Fig. 2

Experimental design for genetic transformation of Liberibacter crescens (Lcr) via E. coli conjugation. A Lomefloxacin effectively suppressed growth of E. coli at all the concentrations (2–12.5 μg/ml) tested with no inhibitory effect on Lcr growth (at 2–10 μg/ml). Iodonitrotetrazolium chloride forms a purple formazan dye on reduction indicative of cell growth and viability in a microplate assay. B The broad-host range mobilizable plasmid pCLL031 is a derivative of pURF071 (RepW, ColE1, Mob⁺, lacZ, Par⁺, Cm^R, Gm^R) containing genes encoding green florescent protein (GFP) driven by a constitutive tryptophan promoter, and glyoxalase A (gloA, B488_RS02175), Kdo2-lipid IVA lauroyltransferase (lpxXL, B488_RS04675) and acyl carrier protein (acpXL, B488_RS04700) with their native promoters. The genotypes of the E. coli strains used for conjugation are, HB101 (helper): F- mcrB mrr hsdS20 (r_B⁻, m_B⁻) recA13 supE44 ara14 galK2 lacY1 proA2 rpsL20 (Sm^R) xy15 λ⁻ leu mtl1; and TOP10 (donor): F-mcrA Δ(mrr-hsdRMS-mcrBC) Φ80lacZΔM15 ΔlacX74 recA1 araD139 Δ(ara leu)7697 galU galK rpsL (Str^R) endA1 nupG. C–D 4 μl aliquots of overnight cultures (Abs₆₀₀ = 1.0) of helper (HB101) and donor (TOP10) E. coli strains were sequentially spotted on top of a 5-day-old 3 ml Lcr culture pellet and cocultured for 8–12 h at 28 °C. E–F The coculture mix was streaked on selective BM7 plates (6 μg/ml lomefloxacin and 2 μg/ml gentamycin) for 12 weeks, and the Lcr exoconjugants were verified for the presence of mobilized pCLL031 by restriction digestion analysis and GFP expression