Abstract
A novel protein family, clostridial hydrophobic with a conserved W (ChW), is specific to Clostridium acetobutylicum, thus, suggesting roles specific to its unique physiology. Functions for members of this protein family have not been characterized. The expression and promoter architecture of two genes, chw14 (CAC1532) and chw16/ 17 (CAC2584), encoding two members of the ChW protein family were characterized and their regulation explored. The genes chw14 and chw16/ 17 behave similarly under every condition tested in the DNA-microarray gene expression studies. Previous protein analysis suggested that the master transcriptional regulator, Spo0A, was required for their accumulation, as ChW14 and ChW16/17 proteins were absent in the spo0A null strain, SKO1. Primer extension assays showed a single transcript for each chw14 and chw16/ 17 detected from mid-exponential phase until early stationary phase. A predicted σ A consensus motif is just upstream of the transcriptional start sites of both chw14 and chw16/ 17, with a single putative Spo0A binding site, 0A box, within the promoter region of both chw14 and chw16/ 17. Using reporter analysis we showed that the promoters of chw14 and chw16/ 17 are highly active during mid-exponential phase in wild type C. acetobutylicum. Analysis of expression in the spo0A deficient SKO1 strain indicated the promoter activity of both chw14 and chw16/ 17 appears constitutive, thus the promoters do not appear to be inactivated in the absence of Spo0A. The relationship among ChW proteins and the ChW domains themselves was delineated. The ChW proteins appear to originate with C. acetobutylicum where the non- C. acetobutylicum ChW proteins are nested within the main C. acetobutylicum protein branch. Examination of the ChW domain alone shows that every third domain clusters together phylogenetically. Additionally, almost all of ChW proteins contain a multiple of three ChW domains. Taken together, these data suggest that ChW domains function in triplets of association.
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