Regulation of clostridium acetobutylicum glutamine synthetase glnA gene
Doctoral Thesis
1994
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University of Cape Town
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The regulation of nitrogen (N) metabolism is being investigated in the Gram positive anaerobic bacterium Clostridium acetobutylicum, which has been utilized in industrial fermentations to produce acetone, butanol and ethanol. The C. acetobutylicum glutamine synthetase (GS) glnA gene region originally cloned in Escherichia coli, is characterized by the glnA structural gene, the upstream promoter sequences P₁ and P₂, a downstream DNA sequence complementary to the 5'end of the glnA gene and the downstream promoter P₃. Transcription of the glnA gene is controlled by the upstream promoters P₁ and P₂. Transcription from the downstream promoter P₃ in the opposite orientation of the glnA gene was demonstrated to express the 43-base glnA antisense (AS) RNA in E. coli and C. acetobutylicum cells. The expression of GS activity or the glnA AS RNA were not regulated by N in the heterologous E. coli host, but the expression of the antisense RNA in these cells was associated with decreased levels of GS activity. The regulation of the glnA gene expression was studied in C. acetobutylicum cells after suitable media for the growth of this bacterium was developed. C. acetobutylicum GS activity, the transcription of glnA mRNA and the glnA AS RNA were regulated by N. In cells grown in N-rich medium GS activity and glnA mRNA were repressed. Repression ratios for GS activity varied from 1.6 to 9.0 depending on the sampling time. The relative number of glnA transcripts was approximately 25%-28% lower in cells grown in N-limiting medium. The expression of the glnA AS RNA was differentially regulated relative to the. GS activity and glnA mRNA levels. The glnA AS RNA was repressed in N-limiting medium and induced in N-rich medium. The relative. number of AS RNA transcripts was approximately 1.5-fold in excess of glnA mRNA transcripts under conditions that repressed GS activity. Under conditions that induced GS activity, glnA mRNA transcripts exceeded AS RNA transcripts. Since differential regulation by N levels of the glnA AS RNA expression relative to the glnA gene was demonstrated in C. acetobutylicum, additional regulatory element(s) affecting the C. acetobutylicum glnA system were investigated. C. acetobutylicum gene libraries were cotransformed in trans with an in-frame glnA-lacZ fusion construct and plasmids from the C. acetobutylicum gene libraries were tested for flgalactosidase expression. No alteration of the lacZ gene expression was detected in the cotransformed colonies. However, DNA sequencing of the region situated downstream of the C. acetobutylicum glnA gene revealed the presence of an open reading frame (ORF) located 199 to 766bp from the 3'end of the glnA structural gene. The glnA AS coding region is located on the putative ribosome binding site and the 5'region of this C. acetobutylicum ORF. The protein encoded by this ORF showed 30% similarity with the carboxy terminus of the Pseudomonas aeruginosa aliphatic amidase regulator encoded by the amiE gene. The amino terminus of this protein also has 28% and 26% similarity with the amino terminal region of the DegU and CheB response regulators, respectively. These regulators belong to the family of the response regulators involving two component signal transduction systems, suggesting that the protein encoded by this ORF may play a role in the mechanism of regulation of the C. acetobutylicum glnA gene in response to nitrogen.
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Bibliography: pages 146-162.
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Fierro-Monti, I. 1994. Regulation of clostridium acetobutylicum glutamine synthetase glnA gene. University of Cape Town.