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Site-directed mutagenesis of conserved inverted repeat sequences in the xylanase C promoter region from Streptomyces sp. EC3

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Abstract

Streptomyces sp. EC3, a strain which was originally isolated from cattle manure compost, was shown to possess a strong xylanolytic activity. One of the genes responsible for this activity, xlnC , encodes a secreted xylanase. In the native strain, as in the heterologous host S. lividans, expression of xlnC was detectable in the presence of xylan but not in the presence of glucose. Induction by xylan was shown to take place at the transcriptional level. The transcriptional start site of xlnC was mapped and likely –35 (5′-TTGACA-3′) and –10 (5′-GAGAAC-3′) motifs were identified. In order to localise putative conserved regulatory sequences, the promoter regions of xylanase-encoding genes from various Streptomyces species were aligned. This alignment revealed the existence of three sets of quite well conserved palindromic AT rich sequences called boxes 1, 2 and 3. Box 3 (5′-CGAAA N TTTCG-3′) is the farthest away from the promoter region (150–200 bp). A shorter version of this palindrome (5′-GAAA NN TTTC-3′) or (5′-CGAAA-3′) constitutes box 1, which is located just upstream of the putative –35 promoter sequence. Box 2, located 5–7 bp upstream of box 1, comprises a shorter palindrome than box 3, with inverted polarity [5′-(G/C)TTTC (N) GAAA(G/C)-3′]. The putative regulatory role of the conserved inverted repeats in boxes 2 and 3 in the promoter region of the xlnC gene from Streptomyces sp. EC3, was assessed. These boxes were modified by site-directed mutagenesis, and the mutant promoter regions, as well as the wild-type promoter region, were separately fused to a β-lactamase reporter gene. Analysis of the expression patterns of these fusions in cultures grown in the presence of glucose, xylan or both carbon sources demonstrated that these motifs were cis -acting negative regulatory elements, each playing a specific role in the regulation of xlnC expression. Box 3 was shown to be critical for the establishment of repression of xlnC expression by glucose, whereas box 2 was shown to play an important role in the induction of xlnC expression by xylan.

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Acknowledgements

We thank Prof D. A. Hopwood and Dr. T. Kieser of the John Innes Center (Norwich, U.K.) and Prof. M. Penninckx of Ceria-ULB (Brussels, Belgium) for the gift of Streptomyces strains. We are especially grateful to Iris Thamm for her excellent technical assistance. This work was supported in part by the Belgian Programme on Interuniversity Poles of Attraction initiated by the Belgian state, Prime Minister's Office, Science Policy programming (PAI P5/33, by the Fund for Joint Basic Research of Belgium, Contract No. 2.4576.97) and by the Ministry of the Walloon Region, Department of Technologies, Research and Energy (Convention No. 981/3709). JD is Research Associate of the National Fund for Scientific Research of Belgium. FG and SR are postdoctoral fellows of the Walloon Region

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Correspondence to F. Giannotta.

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Communicated by W. Goebel

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Giannotta, F., Georis, J., Rigali, S. et al. Site-directed mutagenesis of conserved inverted repeat sequences in the xylanase C promoter region from Streptomyces sp. EC3. Mol Genet Genomics 270, 337–346 (2003). https://doi.org/10.1007/s00438-003-0927-y

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Keywords

  • S1 mapping
  • DNA-binding protein
  • Catabolite repression
  • Transcriptional regulation
  • Operator sequence