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The Escherichia coli eda gene, which encodes the Entner-Doudoroff aldolase, is central to the catabolism of several sugar acids. Here, we show that Eda synthesis is induced by growth on gluconate, glucuronate, or methyl-β- d -glucuronide; phosphate limitation; and carbon starvation. Transcription of eda initiates from three promoters, designated P1, P2, and P4, each of which is responsible for induction under different growth conditions. P1 controls eda induction on gluconate and is regulated by GntR. P2 controls eda induction on glucuronate and galacturonate and is regulated by KdgR. P4 is active under conditions of phosphate starvation and is directly controlled by PhoB. In addition, CsrA activates Eda synthesis, apparently by an indirect mechanism that may be involved in the modest changes in expression level that are associated with carbon starvation. The complex regulation of eda is discussed with respect to its several physiological roles, which apparently accommodate not only sugar acid catabolism but also detoxification of metabolites that could accumulate during starvation-induced stress.

Acid in the stomach is thought to be a barrier to bacterial colonization of the intestine. Escherichia coli , however, has three systems for acid resistance, which overcome this barrier. The most effective of these systems is dependent on transport and decarboxylation of glutamate. GadX regulates two genes that encode isoforms of glutamate decarboxylase critical to this system, but additional genes associated with the glutamate-dependent acid resistance system remained to be identified. The gadX gene and a second downstream araC -like transcription factor gene, gadW , were mutated separately and in combination, and the gene expression profiles of the mutants were compared to those of the wild-type strain grown in neutral and acidified media under conditions favoring induction of glutamate-dependent acid resistance. Cluster and principal-component analyses identified 15 GadX-regulated, acid-inducible genes. Reverse transcriptase mapping demonstrated that these genes are organized in 10 operons. Analysis of the strain lacking GadX but possessing GadW confirmed that GadX is a transcriptional activator under acidic growth conditions. Analysis of the strain lacking GadW but possessing GadX indicated that GadW exerts negative control over three GadX target genes. The strain lacking both GadX and GadW was defective in acid induction of most but not all GadX target genes, consistent with the roles of GadW as an inhibitor of GadX-dependent activation of some genes and an activator of other genes. Resistance to acid was decreased under certain conditions in a gadX mutant and even more so by combined mutation of gadX and gadW . However, there was no defect in colonization of the streptomycin-treated mouse model by the gadX mutant in competition with the wild type, and the gadX gadW mutant was a better colonizer than the wild type. Thus, E. coli colonization of the mouse does not appear to require glutamate-dependent acid resistance.

An important feature of Escherichia coli pathogenesis is an ability to withstand extremely acidic environments of pH 2 or lower. This acid resistance property contributes to the low infectious dose of pathogenic E. coli species. One very efficient E. coli acid resistance system encompasses two isoforms of glutamate decarboxylase ( gadA and gadB ) and a putative glutamate:γ-amino butyric acid (GABA) antiporter ( gadC ). The system is subject to complex controls that vary with growth media, growth phase, and growth pH. Previous work has revealed that the system is controlled by two sigma factors, two negative regulators (cyclic AMP receptor protein [CRP] and H-NS), and an AraC-like regulator called GadX. Earlier evidence suggested that the GadX protein acts both as a positive and negative regulator of the gadA and gadBC genes depending on environmental conditions. New data clarify this finding, revealing a collaborative regulation between GadX and another AraC-like regulator called GadW (previously YhiW). GadX and GadW are DNA binding proteins that form homodimers in vivo and are 42% homologous to each other. GadX activates expression of gadA and gadBC at any pH, while GadW inhibits GadX-dependent activation. Regulation of gadA and gadBC by either regulator requires an upstream, 20-bp GAD box sequence. Northern blot analysis further indicates that GadW represses expression of gadX . The results suggest a control circuit whereby GadW interacts with both the gadA and gadX promoters. GadW clearly represses gadX and, in situations where GadX is missing, activates gadA and gadBC. GadX, however, activates only gadA and gadBC expression. CRP also represses gadX expression. It does this primarily by repressing production of sigma S, the sigma factor responsible for gadX expression. In fact, the acid induction of gadA and gadBC observed when rich-medium cultures enter stationary phase corresponds to the acid induction of sigma S production. These complex control circuits impose tight rein over expression of the gadA and gadBC system yet provide flexibility for inducing acid resistance under many conditions that presage acid stress.

Escherichia coli MG1655 acid-inducible genes were identified by whole-genome expression profiling. Cultures were grown to the mid-logarithmic phase on acidified glucose minimal medium, conditions that induce glutamate-dependent acid resistance (AR), while the other AR systems are either repressed or not induced. A total of 28 genes were induced in at least two of three experiments in which the gene expression profiles of cells grown in acid (pH 5.5 or 4.5) were compared to those of cells grown at pH 7.4. As expected, the genes encoding glutamate decarboxylase, gadA and gadB , were significantly induced. Interestingly, two acid-inducible genes code for small basic proteins with pIs of >10.5, and six code for small acidic proteins with pIs ranging from 5.7 to 4.0; the roles of these small basic and acidic proteins in acid resistance are unknown. The acid-induced genes represented only five functional grouping categories, including eight genes involved in metabolism, nine associated with cell envelope structures or modifications, two encoding chaperones, six regulatory genes, and six unknown genes. It is unlikely that all of these genes are involved in the glutamate-dependent AR. However, nine acid-inducible genes are clustered in the gadA region, including hdeA , which encodes a putative periplasmic chaperone, and four putative regulatory genes. One of these putative regulators, yhiE , was shown to significantly increase acid resistance when overexpressed in cells that had not been preinduced by growth at pH 5.5, and mutation of yhiE decreased acid resistance; yhiE could therefore encode an activator of AR genes. Thus, the acid-inducible genes clustered in the gadA region appear to be involved in glutatmate-dependent acid resistance, although their specific roles remain to be elucidated.

A Bacillus subtilis mutant with a deletion in the citC gene, encoding isocitrate dehydrogenase, the third enzyme of the tricarboxylic acid branch of the Krebs cycle, exhibited reduced growth yield in broth medium and had greatly reduced ability to sporulate compared to the wild type due to a block at stage I, i.e., a failure to form the polar division septum. In early stationary phase, mutant cells accumulated intracellular and extracellular concentrations of citrate and isocitrate that were at least 15-fold higher than in wild-type cells. The growth and sporulation defects of the mutant could be partially bypassed by deletion of the major citrate synthase gene ( citZ ), by raising the pH of the medium, or by supplementation of the medium with certain divalent cations, suggesting that abnormal accumulation of citrate affects survival of stationary-phase cells and sporulation by lowering extracellular pH and chelating metal ions. While these genetic and environmental alterations were not sufficient to allow the majority of the mutant cell population to pass the stage I block (lack of asymmetric septum formation), introduction of the sof-1 mutant form of the Spo0A transcription factor, when coupled with a reduction in citrate synthesis, restored sporulation gene expression and spore formation nearly to wild-type levels. Thus, the primary factor inhibiting sporulation in a citC mutant is abnormally high accumulation of citrate, but relief of this metabolic defect is not by itself sufficient to restore competence for sporulation.

The gntT gene of Escherichia coli is specifically induced by gluconate and repressed via catabolite repression. Thus, gluconate is both an inducer and a repressor of gntT expression since gluconate is a catabolite-repressing sugar. In a gntR deletion mutant, the expression of a chromosomal gntT :: lacZ fusion is both high and constitutive, confirming that GntR is the negative regulator of gntT . Indeed, GntR binds to two consensus gnt operator sites; one overlaps the −10 region of the gntT promoter, and the other is centered at +120 with respect to the transcriptional start site. The binding of GntR to these sites was proven in vitro by gel redardation assays and in vivo by site-directed mutagenesis of the binding sites. Binding of GntR to the operators is eliminated by gluconate and also by 6-phosphogluconate at a 10-fold-higher concentration. Interestingly, when gntR deletion strains are grown in the presence of gluconate, there is a twofold decrease in gntT expression which is independent of catabolite repression and binding of GntR to the operator sites. This novel response of gntR mutants to the inducer is termed ultrarepression. Transcription of gntT is activated by binding of the cyclic AMP (cAMP)-cAMP receptor protein (CRP) complex to a CRP binding site positioned at −71 upstream of the gntT transcription start site.

Although Zymomonas mobilis is prototrophic, glycolytic and fermentative enzymes (ethanologenic enzymes) constitute over half of the cytoplasmic protein. In this study, transcript stability, functional message pools, and the abundance of cytoplasmic products were compared for genes encoding eight of these essential enzymes. The transcripts of all were very stable, with half-lives ranging from 8 to 18 min. This transcript stability is proposed as an important feature in Z. mobilis that may distinguish highly expressed genes for energy generation from biosynthetic genes, which are required at much lower levels. The evolution of multiple promoters to enhance transcription from single-copy genes, of structural features that alter translational efficiency, and of differences in protein turnover is hypothesized to serve a subordinate role in the regulation of Z. mobilis gene expression. Among the eight ethanologenic genes examined, differences in transcript stability were found to directly correlate with differences in functional message pools and cytoplasmic protein levels. These differences in transcript stability are hypothesized to have evolved as a primary mechanism to balance the levels of individual enzymes within the glycolytic and fermentative pathways.

The nucleotide sequence of the entire Escherichia coli edd-eda region that encodes the enzymes of the Entner-Doudoroff pathway was determined. The edd structural gene begins 236 bases downstream of zwf. The eda structural gene begins 34 bases downstream of edd. The edd reading frame is 1,809 bases long and encodes the 602-amino-acid, 64,446-Da protein 6-phosphogluconate dehydratase. The deduced primary amino acid sequences of the E. coli and Zymomonas mobilis dehydratase enzymes are highly conserved. The eda reading frame is 642 bases long and encodes the 213-amino-acid, 22,283-Da protein 2-keto-3-deoxy-6-phosphogluconate aldolase. This enzyme had been previously purified and sequenced by others on the basis of its related enzyme activity, 2-keto-4-hydroxyglutarate aldolase. The data presented here provide proof that the two enzymes are identical. The primary amino acid sequences of the E. coli, Z. mobilis, and Pseudomonas putida aldolase enzymes are highly conserved. When E. coli is grown on gluconate, the edd and eda genes are cotranscribed. Four putative promoters within the edd-eda region were identified by transcript mapping and computer analysis. P1, located upstream of edd, appears to be the primary gluconate-responsive promoter of the edd-eda operon, responsible for induction of the Entner-Doudoroff pathway, as mediated by the gntR product. High basal expression of eda is explained by constitutive transcription from P2, P3, and/or P4 but not P1.

The Zymomonas mobilis gene encoding phosphoglucose isomerase (pgi) was cloned by genetic complementation of an Escherichia coli pgi mutant. An enzyme assay and sodium dodecyl sulfate-polyacrylamide gel electrophoresis confirmed the presence of excess amounts of phosphoglucose isomerase in E. coli clones carrying the Z. mobilis pgi gene. The pgi gene is present in only one copy on the Z. mobilis genome. Nucleotide sequence analysis of the pgi region revealed an open reading frame of 1,524 bp preceded by a strong Shine-Dalgarno sequence. The pgi gene encodes a 507-amino-acid protein with a predicted molecular weight of 55,398. Z. mobilis phosphoglucose isomerase is between 38 and 43% identical to the enzyme from other species. Northern (RNA) blot analysis showed that the pgi transcript is 1.8 kb in length. The level of the pgi transcript was found to be influenced by the phase of growth and by the carbon and energy sources. Transcript levels increased with respect to total RNA during logarithmic growth and were threefold higher when grown on fructose than on glucose. These changes in transcript levels paralleled phosphoglucose isomerase activities in the cultures. Differential mRNA stability was not a factor, since the half-life of the pgi transcript was 6.3 min in glucose-grown cells and 6.0 min in fructose-grown cells. Thus, an increase in the rate of transcription appears to be at least partially responsible for the increased levels of phosphoglucose isomerase observed for Z. mobilis grown on fructose.

Escherichia coli has four gluconate transporters, GntP, GntU, GntT, and IdnT, which are members of the major facilitator superfamily. The physiological function of GntP was previously unknown and is the subject of this study. GntP is not induced by gluconate, and despite being located adjacent to genes involved in glucuronate catabolism, gntP does not encode a glucuronate transporter. Here we identify gntP as the gene which encodes the fructuronate transporter. We show that gntP is induced by fructuronate and is a new member of the UxuR regulon: gntP is derepressed in an uxuR strain, UxuR binds in vitro specifically to an operator site that overlaps the gntP promoter, and UxuR binding is eliminated by fructuronate. Transcription of gntP requires activation by cyclic AMP (cAMP)-cAMP receptor protein. A gntP mutant cannot grow on fructuronate but grows normally on glucuronate and gluconate. Thus, the UxuR regulon is a module of sugar acid catabolism whose physiological role is for growth on fructuronate. Glucuronate, because it proceeds through a fructuronate intermediate, must induce the UxuR regulon and must also induce the ExuR regulon, which encodes the glucuronate transporter, ExuT, and the first step in its catabolism, UxaC. Thus, hexuronate catabolism in E. coli requires both the ExuR and UxuR regulons, while fructuronate catabolism requires only the UxuR regulon.

The genetic organization of the idn genes that encode the pathway for l -idonate catabolism was characterized. The monocistronic idnK gene is transcribed divergently from the idnDOTR genes, which were shown to form an operon. The 215-bp regulatory region between the idnK and idnD genes contains promoters in opposite orientation with transcription start sites that mapped to positions −26 and −29 with respect to the start codons. The regulatory region also contains a single putative IdnR/GntR binding site centered between the two promoters, a CRP binding site upstream of idnD , and an UP element upstream of idnK . The genes of the l -idonate pathway were shown to be under catabolite repression control. Analysis of idnD - and idnK - lacZ fusions in a nonpolar idnD mutant that is unable to interconvert l -idonate and 5-ketogluconate indicated that either compound could induce the pathway. The l -idonate pathway was first characterized as a subsidiary pathway for d -gluconate catabolism (GntII), which is induced by d -gluconate in a GntI (primary gluconate system) mutant. Here we showed that the idnK and idnD operons are induced by d -gluconate in a GntI system mutant, presumably by endogenous formation of 5-ketogluconate from d -gluconate. Thus, the regulation of the GntII system is appropriate for this pathway, which is primarily involved in l -idonate catabolism; the GntII system can be induced by d -gluconate under conditions that block the GntI system.

DNA arrays of the entire set of Escherichia coli genes were used to measure the genomic expression patterns of cells growing in late logarithmic phase on minimal glucose medium and on Luria broth containing glucose. Ratios of the transcript levels for all 4,290 E. coli protein-encoding genes (cds) were obtained, and analysis of the expression ratio data indicated that the physiological state of the cells under the two growth conditions could be ascertained. The cells in the rich medium grew faster, and expression of the majority of the translation apparatus genes was significantly elevated under this growth condition, consistent with known patterns of growth rate-dependent regulation and increased rate of protein synthesis in rapidly growing cells. The cells grown on minimal medium showed significantly elevated expression of many genes involved in biosynthesis of building blocks, most notably the amino acid biosynthetic pathways. Nearly half of the known RpoS-dependent genes were expressed at significantly higher levels in minimal medium than in rich medium, and rpoS expression was similarly elevated. The role of RpoS regulation in these logarithmic phase cells was suggested by the functions of the RpoS dependent genes that were induced. The hallmark features of E. coli cells growing on glucose minimal medium appeared to be the formation and excretion of acetate, metabolism of the acetate, and protection of the cells from acid stress. A hypothesis invoking RpoS and UspA (universal stress protein, also significantly elevated in minimal glucose medium) as playing a role in coordinating these various aspects and consequences of glucose and acetate metabolism was generated. This experiment demonstrates that genomic expression assays can be applied in a meaningful way to the study of whole-bacterial-cell physiology for the generation of hypotheses and as a guide for more detailed studies of particular genes of interest.

Three genes involved in gluconate metabolism, gntR, gntK, and gntU, which code for a regulatory protein, a gluconate kinase, and a gluconate transporter, respectively, were cloned from Escherichia coli K-12 on the basis of their known locations on the genomic restriction map. The gene order is gntU, gntK, and gntR, which are immediately adjacent to asd at 77.0 min, and all three genes are transcribed in the counterclockwise direction. The gntR product is 331 amino acids long, with a helix-turn-helix motif typical of a regulatory protein. The gntK gene encodes a 175-amino-acid polypeptide that has an ATP-binding motif similar to those found in other sugar kinases. While GntK does not show significant sequence similarity to any known sugar kinases, it is 45% identical to a second putative gluconate kinase from E. coli,gntV. The 445-amino-acid sequence encoded by gntU has a secondary structure typical of membrane-spanning transport proteins and is 37% identical to the gntP product from Bacillus subtilis. Kinetic analysis of GntU indicates an apparent Km for gluconate of 212 microM, indicating that this is a low-affinity transporter. Studies demonstrate that the gntR gene is monocistronic, while the gntU and gntK genes, which are separated by only 3 bp, form an operon. Expression of gntR is essentially constitutive, while expression of gntKU is induced by gluconate and is subject to fourfold glucose catabolite repression. These results confirm that gntK and gntU, together with another gluconate transport gene, gntT, constitute the GntI system for gluconate utilization, under control of the gntR gene product, which is also responsible for induction of the edd and eda genes of the Entner-Doudoroff pathway.

The gntP gene, located between the fim and uxu loci in Escherichia coli K-12, has been cloned and characterized. Nucleotide sequencing of a region encompassing the gntP gene revealed an open reading frame of 447 codons with significant homology to the Bacillus subtilis gluconate permease. Northern (RNA) blotting indicated that the gntP gene was monocistronic and was transcribed as an mRNA with an apparent molecular size of 1.54 kb. The transcriptional start point was determined by primer extension analysis. The gntP gene was found to be under catabolite repression and was not induced by gluconate. Also, expression seemed to be stringently controlled. Several observations indicated that the GntP protein is an inner membrane protein; it contains characteristic membrane-spanning regions and was isolated predominantly from the inner-membrane fraction of fractionated host cells. A topology analysis predicted a protein with 14 membrane-spanning segments. The inability of a mutant strain to grow on gluconate minimal medium could be relieved by introduction of a plasmid encoding the gntP gene. Finally, the kinetics of GntP-mediated gluconate uptake were investigated, indicating an apparent Km for gluconate of 25 microM.

Expression of the Zymomonas mobilis glf (glucose facilitator protein) and glk (glucokinase) genes in Escherichia coli ZSC113 (glucose negative) provided a new functional pathway for glucose uptake and phosphorylation. Both genes were essential for the restoration of growth in glucose minimal medium and for acid production on glucose-MacConkey agar plates.

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.

The Zymomonas mobilis gene encoding phosphoglycerate kinase (EC 2.7.3.2), pgk, has been cloned into Escherichia coli and sequenced. It consists of 336 amino acids, including the N-terminal methionine, with a molecular weight of 41,384. This promoterless gene is located 225 base pairs downstream from the gap gene and is part of the gap operon. Previous studies have shown that the specific activities of glyceraldehyde-3-phosphate dehydrogenase and phosphoglycerate kinase do not change coordinately in Z. mobilis, although the two enzymes appear to be under the control of a common promoter. The translated amino acid sequence for the Z. mobilis phosphoglycerate kinase is less conserved than those of eucaryotic genes. A comparison of known sequences for phosphoglycerate kinase revealed a high degree of conservation of structure with 102 amino acid positions being retained by all. In general, the amino acid positions at the boundaries of beta-sheet and alpha-helical regions and those connecting these regions were more highly conserved than the amino acid positions within regions of secondary structure.

The Zymomonas mobilis gene encoding acid phosphatase, phoC, has been cloned and sequenced. The gene spans 792 base pairs and encodes an Mr 28,988 polypeptide. This protein was identified as the principal acid phosphatase activity in Z. mobilis by using zymograms and was more active with magnesium ions than with zinc ions. Its promoter region was similar to the -35 "pho box" region of the Escherichia coli pho genes as well as the regulatory sequences for Saccharomyces cerevisiae acid phosphatase (PHO5). A comparison of the gene structure of phoC with that of highly expressed Z. mobilis genes revealed that promoters for all genes were similar in degree of conservation of spacing and identity with the proposed Z. mobilis consensus sequence in the -10 region. The phoC gene contained a 5' transcribed terminus which was AT rich, a weak ribosome-binding site, and less biased codon usage than the highly expressed Z. mobilis genes.

We have described a procedure for the isolation of lacZ' fusion genes which contain anchor sequences conferring membrane association. This method was used to isolate fragments of DNA from Zymomonas mobilis which contain promoter activity and amino-terminal sequences. The sequences and transcriptional initiation sites of three of these were compared. Both Escherichia coli and Z. mobilis recognized similar regions of DNA for transcriptional initiation. Five to eight consecutive hydrophobic amino acids in the amino terminus served to anchor these hybrid proteins to the membrane in both E. coli and Z. mobilis. General features observed in the Z. mobilis fragments included partial sequence homology with the -35 region sequence of E. coli, repetitive and palindromic A + T-rich regions preceding and adjoining the -10 region, a sequence resembling the consensus sequence of E. coli in the -10 region, and a potential ribosomal-binding site (AGGA) 8 to 12 bases upstream from an in-frame start codon. The level of expression of fusion proteins was generally higher in E. coli than in Z. mobilis. This higher level of expression in E. coli may result from multiple sites of transcriptional initiation and higher plasmid copy number.

The gene that encodes 1,2-propanediol oxidoreductase (fucO) from Escherichia coli was sequenced. The reading frame specified a protein of 383 amino acids (including the N-terminal methionine), with an aggregate molecular weight of 40,642. The induction of fucO transcription, which occurred in the presence of fucose, was confirmed by Northern blot analysis. In E. coli, the primary fucO transcript was approximately 2.1 kilobases in length. The 5' end of the transcript began more than 0.7 kilobase upstream of the fucO start codon within or beyond the fucA gene. Propanediol oxidoreductase exhibited 41.7% identity with the iron-containing alcohol dehydrogenase II from Zymomonas mobilis and 39.5% identity with ADH4 from Saccharomyces cerevisiae. These three proteins did not share homology with either short-chain or long-chain zinc-containing alcohol dehydrogenase enzymes. We propose that these three unusual alcohol dehydrogenases define a new family of enzymes.

DNA sequence analysis showed that pyruvate decarboxylase (one of the most abundant proteins in Zymomonas mobilis) contains 559 amino acids. The promoter for the gene encoding pyruvate decarboxylase was not recognized by Escherichia coli, although the cloned gene was expressed at relatively high levels under the control of alternative promoters. The promoter region did not contain sequences which could be identified as being homologous to the generalized promoter structure for E. coli. Hydropathy plots for the amino acid sequence indicated that pyruvate decarboxylase contains a large number of hydrophobic domains which may contribute to the thermal stability of this enzyme.

The Zymomonas mobilis alcohol dehydrogenase II gene (adhB) was overexpressed 7- to 14-fold on a recombinant plasmid, accompanied by a small decrease in growth rate. A fragment containing the truncated gene with promoter reduced expression from the chromosomal gene as measured immunologically and enzymatically, consistent with the presence of a trans-active regulatory factor and positive regulatory control. Both the complete gene and the promoter fragment increased pyruvate decarboxylase and glucokinase activities, with no effect on alcohol dehydrogenase I or eight glycolytic enzymes. Tandem promoters from adhB expressed beta-galactosidase at higher levels than did either promoter alone in operon fusions. Addition of 50 microM zinc sulfate in minimal medium reduced the expression of adhB and of the operon fusions. Abundant but inactive alcohol dehydrogenase II was produced in iron-limited cells. This inactive enzyme did not form intracellular aggregates, and no morphological changes were apparent by transmission electron microscopy.

In Zymomonas mobilis, the genes encoding glyceraldehyde-3-phosphate dehydrogenase (GAP) and phosphoglycerate kinase (PGK) are encoded in an operon that is transcribed from tandem promoters. The promoter-proximal gap gene is expressed at six- to ninefold higher levels than the pgk gene from chromosomal genes and from multiple copies of plasmid-borne genes. Two dominant transcripts were identified. The smaller, most abundant transcript contained primarily the gap message, whereas the larger, less abundant message contained both genes. The ratio of message levels for gap and pgk was calculated to be 5:1 and is sufficient to account for the observed differences in levels of GAP and PGK. The differences in message abundance are proposed to result from either transcriptional attenuation or preferential degradation of the 3' region encoding pgk. Increases in gene dosage were accompanied by one-third the expected increase in enzymatic activity on the basis of estimates of copy number, consistent with the presence of a limiting, positive regulatory factor. However, GAP and PGK expressions were not reduced from the chromosome in recombinants that contained multiple copies of the gap operon with inactive genes.

The rate at which Z. mobilis (Entner-Doudoroff pathway) converts high concentrations of glucose (20%) into ethanol plus CO2 changes as ethanol accumulates in the surrounding broth. This decline in glycolytic activity (per milligram of cell protein) does not result from inhibitory effects of ethanol, which can be reversed immediately by ethanol removal. The peak of fermentative activity (58 mumol of CO2 evolved per mg of cell protein per h) occurred after the accumulation of 1.1% ethanol (18 h) and declined to one-half this rate after 30 h (6.2% accumulated ethanol), although the cell number continued to increase. These times corresponded to the end of exponential growth and to the onset of the stationary phase (on the basis of measurement of cell protein), respectively. An examination of many of the requirements for fermentation (nucleotides, magnesium, enzyme levels, intracellular pH, delta pH) revealed three possible reasons for this early decline in activity: decreased abundance of nucleotides, a decrease in internal pH from 6.3 to 5.3, and a decrease in the specific activities of two glycolytic enzymes (pyruvate kinase and glyceraldehyde-3-phosphate dehydrogenase). 31P nuclear magnetic resonance spectra of perchlorate extracts from cells fermenting in broth revealed very low levels of glycolytic intermediates (Entner-Doudoroff pathway) in cells examined at the peak of fermentative activity (18-h cells) in comparison with cells examined at a later stage (30-h cells), consistent with limitation of the fermentation rate by glycolytic enzymes near the end of the pathway. It is likely that cell death (loss of colony-forming ability) and the collapse of delta pH also contribute to the further decline in fermentative activity after 30 h.