Karine Lapouge

Summary: The opportunistic pathogen P seudomonas aeruginosa PAO1 has a remarkable capacity to adapt to various environments and to survive with limited nutrients. Here, we report the discovery and characterization of a novel small non‐coding RNA : NrsZ (nitrogen‐regulated sRNA ). We show that under nitrogen limitation, NrsZ is induced by the NtrB / C two‐component system, an important regulator of nitrogen assimilation and P . aeruginosa's swarming motility, in concert with the alternative sigma factor RpoN . Furthermore, we demonstrate that NrsZ modulates P . aeruginosa motility by controlling the production of rhamnolipid surfactants, virulence factors notably needed for swarming motility. This regulation takes place through the post‐transcriptional control of rhlA , a gene essential for rhamnolipids synthesis. Interestingly, we also observed that NrsZ is processed in three similar short modules, and that the first short module encompassing the first 60 nucleotides is sufficient for NrsZ regulatory functions.

In the Gac/Rsm signal transduction pathway of Pseudomonas fluorescens CHA0, the dimeric RNA-binding proteins RsmA and RsmE, which belong to the vast bacterial RsmA/CsrA family, effectively repress translation of target mRNAs containing a typical recognition sequence near the translation start site. Three small RNAs (RsmX, RsmY, RsmZ) with clustered recognition sequences can sequester RsmA and RsmE and thereby relieve translational repression. According to a previously established structural model, the RsmE protein makes optimal contacts with an RNA sequence 5′-A/UCANGGANGU/A-3′, in which the central ribonucleotides form a hexaloop. Here, we questioned the relevance of the hexaloop structure in target RNAs. We found that two predicted pentaloop structures, AGGGA (in pltA mRNA encoding a pyoluteorin biosynthetic enzyme) and AAGGA (in mutated pltA mRNA), allowed effective interaction with the RsmE protein in vivo. By contrast, ACGGA and AUGGA were poor targets. Isothermal titration calorimetry measurements confirmed the strong binding of RsmE to the AGGGA pentaloop structure in an RNA oligomer. Modeling studies highlighted the crucial role of the second ribonucleotide in the loop structure. In conclusion, a refined structural model of RsmE-RNA interaction accommodates certain pentaloop RNAs among the preferred hexaloop RNAs.

Summary: In P seudomonas aeruginosa carbon catabolite repression ( CCR ) is exerted by the CbrA / B ‐ CrcZ ‐ Crc global regulatory system. Crc is a translational repressor that, in the presence of preferred carbon sources, such as C ₄ ‐dicarboxylates, impairs the utilization of less preferred substrates. When non‐preferred substrates are present, the CrcZ sRNA levels increase leading to Crc capture, thereby allowing growth of the bacterium at the expense of the non‐preferred substrates. The C ₄ ‐dicarboxylate transport ( Dct ) system in P . aeruginosa is composed of two main transporters: DctA , more efficient at mM succinate concentrations, and DctPQM , more important at μ M . In this study, we demonstrate that the Dct transporters are differentially regulated by Crc , depending on the concentration of succinate. At high concentrations, Crc positively regulates the expression of the dctA transporter gene and negatively regulates dctPQM post‐transcriptionally. The activation of dctA is explained by a Crc ‐mediated repression of dctR , encoding a transcriptional repressor of dctA . At low succinate concentrations, Crc regulation is impaired. In this condition, CrcZ levels are higher and therefore more Crc proteins are sequestered, decreasing the amount of Crc available to perform CCR on dctR and dctPQM . As a result, expression of dctA is reduced and that of dctPQM is increased.

The opportunistic human pathogen Pseudomonas aeruginosa is able to utilize a wide range of carbon and nitrogen compounds, allowing it to grow in vastly different environments. The uptake and catabolism of growth substrates are organized hierarchically by a mechanism termed catabolite repression control (Crc) whereby the Crc protein establishes translational repression of target mRNAs at CA (catabolite activity) motifs present in target mRNAs near ribosome binding sites. Poor carbon sources lead to activation of the CbrAB two-component system, which induces transcription of the small RNA (sRNA) CrcZ. This sRNA relieves Crc-mediated repression of target mRNAs. In this study, we have identified novel targets of the CbrAB/Crc system in P. aeruginosa using transcriptome analysis in combination with a search for CA motifs. We characterized four target genes involved in the uptake and utilization of less preferred carbon sources: estA (secreted esterase), acsA (acetyl-CoA synthetase), bkdR (regulator of branched-chain amino acid catabolism) and aroP2 (aromatic amino acid uptake protein). Evidence for regulation by CbrAB, CrcZ and Crc was obtained in vivo using appropriate reporter fusions, in which mutation of the CA motif resulted in loss of catabolite repression. CbrB and CrcZ were important for growth of P. aeruginosa in cystic fibrosis (CF) sputum medium, suggesting that the CbrAB/Crc system may act as an important regulator during chronic infection of the CF lung.

Pseudomonas aeruginosa utilizes preferentially C ₄ -dicarboxylates such as malate, fumarate, and succinate as carbon and energy sources. We have identified and characterized two C ₄ -dicarboxylate transport (Dct) systems in P. aeruginosa PAO1. Inactivation of the dctA (PA1183) gene caused a growth defect of the strain in minimal media supplemented with succinate, fumarate or malate, indicating that DctA has a major role in Dct. However, residual growth of the dctA mutant in these media suggested the presence of additional C ₄ -dicarboxylate transporter(s). Tn 5 insertion mutagenesis of the ΔdctA mutant led to the identification of a second Dct system, i.e., the DctPQM transporter belonging to the tr ipartite A TP-independent p eriplasmic (TRAP) family of carriers. The ΔdctA ΔdctPQM double mutant showed no growth on malate and fumarate and residual growth on succinate, suggesting that DctA and DctPQM are the only malate and fumarate transporters, whereas additional transporters for succinate are present. Using lacZ reporter fusions, we showed that the expression of the dctA gene and the dctPQM operon was enhanced in early exponential growth phase and induced by C ₄ -dicarboxylates. Competition experiments demonstrated that the DctPQM carrier was more efficient than the DctA carrier for the utilization of succinate at micromolar concentrations, whereas DctA was the major transporter at millimolar concentrations. To conclude, this is the first time that the high- and low-affinity uptake systems for succinate DctA and DctPQM have been reported to function coordinately to transport C ₄ -dicarboxylates and that the alternative sigma factor RpoN and a DctB/DctD two-component system regulates simultaneously the dctA gene and the dctPQM operon.