Analyses of cis- and trans-acting elements involved in glucose repression and transcriptional regulation of chitin utilization in Streptomyces lividans

To the Gram-positive soil bacteria Streptomyces, catabolite repression is not only a fundamental aspect of their physiology, but also directly related to the most interesting aspects of this bacteria--morphological development and antibiotic production. The chi63 promoter directs glucose-sensitive, chitin-dependent transcription of a gene involved in the utilization of chitin as carbon source. Analysis of {dollar}5spprime{dollar} and {dollar}3spprime{dollar} deletions of the promoter region revealed that a 350 bp segment is sufficient for wild type levels of expression and regulation. The analysis of single base changes identified several sequences to be important for activity and regulation. The sequences centered around {dollar}{lcub}-{rcub}10{dollar} (TATTCT) and {dollar}{lcub}-{rcub}35{dollar} (TTGACC) in this promoter have been identified as the RNA polymerase binding site. Overlapping the RNA polymerase binding site is a perfect 12 base pair direct repeat sequence. Some base changes within this direct repeat resulted in constitutive expression, suggesting that this sequence is an operator for negative regulation. Other base changes resulted in loss of glucose repression while retaining the requirement for chitin induction, suggesting that this sequence is also involved in glucose repression. Since mutations that affect glucose repression and chitin induction fall within the same direct repeat sequence module, the direct repeat sequence apparently facilitates both chitin induction and glucose repression.; The phosphoenolpyruvate:sugar phosphotransferase system (PTS) plays important roles in catabolite repression of most bacteria. The catabolite control protein, CcpA, is a key element in PTS mediated catabolite repression of many Gram-positive bacteria. To investigate whether the HPr-ser-P-CcpA regulatory system is involved in catabolite repression of Streptomyces, PCR primers were designed to amplify a CcpA homologue from Streptomyces lividans. These primers identified reg1, a gene already deposited in the GenBank but with no defined functions. Reg1 encodes a protein that belongs to the LacI/GalR regulatory protein family and has strong amino acid sequence similarity to CcpA. However, disruption of reg1 has no apparent effect on glucose repression of the chi63 and galP1 promoters, indicating that it is not a general regulator of catabolite repression in Streptomyces lividans.