转录调节因子σ38介导铜绿假单胞菌绿脓菌素合成代谢调控

Regulation of pyocyanin biosynthesis by transcriptional factor sigma38 in Pseudomonas aeruginosa PAO1

摘要:

[目的]为了进一步鉴定铜绿假单胞菌转录调控因子σ³⁸对2个拷贝吩嗪合成基因簇（phzA1-G1和phzA2-G2）的具体调控方式并推定介导绿脓菌素合成代谢的可能调控机制。[方法]根据铜绿假单胞菌基因组信息，利用同源重组原理构建rpoS基因缺失突变株ΔrpoS以及克隆全长rpoS基因作互补分析；再以单一吩嗪基因簇缺失突变株Δphz1和Δphz2为出发菌株，分别构建rpoS缺失突变株ΔrpoSphz1和rpoS插入突变株ΔrpoSphz2，测定并比较野生株及相关突变株的绿脓菌素合成量，初步推定σ³⁸因子对2个不同吩嗪基因簇表达的调控方式。[结果]在GA培养基中，突变株ΔrpoS的绿脓菌素合成量比野生株显著增加；互补分析证实，σ³⁸可使突变株ΔrpoS的绿脓菌素降低并接近野生株PAO1水平；与对照株Δphz1相比，突变株ΔrpoSphz1的绿脓菌素合成量因σ³⁸因子缺失而显著减少；而与对照株Δphz2相比，突变株ΔrpoSphz2的绿脓菌素合成量因σ³⁸因子缺失显著增加。[结论]转录调控因子σ³⁸对铜绿假单胞菌绿脓菌素的合成代谢的确具一定的负调控作用；结合已报道的研究结果，初步推定：σ³⁸因子通过负调控吩嗪基因簇phz1，正调控吩嗪基因簇phz2的表达实现对绿脓菌素合成代谢的调控。

Abstract:

Pyocyanin, an important virulence factor, is synthesized and secreted by Pseudomonas aeruginosa PAO1and plays a critical role in pathogen-host interaction during infection. Sigma³⁸ (σ³⁸, σS) is a central regulator for many virulence production in pathogens.[Objective] Our aim is to identify expression and regulation of two phenazine-producing operons mediated by the sigma³⁸ factor in Pseudomonas aeruginosa PAO1.[Methods] We first cloned the flanking fragments of rpoS from the chromosomal DNA of P. aeruginosa PAO1 and constructed the deletion mutant ΔrpoS with the insertion of gentamycin resistance cassette (aacC1). Complementation of rpoS was then carried out after construction and introduction of pME10S (containing the whole rpoS region). Finally, we created the mutant ΔrpoSphz1 and ΔrpoSphz2, and measured pyocyanin production by these mutants in GA medium, using the parental strain Δphz1 and Δphz2 as controls.[Results] In GA medium, pyocyanin production by mutant ΔrpoS increased dramatically in comparison with the wild-type strain PAO1. Production of pyocyanin, however, was decreased to the level of the wild-type strain with complementation of the derivative ΔrpoS harboring pME10S. Mutant ΔrpoSphz2 produced much more pyocyanin than mutant Δphz2. Mutant ΔrpoSphz1, however, produced much less pyocyanin than mutant Δphz1.[Conclusion] By positively regulating the expression of phz2 and negatively regulating the phz1, sigma³⁸ factor exerts negative modulation on pyocyanin biosynthesis in P. aeruginosa PAO1.