus P. maceratum or P. polaris (148–208) at the species level.

Genome similarity of Pectobacterium carotovorum and other species as well as that of P. carotovorum subspecies

The topologies of maximum likelihood tree based on the huge SNP data from the 84 Pectobacterium genomes revealed that the four closely related species formed two major well-resolved groups (Fig. 2a). One group consisted of species P. carotovorum, P. polaris, and Candidatus P. maceratum, while the species P. parmentieri and P. atrosepticum formed the other group. Strains of P. carotovorum were further clustered into four distinct clades: clade 1, for Pcb strains and another one Pcc strain (ATCC 39048); clade 2, for Pcc strains; clade 3, for Pco strains; and clade 4, for Pca strains. Clade 1 was split further as follows: one subclade contained the type strain Pcb LMG 21371T as well as another five published Pcb strains from Brazil, New Zealand, and South Africa, and the other subclade harbored all the strains of the subspecies isolated from China, Russia, Canada, Korea as well as one Pcc strain from USA. This indicates that significant genetic diversity exists among different Pcb strains related to the origins, and the Pcc strain ATCC 39048 appear to be misnamed. It was also showed that a close relationship exists between Pcc and Pco, as they were gathered into a single large group that was separated from the Pcb strains. The Chinese Pco strains isolated from celery and Chinese cabbage were clustered with the type strain of Pco NCPPB 3839T and the other Pco strain isolated from chicory. In addition, two strains P. carotovorum 3–2 and Pcc Ecc71 were clustered with the other published Candidatus P. maceratum strains suggesting an erroneous classification.
Fig. 2

Taxonomy and Identity Score analysis of the 84 Pectobacterium genomes. a Maximum likelihood tree based on SNP data for the 84 Pectobacterium genomes including 56 P. carotovorum (that is 9 Pcc, 29 Pcb, 15 Pco, and 3 Pca genomes), 6 P. parmentieri, 10 P. atrosepticum, 4 P. polaris, and 8 Candidatus P. maceratum genomes, with 3 Dickeya genomes (D. paradisiaca Ech 703, D. zeae Ech586, and D. dadantii 3937) as the outgroup. The tree was generated with 1000 bootstrap replicates. The strains with red letters are the newly sequenced Pectobacterium strains in this study and the three P. carotovorum strains with green letters are the apparently misnamed strains detailed in the text. b Heat map (color code in the ramp, above) of identity scores based on pairwise comparison of these 84 Pectobacterium genomes and 3 Dickeya genomes

To characterize genomic similarity within each of subspecies/species, identity scores (IS) (Fig. 2b) were calculated based on the detected 52,630 SNPs among these tested strains. At the species level, we found P. carotovorum was most similar to Candidatus P. maceratum (90.28–93.49%) and least similar to P. atrosepticum (83.62–84.94%) and P. parmentieri (83.28–85.76%). Within subspecies carotovorum, Pcb was more closely related to Pcc than to Pco and Pca, since Pcb strains had the higher IS values with Pcc strains (91.25–93.39%) than with Pco (89.65–90.63%) and Pca strains (89.01–89.78%). A close relationship was also observed between Pcc and Pco with higher similarity (92.79–93.79%) than that of any other two subspecies. Pca strains also had high similarity with Pcc (91.31–91.80%) and Pco (90.50–90.68%) strains but had low similarity with Pcb (89.01–89.78%). It is noteworthy that the similarity of three Korean Pca strains (KKH3T, ICMP 19971, and ICMP 19972) isolated from kiwi fruit [6, 24] were 100% amongst each other.

Pan-genome analysis identified a srl operon specific to Pco

We performed a pan-genome analysis of Pectobacterium based on 36,634 putative protein-encoding sequences from the 84 Pectobacterium genomes sequences. The Pectobacterium pan-genome can be subdivided into three categories: (i) the core genome (that is, the set of genes present in the genome of at least one strain of each subgroup; not a gene that is shared by all strains), (ii) the accessory genome (the set of genes present in some but not all subgroups), and (iii) the unique genome (genes that are unique to a single subgroup) (Fig. 3). The core genome of Pectobacterium consists of 3171 (17.47%) out of a total of 18,147 orthologous genes. OrthoMCL comparative analysis of shared genes revealed a higher number of genes commonly present in P. carotovorum-Candidatus P. maceratum (4762), and P. carotovorum-P. polaris (4657), than that in common between P. carotovorum-P. atrosepticum (4332), and P. carotovorum-P. parmentieri (4362), supporting our observations from the abovementioned IS analysis that showed a closer genetic relationship between P. carotovorum and Candidatus P. maceratum than with P. polaris, P. atrosepticum, or P. parmentieri (Fig. 3a). The core orthologous genes and subspecies-specific unique genes within a given P. carotovorum species were examined in 9 Pcc, 15 Pco, 29 Pcb, and 3 Pca strains (Fig. 3b). A total of 14,558 homologs were identified. Of these, 3384 orthologs (23.24%) were identified as the P. carotovorum core genome. A total of 5016 genes were shared between Pco and Pcb, while Pcc shared fewer orthologs with Pcb (5009) and Pco (4527) and Pca shared few orthologs with the other three subspecies: Pcb (3763), Pcc (3600), and Pco (3530), highlighting the relatively higher extent of genetic differentiation of Pca compared with the other three subspecies. The differentiation was also presented between different strains within the same subspecies in P. carotovorum (Fig. 3c, d, e, and f).
Fig. 3

Pan- and core-genome analyses of the 84 Pectobacterium genomes. The number of genes in the pan-genomes and core-genomes, and the unique genes among the genomes were classified using CD-HIT, with a threshold of 70% pairwise identity and a 0.6 length difference cut-off in amino acid sequences. a Venn diagram of Pectobacterium genus for 56 P. carotovorum, 4 P. polaris, 8 Candidatus P. maceratum, 10 P. atrosepticum, and 6 P. parmentieri genomes. b Venn diagram of P. carotovorum species for 29 Pcb, 9 Pcc, 15 Pco, and 3 Pca genomes. The core orthologous genes are not the genes that are shared by all of the tested genomes of a given group; rather these are the genes that were present in a minimum of one genome from each of the targeted subgroup. c-f Flower plots of each of four subspecies for 29 Pcb, 9 Pcc, 15 Pco, and 3 Pca genomes, respectively. The figures of 84 Pectobacterium genomes in (g) and (h) were produced using PanGP. g Plot in which the blue line shows the impact on the number clusters for the pan-genome resulting from the addition (simulated, randomized) of further genomes to the analysis, with the asymptotic value of y = AxB + C; the green line shows how such addition influenced the number of core genes, with the asymptotic value of y = AeBx + C. h Histogram showing the increases in the number of new (unique) genes as the number of genomes was increased in our pan-genome analysis. The horizontal dashed line (orange) indicates the asymptotic value with the function of y = AxB

Additionally, the size of the pan-genome and its increase in size upon addition of new strains can be used to predict the future rate of discovering novel genes in a species (Fig. 3g and h): the pan-genome curve fit a Heaps’ law pattern (exponent > 0) and was considered “open”, which is typical of species colonizing multiple environments and having several means of exchanging genetic material [25]. This analysis highlights the large genomic diversity of Pectobacterium species and suggests that these species can very likely adapt to a wide variety of hosts. The COG (Clustering of orthologous groups) analysis of unique genes of P. atrosepticum, P. parmentieri, P. polaris, Candidatus P. maceratum, as well as the subspecies of P. carotovorum showed that the srl operon (BCS7_10600-BCS7_10625) that is known to facilitate sorbitol utilization [26] was found only in the Pco strains, suggesting that this srl operon may be specific to Pco subspecies. The specific distribution of this operon in Pectobacterium genus were future confirmed in a subsequently expanded genome analysis using all of the available genomes from GenBank. These findings highlight substantial divergence among Pectobacterium species and subspecies. Seeking to better understand the genetic basis of pathogenicity and host specificity, our next analyses specifically compared the similarity and divergence of the genes encoding CRISPR-Cas, virulence determinants, regulatory mechanism, and protein secretion systems.

The occurrence of I-E type CRISPR-Cas systems in Pectobacterium

We investigated the CRISPR-Cas bacterial defense systems looking for genome-level variations that may influence the pathogenicity of Pectobacterium [12]. To date, surveys of the CRISPR-Cas systems in Pectobacterium have only been performed for P. atrosepticum SCRI1043, in which a single I-F type system was identified [27]. In the present study, we identified I-F, I-E type systems among these available Pectobacterium genomes, with seven exceptions among the Pectobacterium strains (Pcb LMG 21372, ICMP 19477, CFIA1001, and Y46; Pco NCPPB3841, NCPPB 3839T, and Q3; P. polaris NIBIO1006T) that lacked any CRISPR-Cas system (Table 1). The distribution of these systems varied among Pectobacterium species and subspecies: most of the P. carotovorum, P. polaris, and Candidatus P. maceratum strains contained I-F or I-E systems, and some strains carried both types (28% Pcb, 33% Pcc, 13% Pco, 25% P. polaris, and 50% Candidatus P. maceratum); all of the tested Pca and P. atrosepticum strains have a single I-F type, while both P. parmentieri strains have both the I-F and I-E types (Table 1). Moreover, our analysis identified CRISPR-Cas types (I-F, I-E and I-C) in reference Dickeya genomes, which have not been reported previously, as well as a I-C type in D. dadantii 3937 that is not found in Pectobacterium. The CRISPR-Cas systems from complete genomes of 84 Pectobacterium genomes, as well as three Dickeya genomes, are shown in Fig. 4, and the CRISPR-Cas systems of all the tested genomes in this study are shown in Additional file 1: Table S5.
Table 1

The identified types of CRISPR-Cas system in the 84 Pectobacterium genomes

Species/Subspecies

Strain

Number of each CRISPR-Cas system type

I-F

I-E

I-C

P. carotovorum subsp. brasiliense

PBR1692T = LMG 21371T, BC1, B6, Y3, Y29, Y31, Y60, Y62, Y64, CFIA1009, CFIA1033, PcbHPI01, BD255, ATCC 39048, F157, F152

1

0

0

PCC21, SX309, BZA12, Y21, Y49, Y52, Y65, S2

1

1

0

LMG 21372, ICMP 19477, CFIA1001, Y46

0

0

0

P. carotovorum subsp. carotovorum

Y57

1

0

0

DSM 30168T = NCPPB 312T = ICMP 5702T, B2, Y16

0

1

0

B5, Y39, WPP14

1

1

0

P. carotovorum subsp. odoriferum

Q40, Q106, Q115, Q142, Q166, T4, T5

1

0

0

BCS7, S6, S6–2,

0

1

0

Q32, Q47

1

1

0

NCPPB 3839T, NCPPB3841, Q3

0

0

0

P. carotovorum subsp. actinidiae

ICMP 19972, ICMP 19971, KKH3T

1

0

0

Candidatus P. maceratum

F018, F135, Ecc71

1

0

0

3–2

0

1

0

SCC1T, F131, PB69, PB70

1

1

0

P. polaris

NCPPB 3395, SS28

0

1

0

NIBIO1392

1

1

0

NIBIO1006 T

0

0

0

P. atrosepticum

ICMP 1526T = NCPPB 549T, 36A, 21A, CFBP 6276, NCPPB 3404, PB72, SS26, JG10–08, SCRI1043

1

0

0

P. parmentieri

RNS08.42.1AT, SCC3193, WPP163, SS90, CFIA1002, PB20

1

1

0

D. dadantii

3937

1

0

1

D. zeae

Ech586

1

1

0

D. paradisiaca

Ech703

1

1

0

The strains with bold black font are strains with completely sequenced genomes, whose CRISPR-Cas systems architectures are shown in Fig. 4, and the CRISPR-Cas systems of all strains in this study are shown in Additional file 1: Table S5

Fig. 4

Representative CRISPR-Cas systems types identified in this study. All of the 84 Pectobacterium genomes and 3 out-group Dickeya genomes were identified their CRISPR-Cas systems in this study. The strains with complete genomes were selected to show their CRISPR-Cas systems architectures here, and the detailed information of CRISPR-Cas systems in all of the tested genomes were given in Additional file 1: Table S5. The diagram features in black represented the genes encoding known Cas proteins, features in grey represented the genes predicted to encode hypothetical proteins of unknown function, and features in white represent CRISPR sequences. a I-F type of CRISPR-Cas, b I-E type of CRISPR-Cas, and c I-C type CRISPR-Cas systems in these complete genomes are shown

Inconsistently distributed pathogenicity-related genes among Pectobacterium species and subspecies

To obtain a more comprehensive view of the pathogenicity factors present among Pectobacterium spp., we mined the 84 Pectobacterium genomes to identify previously known pathogenicity determinants, including PCWDEs, polysaccharides, iron uptake proteins, pili, and regulatory systems. Many pathogenicity determinants (e.g., most PCWDEs, enzymes for synthesis of enterobacterial common antigen (ECA) and the 3-hydroxy-2-butanone pathway) (Additional file 1: Table S6 and Table S6–1) are commonly existed among strains due to their importance in ensuring disease development caused by Pectobacterium spp. However, we also found inconsistent distributions of several single genes or large DNA fragments/clusters that possibly contribute to pathogenicity in variable or context-dependent scenarios for several species and subspecies (Table 2). All of the P. carotovorum, P. polaris, and Candidatus P. maceratum strains, but none of P. atrosepticum, and P. parmentieri strains, had the gene fhaB (BASYS01817) whose homolog encodes the filamentous hemagglutinin that influences virulence by human pathogens [28]. No regular distribution was observed on the two clusters (waa and rfb) encoding lipopolysaccharide (LPS) biosynthesis proteins present in P. carotovorum strains. The variation of LPS-related gene composition within the strains in this study likely reflects adaptations by Pectobacterium in the LPS-mediated plant-host interactions at the cell surface [22]. Notably, all of the P. parmentieri strains examined in this study lacked three PCWDEs genes (pel, pelA, and pgl) and a transport system for a ferric citrate cluster (fecABCDE), while possessing unique genes for siderophore biosynthesis and iron transport such as the genes iucABCD [29].
Table 2

The distribution of genes/clusters related to virulence among 84 Pectobacterium genomes

Gene/cluster

Locus tag

Pectobacterium

Reference Dickeya strainsa

Pc (n = 56)

Pa (n = 10)

Pp (n = 6)

CPm (n = 8)

Ppo (n = 4)

D. zeae Ech 586

D. dadanti 3937

D. paradisiaca Ech703

Pcb (n = 29)

Pcc (n = 9)

Pco (n = 15)

Pca (n = 3)

Cellulases (PCWDEs)

celV/celB

BCS7_11780/BCS7_07535

29

9

15

3

10

6

8

4

 putative cellulase

BCS7_10680

29

9

15

3

10

6

8

4

Dd586_2061

Dda3937_00081

Oligogalacturonide (PCWDEs)

ogl

BCS7_09745

29

9

15

3

10

6

8

4

Dd586_1940

Dda3937_03686

Dd703_2056

Pectate_lyase (PCWDEs)

pelX/pelB/pelW

BCS7_21380/BCS7_11020/BCS7_09865

29

9

15

3

10

6

8

4

Dd586_4161/Dd586_3789/Dd586_1962

Dda3937_00143/Dda3937_04192/Dda3937_03361

Dd703_3996/Dd703_0352/Dd703_2033

pel

BCS7_14745

29

9

15

3

10

0

8

4

pelA

BCS7_11155

29

9

15

3

10

0

8

3

Dd586_3083

Dda3937_03370

Dd703_2763

pelL/pelZ

BCS7_08875/BCS7_19275

29

9

15

3

10

6

8

4

Dd586_1488/Dd586_3790

Dda3937_02794/Dda3937_04191

Dd703_1887/Dd703_0351

pel1/pel2/pel3

BCS7_19260-BCS7_19270

29

9

15

3

10

6

8

4

pelI

BCS7_05195

29

9

15

3

10

6

8

4

Dd586_2937

Dda3937_00058

Pectin acetylesterase (PCWDEs)

paeX/paeY

BCS7_09835/BCS7_15435

29

9

15

3

10

6

8

4

Dd586_1956/Dd586_3086

Dda3937_03363/Dda3937_03373

Dd703_2039/Dd703_2765

Pectin lyase

pnl

BCS7_06950

29

9

15

3

10

6

8

4

Dda3937_03551

Dd703_3378

Pectin methylesterase (PCWDEs)

pemA

BCS7_15440

29

9

15

3

10

6

8

4

Dd586_3087

Dda3937_03374

Dd703_2766

pemB

BCS7_00330

29

9

15

3

10

6

8

4

Dda3937_03435

Polygalacturonase (PCWDEs)

pgl

BCS7_17010

29

9

15

3

9

0

8

4

Dd586_3319

Dda3937_00206

Dd703_0972

pehX

BCS7_14740

29

9

15

3

10

6

8

4

Dd586_3904

Dda3937_00261

Dd703_0200

pehN

BCS7_05675

29

9

15

3

10

6

8

4

Dda3937_04155

pehA

BCS7_05200

29

9

15

3

10

6

8

4

Rhamnogalacturonate lyase (PCWDEs)

rhiE

BCS7_03460

29

9

15

3

10

6

7

4

Dd586_2097

Dda3937_01465

Pilus assembly proteins

pilA

BCS7_01135

1

0

15

0

0

0

0

0

cpaABCDEF

BCS7_01140-BCS7_01165

1

0

15

0

0

0

0

0

tadBCDE

BCS7_01170-BCS7_01185

1

0

15

0

0

0

0

0

-

BCS7_01190-BCS7_01230

1

0

15

0

0

0

0

0

papA

BASYS00091

28

9

0

3

10

1

8

3

papC

BASYS00090

28

9

0

3

10

5

8

3

papD

BASYS00089

28

9

0

3

10

6

8

3

fhaB

BASYS01817

29

9

15

3

0

0

8

4

Siderophore synthetase

entB

BASYS04531

29

9

15

3

10

0

8

4

Dd586_2819

Dda3937_02432

Dd703_1488

foxA

BASYS03462

29

9

15

3

10

0

8

4

Dd703_0458

iucABCD

W5S_0838-W5S_0841

0

0

0

0

0

6

0

0

-

W5S_0842

0

0

0

0

10

6

0

0

-/entS/ fepD/fepG

W5S_3869-W5S_3872

0

0

0

0

0

6

0

0

Dd586_2808-Dd586_2811

Dda3937_03032-Dda3937_03035

Dd703_1499-Dd703_1496

fepC

W5S_3873

29

9

15

3

10

6

8

4

Dd586_2812

Dda3937_03036

Dd703_1495

-/mbtH/−/−

W5S_3874-W5S_3877

0

0

0

0

0

6

0

0

Dd586_2813-Dd586_2816

Dda3937_03038-Dda3937_03042

Dd703_1494-Dd703_1491

-

W5S_3878

0

0

0

0

0

6

0

0

fyuA/−

BASYS00782/BASYS00783

28

9

0

3

0

0

8

3

-

BASYS00784

28

5

0

3

0

0

1

0

pchR/msbA

BASYS00785/BASYS00786

28

9

0

3

0

0

8

3

ygaD

BASYS00787

28

9

0

3

0

0

8

4

fecABCDE

BASYS00244-BASYS00240

27

4

0

3

10

0

8

3

nosA

BCS7_07445

0

0

15

3

10

6

8

0

Synthesis of LPS

rfaD/waaFb/waaC

W5S_4520-W5S_4523

29

9

15

3

10

6

8

4

Dd586_0164-Dd586_0162

Dda3937_02041-Dda3937_02039

Dd703_0173-Dd703_0171

waaL1

W5S_4524

18

6

15

3

10

6

2

2

waaL2

W5S_4526

0

0

0

0

7

2

0

0

waaQ/waaG

W5S_4527/W5S_4528

18

6

15

3

10

6

2

2

waaI

W5S_4529

18

6

10

3

10

6

2

2

waaJ

W5S_4530

3

3

3

0

7

2

1

0

-

W5S_4532

7

0

0

0

0

2

0

0

waaX

W5S_4533

0

0

0

0

7

2

0

0

kdtX

W5S_4534

26

6

5

3

7

2

7

3

Dd586_0155

Dda3937_02032

Dd703_0164

-

W5S_4535

6

4

5

0

7

2

1

0

kdtA/kdtB

W5S_4536/W5S_4537

29

9

15

3

10

6

8

4

Dd586_0156/Dd586_0154

Dda3937_02033/Dda3937_02031

Dd703_0165/Dd703_0163

rfbP

ECA1420

4

2

8

0

10

2

0

1

rfbI/rfbF/rfbG/rfbH

ECA1421-ECA1424

0

0

1

0

9

0

1

0

hpcH/−/−/nahO/nahM/−/rfbX/−

ECA1425-ECA1432

0

0

0

0

9

0

0

0

-

ECA1433

0

0

0

0

9

0

0

0

Dd586_0507

−/rfc/rfbU

ECA1434-ECA1436

0

0

0

0

9

0

0

0

rfbN

ECA1437

1

2

6

0

9

0

0

1

Dd586_1241

Dda3937_03925

rfbM

ECA1438

15

3

3

0

9

0

6

1

Dd586_1250

Dda3937_00442

Dd703_3281

rfbK

ECA1439

12

3

2

0

9

0

6

1

Dd586_1251

Dda3937_00443

Dd703_3280

rfbD/rfbC

ECA1440/ECA1441

18

6

15

3

10

6

2

3

Dd586_1246/Dd586_1245

Dda3937_03921/Dda3937_03922

Dd703_3287/Dd703_3288

rfbA

ECA1442

28

9

15

3

10

6

8

4

Dd586_1244

Dda3937_03923

Dd703_3286

Synthesis of ECA

wecG/wzyE/wecF/wzxE/rffA/rffC/rffG/wecC/wecB

BASYS03691-BASYS03699

29

9

15

3

10

6

8

4

Dd586_3890-Dd586_3897

Dda3937_00277-Dda3937_00270

Dd703_0212-Dd703_0205

wecA/wzzE

BASYS03700/BASYS03701

29

9

15

3

10

6

8

4

Dd586_3898/Dd586_3899

Dda3937_00269/Dda3937_00268

Metabolic regulatory protein

metJ

BCS7_20315

29

9

15

3

9

6

8

4

Dd586_3934

Dda3937_03887

Dd703_3792

Necrosis-inducing virulence protein

nip

BCS7_14620

29

9

15

3

10

6

8

4

Dd586_2805

Dda3937_00483

Xanthomonas campestris avirulence protein

svx

BCS7_04170

29

9

15

3

10

6

8

4

Dd586_2807

Dda3937_00480

Citrate transporter

citN

BCS7_06975

29

9

15

3

10

6

8

4

sirB locus

sirB1

BCS7_10775

29

9

15

3

10

6

8

4

Dd586_2024

Dda3937_00039

Dd703_1858

sirB2

BCS7_10780

29

9

15

3

10

6

8

4

The 3-hydroxy-2-butanone pathway

budR/budA

BCS7_03160/BCS7_03165

29

9

15

3

10

6

8

4

Dd586_0623/Dd586_0624

Dda3937_02312/Dda3937_02311

Dd703_3319/Dd703_3318

budB

BCS7_03170

28

9

15

3

10

6

8

4

Dd586_0625

Dda3937_02310

Dd703_3317

budC

BCS7_01530

29

9

15

3

10

6

8

4

Dd586_3192

Dda3937_03162

Dd703_0990

Unknown proteases

 -

BCS7_01920-BCS7_01935

6

0

15

3

10

0

4

0

Dd586_0366-Dd586_0369

Dd703_3577-Dd703_3574

 -

BASYS00142

21

5

0

0

10

0

8

3

yedU

BASYS03033

29

9

15

3

9

0

8

4

Dd586_2787

Dda3937_02122

Dd703_0349

Regulation network

Rsm system–post-transcriptional gene regulation

rsmA

BCS7_16095

29

9

15

3

10

6

8

4

Dd586_3183

Dda3937_03151

Dd703_0999

rsmBc

29

9

15

3

10

6

8

4

1

1

0

rsmC

BCS7_03480

29

9

15

3

10

6

7

4

Dd586_0685

Dda3937_02456

Dd703_3258

ExpI-ExpR Quorum-Sensing system

expI/expR

BCS7_00320/BCS7_00325

29

9

15

3

10

6

8

4

Dd586_0112/Dd586_0111

Dda3937_03219/Dda3937_03218

KdgR repressor

kdgR

BCS7_09750

29

9

15

3

10

6

8

4

Dd586_1941

Dda3937_03685

Dd703_2055

RdgA-RdgB regulate system of pectin lyase production

rdgA/rdgB

BCS7_09700/BCS7_09695

29

9

15

3

10

6

8

4

RNA polymerase sigma factor

rpoS

BCS7_16895

29

9

15

3

9

6

8

4

Dd586_3302

Dda3937_03911

Dd703_0872

HexA repressor of extracellular enzyme and motility

hexA

BCS7_14160

29

9

15

3

10

6

8

4

Dd586_2749

Dda3937_00364

Dd703_2551

Response regulator

expM

BCS7_10215

29

9

15

3

10

6

8

4

Dd586_2148

Dd703_1959

ExpS-ExpA two-component system

expA

BCS7_13455

29

9

15

3

10

6

8

4

Dd586_1482

Dda3937_03960

Dd703_1450

expS

BCS7_17085

29

9

15

3

9

6

8

4

Dd586_3330

Dda3937_00193

Dd703_0847

AepA catalytic protein

aepA

ECA1022

15

7

0

0

10

6

5

1

Flagellar regulon transcriptional activators

flhD/flhC

BCS7_13105/BCS7_13110

29

9

14

3

10

6

8

4

Dd586_1499/Dd586_1498

Dda3937_02784/Dda3937_02785

Dd703_1509/Dd703_1508

The details of these virulence related genes/ RNA fragment in each strain were shown in the Additional file 1: Table S6 and Table S6–1

n number of genomes tested. Numbers indicate the number of genomes that tested positive, Pcc P. carotovorum subsp. carotovorum, Pcb P. carotovorum subsp. brasiliense, Pco P. carotovorum subsp. odoriferum, Pca P. carotovorum subsp. actinidiae, Ppo P. polaris, Pp P. parmentieri, Pa, P. atrosepticum, CPm Candidatus P. maceratum

aReference strains: part of data adapted from Zhou et al. (2015); bwaaF: W5S_4521, W5S_4522; crsmB, encoding a fragment of RNA acting as a global regulator of virulence, has no gene ID

At the subspecies level, Pco exhibited distinctly different gene composition when compared with other closely subspecies Pca, Pcb and Pcc. All Pco strains have the large DNA fragment (BCS7_01135-BCS7_01230) that contains several genes (pli, cpa, tad, and etc.) known to be involved in pili biosynthesis; this fragment is not present in other P. carotovorum strains with the exception of the Pcb strain BZA12. The nosA gene which encoded the out-membrane protein involved in the transport of metals [30] was absent in Pcb and Pcc while was present in Pco and Pca. Similarly, the Pco strains lack several genes carried by other subspecies strains, such as the major part of the pyelonephritis-associated pili (pap) operon (papACD; BASYS00091-BASYS00089), which are involved in the formation of adhesive pili in pathogenic bacteria [31, 32]. We also identified intermittent distribution of AepA exoenzyme regulatory protein among Pcc and Pcb strains, but not at all in Pco and Pca strains. The AepA protein may enable Pectobacterium to use atypical nitrogen sources, which are secreted by the host plant or microorganisms that colonize the host plant’s rhizosphere [33]. Many other genetic variations of pathogenicity related genes among these soft rot pectobacteria were shown in Table 2 and Additional file 1: Table S6. The findings of these unique genes confer to different species/subspecies provided a foundation for further work in elucidation of the genetic basis for pathogenicity in Pectobacterium.

Three T1SS protein secretion systems (has, prt, and lap) in Pectobacterium

The five protein secretion system types (T1SS-T4SS, and T6SS) found in 84 Pectobacterium genomes. Although all six secretion systems were reported to be present in Pectobacterium [11, 21] by Bell et al. and Glasner et al., improvements to annotation algorithms have resulted in the exclusion of T5SS in our study. The TNSSs of 3 Dickeya genomes used as the out-group also exhibited high diversity and were also discussed below.

Three T1SSs (T1SSa, T1SSb, and T1SSc) were identified among the Pectobacterium strains (Fig. 5a and Additional file 1: Table S7). While T1SSb (prtDEF) and T1SSc (lapBCE) were present in all of the Pectobacterium strains in this study, T1SSa (hasDEF) was lost in P. parmentieri. Only T1SSb was found in the Dickeya strains we examined, of which D. paradisiaca Ech703 lacked the T1SSs. T1SSs secrete proteases and translocate molecules such as ions and toxins from the cytoplasm to the extracellular space [34], serving a range of functions. T1SSa of Serratia marcescens, for example, can translocate ions and secrete hemophores such as an extracellular heme-binding protein (HasA) to facilitate iron acquisition [35]. In Pectobacterium, T1SSb has been shown to secrete several Prt metalloproteases that are involved in attacking plant cell walls [36], and the repeats-in-toxin (RTX) protease PrtW mutants of P. parmentieri SCC3193 have altered virulence, suggesting a role for T1SSb in plant pathogenesis [37].
Fig. 5

Gene clusters for TNSSs identified in the 84 Pectobacterium genomes. a Physical maps of Type I, II, III, IV, and VI secretion systems. Red colored arrows indicate known genes involved in the protein secretion systems. b Map showing the presence (blue feature) or absence (grey feature) of each type of secretion systems gene cluster, with a maximum likelihood phylogenetic tree based on the core genes of all the tested strains. a-c represents different subtypes of a given secretion system gene cluster; 1–2 represent the number of the copies of Type IV secretion system gene clusters

Species- and subspecies-specific features of T2SS

In contrast to the various T1SSs in Pectobacterium, only one T2SS cluster (T2SSa) was found among the Pectobacterium strains (Fig. 5b). However, the P. parmentieri strains and three Dickeya strains carried a variant T2SSa (Gsp) lacking gspN compared with that of other Pectobacterium strains. GspN is predicted to have an amino-terminal transmembrane helix, but its function has not yet been determined [38]. We also found that the length of gspG was 441 nt in all of P. atrosepticum and P. parmentieri strains and 456 nt in Candidatus P. maceratum, but found that this varied among the P. carotovorum strains. Moreover, the intergenic sequence length between gspN and gspO was 89 nt in all Pco and Pcc strains, but was 108 nt in all Pcb strains and 88 nt in all Pca strains. This intergenic length was not only specific to each subspecies, but was also specific at the species level, as the intergenic sequence length between gspN and gspO was 63 nt, 123 nt, and 89 nt, respectively, for each of the P. parmentieri, P. atrosepticum, and Candidatus P. maceratum strains. The extra T2SSb (Stt) presented in D. dadantii 3937 was not present in any tested Pectobacterium strains or in the other two Dickeya strains. T2SSb is known to encode a pectate lyase PnlH at the outer face of the outer membrane [39]. T2SS is extremely important for soft rot pathogens because it is responsible for the secretion of the major determinants of pathogenicity for this disease: the pectinases and cellulases that contribute to the characteristic rotting symptoms. It also secretes other factors related to pathogenicity, such as Svx and iron uptake proteins [40]. The inactivation of this secretory system renders Pectobacterium spp. avirulent [41].

T3SSa is absent in Pectobacterium parmentieri but found in other species

T3SSs include the virulence-associated Type III (T3SSa) and the flagellar secretion system (T3SSb). T3SSa clusters were absent from P. parmentieri, but were present in all of the strains of P. carotovorum, Candidatus P. maceratum, and P. atrosepticum (Fig. 5b and Additional file 1: Table S7). These results were in agreement with previous studies that failed to detect T3SSa or any of its surrounding genes in P. parmentieri SCC3193, a strain known to be virulent [22]. It is likely that T3SSa is not strictly required for the virulence and survival of P. parmentieri, although this cluster has been reported to contribute modestly to the virulence of P. atrosepticum and P. carotovorum [42]. A previous study reported some naturally T3SS-deficient P. carotovorum strains [43]; however, one P. polaris strain NCPPB 3395 from the Netherlands, previously assigned to Pcc [9], were identified as a T3SSa-deficient strain in the present study. T3SSa was present in D. zeae Ech586 and D. dadantii 3937, but this was lost in the D. paradisiaca Ech703. Whereas T3SSa had a species-specific distribution, T3SSb (BASYS02177-BASYS02229), which encodes important flagella components [44], was highly conserved among these Pectobacterium spp. and three Dickeya genomes except one Pco strain (NCPPB 3839T).

Irregular distribution of T4SS in Pectobacterium

T4SS is a versatile secretion system that can function in bacterial conjugation, DNA uptake/release, and cargo translocation. Several of the proteins encoded by the T4SS form three major sub-complexes: a cytoplasmic-inner membrane complex (VirB4 and VirB11), a core complex (VirB7, VirB9, and Vir10), and an extracellular pilus-associated complex (VirB2 and VirB5) (Fig. 5a). P. atrosepticum SCRI1043 VirB4 mutants are reported to exhibit reduced pathogenicity [11]. Distribution of T4SS was sporadic among Pectobacterium genomes and the T4SS-deficient strains were present among all five Pectobacterium species (P. carotovorum, P. polaris, P. atrosepticum, P. parmentieri, Candidatus P. maceratum) as well as D. paradisiaca Ech703 (Fig. 5b). The copy number also varied among the strains that carried it. For example, two copies of T4SS (BASYS00894-BASYS00885 and BASYS00696-BASYS00686), were identified in three Pcb strains (BC1, BZA12, and BD255), three Pco strains (Q115, Q142, and Q166), three Pca strains (ICMP 19972, ICMP 19971, and KKH3), as well as D. zeae Ech586, while the other strains only possessed a single copy.

A novel T6SS type (T6SSc) present in Pco strains

Our analysis of 84 Pectobacterium genomes identified three types of T6SSs (T6SSa-c), of which T6SSc is a novel type that identified and termed in this study (Fig. 5a and Additional file 1: Table S7). T6SSa was absent in P. polaris NCPPB 3395, but present in the rest of the P. polaris strains as well as the other four species (P. atrosepticum, P. parmentieri, P. polaris, Candidatus P. maceratum, and P. carotovorum). Similar to T1SS, T3SS, and T4SS, we found that the T6SS gene cluster was not present in the D. paradisiaca Ech703 genome, a finding consistent with results from a study by Zhou et al. [45]. T6SSb was a specific cluster that was only occurred in (all of the) P. parmentieri strains. Interestingly, we also found a novel T6SS that we termed T6SSc (BCS714475-BCS714610) which was only present in the Pco strains; the distribution of each of the genes of the T6SSc cluster varied among the Pco strains (Fig. 6 and Additional file 1: Table S8): nine genes were present in all 15 Pco strains, while the remaining 19 individual genes were variously present among the strains we surveyed. Similarity analysis based on DNA sequences revealed high genetic variation in pairwise identity comparisons of T6SSc between strains, ranging from 40.1% for the most divergent T6SSc of strains to 100% similarity for the most conserved.
Fig. 6

Genes of T6SSc presence in the 61 Enterobacterales genomes. A total of 61 of the 203 Enterobacterales genomes possessed the T6SSc cluster. The strains with red letters were from the newly sequenced genomes in this study; the others strains were from NCBI. The 28 genes of T6SSc of the BCS7 strain was used as a standard; we grouped the genes into two Blocks (Block I includes the first 8 genes (starting from the 5′ end) of the cluster; Block II includes the 20 terminal genes of the cluster at the 3′ end). Map showing the presence (black feature) or absence (grey feature) of each gene of the T6SSc cluster, with a maximum likelihood phylogenetic tree based on the core genes of 61 Enterobacterales genomes. Colored arrows indicate five clusters that divide these strains into different known families (Enterobacteriaceae, Erwiniaceae, Morganellaceae & Yersiniaceae, Budviciaceae & Hafniaceae, and Pectobacteriaceae) of the Enterobacterales order. a and b: two different sequenced genomes (accession numbers: NZ_JIBO00000000 and NZ_FNQS00000000) of the Lonsdalea quercina subsp. quercina strain ATCC 29281T, respectively. The frequency percentage of each gene of T6SSc among these genomes is shown on the right side next to the physical map

An expanded examination of the novel T6SSc and the srl operon

Based on the results of our comparative genomics study of P. carotovorum subspecies and the other four closely related species of Pectobacterium, we found that the novel cluster T6SSc and the srl operon were specific to Pco subspecies. To evaluate the distribution of this operon and T6SSc in other closely related strains, we conducted an expanded genome (203 genomes, Additional file 1: Table S8-S9) analysis with all available genomes data of Pectobacterium downloaded from GenBank in July 2018 as well as other sequences retrieved from GenBank for representative strains from each branch of the phylogenetic trees of the Enterobacterales order from a study by Adeolu et al. [46].

The highly conserved srl operon in Pco subspecies

A total of 203 genomes of Enterobacterales strains were analyzed: only 66 of them possessed this srl operon (Additional file 1: Table S9). In addition to all of the sequenced Pco strains (including 13 from this study and the 2 from NCBI), we did not find any other Pectobacterium spp. or Dickeya spp. which cause similar soft rot diseases that harbored this operon. Besides the Pco strains, the srl operon was also present in two sequenced Brenneria strains and in one of the three sequenced Sodalis strains of the Pectobacteriaceae family. The srl operon occurred among most genomes of the Enterobacteriaceae (81.1%), Yersiniaceae (80.0%), and Erwiniaceae (62.5%) families, but is not present in the Morganellaceae, Budviciaceae, or Hafniaceae families. We found some plant pathogens with a broad host range (e.g., Pantoea ananatis) [47], some that infect hosts containing high levels of sorbitol (e.g., Erwinia spp.) [48, 49, 50], and some growth-promoting rhizobacteria (e.g., nitrogen-fixing strain) [51] also possess this operon. In addition, we aligned the srl operon sequences (protein and DNA) from these 66 strains and conducted phylogenetic and similarity (MUSCLE) analyses. The similarity analysis (protein and DNA) revealed few genetic differences (pairwise similarity > 99.4% in protein sequences; pairwise similarity > 99.5% in DNA sequences) in the srl operon between different Pco strains. Phylogenetic analysis based on DNA and protein sequences of the srl operon (Additional file 3: Figure S2) both showed that Pco strains were clustered into the clade with Erwinia spp., but separated from the clades containing Enterobacteriaceae spp. and Yersiniaceae spp. This result was consistent with the srl operon of Pco strains sharing highest similarity with those of Erwinia spp. (69.1–71.3% in DNA sequence and 78.6–80.0% in protein sequence).

Genetic variation of the novel secretion system T6SSc

Sixty-one Of the 203 Enterobacterales genomes we examined possessed the T6SSc cluster. In contrast to the high conservation of the srl operon among Pco strains, relatively high genetic variation was observed among the Pco T6SSc cluster sequences. Despite of this, the phylogenetic trees (not shown) built based on their T6SSc cluster sequences (protein and DNA) clustered all of the 15 Pco strains sequenced to date into a clade which was clearly separated from the other Enterobacterales strains. The T6SSc cluster was also present in a limited number of Brenneria goodwinii and Lonsdalea quercina strains in the Pectobacteriaceae, as well as being present sporadically among the other 6 families of the Enterobacterales order (Additional file 1: Table S8).

An analysis that screened for the presence/absence of individual genes of the T6SSc cluster in 61 Enterobacterales genomes showed two clear distribution patterns (Fig. 6). It suggested that the cluster consisted of two blocks of genes: the first, Block I, includes the first 8 genes (starting from the 5′ end) of the cluster, while Block II includes the 20 terminal genes of the cluster at the 3′ end. We found that genes of Block I were more highly conserved than those of Block II, a finding that merits further study that may help to clarify the evolutionary history of Pco species within the Pectobacterium genus.

Discussion

Historical assessments which assert that Pcc has the widest host range among the Pectobacterium taxa are due for revision, considering that multiple studies which examined the pathogenicity and host ranges of Pcb and Pco have now established a broad spectrum of hosts for these subspecies as well [5, 52, 53]. Many strains previously known as Pcc have also been re-classified into other species in recent years [8, 9]. Our finding that the P. peruviense, P. polaris, and Candidatus P. maceratum strains did not possess either the T6SSc cluster or the srl operon, viewed alongside our finding that no Pcc strains harbored either cluster, are consistent with the recently proposed adjustments to the classification of Pectobacterium species, which have posited that the newly proposed species are derived from Pcc. We also noticed that the genomic traits of Pca for some pathogenicity-related genes as well as the CRISPR-Cas systems, were not consistent with any of the three-known Pcc, Pcb and Pco subspecies, which is further support to the new subspecies proposal.

Interestingly, an srl operon related to sorbitol metabolism was specific to Pco genomes among the Pectobacterium genus based on the BLAST analysis using both DNA and amino acid sequences. Other studies have reported that mutants with loss-of-function for this operon in another phytopathogen (Erwinia amylovora) with a sorbitol deficiency were unable to grow on minimal medium with sorbitol [26]. Our biochemical test results also confirmed that these Pco strains with the srl operon could produce acid from sorbitol or could utilize this carbon source, while other strains lacking this operon like Pcc and Pcb could not. This is further supported by some previous studies which showed that Pectobacterium strains and the type strains of other species, such as Pcc, P. wasabiae, P. betavasculorum, and P. atrosepticum could not utilize/produce acid from sorbitol but that the type Pco strain as well as other Pco strains could [54, 55, 56, 57]. However, the ability of these Pectobacterium type strains to use sorbitol is still obscure; there are conflicting reports about the biochemical phenotypes for these strains reported in various studies [8, 52, 58].

Moreover, although the sequences of the srl operon from Pco strains are conserved, and are closely related to those present in Erwinia spp., whether the Pco strains acquired this operon from Erwinia spp., or if they acquired it from a common ancestor (and may have subsequently lost it in certain strains) still awaits clarification. Further, although this operon is strictly required for the pathogenicity of E. amylovora [26], its function in Pco is still unknown; it is clearly important considering its high level of conservation in Pco strains.

The other specific cluster to Pco subspecies among Pectobacterium is T6SSc; while the T6SSa and T6SSb type clusters have been reported extensively, we are unaware of other studies reporting a cluster with the gene order we detected in this T6SSc type cluster. T6SS have been shown to function in host cell adherence and in competition with other microbes, but the role of this class of gene cluster in Pectobacterium during infection remains unclear [2]. Our results confirmed the previously reported finding that P. atrosepticum contains only one T6SS (T6SSa), whereas P. parmentieri harbors two such machineries (T6SSa and T6SSb) [22]. However, in contrast to the previous description that P. carotovorum stains only have one T6SS (T6SSa), we detected T6SSc in these species. Although the components of this system were not strictly consistent among the Pco strains, and its function and potential targets must of course be further determined, it will be interesting to explore the possible functional contribution of this novel T6SSc to the pathophysiology of soft rot diseases. Many other genetic variations including pathogenicity-related genes as well as CRISPR-Cas systems that were revealed among the subspecies and species by this study lay a foundation for future work to elucidate the genetic basis of pathogenicity in Pectobacterium. It will also be interesting to determine which among these genomic variations in the different species/subspecies contribute to host ranges and geographic distributions.

Conclusion

Our findings significantly expand the body of knowledge surrounding the genetic basis for pathogenicity of soft rot bacteria in general. The genetic variations catalogued in this study also provide new insights into the evolutionary history of Pectobacterium and give extensive definitive evidence for accurately classifying members of the Pectobacterium species and subspecies. These analyses will provide a foundation for generating hypotheses about pathogenicity, host-bacteria interactions, taxonomy, and phylogenetic relationships among soft rot bacteria and other phytopathogens.

Methods

Collection and phenotyping of 32 Chinese Pectobacterium carotovorum isolates

Thirty-two strains from 3 economically important Chinese vegetables, Chinese cabbage (Brassica rapa subsp. pekinensis), bok choy (Brassica rapa subsp. chinensis), and celery (Apium graveolens) were collected from North China (Beijing area). Typical symptoms of soft rot were observed on the petioles and stems of these plants, which were grown in cultivated field in different districts of Beijing (Additional file 1: Table S3). Isolation of bacteria from diseased samples was carried out according to previous studies [54], were identified by Yu Tian and Xiaoying Li, previously, and were preserved in Beijing Key Laboratory of Agricultural Genetic Resources and Biotechnology, Beijing Academy of Agriculture and Forestry Science. These strains were tested for pathogenicity by inserting one pipette tip containing 10 μL of bacterial suspension (2 × 108 CFU/mL) 2 mm into each sterilized Chinese cabbage petioles surface with sterile distilled water as control. The petioles were then incubated at 28 °C and 95% relative humidity for 24 h before the diameter of the rotting tissue in each petiole was measured. Each strain was represented by three replicate petioles, and the experiment was performed three times. The data were subjected to one-way ANOVAs using SPSS 10.0. Means were compared by the Duncan’s tests, and statistical significance was determined at 5% levels. Strains were re-isolated successfully from symptomatic Chinese cabbages to complete Koch’s postulates. All strains were assayed with several biochemical tests: reducing substances from sucrose; acid production from D-sorbitol, D-arabitol, palatinose, and α-methylglucoside; utilization of citrate, and growth in 5% NaCl and at 37 °C [59]. Moreover, the 94 phenotypes (the oxidation of the 71 carbon sources and chemical susceptibility to 23 compounds) were analyzed using GEN III OmniLog system by the BIOLOG™ GEN III microplates [60].

Genome sequencing, assembly, and annotation of the 32 newly isolated Pectobacterium carotovorum strains

Genomic DNA was extracted and purified using an EasyPure Genomic DNA Kit (Transgen, China) according to the product instructions. Sequencing of the Pcb BC1 genome (accession: CP009769) was performed using the Illumina Hiseq 2000 platform with 2 Kb mate pairs and 200 to 500 bp unique paired-ends insert size libraries. The BC1 sequence was assembled by SOAPdenovo 2.0 software representing a 335-fold average genomic coverage. The scaffold order was based on the PCC21 reference genome, and a PCR-based genomic walking method was used to fill gaps between scaffolds. Sequencing of the Pco BCS7 genome (accession: CP009678) was performed with Pacific Biosciences RS II sequencing technology. A 10 Kb Single-Molecule Real-Time (SMRT) library was prepared from sheared genomic DNA using a 10-kb template library preparation workflow. The library was sequenced on 2 SMRT cells, providing about 137-fold genome coverage. The denovo assembly of the BCS7 reads was performed with the Hierarchical Genome Assembly Process (HGAP) algorithm in SMRT Portal (version 2.3.0). The genomes of the additional 30 P. carotovorum strains were sequenced with MPS (massively parallel sequencing) Illumina technology. A paired-end DNA library with an insert size of 500 bp was constructed and was sequenced using an Illumina HiSeq. Quality control of paired-end reads was performed using an in-house program to filter the low-quality reads, Illumina PCR adapter reads, and duplication reads. After filtering, all 30 of the strains had the sequencing coverage between 65-fold and 200-fold. Denovo assembly of the reads was performed with the SOAPdenovo algorithm (http://soap.genomics.org.cn/soapdenovo.html). All reads were used for further gap closure.

Coding genes of these 32 newly sequenced strains were predicted with GeneMark software by the self-training program GeneMarkS (version: 4.6b, http://topaz.gatech.edu/GeneMark/). Transfer RNA (tRNA) genes were predicted with tRNAscan-SE (version: 1.3.1, http://gtrnadb.ucsc.edu/) [61], Ribosome RNA (rRNA) genes were predicted with rRNAmmer [62], and sRNAs were predicted by BLAST against the Rfam database. Long terminal repeats sequences of all the tested genomes in this study were predicted using Repeat Masker (http://www.repeatmasker.org/) and tandem repeats were analyzed using Tandem Repeat Finder (http://tandem.bu.edu/trf/trf.html). Among these tandem repeats, arrays with repeat units 15 bp to 65 bp will be considered as minisatellites DNA. Gene functional annotations of each of the 32 genomes was performed by BLAST analysis against multiple databases, including NCBI NR (Version: 20150405, http://www.ncbi.nlm.nih.gov/), KEGG (Version: 59, http://www.genome.jp/kegg/), COG (Version: 20090331, http://www.ncbi.nlm.nih.gov/COG/), GO (Version: 20150405, http://geneontology.org/), Swiss-Prot (Version: 20150414, http://www.ebi.ac.uk/uniprot/), PHI (Version: 3.6, http://www.phi-base.org/), VFDB (Version: Tue May 5 10:06:01 2015, http://www.mgc.ac.cn/VFs/main.htm), MvirDB database [63], ARDB (Version: 1.1, http://ardb.cbcb.umd.edu/), CARD (Version: 20140415, http://arpcard.mcmaster.ca/), CAZy (Version: 20141020, http://www.cazy.org/), TCDB (Version: 20110715, http://www.tcdb.org/). For each BLAST hit (a given ORF), the best alignment with the lowest e-value and highest score was retained; to be retained, a hit required at least 0.4 identity for a sequence at 40% of a given alignment’s length. All 32 genomes newly sequenced in this study have been deposited in the NCBI genome database (Accession numbers were shown in Additional file 1: Table S3).

Comparative genomic analysis

A comprehensive comparative genomics study of 84 Pectobacterium genomes (including the 32 newly sequenced genomes in this study and 52 other Pectobacterium genomes published in GenBank by July 2018, for strains including P. carotovorum, P. polaris, Candidatus P. maceratum, P. atrosepticum, and P. parmentieri) was conducted using three complete Dickeya genomes (D. zeae Ech586, D. paradisiaca Ech703, and D. dadantii 3937) as the reference outgroup. Note that the previously reported P. carotovorum strain 3–2, as well as the Pcc strains ATCC 39048 and Ecc71, were reclassified in this study (Fig. 2a); therefore, strains Ecc71 and 3–2 were part of the Candidatus P. maceratum group and strain ATCC 39048 was part of the Pcb group in each of the bioinformatics analyses of this in this comparative genomics study. The verified classification and detailed information for each of the strains are shown in Additional file 1: Table S3. All of these 84 Pectobacterium genomes were used for the following bioinformatics analyses.

Whole-genome SNP detection and identity score analyses

Genome-Wide SNP detection was performed using MUMmer comparison software: the whole-genome sequence of BC1 (the reference) was compared with genome sequence of the other strains to identify differences. BLAT (BLAST-Like Alignment Tool) software was then used to compare the sequences adjacent to the SNP loci to verify a given SNP’s validity [64]. Repeat regions were detected by BLAST, Repeat Masker, and Tandem Repeats Finder software. If the length of the SNP adjacent sequence alignment in the BLAT results was less than 101 bp, or if a SNP was found in a repeat region, the data was considered as suspect and was removed. We performed a phylogenetic analysis based on SNP data: for all species, all SNPs were joined in the same order. PhyML software (Version: 3.0, http://www.atgc-montpellier.fr/phyml/) was then used to construct the phylogenetic tree with a maximum likelihood model. The number of bootstrap replications was set to 1000.

To identify genetic relatedness in any two strains, pairwise Identity Scores (IS) were calculated [65]. The IS is the ratio of unique SNPs between two accessions; this was determined using an in-house Python script and visualized with a heat map which produced with the ggplot2R package.

Pan-genome analyses

Eighty-four Pectobacterium genomes, including 56 P. carotovorum, 6 P. parmentieri, 10 P. atrosepticum, 8 Candidatus P. maceratum, and 4 P. polaris genomes were used in the pan-genome analysis of four closely related species of the Pectobacterium genus (Additional file 1: Table S3). This included the 9 Pcc, 29 Pcb, 15 Pco, and 3 Pca genomes that were used in the aforementioned pan-genome analysis of the P. carotovorum species. Subspecies core orthologous genes and strain-specific unique genes were also examined in the 9 Pcc, 15 Pco, 29 Pcb, and 3 Pca genomes sequences, respectively. Specifically, it should be noted that the five pan-genomes of each species (that is, the pan-genome for 56 P. carotovorum genomes, that of the 6 P. parmentieri, 10 P. atrosepticum, 8 Candidatus P. maceratum, and 4 P. polaris genomes, respectively) were used as the petals for generating the Venn diagram for the Pectobacterium genus.

Likewise, the pan-genomes for each of the four subspecies (that is 29 Pcb, 3 Pcc, 15 Pco, and 3 Pca genomes, respectively) were used as the petals to generate the Venn diagram for the P. carotovorum species. The core and pan genes identified and clustered using CD-HIT rapid clustering of similar proteins software [66, 67], with a threshold of 70% pairwise identity and a 0.6 length difference cut-off in amino acid sequences. Genes that did not reach the threshold were used to initiate a new cluster. Genes from multiple species in each core gene cluster were aligned to each other to make a multi-sequence alignment using MUSCLE (Version: 3.8.31, http://www.drive5.com/muscle). The CD-HIT analysis also identified the genes that were unique to each species/subspecies of Pectobacterium, and their functions differences based on COG annotations were performed. Finally, a core-gene phylogenetic tree of these Pectobacterium strains was constructed with three Dickeya strains as the outgroup. The PhyML (Version: 3.0, http://www.atgc-montpellier.fr/phyml/) software was used with a maximum likelihood model. The number of bootstrap replications was set to 1000.

Gene cluster analysis of orthologs

BLAST analysis using both DNA and amino acid sequences was used to identify the presence of homologs of previously known genes (e.g., pathogenicity-related genes) in the genomes in this study. For each BLAST hit (a given ORF), the best alignment with the lowest e-value and highest score was retained; to be retained, a hit required at least a 0.4 identity for a sequence at 40% of a given alignment’s length. Detection of CRISPR-Cas system components. The CRISPR Finder online tool (http://crispr.i2bc.paris-saclay.fr/Server/) was used to identify all of the predicted CRISPR components for each strain. Predicted cas genes were also identified with BLAST analysis according to Makarova (2011) [68]. The CRISPR and cas results were combined to determine the predicted locations of the CRISPR-Cas system(s) in each strain.

An expanded examination of the novel T6SSc and the srl operon

After confirming the srl operon and T6SSc were two Pco-specific clusters within 84 Pectobacterium and 3 reference Dickeya genome sequences tested above, we examined its distribution in the Pectobacterium genus and even in the order Enterobacterales. This analysis included the 203 genomes, including all of the available Pectobacterium downloaded from GenBank in July 2018, as well as other sequences from GenBank for representative strains from each branch of the phylogenetic trees of Enterobacterales as reported by Adeolu et al. [46]. These 203 Enterobacterales genomes covered 62 species of 31 genera across 7 families (Enterobacteriaceae, Erwiniaceae, Morganellaceae, Yersiniaceae, Budviciaceae, Hafniaceae, and Pectobacteriaceae) of the Enterobacterales order. BLAST analysis using both DNA and amino acid sequences was used to identify the presence of homologs of the genes of the srl operon and T6SSc cluster in these 203 Enterobacterales genomes with the sequences of the BCS7 strain (the only Pco strain with a complete genome) used as a standard. After identifying homologs in these strains, we examined the similarity of the sequences for these two clusters using a maximum likelihood phylogenetic tree based on both the DNA and amino acid sequences. We also calculated the ratio of the similarity based on the total length of alignment of each couple strains in the matrix based on the results of multi-sequence alignment (both the DNA and amino acid sequences) using MUSCLE software.

Notes

Acknowledgements

Not applicable.

Funding

This work was supported by grants from the Earmarked Fund for Beijing Innovation Consortium of Agriculture Research System (BAIC07). The funding source supported the publication of this work and data analysis while had no role in study design and data collection, interpretation of data and in writing the manuscript.

Availability of data and materials

All data generated or analyzed during this study are included in this published article [and its additional information files]. All genome sequences used in the course of this study are available from the National Centre for Biotechnology Information, (NCBI) genome database (https://www.ncbi.nlm.nih.gov). Additionally, all genomes sequenced during the course of this study have been deposited in the NCBI genome database under the following accession numbers: CP009769, CP009678, JUJM00000000, JUJJ00000000, JUJK00000000, JUJL00000000, JUJH00000000, JUJB00000000, JUJC00000000, JUJP00000000, JUJD00000000, JUJE00000000, JUJN00000000, JUJF00000000, JUJT00000000, JUJO00000000, JUJQ00000000, JUJG00000000, JUJR00000000, JUJS00000000, MTAG00000000, MTAI00000000, MTAH00000000, MTAJ00000000, MTAK00000000, MTAL00000000, MTAO00000000, MTAP00000000, MTAN00000000, MTAR00000000, MTAQ00000000, MTAM00000000. Biological materials from the current study are available from the corresponding author on reasonable request.

Authors’ contributions

HX designed the experiment and revised the manuscript; XYL, YLM, and SQL, performed the molecular, biochemical data collection and bioinformatics analysis; XYL, YLM, and SQL wrote the manuscript. SJY, SSX, and YT revised the manuscript. All authors read and approved the final manuscript.

Ethics approval and consent to participate

This study did not involve humans, human data or animals; no ethics approval or consent is required to publish the results.

Consent for publication

Not applicable.

Competing interests

The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

Publisher’s Note

Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

Supplementary material

12864_2018_5269_MOESM1_ESM.xlsx (752 kb)
Additional file 1: Table S1. The biochemical characters and pathogenicity of 32 Chinese Pectobacterium carotovorum strains. Table S2. Selected phenotypic characteristics of Pectobacterium carotovorum subsp. brasiliense, P. carotovorum subsp. carotovorum and P. carotovorum subsp. odoriferum. Table S3. Eighty-four Pectobacterium genomes used in this study, with accession number, host origin and location information. Table S4. The general genomic features of the 84 Pectobacterium genomes. Table S5. The CRISPR-Cas systems identified in the 84 Pectobacterium genomes. Table S6. Presence of genes encoding general virulence factors in the 84 Pectobacterium genomes. Table S6–1. The position of rmsB, the RNA fragment related to virulence in the 84 Pectobacterium genomes. Table S7. Gene clusters for TNSSs identified in the 84 Pectobacterium genomes. Table S8. Presence of the T6SSc gene cluster in the 203 Enterobacterales genomes. Table S9. Presence of the srl operon in the 203 Enterobacterales genomes. (XLSX 751 kb)
12864_2018_5269_MOESM2_ESM.pdf (110.2 mb)
Additional file 2: Figure S1. Soft rot symptoms in Brassica rapa subsp. pekinensis (a), Brassica rapa subsp. chinensis (b), and Apium graveolens (c). (PDF 112808 kb)
12864_2018_5269_MOESM3_ESM.pdf (763 kb)
Additional file 3: Figure S2. Phylogenetic tree based on the amino acid sequences of the srl operon in the 66 Enterobacterales strains. A total of 66 of the 203 Enterobacterales genomes were found to possess the srl operon in this study. The phylogenetic trees based on DNA and amino acid sequences were almost identical. The strains with red letters were the newly sequenced in this study. They were clustered into the clade containing the Ewinia strains. The tree was constructed using a maximum likelihood method and was generated with 1000 bootstrap replicates. (PDF 763 kb)

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© The Author(s). 2018

Open AccessThis article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated.

Authors and Affiliations

  • Xiaoying Li
    • 1
    • 2
  • Yali Ma
    • 1
    • 2
  • Shuqing Liang
    • 3
  • Yu Tian
    • 1
    • 2
  • Sanjun Yin
    • 3
  • Sisi Xie
    • 3
  • Hua Xie
    • 1
    • 2
  1. 1.Beijing Agro-Biotechnology Research CenterBeijing Academy of Agriculture and Forestry SciencesBeijingPeople’s Republic of China
  2. 2.Beijing Key Laboratory of Agricultural Genetic Resources and BiotechnologyBeijingPeople’s Republic of China
  3. 3.Health Time Gene InstituteShenzhenPeople’s Republic of China

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