Study on the Mass Spectrometry Cleavage Pattern of Quinolone Antibiotics.

RATIONALE: Quinolone antibiotics are extensively used clinically for human treatment and in agriculture. However, improper and excessive use can lead to the persistence of quinolone residues in animal tissues, potentially accumulating in the human body and posing health risks. Investigating the correlation between mass spectrometry cleavage patterns and molecular structural features enhances the analytical framework for detecting trace or unknown impurities in quinolones. METHODS: To collect data, we employed triple quadrupole linear ion trap mass spectrometry in electrospray positive ion mode. Primary mass spectrometry scanning was utilized to confirm parent ions, while secondary mass spectrometry scanning enabled the observation of fragment ions. The cleavage characteristics and pathways of the compounds were inferred from accurate mass-to-charge ratios obtained from both primary and secondary mass spectrometry. RESULTS: Under soft ionization conditions, the compounds generally exhibited characteristic fragment ions of [M+H-H2O]+, [M+H-CO]+, and [M+H-H2O-CO]+. Additionally, subtle variations were observed in each compound due to differences in modifying groups. For instance, upon deacidification, the piperazine ring structure underwent breakage and rearrangement, yielding fragment ion peaks devoid of neutral molecules such as C2H5N, C3H7N, or C4H8N. Notably, compounds featuring a cyclopropyl substituent group at the N-1 position typically exhibited characteristic fragments resulting from the loss of the cyclopropyl radical (⋅C3H5). Moreover, substituents at the N-1 and C-8 positions, when linked to form a six-membered carbocyclic ring, were prone to cleavage, releasing the neutral C3H6 molecule. CONCLUSION: Quinolone antibiotics share structural similarities in their parent nuclei, leading to partially similar cleavage pathways. Nevertheless, distinct cleavage patterns emerge due to variations in functional groups. According to the difference of mass spectrometry cleavage patterns, it can provide an identification basis for the measured detection of antibiotics.

Detoxification of phoxim by a gut bacterium of Delia antiqua.

The presence of certain associated bacteria has been reported to increase pest resistance to pesticides, which poses a serious threat to food security and the environment. Researches on the above microbe-derived pesticide resistance would bring innovative approaches for pest management. Investigations into the phoxim resistance of Delia antiqua, one Liliaceae crop pests, revealed the contribution of a phoxim-degrading gut bacterium, D39, to this resistance. However, how the strain degraded phoxim was unknown. In this study, the role of D39 in phoxim degradation and resistance was first confirmed. DT, which had an identical taxonomy but lacked phoxim-degrading activity, was analyzed alongside D39 via comparative genomics to identify the potential phoxim degrading genes. In addition, degradation metabolites were identified, and a potential degradation pathway was proposed. Furthermore, the main gene responsible for degradation and the metabolites of phoxim were further validated via prokaryotic expression. The results showed that D39 contributed to resistance in D. antiqua larva by degrading phoxim. Phoxim was degraded by an enzyme encoded by the novel gene phoD in D39 to O,O-diethyl hydrogen phosphorothioate and 2-hydroxyimino-2-phenylacetonitrile. Finally, downstream products were metabolized in the tricarboxylic acid cycle. Further analysis via prokaryotic expression of phoD confirmed its degradation activity. The mechanisms through which gut microbes promote pesticide resistance are elucidated in this study. These results could aid in the development of innovative pest control methods. In addition, this information could also be used to identify microbial agents that could be applied for the remediation of pesticide contamination.

Adult pancreatoblastoma with atypical histological morphology combined with familial adenomatous polyposis: a rare case report.

Pancreatoblastoma (PB) is a rare malignant pancreatic epithelial tumor that mostly occurs in children and occasionally occurs in adults. The tumor has acinar cell differentiation and squamous corpuscles/squamous epithelial islands, which are frequently separated by fibrous bundles. Familial adenomatous polyposis (FAP) is an autosomal dominant inherited disease characterized by the presence of numerous adenomatous polyps in the colon and rectum. Cases of pancreatoblastoma combined with familial adenomatous polyposis (FAP) are rarely reported. A review of a rare case of adult pancreatoblastoma with atypical histological morphology combined with familial adenomatous polyposis is presented herein. In this case, the patient was first diagnosed with familial adenomatous polyposis and subsequently found to have pancreatoblastoma 1 year and 3 months later. This suggests pancreatoblastoma may occur in patients with familial adenomatous polyposis or a family history of the condition, indicating a possible association between the two tumors. Therefore, pancreatoblastoma should be included in a differential diagnosis for FAP patients with a pancreatic mass. The final diagnosis of pancreatoblastoma depends on the pathological diagnosis. Acinar-like cells and squamous corpuscles/squamous epithelial cell islands under light microscopy are the key diagnostic points. This case report also can improve the awareness of clinicians, radiologists, and pathologists on the presence of rare tumor-adult pancreatoblastoma in patients with familial adenomatous polyposis.

Comparative metaproteomics reveal co-contribution of onion maggot and its gut microbiota to phoxim resistance.

Pesticide resistance inflicts significant economic losses on a global scale each year. To address this pressing issue, substantial efforts have been dedicated to unraveling the resistance mechanisms, particularly the newly discovered microbiota-derived pesticide resistance in recent decades. Previous research has predominantly focused on investigating microbiota-derived pesticide resistance from the perspective of the pest host, associated microbes, and their interactions. However, a gap remains in the quantification of the contribution by the pest host and associated microbes to this resistance. In this study, we investigated the toxicity of phoxim by examining one resistant and one sensitive Delia antiqua strain. We also explored the critical role of associated microbiota and host in conferring phoxim resistance. In addition, we used metaproteomics to compare the proteomic profile of the two D. antiqua strains. Lastly, we investigated the activity of detoxification enzymes in D. antiqua larvae and phoxim-degrading gut microbes, and assessed their respective contributions to phoxim resistance in D. antiqua. The results revealed contributions by D. antiqua and its gut bacteria to phoxim resistance. Metaproteomics showed that the two D. antiqua strains expressed different protein profiles. Detoxifying enzymes including Glutathione S-transferases, carboxylesterases, Superoxide Dismutase, Glutathione Peroxidase, and esterase B1 were overexpressed in the resistant strain and dominated in differentially expressed insect proteins. In addition, organophosphorus hydrolases combined with a group of ABC type transporters were overexpressed in the gut microbiota of resistant D. antiqua compared to the sensitive strain. 85.2% variation of the larval mortality resulting from phoxim treatment could be attributed to the combined effects of proteins from both from gut bacteria and D. antiqua, while the individual contribution of proteins from gut bacteria or D. antiqua alone accounted for less than 10% of the variation in larval mortality caused by phoxim. The activity of the overexpressed insect enzymes and the phoxim-degrading activity of gut bacteria in resistant D. antiqua larvae were further confirmed. This work enhances our understanding of microbiota-derived pesticide resistance and illuminates new strategies for controlling pesticide resistance in the context of insect-microbe mutualism.

Characterization of Antagonistic Bacteria Paenibacillus polymyxa ZYPP18 and the Effects on Plant Growth.

Paenibacillus polymyxa is a plant growth-promoting rhizobacteria (PGPR) that has significant biocontrol properties. Wheat sheath blight caused by Rhizoctonia cerealis is a significant soil-borne disease of wheat that causes significant losses in wheat production, and the biological control against the disease has received extensive attention. P. polymyxa ZYPP18 was identified using morphological and molecular characterization. An antagonistic activity experiment verified that ZYPP18 inhibits the growth of R. cerealis on artificial growth media. A detached leaf assay verified that ZYPP18 inhibits the expansion of wheat sheath blight on the detached leaf. ZYPP18 has been found to possess plant growth-promoting properties, as well as the ability to solubilize phosphate and generate indole-3-acetic acid. Results from hydroponic experiments showed that wheat seedlings treated with ZYPP18 grew faster. Additionally, pot experiments and field experiments demonstrated that ZYPP18 effectively controls the occurrence of wheat sheath blight. ZYPP18 reduced the incidence of wheat sheath blight in wheat seedlings by 37.37% and 37.90%, respectively. The control effect of ZYPP18 on wheat sheath blight was 56.30% and 65.57%, respectively. These findings provide evidence that P. polymyxa ZYPP18 is an effective biological factor that can control disease and promote plant growth.

Effects of different cultivation media on root bacterial community characteristics of greenhouse tomatoes.

Tomato, as a typical greenhouse crop, is commonly first planted as seedlings in a variety of substrates before being transplanted into soil. However, there is rare research on the characteristics of the bacterial community in tomato roots under this planting mode. In this study, tomatoes were planted in pots containing three different cultivation media, including soil and two types of substrates in a greenhouse, followed by a transplanting treatment. After collecting tomato root samples, high-throughput sequencing and bioinformatic analysis were used to compare the differences in bacterial diversity and functions between tomato roots before and after transplanting in different cultivation media. In total, 702776 sequences were obtained, and the OTUs were belonging to 109 genera, 58 families, 41 orders, 14 classes, and 12 phyla. Among the three cultivation media, the ß-diversity was significant, and there was a slight difference in bacterial species diversity along with a large difference in their abundance at the genus level. Soil and both substrates had 79 bacterial genera in common, these genera accounted for 68.70%, 76.70%, and 71.17% of the total genera found in the soil, substrate 1, and substrate 2, respectively. After being transplanted from the two substrates to the soil, the bacterial community structure and abundance exhibited similarities with those found in the soil. Furthermore, based on microbial function prediction, the microbial communities in the two-substrate environment demonstrated a greater potential for promoting growth, while the microbial communities in the soil exhibited a greater tendency to exert their antibacterial potential. Our findings offer theoretical support for the creation of artificially reconstructed microbial communities in greenhouse cultivation.

Solitary Fibrous Tumors of the Lung: A Clinicopathological Analysis of 52 Cases.

OBJECTIVE: To explore the clinicopathological features of solitary fibrous tumors (SFTs) of the lung. METHODS: We collected the clinical data of 52 patients with SFTs of the lung confirmed by pathology, and summarized the clinical, radiological, and morphological features, the immunophenotypes, and the prognosis of SFTs. RESULTS: Fifty-two cases of SFTs of the lung were enrolled in this study, including 51 cases of borderline and 1 case of malignancy, 22 males and 30 females. The average onset age was 52.7 years. The lower lobe of the left lung was the preferred site of SFTs, accounting for 30.77% (16/52). Chest CT showed regular and well-demarcated soft tissue density mass, and the tumor size of most cases (46/52, 88.46%) was 1-10 cm. Morphological features: The distribution of tumor cells showed sparse and dense areas. Tumor cells were spindle-shaped, in whorls or hemangiopericytoma-like conformation. Atypia, mitotic figures, and necrosis were found. Immunohistochemistry showed positive expression of CD34, STAT6, Vimentin, BCL2, and CD99. Ki-67 was ≤ 5% in borderline SFTs and >20% in a malignant SFT. CONCLUSIONS: Solitary fibrous tumors of the lung occur more frequently in middle-aged and elderly people, and there is no significant difference in gender. The lower lobe of the left lung is the preferred site of SFTs. The size of most SFTs is 1-10 cm. Chest CT shows morphologically regular and well-demarcated soft tissue density mass. Pathologically, SFTs of the lung are mostly borderline and occasionally malignant. Immunohistochemistry shows the positive expression of CD34, STAT6, Vimentin, BCL2, and CD99.

Quantitative assessment of selective degradation behavior of etoxazole in different classes of organisms by compound-specific isotope analysis.

In this paper, the stereoselective degradation and quantitative identification of chiral pesticide etoxazole in organisms with different classes of organisms (soil, chlorella algal fluid and mice) were carried out by compound-specific isotope analysis (CSIA). The degradation behavior and stable isotope fractionation effect of etoxazole in soil, chlorella and mice were investigated. The R-etoxazole degraded faster than S-etoxazole in different classes of organisms. The metabolites M1, M2 and M3 were detected in all three substrates. Biodegradation is the main factor for the change of stable isotope ratio of chiral pesticide etoxazole. Furthermore, the relationship between fractionation value of carbon isotope and residual concentration of etoxazole is established by Rayleigh equation, and the biodegradation rate of etoxazole could be calculated by using CSIA without measuring the concentration of etoxazole. Therefore, the use of CSIA can accurately assess the degradation behavior of pesticide pollution in the environment and provide a certain scientific evidence and technical support in the process of environmental remediation.

Ferroptosis Is Involved in Sex-Specific Small Intestinal Toxicity in the Offspring of Adult Mice Exposed to Polystyrene Nanoplastics during Pregnancy.

Nanoplastics are common contaminants in the living environment. Thus far, no investigations have focused on small intestinal injury in the offspring of adult mice that were exposed to nanoplastics through the respiratory system during pregnancy. Here, we evaluated potential intestinal injury in the offspring of adult mice that were subjected to maternal 80 nm polystyrene nanoparticle (PS-NP) exposure during gestation. PS-NP exposure significantly reduced the birth weight of female mice compared with male mice. However, the adult body weights of the female and male offspring were substantially greater in the PS-NP-exposed groups. Additionally, we found that exposure to PS-NPs during pregnancy caused histological changes in the small intestines of both female and male offspring. Mechanistic analysis revealed upregulation of reactive oxygen species in the small intestines, as indicated by changes in the levels of superoxide dismutase (SOD) and malondialdehyde (MDA). Furthermore, exposure to PS-NPs led to downregulation of GPx4, FTH1, and FTL protein levels, indicating initiation of ferroptosis. Notably, the changes in mRNA expression levels of GPx4, FTH1, and FTL differed between female and male offspring. Although all phenotypes failed to demonstrate classic dose-dependent effects, the data imply that small intestinal toxicity is greater in female offspring than in male offspring. Our results suggest that PS-NP exposure during pregnancy causes sex-specific small intestinal toxicity, which might contribute to reactive oxygen species activation and subsequent ferroptosis. Overall, this study showed toxic effects in offspring after PS-NP exposure during pregnancy.

Rearing Axenic Delia antiqua with Half-Fermented Sterile Diets.

Axenic insects are obtained from sterile artificial rearing systems using sterile media. These insects, characterized by their small size, short growth cycle, and low feed requirements, are ideal for studying the relationship between microorganisms and hosts. The gut microbiota significantly influences the physiological characteristics of insect hosts, and introducing specific strains into axenic insects provides a method for verifying gut microbial functions. Delia antiqua, a threatening pest in the order Diptera, family Anthomyiidae, and genus Delia, primarily feeds on onions, garlic, leeks, and other vegetables of the family Liliaceae. Its larvae feed on the bulbs, causing rotting, wilting, and even death of entire plants. By rearing axenic larvae, follow-up studies can be conducted to observe the effects of intestinal microflora on the growth and development of D. antiqua. Unlike the method involving antibiotic elimination of associated microbes, this article presents a low-cost and high-efficiency approach to raising axenic D. antiqua. After surface sterilization of D. antiqua eggs, half-fermented sterile diets were used to raise larvae, and the axenic state of D. antiqua was verified through culture-dependent and culture-independent assays. In conclusion, the combination of insect egg sterilization and the preparation of sterile diets for larval culture has enabled the development of an efficient and simple method for obtaining axenic D. antiqua. This method provides a powerful approach to studying insect-microflora interactions.

Diversity Shifts in the Root Microbiome of Cucumber Under Different Plant Cultivation Substrates.

Application of plant artificial cultivation substrates lead to alteration of rhizosphere environment. Whether this alteration could lead to root microbiome variation was limitedly investigated. This work aims to determine the diversity shifts in the root microbiome of cucumber under different plant cultivation substrates and predict corresponding function of these different root bacterial microbiota. Cucumber root samples cultivated with two artificial cultivation substrates and greenhouse soils were prepared. Subsequently, high throughput sequencing and bioinformatics analysis were applicated to compare the root bacterial diversity of cucumber cultivated in different substrates and their corresponding function. In total, 311,039 sequences were obtained, and they were annotated to 42 operational taxonomic units (OTUs), belonging to 28 genera, 18 families, 12 orders, four classes, and three phyla. The α and ß diversity of samples from the two cultivation substrates and greenhouse soils were significantly different. Only 2-3 bacterial species were found to be discrepancy between cucumber root samples from artificial cultivation substrates and from greenhouse soils. The relative abundance of genus Asticcacaulis, Methylophilus, Massilia, Dyella, and Devosia in samples of artificial cultivation substrates was significantly higher than that of soils, while the relative abundance of genus Phenylobacterium, Noviherbaspirillum, and Arenimonas was significantly lower than that of soils. Besides, compared to cucumber root bacterial community cultivated in soils, the abundance of synthetic pathways for flavonoids and flavonols, bile acids, indole alkaloids, lactose, and neolactose increased by 41.6-, 28.7-, 5.9-, and 5.5-fold, respectively, in the bacterial community of the substrate 1-cultivated roots, and the abundance of clavulanic acid, receptor interaction, sesquiterpenoid, bile acid, flavonoid and flavonol, indole alkaloid, lactose, and neolactose synthetic pathways increased by 42.3-, 32.4-, 32.4-, 13.9-, 10.3-, 6.3-, and 5.2-fold, respectively, in the bacterial community of the substrate two-cultivated roots. This paper verified the diversity shifts in the root microbiome of cucumber under different plant cultivation substrates. Besides, the corresponding function difference of these different root bacterial microbiota was predicted. This work would provide theoretical support for discovering microbial resources and building artificial microbial flora.

Identification of the Phosphorus-Solubilizing Bacteria Strain JP233 and Its Effects on Soil Phosphorus Leaching Loss and Crop Growth.

Phosphorus (P) is one of the most limiting nutrients in global agricultural ecosystems, and phosphorus-solubilizing bacteria (PSB) can convert insoluble P into soluble P, thereby improving the absorption and use of soil P by plants. Increasing leaching loss of soil P due to PSB that could lead to water eutrophication is a major concern, although no direct experimental evidence is available to evaluate these effects. In this study, a highly efficient PSB strain, Pseudomonas sp. JP233, was isolated from soil and its P-solubilizing agent was identified by metabolomics and HPLC analyses. The effects of JP233 on P contents in soil leachates were also analyzed by microcosm leaching experiments in the absence and presence of maize. JP233 could solubilize insoluble P into soluble forms, and the molybdate reactive phosphorus (MRP) content reached 258.07 mg/L in NBRIP medium containing 5 g/L Ca3(PO4)2 within 48 h. Metabolomics analysis demonstrated that the organic acid involved in JP233 P solubilization was primarily 2-keto gluconic acid (2KGA). Further, HPLC analysis revealed that 2KGA contents rapidly accumulated to 19.33 mg/mL within 48 h. Microcosm leaching experiments showed that MRP and total phosphorus (TP) contents in soil leaching solutions were not significantly higher after JP233 inoculation. However, inoculation with JP233 into maize plant soils significantly decreased MRP and TP contents in the soil leaching solutions on days 14 (P < 0.01), 21 (P < 0.01), and 28 (P < 0.05). Inoculation with strain JP233 also significantly increased the biomass of maize aerial components and that of whole plants (P < 0.05). Thus, strain JP233 exhibited a significant plant-growth-promoting effect on maize development. In conclusion, the application of PSB into soils does not significantly increase P leachate loss. Rather, the application of PSB can help reduce P leachate loss, while significantly promoting plant absorption and use of soil P.

First Report of Phytopythium helicoides Causing Stalk Rot on Corn in China.

Corn (Zea mays L.) is one of the most important grain crops in the world, especially in China. Besides, corn stalks are often used in production of bio-fuels (Xue et al., 2017). Recently, the production and quality of corn have been severely influenced by corn stalk rot in China caused by Fusarium spp. (Yu et al., 2017). At the end of June of 2019, a field survey of corn was carried out in Tai'an City, western Shandong Province, China. During the survey, the average day time temperature ranged between 22-28°C with intermittent rainfall, the relative humidity was 50-70%. In this survey, the symptomatic corn plants showed signs of necrosis and rotting on stalks and root collars. Five fields were surveyed and symptomatic corn plants were observed in three fields. The incidence rate of disease was about 5%, and the disease was more of a problem in low-lying areas. A total of twenty-eight symptomatic corn plants (7-12 per field), hybrid Denghai-618, at the 3-4 leaf stage were collected and tested for the presence of pathogens. The diseased tissues were excised, surface-sterilized with 75% ethanol for 30 seconds, rinsed for 3 to 5 times with sterile distilled water, and plated on potato dextrose agar (PDA). All plates were incubated at 28°C for 48 hours, emerging colonies were sub-cultured onto PDA plates. Forty-two isolates were obtained, and twenty-seven isolates were identified as Fusarium spp. The remaining fifteen isolates had similar morphology, with colonies that were white and cottony in texture after incubation at 28°C for three days on PDA. The suitable temperature range for growth of hyphae was between 15°C to 40°C, and sporangia were ellipsoidal, papillate, and 23 - 34×21 - 31 µm in diameter. Oogonia (smooth, 22 - 30 µm in diameter) were present in the cultures after 28 days at 28°C. The isolates were identified using both morphological characteristics and DNA sequencing. Identity of the oomycete was confirmed using the BLAST algorithm available through the GenBank with the DNA sequences of rDNA internal transcribed spacer region (ITS), cytochrome c oxidase Ⅰ (coxⅠ) gene and cytochrome c oxidase Ⅱ (coxⅡ) gene, which were amplified using the primers ITS1/ITS4 (White et al. 1990), FM35/FM59 and FM66/FM58 (Martin 2000), respectively. The fifteen isolates selected for sequence analysis had identical gene sequences, and hence, only sequences for isolate RMSD1 were submitted to GenBank (ITS - MW440691, coxI - MW450815 and cox II - MW450816). The ITS, coxI and coxII sequences of the isolate RMSD1 showed 97% identity (751/774 bp), 99% identity (1087/1098 bp) and 99% identity (548/554 bp) with Phytopythium helicoides Accession nos: HQ643382, FR774199, and AB108014, respectively. The pathogenicity of RMSD1 was tested on the corn hybrid Denghai-618. Three-leaf-stage corn plants (N = 15) were inoculated with mycelial agar disks (3 to 4 mm in diameter) colonized with RMSD1 placed on their root-collars. Sterile PDA disks (3 to 4 mm in diameter) served as the negative control (N = 9). Inoculated plants were placed in the growth chamber at 28°C, 60% relative humidity, 16 h / 8 h light regime cycle. Ten days post-inoculation, the inoculated plants showed necrosis, with symptoms of stem rot similar to those observed in the field. The inoculation experiments were repeated twice with the same results, fulfilling Koch's postulates. The root-collars and stems of negative control remained asymptomatic, and P. helicoides was not isolated. Previously, P. helicoides has been reported as a pathogen of strawberry (Zhan et al. 2020) and kiwi fruits (Wang et al. 2015) from China, but not from corn. To our knowledge, it is the first report of P. helicoides causing corn stalk rot in China. In the future, P. helicoides can be considered as a potential candidate causing stem and collar-rot of corn in China, but not the only one. There are other microbes that can produce similar symptoms on corn, and control methods for pathogenic oomycetes differ from those for fungi.

Diguanylate Cyclase GdpX6 with c-di-GMP Binding Activity Involved in the Regulation of Virulence Expression in Xanthomonas oryzae pv. oryzae.

Cyclic diguanylate monophosphate (c-di-GMP) is a secondary messenger present in bacteria. The GGDEF-domain proteins can participate in the synthesis of c-di-GMP as diguanylate cyclase (DGC) or bind with c-di-GMP to function as a c-di-GMP receptor. In the genome of Xanthomonas oryzae pv. oryzae (Xoo), the causal agent of bacterial blight of rice, there are 11 genes that encode single GGDEF domain proteins. The GGDEF domain protein, PXO_02019 (here GdpX6 [GGDEF-domain protein of Xoo6]) was characterized in the present study. Firstly, the DGC and c-di-GMP binding activity of GdpX6 was confirmed in vitro. Mutation of the crucial residues D403 residue of the I site in GGDEF motif and E411 residue of A site in GGDEF motif of GdpX6 abolished c-di-GMP binding activity and DGC activity of GdpX6, respectively. Additionally, deletion of gdpX6 significantly increased the virulence, swimming motility, and decreased sliding motility and biofilm formation. In contrast, overexpression of GdpX6 in wild-type PXO99A strain decreased the virulence and swimming motility, and increased sliding motility and biofilm formation. Mutation of the E411 residue but not D403 residue of the GGDEF domain in GdpX6 abolished its biological functions, indicating the DGC activity to be imperative for its biological functions. Furthermore, GdpX6 exhibited multiple subcellular localization in bacterial cells, and D403 or E411 did not contribute to the localization of GdpX6. Thus, we concluded that GdpX6 exhibits DGC activity to control the virulence, swimming and sliding motility, and biofilm formation in Xoo.

The RpoN2-PilRX regulatory system governs type IV pilus gene transcription and is required for bacterial motility and virulence in Xanthomonas oryzae pv. oryzae.

The type IV pilus (T4P), a special class of bacterial surface filament, plays crucial roles in surface adhesion, motility, biofilm formation, and virulence in pathogenic bacteria. However, the regulatory mechanism of T4P and its relationship to bacterial virulence are still little understood in Xanthomonas oryzae pv. oryzae (Xoo), the causal pathogen of bacterial blight of rice. Our previous studies showed that the σ54 factor RpoN2 regulated bacterial virulence on rice in a flagellum-independent manner in Xoo. In this study, both yeast two-hybrid and pull-down assays revealed that RpoN2 directly and specifically interacted with PilRX, a homolog of the response regulator PilR of the two-component system PilS-PilR in the pilus gene cluster. Genomic sequence and reverse transcription PCR (RT-PCR) analysis showed 13 regulons containing 25 genes encoding T4P structural components and putative regulators. A consensus RpoN2-binding sequence GGN10 GC was identified in the promoter sequences of most T4P gene transcriptional units. Electrophoretic mobility shift assays confirmed the direct binding of RpoN2 to the promoter of the major pilin gene pilAX, the inner membrane platform protein gene pilCX, and pilRX. Promoter activity and quantitative RT-PCR assays demonstrated direct and indirect transcriptional regulation by RpoN2 of the T4P genes. In addition, individual deletions of pilAX, pilCX, and pilRX resulted in significantly reduced twitching and swimming motility, biofilm formation, and virulence in rice. Taken together, the findings from the current study suggest that the RpoN2-PilRX regulatory system controls bacterial motility and virulence by regulating T4P gene transcription in Xoo.

Transcriptional responses of Xanthomonas oryzae pv. oryzae to type III secretion system inhibitor ortho-coumaric acid.

BACKGROUND: We previously identified a plant-derived phenolic compound ortho-coumaric acid (OCA) as an inhibitor of type III secretion system (T3SS) of Xanthomonas oryzae pv. oryzae (Xoo), the pathogen causing bacterial leaf blight of rice, one of the most devastating bacterial diseases of this staple crop worldwide. However, the molecular mechanisms by which OCA suppresses T3SS and the transcriptional responses to the OCA treatments in Xoo remains unclear. RESULTS: The present study conducted the RNA-seq-based transcriptomic analysis to reveal changes in gene expression in Xoo in response to 30 min, 1 h, 3 h, and 6 h of OCA treatment. Results showed that OCA significantly inhibited the expression of T3SS genes after 30 min, and the inhibition also existed after 1 h, 3 h, and 6 h. After treatment for 30 min, membrane proteins in the functional category of cellular process was the predominant group affected, indicating that Xoo was in the early stress stage. Over time, more differentially-expressed genes (DEGs) gathered in the functional category of biological process. Analysis of common DEGs at all four of time points revealed the core elements of Xoo during the response to OCA treatment. Notable, a multidrug transporter cluster that consisted of a MarR-family protein (PXO_RS13760), a multidrug RND transporter (PXO_RS13755), a multidrug transporter (PXO_RS13750), and an MFS transporter (PXO_RS13745) were significantly up-regulated at all four of the time points. Although these three transporter genes were not upregulated by OCA in the PXO_RS13760 deletion mutant, the deficiency of PXO_RS13760 in Xoo did not affect T3SS transcript, and OCA still had the ability to inhibit the expression of T3SS in the mutant, suggesting that the MarR-family protein was involved in bacterial responses to OCA, but not direct OCA inhibition of T3SS in Xoo. CONCLUSIONS: We analyzed the transcriptome of Xoo during OCA treatment at both early and late stages, which revealed the landscape of Xoo responses to OCA at the whole-genome transcription level. A multidrug transporter cluster was identified to be involved in the response process, but had no direct relation to T3SS in Xoo.

Small Molecule Inhibitors Specifically Targeting the Type III Secretion System of Xanthomonas oryzae on Rice.

The initiative strategy for the development of novel anti-microbial agents usually uses the virulence factors of bacteria as a target, without affecting their growth and survival. The type III secretion system (T3SS), one of the essential virulence factors in most Gram-negative pathogenic bacteria because of its highly conserved construct, has been regarded as an effective target that developed new anti-microbial drugs. Xanthomonas oryzae pv. oryzae (Xoo) causes leaf blight diseases and is one of the most important pathogens on rice. To find potential anti-virulence agents against this pathogen, a number of natural compounds were screened for their effects on the T3SS of Xoo. Three of 34 compounds significantly inhibited the promoter activity of the harpin gene, hpa1, and were further checked for their impact on bacterial growth and on the hypersensitive response (HR) caused by Xoo on non-host tobacco plants. The results indicated that treatment of Xoo with CZ-1, CZ-4 and CZ-9 resulted in an obviously attenuated HR without affecting bacterial growth and survival. Moreover, quantitative reverse transcription-polymerase chain reaction (qRT-PCR) analysis showed that the expression of the Xoo T3SS was suppressed by treatment with the three inhibitors. The mRNA levels of representative genes in the hypersensitive response and pathogenicity (hrp) cluster, as well as the regulatory genes hrpG and hrpX, were reduced. Finally, the in vivo test demonstrated that the compounds could reduce the disease symptoms of Xoo on the rice cultivar (Oryza sativa) IR24.

The Effect of Different Medium on Enantioselectivity in Degradation of Bifenthrin.

Bifenthrin (BF) is a chiral pesticide with two enantiomers. In this work, its stereoselective degradation was investigated in plants and domesticated active sludge. Considering that the degradation to pesticides of plants was effected by external conditions, hence, nanometer materials (gothite) were added into soil to explore the effect of sorption on enantioselectivity in degradation of BF. The microbial community that was responsible for BF-biodegrading in active sludge was studied by polymerase chain reaction and denaturing gradient gel electrophoresis (PCR-DGGE). In plant researches, chiral difference was discovered in the degradation of pesticides that onions resulted in. The existence of gothite in soil not only reduced the amount of BF that plants uptake from the environment, but it also enlarged the chiral difference during the process, indicating that the absorption of gothite to pesticides had enantioselectivity, which may be generated by that adsorption site where BF combined with gothite located in the chiral center of the pesticide. In studies of sludge, BF can be effectively degraded and decline of both isomers followed first-order kinetics. However, there was no obvious stereoselective degradation in domesticated sludge. The analysis of DGGE revealed that active sludge maintained the rich microbial community in the whole process (H > 3). The H index increased in the early domestication, which may because BF had no direct toxicity to the bacteria. The bacteria were able to degrade the small dose of the pesticide and absorbed it as nutrition. The sequence results demonstrated that the variety of bacteria grew, instead of Acidobacteria, Betaproteobacteria, Sphingobacteriia and Alphaproteobacteria, Flavobacteria also appearing in sludge after domestication.

Identification of phenolic compounds that suppress the virulence of Xanthomonas oryzae on rice via the type III secretion system.

The targeting of bacterial type III secretion systems (T3SSs), which are critical virulence factors in most Gram-negative pathogens, is regarded as an alternative strategy for the development of novel anti-microbial drugs. Xanthomonas oryzae pv. oryzae (Xoo) and X. oryzae pv. oryzicola (Xoc) are two of the most important bacterial pathogens on rice, which cause leaf blight and leaf streak diseases, respectively. To identify potential anti-virulence drugs against these two pathogens, we screened a library of plant phenolic compounds and derivatives for their effects on the Xoo T3SS. Ten of 56 compounds significantly inhibited the promoter activity of a harpin gene, hpa1. These inhibitors were further tested for their impact on the hypersensitive response (HR) caused by Xoo on non-host tobacco plants. The results showed that pretreatment of Xoo with TS006 (o-coumaric acid, OCA), TS010, TS015 and TS018 resulted in significantly attenuated HR without affecting bacterial growth or survival. In addition, Cya translocation assays demonstrated that the translocation of two T3 effectors was suppressed by the four inhibitors. Quantitative reverse transcription-polymerase chain reaction (qRT-PCR) analysis showed that mRNA levels of representative genes in the hrp (hypersensitive response and pathogenicity) cluster, as well as the regulatory genes hrpG and hrpX, were reduced by treatment with the four inhibitors, suggesting that expression of the Xoo T3SS was suppressed. The expression of other virulence factors was not suppressed, which indicated possible T3SS-specific inhibition. Finally, we demonstrated that these inhibitors reduced the disease symptoms of Xoo and Xoc on the rice cultivar (Oryza sativa) IR24 to varying extents.