Project description:An important lesson from the war on pathogenic bacteria has been the need to understand the physiological responses and evolution of natural microbial communities. Bacterial populations in the environment are generally forming biofilms subject to some level of phage predation. These multicellular communities are notoriously resistant to antimicrobials and, consequently, very difficult to eradicate. This has sparked the search for new therapeutic alternatives, including phage therapy. This study demonstrates that S. aureus biofilms formed in the presence of a non-lethal dose of phage phiIPLA-RODI exhibit a unique physiological state that could potentially benefit both the host and the predator. Thus, biofilms formed under phage pressure are thicker and have a greater DNA content. Also, the virus-infected biofilm displayed major transcriptional differences compared to an untreated control. Significantly, RNA-seq data revealed activation of the stringent response, which could slow down the advance of the bacteriophage within the biofilm. The end result would be an equilibrium that would help bacterial cells to withstand environmental challenges, while maintaining a reservoir of sensitive bacterial cells available to the phage upon reactivation of the dormant carrier population. Overall design: 6 samples were analyzed, correponding to 3 biological replicates of two different conditions (sample with phage and control biofilm)
Project description:Pseudomonas aeruginosa bacteriophage PhiKZ is the type representative of the ‘giant’ phage genus with unusually large virions and genomes. By unraveling the transcriptional map of the 280 kb genome to single-nucleotide resolution, we show that it encodes 369 genes organized in 134 operons, 20% more than originally annotated. Early transcription is initiated from 28 highly conserved AT-rich promoters distributed over the PhiKZ genome, all located on the same strand. Transcription of middle and late genes is dependent on protein synthesis and mediated by very poorly conserved middle (6) and late (16) promoters. As a result of massive PhiKZ transcription, halfway through infection only 1.5% of all mRNAs in the infected cell remain bacterial. Unique to PhiKZ is its ability to complete its infection in complete absence of bacterial RNA polymerase (RNAP) enzyme activity. Its transcription is performed by the consecutive action of two PhiKZ-encoded, non-canonical RNAPs, one of which is packed within the virion. This unique, rifampicin-resistant transcriptional machinery is conserved among giant viruses, seems to function without auxiliary factors and might have its origin preceding the split between Gram-positive and Gram-negative bacteria. Construction of transcription maps for the PhiKZ phage and analysis of differential expression of host and phage genes using RNA-Seq data from samples taken in duplicate at 0, 5, 10, 15, and 35 minutes into infection.
Project description:The basic biology of bacteriophage–host interactions has attracted increasing attention due to a renewed interest in the therapeutic potential of bacteriophages. In addition, knowledge of the host pathways inhibited by phage may provide clues to novel drug targets. However, the effect of phage on bacterial gene expression and metabolism is still poorly understood. In this study, we tracked phage–host interactions by combining transcriptomic and metabolomic analyses in Pseudomonas aeruginosa infected with a lytic bacteriophage, PaP1. Compared with the uninfected host, 7.1% (399/5655) of the genes of the phage-infected host were differentially expressed genes (DEGs); of those, 354 DEGs were downregulated at the late infection phase. Many of the downregulated DEGs were found in amino acid and energy metabolism pathways. Using metabolomics approach, we then analyzed the changes in metabolite levels in the PaP1-infected host compared to un-infected controls. Thymidine was significantly increased in the host after PaP1 infection, results that were further supported by increased expression of a PaP1-encoded thymidylate synthase gene. Furthermore, the intracellular betaine concentration was drastically reduced, whereas choline increased, presumably due to downregulation of the choline–glycine betaine pathway. Interestingly, the choline–glycine betaine pathway is a potential antimicrobial target; previous studies have shown that betB inhibition results in the depletion of betaine and the accumulation of betaine aldehyde, the combination of which is toxic to P. aeruginosa. These results present a detailed description of an example of phage-directed metabolism in P. aeruginosa. Both phage-encoded auxiliary metabolic genes and phage-directed host gene expression may contribute to the metabolic changes observed in the host.
Project description:We have isolated and characterized several bacteriophages infecting Pseudomonas aeruginosa distantly related to Felix O1 virus and proposed they form a new subfamily named Felixounavirinae. The infectious cycle of bacteriophages belonging to this subfamily has not been studied yet in terms of gene expression. The present study reports the RNA-Seq analysis of bacteriophage PAK_P4 infecting PAK strain of P. aeruginosa. Overall design: RNA profile of Host and Phage at 0min, 3.5min and 13 min after infection of Pseudomonas aeruginosa PAK strain with the Pseudomonas phage PAK P4. Three biological replicates for each time point.
Project description:We have isolated and characterized several bacteriophages infecting Pseudomonas aeruginosa distantly related to Felix O1 virus and proposed they form a new subfamily named Felixounavirinae. The infectious cycle of bacteriophages belonging to this subfamily has not been studied yet in terms of gene expression. The present study reports the RNA-Seq analysis of bacteriophage PAK_P3 infecting PAK strain of P. aeruginosa. RNA profile of Host and Phage at 0min, 3.5min and 13 min after infection of Pseudomonas aeruginosa PAK strain with the Pseudomonas phage PAK P3. Three biological replicates for each time point.
Project description:Biofilms are surface-adhered bacterial communities encased in an extracellular matrix composed of polysaccharides, proteins, and extracelluar (e)DNA, with eDNA being required for the formation and integrity of biofilms. Here we demonstrate that the spatial and temporal release of eDNA is regulated by BfmR, a regulator essential for Pseudomonas aeruginosa biofilm development. The expression of bfmR coincided with localized cell death and DNA release, with high eDNA concentrations localized to the outer part of microcolonies in the form of a ring and as a cap on small clusters. Additionally, eDNA release and cell lysis increased significantly following bfmR inactivation. Genome-wide transcriptional profiling indicated that bfmR was required for repression of genes associated with bacteriophage assembly and bacteriophage-mediated lysis. In order to determine which of these genes were directly regulated by BfmR, we utilized chromatin immunoprecipitation (ChIP) analysis to identify the promoter of PA0691, termed here phdA, encoding a previously undescribed homologue of the prevent-host-death (Phd) family of proteins. Lack of phdA expression coincided with impaired biofilm development, increased cell death and bacteriophage release, a phenotype comparable to ΔbfmR. Expression of phdA in ΔbfmR biofilms restored eDNA release, cell lysis, release of bacteriophages, and biofilm formation to wild type levels. Moreover, overexpression of phdA rendered P. aeruginosa resistant to lysis mediated by superinfective bacteriophage Pf4 which was only detected in biofilms. The expression of bfmR was stimulated by conditions resulting in membrane perturbation and cell lysis. Thus, we propose that BfmR regulates biofilm development by controlling bacteriophage-mediated lysis and thus, cell death and eDNA release, via PhdA. For biofilm growth experiments, three independent replicates of P. aeruginosa strains PAO1 and ΔbfmR were grown as biofilms in a flow-through system using a once-through continuous flow tube reactor system for biofilm sample collection and in flow cells (BioSurface Technologies) for the analysis of biofilm architecture as previously described (Sauer et al., 2002, Sauer et al., 2004, Petrova & Sauer, 2009). Cells were treated with RNAprotect (Qiagen), and total RNA was extracted using an RNeasy mini purification kit (Qiagen) per the manufacturer’s instructions. RNA quality and the presence of residual DNA were checked on an Agilent Bioanalyzer 2100 electrophoretic system pre- and post-DNase treatment. Ten micrograms of total RNA was used for cDNA synthesis, fragmentation, and labeling according to the Affymetrix GeneChip P. aeruginosa genome array expression analysis protocol. Sauer, K., A. K. Camper, G. D. Ehrlich, J. W. Costerton & D. G. Davies, (2002) Pseudomonas aeruginosa displays multiple phenotypes during development as a biofilm. J. Bacteriol. 184: 1140-1154. Sauer, K., M. C. Cullen, A. H. Rickard, L. A. H. Zeef, D. G. Davies & P. Gilbert, (2004) Characterization of nutrient-induced dispersion in Pseudomonas aeruginosa PAO1 biofilm. J. Bacteriol. 186: 7312-7326. Petrova, O. E. & K. Sauer, (2009) A novel signaling network essential for regulating Pseudomonas aeruginosa biofilm development. PLoS Pathogens 5: e1000668.
Project description:The ability of bacteriophages to kill bacteria is well known, as is their potential use as alternatives to antibiotics. As such, bacteriophages reach high doses locally through infection of their bacterial host in the human body. In this study we assessed the gene expression profile, by means of whole transcriptome analysis, of peripheral blood mononuclear cells (PBMCs) derived from a healthy human donor and stimulated with a Pseudomonas aeruginosa phage PNM lysate, or P. aeruginosa strain 573. The PBMCs were stimulated for 20 h, followed by lysis of the cells and RNA extraction. In total, three stimulations were performed: control sample (i.e. not stimulated), P. aeruginosa phage PNM lysate and P. aeruginosa strain 573. Each stimulation was conducted in triplicate. The transcriptome analysis showed that the phage induce a clear immunological responses. Both pro- and anti-inflammatory genes were up-regulated in the PBMCs in the presence of the phage or its bacterial host. Our results indicate that bacteriophages might play a bigger role in the immune response then previously described and might have a broader effect than the clearing of bacterial infections alone, such as the suppression of the immune response to benefit their own survival. Overall design: The transcriptome analysis showed that the phage induce a clear immunological responses. Both pro- and anti-inflammatory genes were up-regulated in the PBMCs in the presence of the phage or its bacterial host. Our results indicate that bacteriophages might play a bigger role in the
Project description:The objective of the current study was to understand the glutaraldehyde resistance mechanisms in P. fluorescens and P. aeruginosa biofilms. Glutaraldehyde is a common biocide used in various industries to control the microbial growth. Recent reports of emergence of glutaraldehyde resistance in several bacterial species motivated this study to understand the genetic factors responsible got glutaraldehyde resistance. Using a combination of phenotypic assays, chemical genetic assays and RNA-seq, we demonstrate that novel efflux pump, polyamine biosynthesis, lipid biosynthesis and phosphonate degradation play significant role in glutaraldehyde resistance and post-glutaraldehyde recovery of Psudomonad biofilms. Examination of P. fluorescens 72 h biofilm transcriptome was elucidated upon exposure to glutaraldehyde. The results were confirmed using qRT--PCR and chemical genetic appraoches in P. fluorescens and P. aeruginosa.
Project description:RNA-sequencing was preformed from RNA isolated from bacteria infected with the bacteriophage. In order to reveal the phage-host interactions between φR1-37 and Yersinia enterocolitica throughout the phage infection cycle, both the transcriptomes were scrutinized during all the stages of infection. Overall design: Fresh cultures (logarythmic phase) of Yersinia enterocolitica YeO3-R1 were infected with bacteriophage R1-37. The RNA samples were taken at following time points post-infection: 0 (negative control), 2, 5, 10, 15, 21, 28, 35, 42 1nd 49 min.
Project description:In our study, we seek to understand the differences in growth physiology between wild type P. aeruginosa PAO1 (F0 strain) and its PB1-phage resistant derivative (F1 strain). F0 and F1 cells were harvested at mid-log for RNA extraction for hybridization to Affymetrix GeneChip P. aeruginosa genome array. A total of 4 biological replicates (4 F0 and 4 F1 samples were used).