Molecular characterization of the Zymomonas mobilis enolase (eno) gene.
The Zymomonas mobilis gene encoding enolase was cloned by genetic complementation of an Escherichia coli eno mutant. An enzyme assay and sodium dodecyl sulfate-polyacrylamide gel electrophoresis confirmed the overexpression of enolase in E. coli clones carrying the Z. mobilis eno gene. The eno gene is present in a single copy of the Z. mobilis genome. Nucleotide sequence analysis of the eno region revealed an open reading frame of 1,293 bp that encodes a protein of 428 amino acids with a predicted molecular weight of 45,813. Comparison of the sequence of Z. mobilis enolase with primary amino acid sequences for other enolases indicates that the enzyme is highly conserved. Unlike all of the previously studied glycolytic genes from Z. mobilis that possess canonical ribosome binding sites, the eno gene is preceded by a modest Shine-Dalgarno sequence. The transcription initiation site was mapped by primer extension and found to be located within a 115-bp sequence that is 55.7% identical to a highly conserved consensus sequence found within the regulatory regions of highly expressed Z. mobilis genes. Northern RNA blot analysis revealed that eno is encoded on a 1.45-kb transcript. The half-life of the eno mRNA was determined to be 17.7 +/- 1.7 min, indicating that it is unusually stable. The abundance of the eno message is proposed to account for enolase being the most prevalent protein in Z. mobilis.
Burnett
M E
ME
School of Biological Sciences, University of Nebraska, Lincoln 68588-0118.
Liu
J
J
Conway
T
T
D000595:Amino Acid Sequence; D001426:Bacterial Proteins / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism; D001483:Base Sequence; D015139:Blotting, Southern; D003001:Cloning, Molecular; D005798:Genes, Bacterial / Q000235:genetics*; D008969:Molecular Sequence Data; D009690:Nucleic Acid Conformation; D010751:Phosphopyruvate Hydratase / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism; D015183:Restriction Mapping; D017355:Zymomonas / Q000201:enzymology* / Q000235:genetics
D001426:Bacterial Proteins; D010751:Phosphopyruvate Hydratase