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Ecoli DH5alpha, pUC overexpression in LB + Amp vs no pUC

ABSTRACT: Responses of Escherichia coli DH5alpha as they overexpress pUC at different ODs in LB + Amp Keywords: time course

ORGANISM(S): Escherichia coli

PROVIDER: GSE4379 | GEO | 2006/03/08

SECONDARY ACCESSION(S): PRJNA94555

REPOSITORIES: GEO

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Project description:E. coli K-12 BW25113 mutant strain hha expression in biofilm cells relative to E. coli wild-type strain expression in biofilm cells. Samples were cultured in LB with glasswool at 37C for 4 hours and in LB glu with glasswool at 37C for 4, 15 and 24 hours. Hha is a temperature- and osmolarity-dependent modulator of gene expression that is induced 30-fold in Escherichia coli biofilms. Here we show through whole-transcriptome analysis that Hha decreases biofilm formation in both LB and LB glu media by (i) repressing fliC encoding the main structural flagellar protein flagellin, (ii) by repressing fimA encoding the major structural subunit of type I fimbriae, (iii) by repressing ihfA encoding a subunit of the transcriptional regulator IHF that induces the transcription of type I fimbriae genes, (iv) by regulating tnaA encoding tryptophanase that inhibits biofilms, and (v) by repressing ybaJ that forms an operon with hha. Corroborating the microarray data, hha deletion increased motility 3.2 ± 0.1-fold, decreased extracellular indole concentrations 12 ± 2-fold, and decreased type I fimbriae (as measured by yeast agglutination). Biofilm tests using single and double mutants of fimA and ihfA and transcriptional studies of the fimA, ihfA and ybaJ-hha promoters confirmed that Hha represses biofilm by inhibiting type I fimbriae production and that it negatively regulates its own transcription and that of ybaJ. Nickel-enhanced DNA microarrays to determine in vivo Hha binding sites confirmed that Hha binds the ybaJ-hha promoter, that it binds fimZ, a positive regulator of fimA, and that it binds to the rare codon tRNAs argU, ileXY, and proL. Sequence analysis of fimZ, fimB, fimE, and the type I fimbriae gene cluster fimAICDFGH revealed a high bias for the rare codons of arginine, isoleucine, proline, leucine, and threonine, and overexpressing Hha leads to cell death. Therefore, it appears Hha decreases biofilms by decreasing type I fimbriae production as a result of inhibiting synthesis of tRNAs for rare codons. Keywords: effect of hha deletion in biofilm formation 4 hr LB and 4,15 and 24 hr LB glu

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Ecoli DH5alpha, pUC overexpression in LB + Amp vs no pUC

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