Pseudomonas putida expression profiles at different stress conditions

The metabolically versatile Pseudomonas putida strain KT2440 is the first Gram-negative soil bacterium certified as a biosafety strain and is being used for applications in agriculture, biotechnology and bioremediation. P. putida has to cope in its niche with numerous abiotic stresses. The stress response to 4°C, pH 4.5, 0.8 M urea or 45 mM sodium benzoate, respectively, was analyzed by the global mRNA expression profile and screening for stress-intolerant Tn5 transposon mutants. In total we identified 49 gene regions to be differentially expressed and 32 genes in 22 operons to be indispensable for growth during exposure to one or the other abiotic stresses. We propose that stress is sensed by the outer membrane proteins OmlA and FepA and the inner membrane constituents PtsP, PhoPQ and CbrAB. The metabolic response is regulated by the cyo operon, the RelA/SpoT modulon, PcnB and VacB that control mRNA stability and BipA that exerts transcript-specific translational control. The adaptation of the membrane barrier, the uptake of phosphate, the maintenance of intracellular pH and redox status and the translational control of metabolism are the indispensable key mechanisms of the P. putida stress response. Keywords: functional genomics Purified DNA of some 4600 overlapping shotgun clones in vector pCR-BluntII-TOPO (Invitrogen, Groningen, The Netherlands) was used as template in standard PCR amplification with the vector-specific primer pair d(TCGGATC CACTAGTAACG) and d(GGCCGCCAGTGTGATG). Shotgun cloning had been performed according to the procedures described by the vector's manufacturer. Each PCR reaction was individually checked by agarose gel electrophoresis. Prior to spotting onto microarrays, the DNA concentration of the fragments was adjusted to about 300 ng μl1. Spotting was done using an SDDC-2 DNA Micro-Arrayer from Engineering Services Inc. (Toronto, Canada) and SMP3 pins (TeleChem International Inc., Sunnyvale, USA) onto glass slides coated with poly L-lysine as described in detail earlier (Diehl et al., 2001). For labelling DNA-samples, random priming (Feinberg and Vogelstein, 1983) was performed on 500 ng genomic Pseudomonas DNA in the presence of 15 μM each of dATP, dGTP and dTTP, and 25 μM Cy3- or Cy5-labelled dCTP (Amersham-Pharmacia, Amersham, UK). Hybridization of such samples to the microarrays was done as described (Diehl et al., 2001). Fluorescence signals were detected on a ScanArray5000 unit (Packard, Billerica, USA) and analysed with the GenePix software package (Axon Instruments, Union City, USA).