Campisano Andrea, Overhage Joerg, Rehm Bernd H A
Institute of Molecular Biosciences, Massey University, Private Bag 11222, Palmerston North, New Zealand.
J Biotechnol. 2008 Feb 29;133(4):442-52. doi: 10.1016/j.jbiotec.2007.11.007. Epub 2007 Nov 23.
The planktonic and biofilm growth mode, the presence of polyhydroxyalkanoate (PHA) granules and the nitrogen availability were considered as parameters to study the regulation of genes involved in PHA biosynthesis in Pseudomonas aeruginosa. The transcriptional start point of the phaIF gene cluster, encoding PHA granule-associated proteins with proposed regulatory function, was experimentally verified. Gfp and lacZ transcriptional reporter fusions, respectively, were used to analyse promoter activities. In planktonic growth under nitrogen limitation, the phaC promoter (PphaC) showed increased induction, while in the PHA-negative mutant the activity of PphaC was reduced to 25% of the wild type and was independent of nitrogen availability. Promoter activity in biofilms was assessed using 2, 0.05 or 0 g/l of NH(4)Cl as nitrogen source, respectively. PphaC activity was increased during early biofilm growth, whereas in mature biofilms PphaC activity was preferentially localised to stalks of microcolonies. Nitrogen starvation led to an increased PphaC activity at the biofilm surface. PHA granule formation was confirmed by electron microscopy in planktonic and in biofilm cells. PphaI activity in planktonic cultures was less dependent on the conditions assayed and presented an oscillatory behaviour. In biofilms, PphaI was strongly activated during early stages of biofilm development, but was inactive in mature biofilms. Under nitrogen starvation PphaI activation resembled that of PphaC. These data suggested a differential regulation of PHA biosynthesis genes in planktonic and biofilm cells, as well as an important regulatory function of PhaF, when considering nitrogen availability. Interestingly, in biofilms PHA biosynthesis gene regulation showed a spatial distribution similar to rhamnolipid biosynthesis genes.
浮游生长模式和生物膜生长模式、聚羟基脂肪酸酯（PHA）颗粒的存在以及氮的可利用性被视为研究铜绿假单胞菌中参与PHA生物合成的基因调控的参数。对编码具有推测调控功能的PHA颗粒相关蛋白的phaIF基因簇的转录起始点进行了实验验证。分别使用绿色荧光蛋白（Gfp）和乳糖操纵子（lacZ）转录报告融合体来分析启动子活性。在氮限制条件下的浮游生长中，phaC启动子（PphaC）的诱导作用增强，而在PHA阴性突变体中，PphaC的活性降至野生型的25%，且与氮的可利用性无关。分别使用2、0.05或0 g/l的氯化铵作为氮源评估生物膜中的启动子活性。在生物膜早期生长过程中，PphaC活性增加，而在成熟生物膜中，PphaC活性优先定位于微菌落的茎部。氮饥饿导致生物膜表面的PphaC活性增加。通过电子显微镜在浮游细胞和生物膜细胞中证实了PHA颗粒的形成。浮游培养物中PphaI的活性对所检测条件的依赖性较小，并呈现出振荡行为。在生物膜中，PphaI在生物膜发育的早期阶段被强烈激活，但在成熟生物膜中无活性。在氮饥饿条件下，PphaI的激活类似于PphaC。这些数据表明，在浮游细胞和生物膜细胞中，PHA生物合成基因存在差异调控，并且在考虑氮的可利用性时，PhaF具有重要的调控功能。有趣的是，在生物膜中，PHA生物合成基因的调控呈现出与鼠李糖脂生物合成基因相似的空间分布。
