Unknown,Transcriptomics,Genomics,Proteomics

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P. aeruginosa and indole and 7-hydroxyindole

ABSTRACT: We used DNA microarrays to investigate the impact of indole and 7-hdroxyindole on P. aeruginosa PAO1.For the microarray experiments, 10 g glass wool (Corning Glass Works, Corning, N.Y.) were used to form biofilms in 250 mL in 1 L Erlenmeyer shake flasks which were inoculated with overnight cultures of P. aeruginosa PAO1 diluted that were 1:100. For P. aeruginosa with 7-hydroxyindole and indole, 500 uM 7-hydroxyindole in 250 uL DMF, 1000 uM indole in 250 uL DMF, or 250 uL DMF alone were added to cells grown in LB. The cells were shaken at 250 rpm and at 37oC for 7 h to form biofilms on the glass wool, and RNA was isolated from the biofilm cells. Keywords: comparison with E. coli signal indole on P. aeruginosa PAO1 For the microarray experiments, 10 g glass wool (Corning Glass Works, Corning, N.Y.) were used to form biofilms in 250 mL in 1 L Erlenmeyer shake flasks which were inoculated with overnight cultures of P. aeruginosa PAO1 diluted that were 1:100. For P. aeruginosa with 7-hydroxyindole and indole, 500 uM 7-hydroxyindole in 250 uL DMF, 1000 uM indole in 250 uL DMF, or 250 uL DMF alone were added to cells grown in LB. The cells were shaken at 250 rpm and at 37oC for 7 h to form biofilms on the glass wool, and RNA was isolated from the biofilm cells.

ORGANISM(S): Pseudomonas aeruginosa PAO1

SUBMITTER: Jintae Lee

PROVIDER: E-GEOD-10065 | biostudies-arrayexpress |

REPOSITORIES: biostudies-arrayexpress

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Project description:The Escherichia coli quorum-sensing regulator, SdiA, belongs to the LuxR family of transcriptional regulators and is responsible for detecting signals from other bacteria. Previously we found that SdiA is necessary for E. coli to control its biofilm formation with indole just as SdiA is necessary for E. coli to alter its biofilm formation in the presence of N-acylhomoserine lactones (AHLs). Here we engineered SdiA by random mutagenesis to further control biofilm formation in the presence of indole and AHLs. After screening of 477?? mutants with indole and two AHLs (N-butyryl-DL-homoserine lactone, and N-(3-oxooctanoyl)-L-homoserine lactone, C6HSL), five SdiA variants were obtained that altered biofilm with and without signals of indole and AHLs. Two truncation variants (1E11 and 14C3) lacking the C-terminal DNA-binding domain of SdiA showed the reduction of biofilm formation by 5-fold and 10-fold in LB and LB glu, respectively. DNA microarrays show that the evolved SdiA (1E11) compared to wild-type SdiA influences indole synthesis genes, AI-2 uptake genes, acid-resistance genes, motility related genes, cold-shock protein genes, and several regulatory protein genes. Corroborating DNA microarrays, SdiA variants produced various amounts of indole which led to different survivals in low pH and influenced swimming motility and final cell density. Also, an AHL sensitive variant (2D10) 2-fold increased biofilm formation in the presence of C6HSL, while another variant (6B12) lowered biofilm formation in the presence of C6HSL. Hence, the results clearly showed that mutation of SdiA itself directly controls biofilm formation and SdiA variants could be further recognized by the inter-species signal AHLs. This is the first random protein engineering to control biofilm formation. Experiment Overall Design: For the microarray experiments, 10 g glass wool (Corning Glass Works, Corning, N.Y.) were used to form biofilms in 250 mL LB ( 1 mM IPTG + Cm30) in 1 L Erlenmeyer shake flasks which were inoculated with overnight cultures diluted that were 1:100. The cells were shaken at 250 rpm and 30C for 12 hours to form biofilms on the glass wool, and RNA was isolated from the the biofilm cells.

Project description:The global transcriptional regulator Hha of Escherichia coli controls hemolysin activity, biofilm formation, and virulence expressions. Earlier, we have reported that Hha represses initial biofilm formation and disperses biofilms as well as controls prophage excision in E. coli. Since biofilm dispersal is a promising area to control biofilms, here we rewired Hha to control biofilm dispersal and formation. The Hha variant Hha13D6 was obtained to have enhanced biofilm dispersal activity along with increased toxicity compared to wild-type Hha (Hha13D6 induces dispersal 60%, whereas wild-type Hha induces dispersal at early biofilms but not at mature biofilms). Toxic Hha13D6 caused cell death probably by the activation of proteases HslUV, Lon, and PrlC, and deletion of protease gene hslV with overproducing Hh13D6 repressed biofilm dispersal, indicating Hha13D6 induces biofilm dispersal through the activity of protease HslV. Furthermore, another Hha variant Hha24E9 was also obtained to decrease biofilm formation 4-fold compared to wild-type Hha by regulation of gadW, glpT, and phnF. However, the dispersal variant Hha13D6 did not decrease biofilm formation, while the biofilm variant Hha24E9 did not induce biofilm dispersal. Hence, Hha may have evolved two ways in response to environmental factors to control biofilm dispersal and formation, but both controlling mechanisms come from different regulatory systems. For the whole-transcriptome study of BW25113 hha/pCA24N-hha13D6 versus BW25113 hha/pCA24N-hha biofilm dispersal, cells were grown in 250 mL of LB glucose (0.2%) for 16 h at 125 rpm with 10 g of glass wool (Corning Glass Works, Corning, NY, USA) in 1 L Erlenmeyer flasks to form a robust biofilm (Ren et al., 2004) and incubated an additional 1 h with 1 mM IPTG to induce wild-type Hha and Hha13D6. Similarly, for the whole transcriptome study of BW25113 hha/pCA24N-hha24E9 versus BW25113 hha/pCA24N-hha biofilm formation, cells were grown in 250 mL of LB glucose (0.2%) containing 1 mM IPTG for 7 h at 250 rpm with 10 g of glass wool to form biofilms. Biofilm cells were obtained by rinsing and sonicating the glass wool in sterile 0.85% NaCl solution at 0Â°C, and RNALater bufferÂ® (Applied Biosystems, Foster City, CA, USA) was added to stabilize RNA during the RNA preparation steps. Total RNA was isolated from biofilm cells using a bead beater (Biospec, Bartlesville, OK, USA). cDNA synthesis, fragmentation, and hybridizations to the E. coli GeneChip Genome 2.0 array (Affymetrix, Santa Clara, CA, USA; P/N 511302). Genes were identified as differentially expressed if the expression ratio was higher than the standard deviation: 2.0-fold (induced and repressed) cutoff for Hha13D6 DNA microarrays (standard deviation 1.3-fold) and 10.0-fold (induced) or 4.0-fold (repressed) for Hha24E9 DNA microarrays (standard deviation 4.0-fold), and if the p-value for comparing two chips was less than 0.05.

Project description:Intercellular signal indole and its derivative hydroxyindoles inhibit Escherichia coli biofilm and diminish Pseudomonas aeruginosa virulence. However, indole and bacterial indole derivatives were unstable in microbial community due to the widespread of diverse oxygenases that could quickly degrade them. Hence, we sought to identify novel non-toxic, stable, and potent indole derivatives from plant sources for inhibiting biofilm formation of E. coli O157:H7 and P. aeruginosa PAO1. Here, plant auxin 3-indolylacetonitrile (IAN) was found to inhibit biofilm formation of both E. coli O157:H7 and P. aeruginosa without affecting its growth. IAN inhibited biofilms more effectively than indole for both E. coli and P. aeruginosa. Additionally, IAN decreased the production of virulence factor pyocyanin in P. aeruginosa. DNA microarray analysis indicated that IAN repressed genes involved in curli formation and glycerol metabolism, while IAN induced indole-related genes and prophage genes in E. coli. It appears that IAN inhibits biofilm formation of E. coli by reducing curli formation and inducing indole production. Furthermore, unlike bacterial indole derivatives, plant-originated IAN was stable in the presence of either E. coli or P. aeruginosa. For the microarray experiments, P. aeruginosa were inoculated in 25 ml of LB medium in 250 ml shake flasks with overnight cultures that were diluted 1:100. IAN (100 μg/ml) dissolved in 25 μl DMSO or 25 μl DMSO alone as a control was added. Cells were cultured in LB at 37C with 250 rpm shakingfor 5 hrs. Cells were immediately chilled with dry ice and 95% ethanol (to prevent RNA degradation) for 30 sec before centrifugation in 50 ml centrifuge tubes at 13,000 g for 2 min; cell pellets were frozen immediately with dry ice and stored -80°C. RNA was isolated using Qiagen RNeasy mini Kit (Valencia, CA, USA). RNA quality was assessed by Agilent 2100 bioanalyser using the RNA 6000 Nano Chip (Agilent Technologies, Amstelveen, The Netherlands), and quantity was determined by ND-1000 Spectrophotometer (NanoDrop Technologies, Inc., DE, USA).

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P. aeruginosa and indole and 7-hydroxyindole

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