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

Renata Moreno,Pilar Fonseca,Fernando Rojo

doi:10.1074/jbc.m110.126615

Abstract

In Pseudomonas putida, the expression of the pWW0 plasmid genes for the toluene/xylene assimilation pathway (the TOL pathway) is subject to complex regulation in response to environmental and physiological signals. This includes strong inhibition via catabolite repression, elicited by the carbon sources that the cells prefer to hydrocarbons. The Crc protein, a global regulator that controls carbon flow in pseudomonads, has an important role in this inhibition. Crc is a translational repressor that regulates the TOL genes, but how it does this has remained unknown. This study reports that Crc binds to sites located at the translation initiation regions of the mRNAs coding for XylR and XylS, two specific transcription activators of the TOL genes. Unexpectedly, eight additional Crc binding sites were found overlapping the translation initiation sites of genes coding for several enzymes of the pathway, all encoded within two polycistronic mRNAs. Evidence is provided supporting the idea that these sites are functional. This implies that Crc can differentially modulate the expression of particular genes within polycistronic mRNAs. It is proposed that Crc controls TOL genes in two ways. First, Crc inhibits the translation of the XylR and XylS regulators, thereby reducing the transcription of all TOL pathway genes. Second, Crc inhibits the translation of specific structural genes of the pathway, acting mainly on proteins involved in the first steps of toluene assimilation. This ensures a rapid inhibitory response that reduces the expression of the toluene/xylene degradation proteins when preferred carbon sources become available.

Highlights

Networks carefully control the numerous catabolic pathways of P. putida
The present results indicate that Crc regulates the expression of the toluene/xylene assimilation (TOL) genes by acting at two different levels
It inhibits the expression of the two transcriptional activators of the pathway, which is reflected in a 2–3 times lower level of the upper and meta polycistronic mRNAs

Summary

EXPERIMENTAL PROCEDURES

Medium contains preferred compounds such as succinate or Bacterial Strains and Culture Media—P. putida KT2440 is glucose or when a complete medium is used, the induction of derived from P. putida mt-2 by the curing of the pWW0 TOL the toluene/xylene assimilation (TOL) pathway is inhibited by a plasmid [34]. The PTSNtr system acts by interfer- Total RNA Purification and Real-time Reverse Transcriptioning, directly or indirectly, with the ability of XylR to activate PCR—Strains KT2442(pWW0) and KT2442C(pWW0) were transcription from promoter Pu [26] It is unclear grown at 30 °C in aerated flasks in LB medium, in the absence or how Crc inhibits the induction of the TOL genes. In the case of xylS, the putative Crc binding site is located on the mRNA originating at the XylR-dependent promoter PS1 but is not present in the mRNA transcribed from the constitutive enzyme activity is defined as the amount of enzyme required to promoter PS2, which initiates immediately downstream of the convert 1 ␮mol of substrate to product in 1 min at 22 °C; spe- presumed Crc target just 10 nt upstream from the AUG start cific activities are given as units/mg of protein

RESULTS

Findings

DISCUSSION