Growth phase‐dependent expression of the Pseudomonas putida KT2440 transcriptional machinery analysed with a genome‐wide DNA microarray

Abstract

Bacterial transcriptional networks are built on a hierarchy of regulators, on top of which lie the components of the RNA polymerase (in particular the sigma factors) and the global control elements, which play a pivotal role. We have designed a genome-wide oligonucleotide-based DNA microarray for Pseudomonas putida KT2440. In combination with real-time reverse transcription polymerase chain reaction (RT-PCR), we have used it to analyse the expression pattern of the genes encoding the RNA polymerase subunits (the core enzyme and the 24 sigma factors), and various proteins involved in global regulation (Crc, Lrp, Fur, Anr, Fis, CsrA, IHF, HupA, HupB, HupN, BipA and several MvaT-like proteins), during the shift from exponential growth in rich medium into starvation and stress brought about by the entry into stationary phase. Expression of the genes encoding the RNA polymerase core and the vegetative sigma factor decreased in stationary phase, while that of sigma(S) increased. Data obtained for sigma(N), sigma(H), FliA and for the 19 extracytoplasmic function (ECF)-like sigma factors suggested that their mRNA levels change little upon entry into stationary phase. Expression of Crc, BipA, Fis, HupB, HupN and the MvaT-like protein PP3693 decreased in stationary phase, while that of HupA and the MvaT-like protein PP3765 increased significantly. Expression of IHF was indicative of post-transcriptional control. These results provide the first global study of the expression of the transcriptional machinery through the exponential stationary-phase shift in P. putida.

The Crc Global Regulator Inhibits the Pseudomonas putida pWW0 Toluene/Xylene Assimilation Pathway by Repressing the Translation of Regulatory and Structural Genes

“…The extension of PCR products was performed at 60°C. Results were normalized relative to those obtained for the rpoN gene because its expression in P. putida remains constant throughout the growth phase and under the conditions used (22,37). The oligonucleotides used for the amplification of each gene are indicated in supplemental Table S1.…”

Fis overexpression enhances Pseudomonas putida biofilm formation by regulating the ratio of LapA and LapF

“…Fis expression does not change so drastically in P. putida. Compared to exponentially growing P. putida, the level of fis mRNA decreases approximately three times in stationary phase cells; however, it still depends on the growth phase (Yuste et al, 2006). In turn, the lapF gene is known to be under the positive regulation of RpoS, the sigma factor required for starvation and stress responses (Martínez-Gil et al, 2010).…”

“…Although Fis is well-studied in enterobacteria, the role of this protein in pseudomonads has only been examined in a few published studies (Jakovleva et al, 2012;Kugelberg et al, 2005;Perron et al, 2005;Teras et al, 2009;Yeung et al, 2009;Yuste et al, 2006). Fis binds and bends DNA, and thereby it participates in several important processes for Escherichia coli, such as modulating DNA supercoiling (Schneider et al, 1999(Schneider et al, , 2001Travers et al, 2001), direct regulation of transcription (Bradley et al, 2007;González-Gil et al, 1996;Xu & Johnson, 1995) and regulation of certain site-specific DNA recombination events (Dorgai et al, 1993;Finkel & Johnson, 1992;Johnson et al, 1986).…”

The Target for the Pseudomonas putida Crc Global Regulator in the Benzoate Degradation Pathway Is the BenR Transcriptional Regulator

“…PCR products were between 100 and 245 bp in length. Results were normalized relative to those obtained for the rpoN gene, since its expression in P. putida under several conditions is known to remain relatively constant throughout the growth phase (7,16,19,46). ␤-Galactosidase assays.…”