The Zymomonas mobilis genes that encode glucose-6-phosphate dehydrogenase (zwj), 6-phosphogluconate dehydratase (edd), and glucokinase (gik) were cloned independently by genetic complementation of specific defects in Escherichia coli metabolism. The identity of these cloned genes was confirmed by various biochemical means. Nucleotide sequence analysis established that these three genes are clustered on the genome and revealed an additional open reading frame in this region that has significant amino acid identity to the E. coli xylose-proton symporter and the human glucose transporter. On the basis of this evidence and structural analysis of the deduced primary amino acid sequence, this gene is believed to encode the Z. mobilis glucose-facilitated diffusion protein, gif. The four genes in the 6-kb cluster are organized in the order gif, zwf, edd, gik. The gif and zwf genes are separated by 146 bp. The zwf and edd genes overlap by 8 bp, and their expression may be translationally coupled. The edd and gik genes are separated by 203 bp. The gik gene is followed by tandem transcriptional terminators. The four genes appear to be organized in an operon. Such an arrangement of the genes that govern glucose uptake and the first three steps of the Entner-Doudoroff glycolytic pathway provides the organism with a mechanism for carefully regulating the levels of the enzymes that control carbon flux into the pathway.Zymomonas mobilis is a gram-negative bacterium which, during evolution, has become highly specialized for growth in plant saps with a high sugar content (37,51,53). This obligately fermentative organism possesses remarkably simple carbon and energy metabolism. Z. mobilis is only able to utilize glucose, fructose, and sucrose, which are converted to the sole fermentation products ethanol and carbon dioxide. Yet, in terms of biosynthetic capabilities, this organism has only two growth factor requirements. Z. mobilis uses the Entner-Doudoroff pathway exclusively for conversion of carbohydrates to pyruvate and the decarboxyclastic mechanism for ethanol production, with the key enzyme pyruvate decarboxylase. The enzymes responsible for this fermentation compose as much as 50% of the total soluble protein. Z. mobilis is totally dependent on substrate-level phosphorylation for energy production and, due to its use of the Entner-Doudoroff pathway, obtains only a single mole of ATP per mole of glucose fermented. For this organism, rapid carbon flux is necessitated by inefficient energy production and is facilitated by high levels of the pathway enzymes.Despite enormous carbon flux, Z. mobilis must keep the levels of toxic metabolic intermediates low while providing sufficient pools of precursor metabolites for biosynthetic pathways. The physiology and biochemistry of this organism are dictated by these constraints on metabolism. The glycolytic enzymes appear to operate near their maximal capacity, and there is no substantial allosteric control of physiologically irreversible enzymes (53). It can be concluded that carbo...

Zymomonas mobilis is known to transport glucose by a facilitated diffusion process. A putative glucose facilitator gene (glf), closely related to a large family of glucose transporters, is located in a cluster of genes that code for enzymes of glucose metabolism. The Z. mobilis glf gene is able to complement glucose transport in an Escherichia coli strain that is defective in native glucose transport and glucokinase. In this study, the recombinant E. coli was shown to be capable of influx counterflow when preloaded with glucose and had an apparent Km for glucose of approximately 1.1-2.9 mM, consistent with the function of Glf as a low-affinity glucose facilitator. The ability of glucokinase mutants expressing glf to transport glucose made it clear that glucokinase activity was not required for Glf-dependent glucose transport. The possibility that glucokinase can interact with Glf to improve the affinity for glucose was not supported since expression of the Z. mobilis glucokinase gene, in addition to glf, did not affect the Km of Glf for glucose in recombinant E. coli. The inability of various sugars to compete with glucose during glucose transport by recombinant E. coli expressing glf indicated that Glf is specific for glucose. While the results of fructose transport assays did not completely rule out the possibility of very low affinity for fructose, the apparent specificity of Glf for glucose makes it possible that Z. mobilis utilizes a different transporter(s) for fructose.

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.

The Zymomonas mobilis gene encoding phosphoglucose isomerase (pgi)

The full-length 6.14-kb polycistronic glf-zwf-edd-glk mRNA from Zymomonas mobilis appears to be processed by endonucleolytic cleavage, resulting in the formation of several discrete transcripts. Northern analysis and transcript mapping revealed that the processed transcripts correspond to functional mono-, di-, or tricistronic messages. The relative abundance of the gene-specific, functional messages was measured. Expression of zwf and edd correlated well with functional message levels. Disproportionally high levels of the glk-specific mRNAs might compensate for the instability of glucokinase by allowing increased translation. The relative abundance of the discrete transcripts was shown to be a function of their respective decay rates. Northern analysis of the fate of the 6.14-kb transcript after inhibition of transcription by rifampin showed that the abundance of shorter, more stable transcripts increased at the expense of longer, less stable transcripts. This is suggestive of endonucleolytic mRNA processing. The most abundant 5' and 3' transcript ends were found to lie within secondary structures that probably impart stability to the most abundant mRNAs.