Direct repeat sequences in the Streptomyces chitinase-63 promoter direct both glucose repression and chitin induction

Abstract

The chi63 promoter directs glucose-sensitive, chitin-dependent transcription of a gene involved in the utilization of chitin as carbon source. Analysis of 5 and 3 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 throughout the promoter region, introduced by random and site-directed mutagenesis, identified several sequences to be important for activity and regulation. Single base changes at ؊10, ؊12, ؊32, ؊33, ؊35, and ؊37 upstream of the transcription start site resulted in loss of activity from the promoter, suggesting that bases in these positions are important for RNA polymerase interaction. The sequences centered around ؊10 (TATTCT) and ؊35 (TTGACC) in this promoter are, in fact, prototypical of eubacterial promoters. Overlapping the RNA polymerase binding site is a perfect 12-bp 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. The fact that cis-acting mutations resulted in glucose resistance but not inducer independence rules out the possibility that glucose repression acts exclusively by inducer exclusion. The fact that mutations that affect glucose repression and chitin induction fall within the same direct repeat sequence module suggests that the direct repeat sequence facilitates both chitin induction and glucose repression.

Transcriptional co-regulation of five chitinase genes scattered on the Streptomyces coelicolor A3(2) chromosome

“…The 12 bp direct repeat in the chi63 promoter region of S. plicatus directs both induction and repression of the chi63 promoter (Delic et al, 1992 ;Ni & Westpheling, 1997). Two copies of the 12 bp putative operator sequence were found in the promoter regions of most of the S. coelicolor chi genes (Fig.…”

“…6) whereas there was no obvious promoter sequence upstream of chiG and chiH. Ni & Westpheling (1997) proposed that the direct repeat sequences in the chi63 promoter region of S. plicatus direct its chitin-induced transcription. In S. coelicolor A3(2), sequences highly similar to the S. plicatus direct repeat are present in the upstream regions of chiA, chiB, chiC, chiD, chiE, chiF and chiH but not of chiG, while only one copy of the repeated sequence is present in the possible promoter region of chiE (Fig.…”

“…Although the molecular mechanisms governing the regulation of expression of chitinase genes in Streptomyces are still unclear, a pair of direct repeat sequences present in the promoter region of chi63, a family 18 chitinase gene of Streptomyces plicatus, was shown to be involved in the induction by chitin and the repression by glucose of the gene expression (Delic et al, 1992 ;Ni & Westpheling, 1997). On the other hand, the glkA gene encoding a glucose kinase (Angell et al, 1992) was shown to be involved in the glucose repression of chitinase production in S. lividans (Saito et al, 1998).…”

Chitinase Inhibitor Allosamidin Is a Signal Molecule for Chitinase Production in Its Producing Streptomyces

“…Presence of the direct repeat sequence at the promoter region of chi65 and two genes encoding a two-component regulatory system suggested that regulation of chi65 expression is not simple. In Streptomyces plicatus, it was shown that the direct repeat sequence region at the promoter of chi63 was essential for regulation of the gene expression by glucose repression [5]. Therefore a repressor-type protein is assumed to bind the region of the direct repeat sequence for the regulation, but its detailed mechanism is still unknown.…”

“…With respect to the regulation mechanism for gene expression of chitinases in Streptomyces, a direct repeat sequence present in the promoter region of the gene is known to be important for its regulation [5]. In the regulation, a repressor-type protein which can bind the sequence is assumed to be present, but the repressor is still unknown [6].…”