Effect of Crc and Hfq proteins on the transcription, processing, and stability of the Pseudomonas putida CrcZ sRNA

Abstract

In Pseudomonas putida, the Hfq and Crc proteins regulate the expression of many genes in response to nutritional and environmental cues, by binding to mRNAs that bear specific target motifs and inhibiting their translation. The effect of these two proteins is antagonized by the CrcZ and CrcY small RNAs (sRNAs), the levels of which vary greatly according to growth conditions. The crcZ and crcY genes are transcribed from promoters PcrcZ and PcrcY, respectively, a process that relies on the CbrB transcriptional activator and the RpoN σ factor. Here we show that crcZ can also be transcribed from the promoter of the immediate upstream gene, cbrB, a weak constitutive promoter. The cbrB-crcZ transcript was processed to render a sRNA very similar in size to the CrcZ produced from promoter PcrcZ The processed sRNA, termed CrcZ*, was able to antagonize Hfq/Crc because, when provided in trans, it relieved the deregulated Hfq/Crc-dependent hyperrepressing phenotype of a ΔcrcZΔcrcY strain. CrcZ* may help in attaining basal levels of CrcZ/CrcZ* that are sufficient to protect the cell from an excessive Hfq/Crc-dependent repression. Since a functional sRNA can be produced from PcrcZ, an inducible strong promoter, or by cleavage of the cbrB-crcZ mRNA, crcZ can be considered a 3'-untranslated region of the cbrB-crcZ mRNA. In the absence of Hfq, the processed form of CrcZ was not observed. In addition, we show that Crc and Hfq increase CrcZ stability, which supports the idea that these proteins can form a complex with CrcZ and protect it from degradation by RNases.

Metabolite profiling of the cold adaptation ofPseudomonas putidaKT2440 and cold‐sensitive mutants

“…These five genes encode central functions in the P. putida cell and our experiments added the information that they are essential to cope with cold stress. BipA is a master regulator of translation (Yuste et al ., ), the 3′,5′ exoribonuclease VacB cleaves poly(A), poly(U) and rRNAs (Hester et al ., ) and the operon cbrA – cbrB – crcZ – pcnB controls the utilization of carbon sources and modulates mRNA stability by polyadenylation (Amador et al ., ; Barroso et al ., ; Fonseca et al ., ; García‐Mauriño et al ., ; Hernández‐Arranz et al ., ; La Rosa et al ., ; Moreno et al ., ; Sánchez‐Hevia et al ., ; Tsipa et al ., Valentini et al ., ). The central role of these genes for P. putida suggests that their inactivation affects numerous features of lifestyle and metabolism.…”

Influence of the Hfq and Crc global regulators on the control of iron homeostasis in Pseudomonas putida

“…However, it should be taken into account that when cells grow in the M9 succinate medium used a large part of the Hfq and Crc molecules are trapped by CrcZ/CrcY and, if Crc is absent, the complexes formed by Hfq with these two sRNAs would be unstable Sonnleitner et al, 2017). In addition, inactivation of the crc gene leads to a strong decrease in CrcZ and CrcY levels (García-Mauriño et al, 2013;Hern andez-Arranz et al, 2016). Thus, the Crc-null strain would have significantly larger amounts of free Hfq protein than the wild type strain.…”

“…To generate a probe against the 5S ribosomal RNA, a DNA fragment corresponding to the gene specifying the 5S RNA was PCRamplified using primers 5S-HindIII-fw and 5S-EcoRI-rv (Supporting Information Table S9), and the resulting fragment cloned between the HindIII and EcoRI sites of plasmid pSPT18, generating plasmid pSPT18-5S. This plasmid was linearized with HindIII and used as a template for an in vitro transcription reaction with T7 RNA polymerase as indicated above (Hern andez-Arranz et al, 2016).…”

“…The activity of Hfq and Crc is antagonized by the CrcZ and CrcY sRNAs. These sRNAs are very similar to one another in sequence and contain several A-rich Hfq target motifs that bind and sequester Hfq and Crc to form a Crc-Hfq-CrcZ/Y complex (Sonnleitner et al, 2009;Moreno et al, 2012;Sonnleitner and Blasi, 2014;Madhushani et al, 2015;Moreno et al, 2015;Hern andez-Arranz et al, 2016). The levels of these two sRNAs vary greatly depending on growth and/or physiological conditions, being lower when cells use rich carbon sources that allow for rapid growth, and much higher when non-preferred carbon sources are used (Valentini et al, 2014, and references cited above).…”

“…CbrB is phosphorylated by CbrA sensor kinase in response to a still unknown signal (Sonnleitner et al, 2009;Moreno et al, 2012;García-Mauriño et al, 2013). The binding of Hfq and Crc to CrcZ and CrcY protects them from degradation by RNases (Hern andez-Arranz et al, 2016).…”