Winteler H V, Schneidinger B, Jaeger K E, Haas D
Laboratoire de Biologie Microbienne, Université de Lausanne, Switzerland.
Appl Environ Microbiol. 1996 Sep;62(9):3391-8. doi: 10.1128/aem.62.9.3391-3398.1996.
The anaerobically inducible arcDABC operon encodes the enzymes of the arginine deiminase pathway in Pseudomonas aeruginosa. Upon induction, the arcAB mRNAs and proteins reach high intracellular levels, because of a strong anaerobically controlled promoter and mRNA processing in arcD, leading to stable downstream transcripts. We explored the usefulness of this system for the construction of expression vectors. The lacZ gene of Escherichia coli was expressed to the highest levels when fused close to the arc promoter. Insertion of lacZ further downstream into arcA or arcB did not stabilize the intrinsically unstable lacZ mRNA. On the contrary, lacZ mRNA appeared to be a vulnerable endonuclease target destabilizing arcAB mRNAs in the 5'-to-3' direction in P. aeruginosa. The native arc promoter was modified for optional expression in the -10 sequence and in the -40 region, which is a binding site for the anaerobic regulator ANR. In P. aeruginosa grown either anaerobically or with oxygen limitation in unshaken cultures, this promoter was stronger than the induced tac promoter. The P. aeruginosa lipAH genes, which encode extracellular lipase and lipase foldase, respectively, were fused directly to the modified arc promoter in an IncQ vector plasmid. Semianaerobic static cultures of P. aeruginosa PAO1 carrying this recombinant plasmid overproduced extracellular lipase 30-fold during stationary phase compared with the production by strain PAO1 without the plasmid. Severe oxygen limitation, in contrast, resulted in poor lipase productivity despite effective induction of the ANR-dependent promoter, suggesting that secretion of active lipase is blocked by the absence of oxygen. In conclusion, the modified arc promoter is useful for driving the expression of cloned genes in P. aeruginosa during oxygen-limited growth and stationary phase.
厌氧诱导型弧菌 arcDABC 操纵子编码铜绿假单胞菌中精氨酸脱亚胺酶途径的酶。诱导后，arcAB 的 mRNA 和蛋白质在细胞内达到高水平，这是由于一个强大的厌氧控制启动子以及 arcD 中的 mRNA 加工，从而产生稳定的下游转录本。我们探究了该系统用于构建表达载体的实用性。当大肠杆菌的 lacZ 基因靠近弧菌启动子融合时，其表达水平最高。将 lacZ 进一步插入 arcA 或 arcB 的下游并不能稳定本质上不稳定的 lacZ mRNA。相反，lacZ mRNA 似乎是一种易受核酸内切酶作用的靶点，会在 5' 到 3' 方向上使铜绿假单胞菌中的 arcAB mRNA 不稳定。对天然弧菌启动子在 -10 序列和 -40 区域（厌氧调节因子 ANR 的结合位点）进行了修饰以实现选择性表达。在厌氧培养或在未振荡培养中存在氧气限制的情况下生长的铜绿假单胞菌中，该启动子比诱导型 tac 启动子更强。分别编码细胞外脂肪酶和脂肪酶折叠酶的铜绿假单胞菌 lipAH 基因，在一个 IncQ 载体质粒中直接与修饰后的弧菌启动子融合。携带该重组质粒的铜绿假单胞菌 PAO1 的半厌氧静态培养物在稳定期产生的细胞外脂肪酶比没有该质粒的 PAO1 菌株多 30 倍。相比之下，尽管 ANR 依赖性启动子有效诱导，但严重的氧气限制导致脂肪酶生产力低下，这表明活性脂肪酶的分泌因缺氧而受阻。总之，修饰后的弧菌启动子可用于在氧气限制生长和稳定期驱动克隆基因在铜绿假单胞菌中的表达。
