First report of crown gall disease caused by Agrobacterium radiobacter on olive plants in Layyah, Punjab, Pakistan.

Crown galls were observed on one-year-old olive plants (Olea europaea cv. Manzanilla) in the District Layyah (30.9693° N, 70.9428° E) of Punjab, Pakistan. Large tumors were evident on collars region, causing growth stunting, leaf yellowing, and overall plant dieback (Supplementary fig. 1). Total 900 of olive plant were grown including 300 young plants in five hectare orchards, around 25% of the young plants in orchard had gall formation with varying in size (2-15cm), majority of the infected plants were grown near the water channel, where soil moisture level were high (90-100%). Other olive orchards in the same area have not crown gall problem and the tumorigenic strains of bacteria can cause crown gall on plants (Nemanja Kuzmanovic et al. 2015). This study was aimed to determine the pathogen of disease. The randomized collected samples were rinsed with tap water and galls were sterilized with 10% sodium hypochlorite solution for 1.5-3.0 min, washed with sterilized Distilled Water (SDW) then chopped and immersed overnight in SDW at room temperature. Isolations were carried out by plating the internal gall tissues on fresh Luria Bertani agar (LB agar) supplemented with natamycin. After incubating at 28°C for 5 days, 10 single colonies were transferred on new LBA plates for further cultivation at 28°C. After 48 to 72 h, three strains showed white to cream-colored, smooth, convex, glistening, circular with entire edges, and mucoid bacterial colonies resembling Agrobacterium spp. These three strains (BAT01, BAT02, BAT03) also showed biochemical and physiological characteristics similar to A. tumefaciens, including oxidase positive, growth at 35°C and in 2% NaCl, and alkalinity from litmus milk. They were tested negative for utilization of citrate and acid production on potato dextrose agar (PDA) supplemented with CaCO3 (Young et al. 2015). Amplification and sequencing of these three strain's 16S rRNA region and chromosomal recA gene with the universal primers fD1/rP2 and F2898/F2899 verified the identification at species level (Weisburg et al. 1991) . BLAST analysis revealed 100% identity for 16S rRNA and recA gene between the olive crown gall strains. Accession No. of deposited sequences were given in table 1 and the reference sequences GenBank Accessions No. of A. tumefaciens is FM209485.1 and KY913787 respectively. Phylogenetic analysis based on 16S rRNA of the strains from the crown gall and reference strains of various species of Agrobacterium by Maximum-likelihood method with Tamura's three-parameter model using the MEGA X software program confirmed the strain from olive was A. tumefaciens (Supplementary fig. 2). Inoculating the crown part of the plant through wounds of sterile needles plunged into young (2 to 3 day) bacterial culture (107 CFU/ml) and sterile distilled water (SDW) was screened for pathogens on 10 one-year-old olive plants cv. Manzanilla. Plants were grown at 23 ± 3°C, and tumor formation was observed 4 weeks after inoculation. Typical tumours formed and no symptoms found in control plants at inoculation sites and Koch's postulates were fulfilled with re-isolation and amplification of bacteria with recA gene region. This data shows that A. tumefaciens causes crown gall in olive plants. though it is reported before in different olive growing region in the world but This is first time reported in Layyah, Punjab, Pakistan.

Agrobacterium radiobacter Endophthalmitis Associated with Baerveldt Tube Exposure.

Agrobacterium radiobacter is a Gram-negative bacillus and a rare cause of endophthalmitis. An 85-year-male presented with late-onset endophthalmitis associated with exposure of an inferonasal Baerveldt tube. The patient was initially treated with anterior chamber paracentesis and intravitreal antibiotics. Aqueous humor culture revealed A. radiobacter resistant to cefazolin, ceftazidime, amikacin, tobramycin, and trimethoprim-sulfamethoxazole. Subsequently, the patient underwent explantation of the glaucoma drainage implant (GDI). After initial improvement, the patient had clinical worsening and was diagnosed with recurrence. Subsequent treatment involved explantation of the second GDI in addition to pars plana vitrectomy with silicone oil infusion, intraocular lens removal, and administration of intravitreal antibiotics. Visual acuity improved but remained at count fingers at 2 weeks. This is the first reported patient with A. radiobacter endophthalmitis associated with an exposed GDI. This report illustrates the resistant nature of this organism in addition to the efficacy of silicone oil administration and intraocular prosthesis explantation.

Genetic Incorporation of Selenotyrosine Significantly Improves Enzymatic Activity of Agrobacterium radiobacter Phosphotriesterase.

Tyrosine plays important roles in many enzymes. To facilitate enzyme design, mechanistic studies and minimize structural perturbation in the active site, here we report the genetic incorporation of a novel unnatural amino acid selenotyrosine (SeHF), which has single-atom replacement in comparison to tyrosine. The arPTE-(Agrobacterium radiobacter Phosphotriesterase) Tyr309SeHF mutant exhibits a significant 12-fold increase in kcat and 3.2-fold enhancement in kcat /KM at pH 7.0. Molecular dynamics simulations show that the SeHF309 mutation results in a conformational switch which opens up the product release pocket and increases the product release rate, thereby elevating the overall enzyme activity. Significant improvement of the catalytic efficiency at neutral pH by single unnatural amino acid (UAA) mutation broadens the application of this enzyme, and provides valuable insights to the mechanism. Our method represents a new approach for designing enzymes with enhanced activity.

Strain ATCC 4720T is the authentic type strain of Agrobacterium tumefaciens, which is not a later heterotypic synonym of Agrobacterium radiobacter.

The original type strains of Agrobacterium radiobacter and Agrobacterium tumefaciens recorded in the eighth edition of Bergey's Manual of Determinative Bacteriology published in 1974 were NCIB 9042T and ATCC 4720T, respectively. However, in the list of the valid names of bacteria compiled in 1980, both strains were changed, A. radiobacter NCIB 9042T to ATCC 19358T and A. tumefaciens ATCC 4720T to ATCC 23308T. These changes were unjustified, particularly in the case of A. tumefaciens whose type strain was replaced by another strain from the same collection, although the original type strain ATCC 4720T was never lost and it is currently available in several culture collections. Therefore, we request that the type strain of A. tumefaciens be corrected from ATCC 23308T to ATCC 4720T.

Endophthalmitis Caused by Agrobacterium radiobacter following Intravitreal Aflibercept for Diabetic Retinopathy.

Agrobacterium (Rhizobium) radiobacter is a gram-negative bacillus rarely implicated in ocular disease. A 79-year-old male who performed extensive yardwork following intravitreal injection with aflibercept for diabetic macular edema developed endophthalmitis caused by Agrobacterium radiobacter on post-injection day 7. The patient was treated with vitreous tap and intravitreal injection of vancomycin and ceftazidime with clearance of the infection and restoration of his baseline visual acuity at 20/80.

Improved genome of Agrobacterium radiobacter type strain provides new taxonomic insight into Agrobacterium genomospecies 4.

The reported Agrobacterium radiobacter DSM 30174T genome is highly fragmented, hindering robust comparative genomics and genome-based taxonomic analysis. We re-sequenced the Agrobacterium radiobacter type strain, generating a dramatically improved genome with high contiguity. In addition, we sequenced the genome of Agrobacterium tumefaciens B6T, enabling for the first time, a proper comparative genomics of these contentious Agrobacterium species. We provide concrete evidence that the previously reported Agrobacterium radiobacter type strain genome (Accession Number: ASXY01) is contaminated which explains its abnormally large genome size and fragmented assembly. We propose that Agrobacterium tumefaciens be reclassified as Agrobacterium radiobacter subsp. tumefaciens and that Agrobacterium radiobacter retains it species status with the proposed name of Agrobacterium radiobacter subsp. radiobacter. This proposal is based, first on the high pairwise genome-scale average nucleotide identity supporting the amalgamation of both Agrobacterium radiobacter and Agrobacterium tumefaciens into a single species. Second, maximum likelihood tree construction based on the concatenated alignment of shared genes (core genes) among related strains indicates that Agrobacterium radiobacter NCPPB3001 is sufficiently divergent from Agrobacterium tumefaciens to propose two independent sub-clades. Third, Agrobacterium tumefaciens demonstrates the genomic potential to synthesize the L configuration of fucose in its lipid polysaccharide, fostering its ability to colonize plant cells more effectively than Agrobacterium radiobacter.

Enhanced catalytic efficiency and enantioselectivity of epoxide hydrolase from Agrobacterium radiobacter AD1 by iterative saturation mutagenesis for (R)-epichlorohydrin synthesis.

Enantioselective hydrolysis of epoxides by epoxide hydrolase (EH) is one of the most attractive approaches for the synthesis of chiral epoxides. So far, attempts to develop an efficient epoxide hydrolase -mediated biotransformation have been limited by either the low activity or insufficient enantioselectivity of epoxide hydrolase. In this study, iterative saturation mutagenesis (ISM) of epoxide hydrolase from Agrobacterium radiobacter AD1 (ArEH) was performed for efficient production of (R)-epichlorohydrin. Six amino acid residues, I108, A110, D131, I133, T247, and G245, were selected for site saturation mutagenesis, and a sequential combination of positive mutants using ISM was constructed. Targeted mutagenesis generated five mutants (T247K, I108L, D131S, T247K/I108L, and T247K/I108L/D131S) with improved activity and enantioselectivity. Kinetics analysis showed that the best mutant, T247K/I108L/D131S, exhibited a 4.5-fold higher catalytic efficiency (k cat/K m) value and a 2.1-fold higher enantioselectivity (E value) towards epichlorohydrin than the wild-type (WT) enzyme. Molecular docking computations support the source of notably improved enantioselectivity. In addition, the triple mutant also displayed a significantly enhanced thermostability, with > 8-fold longer half-life at 50 °C than WT. A gram-scale kinetic resolution of (R,S)-epichlorohydrin was performed using T247K/I108L/D131S mutant as biocatalyst, affording a (R)-epichlorohydrin yield of 40.2% (> 99.9% enantiomeric excess) and an average productivity of 1410 g L-1 d-1. The engineered T247K/I108L/D131S variant is a promising biocatalyst for the enzymatic synthesis of (R)-epichlorohydrin.

Genomic analysis of Agrobacterium radiobacter DSM 30147(T) and emended description of A. radiobacter (Beijerinck and van Delden 1902) Conn 1942 (Approved Lists 1980) emend. Sawada et al. 1993.

Agrobacterium radiobacter is the only known non-phytopathogenic species in Agrobacterium genus. In this study, the whole-genome sequence of A. radiobacter type strain DSM 30147(T) was described and compared to the other available Agrobacterium genomes. This bacterium has a genome size of 7,122,065 bp distributed in 612 contigs, including 6,834 protein-coding genes and 41 RNA genes. It harbors a circular chromosome and a linear chromosome but not a tumor-inducing (Ti) plasmid. To the best of our knowledge, this is the first report of a genome from the A. radiobacter species. In addition, an emended description of A. radiobacter is described. This study reveals information that enhances the current understanding of its non-phytopathogenicity and its phylogenetic position within Agrobacterium genus.

Avaliação da mutagenicidade e antimutagenicidade de um biopolímero extraído do microorganismo Agrobacterium radiobacter em camundongos Swiss / Assessment of mutagenicity and antimutagenicity of a biopolymer extracted from the microorganism Agrobacterium radiobacter in mice

A presente pesquisa avaliou a ação mutagênica e antimutagênica de um biopolímero de glucose extraído da Agrobacterium radiobacter (Biopolímero de Agrobacterium radiobacter). O experimento foi realizado com camundongos Swiss machos divididos em oito grupos. O tratamento com o biopolímero foi realizado por gavage em dose única concomitante a uma dose de solução tampão fosfato nos grupos de avaliação da mutagenicidade, ou ao agente indutor de danos no DNA, ciclofosfamida, na concentração de 50 mg/kg (peso corpóreo - p.c.), nos grupos de avaliação da antimutagenicidade. Utilizou-se o teste de micronúcleo em sangue periférico e a coleta de sangue foi realizada 24 e 48 h após a aplicação das substâncias-teste. A análise estatística demonstrou que o biopolímero não possui atividade mutagênica e que é efetivo em prevenir danos no DNA. As porcentagens de redução de danos nos grupos de antimutagenicidade foram de 83,9 por cento, 89,1 por cento e 103,1 por cento em 24 h e 101,24 por cento, 98,14 por cento e 120,64 por cento em 48 h para as doses de 75, 150 e 300mg/kg (p.c.), respectivamente. A alta porcentagem de redução de danos associada à ausência de efeitos mutagênicos indica, além da atividade quimioprotetora, a possibilidade do biopolímero ser um alimento funcional candidato à utilização como co-adjuvante na quimioterapia para prevenir efeitos colaterais.

This study evaluated the mutagenic and ant mutagenic action of a biopolymer of glucose extracted from Agrobacterium radiobacter (Biopolymer of Agrobacterium radiobacter). The experiment was conducted with Swiss male mice divided into eight groups. Treatment with the biopolymer was performed in a single dose by gavage at a dose of concomitant phosphate buffer groups in the evaluation of mutagenicity, or the agent of inducing DNA damage, cyclophosphamide, the concentration of 50 mg/kg (body weight --b.w.), in groups of assessment ant mutagenic. We used the micronucleus test in peripheral blood. The blood sample was held 24 and 48 h after application of the test substances. Statistical analysis showed that the biopolymer has no mutagenic activity and it is effective in preventing damage to DNA. The percentages of damage reduction in groups of ant mutagenic were 83.9 percent, 89.1 percent and 103.1 percent in 24 h and 101.24 percent, 98.14 percent and 120.64 percent at doses of 48 to 75, 150 and 300 mg/kg (b.w.) respectively. The high percentage of damage reduction associated with the absence of mutagenic effects indicates the possibility of biopolymer chemoprotection action. It can also be considered a functional food candidate to be used as co-adjuvant chemotherapy to prevent side effects.

A Case of Agrobacterium radiobacter Sepsis following Oral Surgery in a Patient of Oral Cancer / 감염과화학요법

Agrobacterium is an aerobic, motile, oxidase-positive, and non-spore-forming gram-negative bacillus. Under laboratory conditions, Agrobacterium can genetically transform a wide range of other eukaryotic species. A plant-pathogenic soil inhabitant, Agrobacterium radiobacter is not characterized as a true human pathogen. It is an opportunistic pathogen of minor clinical significance and has been substantiated as a rare cause of bacteremia, endocarditis, urinary tract infection and peritonitis mostly in catheterized immunocompromised patients. The authors report a case of a 41-year-old female patient with sepsis caused by A. radiobacter bacteremia following wide excisional biopsy of adenoid-cystic carcinoma involving oral cavity. She was suffering from fever and chilling that developed on second post-operation day. Blood cultures yielded a gram-negative bacillus identified as A. radiobacter. She completely recovered with appropriate antibiotics treatment; levofloxacin and isepamicin. We experienced a case of sepsis due to A. radiobacter bacteremia without indwelling foreign body, which was treated successfully with antibiotics therapy.

A Case of Agrobacterium radiobacter Sepsis following Oral Surgery in a Patient of Oral Cancer / 감염과화학요법

Agrobacterium is an aerobic, motile, oxidase-positive, and non-spore-forming gram-negative bacillus. Under laboratory conditions, Agrobacterium can genetically transform a wide range of other eukaryotic species. A plant-pathogenic soil inhabitant, Agrobacterium radiobacter is not characterized as a true human pathogen. It is an opportunistic pathogen of minor clinical significance and has been substantiated as a rare cause of bacteremia, endocarditis, urinary tract infection and peritonitis mostly in catheterized immunocompromised patients. The authors report a case of a 41-year-old female patient with sepsis caused by A. radiobacter bacteremia following wide excisional biopsy of adenoid-cystic carcinoma involving oral cavity. She was suffering from fever and chilling that developed on second post-operation day. Blood cultures yielded a gram-negative bacillus identified as A. radiobacter. She completely recovered with appropriate antibiotics treatment; levofloxacin and isepamicin. We experienced a case of sepsis due to A. radiobacter bacteremia without indwelling foreign body, which was treated successfully with antibiotics therapy.

Isolation and Characterization of a Nicotine-degrading Strain Z7 / 中国生物工程杂志

A nicotine-degrading bacterial strain Z7 was isolated from tobacco growing soil at Zhangjiajie region in Hunan Province.The morphology,physiological and biochemical characteristics of strain Z7 were studied and the results showed that characteristics of this strain were essentially consistent with Agrobacterium radiobacter/tumefaciens.This strain degraded nicotine optimally at 30℃ and initial pH 7.0.It was able to utilize nicotine as its sole carbon source,and it could degrade 71% of nicotine under the optimized incubation conditions for 48 h.Meanwhile,the color of culture medium turned from yellowy to green and dark green,then it turned to brown.This strain might have potential applications in tobacco industry.

A Case of Agrobacterium Radiobacter Endophthalmitis following Cataract Surgery

PURPOSE: We report a case of Agrobacterium radiobacter endophthalmitis following cataract surgery and treated with pars plana vitrectomy and intravitreal antibiotics injection. METHODS: A 63 year-old male patient was transferred for the endophthalmitis following cataract surgery on his left eye. His initial visual acuity was hand motion. Slit lamp examination showed marked inflammatory cells in anterior chamber and vitreous opacity was seen in fundus examination with indirect ophthalmoscopy. We performed the removal of implanted IOL, pars plana vitrectomy and intravitreal antibiotics injection(Vancomycin 1.0 mg/0.1 ml, Ceftazidime 2.0 mg/0.1 ml). RESULTS: Gram stain results revealed the gram negative rods and bacterial culture identification revealed the Agrobacterium radiobacter using the RapID NF plus system. After 8 weeks, his corrected visual acuity is 0.3 and fundus examination shows the localized atrophic change and preretinal membrane in inferotemporal retina.

A Case of Agrobacterium radiobacter Peritonitis in a Patient on CAPD / 대한신장학회잡지

Agrobacterium radiobacter is a rare human pathogen and a few cases were reported in the world. The peritoneal cavity in patient maintained on peritoneal dialysis serves as an excellent incubator and culture medium for microorganisms. We experienced a rare human infection as peritonitis with A. radiobacter in Inha University Hospital, Inchon, Korea. The patient was 36 year-old female and maintained on CAPD for one year. She had two times of CAPD peritonitis. She visited to the hospital because of turbid peritoneal fluid. On initial physical examination, she showed mild abdominal tenderness and elevated body temperature. Laboratory findings showed leukocytosis and anemia with elevated serum creatinine level. The analysis of peritoneal fluid showed white blood cell count as many as 1860/mm3 and it's culture revealed A. radiobacter. She received vancomycin and aminoglycoside antibiotics via intraperitoneal route and the CAPD catheter was removed. She was maintained on hemodialysis and reveived isepamicin 100mg a day via intravenous for 7 days. We report our experience of peritonitis caused by A. radiobacter and review the literature of similar cases of peritonitis.