Molecular analysis and regulation of the glnA gene of the gram-positive anaerobe Clostridium acetobutylicum

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Abstract
The nucleotide sequence of a 2.0-kilobase DNA segment containing the Clostridium acetobutylicum glnA gene was determined. The upstream region of the glnA gene contained two putative extended promoter consensus sequences (p1 and p2), characteristic of gram-positive bacteria. A third putative extended gram-positive promoter consensus sequence (p3), oriented towards the glnA gene, was detected downstream of the structural gene. The sequences containing the proposed promoter regions p1 and p2 or p3 were shown to have promoter activity by subcloning into promoter probe vectors. The complete amino acid sequence (444 residues) of the C. acetobutylicum glutamine synthetase (GS) was deduced, and comparisons were made with the reported amino acid sequences of GS from other organisms. To determine whether the putative promoter p3 and a downstream region with an extensive stretch of inverted repeat sequences were involved in regulation of C. acetobutylicum glnA gene expression by nitrogen in Escherichia coli, deletion plasmids were constructed lacking p3 and various downstream sequences. Deletion of the putative promoter p3 and downstream inverted repeat sequences affected the regulation of GS and reduced the levels of GS approximately fivefold under nitrogen-limiting conditions but did not affect the repression of GS levels in cells grown under nitrogen-excess conditions.