Westfall Landon W, Luna A Marie, Francisco Michael San, Diggle Stephen P, Worrall Kathryn E, Williams Paul, Cámara Miguel, Hamood Abdul N
Department of Microbiology and Immunology, Texas Tech University Health Sciences Center, Lubbock, TX 79430, USA.
Department of Biological Sciences, Texas Tech University, Lubbock, TX 79409, USA.
Microbiology (Reading). 2004 Nov;150(Pt 11):3797-3806. doi: 10.1099/mic.0.27270-0.
Exotoxin A production in Pseudomonas aeruginosa is regulated positively or negatively by several genes. Two such regulatory genes, ptxR and ptxS, which are divergently transcribed from each other, have been described previously. While computer analysis suggested that the ptxR-ptxS intergenic region contains potential binding sites for several regulatory proteins, the mechanism that regulates the expression of either ptxR or ptxS in P. aeruginosa is not known. The presence of a P. aeruginosa protein complex that specifically binds to a segment within this region was determined. In this study the binding region was localized to a 150 bp fragment of the intergenic region and the proteins that constitute the binding complex were characterized as P. aeruginosa HU and MvaT. Recombinant MvaT was purified as a fusion protein (MAL-MvaT) and shown to specifically bind to the ptxR-ptxS intergenic region. A PAO1 isogenic mutant defective in mvaT, PAODeltamvaT, was constructed and characterized. The lysate of PAODeltamvaT failed to bind to the 150 bp probe. The effect of mvaT on ptxS and ptxR expression was examined using real-time PCR experiments. The expression of ptxS was lower in PAODeltamvaT than in PAO1, but no difference was detected in ptxR expression. These results suggest that MvaT positively regulates ptxS expression by binding specifically to the ptxS upstream region.
铜绿假单胞菌中A外毒素的产生受到多个基因的正向或负向调控。此前已描述过两个这样的调控基因ptxR和ptxS,它们彼此反向转录。虽然计算机分析表明ptxR-ptxS基因间区域包含几种调控蛋白的潜在结合位点,但铜绿假单胞菌中调控ptxR或ptxS表达的机制尚不清楚。已确定存在一种能特异性结合该区域内一段序列的铜绿假单胞菌蛋白复合物。在本研究中,结合区域定位于基因间区域的一个150 bp片段,构成结合复合物的蛋白质被鉴定为铜绿假单胞菌HU和MvaT。重组MvaT作为融合蛋白(MAL-MvaT)被纯化,并显示能特异性结合ptxR-ptxS基因间区域。构建并鉴定了mvaT基因缺陷的PAO1同基因突变体PAODeltamvaT。PAODeltamvaT的裂解物未能与150 bp探针结合。使用实时PCR实验检测了mvaT对ptxS和ptxR表达的影响。PAODeltamvaT中ptxS的表达低于PAO1,但ptxR的表达未检测到差异。这些结果表明,MvaT通过特异性结合ptxS上游区域正向调控ptxS的表达。