Differently from the wild-type, the OprB1/OprF ratio for the peripheral and the central cells of the colR mutant

was similar. We suggest that the increased level of OprB1 in OM that is normally induced in response to glucose limitation is unbearable to the colR mutant and therefore does not rise above a certain threshold level. Hunger-induced expression of OprB1 is regulated post-transcriptionally To test the possibility that expression of OprB1 under glucose limitation increases due to enhanced transcription of glucose transport operon (genes gtsA to oprB1), the transcriptional fusion of gtsA with lacZ reporter was constructed and analysed under different glucose concentrations. Results in Figure 7A show that the expression of the gtsA promoter buy PRI-724 is induced by glucose regardless of its concentration. This was also confirmed in the liquid glucose medium by β-galactosidase measurements throughout the growth (data not shown). To find out whether OprB1 expression may be regulated post-transcriptionally we employed the PaWoprB1-tacB1 and PaWcolR-oprB1-tacB1 strains with oprB1 gene under the control of IPTG-inducible tac promoter. We presumed that if the expression of OprB1 is post-transcriptionally suppressed at high glucose and, vice versa, derepressed under glucose limitation, then it should not be possible to artificially overexpress OprB1

from tac promoter in glucose-rich environment, i.e., on 0.8% glucose medium. As predicted, the tac promoter-originated artificial expression of OprB1 was lower at 0.8% glucose compared PJ34 HCl to that at 0.2% glucose (Figure 7B). As a matter of https://www.selleckchem.com/products/Ispinesib-mesilate(SB-715992).html fact, it did not exceed the amount of OprB1 characteristic for the wild-type cells growing on glucose-rich medium. This data strongly suggests that hunger-dependent regulation of OprB1 occurs post-transcriptionally. Here, it is relevant to remind that the amount of OprB1 is slightly reduced in cbrA and cbrB mutants (Figure 3) suggesting that the CbrA-CbrB system is involved in the OprB1 regulation. Recently, CbrA-CbrB system has been shown to act as a positive regulator of CrcZ

which is an antagonist sRNA of catabolite repression control protein Crc [49]. The RNA-binding Crc is a global translational regulator of catabolite repression in pseudomonads [50–52]. Interestingly, if P. putida grows on amino acid-rich LB medium, the glucose transport genes are repressed by Crc [53]. Furthermore, sequences similar to Crc binding consensus were found in the proximity of the AUG start site of gtsA and oprB1 genes [50]. The Crc protein therefore seemed to be a likely candidate for translational repression of OprB1 in the glucose-rich solid medium. Thus, we constructed the crc-deficient strains and analyzed the effect of Crc inactivation on the amount of OprB1 in OM under glucose-rich (0.8%) and glucose-limiting (0.2%) conditions.