Agrobacterium Species Research Articles

Phylogenetic diversity of Rhizobium species recovered from nodules of common beans (Phaseolus vulgaris L.) in fields in Uganda: R. phaseoli, R. etli, and R. hidalgonense.

A total of 75 bacterial isolates were obtained from nodules of beans cultivated across 10 sites in six agro-ecological zones in Uganda. Using recA gene sequence analysis, 66 isolates were identified as members of the genus Rhizobium, while nine were related to Agrobacterium species. In the recA gene tree, most Rhizobium strains were classified into five recognized species. Phylogenetic analysis based on six concatenated sequences (recA-rpoB-dnaK-glnII-gyrB-atpD) placed 32 representative strains into five distinct Rhizobium species, consistent with the species groups observed in the recA gene tree: R. phaseoli, R. etli, R. hidalgonense, R. ecuadorense, and R. sophoriradicis, with the first three being the predominant. The rhizobial strains grouped into three nodC subclades within the symbiovar phaseoli clade, encompassing strains from distinct phylogenetic groups. This pattern reflects the conservation of symbiotic genes, likely acquired through horizontal gene transfer among diverse rhizobial species. The 32 representative strains formed symbiotic relationships with host beans, while the Agrobacterium strains did not form nodules and lacked symbiotic genes. Multivariate analysis revealed that species distribution was influenced by the environmental factors of the sampling sites, emphasizing the need to consider these factors in future effectiveness studies to identify effective nitrogen-fixing strains for specific locations.

Physicochemical and Rheological Properties of Succinoglycan Overproduced by Sinorhizobium meliloti 1021 Mutant.

Commercial bacterial exopolysaccharide (EPS) applications have been gaining interest; therefore, strains that provide higher yields are required for industrial-scale processes. Succinoglycan (SG) is a type of bacterial anionic exopolysaccharide produced by Rhizobium, Agrobacterium, and other soil bacterial species. SG has been widely used as a pharmaceutical, cosmetic, and food additive based on its properties as a thickener, texture enhancer, emulsifier, stabilizer, and gelling agent. An SG-overproducing mutant strain (SMC1) was developed from Sinorhizobium meliloti 1021 through N-methyl-N'-nitro-N-nitrosoguanidine (NTG) mutation, and the physicochemical and rheological properties of SMC1-SG were analyzed. SMC1 produced (22.3 g/L) 3.65-fold more SG than did the wild type. Succinoglycan (SMC1-SG) overproduced by SMC1 was structurally characterized by FT-IR and 1H NMR spectroscopy. The molecular weights of SG and SMC1-SG were 4.20 × 105 and 4.80 × 105 Da, respectively, as determined by GPC. Based on DSC and TGA, SMC1-SG exhibited a higher endothermic peak (90.9 °C) than that of SG (77.2 °C). Storage modulus (G') and loss modulus (G″) measurements during heating and cooling showed that SMC1-SG had improved thermal behavior compared to that of SG, with intersections at 74.9 °C and 72.0 °C, respectively. The SMC1-SG's viscosity reduction pattern was maintained even at high temperatures (65 °C). Gelation by metal cations was observed in Fe3+ and Cr3+ solutions for both SG and SMC1-SG. Antibacterial activities of SG and SMC1-SG against Escherichia coli and Staphylococcus aureus were also observed. Therefore, like SG, SMC1-SG may be a potential biomaterial for pharmaceutical, cosmetic, and food industries.

Biomedical Relevance of Biological Smart Materials Approach for Synthesis of Selenium Nanoparticles by Agrobacterium

Nanotechnologies are making more efforts to develop new materials to use in different ways to be beneficial to human life. Their unique properties are constantly being improved to enhance more effective and perfect activity as per the requirement. Researchers are continuously producing highly useful materials with the help of different types of microorganisms and green technology for the synthesis of nanoparticles as per the requirement. Primarily Agrobacterium species are the most useful microorganisms for the synthesis of selenium nanoparticles in the diameter range of nanoscale. These are capable and more effective in making nanomaterials from selenium to use in medicinal and other applications. These are opening a new dimension in the field of medical science which is beneficial for multipurpose use directly or indirectly.

Impact of Using Date Juice as a Carbon Source on Curdlan Produced by a Local Isolate of Agrobacterium leguminum

Agrobacterium species are responsible for the production of the polysaccharide known as curdlan. The curdlan was produced from 10 isolates that were collected from a variety of local sources, including as agricultural soils, root nodules, and plant roots. The isolates were identified by examinations using morphological, microscopic, and biochemical tests. After testing the isolates to see which ones could produce the most curdlan, the A2 isolate finally emerged with a production capacity of 29.2 g.L-1. According to the findings, the production of curdlan was increased using the modified medium that contained date juice at a concentration of 26.4 mL.100 mL-1 of the production medium. The resultant yield was 30.7 g.L-1, which was the highest possible yield. The identification of curdlan was validated by the utilization of FTIR, NMR, and HPLC techniques. The modified medium had the capability of being utilized in the production of curdlan from agricultural waste products.

Genome Sequencing of Efficient Plant Growth-Promoting Bacteria for Lowland Rice.

The genomes of five elite strains identified as growth promoters of lowland rice (Oryza sativa L.) in Brazil were sequenced. They ranged in size from 3,695,387 bp to 5,682,101 bp, encompassing genes of saprophytic ability and stress tolerance. Genome taxonomy enabled their classification as Priestia megaterium, Bacillus altitudinis, and three putative new species of Pseudomonas, Lysinibacillus, and Agrobacterium.

Phylogenomics reveals insights into the functional evolution of the genus Agrobacterium and enables the description of Agrobacterium divergens sp. nov

The genus Agrobacterium was initially described as mainly phytopathogenic strains. Nowadays, the genus includes phytopathogenic and non-phytopathogenic bacteria that are distinctive among the Rhizobiaceae family. Recently we have isolated two closely related strains, LMG 31531T and LMG 31532, from soil and plant roots, respectively. Both strains differ from previously reported species based on the genomic and phenotypic data. A. arsenijevicii KFB 330T and A. fabacearum LMG 31642T showed the highest 16S rRNA similarity (98.9 %), followed by A. nepotum LMG 26435T (98.7 %). A clear genomic feature that distinguishes LMG 31531T and LMG 31532 from other Agrobacterium species is the absence of a linear chromid. Nevertheless, typical values of the core-proteome Average Amino Acid Identity (cpAAI > 85 %) and 16S rRNA gene sequence similarity (>96 %) when compared to other members of the genus confirm the position of these two strains as part of the Agrobacterium genus. They are therefore described as Agrobacterium divergens sp. nov. Besides, our comparative genomic study and survey for clade-specific markers resulted in the discovery of conserved proteins that provide insights into the functional evolution of this genus.

Agrobacterium cucumeris sp. nov. isolated from crazy roots on cucumber (Cucumis sativus)

Three plant rhizogenic strains O132T, O115 and O34 isolated from Cucumis sp. L. were assessed for taxonomic affiliation by using polyphasic taxonomic methods. Based on the results of the sequence analysis of the 16S rRNA and multilocus sequence analysis (MLSA) of the three housekeeping genes atpD, recA and rpoB, all the strains were clustered within the genus Agrobacterium where they form a novel branch. Their closest relative was Agrobacterium tomkonis (genomospecies G3). Moreover, digital DNA-DNA hybridization (dDDH) and average nucleotide identity (ANI) comparisons between strains O132T and O34 and their closest relatives provided evidence that they constitute a new species, because the obtained values were significantly below the threshold considered as a borderline for the species delineation. Whole-genome phylogenomic analysis also indicated that the cucumber strains are located within the separate, well-delineated biovar 1 sub-clade of the genus Agrobacterium. Furthermore, the physiological and biochemical properties of these strains allowed to distinguish them from their closest related species of the genus Agrobacterium. As a result of the performed overall characterization, we propose a new species as Agrobacterium cucumeris sp. nov., with O132T (=CFBP 8997T = LMG 32451T) as the type strain.

CRISPR RNA-guided integrase enables high-efficiency targeted genome engineering in Agrobacterium tumefaciens.

Summary Agrobacterium tumefaciens, the causal agent of plant crown gall disease, has been widely used to genetically transform many plant species. The inter‐kingdom gene transfer capability made Agrobacterium an essential tool and model system to study the mechanism of exporting and integrating a segment of bacterial DNA into the plant genome. However, many biological processes such as Agrobacterium‐host recognition and interaction are still elusive. To accelerate the understanding of this important plant pathogen and further improve its capacity in plant genetic engineering, we adopted a CRISPR RNA‐guided integrase system for Agrobacterium genome engineering. In this work, we demonstrate that INsertion of Transposable Elements by Guide RNA–Assisted TargEting (INTEGRATE) can efficiently generate DNA insertions to enable targeted gene knockouts. In addition, in conjunction with Cre‐loxP recombination system, we achieved precise deletions of large DNA fragments. This work provides new genetic engineering strategies for Agrobacterium species and their gene functional analyses.

Abstract 3058: Characterization of the tumor microbiome in patients with locally advanced rectal cancer treated with neoadjuvant therapy

Abstract The gut microbiota is a component of the tumor microenvironment that has the potential to influence sensitivity or resistance to neoadjuvant therapy through modulation of the host immune response in patients diagnosed with locally advanced rectal cancer (LARC, stage II-III). We performed DNA and RNA sequencing of pre-treatment endoscopic biopsies from 89 LARC patients treated with neoadjuvant therapy (NAT). Our cohort included 84 microsatellite stable and 5 microsatellite unstable (MSI) patients. We identified the presence of microbial species by mapping non-human RNA read sequences to a large catalogue of viral and bacterial microorganisms. Our methodology has been validated and determined to have comparable accuracy to reference microbial tests for both viruses and bacteria. We analyzed correlations between presence of specific microbial species and (1) clinicopathological variables such as age, stage, anatomic location and MSI status, (2) genomic variables such as tumor mutational burden, chromosomal instability and somatic alterations and (3) response to NAT. We identified 152 species with at least 100 mapped reads in 10% or more of our cohort. The median number of species per patient was 37 [IQR 16-66], with only 6/89, 7% patients having no detected species at all. The most frequently detected species included well-known intestinal bacteria such as Escherichia coli, Fusobacterium nucleatum, Faecalibacterium prausnitzii and several members of the Bacteroides genus. The Parvimonas micra coccus was more often detected in tumors from the lower rectum (13/22, 59%, 0-4 cm from the anal verge) than the middle (8/36, 22%, 4-8 cm) and upper rectum (5/30, 17%, 8-12 cm) (p<0.01). Several species from the Gordonia and Rhodococcus genera were associated with increased tumor mutational burden (p<0.001) and significantly higher numbers of mapped reads in samples from the CMS1 molecular subtype (p<0.01), which is enriched in MSI tumors. RNA-Seq based estimates of neutrophil abundance were positively correlated with detection of several Ruminococcus and Prevotella species (p<0.001), highlighting the cross-talk between host immunity and microbiota. Opportunistic bacteria previously reported to benefit from a patient’s weakened immune system such as Williamsia muralis and several species of Agrobacterium were associated with lower rates of complete response (30% vs. 15%, n.s.) and significantly shorter disease free survival following NAT (3-year disease free survival was 25% in 8 Williamsia positive patients vs. 42% in 12 Agrobacterium fabrum patients vs. 87% in the rest of the cohort; pairwise log-rank p<0.001). Our data shows that standard RNA sequencing platforms can be used to detect microbial species in endoscopic biopsies from LARC patients and that the presence of specific species is associated with clinical and genomic features of translational relevance. Citation Format: Chad M. Vanderbilt, Walid K. Chatila, Danny N. Khalil, Chin-Tung Chen, Jin K. Kim, Fan Wu, Nikolaus Schultz, Martin R. Weiser, Julio Garcia-Aguilar, Francisco Sanchez-Vega. Characterization of the tumor microbiome in patients with locally advanced rectal cancer treated with neoadjuvant therapy [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 3058.

Contrasting effects of the inoculation time with passenger endophytic Agrobacterium sp.10C2 on the nodule functioning and growth of Medicago truncatula

Agrobacterium species are one of the most frequently isolated endophytes from root nodules of a wide range of wild and cultivated legumes. Many studies have investigated the role of endophytes in phytoprotection and phytoremediation enhancement. However, their biological significance and their implication in nodule functioning have been scarcely studied, especially, the effect of the inoculation time on nodule activity and plant growth which still unknown. Accordingly, in this study, nodule-endophytic Agrobacterium sp.10C2 was inoculated before and after nodule formation, to determine the effect of the inoculation time on the plant growth and nodule functioning in the reference line Jemalong A17 of the model legume Medicago truncatula in symbiotic association with Sinorhizobium meliloti TII7. The results revealed that nodule functioning was strongly influenced by the inoculation time of the endophytic Agrobacterium sp.10C2. In fact, the early inoculation with Agrobacterium sp.10C2 induced an increase of the highest nodules nitrogenase activity, recorded at the day 66 after the inoculation, by 66%, the leghemoglobin concentration by 26% and the total sugar content by 67%, in addition to a higher photosynthesis activity and biomass plant production compared to the control. Interestingly, when inoculation occurred after nodule formation, nodules have induced a defense response against Agrobacterium sp. 10C2 evidenced by an overproduction of guaicol peroxidase which increased from 1.04 μmol H2O2 min-1 mg-1 proteins in the control to 2.36 μmol H2O2 min-1 mg-1 proteins in the late inoculated plants, leading to a loss of the beneficial impact of the endophytic Agrobacterium on both biomass production and nodule functioning parameters. We showed that nodule response to endophytic agrobacteria change according to the time of inoculation. When inoculated at early stage, 10C2 plays the role of a Plant Growth Promoting Rhizobacteria (PGPR), however, after nodule formation, 10C2 could act as a pathogen agent. Thus, the inoculation time could be considered as a crucial parameter for the nodule functioning and plant growth.

Agrobacterium vaccinii sp. nov. isolated from galls on blueberry plants (Vaccinium corymbosum)

Four non-pathogenic strains isolated from the galls on blueberry plants (Vaccinium corymbosum) were characterized by using polyphasic taxonomic methods. Based on 16S rRNA gene phylogeny, strains were clustered within the genus Agrobacterium. Furthermore, multilocus sequence analysis (MLSA) based on the partial sequences of atpD, recA and rpoB housekeeping genes and whole-genome-based phylogeny indicated that the strains studied form a novel Agrobacterium species. Analyses showed that the strains belong to “rubi” sub-clade of Agrobacterium genus and their closest relatives are Agrobacterium rubi and “Agrobacterium bohemicum”. Average nucleotide identity (ANI) and digital DNA–DNA hybridization (dDDH) comparisons between genome sequences of representative strains B7.6T and B19.1.4, and their closest relatives, confirmed the distinct phylogenetic position of studied strains, because obtained values were considerably below the proposed thresholds for the species delineation. The four strains studied were phenotypically distinguishable from other species of the genus Agrobacterium. Overall, polyphasic characterization showed that the strains studied represent a novel species of the genus Agrobacterium, for which the name Agrobacterium vaccinii sp. nov. is proposed. The type strain of A. vaccinii is B7.6T (=CFBP 8740T = LMG 31849T).

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 Kuzmanović 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.

Occurrence of Crown Gall Disease on Japanese Spindle (Euonymus japonicas var. Green Rocket) Caused by Agrobacterium rosae in Iran.

Crown gall disease caused by diverse Agrobacterium species is one of the main biotic constraints in the ornamental plants industry in Iran (Mafakheri et al. 2017). In August 2019, Japanese spindle (Euonymus japonicus var. Green Rocket) plants showing crown gall symptoms were observed in a commercial greenhouse in Tehran, Iran. Infected plants were characterized by a visible overgrowth on their stems and crown. Bacterial isolation from the gall tissues was performed on nutrient agar (NA) and 1A media as described by Moore et al. (2001). The six resulted bacterial strains (A.E1 to A.E6) were evaluated using PCR primer pair F8360/F8361 amplifying a 453 bp DNA fragment in recA gene and confirmed as Agrobacterium sp. (Shams et al. 2013). Pathogenicity of the strains was evaluated in two independent assays on Japanese spindle plantlets as well as 10-15 day old tomato (Solanum lycopersicum cv. Sunseed 6189) and sunflower (Helianthus annuus cv. Armavirski) plants in greenhouse conditions using the needle prick method as described previously (Mafakheri et al. 2019). The reference strain A. radiobacter ICMP 5856 and sterile distilled water were used as positive and negative controls, respectively. Crown gall symptoms appeared 20-25 days post inoculation on the Japanese spindle plantlets as well as tomato and sunflower plants inoculated with the strains isolated in this study, while the negative control plants remained asymptomatic. Koch's postulates were accomplished by re-isolating on NA medium and PCR-based identification of the inoculated strains from the symptomatic plants. The representative strain A.E1 was subjected to multilocus sequence analysis (MLSA) using the sequences of four housekeeping genes (i.e. atpD, gyrB, recA, and rpoB) as described previously (Mafakheri et al. 2019). MLSA results revealed that the strain A.E1 is phylogenetically closely related to A. rosae. The sequences were deposited into GenBank under the accession numbers MT007962 to MT007965 for atpD, gyrB, recA, and rpoB, respectively. Further, the strain A.E1 was subjected to whole genome sequencing using Illumina HiSeq X platform. DNA extraction was performed using NucleoSpin Microbial DNA kit (Macherey-Nagel, Germany), DNA libraries were obtained with Nextera XT DNA Library Prep Kit (Illumina, USA), and de novo sequence assembly was performed using SPAdes genome assembler. The resulting whole genome sequence was deposited into the GenBank database under the accession number JAFJZW000000000. Average nucleotide identity (ANI) and digital DNA-DNA hybridization (dDDH) values were calculated among all the type strains of Agrobacterium species/genomospecies using standard criteria as detailed previously (Osdaghi et al. 2020; Chen et al. 2021). The strain A.E1 had 97% ANI and 72% dDDH values with A. rosae strain NCPPB 1650, suggesting that the bacterial strains isolated from Japanese spindle in Iran belong to A. rosae. This is the first report of A. rosae causing crown gall disease on Japanese spindle in Iran. The new crown gall disease could negatively affect the ornamental shrub production industry in central Iran unless strict sanitary measures are taken into the account in the nurseries in these areas. Further nationwide surveys and samplings are warranted to elucidate the economic impact of the pathogen on ornamental plant industry in the country.

Comparative Genomics of Novel Agrobacterium G3 Strains Isolated From the International Space Station and Description of Agrobacterium tomkonis sp. nov.

Strains of Agrobacterium genomospecies 3 (i.e., genomovar G3 of the Agrobacterium tumefaciens species complex) have been previously isolated from diverse environments, including in association with plant roots, with algae, as part of a lignocellulose degrading community, from a hospital environment, as a human opportunistic pathogen, or as reported in this study, from a surface within the International Space Station. Polyphasic taxonomic methods revealed the relationship of Agrobacterium G3 strains to other Agrobacterium spp., which supports the description of a novel species. The G3 strains tested (n = 9) were phenotypically distinguishable among the strains from other genomospecies of the genus Agrobacterium. Phylogenetic analyses of the 16S rRNA gene, gyrB gene, multi-locus sequence analysis, and 1,089-gene core-genome gene concatenate concur that tested G3 strains belong to the Agrobacterium genus and they form a clade distinct from other validly described Agrobacterium species. The distinctiveness of this clade was confirmed by average nucleotide identity (ANI) and in silico digital DNA–DNA hybridization (dDDH) comparisons between the G3 tested strains and all known Agrobacterium species type strains, since obtained values were considerably below the 95% (ANI) and 70% (dDDH) thresholds used for the species delineation. According to the core-genome phylogeny and ANI comparisons, the closest relatives of G3 strains were Agrobacterium sp. strains UGM030330-04 and K599, members of a novel genomospecies we propose to call genomovar G21. Using this polyphasic approach, we characterized the phenotypic and genotypic synapomorphies of Agrobacterium G3, showing it is a bona fide bacterial species, well separated from previously named Agrobacterium species or other recognized genomic species. We thus propose the name Agrobacterium tomkonis for this species previously referred to as Agrobacterium genomospecies 3. The type strain of A. tomkonis is IIF1SW-B1T (= LMG 32164 = NRRL B-65602). Comparative genomic analysis show A. tomkonis strains have species-specific genes associated with secretion of secondary metabolites, including an exopolysaccharide and putative adhesins and resistance to copper. A. tomkonis specific gene functions notably relate to surface adhesion and could be involved to colonize nutrient-poor and harsh habitats. The A. tomkonis strains from the ISS showed presence of a 40-kbp plasmid and several other potential mobile genetic elements detected that could also be part of conjugative elements or integrated prophages.

Agrobacterium leguminum sp. nov., isolated from nodules of Phaseolus vulgaris in Spain.

Two endophytic strains, coded MOVP5T and MOPV6, were isolated from nodules of Phaseolus vulgaris plants grown on agricultural soil in Southeastern Spain, and were characterized through a polyphasic taxonomy approach. Their 16S rRNA gene sequences showed 99.3 and 99.4 %, 98.9 and 99.6 %, and 99.0 and 98.7% similarity to 'A. deltaense' YIC 4121T, A. radiobacter LGM 140T, and A. pusense NRCPB10T, respectively. Multilocus sequence analysis based on sequences of recA and atpD genes suggested that these two strains could represent a new Agrobacterium species with less than 96.5 % similarity to their closest relatives. PCR amplification of the telA gene, involved in synthesis of protelomerase, confirmed the affiliation of strains MOPV5T and MOPV6 to the genus Agrobacterium. Whole genome average nucleotide identity and digital DNA-DNA hybridization average values were less than 95.1 and 66.7 %, respectively, with respect to its closest related species. Major fatty acids in strain MOPV5T were C18 : 1 ω7c/C18 : 1 ω6c in summed feature 8, C19 : 0 cyclo ω8c, C16 : 0 and C16 : 0 3-OH. Colonies were small to medium, pearl-white coloured on YMA at 28 °C and growth was observed at 10-42 °C, pH 5.0-10.0 and with 0.0-0.5 % (w/v) NaCl. The DNA G+C content was 59.9 mol%. These two strains differ from all other genomovars of Agrobacterium found so far, including those that have not yet given a Latin name. The combined genotypic, phenotypic and chemotaxonomic data support the classification of strain MOPV5T as representing a novel species of Agrobacterium, for which the name Agrobacterium leguminum sp. nov. is proposed. The type strain is MOPV5T (=CECT 30096T=LMG 31779T).

The Azospirillum brasilense type VI secretion system promotes cell aggregation, biocontrol protection against phytopathogens and attachment to the microalgae Chlorella sorokiniana.

The plant-growth-promoting bacterium Azospirillum brasilense is able to associate with the microalgae Chlorella sorokiniana. Attachment of A. brasilense increases the metabolic performances of the microalgae. Recent genome analyses have revealed that the A. brasilense Az39 genome contains two complete sets of genes encoding type VI secretion systems (T6SS), including the T6SS1 that is induced by the indole-3-acetic acid (IAA) phytohormone. The T6SS is a multiprotein machine, widespread in Gram-negative bacteria, that delivers protein effectors in both prokaryotic and eukaryotic cells. Here we show that the A. brasilense T6SS is required for Chlorella-Azospirillum synthetic mutualism. Our data demonstrate that the T6SS is an important determinant to promote production of lipids, carbohydrates and photosynthetic pigments by the microalgae. We further show that this is likely due to the role of the T6SS during the attachment stage and for the production of IAA phytohormones. Finally, we demonstrate that the A. brasilense T6SS provides antagonistic activities against a number of plant pathogens such as Agrobacterium, Pectobacterium, Dickeya and Ralstonia species in vitro, suggesting that, in addition to promoting growth, A. brasilense might confer T6SS-dependent bio-control protection to microalgae and plants against bacterial pathogens.

Plant DNA Repair and Agrobacterium T-DNA Integration.

Agrobacterium species transfer DNA (T−DNA) to plant cells where it may integrate into plant chromosomes. The process of integration is thought to involve invasion and ligation of T-DNA, or its copying, into nicks or breaks in the host genome. Integrated T−DNA often contains, at its junctions with plant DNA, deletions of T−DNA or plant DNA, filler DNA, and/or microhomology between T-DNA and plant DNA pre-integration sites. T−DNA integration is also often associated with major plant genome rearrangements, including inversions and translocations. These characteristics are similar to those often found after repair of DNA breaks, and thus DNA repair mechanisms have frequently been invoked to explain the mechanism of T−DNA integration. However, the involvement of specific plant DNA repair proteins and Agrobacterium proteins in integration remains controversial, with numerous contradictory results reported in the literature. In this review I discuss this literature and comment on many of these studies. I conclude that either multiple known DNA repair pathways can be used for integration, or that some yet unknown pathway must exist to facilitate T−DNA integration into the plant genome.

Methionine biosynthesis pathway genes affect curdlan biosynthesis of Agrobacterium sp. CGMCC 11546 via energy regeneration

Curdlan is a water-insoluble exopolysaccharide produced by Agrobacterium species under nitrogen starvation. The curdlan production in the ΔmdeA, ΔmetA, ΔmetH, and ΔmetZ mutants of methionine biosynthesis pathway of Agrobacterium sp. CGMCC 11546 were significantly impaired. Fermentation profiles of four mutants showed that the consumption of ammonia and sucrose was impaired. Transcriptome analysis of the ΔmetH and ΔmetZ mutants showed that numerous differentially expressed genes involved in the electron transfer chain (ETC) were significantly down-regulated, suggesting that methionine biosynthesis pathway affected the production of energy ATP during the curdlan biosynthesis. Furthermore, metabolomics analysis of the ΔmetH and ΔmetZ mutants showed that ADP and FAD were significantly accumulated, while acetyl-CoA was diminished, suggesting that the impaired curdlan production in the ΔmetH and ΔmetZ mutants might be caused by the insufficient supply of energy ATP. Finally, the addition of both dibasic sodium succinate as a substrate of FAD recycling and methionine significantly restored the curdlan production of four mutants. In conclusion, methionine biosynthesis pathway plays an important role in curdlan biosynthesis in Agrobacterium sp. CGMCC 11546, which affected the sufficient supply of energy ATP from the ETC during the curdlan biosynthesis.

Bacillus velezensis strain MBY2, a potential agent for the management of crown gall disease.

The reduction of the use chemical pesticides in agriculture is gaining importance as an objective of decision-makers in both politics and economics. Consequently, the development of technically efficient and economically affordable alternatives as, e.g., biological control agents or practices is highly solicited. Crown gall disease of dicotyledonous plants is caused by ubiquitous soil borne pathogenic bacteria of the Agrobacterium tumefaciens species complex, that comprises the species Agrobacterium fabrum and represents a globally relevant plant protection problem. Within the framework of a screening program for bacterial Agrobacterium antagonists a total of 14 strains were isolated from Tunisian soil samples and assayed for antagonistic activity against pathogenic agrobacteria. One particularly promising isolate, termed strain MBY2, was studied more in depth. Using a Multilocus Sequence Analysis (MLSA) approach, the isolate was assigned to the taxonomic species Bacillus velezensis. Strain MBY2 was shown to display antagonistic effects against the pathogenic A. fabrum strain C58 in vitro and to significantly decrease pathogen populations under sterile and non-sterile soil conditions as well as in the rhizosphere of maize and, to a lower extent, tomato plants. Moreover, the ability of B. velezensis MBY2 to reduce C58-induced gall development has been demonstrated in vivo on stems of tomato and almond plants. The present study describes B. velezensis MBY2 as a newly discovered strain holding potential as a biological agent for crown gall disease management.

Single stranded DNA annealing is a conserved activity of telomere resolvases.

Bacterial species of the genera Agrobacterium and Borrelia possess chromosomes terminated by hairpin telomeres. Replication produces dimeric replication intermediates fused via replicated telomere junctions. A specialized class of enzymes, referred to as telomere resolvases, promotes the resolution of the replicated intermediate into linear monomers terminated by hairpin telomeres. Telomere resolution is catalyzed via DNA cleavage and rejoining events mechanistically similar to those promoted by topoisomerase-IB and tyrosine recombinase enzymes. Examination of the borrelial telomere resolvase, ResT, revealed unanticipated multifunctionality; aside from its expected telomere resolution activity ResT possessed a singled-stranded DNA (ssDNA) annealing activity that extended to both naked ssDNA and ssDNA complexed with its cognate single-stranded DNA binding protein (SSB). At present, the role this DNA annealing activity plays in vivo remains unknown. We have demonstrated here that single-stranded DNA annealing is also a conserved property of the agrobacterial telomere resolvase, TelA. This activity in TelA similarly extends to both naked ssDNA and ssDNA bound by its cognate SSB. TelA’s annealing activity was shown to stem from the N-terminal domain; removal of this domain abolished annealing without affecting telomere resolution. Further, independent expression of the N-terminal domain of TelA produced a functional annealing protein. We suggest that the apparent conservation of annealing activity in two telomere resolvases, from distantly related bacterial species, implies a role for this activity in hairpin telomere metabolism. Our demonstration of the separation of the telomere resolution and annealing activities of TelA provides a platform for future experiments aimed at identifying the role DNA annealing performs in vivo.