Figure 1.
Effects of RsmA on PCA production and the growth of strain M18 and its mutants.
Growth curves (open symbols) and PCA production (solid symbols) were determined for (A) the wild-type strain M18 (squares) and its rsmA mutant M18ΔRA (triangles), (B) M18ΔRA/p-rsmA (squares) and M18ΔRA/pME6000 (triangles), (C) triple-mutant M18ΔMSP1 (squares) and quadruple-mutant M18ΔMSAP1 (triangles), and (D) triple-mutant M18ΔMSP2 (squares) and quadruple-mutant M18ΔMSAP2 (triangles) in PPM broth at 28°C. M18ΔRA/pME6000 indicates rsmA mutant M18ΔRA harbouring an empty pME6000 plasmid. M18ΔRA/p-rsmA indicates rsmA mutant M18ΔRA harbouring recombinant pME6000 that expressed the rsmA gene. Values are the means ± standard deviations of triplicate cultures.
Figure 2.
Effect of the Gac/Rsm system on the cell growth and PCA production.
Cell growth assay (A) and PCA production (B) of the wild-type strain M18 and its derivative mutants were determined in PPM broth at 28°C. M18 (wild-type strain; solid squares), M18G (gacA mutant strain; solid circles), M18RYZ (rsmY/Z double mutant strain; open circles) and M18ΔRA (rsmA mutant strain; solid triangles).
Figure 3.
Activities of transcriptional and translational lacZ fusions for phzA1 and phzA2 in strain M18 and its rsmA mutant M18ΔRA.
β-gal activities of the two transcriptional fusions of pMP1C (phzA1-lacZ) and pMP2C-2 (phzA2-lacZ) are shown in (A) and (B), and the activities of the two translational fusions of pMP1L (phzA1′-’lacZ) and pMP2L (phzA2′-’lacZ) are shown in (C) and (D) both in wild-type strain M18 (squares) and rsmA mutant M18ΔRA (triangles) in PPM broth at 28°C. Values are the means ± standard deviations of triplicate cultures.
Figure 4.
5′-UTR sequence of the two phz gene clusters.
The 5′-untranslated nucleotide sequences between the transcriptional start site (TSS) and the translation start codon (ATG) in the phzA1 (A) and phzA2 (B) gene. B1–7 and S1–4 (GGA motifs): potential RsmA binding sites.
Table 1.
Relative phzA2 transcript levels in the M18 and M18ΔRA strains during the exponential and stationary phases as assessed by qRT-PCR.
Figure 5.
Gel mobility shift assays for RsmA protein direct binding to the 5′-UTR of the phz2 transcript.
3′ end-labeled WT phz2-S1, phz2-S2, phz2-S3, phz2-S4 and mutant phz2-S3M transcripts (1 nM) were incubated with RsmA (concentration indicated at the bottom of each lane). Positions of bound (B) and free (F) phz2 leader RNA are marked. For an RNA competition assay, labeled phz2-S3 RNA was incubated with RsmA ±100- or 500-fold excess of non-specific (trp) and specific (rsmY and phz2-S3) competitor RNA. The concentrations of RsmA and competitor RNA were shown at the bottom of the corresponding lanes. The equilibrium binding constant (KD) of RsmA binding to WT phz2-S3 was calculated to be 54.5 nM using surface plasmon resonance assays.
Figure 6.
Positive regulation of phz2 expression by RsmA and identification of a target regulatory sequence within the phz2 operon leader.
Construction map for a series of translational and post-transcriptional lacZ fusions for phzA2 and the β-gal activities of the four fusions were determined in wild-type strain M18 and mutant M18ΔRA in PPM broth at 28°C. The possible regulatory site of RsmA on the activating region was located at +122 to +228 nt downstream of the phzA2 TSS. Values are the means ± standard deviations of triplicate cultures.
Figure 7.
Structure prediction of the phz2 transcript and putative model of RsmA mediated activation of phz2 translation.
(A) A predicted 5′-UTR secondary structure from +160 to +205 nucleotides of the phz2 transcript generated by RNA structure. (B) A putative direct activation model of the phz2 transcript mediated by RsmA in P. aeruginosa M18. In the rsmA-deleted mutant (right), base-paired nucleotides between the SD with its flanking sequence and the RABS resulted in a relatively stable stem-loop structure, which prevented ribosome access and translation initiation. However, in the WT strain (left), the loose stem-loop structure caused by RsmA binding resulted in easy access to the ribosome and translation activation of the phz2 transcript. RABS: RsmA binding site.
Figure 8.
Functional analysis of the putative RsmA-binding region and its flanking sequences of phz2 transcripts.
(A) Construction of a WT phzA2′-‘lacZ translational fusion and two mutant fusions for pMP2L-M1 and pMP2L-M2 in which the RsmA-binding site (RABS) and both RABS along with its flanking sequences for the phz2 leader region were replaced, respectively. (B) β-gal activities of the three fusions were determined in strain M18 and mutant M18ΔRA in PPM broth at 28°C. Symbols: pMP2L, translational fusion of WT phz2 cluster; pMP2L-M1 fusion, containing a substitution of the RsmA binding site; pMP2L-M2 fusion, containing a substitution of the paired-base region. Values are the means ± standard deviations of triplicate cultures.
Figure 9.
Secondary structures and free energies of WT and mutant phz2 leader regions.
The RNA structures of the phz2 upstream region from nt +160 to +205 in the three plasmids of pMP2L (A), pMP2L-M1 (B), pMP2L-M2 (C) were predicted by M-fold and their folding free energies were -18.5 kcal/mol, -14.5 kcal/mol, and-7.1 kcal/mol, respectively. RABS: RsmA binding site.