Positive control of swarming, rhamnolipid synthesis, and lipase production by the posttranscriptional RsmA/RsmZ system in Pseudomonas aeruginosa PAO1

Abstract

In Pseudomonas aeruginosa, the small RNA-binding, regulatory protein RsmA is a negative control element in the formation of several extracellular products (e.g., pyocyanin, hydrogen cyanide, PA-IL lectin) as well as in the production of N-acylhomoserine lactone quorum-sensing signal molecules. RsmA was found to control positively the ability to swarm and to produce extracellular rhamnolipids and lipase, i.e., functions contributing to niche colonization by P. aeruginosa. An rsmA null mutant was entirely devoid of swarming but produced detectable amounts of rhamnolipids, suggesting that factors in addition to rhamnolipids influence the swarming ability of P. aeruginosa. A small regulatory RNA, rsmZ, which antagonized the effects of RsmA, was identified in P. aeruginosa. Expression of the rsmZ gene was dependent on both the global regulator GacA and RsmA, increased with cell density, and was subject to negative autoregulation. Overexpression of rsmZ and a null mutation in rsmA resulted in quantitatively similar, negative or positive effects on target genes, in agreement with a model that postulates titration of RsmA protein by RsmZ RNA.

FIG. 1.

Influence of an rsmA mutation on swarming ability in the absence or presence of rhamnolipids. Inocula of 2 μl from overnight cultures of PAO1 (wild type), PAZH13 (ΔrsmA), or PT712 (rhlA′::Ω-Gm) with the control vector pME6032 (A) or plasmid pME3839 carrying the rhlAB genes under the control of the inducible tac promoter (B) were spotted onto 0.5% agar supplemented with 1 mM IPTG and incubated at 37°C overnight. The amounts of rhamnolipids (Rhl) produced by each strain were assayed in a separate experiment as described in Materials and Methods and are indicated below each swarming plate. The value obtained for PAO1/pME6032 (1.21 ± 0.04 μg/ml) was set at 100%.

FIG. 2.

Influence of rsmA and rsmZ mutations on rhlA expression. β-Galactosidase expression from a translational rhlA′-′lacZ fusion on pECP60 (A) and a transcriptional fusion on pME3838 (B) was determined in PAO1 (□), PAO6354 (rsmZ) (⋄), and PAZH13 (rsmA) (○). Each result is the mean ± standard deviation of the results from three measurements. Bacterial growth in NYB medium reached a plateau at an OD₆₀₀ of about 3.

FIG. 3.

(A) The 1.8-kb region of P. aeruginosa PAO1 with rpoS, rsmZ, and fdxA (50). Nucleotide sequence identities with the rsmZ region of P. fluorescens CHA0 (18) are represented. The transcription start site (+1) and the −35/−10 promoter sequences are deduced from the similar prrB (rsmZ) gene of P. fluorescens F113 (1). T1 and T2 are rho-independent terminators. A well-conserved upstream activation sequence pointed out by Heeb et al. (18) located around position −180 is boxed. The rsmZ deletion of strain PAO6354 extends from −177 to +70 and was created between the two artificial HindIII restriction sites in primers PRSMPAO8 and PRSMPAO9 indicated below the sequence. (B) Predicted secondary structure of RsmZ at 37°C obtained by using the M-fold program (65).

FIG. 4.

Northern blot revealing RsmZ RNA. Total RNA (4 μg) from P. aeruginosa PAO1 and PAO6354 was electrophoresed in a urea-8% acrylamide-0.4% bisacrylamide gel and hybridized with an rsmZ digoxigenin-labeled probe (see Materials and Methods). (A) Lane 1, PAO1; lane 2, PAO6354 (ΔrsmZ). (B) Lane 1, PAO1/pME6000 vector control; lane 2, PAO1/pME3337.1 (rsmZ⁺⁺). nt, nucleotides.

FIG. 5.

Influence of an rsmZ mutation on lectin production. Cells were grown in NYB to an OD₆₀₀ of 2.5, and lectin was visualized by Western blotting as previously described (43). (A) Lane 1, PAO1 (wild type); lane 2, PAZH13 (ΔrsmA). (B) Lane 1, PAO1 (wild type); lane 2, PAO6354 (ΔrsmZ); lane 3, PAO1/pME6000 (control for pME3337.1); lane 4, PAO1/pME3337.1 (rsmZ⁺⁺).

FIG. 6.

Influence of different mutations on rsmZ expression. (A) β-Galactosidase expression from the transcriptional fusion rsmZ-lacZ on pME3331 was determined in the wild type, PAO1 (□), the rsmZ mutant PAO6354 (⋄), the rsmA mutant PAZH13(○), and the gacA mutant PAO6281 (▵). Each result is the mean ± standard deviation of the results from three measurements. Bacterial growth reached a plateau at an OD₆₀₀ of 2 to 3. (B) Northern blot showing RsmZ RNA. Total RNA (4 μg) from P. aeruginosa PAO1 was prepared from cultures grown to different densities, as indicated by OD₆₀₀ values. (C) Northern blot showing RsmZ RNA in different genetic contexts. T, pME6001 vector control; nt, nucleotides; wt, wild type.

FIG. 7.

Influence of an rsmA mutation on gacA expression. β-Galactosidase activities from a translational gacA′-′lacZ fusion inserted at the Tn7 attachment site of the chromosome were determined in the wild type, PAO1 (□), and the rsmA mutant PAZH13 (▵). Each result is the mean ± standard deviation of the results from three measurements. Bacterial growth reached a plateau at an OD₆₀₀ of about 3.

FIG. 8.

Model of the GacA/RsmA signal transduction pathway in P. aeruginosa PAO1. Expression of the untranslated regulatory RNA RsmZ depends on the presence of GacA. The function of RsmZ is to antagonize the action of the small RNA-binding protein RsmA. RsmA positively controls rsmZ expression, thus forming a negative autoregulatory circuit whose mechanism is not understood at present. RsmA also negatively controls AHL-dependent quorum sensing as well as a number of quorum-sensing-dependent genes, some of which code for secondary metabolites and virulence determinants; these are regulated indirectly at the transcriptional level via quorum sensing but probably also directly at the translational level, as is the case for hcnA (42). Lipase and rhamnolipid production are controlled positively by RsmA, independently of the quorum-sensing control. Dotted line, modulating negative effect; solid bar, negative effect; arrow, positive effect.