以轉位子突變法進行大規模篩選 Azorhizobium caulinodans ORS571 根瘤菌之第六型分泌系統調控因子

Abstract

Type VI secretion system (T6SS) is molecular machine that is widespread among phylum Proteobacteria to mediate interactions with neighboring eukaryotic and prokaryotic cells. The legume symbiont, Azorhizobium caulinodans ORS571 (α-proteobacteria), possess one deduced T6SS cluster harboring impA~impL genes on the genome. However, the T6SS hallmark protein, Hcp (hemolysin-coregulated protein) is not detectable in the extracellular matrix of ORS571 under current culture conditions. To facilitate the study of the role and regulatory pathway of T6SS in ORS571 life cycle, I screened for T6SS-active mutants from a transposon mutant library using colony lift immunoassay. The genome-wide mutant library was constructed by conjugative transfer of mini-Tn5 vector (pFAJ1819) into ORS571 cells followed by irreversible random Tn5 insertions into the genome. Efficiency of the transposon-based mutagenesis was increased 3~20 fold by refrigerating (3~11oC) the post-conjugation suspension for 2 days ~ 4 weeks before spreading on antibiotic selection plates. The improved mutation efficiency enabled colony development at high density, and these mutant colonies were directly transferred onto membrane and incubated overnight for accumulation of secreted Hcp protein. Background noise caused by adherent colonies was effectively reduced by using the dispersing chemical NaIO4 followed by a thorough vortex step. Accordingly, the detection limit was improved down to 6.3 ng Hcp protein. Approximately 68,000 mutants were analyzed, and 4 Hcp-secreting mutants and 1 Hcp-overexpression mutant were identified. Among the 4 Hcp-secreting mutants, strain Tn-A3 displayed decreased viability and imp-independent Hcp release caused by overexpression of phage-related loci (Azc_2137-2139); strain Tn-A2 and Tn-h91 showed compromised vitality and imp-independent Hcp secretion/release caused by C-terminal disruption of alanine racemase (alr, Azc_2065); strain Tn-A4 was disrupted in hypothetical gene Azc_0044, so it was pending further investigation. On the other hand, Hcp-overexpression strain Tn-A1 was unexpectedly identified by its phenotype of hyper-adherence presumably due to phosphate dyshomeostasis (pstS, Azc_4037). Further genetic verifications are required to assert the regulatory role of phosphate homeostasis on T6SS. Overall, the near-saturation screen suggests pathway(s) other than the deduced T6SS (Azc_2586-2605) participating in Hcp secretion/ release in A. caulinodans ORS571, and the mechanism(s) await future elucidation.