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.

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:Two lineages of enterohemorrhagic (EHEC) Escherichia coli O157:H7 (EDL933, Stx1+ and Stx2+) and 86-24 (Stx2+) were investigated in regards to biofilm formation on an abiotic surface. Strikingly, EDL933 strain formed a robust biofilm while 86-24 strain formed no biofilm on either a polystyrene plate or a polyethylene tube. To identify the genetic mechanisms of different biofilm formation in two EHEC strains, DNA microarrays were first performed and phenotypic assays were followed. In the comparison of the EDL933 strain versus 86-24 strain, genes (csgBAC and csgDEFG) involved in curli biosynthesis were significantly induced while genes (trpLEDCB and mtr) involved in indole signaling were repressed. Additionally, a dozen of phage genes were differentially present between two strains. Curli assays using a Congo red plate and scanning electron microscopy corroborate the microarray data as the EDL 933 strain produces a large amount of curli, while 86-24 forms much less curli. Also, the indole production in the EDL933 was 2-times lower than that of 86-24. It was known that curli formation positively regulates and indole negatively regulates biofilm formation of EHEC. Hence, it appears that less curli formation and high indole production in the 86-24 strain are majorly responsible for no biofilm formation. For the microarray experiments, E. coli O157:H7 EDL933 and 86-24 were inoculated in 25 ml of LB in 250 ml shake flasks with overnight cultures that were diluted 1:100. Cells were shaken at 250 rpm and 37°C for an absorbance of 4.0 at 600 nm. 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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Transcriptional changes involved in inhibition of biofilm formation by ε-Polylysine in Salmonella Typhimurium

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