Project description:Hfq is an RNA chaperone and an important post-transcriptional regulator in bacteria. Using chromatin immunoprecipitation together with DNA sequencing (ChIP-Seq), we show that Hfq associates with hundreds of different regions of the Pseudomonas aeruginosa chromosome. These associations are abolished when transcription is inhibited, indicating they reflect Hfq binding to transcripts during their synthesis. Analogous ChIP-Seq analyses with the post-transcriptional regulator Crc reveal that it associates with many of the same nascent transcripts as Hfq, an activity we show is Hfq dependent. Our findings indicate that Hfq binds many transcripts co-transcriptionally in P. aeruginosa, often in concert with Crc, and uncover direct regulatory targets of these proteins. They also highlight a general approach for studying the interactions of RNA-binding proteins with nascent transcripts in bacteria. The binding of post-transcriptional regulators to nascent mRNAs may represent a prevalent means of controlling translation in bacteria where transcription and translation are coupled.
Project description:ParA and ParB homologs are involved in accurate chromosome segregation in bacteria. ParBs participate in proper folding and initial separation of ori domains by binding to specific parS sites (palindromic centromere-like sequences), mainly localized close to oriC. Bioinformatic analyses identified 10 parS sequences in the Pseudomonas aeruginosa PAO1 genome. One parS from the parS1-parS4 cluster is required for ParB mediated chromosome segregation. To verify the binding of ParB to all known parSs in vivo as well as to identify additional ParB binding sites we performed chromation immunoprecipitation (ChIP) using polyclonal anti-ParB antibodies followed by high throughput sequencing. ChIP was performed with P. aeruginosa PAO1161 (WT) cells, PAO1161 pKB9 (ParB+++) cells with a slight, non-toxic ParB overproduction as well as with 3 strains containing parS modifications impairing ParB binding to these sites. The data confirmed ParB binding to all known parS sequences with the exception of parS5. Moreover, we identified more than a 1000 of new ParB-bound regions, majority of which contained a DNA motif corresponding to inner 7 nt from one arm of the parS palindrome. ParB interactions with these numerous sites could affect chromosome topology, compaction and gene expression classifying P. aeruginosa ParB as a Nucleoid Associated Protein (NAP).
Project description:We used the AHL-degrading enzyme AiiA-lactonase to interrogate the evolution of the LasR-dependent QS regulon of P. aeruginosa in conditions where QS is required to obtain carbon and energy, and show that populations of P. aeruginosa adaptively reduced the size of their QS regulon over ~1000 generations.
Project description:Bacteriophages (hereafter “phages”) are ubiquitous predators of bacteria in the natural world, but interest is growing in their development into antibacterial therapy as complement or replacement for antibiotics. However, bacteria have evolved a huge variety of anti-phage defense systems allowing them to resist phage lysis to a greater or lesser extent, and in pathogenic bacteria these inevitably impact phage therapy outcomes. In addition to dedicated phage defense systems, some aspects of the general stress response also impact phage susceptibility, but the details of this are not well known. In order to elucidate these factors in the opportunistic pathogen Pseudomonas aeruginosa, we used the laboratory-conditioned strain PAO1 as host for phage infection experiments as it is naturally poor in dedicated phage defense systems. Screening by transposon insertion sequencing indicated that the uncharacterized operon PA3040-PA3042 was potentially associated with resistance to lytic phages. However, we found that its primary role appeared to be in regulating biofilm formation. Its expression was highly growth-phase dependent and responsive to phage infection and cell envelope stress.
Project description:We performed ChIP-seq analyses of RhlR to map the C4-homoserine lactone-dependent and PqsE-dependent RhlR binding sites in the P. aeruginosa genome.
Project description:Anthropogenic pollution has increased the levels of heavy metals in the environment. Bacterial populations continue to thrive in highly polluted environments and bacteria must have mechanisms to counter heavy metal stress. We chose to examine the response of the environmentally-relevant organism Pseudomonas aeruginosa to two different copper treatments. A short, 45 min exposure to copper was done in the Cu shock treatment to examine the immediate transcriptional profile to Cu stress. The Cu adapted treatment was designed to view the transcriptional profile of cells that were actively growing in the presence of Cu. Keywords: stress response
Project description:The response of bacteria to the conditions at the site of infection is a key part of the transcriptional program that will determine the sucess of the infectious agent. To model the environment of the distal airway, we used bovine pulmonary surfactant (Survanta). P. aeruginosa transcript levels were measured in the presence or absence of Survanta in MOPS minimal medium to identify transcripts altered in response to surfactant. The most highly induced transcript in Survanta was PA5325, renamed sphA based on our findings that the gene was specifically induced by sphingosine derived from the sphingomyelin present in pulmonary surfactant. A divergently transcribed transcription factor, PA5324, was demonstrated to be critical for the sphingosine dependent induction of sphA and was therefore renamed SphR. Microarrays of the sphR mutant cells were compared to wild type to determine the likely SphR regulon.