The response of Pseudomonas aeruginosa strains PAO1 and FRD1 to 10 mM calcium under planktonic and biofilm conditions
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ABSTRACT:
ORGANISM(S): Pseudomonas aeruginosa
PROVIDER: GSE74491 | GEO | 2015/12/02
SECONDARY ACCESSION(S): PRJNA300549
REPOSITORIES: GEO
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Project description:PAO1 and FRD1 were cultured planktonically and in biofilms with 10 mM calcium and no added calcium. The transcriptional response to calcium addition was determined for PAO1 cultured planktonically, for FRD1 cultured planktonically, for PAO1 cutured in biofilms, and for FRD1 cultured in biofilms. Pseudomonas aeruginosa is an opportunistic human pathogen that causes severe, life threatening infections in patients with cystic fibrosis (CF), endocarditis, wounds, or with artificial implants. During CF pulmonary infections, P. aeruginosa often encounters environments where the levels of calcium (Ca2+) are elevated. Previously, we showed that P. aeruginosa responds to externally added Ca2+ through enhanced biofilm formation, increased production of several secreted virulence factors, and by developing a transient increase in the intracellular Ca2+ followed by its removal to the basal sub-micromolar level. However, the molecular mechanisms responsible for regulating Ca2+-induced virulence factor production and Ca2+ homeostasis are not known. Here, we characterized the genome-wide transcriptional response of P. aeruginosa strains PAO1 and FRD1 to elevated [Ca2+] in both planktonic cultures and in biofilms. Among the genes induced by CaCl2 in PAO1 was an operon containing the two-component regulator PA2656-PA2657 (here called carS and carR), while the closely related two-component regulators, phoPQ and pmrAB were repressed by CaCl2 addition. To identify the regulatory targets of CarSR, we constructed a deletion mutant of carR, and performed transcriptome analysis of the mutant strain at low and high [Ca2+]. Among the genes regulated by CarSR in response to CaCl2 are the predicted periplasmic OB-fold protein, PA0320 and the inner membrane-anchored five-bladed -propeller protein, PA0327. Mutations in both PA0320 and PA0327 affected Ca2+ homeostasis, reducing the ability of P. aeruginosa to export excess Ca2+. In addition, a mutation in PA0327 had a pleotrophic effect in a Ca2+-dependent manner, altering swarming motility, pyocyanin production, and tobramycin sensitivity. Overall, the results indicate that the two-component system CarSR is responsible for sensing high levels of external Ca2+, and responding through its regulatory targets that modulate Ca2+ homeostasis, surface-associated motility, and production of the virulence factor, pyocyanin.
2015-12-02 | GSE74445 | GEO
Project description:PAO1 was cultured planktonically to stationary phase with 10 mM calcium and no added calcium. The transcriptional response to calcium addition was determined. Pseudomonas aeruginosa is an opportunistic human pathogen that causes severe, life threatening infections in patients with cystic fibrosis (CF), endocarditis, wounds, or with artificial implants. During CF pulmonary infections, P. aeruginosa often encounters environments where the levels of calcium (Ca2+) are elevated. Previously, we showed that P. aeruginosa responds to externally added Ca2+ through enhanced biofilm formation, increased production of several secreted virulence factors, and by developing a transient increase in the intracellular Ca2+ followed by its removal to the basal sub-micromolar level. However, the molecular mechanisms responsible for regulating Ca2+-induced virulence factor production and Ca2+ homeostasis are not known. Here, we characterized the genome-wide transcriptional response of P. aeruginosa strains PAO1 and FRD1 to elevated [Ca2+] in both planktonic cultures and in biofilms. Among the genes induced by CaCl2 in PAO1 was an operon containing the two-component regulator PA2656-PA2657 (here called carS and carR), while the closely related two-component regulators, phoPQ and pmrAB were repressed by CaCl2 addition. To identify the regulatory targets of CarSR, we constructed a deletion mutant of carR, and performed transcriptome analysis of the mutant strain at low and high [Ca2+]. Among the genes regulated by CarSR in response to CaCl2 are the predicted periplasmic OB-fold protein, PA0320 and the inner membrane-anchored five-bladed -propeller protein, PA0327. Mutations in both PA0320 and PA0327 affected Ca2+ homeostasis, reducing the ability of P. aeruginosa to export excess Ca2+. In addition, a mutation in PA0327 had a pleotrophic effect in a Ca2+-dependent manner, altering swarming motility, pyocyanin production, and tobramycin sensitivity. Overall, the results indicate that the two-component system CarSR is responsible for sensing high levels of external Ca2+, and responding through its regulatory targets that modulate Ca2+ homeostasis, surface-associated motility, and production of the virulence factor, pyocyanin.
2015-12-02 | GSE74444 | GEO
Project description:The response of Pseudomonas aeruginosa strains PAO1 and FRD1 to 10 mM calcium under planktonic and biofilm conditions
| PRJNA300549 | ENA
Project description:Pseudomonas aeruginosa PAO1 is the most commonly used strain for research on this ubiquitous and metabolically versatile opportunistic pathogen. Strain PAO1, a derivative of the original Australian PAO isolate, has been distributed worldwide to laboratories and strain collections. Over decades discordant phenotypes of PAO1 sublines have emerged. Taking the existing PAO1-UW genome sequence (named after the University of Washington, which led the sequencing project) as a blueprint, the genome sequences of reference strains MPAO1 and PAO1-DSM (stored at the German Collection for Microorganisms and Cell Cultures [DSMZ]) were resolved by physical mapping and deep short read sequencing-by-synthesis. MPAO1 has been the source of near-saturation libraries of transposon insertion mutants, and PAO1-DSM is identical in its SpeI-DpnI restriction map with the original isolate. The major genomic differences of MPAO1 and PAO1-DSM in comparison to PAO1-UW are the lack of a large inversion, a duplication of a mobile 12-kb prophage region carrying a distinct integrase and protein phosphatases or kinases, deletions of 3 to 1,006 bp in size, and at least 39 single-nucleotide substitutions, 17 of which affect protein sequences. The PAO1 sublines differed in their ability to cope with nutrient limitation and their virulence in an acute murine airway infection model. Subline PAO1-DSM outnumbered the two other sublines in late stationary growth phase. In conclusion, P. aeruginosa PAO1 shows an ongoing microevolution of genotype and phenotype that jeopardizes the reproducibility of research. High-throughput genome resequencing will resolve more cases and could become a proper quality control for strain collections.
| S-EPMC2812968 | biostudies-literature
Project description:Chronic polymicrobial lung infections are the chief complication in patients with cystic fibrosis. The dominant pathogen in late-stage disease is Pseudomonas aeruginosa, which forms recalcitrant, structured communities known as biofilms. Many aspects of biofilm biology are poorly understood; consequently, effective treatment of these infections is limited, and cystic fibrosis remains fatal. Here we combined in-solution protein digestion of triplicate growth-matched samples with a high-performance mass spectrometry platform to provide the most comprehensive proteomic dataset known to date for whole cell P. aeruginosa PAO1 grown in biofilm cultures. Our analysis included protein-protein interaction networks and PseudoCAP functional information for unique and significantly modulated proteins at three different time points. Secondary analysis of a subgroup of proteins using extracted ion currents validated the spectral counting data of 1884 high-confidence proteins. In this paper we demonstrate a greater representation of proteins related to metabolism, DNA stability, and molecular activity in planktonically grown P. aeruginosa PAO1. In addition, several virulence-related proteins were increased during planktonic growth, including multiple proteins encoded by the pyoverdine locus, uncharacterized proteins with sequence similarity to mammalian cell entry protein, and a member of the hemagglutinin family of adhesins, HecA. Conversely, biofilm samples contained an uncharacterized protein with sequence similarity to an adhesion protein with self-association characteristics (AidA). Increased levels of several phenazine biosynthetic proteins, an uncharacterized protein with sequence similarity to a metallo-beta-lactamase, and lower levels of the drug target gyrA support the putative characteristics of in situ P. aeruginosa infections, including competitive fitness and antibiotic resistance. This quantitative whole cell approach advances the existing P. aeruginosa subproteomes and provides a framework for identifying and studying entire pathways critical to biofilm biology in this model pathogenic organism. The identification of novel protein targets could contribute to the development of much needed antimicrobial therapies to treat the chronic infections found in patients with cystic fibrosis.
| S-EPMC3977187 | biostudies-literature
Project description:Coumarin has been reported as a quorum sensing inhibitor for Pseudomonas aeruginosa. The goal of this transcriptomic analysis is to elucidate the effect of coumarin on gene expression of P. aeruginosa. Therefore, planktonic cells of P. aeruginosa were treated by coumarin for 1h and biofilms were formed in the presence of coumarin for 24h. Unreated controls (with dimethyl sulfoxide ) for both planktonic and biofilm samples were also included. Three biological replicates per treatment were performed with RNA sequencing.
2018-04-29 | E-MTAB-6629 | biostudies-arrayexpress
Project description:The processes associated with early events in biofilm formation have become a major research focus over the past several years. Events associated with dispersion of cells from late stage biofilms have, however, received little attention. We demonstrate here that dispersal of Pseudomonas aeruginosa PAO1 from biofilms is inducible by a sudden increase in carbon substrate availability. Most efficient at inducing dispersal were sudden increases in availability of succinate > glutamate > glucose that led to approximately 80% reductions in surface-associated biofilm biomass. Nutrient-induced biofilm dispersion was associated with increased expression of flagella (fliC) and correspondingly decreased expression of pilus (pilA) genes in dispersed cells. Changes in gene expression associated with dispersion of P. aeruginosa biofilms were studied by using DNA microarray technology. Results corroborated proteomic data that showed gene expression to be markedly different between biofilms and newly dispersed cells. Gene families that were upregulated in dispersed cells included those for flagellar and ribosomal proteins, kinases, and phage PF1. Within the biofilm, genes encoding a number of denitrification pathways and pilus biosynthesis were also upregulated. Interestingly, nutrient-induced dispersion was associated with an increase in the number of Ser/Thr-phosphorylated proteins within the newly dispersed cells, and inhibition of dephosphorylation reduced the extent of nutrient-induced dispersion. This study is the first to demonstrate that dispersal of P. aeruginosa from biofilms can be induced by the addition of simple carbon sources. This study is also the first to demonstrate that dispersal of P. aeruginosa correlates with a specific dispersal phenotype.
| S-EPMC523207 | biostudies-literature
Project description:Biofilm development, specifically the fundamentally adaptive switch from acute to chronic infection phenotypes, requires global regulators and small non-coding regulatory RNAs (sRNAs). This work utilized RNA-sequencing (RNA-seq) to detect sRNAs differentially expressed in Pseudomonas aeruginosa biofilm versus planktonic state.A computational algorithm was devised to detect and categorize sRNAs into 5 types: intergenic, intragenic, 5'-UTR, 3'-UTR, and antisense. Here we report a novel RsmY/RsmZ-type sRNA, termed RsmW, in P. aeruginosa up-transcribed in biofilm versus planktonic growth. RNA-Seq, 5'-RACE and Mfold predictions suggest RsmW has a secondary structure with 3 of 7 GGA motifs located on outer stem loops. Northern blot revealed two RsmW binding bands of 400 and 120 bases, suggesting RsmW is derived from the 3'-UTR of the upstream hypothetical gene, PA4570. RsmW expression is elevated in late stationary versus logarithmic growth phase in PB minimal media, at higher temperatures (37 °C versus 28 °C), and in both gacA and rhlR transposon mutants versus wild-type. RsmW specifically binds to RsmA protein in vitro and restores biofilm production and reduces swarming in an rsmY/rsmZ double mutant. PA4570 weakly resembles an RsmA/RsmN homolog having 49 % and 51 % similarity, and 16 % and 17 % identity to RsmA and RsmN amino acid sequences, respectively. PA4570 was unable to restore biofilm and swarming phenotypes in ?rsmA deficient strains.Collectively, our study reveals an interesting theme regarding another sRNA regulator of the Rsm system and further unravels the complexities regulating adaptive responses for Pseudomonas species.
| S-EPMC4950607 | biostudies-literature
Project description:Pseudomonas aeruginosa responds to environmental changes and regulates its life cycle from planktonic to biofilm modes of growth. The control of cell attachment to surfaces is one of the critical processes that determine this transition. Environmental signals are typically relayed to the cytoplasm by second messenger systems. We here demonstrated that the second messenger, cAMP, regulated the attachment of cells. Our results suggest cAMP inhibited the transition from reversible to irreversible attachment. Further analyses revealed that cell surface hydrophobicity, one of the key factors in cell attachment, was altered by cAMP.
| S-EPMC4041239 | biostudies-literature
Project description:Peptidoglycan (PG) is a critical component of the bacterial cell wall and is composed of a repeating ?-1,4-linked disaccharide of N-acetylglucosamine and N-acetylmuramic acid appended with a highly conserved stem peptide. In Gram-negative bacteria, PG is assembled in the cytoplasm and exported into the periplasm where it undergoes considerable maturation, modification, or degradation depending on the growth phase or presence of environmental stressors. These modifications serve important functions in diverse processes, including PG turnover, cell elongation/division, and antibiotic resistance. Conventional methods for analyzing PG composition are complex and time-consuming. We present here a streamlined MS-based method that combines differential analysis with statistical 1D annotation approaches to quantitatively compare PGs produced in planktonic- and biofilm-cultured Pseudomonas aeruginosa We identified a core assembly of PG that is present in high abundance and that does not significantly differ between the two growth states. We also identified an adaptive PG assembly that is present in smaller amounts and fluctuates considerably between growth states in response to physiological changes. Biofilm-derived adaptive PG exhibited significant changes compared with planktonic-derived PG, including amino acid substitutions of the stem peptide and modifications that indicate changes in the activity of amidases, deacetylases, and lytic transglycosylases. The results of this work also provide first evidence of de-N-acetylated muropeptides from P. aeruginosa The method developed here offers a robust and reproducible workflow for accurately determining PG composition in samples that can be used to assess global PG fluctuations in response to changing growth conditions or external stimuli.
| S-EPMC6956531 | biostudies-literature