Isolation and characterization of Zymomonas mobilis DNA fragments acting as promoter transcriptional elements in Escherichia coli.

Abstract

Promoter libraries of Zymomonas mobilis were generated in Escherichia coli using the CAT promoterless vector, pKK232-8. Among the 15 clones retained for further study, quantitative analysis of promoter strength revealed a 140-fold difference between the weakest and strongest promoter fragment. Nucleotide sequence determination and primer extension analysis were used to locate possible promoter-like sequences. Inspection of the DNA sequence surrounding the mRNA start-site, revealed regions similar to the E. coli sigma 70 consensus sequence (TTGACA-17 bps-TATAAT), approximately 4-8 bps upstream. Analogous to E. coli upstream activators, regions high in AT content (70-80%) and rich in static DNA bands, were found preceding the -35 hexamer. Only one promoter fragment clone (Zi) was found to carry an open reading frame preceded by a sequence resembling a ribosome-binding site with a free energy of SD-anti-SD binding (dG°) of -12.4 kcal/mol.The general features of gene regulation in Zymomonas mobilis are not well established, nor are those affecting the expression of the Z. mobilis genes after transfer into Escherichia coll. According to the literature, there appears to be two types of promoter classes in Z. mobilis: those that function well in Z. mobilis but poorly in E. coli, and those that initiate transcription in both Z. mobilis and E. coil at relatively similar frequencies. The latter group of transcriptional elements are less well studied. Conway et al. (5) isolated DNA fragments from Z. mobilis which contained promoter activity and amino-terminal sequences. Although both E, coil and Z. mobilis recognized similar regions of DNA for transcriptional initiation, comparisons revealed that two of the three fragments did not contain sequences