Posttranscriptional Repression of GacS/GacA-Controlled Genes by the RNA-Binding Protein RsmE Acting Together with RsmA in the Biocontrol Strain Pseudomonas fluorescens CHA0

Impact of rsmE, rsmA, and gacS mutations on the expression of hcnA, aprA, and phlA. (A) β-Galactosidase expression of a chromosomal hcnA′-′lacZ fusion was determined in CHA207 (solid squares, wild-type context), CHA1022 (open squares, gacS mutant), CHA1023 (open circles, rsmA mutant), CHA1025 (solid circles, rsmE mutant), CHA1027 (solid diamonds, rsmA rsmE double mutant), and CHA1028 (open diamonds, rsmA rsmE gacS triple mutant). (B) Expression of a chromosomal aprA′-′lacZ fusion in CHA805 (solid squares), CHA806 (open squares), CHA1020 (open circles), CHA1005 (solid circles), CHA1021 (solid diamonds), and CHA1007 (open diamonds). (C) Expression of a phlA′-′lacZ fusion on pME6702 in CHA0 (solid squares), CHA19 (open squares), CHA1076 (open circles), CHA1003 (solid circles), CHA1009 (solid diamonds), and CHA1008 (open diamonds). Each value is the average from three different cultures ± standard deviation.

Production of the RsmE and RsmA proteins in P. fluorescens mutants. (A) Western blot detection of the RsmE and RsmA proteins from 20-ml cultures grown in 125-ml flasks. Samples from strains CHA0 (wild type, wt), CHA89 (gacA), CHA1076 (rsmA), CHA1003 (rsmE), and CHA1009 (rsmA rsmE) were taken in late exponential phase (4 h after inoculation) and in stationary phase (8 h after inoculation) for gel electrophoresis and immunodetection. (B) Control of protein load. A portion of the gel showing polypeptides of 6 to 20 kDa, after protein transfer and staining with Coomassie blue. Note that under the conditions used, the slightly larger but more hydrophobic RsmE protein migrated ahead of RsmA.

Cellular levels of RsmE and RsmA proteins in P. fluorescens CHA0 during growth in 50-ml flasks containing 20 ml of NYB and 0.05% Triton X-100. Samples for immunoblot analysis were taken at increasing cell densities. The protein load in each well was similar (not shown).

Expression of a chromosomal rsmE′-′lacZ fusion in the wild-type context (CHA1134, solid squares), in a gacA mutant (CHA1136, open squares), in an rsmA mutant (CHA1161, open circles), in an rsmE mutant (CHA1138, solid circles), and in an rsmA rsmE double mutant (CHA1162, solid diamonds). Each value is the average from three different cultures ± standard deviation.

Interaction of RsmA6H and RsmE6H with the regulatory RNAs RsmY and RsmZ. [α-³³P]UTP-labeled RsmY (5 nM) and RsmZ (6.5 nM) were incubated with different concentrations of purified RsmA6H or RsmE6H before fractionation on nondenaturing gels and autoradiography. The positions of free (F) and bound (B) RNA species are indicated. (A) RsmY versus RsmA6H. (B) RsmY versus RsmE6H. (C) RsmZ versus RsmA6H. (D) RsmZ versus RsmE6H.

Competition of RsmY and RsmZ RNAs for binding to RsmE6H. [α-³³P]UTP-labeled RsmY (5 nM) and RsmZ (6.5 nM) and different unlabeled RNA competitors (RsmY, RsmZ, and the leader of carA mRNA) were incubated with RsmE6H (275 nM) before fractionation on nondenaturing gels and autoradiography. (A) Competition of unlabeled RNAs with RsmY-RsmE6H complexes. (B) Competition of unlabeled RNAs with RsmZ-RsmE6H complexes. F, free transcripts; B, transcripts bound to RsmE6H.

Impact of rsmE, rsmA, and gacA mutations on rsmY and rsmZ transcription. (A) β-Galactosidase expression of an rsmY-lacZ transcriptional fusion on pME6916 in the wild-type CHA0 (solid squares), the gacA mutant CHA89 (open squares), the rsmE mutant CHA1003 (solid circles), the rsmA mutant CHA1076 (open circles), the rsmA rsmE double mutant CHA1009 (solid diamonds), and the rsmA rsmE gacS triple mutant CHA1008 (open diamonds). (B) β-Galactosidase expression of an rsmZ-lacZ transcriptional fusion on pME6091 in CHA0 (solid squares), CHA89 (open squares), CHA1003 (solid circles), CHA1076 (open circles), CHA1009 (solid diamonds), and CHA1008 (open diamonds).

RsmA and RsmE proteins stabilize RsmY and RsmZ RNAs. (A) RsmY and RsmZ transcript decay in the wild-type strain P. fluorescens CHA0 (wt) and in the rsmA rsmE double mutant CHA1009 was determined by Northern blotting after blocking transcription with rifampin. The amount of RNA loaded was 0.5 μg for the wild type and 3 μg for the rsmA rsmE double mutant. As both rsmY and rsmZ are poorly expressed in the rsmA rsmE background, the stability of RsmY and RsmZ was studied in CHA1009 expressing rsmY or rsmZ from the tac promoter of pME6918 (rsmY) or pME6359 (rsmZ). (B) Densitometric analysis of RsmY stability in CHA0 (solid circles) and CHA1009 (open circles). (C) Densitometric analysis of RsmZ stability in CHA0 (solid circles) and CHA1009 (open circles).