PMID:10760150
| Citation |
Francetic, O, Badaut, C, Rimsky, S and Pugsley, AP (2000) The ChiA (YheB) protein of Escherichia coli K-12 is an endochitinase whose gene is negatively controlled by the nucleoid-structuring protein H-NS. Mol. Microbiol. 35:1506-17 |
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| Abstract |
The chromosome of Escherichia coli K-12 contains a putative gene, yheB (chiA), at centisome 74.7, whose product shows sequence similarity with chitinases of bacterial and viral origin. We cloned the chiA (yheB) gene and demonstrated that it codes for a 94.5 kDa periplasmic protein with endochitinase/lysozyme activity. Under standard laboratory growth conditions, chiA expression is very low, as shown by the Lac- phenotype of a chiA transcriptional fusion to a promoterless lacZ reporter. To identify factors that control chitinase gene expression, we generated random Tn10 insertions in the chromosome of the fusion-containing strain, selecting for a Lac+ phenotype. The majority of the mutations that caused a Lac+ phenotype mapped to the hns gene, encoding the nucleoid-structuring protein H-NS. Transcription of chiA in vivo is driven by a single sigma70 promoter and is derepressed in an hns mutant. Using a competitive gel retardation assay, we demonstrated that H-NS binds directly and with high affinity to the chiA promoter region. In addition to hns, other E. coli mutations causing defects in global regulatory proteins, such as fis, crp or stpA in combination with hns, increased chiA expression to different extents, as did decreasing the growth temperature from 37 degrees C to 30 degrees C. A possible physiological function of ChiA (YheB) endochitinase in E. coli K-12 is discussed. |
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| Keywords |
Amino Acid Sequence; Bacterial Proteins; Base Sequence; Chitinase/genetics; Chitinase/metabolism; Cloning, Molecular; DNA Transposable Elements; DNA-Binding Proteins/genetics; DNA-Binding Proteins/metabolism; Escherichia coli/genetics; Gene Expression Regulation, Bacterial; Molecular Sequence Data; Mutation; Promoter Regions, Genetic; Subcellular Fractions; Substrate Specificity; beta-Galactosidase/genetics |
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