Characterization of the LysP2110-HolP2110 Lysis System in Ralstonia solanacearum Phage P2110.

Ralstonia solanacearum, a pathogen causing widespread bacterial wilt disease in numerous crops, currently lacks an optimal control agent. Given the limitations of traditional chemical control methods, including the risk of engendering drug-resistant strains and environmental harm, there is a dire need for sustainable alternatives. One alternative is lysin proteins that selectively lyse bacteria without contributing to resistance development. This work explored the biocontrol potential of the LysP2110-HolP2110 system of Ralstonia solanacearum phage P2110. Bioinformatics analyses pinpointed this system as the primary phage-mediated host cell lysis mechanism. Our data suggest that LysP2110, a member of the Muraidase superfamily, requires HolP2110 for efficient bacterial lysis, presumably via translocation across the bacterial membrane. LysP2110 also exhibits broad-spectrum antibacterial activity in the presence of the outer membrane permeabilizer EDTA. Additionally, we identified HolP2110 as a distinct holin structure unique to the Ralstonia phages, underscoring its crucial role in controlling bacterial lysis through its effect on bacterial ATP levels. These findings provide valuable insights into the function of the LysP2110-HolP2110 lysis system and establish LysP2110 as a promising antimicrobial agent for biocontrol applications. This study underpins the potential of these findings in developing effective and environment-friendly biocontrol strategies against bacterial wilt and other crop diseases.

The Xanthomonas citri Reverse Fitness Deficiency by Activating a Novel ß-Glucosidase Under Low Osmostress.

Bacteria can withstand various types of environmental osmostress. A sudden rise in osmostress affects bacterial cell growth that is countered by activating special genes. The change of osmostress is generally a slow process under the natural environment. However, the collective response of bacteria to low osmostress remains unknown. This study revealed that the deletion of phoP (ΔphoP) from X. citri significantly compromised the growth and virulence as compared to the wild-type strain. Interestingly, low osmostress reversed physiological deficiencies of X. citri phoP mutant related to bacterial growth and virulence. The results also provided biochemical and genetic evidence that the physiological deficiency of phoP mutant can be reversed by low osmostress induced ß-glucosidase (BglS) expression. Based on the data, this study proposes a novel regulatory mechanism of a novel ß-glucosidase activation in X. citri through low osmostress to reverse the fitness deficiency.

First report of bacterial leaf spot disease on Pueraria montana var. thomsonii caused by Robbsia andropogonis in China.

Pueraria montana var. thomsonii (Hereinafter referred to as Pmt) belongs to the Leguminosae and is widely distributed in China, Laos, Thailand, Myanmar, Bhutan and other Asian countries. The plant is called "Fenge" in China, and its root is widely used in medicine and food. In recent years, an unknown leaf spot disease of Pmt has occurred in Gaoming, Zhaoqing and Yunfu districts of Guangdong Province in China, where 1,600 hectares of Pmt plants were affected. The incidence rate of plants were more than 80% and led to 10-15% death of Pmt plants in Gaoming district. . In the early stage of the disease, radiating and water-soaking lesions appeared between the main veins and side veins of Pmt leaves. After the spread of the lesions, they formed brown and short strips with yellow haloes around them, which led to leaf shedding, plant death and decline of production. To isolate bacteria, diseased leaves were surface sterilized with 0.6% sodium hypochlorite solution for 30 s, followed by three consecutive rinses in distilled water. The leaves were aseptically macerated, and the macerate streaked on PDA medium. Whitish to dull white, mucoid, raised, round, and translucent colonies were obtained. All isolates were gram-negative and had a single, polar, sheathed flagellum. Sequences (approx. 1,458 bp each) of the 16S rRNA gene amplified from five isolates (FG2, FG3, FG9, FG12 and FG17) using primer pair 27F/1492R (Lane et al,1991) (GenBank Accession Nos. OL677034, OL677351, OL677352, OL677353 and OL677354 respectively) shared 99.93% sequence identity with that of Robbsia andropogonis (Synonyms: Burkholderia andropogonis) (Lopes-Santos et al,2017) type strain LMG2129 (NR104960.1). The specific 410-bp and 704-bp target fragments were also amplified from isolates using R. andropogonis-specific primers Pf/Pr (Bagsic et al,1995) and LJ23f/LJ24R (Duan et al,2009). The four housekeeping genes atpD, lepA, gyrB and rpoD were partially sequenced for FG9 isolates using primers atpD-F3/atpD-R3, lepA-F2/lepA-R, LJ23f/LJ24R and LJ25f/LJ26r (Duan et al,2009; Estrada-De et al,2013) respectively. Multilocus sequence analyses confirmed the isolates from Pmt as R. andropogonis. Physiological and biochemical tests revealed the isolates are negative for oxidase, arginine dihydrolase, saccharose and betaine, and positive for sorbitol, lactose and galactose (Gillis et al,1995; Lopes-Santos et al,2017). In addition, all isolates caused a hypersensitive reaction on leaves of Nicotiana benthamiana and were pathogenic to some crops, including maize (Zea mays), sorghum (Sorghum bicolor), carnation (Dianthus caryophilus), common bean (Phaseolus vulgaris), tomato. Five isolates (FG2, FG3, FG9, FG12 and FG17) pathogenicity were tested twice with a total of three replications per isolate. Two young leaves each of 3-month-old Pmt plants grow in greenhouse were sprayed a bacterial suspension at 108 CFU/ml, then covered the inoculated leaves individually with plastic bags for 24 h, and incubated at 100% relative humidity with 16 h of daylight at 30°C and 8 h of darkness at 22°C in a greenhouse. Radiating and water-soaked lesions with yellow haloes were observed between the main veins and side veins of Pmt leaves 5 days after inoculation and were similar to those caused by R. andropogonis in the field. Koch's postulates were fulfilled by reisolating bacteria from typical lesions on inoculated plants. And the reisolated bacteria were identical to the inoculated ones. To our knowledge, this is the first report of R. andropogonis on Pueraria montana var. thomsonii in China.

The GacA-GacS Type Two-Component System Modulates the Pathogenicity of Dickeya oryzae EC1 Mainly by Regulating the Production of Zeamines.

The GacS-GacA type two-component system (TCS) positively regulates pathogenicity-related phenotypes in many plant pathogens. In addition, Dickeya oryzae EC1, the causative agent of soft rot disease, produces antibiotic-like toxins called zeamines as one of the major virulence factors that inhibit the germination of rice seeds. The present study identified a GacS-GacA type TCS, named TzpS-TzpA, that positively controls the virulence of EC1, mainly by regulating production of the toxin zeamines. RNA-seq analysis of strain EC1 and its tzpA mutant showed that the TCS regulated a wide range of virulence genes, especially those encoding zeamines. Protein-protein interaction was detected between TzpS and TzpA through the bacterial two-hybrid system and pull-down assay. In trans expression of tzpA failed to rescue the defective phenotypes in both the ΔtzpS and ΔtzpSΔtzpA mutants. Furthermore, TzpA controls target gene expression by direct binding to DNA promoters that contain a Gac-box motif, including a regulatory RNA rsmB and the vfm quorum-sensing system regulator vfmE. These findings therefore suggested that the EC1 TzpS-TzpA TCS system mediates the pathogenicity of Dickeya oryzae EC1 mainly by regulating the production of zeamines.[Formula: see text] Copyright © 2022 The Author(s). This is an open access article distributed under the CC BY-NC-ND 4.0 International license.

RNA-seq analysis provides insights into cold stress responses of Xanthomonas citri pv. citri.

BACKGROUND: Xanthomonas citri pv. citri (Xcc) is a citrus canker causing Gram-negative bacteria. Currently, little is known about the biological and molecular responses of Xcc to low temperatures. RESULTS: Results depicted that low temperature significantly reduced growth and increased biofilm formation and unsaturated fatty acid (UFA) ratio in Xcc. At low temperature Xcc formed branching structured motility. Global transcriptome analysis revealed that low temperature modulates multiple signaling networks and essential cellular processes such as carbon, nitrogen and fatty acid metabolism in Xcc. Differential expression of genes associated with type IV pilus system and pathogenesis are important cellular adaptive responses of Xcc to cold stress. CONCLUSIONS: Study provides clear insights into biological characteristics and genome-wide transcriptional analysis based molecular mechanism of Xcc in response to low temperature.

Global Regulator PhoP is Necessary for Motility, Biofilm Formation, Exoenzyme Production and Virulence of Xanthomonas citri Subsp. citri on Citrus Plants.

Citrus canker caused by Xanthomonas citri subsp. citri is one of the most important bacterial diseases of citrus, impacting both plant growth and fruit quality. Identifying and elucidating the roles of genes associated with pathogenesis has aided our understanding of the molecular basis of citrus-bacteria interactions. However, the complex virulence mechanisms of X. citri subsp. citri are still not well understood. In this study, we characterized the role of PhoP in X. citri subsp. citri using a phoP deletion mutant, ΔphoP. Compared with wild-type strain XHG3, ΔphoP showed reduced motility, biofilm formation, as well as decreased production of cellulase, amylase, and polygalacturonase. In addition, the virulence of ΔphoP on citrus leaves was significantly decreased. To further understand the virulence mechanisms of X. citri subsp. citri, high-throughput RNA sequencing technology (RNA-Seq) was used to compare the transcriptomes of the wild-type and mutant strains. Analysis revealed 1017 differentially-expressed genes (DEGs), of which 614 were up-regulated and 403 were down-regulated in ΔphoP. Gene ontology functional enrichment and Kyoto Encyclopedia of Genes and Genomes pathway analyses suggested that the DEGs were enriched in flagellar assembly, two-component systems, histidine metabolism, bacterial chemotaxis, ABC transporters, and bacterial secretion systems. Our results showed that PhoP activates the expression of a large set of virulence genes, including 22 type III secretion system genes and 15 type III secretion system effector genes, as well as several genes involved in chemotaxis, and flagellar and histidine biosynthesis. Two-step reverse-transcription polymerase chain reaction analysis targeting 17 genes was used to validate the RNA-seq data, and confirmed that the expression of all 17 genes, except for that of virB1, decreased significantly. Our results suggest that PhoP interacts with a global signaling network to co-ordinate the expression of multiple virulence factors involved in modification and adaption to the host environment during infection.

Integration Host Factor Is Essential for Biofilm Formation, Extracellular Enzyme, Zeamine Production, and Virulence in Dickeya zeae.

Dickeya zeae is a globally important pathogenic bacterium that infects many crops, including rice, maize, potato, and banana. Bacterial foot rot of rice caused by D. zeae is one of the most important bacterial diseases of rice in China and some Southeast Asian countries. To investigate the functions of integration host factor (IHF) in D. zeae, we generated knockout mutants of ihfA and ihfB. Phenotypic assays showed that both the ΔihfA and ΔihfB strains had greatly reduced mobility, biofilm formation, extracellular protease, and pectinase activities, and toxin production compared with the wild-type strain. In addition, the mutants did not inhibit the germination of rice seeds, failed to cause soft rot in potatoes and a hypersensitive response in tobacco, and were avirulent in rice. Quantitative reverse-transcription polymerase chain reaction analysis demonstrated that IHF positively regulates the expression of zmsA, hrpN/Y, pelA/B/C, pehX, celZ, prtG, fliC, and DGC (diguanylate cyclase). Electrophoretic mobility shift assays further confirmed that IhfA binds to the promoter region of the DGC gene and may alter the levels of a second bacterial messenger, c-di-GMP, to regulate the pathogenicity or other physiological functions of D. zeae. In summary, IHF is an important integrated regulator of pathogenicity in D. zeae.

Pathotypes of Xanthomonas axonopodis pv. dieffenbachiae Isolated from Anthurium andraeanum in China.

Anthurium blight, caused by Xanthomonas axonopodis pv. dieffenbachiae (Xad), is one of the most serious diseases of Anthurium andraeanum. However, little is known about variations in virulence between Xad pathotypes. Here, we examined the virulence of 68 Xad strains isolated from 30 anthurium plants from five regions of China against five different anthurium cultivars. Seven bacterial pathotypes were identified based on disease index and incidence analyses following foliar spray or leaf-clip inoculation. The resulting disease susceptibility patterns for pathotypes I⁻VII were RRRSS, RRSRS, RSRSR, RRSSS, RSSRS, RSSSS, and SSSSS, respectively. Overall, 72% of tested strains belonged to pathotypes VI or VII and were highly virulent. A further 22.1% of strains showed medium-level virulence and were classed as pathotype III, IV, or V, while the remaining 5.9% of strains were pathotype I or II, showing low virulence. Further analysis revealed differences in the virulence of Xad strains from the same anthurium cultivar, with variation also observed in pathovars associated with the same cultivar from different areas. Our results reveal the diversity and complexity of the Xad population structure in China and suggest that investigation of Xad pathotypes provides useful information to guide the identification and use of resistant varieties of A. andraeanum.

A nonribosomal peptide synthase containing a stand-alone condensation domain is essential for phytotoxin zeamine biosynthesis.

Dickeya zeae is the causal agent of rice foot rot and maize stalk rot diseases, which could cause severe economic losses. The pathogen is known to produce two phytotoxins known as zeamine and zeamine II which are also potent antibiotics against both gram-positive and gram-negative bacteria pathogens. Zeamine II is a long-chain aminated polyketide and zeamine shares the same polyketide structure as zeamine II, with an extra valine derivative moiety conjugated to the primary amino group of zeamine II. In this study, we have identified a gene designated as zmsK encoding a putative nonribosomal peptide synthase (NRPS) by screening of the transposon mutants defective in zeamine production. Different from most known NRPS enzymes, which are commonly multidomain proteins, ZmsK contains only a condensation domain. High-performance liquid chromatography and mass spectrometry analyses showed that the ZmsK deletion mutant produced only zeamine II but not zeamine, suggesting that ZmsK catalyzes the amide bond formation by using zeamine II as a substrate to generate zeamine. We also present evidence that a partially conserved catalytic motif within the condensation domain is critical for zeamine production. Furthermore, we show that deletion of zmsK substantially decreased the total antimicrobial activity and virulence of D. zeae. Our findings provide a new insight into the biosynthesis pathway of zeamines and the virulence mechanisms of the bacterial pathogen D. zeae.

A novel multidomain polyketide synthase is essential for zeamine production and the virulence of Dickeya zeae.

Dickeya zeae is the causal agent of the rice foot rot disease, but its mechanism of infection remains largely unknown. In this study, we identified and characterized a novel gene designated as zmsA. The gene encodes a large protein of 2,346 amino acids in length, which consists of multidomains arranged in the order of N-terminus, ß-ketoacyl synthase, acyl transferase, acyl carrier protein, ß-ketoacyl reductase, dehydratase. This multidomain structure and sequence alignment analysis suggest that ZmsA is a member of the polyketide synthase family. Mutation of zmsA abolished antimicrobial activity and attenuated the virulence of D. zeae. To determine the relationship between antimicrobial activity and virulence, active compounds were purified from D. zeae EC1 and were structurally characterized. This led to identification of two polyamino compounds, i.e., zeamine and zeamine II, that were phytotoxins and potent antibiotics. These results have established the essential role of ZmsA in zeamine biosynthesis and presented a new insight on the molecular mechanisms of D. zeae pathogenicity.

[Genetic and molecular characters of toxin producing Erwinia chrysanthemi pv. zeae].

OBJECTIVE: Bacterial foot rot, caused by Erwinia chrysanthemi pv. zeae, is one of the most important diseases in rice. Genetic and molecular characters of toxin producting for Erwinia chrysanthemi pv. zeae were conducted in this paper. METHODS: A plasmid-deficient strain, Ech7-mul, was obtained by chemical mutation,and the relative specific molecular mark with toxin was screened from Random Amplified Polymorphic DNA (RAPD) by PCR. RESULTS: The wild strain Ech7 and the plasmid-deficient strain Ech7-mul could both produce toxin.We screened 260 random primers in PCR, and found that a specific fragment (2139bp) could be amplified with K10 primer from theminus-toxin strain Ech7-4 DNA, but could not from the wild strain Ech7 DNA. The amplified fragment DNA was cloned and sequenced, and specific primers were designed to amplify it. The 2139bp fragment DNA could be a specific molecular mark with 100% SCAR identity between wild strain and the toxin mutant strain. Sequence analysis showed that there were five open reading frame (ORF), two of them were NADH-flavin reductase and N-acetyltransferase,respectively. Another ORF, located in the end region of 2139bp fragment, had 66% and 46% homologies with permeases of the drug/metabolite transporter (DMT) from Pseudomonas aerginosa (ZP00136947) and Yersinia Pestis (ZP01177873). CONCLUSION: Toxin biosynthesis in E. chrysanthemi pv. zeae might be coded by chromosome, but not by the bacterial plasmid.The position of gene mutation in the mutant Ech7-4 might be at the 3' region of toxin-relation SCAR DNA fragment.

[Separation, characters and biological functions of Erwinia chrysanthemi pv. zeae toxin].

OBJECTIVE: The toxin produced by Erwinia chrysanthemi pv. zeae has not been reported so far. Toxin is one of the important pathogenic factors for plant pathogenic bacteria. The separation and purification of toxin are the key and basal work for toxin functional study. METHODS: We used several chromatography columns, chemical and biochemical methods for Erwinia chrysanthemi pv. zeae toxin separation and its characterization. RESULTS: We obtained a pure ingredient T3 of Erwinia chrysanthemi pv. zeae toxin . It was a yellow solid and dissolved in methanol, N-butyl alcohol(NBA), water and formic acid. It dissolved weakly in acetone but did not dissolve in trichloromethane and ethyl acetate. The results showed that T3 toxin ingredient was neither carbohydrate nor protein, and was sensitive to ultraviolet ray. Biological assays of the toxin showed that it could inhibit rice growth, cause rice seedlings to wilt and make tobacco cells necrosis. Toxin with high content could inhibit buds and roots of rice, corn, tomato and tobacco to grow, whereas toxin with low content could promote their growth. In addition, the toxin inhibited 10 plant pathogenic bacteria with 5 genera. Furthermore, toxin T3 induced the activities of phenylalanine ammonia-lysae(PAL) and peroxidase(POD) in rice. CONCLUSIONS: It is the first report about the separation and purification of E. chrysanthemi pv.zeae toxin. The T3 toxin of E. chrysanthemi pv.zeae had the biological characters with inhibiting plant seeds germination, causing rice seedlings wilt, inhibiting some plant pathogenic bacteria and inducing defense enzyme activities in rice.

The acyl-homoserine lactone-type quorum-sensing system modulates cell motility and virulence of Erwinia chrysanthemi pv. zeae.

Erwinia chrysanthemi pv. zeae is one of the Erwinia chrysanthemi pathovars that infects on both dicotyledons and monocotyledons. However, little is known about the molecular basis and regulatory mechanisms of its virulence. By using a transposon mutagenesis approach, we cloned the genes coding for an E. chrysanthemi pv. zeae synthase of acyl-homoserine lactone (AHL) quorum-sensing signals (expI(Ecz)) and a cognate response regulator (expR(Ecz)). Chromatography analysis showed that expI(Ecz) encoded production of the AHL signal N-(3-oxo-hexanoyl)-homoserine lactone (OHHL). Null mutation of expI(Ecz) in the E. chrysanthemi pv. zeae strain EC1 abolished AHL production, increased bacterial swimming and swarming motility, disabled formation of multicell aggregates, and attenuated virulence of the pathogen on potato tubers. The mutation also marginally reduced the inhibitory activity of E. chrysanthemi pv. zeae on rice seed germination. The mutant phenotypes were rescued by either exogenous addition of AHL signal or in trans expression of expI(Ecz). These data demonstrate that the AHL-type QS signal plays an essential role in modulation of E. chrysanthemi pv. zeae cell motility and the ability to form multicell aggregates and is involved in regulation of bacterial virulence.

[Capacity evaluation of different media for isolating bacteria populations from the rhizosphere of tomato plants with DGGE].

Bacteria from the rhizosphere of tomato plants were cultured on nutrient broth, YG, soil extract and root exudate media. The capabilities of these 4 media for recovering the bacterium communities were evaluated with denaturing gradient gel electrophoresis (DGGE) technique. Results showed that there were certain differences in the obtained bacterial communities when different media and culture tempitures were applied. Numbers of CFU on solid media indicated that the YG agar medium incubated at 20 degrees C could recover more populations than the high-nutrient-concentration Nutrient agar medium; while the root exudate based medium recovered the highest numbers of bacteria from the rhizosphere of tomato plants. 16S rDNA fragments amplified by PCR from different media and rhizosphere soil bacterium DNA were analyzed with DGGE. The DGGE fingerprints showed that there was a discrepancy between bacterial populations observed by cultivation techniques and molecular retrieval. The rhizosphere pattern consisted of more strong bands than the media. The DGGE patterns of the 4 different media incubated at 28 degrees C showed a relatively high level of similarity. In contrast, those of the 4 media incubated at 20 degrees C were significantly different from each other. Shannon-Wiener index (H), species richness (S), Cs values and Cluster dendrogram analysis of different samples revealed that the YG agar medium and the root exudate based medium recovered the highest proportion of predominant populations from the rhizosphere. In the study a new strategy has been proposed for evaluating the capacity of different media in terms of recovering the predominant populations.