Unknown,Transcriptomics,Genomics,Proteomics

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Transcription profiling of S. cerevisiaeAir-liquid interfacial biofilm vs planktonic S. cerevisiae cells

ABSTRACT: Goal was to identify yeast genes whose expression changed as a function of the shift from growth in bulk culture to growth in an air-liquid interfacial biofilm. Experiment Overall Design: Cells were grown 24 h at 30 C in YEPD to a density of about 500,000,000 cells/ml. At harvest, sugar was found to be depleted as measured by an enzymatic dip stick (Diastic, Bayer). Cells were pelleted and washed twice in sterile distilled water by centrifugation and then diluted 10-fold into 100 ml of Flor medium (YNB + 4% ethanol + leucine + histidine + uracil) in triplicate 250 ml beakers. Cultures were then grown statically in Flor medium at 27 C. Within a few hours following inoculation, the bulk liquid appeared to be clear, no biofilm was evident by visual observation, and a layer of settled cells was evident at the bottom of the beakers. After 48 h, a visible air-liquid interfacial biofilm covered the entire surface while the thickness of the layer of settled cells appeared unchanged. After 48 h, biofilm cells (floaters) were collected by aseptic aspiration. Once the biofilm cells were removed, cells at the bottom of the beaker (sinkers) were collected similarly. Cells from both populations were washed once in sterile distilled water by centrifugation prior to RNA isolation. Significant cell clumping was evident in both populations of cells by microscopic observation. While cell viability was estimated by plating on YEPD, the resultant cfu/ml values could not be directly correlated with cell counts in a hemacytometer because clumps of cells containing at least one viable cell presumably produced only a single colony. Further, counting individual cells accurately in the numerous clumps containing large numbers of cells was not possible. Nonetheless, when cells were counted (clumps were counted as single cells) and compared to cfu/ml for the same suspension, the cfu/ml values were about 2-fold higher than the corresponding values for cell counts using the hemacytometer for both biofilm and bottom layer cells.

ORGANISM(S): Saccharomyces cerevisiae

SUBMITTER: Alan Bakalinsky

PROVIDER: E-GEOD-19156 | biostudies-arrayexpress |

REPOSITORIES: biostudies-arrayexpress

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Project description:Background: Biofilm formation by Salmonella species enhances the capacity of these pathogenic bacteria to survive stresses that are commonly encountered within food processing and during host infection. The persistence of Salmonella within the food chain has become a major health concern, as biofilms can serve as a reservoir to contaminate food products. While the molecular mechanisms required for the survival of bacteria on surfaces are not fully understood, transcriptional studies of other bacteria have demonstrated that biofilm growth triggers the expression of specific sets of genes, compared with planktonic cells. Until now, most transcriptomic studies of Salmonella have focused on the effect of infection-relevant stressors on virulence and on comparing mutant and wild-type (WT). However little is known about the physiological responses taking place inside a Salmonella biofilm. Results: We have determined the transcriptomic and proteomic profiles of biofilms of Salmonella enterica serovar Typhimurium. We discovered that 124 of detectable proteins were differentially expressed in the biofilm compared with planktonic cells, and that 10% (433 genes) of S. Typhimurium genes showed a biofilm-specific pattern of transcription (i.e. a 2-fold or more change in the biofilm compared with planktonic cells). The genes that were significantly up-regulated implicated specific cellular processes in biofilm development including amino acid metabolism, cell motility, global regulation and tolerance to stress. We found that the most highly down-regulated genes in the biofilm were located on Salmonella Pathogenicity Island 2 (SPI2), but that a functional SPI2 secretion system regulator (ssrA) was required for WT biofilm formation. We identified STM0341 as a gene of unknown function that was needed for WT biofilm growth. Genes involved in tryptophan (trp) biosynthesis and transport were up-regulated in the biofilm. Deletion of trpE led to a decrease in bacterial attachment and we show that this biofilm defect is restored by exogenous tryptophan or indole. Conclusion: Biofilm growth of S. Typhimurium causes distinct changes in gene and protein expression. Our results show that aromatic amino acids make an important contribution to biofilm formation, and reveal that SPI2 genes, required for virulence in host cells, show opposing patterns of expression to biofilm-specific genes in S. Typhimurium. Overnight cultures of SL1344 grown in CFA broth were standardized to achieve an initial concentration of 10^6 CFU ml-1 and injected into a stirring influent flask containing 5 L of pre-warmed (25C) sterile CFA medium. The inoculated influent was then pumped through silicon tubing (5 mm internal diameter, Samco Silicon Products Ltd.) at 60 ml h-1 using a peristaltic pump (Minipulse 3, Gilson). The bacteria were allowed to flow through the closed system and either attach to a vertical piece of silicon tubing (1 meter length, 16 mm internal diameter, Samco Silicon Products Ltd.) or flow out into a waste reservoir. The tubing was positioned vertically to collect biofilm cells adherent to the tubing and minimise collecting bacteria that had simply sedimented. Possible backflow of media or bacteria into the influent from the silicon tubing was eliminated using media breaks. Samples taken to determine the pH of the medium or bacterial cell counts were removed via a media port located near the effluent and influent vessel, respectively. Planktonic cells were removed after 72 h of growth from the influent vessel to avoid any contamination with biofilm cells. The entire system was kept at a constant temperature of 25C and biofilm cells were isolated after 72 h of growth. All planktonic and biofilm samples were isolated from the same experimental system and used for both RNA and protein isolation. For this study, four biological replicates were performed at four different dates (Jun25, Jul5, Jul16, Oct11; as indicated in the sample title). For the dual-colour microarray hybridisations, we used Salmonella genomic DNA as the comparator which also acted as the control for spot quality. Individual labelled cDNA samples (RNA) were hybridised up to 4 times (technical replicates).

Project description:For Streptococcus pneumoniae, biofilms have been suggested to promote long-term colonization of the nasopharynx and contribute to the pathology of recurrent middle ear infections. To date numerous studies have investigated the contribution of specific genetic determinants for the development of pneumococcal biofilms, however, studies examining the global changes that occur during biofilm development and how they contribute to disease are lacking. Using Scanning and Transmission electron microscopy we examined development of a mature pneumococcal biofilm in a continuous flow through reactor. We determined that a mature biofilm is formed in discrete stages, is marked by the formation of complex 3-dimensional structures, and is primarily composed of dead pneumococci. Using genomic microarrays we determined that pneumococci in mature biofilms down regulate genes involved in protein synthesis, energy production, metabolism, capsular polysaccharide production, and virulence. We confirmed these changes by testing bacterial resistance to antimicrobials, measuring capsule production by ELSIA, and immunoblotting for pneumolysin production. We determined that biofilm pneumococci are hyper-adhesive, binding to cell lines at levels 9 to 11-fold greater than planktonic counterparts. Using Western blot and ELISA, we determined that biofilm bacteria produce greater amounts of the adhesins PsrP, CbpA, and surface exposed phosphorylcholine. We subsequently determined that the hyper-adhesive phenotype was in part due to selection of the transparent phase variant during biofilm growth. Intranasal, intratracheal and intraperitoneal challenge of mice with biofilm and planktonic pneumococci determined that biofilm bacteria were highly attenuated for invasive disease but not nasopharyngeal colonization. Immunization of mice with ethanol-killed biofilm pneumococci of serotype 4 conferred protection against challenge with same isolate but not a serotype 3. ELISA for reactive IgG levels subsequently determined that biofilm pneumococci do not provide high levels of cross-reactive protein antigens. Together these studies suggest that biofilms do not directly contribute to disease but instead confer a protected mode of growth for the pneumococcus. Pneumococcal biofilms compared to planktonic control at 4, 12, 24, 48 hours. 3 biological replicates each of 4 and 12 hour time points, and 2 biological replicates each of 24 and 48 hour time points. Flip dye (technical replicates) performed for 4, 12, and 24 hour time points; no technical replicate performed for 48 hour time point due to limiting material. Ratios were determined by averaging across technical and biological replicates. The following hybridizations made up each biological replicate: 14090167.tav.annot and 14090190.tav.annot (4hr biol rep 1); 14090169.tav.annot and 14090176.tav.annot (4hr biol rep 2); 14090192.tav.annot and 14090188.tav.annot (4hr biol rep 3); 14087688.tav.annot and 14090180.tav.annot (12hr biol rep 1); 14090185.tav.annot and 14090168.tav.annot (12hr biol rep 2); 14090191.tav.annot and 14090174.tav.annot (12hr biol rep 3); 14090170.tav.annot and 14090175.tav.annot (24hr biol rep 2); 14090193.tav.annot and 14087687.tav.annot (24hr biol rep 3); 14090181.tav.annot (48hr biol rep 1); 14090187.tav.annot (48hr biol rep 2)

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Transcription profiling of S. cerevisiaeAir-liquid interfacial biofilm vs planktonic S. cerevisiae cells

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