Pseudomonas putida KT2440 Genome sequencing and assembly

In Pseudomonas putida KT2440, the clustered distribution of genes encoding glucose catabolic enzymes and four related transcriptional factors (TFs) may facilitate the tight regulation of glucose catabolism. However, the genes under the direct control of these TFs remain unidentified, so their specific contributions to glucose metabolism remain elusive. Furthermore, the carbon source gluconate was metabolized similarly to glucose in P. putida KT2440, but the response of these catabolic genes and TFs genes to gluconate was unclear. Here, we aim to unravel these mysteries through multi-omics analysis combined with physiological studies. We first used RNA-seq analysis to study the expression of these catabolic enzymes of glucose metabolism and the four related TFs in P. putida KT2440 grown on several carbon sources and found that both glucose and gluconate could significantly induce the expression of these genes. We then used ChIP-seq combined with RNA-seq to define the regulon of GnuR, one of the four TFs induced by both glucose and gluconate, and discovered that the primary role of GnuR is to directly repress the expression of catabolic genes involved in the peripheral pathways and the Entner-Doudoroff (ED) pathway of glucose and gluconate metabolism. The repressive effects of GnuR on the expression of glucose and gluconate catabolic genes were further validated by physiological studies of cell growth. Finally, a regulatory mode of an incoherent feedforward loop formed by GnuR, glucose or gluconate and these catabolic genes was proposed and discussed, suggesting a delicate control of glucose and gluconate metabolism in P. putida. Our present research provides new insights into the metabolic regulation of glucose and gluconate in P. putida.