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. 2020 Nov 4;5(6):e00864-20. doi: 10.1128/mSphere.00864-20

FIG 3.

FIG 3

Transposition mutagenesis in S. glossinidius. (A) Serial dilutions of conjugation mixtures of S. glossinidius and E. coli dapA hemA (MP1554) harboring the suicide vector encoding a Mariner transposon system (Himar1), pMarC9-R6k. Five microliters of cell suspension was spotted on BHIB agar (left panel) or BHIB agar supplemented with kanamycin (middle panel). Individually grown conjugation partners, S. glossinidius and E. coli dapA hemA (MP1554) pMarC9-R6k, were also spotted on BHIB agar supplemented with kanamycin (right panel). The red box indicates plates containing kanamycin. Note that dots at the donor lane correspond to locations where pipette tips punctured the agar. Similar dots are present in lane R2 and on some lanes of panel B. (B) Serial dilutions of conjugation mixtures of S. glossinidius and E. coli dapA hemA (MP1554) harboring the suicide vector encoding the Tn5-based promoter-probe transposition system, pUTmini-Tn5-luxCDABE-Spc. Five microliters of cell suspension was spotted on BHIB agar (left panel) or BHIB agar supplemented with spectinomycin (middle panel). Individually grown S. glossinidius and E. coli dapA hemA (MP1554) pUTmini-Tn5-luxCDABE-Spc were spotted on BHIB agar supplemented with spectinomycin (right panel). Red box indicates plates containing spectinomycin. (C) Transconjugants obtained in a conjugation experiment described for panel B were purified on BHIB agar supplemented with spectinomycin. Luminescence signals of four distinct clones are depicted on the right side of the figure. Plates were incubated for 8 days at 27°C under microaerophilic conditions. Images depict representative plates of at least 3 independent experiments. (D) Quantification of luminescence signals derived from selected S. glossinidius mini-Tn5-luxCDABE-spcR transconjugants obtained as described for panel B. Error bars represent standard deviations from three technical replicates. (E) Schematic illustration depicting locations of mini-Tn5-luxCDABE-spcR transposition insertions in selected S. glossinidius clones—hnh (SGGMMB4_03814), clpX (SGGMMB4_01523), pld (SGGMMB4_05728), and amsH (SGGMMB4_02193).