Modulation of plant DNA damage response gene expression during Agrobacterium infection.

Agrobacterium T-DNA (transfer DNA) integration into the plant genome relies mostly on host proteins involved in the DNA damage repair pathways. However, conflicting results have been obtained using plants with mutated or down-regulated genes involved in these pathways. Here, we chose a different approach by following the expression of a series of genes, encoding proteins involved in the DNA damage response, during early stages of Agrobacterium infection in tobacco. First, we identified tobacco homologs of Arabidopsis genes induced upon DNA damage and demonstrated that their expression was activated by bleomycin, a DNA-break causing agent. Then, we showed that Agrobacterium infection induces the expression of several of these genes markers of the host DNA damage response, with different patterns of transcriptional response. This induction largely depends on Agrobacterium virulence factors, but not on the T-DNA, suggesting that the DNA damage response activation may rely on Agrobacterium-encoded virulence proteins. Our results suggest that Agrobacterium modulates the plant DNA damage response machinery, which might facilitate the integration of the bacterial T-DNA into the DNA breaks in the host genome.

[The Microbiological Analysis of a Rhizobium radiobacter Outbreak After Intravitreal Injection]. / Intravitreal Enjeksiyon Sonrasi Ortaya Çikan Rhizobium radiobacter Salgininin Mikrobiyolojik Analizi.

Rhizobium radiobacter, which is found in nature and causes tumorigenic plant diseases can lead to opportunistic infections, especially in people with underlying diseases. In our study, endophthalmitis that observed in ten patients caused by R.radiobacter bacteria after intravitreal ranibizumab injection in Ophthalmology Clinic were examined microbiologically. Vitreous fluid samples of 13 patients who received intravitreal ranibizumab injection were sent to the Microbiology Laboratory from Van Yuzuncu Yil University Faculty of Medicine's Ophthalmology Clinic for microbiological examination in December 21, 2016. Samples were examined under microscope after staining with Gram and cultured with 5% sheep blood agar and Eosin Methylene Blue (EMB) agar. The culture plates were incubated for 18-24 hours at 37°C in 5% CO2. At the end of this period, catalase, oxidase, and urease tests were performed on the colonies. The identification and antibiotic susceptibility tests of microorganisms growing in vitreous fluid samples were performed using BD Phoenix (Becton Dickinson, USA), Vitek 2 Compact (BioMerieux, France), and Vitek MS (BioMerieux, France) systems. In addition, 16S rDNA sequence analysis was performed and the pulsed field gel electrophoresis (PFGE) method was used to determine the clonal relationship between the isolates. After growing in cultures (one day after the procedure), culture samples were collected from the objects, medical tools and equipment, hands of healthcare staff and a new injection solution in the area where the procedure was performed. R.radiobacter was isolated in 10 of the vitreous fluid samples of 13 patients, and no bacterial growth was detected in 3. The microorganisms were found to be gram-negative bacilli, non-fermenter, motile, catalase/oxidase/urease positive, in compliance with R.radiobacter. All isolates were identified as R.radiobacter by BD Phoenix (Becton Dickinson, USA), Vitek 2 Compact (BioMerieux, France), and Vitek MS (BioMerieux, France) (database v2.0) systems. R.radiobacter isolates were found to be resistant to ampicillin, amoxicillin/clavulanate, trimethoprim/ sulfamethoxazole, cefotaxime and ceftazidime; susceptible to cefuroxime, cefepime, amikacin, gentamicin, imipenem, meropenem, ciprofloxacin, levofloxacin and piperacillin/tazobactam. The isolates were identified as R.radiobacter by 16S rDNA sequence analysis. PFGE showed that all isolates had the same band profile. R.radiobacter isolates with the same band profile likely revealed that the contamination was from the same source. However, the growth of R.radiobacter was not detected in the cultures made from the objects, medical instruments and supplies, the hands of healthcare professionals and the new injection solution in the area where the procedure was performed, and the source of the agent could not be determined. The results have shown that intravitreal injection procedure carries a risk for R.radiobacter infection. Disinfection and antisepsis conditions, before and during the procedure, is important for the prevention of such infections. This study is the first epidemic outbreak report of endophthalmitis caused by the same strain of R.radiobacter and the second article in which R.radiobacter was reported as the cause of endophthalmitis after intravitreal injection.

Septic shock caused by Rhizobium radiobacter in an elderly woman: A case report.

RATIONALE: Rhizobium radiobacter is a Gram-negative pathogen present in soil and plants. Cases of R radiobacter infection in immunocompromised hosts have been sporadically reported. However, septic shock caused by R radiobacter is rarely seen. PATIENT CONCERNS: Here, we describe an elderly patient with a rapid progression of watery diarrhea, anorexia, fever, weakness, oliguria, and shock. Blood results showed increased total white blood cell count and C-reactive protein. Arterial blood gas results showed hypoxia and elevated lactate level. The Sequential Organ Failure Assessment score was 11. Blood culture at admission showed Gram-negative bacteria, which were later confirmed as R radiobacter. DIAGNOSIS: Septic shock caused by R Radiobacter. INTERVENTIONS: The patient was treated with intravenous cefoperazone/sulbactam and sequential oral levofloxacin. OUTCOMES: The patient recovered completely. CONCLUSION: R radiobacter may be considered as a potential opportunistic pathogen that may cause severe sepsis in elderly patients, especially those with multiple underlying diseases.

Isolation, characterization, and selection of heavy metal-resistant and plant growth-promoting endophytic bacteria from root nodules of Robinia pseudoacacia in a Pb/Zn mining area.

Multiple heavy metals (HMs) commonly coexist in mining areas, which highlights the necessity to select multiple HM-resistant plant growth-promoting bacteria for improving phytoremediation efficiency. In this study, we isolated and characterized 82 endophytic bacteria from the root nodules of black locust (Robinia pseudoacacia) grown in a Pb-Zn mining area. There were 80 isolates showing resistance to four HMs, 0.01-18.0 mM/L for Cd, 0.2-40.0 mM/L for Zn, 0.3-2.2 mM/L for Pb, and 0.2-1.4 mM/L for Cu. Indole-3-acetic acid production, siderophore production, and 1-aminocyclopropane-1-carboxylate deaminase activity were detected in 43, 50, and 17 isolates, respectively. Two symbiotic isolates selected with the highest potential for HM resistance and PGP traits, designated Mesorhizobium loti HZ76 and Agrobacterium radiobacter HZ6, were evaluated for promotion of plant growth and metal uptake by R. pseudoacacia seedlings grown in pots containing different levels of Cd, Zn, Pb, or Cu. HZ76 significantly increased plant shoot biomass, while HZ6 did not, compared with non-inoculated controls. The results indicate that inoculation with HZ76 or HZ6 relieved HM stress in the plants, depending on the type and concentration of HM in the treatment. Mesorhizobium loti HZ76 may be a better candidate for application in phytoremediation than A. radiobacter HZ6. The microsymbiosis between HM-resistant rhizobia and R. pseudoacacia is an interesting mutualistic system for phytoremediation in mining areas contaminated with multiple HMs.

Molecular investigation of isolates from a multistate polymicrobial outbreak associated with contaminated total parenteral nutrition in Brazil.

BACKGROUND: Between November 2013 and June 2014, 56 cases of bacteremia (15 deaths) associated with the use of Total Parenteral Nutrition (TPN) and/or calcium gluconate (CG) were reported in four Brazilian states. METHODS: We analyzed 73 bacterial isolates from four states: 45 from blood, 25 from TPN and three from CG, originally identified as Acinetobacter baumannii, Rhizobium radiobacter, Pantoea sp. or Enterobacteriaceae using molecular methods. RESULTS: The first two bacterial species were confirmed while the third group of species could not be identified using standard identification protocols. These isolates were subsequently identified by Multi-Locus Sequence Analysis as Phytobacter diazotrophicus, a species related to strains from similar outbreaks in the United States in the 1970's. Within each species, TPN and blood isolates proved to be clonal, whereas the R. radiobacter isolates retrieved from CG were found to be unrelated. CONCLUSION: This is the first report of a three-species outbreak caused by TPN contaminated with A. baumannii, R. radiobacter and P. diazotrophicus. The concomitant presence of clonal A. baumannii and P. diazotrophicus isolates in several TPN and blood samples, as well as the case of one patient, where all three different species were isolated simultaneously, suggest that the outbreak may be ascribed to a discrete contamination of TPN. In addition, this study highlights the clinical relevance of P. diazotrophicus, which has been involved in outbreaks in the past, but was often misidentified as P. agglomerans.

The plant defense signal galactinol is specifically used as a nutrient by the bacterial pathogen Agrobacterium fabrum.

The bacterial plant pathogen Agrobacterium fabrum uses periplasmic-binding proteins (PBPs) along with ABC transporters to import a wide variety of plant molecules as nutrients. Nonetheless, how A. fabrum acquires plant metabolites is incompletely understood. Using genetic approaches and affinity measurements, we identified here the PBP MelB and its transporter as being responsible for the uptake of the raffinose family of oligosaccharides (RFO), which are the most widespread d-galactose-containing oligosaccharides in higher plants. We also found that the RFO precursor galactinol, recently described as a plant defense molecule, is imported into Agrobacterium via MelB with nanomolar range affinity. Structural analyses and binding mode comparisons of the X-ray structures of MelB in complex with raffinose, stachyose, galactinol, galactose, and melibiose (a raffinose degradation product) revealed how MelB recognizes the nonreducing end galactose common to all these ligands and that MelB has a strong preference for a two-unit sugar ligand. Of note, MelB conferred a competitive advantage to A. fabrum in colonizing the rhizosphere of tomato plants. Our integrative work highlights the structural and functional characteristics of melibiose and galactinol assimilation by A. fabrum, leading to a competitive advantage for these bacteria in the rhizosphere. We propose that the PBP MelB, which is highly conserved among both symbionts and pathogens from Rhizobiace family, is a major trait in these bacteria required for early steps of plant colonization.

Complete sequence of the tumor-inducing plasmid pTiChry5 from the hypervirulent Agrobacterium tumefaciens strain Chry5.

Agrobacterium tumefaciens strain Chry5 is hypervirulent on many plants including soybean that are poorly transformed by other A. tumefaciens strains. Therefore, it is considered as a preferred vector for genetic transformation of plants. Here we report the complete nucleotide sequence of its chrysopine-type Ti-plasmid pTiChry5. It is comprised of 197,268 bp with an overall GC content of 54.5%. Two T-DNA regions are present and 219 putative protein-coding sequences could be identified in pTiChry5. Roughly one half of the plasmid is highly similar to the agropine-type Ti plasmid pTiBo542, including the virulence genes with an identical virG gene, which is responsible for the supervirulence caused by pTiBo542. The remaining part of pTiChry5 is less related to that of pTiBo542 and embraces the trb operon of conjugation genes, genes involved in the catabolism of Amadori opines and the gene for chrysopine synthase, which replaces the gene for agropine synthase in pTiBo542. With the exception of an insertion of IS869, these Ti plasmids differ completely in the set of transposable elements present, reflecting a different evolutionary history from a common ancestor.

Rhizobium radiobacter en absceso pulmonar asociado a neumonía necrotizante postgripal / Rhizobium radiobacter in pulmonary abscess associated with postgripal necrotizing pneumonia

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Rhizobium radiobacter Endocarditis in an Intravenous Drug User: Clinical Presentation, Diagnosis, and Treatment.

Rhizobium radiobacter, a soil-based organism, is not, usually, pathogenic unless in the immunecompromised. Endocarditis, in the immunocompromised, is a typical presentation generally as a result of catheter-based infections. We describe the presentation of R. radiobacter prosthetic valve endocarditis and the inherent challenges in its presentation and diagnosis. A patient presented with acute limb ischemia secondary to R. radiobacter-mediated endocarditis and subsequent thromboembolization of the distal superior femoral and proximal popliteal arteries in the left lower limb. He underwent an uneventful thrombolectomy that restored blood flow distal to the occlusion and restored the patency of the affected arteries. Postoperatively, the patient maintained several unexplained febrile episodes. Blood cultures remained negative for infection. A cardiac work-up demonstrated the presence of vegetative growth on the prosthetic mitral and native aortic valves. Histopathologic analysis of the extracted thrombus confirmed the presence of R. radiobacter. On further history, it was elucidated that the patient was an intravenous drug user who routinely stored drug paraphernalia in plant beds. The patient recovered uneventfully after Piptazobactam was administered. R. radiobacter, and similarly other soil-based pathogens, should be considered as a potential source of endocarditic infection and thromboembolization in patients who similarly describe a history of intravenous drug use.

Rhizobium Radiobacter Infection in a 27-Year-Old African American Woman With Munchausen Syndrome.

Rhizobium radiobacter is an opportunistic, usually saprophytic, gram-negative bacillus found in agricultural soil. Isolation from blood has been reported most often in hospitalized patients harboring malignant neoplasms or human immunodeficiency virus (HIV) associated immunosuppression, who have catheter or medical device-related febrile neutropenia; treatment involves removal of the catheter or implanted medical device.(1)Herein, we report a case of a 27-year-old African American woman with sickle cell anemia who sought treatment of generalized body pain, shaking, chills, dyspnea, and fever, suggestive of sickle cell crisis. As part of her work up, routine blood cultures were drawn, revealing the presence of a Gram negative bacillus that was identified as the nonfermenter bacillus R. radiobacter The patient displayed a unique infection with R. radiobacter sepsis in a patient secondary to self-injection of organic material into a peripheral line during hospitalization. The growth of an unusual organism in the blood of a patient, without the usual risk factors of R. radiobacter, raised suspicion of a factitious psychiatric disorder known as Munchausen syndrome, which was confirmed when we discovered self-injection of feces and dirt into a central intravenous (IV) line.

Rhizobium radiobacter: A Recently Recognized Cause of Bacterial Keratitis.

PURPOSE: To present the first reported cases of keratitis caused by Rhizobium radiobacter. METHODS: A retrospective review of an observational case series from 2 institutions. RESULTS: There were 4 cases of microbiologically proven R. radiobacter. Three of these patients were contact lens wearers. The patient in case 4 defaulted from follow-up after receipt of the culture result. Keratitis in the other cases resolved with variable clinical courses once culture-directed therapy was instituted. CONCLUSIONS: R. radiobacter is a member of the genus Rhizobium. It is a gram-negative bacillus and was previously identified as an opportunistic pathogen in nonophthalmic infections and in a few cases of endophthalmitis. To our knowledge, we have described the clinical presentation, management, and treatment outcomes of the first reported cases of keratitis caused by R. radiobacter.

Endophthalmitis Caused by Rhizobium radiobacter After Posterior Chamber Phakic Intraocular Lens Implantation to Correct Myopia.

PURPOSE: To describe a 29-year-old man presenting with acute-onset endophthalmitis caused by Rhizobium (formerly Agrobacterium) radiobacter after uneventful implantable collamer lens implantation for myopia. METHODS: Case report. RESULTS: The patient was treated with intravitreal injections of vancomycin and ceftazidime, as well as topical and systemic antibiotics. The patient had a good response with a final visual acuity of 20/50 at the last follow-up visit 25 months postoperatively. CONCLUSIONS: This case represents the first case report of endophthalmitis caused by Rhizobium radiobacter following implantable collamer lens implantation.

Comparison of Soybean Transformation Efficiency and Plant Factors Affecting Transformation during the Agrobacterium Infection Process.

The susceptibility of soybean genotype to Agrobacterium infection is a key factor for the high level of genetic transformation efficiency. The objective of this study is to evaluate the plant factors related to transformation in cotyledonary nodes during the Agrobacterium infection process. This study selected three genotypes (Williams 82, Shennong 9 and Bert) with high transformation efficiency, which presented better susceptibility to Agrobacterium infection, and three low transformation efficiency genotypes (General, Liaodou 16 and Kottman), which showed a relatively weak susceptibility. Gibberellin (GA) levels and soybean GA20ox2 and CYP707A2 transcripts of high-efficiency genotypes increased and were higher than those of low-efficiency genotypes; however, the opposite performance was shown in abscisic acid (ABA). Higher zeatin riboside (ZR) content and DNA quantity, and relatively higher expression of soybean IPT5, CYCD3 and CYCA3 were obtained in high-efficiency genotypes. High-efficiency genotypes had low methyl jasmonate (MeJA) content, polyphenol oxidase (PPO) and peroxidase (POD) activity, and relatively lower expression of soybean OPR3, PPO1 and PRX71. GA and ZR were positive plant factors for Agrobacterium-mediated soybean transformation by facilitating germination and growth, and increasing the number of cells in DNA synthesis cycle, respectively; MeJA, PPO, POD and ABA were negative plant factors by inducing defence reactions and repressing germination and growth, respectively.

Molecular and phenotypic characterization of Agrobacterium species from vineyards allows identification of typical Agrobacterium vitis and atypical biovar 1 strains.

AIM: To molecularly and phenotypically characterize a selection of Agrobacterium-like isolates from grapevine canes, crowns, soil and tumours in plants grown under cold conditions. METHODS AND RESULTS: Most of the strains were biovar 3 (Agrobacterium vitis), and the remaining were atypical biovar 1 (Agrobacterium tumefaciens). All of them were tumourigenic on grapevine plants but differences in other hosts were observed. Chromosomal and plasmid-borne traits were analysed by gene amplification with four primer sets. Detection of the pectin enzyme hydrolase gene clearly distinguished A. vitis from the atypical A. tumefaciens. Regarding the virulence sensor gene, limited host range tumour-inducing plasmids were found in the atypical isolates. About opine utilization, most A. vitis and some A. tumefaciens contained octopine/cucumopine plasmids, but the nopaline-type was only detected in one A. tumefaciens. CONCLUSIONS: The A. vitis strains were molecularly and phenotypically more homogeneous than those of A. tumefaciens, the latter displaying some typical A. vitis characteristics, suggesting an adaptation to life in grapevine. SIGNIFICANCE AND IMPACT OF THE STUDY: The findings of this work will help to improve detection procedures of the pathogen, and demonstrate the pathogen diversity in cold vineyards, laying the groundwork for epidemiological studies and development of control strategies of the crown and cane gall disease.

Identification of effective Pb resistant bacteria isolated from Lens culinaris growing in lead contaminated soils.

Soil bacteria are a new phytoremediation system for the removal of heavy metals from soils. In this study, fifteen soil bacteria were isolated from root nodules of lentil growing in heavy metals contaminated soils, particularly by lead. Molecular characterization of the collection showed a large diversity, including Agrobacterium tumefaciens, Rahnella aquatilis, Pseudomonas, and Rhizobium sp. These soil bacteria had a wide range of tolerance to heavy metals. Among them, strains of A. tumefaciens and R. aquatilis tolerated up to 3.35 mM Pb; whereas Pseudomonas tolerated up to 3.24 mM Pb. The inoculation of lentil grown hydroponically with inoculums formed by these efficient and Pb resistant bacteria enhanced plant biomass. The treatment of this symbiosis by 1 mM Pb for 10 days or by 2 mM Pb for 3 days demonstrated that lentil had Pb accumulation capacity and can be considered a Pb accumulator plant, elsewhere, roots accumulated more Pb than shoots, and the inoculation decreased the Pb up take by the plants, suggesting that this symbiosis should be investigated for use in phytostabilization of Pb-contaminated soils. At the same time, a modulation in the antioxidant enzyme activity and a specific duration was required for the induction of the superoxide dismutase (SOD), peroxidase (POX), and ascorbate peroxidase (APX) response and to adapt to Pb stress. These results suggested that these enzymes may be involved in the main mechanism of antioxidative defense in lentil exposed to Pb oxidative stress.

Agrobacterium uses a unique ligand-binding mode for trapping opines and acquiring a competitive advantage in the niche construction on plant host.

By modifying the nuclear genome of its host, the plant pathogen Agrobacterium tumefaciens induces the development of plant tumours in which it proliferates. The transformed plant tissues accumulate uncommon low molecular weight compounds called opines that are growth substrates for A. tumefaciens. In the pathogen-induced niche (the plant tumour), a selective advantage conferred by opine assimilation has been hypothesized, but not experimentally demonstrated. Here, using genetics and structural biology, we deciphered how the pathogen is able to bind opines and use them to efficiently compete in the plant tumour. We report high resolution X-ray structures of the periplasmic binding protein (PBP) NocT unliganded and liganded with the opine nopaline (a condensation product of arginine and α-ketoglurate) and its lactam derivative pyronopaline. NocT exhibited an affinity for pyronopaline (K(D) of 0.6 µM) greater than that for nopaline (KD of 3.7 µM). Although the binding-mode of the arginine part of nopaline/pyronopaline in NocT resembled that of arginine in other PBPs, affinity measurement by two different techniques showed that NocT did not bind arginine. In contrast, NocT presented specific residues such as M117 to stabilize the bound opines. NocT relatives that exhibit the nopaline/pyronopaline-binding mode were only found in genomes of the genus Agrobacterium. Transcriptomics and reverse genetics revealed that A. tumefaciens uses the same pathway for assimilating nopaline and pyronopaline. Fitness measurements showed that NocT is required for a competitive colonization of the plant tumour by A. tumefaciens. Moreover, even though the Ti-plasmid conjugal transfer was not regulated by nopaline, the competitive advantage gained by the nopaline-assimilating Ti-plasmid donors led to a preferential horizontal propagation of this Ti-plasmid amongst the agrobacteria colonizing the plant-tumour niche. This work provided structural and genetic evidences to support the niche construction paradigm in bacterial pathogens.