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The E. coli pathobiont LF82 encodes a unique variant of sigma 70 that results in global gene expression changes and altered phenotypes

ABSTRACT: Purpose: To investigave how a unique rpoD variant of LF82 contributes to its biology by seeing how it affects phenotype and global gene expression Method: Isogenic E. coli MG1655 and D445V mutant were grown to a cell density of OD600 ~0.5-0.6 in LB broth at either 37oC or 23oC. RNA was isolated at 5 post-infection using method II of (Hinton 1989). rRNA subtraction was performed with the bacterial RiboMinus Kit (Ambion) according to manufacturer instructions. cDNA was prepared using the NEBNext strand specific kit (New England BioLabs) according to manufacturer instruction for libraries with 300-450 bp insert size with the following modifications. Illumina adaptors sequences based on TruSeq HT Sample Prep Kits were purchase from Integrated DNA Technologies and used in the ligation step. TruSeq-1 and TruSeq-2 primer were used for PCR enrichment of adaptor ligated DNA. Library size was verified with a Bioanalyzer using an Agilent High Sensitivity DNA kit. The concentration of each library was determined using the KAPA Library Quantification Kit for Illumina platforms. Sequencing was performed by the NIDDK Genomics Core facility using a MiSeq system with the MiSeq 2 x 250 bp Sequencing Kit (Illumina). Result: RNA-seq data revealed that the single D445V subsitition changes the expression of multiple genes in E. coli. We see a total of 137 genes up-regulated at 23oC and 75 gene up-regulated at 37oC in the mutant compared the WT. We saw an increase in genes that lead to beta-lactam resistance, biofilm formation, methionine biosynsesis. We saw a total of 236 genes that were down regulated at 23oC and 57 genes down-regulated at 37oC in the mutant compared to the WT, including a decrease in flagella gene expression. Conclusion: The rpoD D445V mutation affects the expression level of multiple specific genes, consistent with the LF82 lifestyle.

ORGANISM(S): Escherichia coli str. K-12 substr. MG1655

PROVIDER: GSE222248 | GEO | 2023/01/10

REPOSITORIES: GEO

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The E. coli pathobiont LF82 encodes a unique variant of sigma 70 that results in global gene expression changes and altered phenotypes

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