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DNA-seq of differentially induced promoter regions in E. coli UTI89 strain grown in either J82 bladder epithelial cells, urine, DMEM or LB medium.

ABSTRACT: DNA-seq of differentially induced promoter regions in E. coli UTI89 strain grown in either J82 bladder epithelial cells, urine, DMEM or LB medium.

PROVIDER: PRJEB20312 | ENA |

REPOSITORIES: ENA

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			Action	DRS
	ERR1916462.fastq.gz	Fastqsanger.gz
	ERR1916463.fastq.gz	Fastqsanger.gz
	ERR1916464.fastq.gz	Fastqsanger.gz
	ERR1916465.fastq.gz	Fastqsanger.gz

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DNA-seq of differentially induced promoter regions in E. coli UTI89 strain grown in either J82 bladder epithelial cells, urine, DMEM or LB medium.

Project description:The method DFI-seq was developed to enable identification of differentially expressed genes in uropathogenic E. coli strain UTI89 during growth in human urine and in bladder epithelial cells. By utilising this new method, the aim was to identify novel virulence genes in UTI89. DFI-seq is a combination of differential fluorescence induction (DFI) with next-generation sequencing. DFI-seq was compared to DFI by analysing gene expression of UPEC in human urine and thereby confirming that DFI-seq gives a better overview of gene expression. DFI-seq was hereafter used to look at gene expression in UTI89 while infecting bladder epithelial cells. We demonstrate the usefulness of DFI-seq for identification of genes required for optimal growth of UPEC in human urine, as well as potential virulence genes upregulated during infection of bladder epithelial cells. DFI-seq holds potential for the study of bacterial gene expression in live-animal infection systems.

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Proteomes of uropathogenic Escherichia coli growing in human urine and in J82 urinary bladder cell.

Project description:Uropathogenic Escherichia coli (UPEC) are the most common cause of urinary tract infection (UTI). UPEC normally reside in the intestine, and during establishment of UTI, it undergoes metabolic adaptations, first to urine and then upon tissue invasion to the bladder cell interior. In order to understand these adaptations, we used quantitative proteomic profiling to characterize protein expression of UPEC strain UTI89 growing in human urine and when inside J82 bladder cells. In order to facilitate detection of UPEC proteins over the excess amount of eukaryotic proteins in bladder cells, we developed a method where proteins from UTI89 grown in MOPS and urine was spiked-in to enhance detection of bacterial proteins. More than 2000 E. coli proteins were detected. During growth in urine, proteins associated with iron acquisition and several amino acid uptake and biosynthesis systems, in particular arginine metabolism, were significantly upregulated. During growth in J82 cells, proteins related to iron uptake and arginine metabolisms were upregulated together with proteins involved in sulphur compound turnover. Results suggested that UPEC experience a richer environment in bladder cells compared to urine. There was no direct correlation between upregulated proteins and proteins reported to be essential for infections, showing that upregulation during growth does not signify that the proteins are essential for growth under a condition.

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Impact of urine from cranberry-treated volunteers on global gene expression of uropathogenic Escherichia coli

Project description:Growth in urine of cranberry extract-treated volunteers did not markedly affect the transcirptome of UPEC strain UTI89. Expression of bacterial adhesin determinants (P fimbriae, S fimbrae, curli) was not markedly changed upon growth in the urine samples, while a slight, but non-significant upregulation of type 1 fimbriae was observed.

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Project description:RNA-seq of Klebsiella pneumoniae SGH10 grown in LB or DMEM medium

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RNA-seq of Klebsiella pneumoniae SGH10 grown in LB or DMEM medium

Project description:SGH10 bacteria were grown under DMEM (Dulbecco's Modified Eagle Medium) and LB(Lysogeny broth). Bacteria were harvested by centrifugation, the pellet was resuspended in TE buffer, mixed with lysosyme (SigmaAldrich) in a final concentration of 0.9 mg/ml, and immediately frozen in liquid nitrogen. The frozen pellet was subjected to two cycles of thaw and freeze at 37°C and in liquid nitrogen, respectively, and RNA was extracted using Tri-Reagent (Sigma-Aldrich). RNA-seq libraries were constructed by the RNAtag-seq method. RNA-seq libraries were sequenced by single end sequencing using Nextseq 500 Sequencer (Illumina). sequence fragments were mapped to SGH10 which contains a chromosome NZ_CP025080.1 and a plasmid NZ_CP025081.1. For each growth condition, three biological repeats were done.

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