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Pseudomonas aeruginosa is an opportunistic human pathogen that is able to sense and adapt to numerous environmental stimuli by the use of transcriptional regulators, including two-component regulatory systems. In this study, we demonstrate that the sensor kinase PA4398 is involved in the regulation of swarming motility and biofilm formation in P. aeruginosa PA14. APA4398 mutant strain was considerably impaired in swarming motility, while biofilm formation was increased by approximately 2-fold. The PA4398 mutant showed no changes in growth rate, rhamnolipid synthesis, or the production of the Pel exopolysaccharide but exhibited levels of the intracellular second messenger cyclic dimeric GMP (c-di-GMP) 50% higher than those in wild-type cells. The role of PA4398 in gene regulation was investigated by comparing the PA4398 mutant to the wildtype strain by using microarray analysis, which demonstrated that 64 genes were up- or downregulated more than 1.5-fold (P<0.05) under swarming conditions. In addition, more-sensitive real-time PCR studies were performed on genes known to be involved in c-di-GMP metabolism. Among the dysregulated genes were several involved in the synthesis and degradation of c-di-GMP or in the biosynthesis, transport, or function of the iron-scavenging siderophores pyoverdine and pyochelin, in agreement with the swarming phenotype observed. By analyzing additional mutants of selected pyoverdine- and pyochelin-related genes,we were able to show that not only pvdQ but also pvdR, fptA, pchA, pchD, and pchH are essential for the normal swarming behavior of P. aeruginosa PA14 and may also contribute to the swarming-deficient phenotype of the PA4398 mutant in addition to elevated c-di-GMP levels.

A multitude of different virulence factors as well as the ability to rapidly adapt to adverse environmental conditions are important features for the high pathogenicity of Pseudomonas aeruginosa. Both virulence and adaptive resistance are tightly controlled by a complex regulatory network and respond to external stimuli, such as host signals or antibiotic stress, in a highly specific manner. Here, we demonstrate that physiological concentrations of the human host defense peptide LL-37 promote virulence factor production as well as an adaptive resistance against fluoroquinolone and aminoglycoside antibiotics in P. aeruginosa PAO1. Microarray analyses of P. aeruginosa cells exposed to LL-37 revealed an upregulation of gene clusters involved in the production of quorum sensing molecules and secreted virulence factors (PQS, phenazine, hydrogen cyanide (HCN), elastase and rhamnolipids) and in lipopolysaccharide (LPS) modification as well as an induction of genes encoding multidrug efflux pumps MexCD-OprJ and MexGHI-OpmD. Accordingly, we detected significantly elevated levels of toxic metabolites and proteases in bacterial supernatants after LL-37 treatment. Pre-incubation of bacteria with LL-37 for 2 h led to a decreased susceptibility towards gentamicin and ciprofloxacin. Quantitative Realtime PCR results using a PAO1-pqsE mutant strain present evidence that the quinolone response protein and virulence regulator PqsE may be implicated in the regulation of the observed phenotype in response to LL-37. Further experiments with synthetic cationic antimicrobial peptides IDR-1018, 1037 and HHC-36 showed no induction of pqsE expression, suggesting a new role of PqsE as highly specific host stress sensor.

In this study, we evaluated how gene expression differs in mature Pseudomonas aeruginosa biofilms as opposed to planktonic cells by the use of RNA sequencing technology that gives rise to both quantitative and qualitative information on the transcriptome. Although a large proportion of genes were consistently regulated in both the stationary phase and biofilm cultures as opposed to the late exponential growth phase cultures, the global biofilm gene expression pattern was clearly distinct indicating that biofilms are not just surface attached cells in stationary phase. A large amount of the genes found to be biofilm specific were involved in adaptation to microaerophilic growth conditions, repression of type three secretion and production of extracellular matrix components. Additionally, we found many small RNAs to be differentially regulated most of them similarly in stationary phase cultures and biofilms. A qualitative analysis of the RNA-seq data revealed more than 3000 putative transcriptional start sites (TSS). By the use of rapid amplification of cDNA ends (5'-RACE) we confirmed the presence of three different TSS associated with the pqsABCDE operon, two in the promoter of pqsA and one upstream of the second gene, pqsB. Taken together, this study reports the first transcriptome study on P. aeruginosa that employs RNA sequencing technology and provides insights into the quantitative and qualitative transcriptome including the expression of small RNAs in P. aeruginosa biofilms.

Cell aggregation is a stress response and serves as a survival strategy for Pseudomonas aeruginosa strain PAO1 during growth with the toxic detergent Na-dodecylsulfate (SDS). This process involves the psl operon and is linked to c-di-GMP signalling. The induction of cell aggregation in response to SDS was studied. Transposon and site-directed mutagenesis revealed that the cupA-operon and the co-transcribed genes siaA (PA0172) and siaD (PA0169) were essential for SDS-induced aggregation. While siaA encodes a putative membrane protein with a HAMP and a PP2C-like phosphatase domain, siaD encodes a putative diguanylate cyclase involved in the biosynthesis of c-di-GMP. Complementation studies uncovered that the loss of SDS-induced aggregation in the formerly isolated spontaneous mutant strain N was caused by a non-functional siaA allele. DNA-microarray analysis of SDS-grown cells revealed consistent activation of eight genes, including cupA1, with known or presumptive important functions in cell aggregation in the parent strain compared with non-aggregating siaA and siaD mutants. A siaAD-dependent increase of cupA1 mRNA levels in SDS-grown cells was also shown by Northern blots. These results clearly demonstrate that SiaAD are essential for inducing cell aggregation as a specific response to SDS and suggest that they are responsible for perceiving and transducing SDS-related stress.

Background: Whole genome sequencing techniques have added a new dimension to studies on bacterial adaptation, evolution and diversity in chronic infections. By using this powerful approach it was demonstrated that Pseudomonas aeruginosa undergoes intense genetic adaptation processes, crucial in the development of persistent disease. The challenge ahead is to identify universal infection relevant adaptive bacterial traits as potential targets for the development of alternative treatment strategies., Results: We developed a microarray-based method applicable for discovery of single nucleotide polymorphisms (SNPs) in P. aeruginosa as an easy and economical alternative to whole genome sequencing. About 50% of all SNPs theoretically covered by the array could be detected in a comparative hybridization of PAO1 and PA14 genomes at high specificity (> 0.996). Variations larger than SNPs were detected at much higher sensitivities, reaching nearly 100% for genetic differences affecting multiple consecutive probe oligonucleotides. The detailed comparison of the in silico alignment with experimental hybridization data lead to the identification of various factors influencing sensitivity and specificity in SNP detection and to the identification of strain specific features such as a large deletion within the PA4684 and PA4685 genes in the Washington Genome Center PAO1., Conclusion: The application of the genome array as a tool to identify adaptive mutations, to depict genome organizations, and to identify global regulons by the "ChIP-on-chip" technique will expand our knowledge on P. aeruginosa adaptation, evolution and regulatory mechanisms of persistence on a global scale and thus advance the development of effective therapies to overcome persistent disease.

The function unknown gene PA2384 of Pseudomonas aeruginosa PAO1 has been previously shown dramatically responsive to iron limitation. In the present study, a bioinformatics analysis showed that PA2384 has a weak similarity to the N-terminus DNA-binding domain of Fur, the well-known ferric uptake regulator. To investigate the potential function of PA2384 in iron regulation a P. aeruginosa PAO1 recombinant (pUCP20::PA2384) over-expressing PA2384 and a PA2384 disrupted mutant PAO1*PA2384 were constructed. Physiological characterization showed that the knockout mutant had a longer lag phase. Genome-scale transcriptional profiles at different growth stages were compared between the wild type and the DeltaPA2384 mutant grown under iron-limiting conditions. The expression of more than 350 genes was affected by the knockout of PA2384. Among them, 71 genes involved in iron uptake were significantly down-regulated in the absence of PA2384. One hundred two quorum sensing (QS) dependent genes displayed differential transcriptions, including genes involved in the biosynthesis of some important virulence factors such as pyocyanin, rhamnolipids and hydrogen cyanide. The transcription of genes responsible for the synthesis of Pseudomonas quinolone signal (PQS) was greatly enhanced by the knockout of PA2384. Furthermore, the knockout of PA2384 also resulted in an altered expression of genes involved in electron transfer, central metabolism, phosphorus starvation and translation. It implies that PA2384 might affect more physiological processes than iron acquisition in P. aeruginosa.

Virulence factor production and the development of biofilms in Pseudomonas aeruginosa have been shown to be regulated by two hierarchically organized quorum-sensing systems activated by two types of small acyl-homoserine lactone signal molecules. Recently, a third type of bacterial signal molecule, the Pseudomonas quinolone signal (PQS), has been identified, which positively regulates a subset of genes dependent on the quorum-sensing systems. However, the molecular mechanism underlying PQS signalling has remained poorly understood. In this study the global transcriptional profile of P. aeruginosa in response to PQS revealed a marked upregulation of genes belonging to the tightly interdependent functional groups of the iron acquisition and the oxidative stress response. Remarkably, most of the differentially regulated genes, as well as the induction of rhlR, could be traced back to an iron-chelating effect of PQS. Our results amount to the elucidation of how PQS affects P. aeruginosa and have important implications for the understanding of the complex regulatory circuits involved in P. aeruginosa gene regulation.

The administration of macrolides such as azithromycin for chronic pulmonary infection of cystic fibrosis patients has been reported to be of benefit. Although the mechanisms of action remain obscure, anti-inflammatory effects as well as interference of the macrolide with Pseudomonas aeruginosa virulence factor production have been suggested to contribute to an improved clinical outcome. In this study we used a systematic approach and analyzed the impact of azithromycin on the global transcriptional pattern and the protein expression profile of P. aeruginosa PAO1 cultures versus those in untreated controls. The most remarkable result of this study is the finding that azithromycin exhibited extensive quorum-sensing antagonistic activities. In accordance with the inhibition of the quorum-sensing systems, virulence factor production was diminished and the oxidative stress response was impaired, whereas the type III secretion system was strongly induced. Moreover, P. aeruginosa motility was reduced, which probably accounts for the previously observed impaired biofilm formation capabilities of azithromycin-treated cultures. The interference of azithromycin with quorum-sensing-dependent virulence factor production, biofilm formation, and oxidative stress resistance in P. aeruginosa holds great promise for macrolide therapy in cystic fibrosis. Clearly quorum-sensing antagonist macrolides should be paid more attention in the management of chronic P. aeruginosa infections, and as quorum-sensing antagonists, macrolides might gain vital importance for more general application against chronic infections.

The Liverpool epidemic strain (LES) of Pseudomonas aeruginosa is a transmissible aggressive pathogen of cystic fibrosis (CF) patients. We compared transcriptome profiles of two LES isolates with each other and with a laboratory and genetic reference strain (PAO1) after growth to late exponential phase and following exposure to oxidative stress. Both LES isolates exhibited enhanced antimicrobial resistances linked to specific mutations in efflux pump genes. Although transcription of AmpC beta-lactamase was up-regulated in both, one LES isolate contained a specific mutation rendering the ampC gene untranslatable. The virulence-related quorum-sensing (QS) regulon of LES431, an isolate that caused pneumonia in the non-CF parent of a CF patient, was considerably up-regulated in comparison to either isolate LES400, associated with a chronic CF infection, or strain PAO1. Premature activation of QS genes was detected in isolates from both non-CF parents and the CF patient in a previously reported infection episode. LES isolates lacking the up-regulated QS phenotype contained different frameshift mutations in lasR. When fed to Drosophila melanogaster, isolate LES431 killed the fruit flies more readily than either isolate LES400 or strain PAO1, indicating that virulence varies intraclonally. The LES may represent a clone with enhanced virulence and antimicrobial resistance characteristics that can vary or are lost due to mutations during long-term colonization but have contributed to the successful spread of the lineage throughout the CF population of the United Kingdom.

In this paper, we report the synthesis of Pseudomonas aeruginosa cDNA in the presence of oligo(dT) primers. Hybridization of oligonucleotide DNA microarrays indicates that under the experimental conditions used, at least 43.7% of the expressed genes from P. aeruginosa PAO1, representing many different functional classes, can be detected by using oligo(dT)-primed cDNAs.