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Plant Disease Mar 2023(satin pothos or silver vine) is an evergreen climbing plant belonging to the Araceae family, subfamily Monstereae (Bown, 2000), which is also cultivated as a foliage...
(satin pothos or silver vine) is an evergreen climbing plant belonging to the Araceae family, subfamily Monstereae (Bown, 2000), which is also cultivated as a foliage ornamental (Masnira et al. 2019). In September of 2022, soft rot symptoms were observed on potted . plants grown in a greenhouse in Nantun District, Taichung, Taiwan, in which soft rot of another aroid (philodendron) has also been reported (Wu et al. 2023). The symptoms appeared on the petioles and most of them tended to extend to the leaf blades; the colors of leaf lesions ranged from dark brown to gray (Fig. S1). Some 70% of the plants in the greenhouse showed similar symptoms and losses were estimated to be 15-30%. Four symptomatic plants were sampled. Macerated tissues from rotting petioles were soaked in 10 mM MgCl and observed under a light microscope (Nikon, Japan) at 400 x magnification. Motile, rod-shaped bacteria were observed, and 1-2 loopfuls of undiluted sample suspension were streaked onto nutrient agar (NA; Gibco, USA). After culturing at 28°C for 1 day, all samples yielded round, creamy-white colonies (0.9 mm in diameter) and from each of the four samples a pure culture was obtained (Spi1-Spi4). All isolates exhibited oxidative and fermentative metabolism of glucose (Schaad et al. 2001). They caused pitting on crystal violet pectate agar, induced maceration on potato tuber and were tested positive for phosphatase activity and indigoidine production (Lee and Yu 2006; Schaad et al. 2001). Polymerase chain reaction tests using -specific primers 5A and 5B (Chao et al. 2006) amplified the expected amplicon (0.5 kb) in extracted DNA samples of all isolates. Identification of the strains was achieved by amplifying and sequencing fragments of the housekeeping genes , , , , and (Marrero et al. 2013); the lengths of the five gene fragments analyzed were 822, 762, 720, 672, and 450 bp, respectively (accession nos. OP985528-OP985532). The five sequences were concatenated for every isolate; the resulting 3,426 bp sequences were aligned with ClustalW and found to be identical. A maximum-likelihood analysis was conducted using the 3,426-bp sequences and those of known species' type strains. Spi1 to Spi4 clustered with . subsp. NCPPB 2976 and . subsp. CFBP 1269 (Fig. S2) with sequence identities of 98.4 and 98%, respectively. To fulfil Koch's Postulates, stab inoculations of the four isolates into the petioles of cutting propagated, 38-day-old . plants (3 plants per isolate) were conducted using sterile toothpicks. The amounts of bacteria used was approximately 10 cfu per toothpick; the bacterial loads were estimated by suspending the cells in 10 mM MgCl and spread-plating diluted suspensions on NA. Sterile toothpicks were used as control. All tested plants were sealed in plastic bags (containing wet paper towel) and kept in a growth chamber (28°C; 12-h photoperiod). After 1 day, all isolates induced soft rot symptoms resembling those observed under natural conditions in the greenhouse. Bacteria were re-isolated, and they all shared the same sequence with strains Spi1 to Spi4. This is the first report of . affected by . in Taiwan. Further investigation is needed to determine whether Spi1-Spi4 belong to . subsp. . has been found infecting different aroids (Lee and Chen 2021; Lin et al. 2012). The species has also been reported in Taiwan on poinsettia (Wei et al., 2019) and philodendron (Wu et al. 2023). Because these plants are often grown closely in the same facilities, growers should be wary of . 's spread among these plants. Reduction of environmental humidity and avoiding overhead irrigation may be effective in preventing the pathogen's transmission.
PubMed: 36947837
DOI: 10.1094/PDIS-12-22-2885-PDN -
Metallomics : Integrated Biometal... Mar 2023Bacteria secrete siderophores whose function is to acquire iron. In recent years, the siderophores of several Chryseobacterium species were shown to promote the health...
Bacteria secrete siderophores whose function is to acquire iron. In recent years, the siderophores of several Chryseobacterium species were shown to promote the health and growth of various plants such as tomato or rice. However, the chemical nature of Chryseobacterium siderophores remained unexplored despite great interest. In this work, we present the purification and structure elucidation by nuclear magnetic resonance (NMR) spectroscopy and tandem mass spectrometry (MS/MS) of chryseochelin A, a novel citrate-based siderophore secreted by three Chryseobacterium strains involved in plant protection. It contains the unusual building blocks 3-hydroxycadaverine and fumaric acid. Furthermore, the unstable structural isomer chryseochelin B and its stable derivative containing fatty acid chains, named chryseochelin C, were identified by mass spectrometric methods. The latter two incorporate an unusual ester connectivity to the citrate moiety showing similarities to achromobactin from the plant pathogen Dickeya dadantii. Finally, we show that chryseochelin A acts in a concentration-dependent manner against the plant-pathogenic Ralstonia solanacearum strain by reducing its access to iron. Thus, our study provides valuable knowledge about the siderophores of Chryseobacterium strains, which have great potential in various applications.
Topics: Siderophores; Citric Acid; Chryseobacterium; Tandem Mass Spectrometry; Iron; Citrates
PubMed: 36792066
DOI: 10.1093/mtomcs/mfad008 -
The Journal of Biological Chemistry Dec 2022Succination is the spontaneous reaction between the respiratory intermediate fumarate and cellular thiols that forms stable S-(2-succino)-adducts such as...
Succination is the spontaneous reaction between the respiratory intermediate fumarate and cellular thiols that forms stable S-(2-succino)-adducts such as S-(2-succino)cysteine (2SC). 2SC is a biomarker for conditions associated with elevated fumarate levels, including diabetes, obesity, and certain cancers, and succination likely contributes to disease progression. Bacillus subtilis has a yxe operon-encoded breakdown pathway for 2SC that involves three distinct enzymatic conversions. The first step is N-acetylation of 2SC by YxeL to form N-acetyl-2SC (2SNAC). YxeK catalyzes the oxygenation of 2SNAC, resulting in its breakdown to oxaloacetate and N-acetylcysteine, which is deacetylated by YxeP to give cysteine. The monooxygenase YxeK is key to the pathway but is rare, with close homologs occurring infrequently in prokaryote and fungal genomes. The existence of additional 2SC breakdown pathways was not known prior to this study. Here, we used comparative genomics to identify a S-(2-succino) lyase (2SL) that replaces yxeK in some yxe gene clusters. 2SL genes from Enterococcus italicus and Dickeya dadantii complement B. subtilis yxeK mutants. We also determined that recombinant 2SL enzymes efficiently break down 2SNAC into fumarate and N-acetylcysteine, can perform the reverse reaction, and have minor activity against 2SC and other small molecule thiols. The strong preferences both YxeK and 2SL enzymes have for 2SNAC indicate that 2SC acetylation is a conserved breakdown step. The identification of a second naturally occurring 2SC breakdown pathway underscores the importance of 2SC catabolism and defines a general strategy for 2SC breakdown involving acetylation, breakdown, and deacetylation.
Topics: Cysteine; Lyases; Acetylcysteine; Sulfhydryl Compounds; Fumarates
PubMed: 36309089
DOI: 10.1016/j.jbc.2022.102639 -
Mobile DNA Aug 2022ICEs are mobile genetic elements found integrated into bacterial chromosomes that can excise and be transferred to a new cell. They play an important role in horizontal...
BACKGROUND
ICEs are mobile genetic elements found integrated into bacterial chromosomes that can excise and be transferred to a new cell. They play an important role in horizontal gene transmission and carry accessory genes that may provide interesting phenotypes for the bacteria. Here, we seek to research the presence and the role of ICEs in 300 genomes of phytopathogenic bacteria with the greatest scientific and economic impact.
RESULTS
Seventy-eight ICEs (45 distinct elements) were identified and characterized in chromosomes of Agrobacterium tumefaciens, Dickeya dadantii, and D. solani, Pectobacterium carotovorum and P. atrosepticum, Pseudomonas syringae, Ralstonia solanacearum Species Complex, and Xanthomonas campestris. Intriguingly, the co-occurrence of four ICEs was observed in some P. syringae strains. Moreover, we identified 31 novel elements, carrying 396 accessory genes with potential influence on virulence and fitness, such as genes coding for functions related to T3SS, cell wall degradation and resistance to heavy metals. We also present the analysis of previously reported data on the expression of cargo genes related to the virulence of P. atrosepticum ICEs, which evidences the role of these genes in the infection process of tobacco plants.
CONCLUSIONS
Altogether, this paper has highlighted the potential of ICEs to affect the pathogenicity and lifestyle of these phytopathogens and direct the spread of significant putative virulence genes in phytopathogenic bacteria.
PubMed: 35962419
DOI: 10.1186/s13100-022-00275-1 -
Nucleic Acids Research Sep 2022DNA supercoiling is an essential mechanism of bacterial chromosome compaction, whose level is mainly regulated by topoisomerase I and DNA gyrase. Inhibiting either of...
DNA supercoiling is an essential mechanism of bacterial chromosome compaction, whose level is mainly regulated by topoisomerase I and DNA gyrase. Inhibiting either of these enzymes with antibiotics leads to global supercoiling modifications and subsequent changes in global gene expression. In previous studies, genes responding to DNA relaxation induced by DNA gyrase inhibition were categorised as 'supercoiling-sensitive'. Here, we studied the opposite variation of DNA supercoiling in the phytopathogen Dickeya dadantii using the non-marketed antibiotic seconeolitsine. We showed that the drug is active against topoisomerase I from this species, and analysed the first transcriptomic response of a Gram-negative bacterium to topoisomerase I inhibition. We find that the responding genes essentially differ from those observed after DNA relaxation, and further depend on the growth phase. We characterised these genes at the functional level, and also detected distinct patterns in terms of expression level, spatial and orientational organisation along the chromosome. Altogether, these results highlight that the supercoiling-sensitivity is a complex feature, which depends on the action of specific topoisomerases, on the physiological conditions, and on their genomic context. Based on previous in vitro expression data of several promoters, we propose a qualitative model of SC-dependent regulation that accounts for many of the contrasting transcriptomic features observed after DNA gyrase or topoisomerase I inhibition.
Topics: DNA Gyrase; DNA Topoisomerases, Type I; DNA, Superhelical; DNA, Bacterial; Enterobacteriaceae; Anti-Bacterial Agents
PubMed: 35950487
DOI: 10.1093/nar/gkac679 -
Polymers Jul 2022In order to obtain a thermostable pectate lyase for ramie degumming, a rational design based on structural analysis was carried out on a novel pectate lyase (Pel419)...
In order to obtain a thermostable pectate lyase for ramie degumming, a rational design based on structural analysis was carried out on a novel pectate lyase (Pel419) derived from the DCE-01 for high-efficiency ramie degumming. A total of five potential amino acid sites were chosen to replace residues. Then, the mutant enzymes were subjected to the heterologous expressions in and their enzymatic characteristics were determined. The optimal reaction temperature for the five mutants kept consistent with that for the wild type. The enzyme activity and thermal stability of mutant V52A were significantly improved. Meanwhile, the weight loss rate obtained by V52A with the best enzymatic characteristics in the ramie degumming process at 50 °C is comparable with that obtained by commercial cotton-ramie processing pectinases, indicating that V52A was a potential industrial enzyme that could be applied to large-scale ramie degumming. In this study, the biological functions of conservative residues of Pel419 were preliminarily explored. The mutant V52A with both enzymatic activity and improved heat resistance was acquired, providing a superior material for developing enzyme preparations of ramie degumming, and rendering an effective method for the rational design aiming to improve the thermostability of pectate lyase.
PubMed: 35890653
DOI: 10.3390/polym14142878 -
Plant Disease Jun 2022Philodendrons are important foliage ornamentals planted worldwide (Chen et al. 2010). In November 2021, soft rot symptoms were observed on (now known as ; Sakuragui et...
Philodendrons are important foliage ornamentals planted worldwide (Chen et al. 2010). In November 2021, soft rot symptoms were observed on (now known as ; Sakuragui et al. 2018) grown in a nursery in Taichung, Taiwan. On symptomatic plants, the petioles were macerated; leaf lesions were also found on some plants (Figure S1). About 60% of the plants on site were symptomatic; these plants tended to cluster together. Four plants were sampled. Infected tissues were soaked and cut into pieces in 10 mM MgCl (using scalpels); undiluted samples were streak-plated onto nutrient agar (NA) and grown for 24 h at 28°C. Translucent, creamy-white colonies were isolated from all of the tissues examined, and 4 isolates, PHIL1 to PHIL4, were obtained (each from a different plant). All isolates exhibited typical phenotypes of bacteria belonging to ; they could cause maceration symptoms on potato slices, ferment glucose and produce phosphatase (Schaad et al. 2001); they could also produce indigoidine on NGM medium (NA added with glycerol and MnCl; Lee and Yu. 2006). Polymerase chain reactions using -specific primers 5A and 5B (Chao et al. 2006) amplified the expected amplicon in all 4 isolates. The 16S rDNA of PHIL1 to PHIL4 were amplified using primer pair 27f/1492r (Lane 1991) and the amplicons were sequenced; all 4 isolates shared the same 1,395-bp sequence (accession nos. ON203122, ON479664-ON479666). Among the strains belonging to known species (in GenBank), PHIL1 to PHIL4 shared the highest sequence identity (99.93%) with . 3937; they also shared 98.78% sequence identity with . CFBP 1269. Multilocus sequence analysis (MLSA) targeting fragments of PHIL1 to PHIL4's A (720 bp), J (672 bp), X (450 bp), B (822 bp), and N (762 bp) genes (Marrero et al. 2013) were conducted. The five-gene concatenated sequences (3,426 bp) of the 4 isolates (accession nos. ON227444-ON227448, ON494509-ON494523) were identical. A maximum-likelihood phylogenetic analysis including these sequences and those of type strains of other known species revealed that PHIL1 to PHIL4 clustered with strains belonging to . (Figure S2). Koch's postulates were fulfilled with an inoculation test conducted on . (17 cm in aboveground height; 7-months-old). Stab inoculation using sterile toothpicks was conducted on petioles. Three plants were tested for each isolate and 2 petioles were inoculated for each plant; all 4 isolates were included in the assay. The pathogen loads inoculated were quantified by the spread plate method and were 3.22 - 4.81 x 10 colony forming units. Three plants were stabbed with bacteria-free toothpicks, serving as controls. All plants were bagged post inoculation and kept in a growth chamber (28°C; 14 h light). After 72 h, all of the inoculated petioles exhibited symptoms resembling those observed in the nursery. Bacteria were re-isolated from the symptomatic tissues (one isolate from each treatment), and all of their five-gene concatenated sequences were the same as those of PHIL1 to PHIL4. This is the first formal report of the occurrence of . infecting . in Taiwan. Studies have shown that . could affect other Araceae plants in Taiwan (Lee and Chen 2021). Since different Araceae ornamentals are often planted together in gardens and nurseries, growers should be aware of potential transmission of . among them.
PubMed: 35771110
DOI: 10.1094/PDIS-04-22-0924-PDN -
Nature Communications Jun 2022CRISPR SWAPnDROP extends the limits of genome editing to large-scale in-vivo DNA transfer between bacterial species. Its modular platform approach facilitates species...
CRISPR SWAPnDROP extends the limits of genome editing to large-scale in-vivo DNA transfer between bacterial species. Its modular platform approach facilitates species specific adaptation to confer genome editing in various species. In this study, we show the implementation of the CRISPR SWAPnDROP concept for the model organism Escherichia coli, the fast growing Vibrio natriegens and the plant pathogen Dickeya dadantii. We demonstrate the excision, transfer and integration of large chromosomal regions between E. coli, V. natriegens and D. dadantii without size-limiting intermediate DNA extraction. CRISPR SWAPnDROP also provides common genome editing approaches comprising scarless, marker-free, iterative and parallel insertions and deletions. The modular character facilitates DNA library applications, and recycling of standardized parts. Its multi-color scarless co-selection system significantly improves editing efficiency and provides visual quality controls throughout the assembly and editing process.
Topics: CRISPR-Cas Systems; DNA; Escherichia coli; Gene Editing; Genetic Therapy; Genome, Bacterial
PubMed: 35701417
DOI: 10.1038/s41467-022-30843-1 -
MBio Jun 2022The phytopathogenic proteobacterium Dickeya dadantii secretes an array of plant cell wall-degrading enzymes and other virulence factors via the type 2 secretion system...
Scaffolding Protein GspB/OutB Facilitates Assembly of the Dickeya dadantii Type 2 Secretion System by Anchoring the Outer Membrane Secretin Pore to the Inner Membrane and to the Peptidoglycan Cell Wall.
The phytopathogenic proteobacterium Dickeya dadantii secretes an array of plant cell wall-degrading enzymes and other virulence factors via the type 2 secretion system (T2SS). T2SSs are widespread among important plant, animal, and human bacterial pathogens. This multiprotein complex spans the double membrane cell envelope and secretes fully folded proteins through a large outer membrane pore formed by 15 subunits of the secretin GspD. Secretins are also found in the type 3 secretion system and the type 4 pili. Usually, specialized lipoproteins termed pilotins assist the targeting and assembly of secretins into the outer membrane. Here, we show that in , the pilotin acts in concert with the scaffolding protein GspB. Deletion of profoundly impacts secretin assembly, pectinase secretion, and virulence. Structural studies reveal that GspB possesses a conserved periplasmic homology region domain that interacts directly with the N-terminal secretin domain. Site-specific photo-cross-linking unravels molecular details of the GspB-GspD complex . We show that GspB facilitates outer membrane targeting and assembly of the secretin pores and anchors them to the inner membrane while the C-terminal extension of GspB provides a scaffold for the secretin channel in the peptidoglycan cell wall. Phylogenetic analysis shows that in other bacteria, GspB homologs vary in length and domain composition and act in concert with either a cognate ATPase GspA or the pilotin GspS. Gram-negative bacteria have two cell membranes sandwiching a peptidoglycan net that together form a robust protective cell envelope. To translocate effector proteins across this multilayer envelope, bacteria have evolved several specialized secretion systems. In the type 2 secretion system and some other bacterial machineries, secretins form large multimeric pores that allow transport of effector proteins or filaments across the outer membrane. The secretins are essential for nutrient acquisition and pathogenicity and constitute a target for development of new antibacterials. Targeting of secretin subunits into the outer membrane is often facilitated by a special class of lipoproteins called pilotins. Here, we show that in and some other bacteria, the scaffolding protein GspB acts in concert with pilotin, facilitating the assembly of the secretin pore and its anchoring to both the inner membrane and the bacterial cell wall. GspB homologs of varied domain composition are present in many other T2SSs.
Topics: Bacterial Outer Membrane Proteins; Bacterial Proteins; Cell Wall; Dickeya; Enterobacteriaceae; Lipoproteins; Peptidoglycan; Phylogeny; Secretin; Type II Secretion Systems
PubMed: 35546537
DOI: 10.1128/mbio.00253-22 -
MBio Jun 2022Dickeya dadantii is a phytopathogenic bacterium that causes soft rot in a wide range of plant hosts worldwide and a model organism for studying virulence gene...
Dickeya dadantii is a phytopathogenic bacterium that causes soft rot in a wide range of plant hosts worldwide and a model organism for studying virulence gene regulation. The present study provides a comprehensive and annotated transcriptomic map of obtained by a computational method combining five independent transcriptomic data sets: (i) paired-end RNA sequencing (RNA-seq) data for a precise reconstruction of the RNA landscape; (ii) DNA microarray data providing transcriptional responses to a broad variety of environmental conditions; (iii) long-read Nanopore native RNA-seq data for isoform-level transcriptome validation and determination of transcription termination sites; (iv) differential RNA sequencing (dRNA-seq) data for the precise mapping of transcription start sites; (v) DNA microarray data for a comparison of gene expression profiles between experiments and the early stages of plant infection. Our results show that transcription units sometimes coincide with predicted operons but are generally longer, most of them comprising internal promoters and terminators that generate alternative transcripts of variable gene composition. We characterize the occurrence of transcriptional read-through at terminators, which might play a basal regulation role and explain the extent of transcription beyond the scale of operons. We finally highlight the presence of noncontiguous operons and excludons in the genome, novel genomic arrangements that might contribute to the basal coordination of transcription. The highlighted transcriptional organization may allow to finely adjust its gene expression program for a rapid adaptation to fast-changing environments. This is the first transcriptomic map of a species. It may therefore significantly contribute to further progress in the field of phytopathogenicity. It is also one of the first reported applications of long-read Nanopore native RNA-seq in prokaryotes. Our findings yield insights into basal rules of coordination of transcription that might be valid for other bacteria and may raise interest in the field of microbiology in general. In particular, we demonstrate that gene expression is coordinated at the scale of transcription units rather than operons, which are larger functional genomic units capable of generating transcripts with variable gene composition for a fine-tuning of gene expression in response to environmental changes. In line with recent studies, our findings indicate that the canonical operon model is insufficient to explain the complexity of bacterial transcriptomes.
Topics: Bacteria; Dickeya; Enterobacteriaceae; Gene Expression Regulation, Bacterial
PubMed: 35491820
DOI: 10.1128/mbio.00524-22