Picture from Alani OS et al. (2023) MicroPubl Biol "Conjugation and transposon mutagenesis of Xenorhabdus griffiniae ...."

Figure 1. Creation of Xenorhabdus griffinae transposon mutants and screening for promoter-lacZ expression and auxotrophies:A) A region of the pKV124 plasmid carrying a mini-Tn10 transposon with a promoter-less lacZ gene, the pir-dependent oriR6K and a chloramphenicol resistance cassette flanked by inverted repeats (IR) consisting of the outermost 70-bp of IS10R. B) Examples of four types of possible mini-Tn10 transposon insertions (black triangle with orientation of lacZ denoted by arrow), relative to coding regions (block arrows) and promoters (purple boxes with arrows indicating direction of transcription) of endogenous genes. Insertions 1, 3, and 4 may give rise to lacZ expression driven by the upstream promoters. Insertion 2 is not expected to yield lacZ expression, as it is not downstream of a promoter in the genome. C) Schematic representation of the types of lacZ expression and growth phenotypes that can be observed among mini-Tn10-lacZ mutants. Whether lacZ is expressed or not is indicated by blue or tan colony color, respectively, when grown on media (e.g., glucose minimal media [GMM] or LB pyruvate [LBP] containing the X-gal substrate for β-galactosidase. Colony color comparisons among media types can indicate whether a promoter is constitutive (expressed on all media tested), inducible (expressed only on certain media) or not expressed. Growth of a mutant on LBP but not GMM suggests the transposon insertion has disrupted a locus required for the biosynthesis of essential nutrients present in LBP, but not GMM. Shown are the numbers of mutants among the 192 X. griffiniae mutants tested that exhibited each of the phenotypes observed in this study. D) Conjugation conditions were optimized by testing different ratios of two X. griffiniae HGB2507 and HGB2511 with the E. coli donor strain. Based on the observation that the highest numbers of exconjugants were obtained at a ratio of 1:10 (X. griffiniae:E. coli), this ratio was used for subsequent isolation of X. griffiniae HGB2511. E) Representative GMM and LBP plates on which individual mutants were patched in parallel to compare phenotypes. The mutants patched included those with phenotype classes of constitutive lacZ expression (class 1), no lacZ expression (class 2), GMM inducible lacZ expression (class 3), LB inducible lacZ expression (class 4), and auxotrophy (class 5).