title: Profiling antibiotic resistance and electrotransformation potential of Ensifer adhaerens OV14; a non-Agrobacterium species capable of efficient rates of plant transformation.
abstract: Ensifer adhaerens OV14 underpins the successful crop transformation protocol termed Ensifer-mediated transformation but issues exist in regard to addressing the pleomorphic tendency of the bacterium in suboptimal conditions, identifying the optimal parameters for electrotransformation and defining the strain's antibiotic profile. Here, modifications made to growth medium composition addressed the pleomorphic trait of E. adhaerens OV14, delivering uniform E. adhaerens OV14 growth to ensure efficient rates of electroporation with plasmids up to 42.2 kb in size. Separately, 63 putative antibiotic resistance coding sequences were identified across the E. adhaerens OV14 genome, with testing confirming the strain's susceptibility to gentamicin (≥10 mg L(-1)), tetracycline (≥10 mg L(-1)), chloramphenicol (≥100 mg L(-1)) and cefotaxime (≥500 mg L(-1)) and resistance to ampicillin, paramomycin, streptomycin, spectinomycin, ticarcillin-clavulanate and kanamycin. Partial resistance against carbenicillin, rifampicin, hygromycin-B and neomycin was also recorded. Resistance to kanamycin was supported by seven independent nptII-like homologs located within the E. adhaerens OV14 genome. Transcriptional analysis of these targets highlighted two homologs (AHK42288 and AHK42618) whose transcription was significantly elevated within 2 h exposure to kanamycin and which in the case of AHK42288 was maintained out to 6 h post-exposure. In conclusion, our results have identified optimal conditions for the handling of E. adhaerens and have identified a future genome editing target (AHK42288) to negate the kanamycin-resistant phenotype of E. adhaerens.
authors.affiliation: Dept Crop Science, Teagasc Crops Research Centre, Oak Park, Carlow, Ireland Earth Institute and School of Biology and Environmental Sciences, University College Dublin, Belfield, Dublin 4, Ireland.
authors.forename: Dheeraj Singh
authors.initials: DS
authors.lastname: Rathore
authors.affiliation: Dept Crop Science, Teagasc Crops Research Centre, Oak Park, Carlow, Ireland.
authors.forename: Manuel A
authors.initials: MA
authors.lastname: Lopez-Vernaza
authors.affiliation: Earth Institute and School of Biology and Environmental Sciences, University College Dublin, Belfield, Dublin 4, Ireland.
authors.forename: Fiona
authors.initials: F
authors.lastname: Doohan
authors.affiliation: Dept of Science and Health, Institute of Technology Carlow, Kilkenny Road, Carlow, Ireland.
authors.forename: Danielle O
authors.initials: DO
authors.lastname: Connell
authors.affiliation: Dept of Science and Health, Institute of Technology Carlow, Kilkenny Road, Carlow, Ireland.
authors.forename: Andrew
authors.initials: A
authors.lastname: Lloyd
authors.affiliation: Dept Crop Science, Teagasc Crops Research Centre, Oak Park, Carlow, Ireland ewen.mullins@teagasc.ie.
authors.forename: Ewen
authors.initials: E
authors.lastname: Mullins
mesh_terms: D004352:Drug Resistance, Microbial / Q000235:genetics*; D024901:Drug Resistance, Multiple, Bacterial / Q000235:genetics*; D018274:Electroporation; D005822:Genetic Vectors; D016680:Genome, Bacterial; D010641:Phenotype; D010944:Plants / Q000382:microbiology; D010957:Plasmids; D012230:Rhizobiaceae / Q000235:genetics* / Q000254:growth & development*; D014158:Transcription, Genetic; D014169:Transformation, Bacterial; D014170:Transformation, Genetic
keywords: Ensifer adhaerens OV14; antibiotic resistance; electroporation; plasmid; transformation