New Insights and Approach Toward the Genetic Diversity and Strain Typing of Erwinia pyrifoliae Based on rsxC, an Electron Transport Gene.

Erwinia pyrifoliae, a causal agent of black shoot blight in apple and pear trees, is a plant pathogenic bacterium first reported in South Korea. The symptoms of black shoot blight are very similar to those of the fire blight disease in apple and pear trees caused by E. amylovora, as E. pyrifoliae has a genetically very close relationship with E. amylovora. Recently, there have been reports that E. pyrifoliae causes disease in European strawberries, resulting in severe fruit loss that aroused great concern about its spread, distribution, and host range. Therefore, it is essential to establish a trustworthy approach to understanding the distribution patterns of E. pyrifoliae based on the genetic background to strengthen the barrier of potential spreading risks, although advanced methods have been provided to accurately detect E. pyrifoliae and E. amylovora. Consequently, this study discovered a noble and noteworthy gene, rsxC, capable of providing the pathogen genotype by comparing E. pyrifoliae genomic sequences in the international representative genome archive. Different numbers of 40-unit amino acid repeats in this gene among the strains induced intraspecific traits in RsxC. By comparing their repeat pattern, E. pyrifoliae isolates were divided into two main groups, branching into several clades via sequence alignment of 35 E. pyrifoliae isolates from various apple orchards from 2020 to 2021 in South Korea. The newly discovered quadraginta amino acid repeat within this gene would be a valuable genetic touchstone for determining the genotype and distribution pattern of E. pyrifoliae strains, ultimately leading to exploring their evolution. The function of amino acid repeats and the biological significance of strains need to be elucidated further.

Determination and Kinetic Characterization of a New Potential Inhibitor for AmsI Protein Tyrosine Phosphatase from the Apple Pathogen Erwinia amylovora.

Erwinia amylovora is a Gram-negative bacterium, responsible for the fire blight disease in Rosaceae plants. Its virulence is correlated with the production of an exopolysaccharide (EPS) called amylovoran, which protects the bacterium from the surrounding environment and helps its diffusion inside the host. Amylovoran biosynthesis relies on the expression of twelve genes clustered in the ams operon. One of these genes, amsI, encodes for a Low Molecular Weight Protein Tyrosine Phosphatase (LMW-PTP) called EaAmsI, which plays a key role in the regulation of the EPS production pathway. For this reason, EaAmsI was chosen in this work as a target for the development of new antibacterial agents against E. amylovora. To achieve this aim, a set of programs (DOCK6, OpenEye FRED) was selected to perform a virtual screening using a database of ca. 700 molecules. The six best-scoring compounds identified were tested in in vitro assays. A complete inhibition kinetic characterization carried out on the most promising molecule (n-Heptyl ß-D-glucopyranoside, N7G) showed an inhibition constant of 7.8 ± 0.6 µM. This study represents an initial step towards the development of new EaAmsI inhibitors able to act as antibacterial agents against E. amylovora infections.

Identifying the core genome of the nucleus-forming bacteriophage family and characterization of Erwinia phage RAY.

We recently discovered that some bacteriophages establish a nucleus-like replication compartment (phage nucleus), but the core genes that define nucleus-based phage replication and their phylogenetic distribution were still to be determined. Here, we show that phages encoding the major phage nucleus protein chimallin share 72 conserved genes encoded within seven gene blocks. Of these, 21 core genes are unique to nucleus-forming phage, and all but one of these genes encode proteins of unknown function. We propose that these phages comprise a novel viral family we term Chimalliviridae. Fluorescence microscopy and cryoelectron tomography studies of Erwinia phage vB_EamM_RAY confirm that many of the key steps of nucleus-based replication are conserved among diverse chimalliviruses and reveal variations on this replication mechanism. This work expands our understanding of phage nucleus and PhuZ spindle diversity and function, providing a roadmap for identifying key mechanisms underlying nucleus-based phage replication.

Examination of Large Chromosomal Inversions in the Genome of Erwinia amylovora Strains Reveals Worldwide Distribution and North America-Specific Types.

Erwinia amylovora is a relatively homogeneous species with low genetic diversity at the nucleotide level. However, phenotypic differences and genomic structural variations among E. amylovora strains have been documented. In this study, we identified 10 large chromosomal inversion (LCI) types in the Spiraeoideae-infecting (SI) E. amylovora strains by combining whole genome sequencing and PCR-based molecular markers. It was found that LCIs were mainly caused by homologous recombination events among seven rRNA operons (rrns) in SI E. amylovora strains. Although ribotyping results identified inter- and intra-variations in the internal transcribed spacer (ITS1 and ITS2) regions among rrns, LCIs tend to occur between rrns transcribed in the opposite directions and with the same tRNA content (tRNA-Glu or tRNA-Ile/Ala) in ITS1. Based on the LCI types, physical/estimated replichore imbalance (PRI/ERI) was examined and calculated. Among the 117 SI strains evaluated, the LCI types of Ea1189, CFBP1430, and Ea273 were the most common, with ERI values at 1.31, 7.87, and 4.47°, respectively. These three LCI types had worldwide distribution, whereas the remaining seven LCI types were restricted to North America (or certain regions of the United States). Our results indicated ongoing chromosomal recombination events in the SI E. amylovora population and showed that LCI events are mostly symmetrical, keeping the ERI less than 15°. These findings provide initial evidence about the prevalence of certain LCI types in E. amylovora strains, how LCI occurs, and its potential evolutionary advantage and history, which might help track the movement of the pathogen.

Three novel Erwinia billingiae phages isolated from organic waste represent three new genera.

Despite the ecological significance of viral communities, phages remain insufficiently studied. Current genomic databases lack high-quality phage genome sequences linked to specific bacteria. Bacteria of the genus Erwinia are known to colonize the phyllosphere of plants, both as commensals and as pathogens. We isolated three Erwinia billingiae phages-Zoomie, Pecta, and Snitter-from organic household waste. Based on sequence similarity to their closest relatives, we propose that they represent three new genera: "Pectavirus" within the family Zobellviridae, "Snittervirus" in the subfamily Tempevirinae, family Drexlerviridae, and "Zoomievirus" within the family Autographiviridae, which, together with the genus Limelightvirus, may constitute a new subfamily.

Isolation of Streptomycin-Resistant Erwinia pyrifoliae in Korea.

As a black shoot blight disease-causing agent, Erwinia pyrifoliae was first reported in 1995 in Korea. A total of 101 isolates of E. pyrifoliae were isolated from samples showing bacterial symptoms collected from apple and pear orchards between 2020 and 2021. These isolates were screened for streptomycin resistance, with one from an orchard in Gwangju showing resistance at 100 µg/ml streptomycin. This streptomycin-resistant E. pyrifoliae (EpSmR) isolate was identified via polymerase chain reaction amplification of the strA/strB gene and an internal region of the ribosomal rpsL gene containing codon 43. EpSmR has a point mutation that altered this codon from lysine (AAA) to threonine (ACA). The strA and strB genes were not identified in EpSmR. EpSmR showed a high resistance to streptomycin (>50,000 µg/ml). This is the first study reporting EpSmR, which emerged due to a mutation in codon 43 of the rpsL gene.

Dynamic interactions between the symbiont Candidatus Erwinia dacicola and its olive fruit fly host Bactrocera oleae.

The olive fruit fly, Bactrocera oleae, the most serious pest of olives, requires the endosymbiotic bacterium Candidatus Erwinia dacicola in order to complete its development in unripe green olives. Hence, a better understanding of the symbiosis of Ca. E. dacicola and its insect host may lead to new strategies for B. oleae control. The relative abundance of bacteria during the fly life cycle comparing black and green olives was estimated by real time quantitative PCR revealing significant fluctuations during development in black olives with a peak of the bacteria in the second instar larvae. By microscopy analysis of larvae, we show that the bacteria reside extracellularly in the gastric caeca. During the transition to late third instar larvae, the bacteria were discharged into the midgut concomitant with a change in caeca size and morphology due to the contraction of the muscles surrounding the caeca. A similar alteration was also observed in a laboratory strain devoid of bacteria. To further investigate the symbiotic interaction and the change in caeca morphology a comparative transcriptomics analysis was undertaken. Samples of dissected caeca from second and third instar larvae collected from the field as well as second instar larvae from a laboratory strain devoid of symbionts showed significant changes in transcript expression. This highlighted genes associated with the developmental changes revealed by the microscopic analysis as well as responses to microorganisms.

Draft Genomes of Six Philippine Erwinia mallotivora Isolates: Comparative Genomics and Genome-Wide Analysis of Candidate Secreted Proteins.

Erwinia mallotivora is one of the most important bacterial pathogens of papaya and causes bacterial crown rot disease in the Philippines. In this paper, we present the draft genome sequences of six Philippine E. mallotivora isolates to provide insights into the genes involved in host-pathogen interactions and compare their genomes to other Erwinia species. The genomes were sequenced using Illumina Miseq platform. The draft whole-genome assemblies of the E. mallotivora isolates are composed of 36-64 contigs with N50 value ranging from 285 to 332 kbp and cover 96.2-100% of the estimated genome size. Structural genome annotation of these assemblies has predicted 4489-4749 protein-coding genes. Comparative genomic analysis using orthologous gene sets led to the identification of conserved genes within the genus and species-specific gene orthologous groups, which collectively provide a baseline for functional genomic studies to determine genes affecting virulence and host specificity. Secreted proteins of E. mallotivora were also predicted and characterized to unravel putative genes involved in plant-pathogen interactions. This study provides the first draft whole-genome sequences of Philippine isolates of E. mallotivora, thus expanding the genomic knowledge for this species in comparison with other members of the genus Erwinia.

Tenebrionicola larvae gen. nov., sp. nov., isolated from larvae of mealworm Tenebrio molitor L., and a proposal to transfer Erwinia teleogrylli Liu et al. 2016 to a new genus Entomohabitans as Entomohabitans teleogrylli comb. nov.

Two enterobacterial strains, designated YMB-R21T and YMB-R22, were isolated from larvae of mealworm Tenebrio molitor L. and examined for their taxonomic characteristics. A 16S rRNA gene-based neighbour-joining tree showed that the two isolates formed two distinct sublineages within the family Enterobacteriaceae and were separated from other genera of the family. The isolates showed 16S rRNA gene sequence similarity of 98.9 % to each other and ≤96.5 % to members of the order Enterobacteriales. The phylogenomic analysis based on 92 singly-copy core genes showed that the two isolates belonged to the family Enterobacteriaceae and formed a distinct sublineage at a position located remotely from the genera of the family. The loosely associated members were the type strain of Erwinia teleogrylli and members of the genus Shimwellia. Average nucleotide identity and digital DNA-DNA hybridization values showed that the isolates represented members of a novel species in the family Enterobacteriaceae. The values of amino acid identity between the two isolates and the closest relatives were 74.5-75.0 % with the type strain of E. teleogrylli and 74.5-74.8 % with the type strains of two Shimwellia species, while E. teleogrylli showed the amino acid identity values of 76.3-76.5 % with two Shimwellia species. Based on the results obtained here, we propose a new genus Tenebrionicola with the description of Tenebrionicola larvae sp. nov. (type strain YMB-R21T=KCTC 82597T=CCM 9152T and strain YMB-R22=KCTC 82598=CCM 9153), with the transfer of Erwinia teleogrylli Liu et al. 2016 to a new genus Entomohabitans as Entomohabitans teleogrylli comb. nov. (type strain SCU-B244T=CGMCC 1.12772T=DSM 28222T=KCTC 42022T).

Expression and Functional Analysis of the Type III Secretion System Effector Repertoire of the Xylem Pathogen Erwinia tracheiphila on Cucurbits.

The predicted repertoire of type III secretion system effectors (T3SEs) in Erwinia tracheiphila, causal agent of cucurbit bacterial wilt, is much larger than in xylem pathogens in the closely related genera Erwinia and Pantoea. The genomes of strains BHKY and SCR3, which represent distinct E. tracheiphila clades, encode at least 6 clade-specific and 12 shared T3SEs. The strains expressed the majority of the T3SE genes examined in planta. Among the shared T3SE genes, eop1 was expressed most highly in both strains in squash (Cucurbita pepo) and muskmelon (Cucumis melo) but the clade-specific gene avrRpm2 was expressed 40- to 900-fold more than eop1 in BHKY. The T3SEs AvrRpm2, Eop1, SrfC, and DspE contributed to BHKY virulence on squash and muskmelon, as shown using combinatorial mutants involving six T3SEs, whereas OspG and AvrB4 contributed to BHKY virulence only on muskmelon, demonstrating host-specific virulence functions. Moreover, Eop1 was functionally redundant with AvrRpm2, SrfC, OspG, and AvrB4 in BHKY, and BHKY mutants lacking up to five effector genes showed similar virulence to mutants lacking only two genes. The T3SEs OspG, AvrB4, and DspE contributed additively to SCR3 virulence on muskmelon and were not functionally redundant with Eop1. Rather, loss of eop1 and avrB4 restored wild-type virulence to the avrB4 mutant, suggesting that Eop1 suppresses a functionally redundant effector in SCR3. These results highlight functional differences in effector inventories between two E. tracheiphila clades, provide the first evidence of OspG as a phytopathogen effector, and suggest that Eop1 may be a metaeffector influencing virulence. [Formula: see text] Copyright © 2022 The Author(s). This is an open access article distributed under the CC BY 4.0 International license.

Phylogenomic analysis of the Erwiniaceae supports reclassification of Kalamiella piersonii to Pantoea piersonii comb. nov. and Erwinia gerundensis to the new genus Duffyella gen. nov. as Duffyella gerundensis comb. nov.

To better understand the taxonomy of Erwinia in the context of the Erwiniaceae family, we carried out a taxogenomic analysis of the Erwiniaceae, a family that was created following the taxonomic revision of the family, Enterobacteriaceae. There has been no systematic analysis of this family, including the agriculturally relevant genus, Erwinia. Our analyses focused on 80 strains of Erwinia along with 37 strains representing 7 other genera in the family. We identified 308 common proteins, generated a genome-level phylogeny and carried out Average Nucleotide Identity, Average Amino Acid Identity and Percentage of Conserved Protein analyses. We show that multiple strains of Erwinia cannot be assigned to established species groups and that both Erwinia gerundensis and "Erwinia mediterraneensis" are not members of Erwinia. We propose the creation of the genus Duffyella gen. nov. and the reclassification of Erwinia gerundensis to this genus as the type species, Duffyella gerundensis comb. nov. Furthermore, divergence between other species within Erwinia as measured by Average Amino Acid Identity is greater than the divergence between Erwinia and other genera, supporting the possible subdivision of the genus Erwinia into at least two genera. Our analyses also suggest that there is no basis for the establishment of the genus Kalamiella within the Erwiniaceae or the taxonomic revision of the Pantoea septica lineage. Therefore, we propose reclassifying Kalamiella piersonii as Pantoea piersonii comb. nov. Our study provides new insight into the diversity of the Erwiniaceae and provides a solid foundation for advancing taxonomic revision of this broadly relevant family.

Comparative genome analysis reveals the presence of multiple quorum-sensing systems in plant pathogenic bacterium, Erwinia rhapontici.

We present the complete genome sequences of 3 Erwinia rhapontici strains, MAFF 311153, 311154, and 311155. These chromosome sequences contained variety types of luxI/luxR gene pair involved in acylhomoserine lactone biosynthesis and reception. Large-scale insertion sequence was observed in the indigenous plasmid of MAFF 311154 and contained eraI3/eraR3 gene pair that make possible to produce acylhomoserine lactone.

Characterization of Erwinia tracheiphila Bacteriophage FBB1 Isolated from Spotted Cucumber Beetles that Vector E. tracheiphila.

Erwinia tracheiphila, the causal pathogen of bacterial wilt of cucurbit crops, is disseminated by cucumber beetles. A bacteriophage, designated FBB1 (Fu-Beattie-Beetle-1), was isolated from spotted cucumber beetles (Diabrotica undecimpunctata) that were collected from a field in which E. tracheiphila is endemic. FBB1 was classified into the Myoviridae family based on its morphology, which includes an elongated icosahedral head (106 × 82 nm) and a putatively contractile tail (120 nm). FBB1 infected all 62 E. tracheiphila strains examined and three Pantoea spp. strains. FBB1 virions were stable at 55°C for 1 h and tolerated a pH range from 3 to 12. FBB1 has a genome of 175,994 bp with 316 predicted coding sequences and a GC content of 36.5%. The genome contains genes for a major bacterial outer-membrane protein, a putative exopolysaccharide depolymerase, and 22 predicted transfer RNAs. The morphology and genome indicate that FBB1 is a T4-like virus and thus in the Tevenvirinae subfamily. FBB1 is the first virulent phage of E. tracheiphila to be reported and, to date, is one of only two bacteriophages to be isolated from insect vectors of phytopathogens. Collectively, the results support FBB1 as a promising candidate for biocontrol of E. tracheiphila based on its virulent (lytic) rather than lysogenic lifestyle, its infection of all E. tracheiphila strains examined to date, and its infection of a few nonpathogenic bacteria that could be used to support phage populations when pathogen numbers are low.

Characterization of a recombinant sucrose isomerase and its application to enzymatic production of isomaltulose.

OBJECTIVE: To characterize a recombinant isomerase that can catalyze the isomerization of sucrose into isomaltulose and investigate its application for the enzymatic production of isomaltulose. RESULTS: A sucrose isomerase gene from Erwinia sp. Ejp617 was synthesized and expressed in Escherichia coli BL21(DE3). The enzymatic characterization revealed that the optimal pH and temperature of the purified sucrose isomerase were 6.0 and 40 °C, respectively. The enzyme activity was slightly activated by Mn2+and Mg2+, but partially inhibited by Ca2+, Ba2+, Cu2+, Zn2+ and EDTA. The kinetic parameters of Km and Vmax for sucrose were 69.28 mM and 118.87 U/mg, respectively. The time course showed that 240.9 g/L of isomaltulose was produced from 300 g/L of sucrose, and the yield reached 80.3% after bioreaction for 180 min. CONCLUSIONS: This recombinant enzyme showed excellent capability for biotransforming sucrose to isomaltulose at the substrate concentration of 300 g/L. Further investigations should be carried out focusing on selection of suitable heterologous expression system with the aim to improve its expression level.

Biological Control and Microbial Ecology Draft Genome Sequence Data of Glutamicibacter sp. FBE-19, a Bacterium Antagonistic to the Plant Pathogen Erwinia tracheiphila.

Glutamicibacter sp. FBE-19 was isolated based on its strong antagonism to the cucurbit bacterial blight pathogen Erwinia tracheiphila on plates. Members of the Glutamicibacter genus can promote plant growth under saline conditions and antagonize fungi on plates via chitinolytic activity; however, their production of antibacterial compounds has not been examined. Here, we report the genome sequence of strain FBE-19. The genome is 3.85 Mbp with a G+C content of 60.1% and comprises 3,791 genes. Genes that may contribute to its antagonistic activity include genes for the secondary metabolites stenothricin, salinosporamide A, a second ß-lactone compound, and a carotenoid. The Glutamicibacter sp. FBE-19 genome data may be a useful resource if this strain proves to be an effective biocontrol agent against E. tracheiphila.

A horizontally acquired expansin gene increases virulence of the emerging plant pathogen Erwinia tracheiphila.

Erwinia tracheiphila is a bacterial plant pathogen that causes a fatal wilt infection in some cucurbit crop plants. Wilt symptoms are thought to be caused by systemic bacterial colonization through xylem that impedes sap flow. However, the genetic determinants of within-plant movement are unknown for this pathogen species. Here, we find that E. tracheiphila has horizontally acquired an operon with a microbial expansin (exlx) gene adjacent to a glycoside hydrolase family 5 (gh5) gene. Plant inoculation experiments with deletion mutants in the individual genes (Δexlx and Δgh5) and the full operon (Δexlx-gh5) resulted in decreased severity of wilt symptoms, decreased mortality rate, and impaired systemic colonization compared to the Wt strain. Co-inoculation experiments with Wt and Δexlx-gh5 rescued the movement defect of the mutant strain, suggesting that expansin and GH5 function extracellularly. Together, these results show that expansin-GH5 contributes to systemic movement through xylem, leading to rapid wilt symptom development and higher rates of plant death. The presence of expansin genes in diverse species of bacterial and fungal wilt-inducing pathogens suggests that microbial expansin proteins may be an under-appreciated virulence factor for many pathogen species.

Modulation of the Erwinia ligand-gated ion channel (ELIC) and the 5-HT3 receptor via a common vestibule site.

Pentameric ligand-gated ion channels (pLGICs) or Cys-loop receptors are involved in fast synaptic signaling in the nervous system. Allosteric modulators bind to sites that are remote from the neurotransmitter binding site, but modify coupling of ligand binding to channel opening. In this study, we developed nanobodies (single domain antibodies), which are functionally active as allosteric modulators, and solved co-crystal structures of the prokaryote (Erwinia) channel ELIC bound either to a positive or a negative allosteric modulator. The allosteric nanobody binding sites partially overlap with those of small molecule modulators, including a vestibule binding site that is not accessible in some pLGICs. Using mutagenesis, we extrapolate the functional importance of the vestibule binding site to the human 5-HT3 receptor, suggesting a common mechanism of modulation in this protein and ELIC. Thus we identify key elements of allosteric binding sites, and extend drug design possibilities in pLGICs with an accessible vestibule site.

Economical production of isomaltulose from agricultural residues in a system with sucrose isomerase displayed on Bacillus subtilis spores.

A safe, efficient, environmentally friendly process for producing isomaltulose is needed. Here, the biocatalyst, sucrose isomerase (SIase) from Erwinia rhapontici NX-5, displayed on the surface of Bacillus subtilis 168 spores (food-grade strain) was applied for isomaltulose production. The anchored SIase showed relatively high bioactivity, suggesting that the surface display system using CotX as the anchoring protein was successful. The stability of the anchored SIase was also significantly better. Thermal stability analysis showed that 80% of relative activity was retained after incubation at 40 °C and 45 °C for 60 min. To develop an economical industrial fermentation medium, untreated beet molasses (30 g/L) and cold-pressed soybean powder (50 g/L) were utilised as the main broth components for SIase pilot-scale production. Under the optimal conditions, the productive spores converted 92% of sucrose after 6 h and the conversion rate was 45% after six cycles. Isomaltulose production with this system using the agricultural residues, untreated beet molasses and soybean powder, as substrates is cost-effective and environmentally friendly and can help to overcome issues due to the genetic background.

Serial horizontal transfer of vitamin-biosynthetic genes enables the establishment of new nutritional symbionts in aphids' di-symbiotic systems.

Many insects depend on obligate mutualistic bacteria to provide essential nutrients lacking from their diet. Most aphids, whose diet consists of phloem, rely on the bacterial endosymbiont Buchnera aphidicola to supply essential amino acids and B vitamins. However, in some aphid species, provision of these nutrients is partitioned between Buchnera and a younger bacterial partner, whose identity varies across aphid lineages. Little is known about the origin and the evolutionary stability of these di-symbiotic systems. It is also unclear whether the novel symbionts merely compensate for losses in Buchnera or carry new nutritional functions. Using whole-genome endosymbiont sequences of nine Cinara aphids that harbour an Erwinia-related symbiont to complement Buchnera, we show that the Erwinia association arose from a single event of symbiont lifestyle shift, from a free-living to an obligate intracellular one. This event resulted in drastic genome reduction, long-term genome stasis, and co-divergence with aphids. Fluorescence in situ hybridisation reveals that Erwinia inhabits its own bacteriocytes near Buchnera's. Altogether these results depict a scenario for the establishment of Erwinia as an obligate symbiont that mirrors Buchnera's. Additionally, we found that the Erwinia vitamin-biosynthetic genes not only compensate for Buchnera's deficiencies, but also provide a new nutritional function; whose genes have been horizontally acquired from a Sodalis-related bacterium. A subset of these genes have been subsequently transferred to a new Hamiltonella co-obligate symbiont in one specific Cinara lineage. These results show that the establishment and dynamics of multi-partner endosymbioses can be mediated by lateral gene transfers between co-ocurring symbionts.