Cloning, characterization and expression of the Zymononas mobilis eda gene that encodes 2‐keto‐3‐deoxy‐6‐phosphogluconate aldolase of the Entner‐Doudoroff pathway

Abstract

The eda gene that encodes 2-keto-3-deoxy-6-phosphogluconate aldolase of the Entner-Doudoroff pathway was cloned from Zymomonas mobilis by genetic complementation of an Escherichia coli mutant. The gene is present in a single copy on the Z. mobilis genome and is not tightly linked to the edd gene. Nucleotide sequence analysis of the eda region revealed that the structural gene is 627 bp long and capable of encoding a protein of 208 amino acids with a deduced molecular weight of 21,505. The eda gene is monocistronic and is transcribed from a single promoter. The transcriptional initiation site was determined and an improved consensus promoter sequence for Z. mobilis was derived. High-level expression of the eda gene can be attributed to very efficient translational initiation caused by the high quality of the ribosome-binding site and stability of the mRNA, which has a decay rate of 7.6 min. A comparison of highly expressed Z. mobilis genes indicated that the relative quality of the ribosome-binding sites of these genes might play an important role in determining the level of enzyme synthesis. This possibility is discussed with regard to the role of gene expression in co-ordinating the enzyme levels of the Entner-Doudoroff glycolytic pathway.

Molecular characterization of the Entner-Doudoroff pathway in Escherichia coli: sequence analysis and localization of promoters for the edd-eda operon

“…Genetic and physical analyses of zwf expression have established that the zwf gene is monocistronic and subject to growth rate-dependent regulation (29). Expression of edd, as well as gluconate transport and gluconokinase, is controlled in a negative fashion by the product of gntR, which presumably encodes a repressor CHARACTERIZATION OF E. COLI edd-eda OPERON 4639 accomplished by complementation of a defect in glucuronic acid metabolism in E. coli DF214 as described previously (5). A subclone of a portion of the eda region, pTC196, was made by ligating a 0.3-kb HincII-to-PstI restriction fragment (see Fig.…”

“…The molecular biology of the Entner-Doudoroff pathway has been investigated in some detail in Zymomonas mobilis (2,5), but few molecular studies of the Entner-Doudoroff pathway in E. coli have been conducted (14). In E. coli, the edd and eda genes are tightly linked to the zwf gene, which codes for glucose-6-phosphate dehydrogenase (13).…”

“…Primer extension analysis of transcriptional initiation sites was accomplished as described previously (5). The oligonucleotide used for analysis of edd was 26 bases long, spanning nucleotides 1718 to 1743 (Fig.…”

“…6-Phosphogluconate dehydratase was assayed by published methods (41). KDPG aldolase was assayed as described previously (5).…”

Computational and functional analysis of β-lactam resistance in Zymomonas mobilis

“…pomaceae lectotype ATCC 29192 and Z. mobilis B14023 (NRRL, Peoria, Illinois) with various β-lactam antibiotics in order to determine their antibiotic sensitivity/resistance profile. For transcript analysis, the total RNA was isolated from mid-log phase cultures (OD600 0.7) of Z. mobilis grown in rich medium with/without penicillin (500 µg/mL) as described previously (Conway et al 1991). The RNA integrity was analyzed by formaldehyde agarose gel electrophoresis (Sambrook & Russell 2001).…”