GeneChip® expression analysis of the iron starvation response in Pseudomonas aeruginosa:: identification of novel pyoverdine biosynthesis genes

Upon iron restriction, the opportunistic pathogen Pseudomonas aeruginosa produces various virulence factors, including siderophores,exotoxin, proteases and haemolysin. The ferric uptake regulator (Fur) plays a central role in this response and also controls other regulatorygenes, such as pvdS, which encodes an alternative sigma factor. This circuit leads to a hierarchical cascade of direct and indirectiron regulation. We used the GeneChip(R) to analyse the global gene expression profiles in response to iron. In iron-starved cells,the expression of 118 genes was increased at least fivefold compared with that in iron-replete cells, whereas the expression of 87 geneswas decreased at least fivefold. The GeneChip(R) data correlated well with results obtained using individual lacZ genefusions. Strong iron regulation was observed for previously identified genes involved in biosynthesis or uptake of the siderophores pyoverdine and pyochelin, utilization of heterologous siderophores and haem and ferrous iron transport. A low-iron milieu led to increased expression of the genes encoding TonB, alkaline protease,PrpL protease, exotoxin A, as well as fumarase C, Mn-dependent superoxide dismutase SodA, a ferredoxin and ferredoxin reductase and several oxidoreductases and dehydrogenases. Iron-controlledregulatory genes included seven alternative sigma factors and five other transcriptional regulators. Roughly 20% of the iron-regulated genes encoded proteins of unknown function andlacked any conclusive homologies. Under low-iron conditions, expression of 26 genes or operons was reduced in a Delta pvdS mutant comparedwith wild type, including numerous novel pyoverdine biosynthetic genes. The GeneChip(R) proved to be a very useful tool forrapid gene expression analysis and identification of novel genes controlled by Fur or PvdS.