RNASeq Based Transcriptional Profiling of Pseudomonas aeruginosa PA14 after Short- and Long-Term Anoxic Cultivation in Synthetic Cystic Fibrosis Sputum Medium

The opportunistic human pathogen Pseudomonas aeruginosa can thrive under microaerophilic to anaerobic conditions in the lungs of cystic fibrosis patients. RNA$_{Seq}$ based comparative RNA profiling of the clinical isolate PA14 cultured in synthetic cystic fibrosis medium was performed after planktonic growth (OD$_{600}$ = 2.0; P), 30 min after shift to anaerobiosis (A-30) and after anaerobic biofilm growth for 96h (B-96) with the aim to reveal differentially regulated functions impacting on sustained anoxic biofilm formation as well as on tolerance towards different antibiotics. Most notably, functions involved in sulfur metabolism were found to be up-regulated in B-96 cells when compared to A-30 cells. Based on the transcriptome studies a set of transposon mutants were screened, which revealed novel functions involved in anoxic biofilm growth.In addition, these studies revealed a decreased and an increased abundance of the oprD and the mexCD-oprJ operon transcripts, respectively, in B-96 cells, which may explain their increased tolerance towards meropenem and to antibiotics that are expelled by the MexCD-OprD efflux pump. The OprI protein has been implicated as a target for cationic antimicrobial peptides, such as SMAP-29. The transcriptome and subsequent Northern-blot analyses showed that the abundance of the oprI transcript encoding the OprI protein is strongly decreased in B-96 cells. However, follow up studies revealed that the susceptibility of a constructed PA14ΔoprI mutant towards SMAP-29 was indistinguishable from the parental wild-type strain, which questions OprI as a target for this antimicrobial peptide in strain PA14

Negative Control of RpoS Synthesis by the sRNA ReaL in Pseudomonas aeruginosa

Pseudomonas aeruginosa (Pae) is an opportunistic human pathogen, able to resist host defense mechanisms and antibiotic treatment. In Pae, the master regulator of stress responses RpoS (σS) is involved in the regulation of quorum sensing and several virulence genes. Here, we report that the sRNA ReaL translationally silences rpoS mRNA, which results in a decrease of the RpoS levels. Our studies indicated that ReaL base-pairs with the Shine-Dalgarno region of rpoS mRNA. These studies are underlined by a highly similar transcription profile of a rpoS deletion mutant and a reaL over-expressing strain

Improper Coordination of BamA and BamD Results in Bam Complex Jamming by a Lipoprotein Substrate

The β-barrel assembly machinery, the Bam complex, consists of five components, BamA to -E, among which BamA and BamD are highly conserved and essential. The nonessential components are believed to play redundant roles simply by improving the rate of β-barrel folding. Here we show that BamE contributes a specific and nonoverlapping function to the Bam complex. BamE coordinates BamA and BamD to form a complex between the lipoprotein RcsF and its partner outer membrane β-barrel protein, allowing RcsF to reach the cell surface. In the absence of BamE, RcsF accumulates on BamA, thus blocking the activity of the Bam complex. As the Bam complex is a major antibiotic target in Gram-negative bacteria, the discovery that a lipoprotein can act as a jamming substrate may open the door for development of novel Bam complex inhibitors.The β-barrel assembly machinery, the Bam complex, is central to the biogenesis of integral outer membrane proteins (OMPs) as well as OMP-dependent surface-exposed lipoproteins, such as regulator of capsule synthesis protein F (RcsF). Previous genetic analysis established the model that nonessential components BamE and BamB have overlapping, redundant functions to enhance the kinetics of the highly conserved BamA/BamD core. Here we report that BamE plays a specialized nonredundant role in the Bam complex required for surface exposure of RcsF. We show that the lack of bamE, but not bamB, completely abolishes assembly of RcsF/OMP complexes and establish that the inability to assemble RcsF/OMP complexes is a molecular reason underlying all synthetic lethal interactions of ΔbamE. Our genetic analysis and biochemical cross-linking suggest that RcsF accumulates on BamA when BamA cannot engage with BamD because of its limited availability or the incompatible conformation. The role of BamE is to promote proper coordination of RcsF-bound BamA with BamD to complete OMP assembly around RcsF. We show that in the absence of BamE, RcsF is stalled on BamA, thus blocking its function, and we identify the lipoprotein RcsF as a bona fide jamming substrate of the Bam complex

Cross-regulation by CrcZ RNA controls anoxic biofilm formation in Pseudomonas aeruginosa.

Pseudomonas aeruginosa (PA) can thrive in anaerobic biofilms in the lungs of cystic fibrosis (CF) patients. Here, we show that CrcZ is the most abundant PA14 RNA bound to the global regulator Hfq in anoxic biofilms grown in cystic fibrosis sputum medium. Hfq was crucial for anoxic biofilm formation. This observation complied with an RNAseq based transcriptome analysis and follow up studies that implicated Hfq in regulation of a central step preceding denitrification. CrcZ is known to act as a decoy that sequesters Hfq during relief of carbon catabolite repression, which in turn alleviates Hfq-mediated translational repression of catabolic genes. We therefore inferred that CrcZ indirectly impacts on biofilm formation by competing for Hfq. This hypothesis was supported by the findings that over-production of CrcZ mirrored the biofilm phenotype of the hfq deletion mutant, and that deletion of the crcZ gene augmented biofilm formation. To our knowledge, this is the first example where competition for Hfq by CrcZ cross-regulates an Hfq-dependent physiological process unrelated to carbon metabolism

The Anaerobically Induced sRNA PaiI Affects Denitrification in Pseudomonas aeruginosa PA14

Pseudomonas aeruginosa is an opportunistic pathogen that can thrive by anaerobic respiration in the lungs of cystic fibrosis patients using nitrate as terminal electron acceptor. Here, we report the identification and characterization of the small RNA PaiI in the P. aeruginosa strain 14 (PA14). PaiI is anaerobically induced in the presence of nitrate and depends on the two-component system NarXL. Our studies revealed that PaiI is required for efficient denitrification affecting the conversion of nitrite to nitric oxide. In the absence of PaiI anaerobic growth was impaired on glucose, which can be reconciled with a decreased uptake of the carbon source under these conditions. The importance of PaiI for anaerobic growth is further underlined by the observation that a paiI deletion mutant was impaired in growth in murine tumors

Meta-analysis of normalized expression of differentially abundant transcripts under the conditions A-30 and B-96 when compared with condition P.

<p>The genes are grouped into the corresponding pathways (<a href="http://www.kegg.jp/kegg-bin/show_organism?org=pau" target="_blank">http://www.kegg.jp/kegg-bin/show_organism?org=pau</a>). For each group the overall behavior was summarized by the averaged log₂ fold change of its significantly modulated members. The column denotes A-30 versus P and B-96 versus P, respectively. The color code shown in the scale at the right denotes log₂-fold changes. Red indicates an overall decrease and green indicates an overall increase in the mRNA levels of genes in a particular pathway. The numbers of genes within each group are indicated by the numbers given in parenthesis.</p

OprI is not required for susceptibility of PA14 towards the antimicrobial peptide SMAP-29.

<p><b>A)</b> Determination of the levels of <i>oprI</i> mRNA in P cells (P) and B-96 cells (B-96) by Northern-blot analysis. <i>In-vitro</i> transcribed <i>oprI</i> mRNA (0.5 ng) was used as a control (C). 5S rRNA served as a loading control. <b>B)</b> The strains were grown planktonically to an OD₆₀₀ of 2.0 in SCFM (P) and for 96 hours under anaerobic conditions (B-96). Then, the cultures were harvested and the β-galactosidase activities were determined. The bars depict β-galactosidase values conferred by the translational OprI-LacZ protein in strain PA14(pTLoprI) under the conditions P and B-96. The error bars represent standard deviations from three independent experiments. <b>C)</b> Susceptibility of PA14 (red) and PA14Δ<i>oprI</i> (green) towards the cationic peptide, SMAP-29, under aerobic conditions. The experiment was performed as outlined in Materials and Methods. <b>D)</b> Determination of the <i>oprI</i> mRNA levels by Northern-blot analysis in strains PA14 and PA14∆<i>oprI</i> at the time of addition of SMAP-29 to the cultures. <i>In vitro</i> transcribed <i>oprI</i> mRNA (1 ng) was used as a control (C). 5S rRNA served as a loading control.</p

Biofilm formation of PA14 and transposon mutants thereof after anaerobic growth in SCFM medium after 96h.

<p>Biofilm formation was quantified by measuring the absorbance at 595 nm after crystal violet staining. The results are averaged from three independent experiments.</p

Altered abundance of transcripts encoding antibiotic tolerance functions.

<p>Fold change under the condition B-96 when compared with P. Only ≥ ± 5 fold change was considered.</p

MICs of different antibiotics for B-96 cells versus P cells.

<p>MICs of different antibiotics for B-96 cells versus P cells.</p