Background

The bacterial genus Pectobacterium (formerly classified as the Erwinia genus) is a group of facultative anaerobic, Gram-negative, non-sporulating, motile bacteria belonging to the Pectobacteriaceae family [1,5: Table S3. The gene sequences were aligned using MUSCLE software and trimmed to remove ambiguously aligned regions. Subsequently, six housekee** gene sequences were concatenated in the same order using SequenceMatrix. The phylogenetic tree was constructed using the maximum likelihoods method derived from MEGA 6.0 software [35], and 1,000 bootstrap replicates were included in a heuristic search with a random tree and the tree bisection-reconnection branch-swap** algorithm.

Comparative analysis

According to the phylogenetic analysis, we selected eight closely related species or subspecies with released complete genomes including P. atrosepticum SCRI1043, P. parmentieri RNS08.42.1A, P. parmentieri SCC3193, P. wasabiae CFBP 3304, P. carotovorum subsp. brasiliense BC1, P. carotovorum subsp. brasiliense BZA12, P. carotovorum subsp. carotovorum PCC21, and P. carotovorum subsp. odoriferum BC S7 for genome comparison. Average nucleotide identities (ANI) values were computed for pairwise genome comparison using the OrthoANIu Algorithm (https://www.ezbiocloud.net/tools/orthoaniu) [36]. In silico DNA-DNA hybridization (DDH) was calculated using the Genome-to-Genome Distance Calculator (GGDC) (http://ggdc.dsmz.de/ggdc.php#) [37]. Complete genome comparisons were conducted using the progressive alignment option of the Mauve 2.3.1 comparison software [38] with the SX309 genome as the reference genome. Furthermore, synteny plots were also generated as alignments of the complete genome nucleotide sequences using MUMmer 3.22 [39]. To identify the set of common genes for the Pectobacterium genus and the set of genes unique to each species or subspecies, comparative analyses at the protein level were performed using an all-against-all comparison of the annotated genomes using BLASTP [40], and ortholog gene clustering analysis was implemented with the default settings [41]. Venn diagrams were created using R project language [42]. The comparative analysis of the T3SS effectors, QS system, TCS, and CRISPR/Cas system were BLASTed at the protein level using T3DB [43], SigMol [44], P2CS database [45], and CRISPRs Finder tool [46], respectively. The targets of the spacers were identified using ViroBLAST (https://indra.mullins.microbiol.washington.edu/viroblast/viroblast.php) and local BLAST analysis against NCBI plasmid genomes (ftp://ftp.ncbi.nih.gov/refseq/release/plasmid/).

Extracellular enzyme assays

Plate assays for the activity of Pel, Peh, Cel, and Prt were conducted as described by Chatterjee et al. [17] (1995) with slight modifications. Wells were bored in the agarose medium with a No. 2 cork borer, and the bottoms were sealed with 0.8% (w/v) of molten agarose. Bacterial cells were grown in NB liquid medium overnight at 28°C and adjusted to OD600 = 0.8. Samples were applied to the wells, and the plates were incubated for 24 h at 28°C for Pel, Peh, and Cel and for 48 h for Prt. The Pel and Peh plates were developed with 4 N HCl, and the Cel plates were stained with 0.1% (w/v) Congo red solution for 10 min and then washed with 1 M NaCl solution three times. Haloes in the Prt plates became visible without any further treatment. Each treatment was repeated three times, and all of the experiments were repeated three times.

Virulence assays

The virulence and symptom development caused by P. carotovorum subsp. brasiliense SX309 were assessed in cucumber plants (Cucumis sativus) and potato plants (Solanum tuberosum). Cucumber and potato stems were stab-inoculated with 10 μL of approximately 1 × 108 CFU/mL bacterial suspensions of the SX309 strain. They were then incubated in a moist chamber at 28°C, and the appearance of the symptoms was periodically observed. Sterilized distilled water was used for the negative control inoculations. For each inoculation experiment, three plants were used, and the experiments were repeated three times.

Microscopic analysis

For transmission electron microscope (TEM) observation, bacterial cells were negatively stained using 1% uranium acetate on collodion-coated 100-mesh grids. The samples were visualized using a transmission electron microscope Hitachi-7700 (Hitachi High-Technologies Corporation, Tokyo). For fluorescence electron microscope (FEM) observation, the plasmid pSMC21 containing the gfp gene was used to generate a GFP-tagged P. carotovorum subsp. brasiliense strain [47]. The plasmid was introduced into the bacterial cells using electroporation. The GFP-tagged SX309 strain was then visualized using a fluorescence microscope Olympus BX51. For scanning electron microscope (SEM) observation, bacterial cells in exponential and stationary phases were fixed using 2.5% glutaraldehyde. Samples were observed using a scanning electron microscope Hitachi-S3400N.

Results

Organism information

P. carotovorum subsp. brasiliense SX309 is a facultative anaerobic, Gram-negative, non-sporulating bacterium belonging to the Pectobacteriaceae family (Additional file 1: Table S1). SX309 strain is rod-shaped with a length of 1.5-2 μm and a diameter of 0.5-0.8 μm. It is motile by using peritrichous flagella (Additional file 2: Figure S1). Strain SX309 can utilize several carbon sources and grow in 5% NaCl [7: Table S4 and Additional file 8: Figure S4). As expected, the twenty Pectobacterium strains, seven Dickeya strains, and eight Erwinia strains were clustered into three major clades. In practice, Pectobacterium spp. are considered as broad-host range pathogens, except that P. atrosepticum has been reported almost exclusively from potatoes (Solanum tuberosum) and P. betavasculorum exclusively from sugar beets (Beta vulgaris). P. carotovorum has a broader host range and less restricted survival conditions than P. atrosepticum, P. parmentieri, and P. wasabiae, which are specialized to cause disease in one or few host plants only [49]. Strain SX309 is able to infect a wide range of plant species [62]. The role of T2SS in Pcb SX309 remains to be determined in the future.

T3SSs are used by many Gram-negative pathogenic bacteria to deliver virulence proteins (known as effectors) into host cells. Once inside host cells, the effectors manipulate host defenses and promote bacterial growth [63]. Unlike in many other plant bacterial pathogens, the T3SS in P. carotovorum subsp. carotovorum appears to secrete only one effector protein, DspE [64]. Therefore, Pectobacterium seems do not require the T3SS for pathogenicity [21]. T3SS contributes to P. carotovorum growth in the leaves of Arabidopsis thaliana [65] at the early stages of infection and contributes to the virulence of P. atrosepticum on Solanum tuberosum [66]. However, it need to be determined whether the virulence of Pcb partly depend on T3SS during infection of the host plant.

A promiscuous secretion system, possibly participated in bacterial pathogenicity, is the recently identified type VI secretion systems (T6SS) in diverse Gram-negative bacteria [67]. T6SS gene clusters consist of 13 core genes that are hypothesized to be minimally necessary for function and conserved genes that vary in composition between species [68]. The vgrG (encoding valine/glycine-repeat protein G) gene contribute to the virulence in Acinetobacter baumannii ATCC 19606 [69]. In Acidovorax avenae subsp. avenae strain RS-2, disruption of the genes pppA, clpB, icmF, impJ and impM caused the reduction of biofilm formation, and mutation of pppA, clpB, icmF and hcp resulted in the reduction in motility. The vital roles of T6SS in the virulence of strain RS-2 may be partially attributed to the reductions in Hcp secretion, biofilm formation and motility. In the Pectobacterium genus, for the biological functions of the T6SS, researchers have not yielded a generalizable conclusion. In Pcc S1, impG strongly influences the virulence and hypersensitive response [70]. It was demonstrated that the PCWDEs genes (pelA and prtF) and T6SS genes (vgrG and hcp3) had the same expression profiles regulated by QS. In P. atrosepticum SCRI1043, and the hcp and vgrG genes are induced in response to potato extracts. However, the virulence of a single gene defective mutant that was interfered in the secretion of Hcp was reported to be stronger than that of the wild-type pathogen in potato tubers [71]. A mutant with double deletions of two machinery encoding clusters spanning 16 (W5S_0962-W5S_0978) and 23 (W5S_2418-W5S_2441) genes that included the two putative T6SS encoding loci was modestly affected in its virulence in the potato tuber slice assay [56]. To date, T6SSs in many bacteria may be involved in pathogenic or symbiotic interactions with their hosts. However, more work are needed to define the function of this intriguing system in Pcb.

Quorum Sensing is a special type of regulation of bacterial gene expression, usually active in conditions of a high population density of bacterial cells. QS systems are widespread among the plant soft-rotting bacteria [7].Previous research showed that Pectobacterium spp. produces two AHL family quorum sensing signals, i.e., N-3-oxooctanoyl-L-homoserine lactone (3-oxo-C8-AHL) and 3-oxohexanoyl-L-homoserine lactone (3-oxo-C6-AHL), which are encoded by the luxI homolog expI [72, 73]. The AHL signal was detected by ExpR that belongs to the LuxR family of proteins and was transduced into cellular responses. The inactivation of expI resulted in the decreased production of PCWDEs and decreased virulence [19].

The second QS system, based on the production of the AI-2 signal molecules and controlled by the S-ribosylhomocysteine lyase LuxS protein, exists in a wide variety of both Gram-negative and Gram-positive bacteria and is involved in bacterial interspecies communication [74]. The LuxS/AI-2 type QS plays a strain-dependent role in virulence of different Pectobacterium strains. A luxS homolog from a Pectobacterium was first reported in a derivative of P. carotovorum subsp. carotovorum ATTn10 and in P. atrosepticum SCRI1043 [75]. Previous study revealed that there is a correlation between the AI-2 level and the production of pectinolytic enzymes. But it lacks orthologs for both known AI-2 receptors: the LuxPQ-receptor and the Lsr ABC-transporter [76]. We hypothesize that RbsB is an alternative to the AI-2 receptors in the Pectobacterium strain. However, the function of the rbsB gene still needs to be validated.

Interestingly, a new kind of autoinducer (AI-3) was discovered in Enterohemorrhagic Escherichia coli (EHEC). AI-3 is perceived by the sensor kinase QseC and its cognate response regulator QseB [77]. Meanwhile, it was found that qseC and qseB were both in PcbSX309, Overall, the biological significance of various QS systems, especially the LuxS/AI-2 QS system in SX309 and other Pectobacterium species, remains to be studied further. Previous studies show that the expression of the rsmA/rsmB genes involved in the regulation of PCWDE biosynthesis is also dependent upon the global regulatory GacA/GacS system [18].

To survive, colonize and cause disease, plant-pathogenic bacteria often modulate the expression of their genes using two-component signal transduction systems (TCSs). These systems typically consist of a sensor histidine kinase (HK) and a response regulator (RR) performing a His-Asp phosphotransfer [78].It has been reported that virulence, resistance to magainin II, and the expression of pectate lyase in D. chrysanthemi 3937 were mediated by the response of the PhoP-PhoQ TCSs to pH and magnesium [79]. Additionally, the GacS/GacA two-component regulators are involved in the global control of virulence in P. carotovorum subsp. carotovorum [80]. However, the functions of these TCSs still need to be addressed.

Bacterial flagella are complex and originated very early as organelles that provide swimming and swarming motilities and play a central role in adhesion, biofilm formation, and host invasion [81]. Flagellar proteins are normally responsible for cell motility and intracellular trafficking, secretion and vesicular transport, while the chemotactic proteins are involved in cell motility and signal transduction [82].In D. dadantii 3937, the mutation of fliA encoding a sigma factor eliminated the bacterial motility, and significantly reduced Pel production and the bacterial attachment to plant tissues [82]. Similarly, the inactivation of flgA, fliA, and flhB gene abolished the bacterial motility and significantly reduced the bacterial virulence in P. carotovorum subsp. carotovorum PCC21 [83]. We have observed that Pectobacterium cells are motile in diseased plant tissues (data not published), but whether the production of PCWDEs and secretion systems that contribute to virulence is coordinated with motility is still unclear. Thus, the functions of flagellar and chemotactic genes in Pectobacterium pathogenicity, especially in pathogen-host plant interactions, remain to be explored.

LPSs were shown to have complex and differing roles depending on their origin and the challenged plant. Previous research reported that different defense response patterns could be induced by the LPS of P. atrosepticum and Pseudomonas corrugata in three Solanaceae species, including tobacco, tomato, and potato [84]. Additionally, different signaling pathways could also be activated by LPS in Arabidopsis thaliana cells [85].A previous study showed that LPS are crucial for the optimal growth, survival and virulence of P. atrosepticum [86], but the roles of LPS in the SX309 strain remain to be determined.

The CRISPR-Cas system mediate immunity to invading genetic elements such as bacteriophages, viruses and plasmids [87]. Based on the presence of the Cas3, Cas9, and Cas10 proteins, different CRISPR-Cas systems were classified into three major types, type I, II, and III. The major types comprise further subtypes (e.g., I-A to I-F), each is characterized by a specific set of proteins [90]. Cas1 is the protein hallmark of CRISPR-mediated immunity, and Cas 1 and Cas2 were found in all CRISPR-containing organisms [23].

The key factors of the CRISPR-mediated immunity system are small CRISPR RNAs that guide nucleases to complementary target nucleic acids of invading genetic material, generally followed by the degradation of the invader [88, 89]. Previous studies revealed that the P. atrosepticum SCRI1043 CRISPR-Cas system contains six proteins, including Cas1, Cas3, and the four subtype I-F specific proteins Csy1, Csy2, Csy3, and Csy4, and three CRISPR repeats [26]. In P. atrosepticum, the Csy4 protein was identified to be responsible for processing the CRISPR RNAs into crRNAs and appears to interact with itself in the absence of other Cas proteins [90]. In our study, we found Pcc, Pco, and Pcb all harbor two subtypes of CRISPR/CAS system (Type I-E, I-F). In Escherichia coli, primed adaptation by type I-E CRISPR-Cas system occurs after the Cascade-crRNA complex interacts with a fully matching protospacer that is subject to interference [25]. However, there are relatively few reports concerning the CRISPR-Cas system in Pectobacterium species.

T6SS can be deployed as versatile weapons to compete with other bacterial cells or attack simple or higher eukaryotic cells and likely plays an important role in mediating a pathogenic or a symbiotic relationship between bacteria and eukaryotes in various environmental niches [68, 91,92,93,94]. Antibacterial effector toxins secreted by T6SSs contributed to the antibacterial functions, which could be neutralized by corresponding antagonistic immunity proteins to preventing self-killing or sibling-intoxication. In Vibrio cholerae, VgrG-3 was found to degrade peptidoglycan and hydrolyse the cell wall of Gram-negative bacteria, and the TsaB (type six secretion antitoxin B) was identified as the immunity protein. In Dickeya dadantii 3937, Rhs played an important role in intercellular competition, which is linked with the VgrG component of T6SS [94]. The functions of T6SSs should be determined in future research.

Conclusion

This study provided a comprehensive analysis of the complete genome of P. carotovorum subsp. brasiliense strain SX309, a causative agent of bacterial soft rot disease. The genomic analysis of strain SX309 has shown that this bacterium belongs to the P. carotovorum subsp. brasiliense. The chromosome organization and structure in P. carotovorum subsp. brasiliense strain SX309 is most similar to that of P. carotovorum subsp. brasiliense BC1, which is consistent with the finding that SX309 and BC1 are closely related based on multilocus sequence analysis. To our knowledge, the primary pathogenicity determinants of Pectobacterium are the coordinated production of PCWDEs that macerate host tissue and release nutrients for bacterial growth. These extracellular enzymes are secreted by the T2SS under the control of QS system [17]. Type III secretion system (T3SS) genes are not involved in the secretion of exoenzymes, but they still play a crucial role in Pectobacterium pathogenicity [21]. Compared to other pathogenic bacteria belonging to the Pectobacterium genus, the genome of P. carotovorum subsp. brasiliense SX309 encodes many similar virulence factors, including the PCWDE biosynthetic genes, T2SS and T3SS genes, bacterial QS genes, flagella and chemotactic genes. Moreover, comparative analysis revealed that the Pectobacterium strains harbor the type VI secretion system and CRISPR-Cas immune system genes, which were suggested to contribute to bacterial virulence and adaptive immunity. However, the functions of these genes remain to be elucidated in P. carotovorum subsp. brasiliense.

In summary, the comprehensive analyses of the genomes of Pectobacterium strains provide new insights for the conservation and evolution processes of virulence elements in these important bacterial pathogens. Knowledge of the variability and specificities of the Pectobacterium organisms could contribute to a better understanding of the molecular mechanisms of unique genetic features and pathogenesis.