Jurado Paola, Fernández Luis A, de Lorenzo Víctor
Centro Nacional de Biotecnología del Consejo Superior de Investigaciones Científicas, Campus de Cantoblanco, 28049 Madrid, Spain.
J Bacteriol. 2003 Jun;185(11):3379-83. doi: 10.1128/JB.185.11.3379-3383.2003.
The cellular levels of the alternative sigma factor sigma(54) of Pseudomonas putida have been examined in a variety of growth stages and culture conditions with a single-chain Fv antibody tailored for detection of scarce proteins. The levels of sigma(54) were also monitored in P. putida strains with knockout mutations in ptsO or ptsN, known to be required for the C-source control of the sigma(54)-dependent Pu promoter of the TOL plasmid. Our results show that approximately 80 +/- 26 molecules of sigma(54) exist per cell. Unlike that in relatives of Pseudomonas (e.g., Caulobacter), where fluctuations of sigma(54) determine adaptation and differentiation when cells face starvation, sigma(54) in P. putida remains unexpectedly constant at different growth stages, in nitrogen starvation and C-source repression conditions, and in the ptsO and ptsN mutant strains analyzed. The number of sigma(54) molecules per cell in P. putida is barely above the predicted number of sigma(54)-dependent promoters. These figures impose a framework on the mechanism by which Pu (and other sigma(54)-dependent systems) may become amenable to physiological control.
利用为检测稀有蛋白质量身定制的单链Fv抗体，已在多种生长阶段和培养条件下检测了恶臭假单胞菌替代σ因子σ⁵⁴的细胞水平。还在ptsO或ptsN发生敲除突变的恶臭假单胞菌菌株中监测了σ⁵⁴的水平，已知ptsO或ptsN是TOL质粒中依赖σ⁵⁴的Pu启动子碳源控制所必需的。我们的结果表明，每个细胞中约存在80±26个σ⁵⁴分子。与假单胞菌的亲缘菌（如柄杆菌）不同，在柄杆菌中，当细胞面临饥饿时，σ⁵⁴的波动决定适应性和分化，而恶臭假单胞菌中的σ⁵⁴在不同生长阶段、氮饥饿和碳源抑制条件下以及在所分析的ptsO和ptsN突变菌株中出乎意料地保持恒定。恶臭假单胞菌每个细胞中σ⁵⁴分子的数量仅略高于预测的依赖σ⁵⁴的启动子数量。这些数据为Pu（以及其他依赖σ⁵⁴的系统）可能接受生理控制的机制提供了一个框架。
