Karas, Bogumil

Meaney, Rebecca

Wilcox, Aiden J

English

Scholarship@Western

RESEARCH POSTER PRESENTATION TEMPLATE © 2019www.PosterPresentations.comAssessing selective plasmids for Bradyrhizobium sp. DOA9 and Mesorhizobium lotiAiden Wilcox, Rebecca Meaney, Dr. Bogumil KarasWestern University USRIINTRODUCTION• Plant growth is limited by the availability of usable forms ofnitrogen, which they obtain from the soil where dinitrogen isreduced to ammonia by nitrogen-fixing bacteria.• Synthetic nitrogen fertilizers produced through the Haber-Boschprocess drove the green agricultural revolution throughout the19th and 20th centuries, and have become essential for sustainingfood supply for the growing global population.• Excess production and use of nitrogen fertilizers can cause algalblooms, decreased coastal ocean oxygen levels, nitrous oxidegreenhouse gasses, and soil acidification1.• Biological nitrogen fixation (BNF) in diazotrophic bacteria mayprovide a sustainable means of providing plants with nitrogen, butattempts to artificially engineer these features in plants haveproven extremely difficult2.• Rhizobia are bacteria that intracellularly reside in specialized rootnodules of legumes where they can perform BNF.• The Karas team proposed that the engineering of Rhizobia toform intracellular symbioses with other eukaryotes could lead tosynthetic nitrogen-fixing organelles, or SyNOrganelles3.• An important tool for engineering Rhizobia will be vectors that arecompatible with Rhizobia, in addition to other model organisms foreasier manipulation with existing tools.• Brumwell et. al of the Karas team successfully developed multi-host shuttle (MHS) vectors that replicate in Sinorhizobium melilotiusing origins from either of S. meliloti’s megaplasmids (Figure 1)4.OBJECTIVEDevelop and assess tools for the engineering of additionalRhizobia species, Bradyrhizobium sp. DOA9 andMesorhizobium loti, beginning with selective plasmids.METHODS• Bradyrhizobium sp. DOA9 was grown on Yeast Extract Mannitol(YEM) media5, and M. loti on Tryptone Yeast Extract Mannitol (TY-M). 50 mL cultures were grown to an optical density (OD600) of 1,centrifuged at 3000 rcf for 15 minutes, and resuspended in 1 mLof water.• Wildtype sensitivity to antibiotics was tested by spot plating 5 μLaliquots from a 1 to 10−6 dilution series of 3 biological replicatesfor both Rhizobia species on various antibiotic concentrations.Plates were grown at 30℃ and examined after 1-5 days.• Plasmid and antibiotic marker functionality was assessed bytrans-conjugation from E. coli ECGE101 ΔdapA strains with therelevant constructs (Table 1) into Rhizobia.RESULTSDISCUSSIONREFERENCES1. Robertson, G. P. & Vitousek, P. M. Nitrogen in Agriculture: Balancing the Cost of anEssential Resource. Annu. Rev. Environ. Resour. 34, 97–125 (2009).2. Mus, F. et al. Symbiotic Nitrogen Fixation and the Challenges to Its Extension toNonlegumes. Appl. Environ. Microbiol. 82, 3698–3710 (2016).3. Meaney, R. S., Hamadache, S., Soltysiak, M. P. M. & Karas, B. J. Designerendosymbionts: Converting free-living bacteria into organelles. Current Opinion inSystems Biology vol. 24 41–50 (2020).4. Brumwell, S. L. et al. Designer Sinorhizobium meliloti strains and multi-functionalvectors enable direct inter-kingdom DNA transfer. PLoS One 14, (2019).5. Schwartz, W. J. M. Vincent, A Manual for the Practical Study of the Root-NoduleBacteria (IBP Handbuch No. 15 des International Biology Program, London). XI u. 164S., 10 Abb., 17 Tab., 7 Taf. Oxford-Edinburgh 1970: Blackwell Scientific Publ., 45 s. Z.Allg. Mikrobiol. 12, 440–440 (1972).ACKNOWLEDGEMENTSWestern University Dr. Bogumil Karas Karas Lab TeamUndergraduate Summer Research Internship program• Transconjugant colonies were passed on new plates three times, then screened by multiplex PCR (MPX) of DNA isolatedfrom Rhizobia by alkaline lysis.• MPX amplicons were analysed by agarose gel electrophoresis, with expected band sizes of 294 and 226 bp for the pAGE and pBGE origins, and 504, 456, and 390 bp for the Spec, Tet, and Neo markers, respectively.Table 1. Constructs for testing delivery and antibiotic selection in Bradyrhizobiumsp. DOA9 and Mesorhizobium loti. Escherichia coli ECGE101 ΔdapA strains were used to deliver constructs, and each contained pTA-Mob to encode conjugation machinery (except for with pTA-Mob2.0 which contains its own mobilization and can replicate in S. meliloti). Constructs contain repABC origins from S meliloti.Construct​ Antibiotic Resistance​ Origin of replicationpAGE1.0 Spectinomycin (Spec) pSymApAGE2.0 Tetramycin (Tet) pSymApAGE3.0 Neomycin (Nm) pSymApBGE1.0 Spec​ pSymBpBGE2.0 Tet​ pSymBpBGE3.0 Nm pSymBpTA-Mob2.0 Gentamycin (Gm) unknown​• Conjugation experiments (Figure 2) were performed in groupsbased on antibiotic selection, with controls (per antibiotic) of aconjugation to DOA9 from E. coli with no construct (Mobnegative), and a plate with water in place of E. coli (negative).Figure 3. Growth levels of wildtype Bradyrhizobium sp DOA9 (A) andMesorhizobium loti (B) under selection with various antibiotics. Spot plates (from a 1to 10−6 dilution series of 3 biological replicates) were grown at 30℃ for ~4 days, and thehighest dilution factor to which growth was observed is shown. Antibiotic concentrations(μg/mL) are indicated to the right of each bar...2111111502.525202525251005504050505020010100801001001000 2 4 6A2111111502.525202525251005504050505020010100801001001000 2 4 6SpecTetNmGmKanCefAmpGrowth Level [-Log(Dilution Factor)]Antibiotic SelectionBCells were preprepared and frozen at -80℃ by growing 50 mL cultures to OD600 ~1 and resuspending in 1 mL 10% glycerolRhizobia and E. coli were mixed in 1:1 ratio and plated on diaminopimelic acid (DAP, 60 μg/mL) media to support ΔdapA E. coli during conjugation.Cells conjugated at 30 ℃ for 3 hours.Cells were scraped from plates with 1 mL water, then a 10−1 to 10−5 dilution series was made for each conjugation sampleSelection plates were incubated at 30 ℃ and colonies were counted after ~3 daysFigure 2. General protocol for Rhizobia conjugation experiments. Growthmedia were YEM for B. sp. DOA9 and TY-M for M. loti. Appropriate antibioticconcentrations were determined based on antibiotic assay results.ChrpSymApSymBBAFigure 1. Derivation of multi-host (MHS) shuttle vectors for Rhizobia. A) The tripartiteS.meliloti genome composed of a chromosome (Chr) and two megaplasmids (pSymA,pSymB). B) Components of MHS vectors derived from S. meliloti repABC origins, a Yeastand Bacteria Artificial Chromosome (YAC/BAC) backbone, an origin of transfer (oriT), adiatom selectable marker (Ntc), and Rhizobia selection markers (Spec, Tet, or Neo).repA1B1C1repA2B2C2YAC/BACNtcoriTSpec Tet Neo• The Karas team aims to approach the engineering of Rhizobiafrom different angles by experimenting with multiple strains. LikeS. meliloti, Mesorhizobium loti was chosen for its existing researchand potential as a model organism, and the less well-characterizedBradyrhizobium sp. DOA9 was chosen due to its unique ability toperform BNF under free-living conditions (outside of root nodules).• Hence, the functionality of pAGE/pBGE in these additional strainswas tested by determining appropriate antibiotic concentrationsand delivering constructs by conjugation with the aid of pTA-Mob.pAGE /pBGETable 2. Colony counts for conjugation of Rhizobia multi-host shuttle (MHS) vectors from Escherichia coli to Bradyrhizobium sp. DOA9 (A) and Mesorhizobium loti (B), using various antibiotic markers. Controls consisted of conjugations with no MHS construct in E. coli (“Mob”), or no E. coli present (“-”). Confluent growth, colonies that are to high to count, and no growth are denoted by “conf”, “thtc”, and “✗”.Antibiotic (μg/mL)Sample​Colony Counts10-1 10-2 10-3 10-4 10-5Spec 200 pAGE1.0​ 8​ ✗ ✗ ✗ ✗pBGE1.0​ 8​ ✗ ✗ ✗ ✗Mob ✗ ✗ ✗ ✗ ✗- ✗ ✗ ✗ ✗ ✗Tet 10 pAGE2.0​ conf​ conf​ thtc thtc 76​pBGE2.0​ conf​ conf​ thtc​ 1040​ 128​Mob​ ✗ ✗ ✗ ✗ ✗- ✗ ✗ ✗ ✗ ✗Nm 100 pAGE3.0​ 4​ ✗ ✗ ✗ ✗pBGE3.0​ 7​ ✗ ✗ ✗ ✗Mob​ ✗ ✗ ✗ ✗ ✗- ✗ ✗ ✗ ✗ ✗Gm 60 pTA-Mob2.0​ 167​ 11​ ✗ ✗ ✗Mob​ 50​ 20​ 8​ ✗ ✗- 75​ 25​ 5​ ✗ ✗Spec 100 pAGE1.0​ conf​ conf​ thtc​ 1369​ 214​pBGE1.0​ ​conf conf​ thtc​ 751​ 111​Mob​ ✗ ✗ ✗ ✗ ✗- ✗ ✗ ✗ ✗ ✗Tet 5 pAGE2.0​ ​conf thtc​ 156​ 22​ 3​pBGE2.0​ ​conf thtc​ 382​ 79​ 3​Mob​ ✗ ✗ ✗ ✗ ✗- ✗ ✗ ✗ ✗ ✗ABFigure 4. Agarose gel electrophoresis results forBradyrhizobium sp. DOA9 Tet transconjugants. MPXwas performed on DNA isolated by alkaline lysis from 3colonies each (C1-3) on the 10−5 dilution plates of thepAGE2.0 and pBGE2.0 experimental samples. A Purple 1kb plus DNA ladder is included on both sides, with thesizes indicated in bp. Expected band sizes are 294 and226 bp for the pAGE and pBGE origins, and 504, 456,and 390 bp for the Spec, Tet, and Neo markers,respectively.100200300400600• Of the vector antibiotics tested in DOA9, Spec, Tet, and Nm allappear effective in both Rhizobia but slightly more-so in M. loti,except for Gm which was only partially effective in DOA9.• Kanamycin (Kan), Cefotaxime (Cef), and Ampicillin (Amp) were alsotested in case of future use in constructs, and all 3 appeared veryeffective, except for Kan in M. loti which performed poorly.• Hence, high antibiotic concentrations were used to sufficientlyprevent background growth in DOA9 conjugations, amd lowerconcentrations were used with M. loti since they are more potent.• For DOA9 conjugations, only Tet constructs resulted in substantialgrowth, suggesting that Spec200 and Nm100 were too strong.Future experiments will attempt conjugations with lower doses.• The pTA-Mon2.0 conjugation resulted in substantial growth on thenegataive controls, in accordance with the poor effect of Gm onDOA9. Future experiments may use a different antibiotic marker.• Screening of Tet transconjugants results in bands matching theexpected marker size, but the pAGE and pBGE origins appearreversed, possibly due to a switch while preparing cultures. Follow-upexperiments are being performed to confirm this.• Both Spec and Tet constructs appeared functional in M. loti.Nm and Gm constructs remain to be tested, and transconjugantsremain to be screened.• In summary, it has been shown that Rhizobia MHS vectors can bedelivered by conjugation to Bradyrhizobium sp. DOA9 andMesorhizobium loti, using Tet for DOA9, and Spec or Tet for M. lotias effective antibiotic markers.

Assessing selective plasmids for Bradyrhizobium sp. DOA9 and Mesorhizobium loti

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Assessing selective plasmids for Bradyrhizobium sp. DOA9 and Mesorhizobium loti

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