A peptidoglycan N-deacetylase specific for anhydroMurNAc chain termini in Agrobacterium tumefaciens.

During growth, bacteria remodel and recycle their peptidoglycan (PG). A key family of PG-degrading enzymes is the lytic transglycosylases, which produce anhydromuropeptides, a modification that caps the PG chains and contributes to bacterial virulence. Previously, it was reported that the polar-growing Gram-negative plant pathogen Agrobacterium tumefaciens lacks anhydromuropeptides. Here, we report the identification of an enzyme, MdaA (MurNAc deacetylase A), which specifically removes the acetyl group from anhydromuropeptide chain termini in A. tumefaciens, resolving this apparent anomaly. A. tumefaciens lacking MdaA accumulates canonical anhydromuropeptides, whereas MdaA was able to deacetylate anhydro-N-acetyl muramic acid in purified sacculi that lack this modification. As for other PG deacetylases, MdaA belongs to the CE4 family of carbohydrate esterases but harbors an unusual Cys residue in its active site. MdaA is conserved in other polar-growing bacteria, suggesting a possible link between PG chain terminus deacetylation and polar growth.

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.

[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.

Two Novel Genomospecies in the Agrobacterium tumefaciens Species Complex Associated with Rose Crown Gall.

In this study, we explored the pathogenicity and phylogenetic position of Agrobacterium spp. strains isolated from crown gall tissues on annual, perennial, and ornamental plants in Iran. Of the 43 strains studied, 10 strains were identified as Allorhizobium vitis (formerly Agrobacterium vitis) using the species-specific primer pair PGF/PGR. Thirty-three remaining strains were studied using multilocus sequence analysis of four housekeeping genes (i.e., atpD, gyrB, recA, and rpoB), from which seven strains were identified as A. larrymoorei and one strain was identified as A. rubi (Rer); the remaining 25 strains were scattered within the A. tumefaciens species complex. Two strains were identified as genomospecies 1 (G1), seven strains were identified as A. radiobacter (G4), seven strains were identified as A. deltaense (G7), two strains were identified as A. nepotum (G14), and one strain was identified as "A. viscosum" (G15). The strains Rnr, Rnw, and Rew as well as the two strains OT33 and R13 all isolated from rose and the strain Ap1 isolated from apple were clustered in three atypical clades within the A. tumefaciens species complex. All but eight strains (i.e., Nec10, Ph38, Ph49, fic9, Fic72, R13, OT33, and Ap1) were pathogenic on tomato and sunflower seedlings in greenhouse conditions, whereas all but three strains (i.e., fic9, Fic72, and OT33) showed tumorigenicity on carrot root discs. The phylogenetic analysis and nucleotide diversity statistics suggested the existence of two novel genomospecies within the A. tumefaciens species complex, which we named "G19" and "G20." Hence, we propose the strains Rew, Rnw, and Rnr as the members of "G19" and the strains R13 and OT33 as the members of G20, whereas the phylogenetic status of the atypical strain Ap1 remains undetermined.

An extra repABC locus in the incRh2 Ti plasmid pTiBo542 exerts incompatibility toward an incRh1 plasmid.

Ti/Ri plasmids in pathogenic Agrobacterium species are repABC replicons that are stably maintained by the function of repABC genes. Two Ti plasmids, pTiBo542 and pTiS4, belonging to incRh2 and incRh4 incompatibility groups, respectively, were reported to carry two repABC loci. In the present study, to reveal the roles of the two repABC loci in the two plasmids, we constructed mini-replicons carrying any one or both of the repABC loci (referred to as repABC1 and repABC2 here) and examined their replication and incompatibility properties. The introduction of mini-replicons into A. tumefaciens C58C1 strains suggested that repABC1 functions as replicator genes but repABC2 does not in both the Ti plasmids. Because the components of repABC2 of pTiBo542 have highly similar amino acid and nucleotide sequences to those of the incRh1-type repABC replicon, we introduced repABC2-containing replicons into cells harboring an incRh1 plasmid in order to check their incompatibility traits. As a result, the repABC2-containing replicon expelled the resident incRh1 plasmid, indicating that the extra repABC locus is dispensable for replication and could work as an incompatibility determinant against incRh1 group plasmids. We suggest that the locus contributes to plasmid retention by eliminating the burden of co-existing competitive plasmids in host cells through its incompatibility.

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.

Agrobacterium radiobacter (Beijerinck and van Delden 1902) Conn 1942 has priority over Agrobacterium tumefaciens (Smith and Townsend 1907) Conn 1942 when the two are treated as members of the same species based on the principle of priority and Rule 23a, Note 1 as applied to the corresponding specific epithets. Opinion 94. Judicial Commission of the International Committee on Systematics of Prokaryotes.

The Judicial Commission affirms that, according to the Rules of the International Code of Nomenclature of Bacteria (including changes made to the wording), the combination Agrobacterium radiobacter (Beijerinck and van Delden 1902) Conn 1942 has priority over the combination Agrobacterium tumefaciens (Smith and Townsend 1907) Conn 1942 when the two are treated as members of the same species based on the principle of priority as applied to the corresponding specific epithets. The type species of the genus is Agrobacterium tumefaciens (Smith and Townsend 1907) Conn 1942, even if treated as a later heterotypic synonym of Agrobacterium radiobacter (Beijerinck and van Delden 1902) Conn 1942. Agrobacterium tumefaciens (Smith and Townsend 1907) Conn 1942 is typified by the strain defined on the Approved Lists of Bacterial Names and by strains known to be derived from the nomenclatural type.

Recruitment of conjugative DNA transfer substrate to Agrobacterium type IV secretion apparatus.

Bacterial type IV secretion system (T4SS) belongs to a growing class of evolutionarily conserved transporters that translocate DNA and proteins into a wide variety of organisms including bacterial and eukaryotic cells. Archetypal is the Agrobacterium tumefaciens VirB/D4 T4SS that transfers oncogenic T-DNA to various eukaryotic cells, which is transferred as a nucleoprotein T-complex with VirD2 as the pilot protein. As a derivative of plasmid conjugation systems, the VirB/D4 T4SS can also transfer certain mobilizable plasmids and bacterial proteins like VirE2 and VirF, although it is unknown how the membrane-bound T4SS recruits different transfer substrates. Here, we show that a cytoplasmic VirD2-binding protein (VBP) is involved in the recruitment of the T-complex to the energizing components of the T4SS, including VirD4, VirB4, and VirB11. VBP is also important for the recruitment of a conjugative plasmid to a different transfer system independent of VirB/D4. These data indicate that VBP functions as a previously unrecognized recruiting protein that helps couple nucleoprotein substrates to the appropriate transport sites for conjugative DNA transfers. VBP has three functionally redundant homologs, and similar homologs can be found in different bacterial genomes, suggesting a previously uncharacterized class of proteins involved in conjugative DNA transfers.

Pseudo-outbreak of Rhizobium radiobacter infection resulting from laboratory contamination of saline solution.

We report a pseudo-outbreak of Rhizobium radiobacter infections resulting from contamination by a saline dispenser in the microbiology laboratory. Isolates from clinical specimens had identical antimicrobial susceptibilities and electrophoretic fingerprints. The episode resolved with autoclaving of the dispenser. This demonstrates the importance of timely, thorough investigation of unusual organisms, particularly when they appear as a cluster.

Genome sequence of the plant pathogen and biotechnology agent Agrobacterium tumefaciens C58.

Agrobacterium tumefaciens is a plant pathogen capable of transferring a defined segment of DNA to a host plant, generating a gall tumor. Replacing the transferred tumor-inducing genes with exogenous DNA allows the introduction of any desired gene into the plant. Thus, A. tumefaciens has been critical for the development of modern plant genetics and agricultural biotechnology. Here we describe the genome of A. tumefaciens strain C58, which has an unusual structure consisting of one circular and one linear chromosome. We discuss genome architecture and evolution and additional genes potentially involved in virulence and metabolic parasitism of host plants.

The genome of the natural genetic engineer Agrobacterium tumefaciens C58.

The 5.67-megabase genome of the plant pathogen Agrobacterium tumefaciens C58 consists of a circular chromosome, a linear chromosome, and two plasmids. Extensive orthology and nucleotide colinearity between the genomes of A. tumefaciens and the plant symbiont Sinorhizobium meliloti suggest a recent evolutionary divergence. Their similarities include metabolic, transport, and regulatory systems that promote survival in the highly competitive rhizosphere; differences are apparent in their genome structure and virulence gene complement. Availability of the A. tumefaciens sequence will facilitate investigations into the molecular basis of pathogenesis and the evolutionary divergence of pathogenic and symbiotic lifestyles.

Cyanuric acid biodegradation by a mixed bacterial culture of Agrobacterium tumefaciens and Acinetobacter sp. in a packed bed biofilm reactor.

Cyanuric acid (1,3,5-triazine-2,4,6-triol [OOOT]) is a common biodegradation byproduct of triazinic herbicides, frequently accumulated in soils or water when supplementary carbon sources are absent. A binary bacterial culture able to degrade OOOT was selected through a continuous selection process accomplished in a chemostat fed with a mineral salt (MS) medium containing cyanuric acid as the sole carbon and nitrogen source. By sequence comparison of their 16S rDNA amplicons, bacterial strains were identified as Agrobacterium tumefaciens, and Acinetobacter sp. When the binary culture immobilized in a packed bed reactor (PBR) was fed with MS medium containing OOOT (50 mg L(-1)), its removal efficiencies were about 95%; when it was fed with OOOT plus glucose (120 mg L(-1)) as a supplementary carbon source, its removal efficiencies were closer to 100%. From sessile cells, attached to PBR porous support, or free cells present in the outflowing medium, DNA was extracted and used for Random Amplification of Polymorphic DNA analysis. Electrophoretic patterns obtained were compared to those of pure bacterial strains, a clear predominance of A. tumefaciens in PBR was observed. Although in continuous suspended cell culture, a stable binary community could be maintained, the attachment capability of A. tumefaciens represented a selective advantage over Acinetobacter sp. in the biofilm reactor, favoring its predominance in the porous stone support.

Efficient generation of transgenic barley: the way forward to modulate plant-microbe interactions.

Stable genetic transformation represents the gold standard approach to the detailed elucidation of plant gene functions. This is particularly relevant in barley, an important experimental model widely employed in applied molecular, genetic and cell biological research, and biotechnology. Presented are details of the establishment of a protocol for Agrobacterium-mediated gene transfer to immature embryos, which enables the highly efficient generation of transgenic barley. Advancements were achieved through comparative experiments on the influence of various explant treatments and co-cultivation conditions. The analysis of representative numbers of transgenic lines revealed that the obtained T-DNA copy numbers are typically low, the generative transmission of the recombinant DNA is in accordance with the Mendelian rules and the vast majority of the primary transgenics produce progeny that expresses the respective transgene product. Moreover, the newly established protocol turned out to be useful to transform not only the highly amenable cultivar (cv.) 'Golden Promise' but also other spring and winter barley genotypes, albeit with substantially lower efficiency. As a major result of this study, a very useful tool is now available for future functional gene analyses as well as genetic engineering approaches. With the aim to modify the expression of barley genes putatively involved in plant-fungus interactions, numerous transgenic plants have been generated using diverse expression cassettes. These plants represent an example of how transformation technology may contribute to further our understanding of important biological processes.

Primary bacteraemia caused by Rhizobium radiobacter in a patient with solid tumours.

A case of Rhizobium radiobacter primary bacteraemia in a patient with solid tumours is reported. Corticosteroid therapy and diabetes mellitus were the predisposing factors. The patient was treated successfully with amikacin and piperacillin/tazobactam. The clinical isolate was identified as R. radiobacter by 16S rRNA gene sequencing. Phytopathogenicity tests and a PCR assay demonstrated that the organism was not a plant pathogen.

Systematic determination of the mosaic structure of bacterial genomes: species backbone versus strain-specific loops.

BACKGROUND: Public databases now contain multitude of complete bacterial genomes, including several genomes of the same species. The available data offers new opportunities to address questions about bacterial genome evolution, a task that requires reliable fine comparison data of closely related genomes. Recent analyses have shown, using pairwise whole genome alignments, that it is possible to segment bacterial genomes into a common conserved backbone and strain-specific sequences called loops. RESULTS: Here, we generalize this approach and propose a strategy that allows systematic and non-biased genome segmentation based on multiple genome alignments. Segmentation analyses, as applied to 13 different bacterial species, confirmed the feasibility of our approach to discern the 'mosaic' organization of bacterial genomes. Segmentation results are available through a Web interface permitting functional analysis, extraction and visualization of the backbone/loops structure of documented genomes. To illustrate the potential of this approach, we performed a precise analysis of the mosaic organization of three E. coli strains and functional characterization of the loops. CONCLUSION: The segmentation results including the backbone/loops structure of 13 bacterial species genomes are new and available for use by the scientific community at the URL: http://genome.jouy.inra.fr/mosaic.

Diverse bacteria isolated from root nodules of Phaseolus vulgaris and species within the genera Campylotropis and Cassia grown in China.

Eighty bacterial isolates from root nodules of the leguminous plants Phaseolus vulgaris, Campylotropis spp. and Cassia spp. grown in China were classified into five groups by phenotypic analyses, SDS-PAGE of whole-cell proteins, PCR-based 16S rRNA gene restriction-fragment-length-polymorphism and sequencing. Thirty-three isolates from the three plant genera were identified as Agrobacterium tumefaciens because they are closely related to the type strain of A. tumefaciens. Fourteen isolates from P. vulgaris grown in Yunnan and Inner Mongolia were classified as R. leguminosarum bv. phaseoli based on their close relationship with the type strain in numerical taxonomy and in 16S rDNA phylogeny. Twenty-seven isolates from Campylotropis delavayi, P. vulgaris and four species of Cassia grown in the central zones of China were classified into three groups within the genus Bradyrhizobium. One of these three groups could be defined as Bradyrhizobium japonicum. Our results demonstrated that P. vulgaris and the species of Campylotropis and Cassia could form nodules with diverse rhizobia in Chinese soils, including novel lineages associated with P. vulgaris. These results also offered information about the convergent evolution between rhizobia and legumes since the rhizobial populations associated with P. vulgaris in Chinese soils were completely different from those in Mexico, the original cite of this plant. Some rhizobial species could be found in all of the three leguminous genera.

Development of auxotrophic agrobacterium tumefaciens for gene transfer in plant tissue culture.

Auxotrophic strains of Agrobacterium tumefaciens were generated for use in liquid co-culture with plant tissue for transient gene expression. Twenty-one auxotrophs were recovered from 1,900 tetracycline-resistant insertional mutants generated with a suicide vector transposon mutagenesis system. Twelve of these auxotrophs were characterized on a nutrient matrix. Isolates were screened for growth in plant cell and root culture, and three auxotrophs were identified that had limited growth: adenine (ade-24), leucine (leu-27), and cysteine (cys-32). Ade-24 displayed poor T-DNA delivery in a transient expression test delivering GUS from a binary vector, while cys-32 displayed the best ability to deliver DNA of these three auxotrophs. The growth yield of cys-32 on cysteine was assessed to provide a quantitative basis for co-culture nutrient supplementation. The utility of cys-32 for delivering T-DNA to plant tissues is demonstrated, where an 85-fold enhancement in GUS expression over wild-type A. tumefaciens was achieved.

[Isolation and identification of Agrobacterium spp. from cherry crown galls and their sensitivities to agrocin 84].

Crown galls were sampled from cherry yards of Shandong, Hebei, and Liaoning provinces. 46 pathogenic strains were isolated. Physiological and biochemical tests revealed that 4 strains were Agrobacterium tumefaciens(bio. 1) and that the other 42 strains were A. rhizogenes (bio. 2), according to the classifications and nomenclatures of the genus Agrobacterium and the species revised by Sawada et al and Bouzar. The Ti plasmids of all strains were nopaline type. All strains were sensitive to agrocin 84, which suggested that crown gall disease of cherry could be controlled by K84 strain.

Genetic diversity and symbiotic evolution of rhizobia from root nodules of Coronilla varia.

Ninety symbiotic rhizobial isolates from root nodules of Coronilla varia growing in the Shaanxi province of China were characterized. Combined with the results of RFLP patterns, six genotypes were defined among the rhizobial strains and they were divided into three genomic genera. These included Mesorhizobium sp., M. alhagi, M. amorphae, M. metallidurans/M. gobiense as the dominant group (86.7%), and Rhizobium yanglingense and Agrobacterium tumefaciens as the minor groups, according to analysis of the corresponding 16S rRNA, nodC and nifH genes. Five nodC types, which mainly grouped into the Mesorhizobium genus, were obtained from all the isolates examined, implying that nodC genes probably occurred from the native habitat through lateral transfer and long-term adaptation, finally evolving toward M. alhagi. Four different nifH types, displaying obvious differences compared to those of 16S rRNA and nodC, implied that possible lateral transfer of the symbiotic genes occurred between different genera. The association between soil components and the genetic diversity of the rhizobial population demonstrated that combined genotypes were positively correlated with the pH of soil samples.