Beyond good and evil in the oral cavity: insights into host-microbe relationships derived from transcriptional profiling of gingival cells.

In many instances, the encounter between host and microbial cells, through a long-standing evolutionary association, can be a balanced interaction whereby both cell types co-exist and inflict a minimal degree of harm on each other. In the oral cavity, despite the presence of large numbers of diverse organisms, health is the most frequent status. Disease will ensue only when the host-microbe balance is disrupted on a cellular and molecular level. With the advent of microarrays, it is now possible to monitor the responses of host cells to bacterial challenge on a global scale. However, microarray data are known to be inherently noisy, which is caused in part by their great sensitivity. Hence, we will address several important general considerations required to maximize the significance of microarray analysis in depicting relevant host-microbe interactions faithfully. Several advantages and limitations of microarray analysis that may have a direct impact on the significance of array data are highlighted and discussed. Further, this review revisits and contextualizes recent transcriptional profiles that were originally generated for the specific study of intricate cellular interactions between gingival cells and 4 important plaque micro-organisms. To our knowledge, this is the first report that systematically investigates the cellular responses of a cell line to challenge by 4 different micro-organisms. Of particular relevance to the oral cavity, the model bacteria span the entire spectrum of documented pathogenic potential, from commensal to opportunistic to overtly pathogenic. These studies provide a molecular basis for the complex and dynamic interaction between the oral microflora and its host, which may lead, ultimately, to the development of novel, rational, and practical therapeutic, prophylactic, and diagnostic applications.

Glycolytic gene expression in Saccharomyces cerevisiae: nucleotide sequence of GCR1, null mutants, and evidence for expression.

In Saccharomyces cerevisiae, the gcr mutation is known to have a profound effect on the levels of most glycolytic enzymes, reducing them to 5% of normal or less in growth on noncarbohydrates. Here I report the preparation of chromosomal gcr insertion and deletion mutations. The null mutations were recessive, were not lethal, and caused a pattern of glycolytic enzyme deficiency similar to that seen earlier for the gcr1-1 allele, including the partial inducibility by glucose of the residual enzyme activities. DNA sequence analysis showed that GCR1 encoded a protein of molecular weight 94,414, with a very low codon bias index, characteristic of several S. cerevisiae regulatory genes; adjacent 5' and 3' sequences contained elements suggesting that it was transcribed, polyadenylated, and translated. RNA gel transfer hybridization experiments with purified polyadenylated RNA and a probe complementary to the 5' portion of the open reading frame showed that Ger was expressed as a polyadenylated transcript. Together with previous work, the present results suggest that the Gcr product may be a transcriptional factor necessary specifically for the high-level transcription of a limited set of genes whose products, the enzymes of glycolysis, constitute a substantial fraction of cell proteins and are responsible for the primary metabolic flux in many cells.

Concerted action of the transcriptional activators REB1, RAP1, and GCR1 in the high-level expression of the glycolytic gene TPI.

In Saccharomyces cerevisiae, the TPI gene product, triosephosphate isomerase, makes up about 2% of the soluble cellular protein. Using in vitro and in vivo footprinting techniques, we have identified four binding sites for three factors in the 5' noncoding region of TPI: a REB1-binding site located at positions -401 to -392, two GCR1-binding sites located at positions -381 to -366 and -341 to -326, and a RAP1-binding site located at positions -358 to -346. We tested the effects of mutations at each of these binding sites on the expression of a TPI::lacZ gene fusion which carried 853 bp of the TPI 5' noncoding region integrated at the URA3 locus. The REB1-binding site is dispensable when material 5' to it is deleted; however, if the sequence 5' to the REB1-binding site is from the TPI locus, expression is reduced fivefold when the site is mutated. Because REB1 blocks nucleosome formation, the most likely function of its binding site in the TPI controlling region is to prevent the formation of nucleosomes over the TPI upstream activation sequence. Mutations in the RAP1-binding site resulted in a 10-fold reduction in expression of the reporter gene. Mutating either GCR1-binding site alone had a modest effect on expression of the fusion. However, mutating both GCR1-binding sites resulted in a 68-fold reduction in the level of expression of the reporter gene. A LexA-GCR1 fusion protein containing the DNA-binding domain of LexA fused to the amino terminus of GCR1 was able to activate expression of a lex operator::GAL1::lacZ reporter gene 116-fold over background levels. From this experiment, we conclude that GCR1 is able to activate gene expression in the absence of REB1 or RAP1 bound at adjacent binding sites. On the basis of these results, we suggest that GCR1 binding is required for activation of TPI and other GCR1-dependent genes and that the primary role of other factors which bind adjacent to GCR1-binding sites is to facilitate of modulate GCR1 binding in vivo.

Activation mechanism of the multifunctional transcription factor repressor-activator protein 1 (Rap1p).

Transcriptional activation in eukaryotic organisms normally requires combinatorial interactions of multiple transcription factors. In most cases, the precise role played by each transcription factor is not known. The upstream activating sequence (UAS) elements of glycolytic enzyme genes in Saccharomyces cerevisiae are excellent model systems for the study of combinatorial interactions. The yeast protein known as Rap1p acts as both a transcriptional repressor and an activator, depending on sequence context. Rap1p-binding sites are found adjacent to Gcr1p-binding sites in the UAS elements of glycolytic enzyme genes. These UAS elements constitute some of the strongest activating sequences known in S. cerevisiae. In this study, we have investigated the relationship between Rap1p- and Gcr1p-binding sites and the proteins that bind them. In vivo DNA-binding studies with rap1ts mutant strains demonstrated that the inability of Rap1p to bind at its site resulted in the inability of Gcr1p to bind at adjacent binding sites. Synthetic oligonucleotides, modeled on the UAS element of PYK1, in which the relative positions of the Rap1p- and Gcr1p-binding sites were varied prepared and tested for their ability to function as UAS elements. The ability of the oligonucleotides to function as UAS elements was dependent not only on the presence of both binding sites but also on the relative distance between the binding sites. In vivo DNA-binding studies showed that the ability of Rap1p bind its site was independent of Gcr1p but that the ability of Gcr1p to bind its site was dependent on the presence of an appropriately spaced and bound Rap1p-binding site. In vitro binding studies showed Rap1p-enhanced binding of Gcr1p on oligonucleotides modeled after the native PYK1 UAS element but not when the Rap1p- and Gcr1p-binding sites were displaced by 5 nucleotides. This work demonstrates that the role of the Rap1p in the activation of glycolytic enzyme genes is to bind in their UAS elements and to facilitate the binding of Gcr1p at adjacent binding sites.

Characterization of the DNA-binding activity of GCR1: in vivo evidence for two GCR1-binding sites in the upstream activating sequence of TPI of Saccharomyces cerevisiae.

GCR1 gene function is required for high-level glycolytic gene expression in Saccharomyces cerevisiae. Recently, we suggested that the CTTCC sequence motif found in front of many genes encoding glycolytic enzymes lay at the core of the GCR1-binding site. Here we mapped the DNA-binding domain of GCR1 to the carboxy-terminal 154 amino acids of the polypeptide. DNase I protection studies showed that a hybrid MBP-GCR1 fusion protein protected a region of the upstream activating sequence of TPI (UASTPI), which harbored the CTTCC sequence motif, and suggested that the fusion protein might also interact with a region of the UAS that contained the related sequence CATCC. A series of in vivo G methylation protection experiments of the native TPI promoter were carried out with wild-type and gcr1 deletion mutant strains. The G doublets that correspond to the C doublets in each site were protected in the wild-type strain but not in the gcr1 mutant strain. These data demonstrate that the UAS of TPI contains two GCR1-binding sites which are occupied in vivo. Furthermore, adjacent RAP1/GRF1/TUF- and REB1/GRF2/QBP/Y-binding sites in UASTPI were occupied in the backgrounds of both strains. In addition, DNA band-shift assays were used to show that the MBP-GCR1 fusion protein was able to form nucleoprotein complexes with oligonucleotides that contained CTTCC sequence elements found in front of other glycolytic genes, namely, PGK, ENO1, PYK, and ADH1, all of which are dependent on GCR1 gene function for full expression. However, we were unable to detect specific interactions with CTTCC sequence elements found in front of the translational component genes TEF1, TEF2, and CRY1. Taken together, these experiments have allowed us to propose a consensus GCR1-binding site which is 5'-(T/A)N(T/C)N(G/A)NC(T/A)TCC(T/A)N(T/A)(T/A)(T/G)-3'.

The role of Gcr1p in the transcriptional activation of glycolytic genes in yeast Saccharomyces cerevisiae.

To study the interdependence of Gcr1p and Rap1p, we prepared a series of synthetic regulatory sequences that contained various numbers and combinations of CT-boxes (Gcr1p-binding sites) and RPG-boxes (Rap1p-binding sites). The ability of the synthetic oligonucleotides to function as regulatory sequences was tested using an ENO1-lacZ reporter gene. As observed previously, synthetic oligonucleotides containing both CT- and RPG-boxes conferred strong UAS activity. Likewise, a lone CT-box did not show any UAS activity. By contrast, oligonucleotides containing tandem Ct-boxes but no RPG-box conferred strong promoter activity. This UAS activity was not dependent on position or orientation of the oligonucleotides in the 5' noncoding region. However, it was dependent on both GCR1 and GCR2. These results suggest that the ability of Gcr1p to bind Gcr1p-binding sites in vivo is not absolutely dependent on Rap1p. Eleven independent mutants of GCR1 were isolated that conferred weak UAS activity to a single CT-box. Five mutants has single mutations in Gcr1p's DNA-binding domain and displayed slightly higher affinity for the CT-box. These results support the hypothesis that Gcr1p and Gcr2p play the central role in glycolytic gene expression and that the function of Rap1p is to facilitate the binding of Gcr1p to its target.

DNA sequence of the Escherichia coli gene, gnd, for 6-phosphogluconate dehydrogenase.

Expression of gnd of Escherichia coli, which encodes 6-phosphogluconate dehydrogenase, an enzyme of the hexose monophosphate shunt, is subject to growth rate-dependent regulation and is gene dosage-dependent: the level of the enzyme increases in direct proportion to the cellular growth rate at both low and high gene copy numbers. We have determined the nucleotide sequence of gnd and flanking control regions, the 5'-end of in vivo gnd mRNA, and the start codon of the structural gene. Analysis of the sequence indicated that: (i) the gnd promoter is typical of other E. coli promoters and the structural gene is followed by a rho-independent transcription termination signal; (ii) the 56-nucleotide leader of gnd mRNA does not contain a rho-independent transcription termination signal, so growth rate-dependent regulation of 6-phosphogluconate dehydrogenase level is not carried out by an attenuation mechanism analogous to the one that controls expression of the E. coli ampC gene; (iii) the codon composition of the structural gene resembles that of other highly expressed E. coli genes and thus is not responsible for the regulation either; (iv) the structural gene is preceded at an optimal distance by a strong Shine-Dalgarno (SD) sequence, AGGAG ; (v) the leader region of the mRNA contains regions of dyad symmetry that have the potential to sequester the SD sequence and the start codon. This latter feature of the gene suggests that growth rate-dependent regulation may involve regulation of translation initiation frequency.

The E-box DNA binding protein Sgc1p suppresses the gcr2 mutation, which is involved in transcriptional activation of glycolytic genes in Saccharomyces cerevisiae.

Glycolytic gene expression is mediated by the Gcr1p-Gcr2p transcriptional activation complex. A screen for multicopy suppressors of gcr2 yielded SGC1. SGC1's suppression activity was specific to gcr2, it did not extend to gcr1. Disruption of SGC1 moderately affected glycolytic enzyme activities, although no growth defect was evident. Sgc1p exhibits a bHLH motif which is characteristic of E-box DNA-binding proteins. DNA footprinting experiments demonstrated Sgc1p's ability to bind at an E-box. However, its binding specificity was less than 10-fold, which is also characteristic of E-box binding proteins. LexA fusion experiments demonstrated that Sgc1p has weak intrinsic activating activity independent of GCR1 and GCR2. We propose that Sgc1p binds at E-boxes of glycolytic genes and contributes to their activation.

AP-1 transcription factor binding activity in rat adrenal medulla and hypothalamus with age and cold exposure.

The AP-1 regulatory element has been implicated in the cold-induced expression of tyrosine hydroxylase in the adrenal medulla. Since in this tissue, the cold-induced increase in tyrosine hydroxylase expression is impaired with age and in other tissues, there is some evidence that AP-1 transcription factor binding is diminished with age, we examined the cold-stimulated AP-1 transcription factor binding to an oligonucleotide with the consensus sequence of the AP-1 response element in nuclear extracts from adrenal medulla and hypothalamus of young and senescent rats. AP-1 transcription factor binding activity diminished by 38% with age in unstimulated adrenal medulla. Following cold stimulation, the AP-1 binding activity increased by 21-25% in the adrenal medulla of both young and senescent rats. However, the level of AP-1 binding in cold-stimulated senescent rats was still less than in cold-stimulated younger rats. There were no changes in AP-3 binding activity with either age or cold exposure in the adrenal medulla. Similarly, in the hypothalamus, there was a 25% decrease with age and a 25% increase following cold stimulation in the level of AP-1 binding. There was a 62% age-related increase in AP-3 binding activity but no change with cold exposure. These data indicate that there is reduced AP-1 binding activity in senescent control rats. Moreover, the demonstration that cold stimulus evokes similar increases in AP-1 binding activity in both young and old rats suggests that the stimulation pathway that increases AP-1 transcription factor is maintained in the senescent animal.

Transcriptome analysis of LuxS-deficient Streptococcus mutans grown in biofilms.

We previously reported that LuxS in Streptococcus mutans is involved in stress tolerance and biofilm formation. In this study, flowcells and confocal laser scanning microscopy were used to further examine the effects of LuxS-deficiency on biofilm formation. Similar to the wild-type strain (UA159), a strain deficient in LuxS (TW26D) bound efficiently to the flowcells and formed microcolonies 4 h after inoculation. Unlike UA159, which accumulated and formed compact, evenly distributed biofilms after 28 h, TW26D showed only loose, sporadic, thin biofilms. DNA microarray analysis revealed alterations in transcription of more than 60 genes in TW26D biofilms by at least 1.5-fold (P < 0.001). Among the upregulated genes were those for sugar-specific enzymes II of the phosphotransferase (PTS) system and the atp operon, which codes for the proton-pumping F-ATPase. Of the downregulated genes, several encode proteins with putative functions in DNA repair. Mutation of selected genes caused severe defects in the ability of the mutants to tolerate low pH and oxidative stress. These results provide additional proof that LuxS-deficiency causes global alterations in the expression of genes central to biofilm formation and virulence of S. mutans, including those involved in energy metabolism, DNA repair and stress tolerance.

Polymicrobial periodontal pathogen transcriptomes in calvarial bone and soft tissue.

Porphyromonas gingivalis, Treponema denticola, and Tannerella forsythia are consistently associated with adult periodontitis. This study sought to document the host transcriptome to a P. gingivalis, T. denticola, and T.forsythia challenge as a polymicrobial infection using a murine calvarial model of acute inflammation and bone resorption. Mice were infected with P. gingivalis, T. denticola, and T. forsythia over the calvaria, after which the soft tissues and calvarial bones were excised. A Murine GeneChip(®) array analysis of transcript profiles showed that 6997 genes were differentially expressed in calvarial bones (P < 0.05) and 1544 genes were differentially transcribed in the inflamed tissues after the polymicrobial infection. Of these genes, 4476 and 1035 genes in the infected bone and tissues were differentially expressed by upregulation. Biological pathways significantly impacted by the polymicrobial infection in calvarial bone included leukocyte transendothelial migration (LTM), cell adhesion molecules, adherens junction, major histocompatibility complex antigen, extracellular matrix-receptor interaction, and antigen processing and presentation resulting in inflammatory/cytokine/chemokine transcripts stimulation in bone and soft tissue. Intense inflammation and increased activated osteoclasts were observed in calvarias compared with sham-infected controls. Quantitative real-time RT-PCR analysis confirmed that the mRNA level of selected genes corresponded with the microarray expression. The polymicrobial infection regulated several LTM and extracellular membrane pathway genes in a manner distinct from mono-infection with P. gingivalis, T. denticola, or T. forsythia. To our knowledge, this is the first definition of the polymicrobially induced transcriptome in calvarial bone and soft tissue in response to periodontal pathogens.

Porphyromonas gingivalis infection-induced tissue and bone transcriptional profiles.

Porphyromonas gingivalis has been associated with subgingival biofilms in adult periodontitis. However, the molecular mechanisms of its contribution to chronic gingival inflammation and loss of periodontal structural integrity remain unclear. This investigation aimed to examine changes in the host transcriptional profiles during a P. gingivalis infection using a murine calvarial model of inflammation and bone resorption. P. gingivalis FDC 381 was injected into the subcutaneous soft tissue over the calvaria of BALB/c mice for 3 days, after which the soft tissues and calvarial bones were excised. RNA was isolated from infected soft tissues and calvarial bones and was analysed for transcript profiles using Murine GeneChip((R)) arrays to provide a molecular profile of the events that occur following infection of these tissues. After P. gingivalis infection, 6452 and 2341 probe sets in the infected soft tissues and calvarial bone, respectively, were differentially expressed (P

Molecular characterization of Treponema denticola infection-induced bone and soft tissue transcriptional profiles.

Treponema denticola is associated with subgingival biofilms in adult periodontitis and with acute necrotizing ulcerative gingivitis. However, the molecular mechanisms by which T. denticola impacts periodontal inflammation and alveolar bone resorption remain unclear. Here, we examined changes in the host transcriptional profiles during a T. denticola infection using a murine calvarial model of inflammation and bone resorption. T. denticola was injected into the subcutaneous soft tissue over the calvaria of BALB/c mice for 3 days, after which the soft tissues and the calvarial bones were excised. RNA was isolated and analysed for transcript profiling using Murine GeneChip arrays. Following T. denticola infection, 2905 and 1234 genes in the infected calvarial bones and soft tissues, respectively, were differentially expressed (P

Tannerella forsythia infection-induced calvarial bone and soft tissue transcriptional profiles.

Tannerella forsythia is associated with subgingival biofilms in adult periodontitis, although the molecular mechanisms contributing to chronic inflammation and loss of periodontal bone remain unclear. We examined changes in the host transcriptional profiles during a T. forsythia infection using a murine calvarial model of inflammation and bone resorption. Tannerella forsythia was injected into the subcutaneous soft tissue over calvariae of BALB/c mice for 3 days, after which the soft tissues and calvarial bones were excised. RNA was isolated and Murine GeneChip (Affymetrix, Santa Clara, CA) array analysis of transcript profiles showed that 3226 genes were differentially expressed in the infected soft tissues (P < 0.05) and 2586 genes were differentially transcribed in calvarial bones after infection. Quantitative real-time reverse transcription-polymerase chain reaction analysis of transcription levels of selected genes corresponded well with the microarray results. Biological pathways significantly impacted by T. forsythia infection in calvarial bone and soft tissue included leukocyte transendothelial migration, cell adhesion molecules (immune system), extracellular matrix-receptor interaction, adherens junction, and antigen processing and presentation. Histologic examination revealed intense inflammation and increased osteoclasts in calvariae compared with controls. In conclusion, localized T. forsythia infection differentially induces transcription of a broad array of host genes, and the profiles differ between inflamed soft tissues and calvarial bone.

Gene expression profiles are influenced by ISS, MOF, and clinical outcome in multiple injured patients: a genome-wide comparative analysis.

BACKGROUND: Posttraumatic immune system activation in major trauma patients is linked to systemic inflammatory response syndrome, multiple organ failure (MOF), and mortality. Recent studies suggest that genome-wide expression is altered in response to distinct clinical parameters; however, the functional allocation of theses genes remains unclear. PATIENTS AND METHODS: Thirteen patients after major trauma (Injury Severity Score < 16) were studied. Monocytes were obtained on admission (within 90 min) and at 6, 12, 24, 48, and 72 h after trauma. Complementary ribonucleic acid (RNA) targets were hybridized to Affymetrix HG U 133A microarrays. Searching for genes that are differentially expressed, the patients were dichotomously assigned depending upon survival, injury severity, and MOF. The data were analyzed by supervised analysis, clustering, and comparative pathway analysis. RESULTS: Gene expression profiles of patients with adverse outcomes (763 probe sets) mainly consist of those involved in "immunological activation" or "cellular movement," whereas the gene set associated with MOF (660) is associated with "cancer" and "cell death." Injury severity (295) leads to an overexpression of genes involved in inflammatory disease. CONCLUSION: We demonstrate for the first time a serial, sequential screening analysis of monocyte messenger RNA expression patterns after multiple injury indicating a strongly significant connection between the patients' expression profile and different clinical parameters. The latter provoke a characteristic overexpression of specific functional gene ontologies. Further studies to clarify clinical consequence of this differential gene regulation are currently anticipated.

Distinctive characteristics of transcriptional profiles from two epithelial cell lines upon interaction with Actinobacillus actinomycetemcomitans.

Transcriptional profiling and gene ontology analyses were performed to investigate the unique responses of two different epithelial cell lines to an Actinobacillus actinomycetemcomitans challenge. A total of 2867 genes were differentially regulated among all experimental conditions. The analysis of these 2867 genes revealed that the predominant specific response to infection in HeLa cells was associated with the regulation of enzyme activity, RNA metabolism, nucleoside and nucleic acid transport and protein modification. The predominant specific response in immortalized human gingival keratinocytes (IHGK) was associated with the regulation of angiogenesis, chemotaxis, transmembrane receptor protein tyrosine kinase signaling, cell differentiation, apoptosis and response to stress. Of particular interest, stress response genes were significantly - yet differently - affected in both cell lines. In HeLa cells, only three regulated genes impacted the response to stress, and the response to unfolded protein was the only term that passed the ontology filters. This strikingly contrasted with the profiles obtained for IHGK, in which 61 regulated genes impacted the response to stress and constituted an extensive network of cell responses to A. actinomycetemcomitans interaction (response to pathogens, oxidative stress, unfolded proteins, DNA damage, starvation and wounding). Hence, while extensive similarities were found in the transcriptional profiles of these two epithelial cell lines, significant differences were highlighted. These differences were predominantly found in pathways that are associated with host-pathogen interactions.

GCR1 of Saccharomyces cerevisiae encodes a DNA binding protein whose binding is abolished by mutations in the CTTCC sequence motif.

In Saccharomyces cerevisiae, glycolysis enzymes constitute 30-60% of the soluble protein. GCR1 gene function is required for high-level glycolytic gene expression. In gcr1 mutant strains the levels of most glycolytic enzymes are between 2% and 10% of wild type. Binding sites for the global regulatory protein known as repressor activator protein 1 (RAP1)/general regulatory factor 1 (GRF1)/translation upstream factor (TUF) are found in close proximity to one or more CTTCC sequence motifs in the controlling region of GCR1-dependent genes. RAP1/GRF1/TUF-binding sites are known to be essential elements of upstream activating sequences that control expression of many glycolytic genes. In this report, I demonstrate that GCR1 encodes a DNA binding protein whose ability to bind DNA is dependent on the CTTCC sequence motif. This finding, in addition to the work of others, suggests that the GCR1 gene product and the RAP1/GRF1/TUF gene product act in concert to mediate high-level glycolytic gene expression.

Understanding the growth phenotype of the yeast gcr1 mutant in terms of global genomic expression patterns.

The phenotype of an organism is the manifestation of its expressed genome. The gcr1 mutant of yeast grows at near wild-type rates on nonfermentable carbon sources but exhibits a severe growth defect when grown in the presence of glucose, even when nonfermentable carbon sources are available. Using DNA microarrays, the genomic expression patterns of wild-type and gcr1 mutant yeast growing on various media, with and without glucose, were compared. A total of 53 open reading frames (ORFs) were identified as GCR1 dependent based on the criterion that their expression was reduced twofold or greater in mutant versus wild-type cultures grown in permissive medium consisting of YP supplemented with glycerol and lactate. The GCR1-dependent genes, so defined, fell into three classes: (i) glycolytic enzyme genes, (ii) ORFs carried by Ty elements, and (iii) genes not previously known to be GCR1 dependent. In wild-type cultures, GCR1-dependent genes accounted for 27% of the total hybridization signal, whereas in mutant cultures, they accounted for 6% of the total. Glucose addition to the growth medium resulted in a reprogramming of gene expression in both wild-type and mutant yeasts. In both strains, glycolytic enzyme gene expression was induced by the addition of glucose, although the expression of these genes was still impaired in the mutant compared to the wild type. By contrast, glucose resulted in a strong induction of Ty-borne genes in the mutant background but did not greatly affect their already high expression in the wild-type background. Both strains responded to glucose by repressing the expression of genes involved in respiration and the metabolism of alternative carbon sources. Thus, the severe growth inhibition observed in gcr1 mutants in the presence of glucose is the result of normal signal transduction pathways and glucose repression mechanisms operating without sufficient glycolytic enzyme gene expression to support growth via glycolysis alone.

Essential site for growth rate-dependent regulation within the Escherichia coli gnd structural gene.

Expression of gnd of Escherichia coli, which encodes the hexose monophosphate shunt enzyme, 6-phosphogluconate dehydrogenase (6PGD; EC 1.1.1.44), is subject to growth rate-dependent regulation: the level of the enzyme is directly proportional to growth rate under a variety of growth conditions. Previous results obtained with strains carrying transcriptional fusions of gnd to the structural genes of the lactose operon suggested that the growth rate-dependent regulation of gnd expression is at the post-transcriptional level. To characterize the regulation further, we prepared with phage MudII a set of eight independent gnd-lac gene (protein) fusions. We showed through genetic analysis and DNA sequencing that each fusion joint was located within the 6PGD-coding sequence between the first and second base pair of a codon, the reading frame required for production of a hybrid 6PGD-beta-galactosidase. Strains harboring the gnd-lac fusion plasmids produced proteins whose mobility in a NaDodSO4/polyacrylamide gel agreed with the molecular weights predicted from the DNA sequence for the respective hybrid proteins. The level of beta-galactosidase was high and relatively growth rate-independent in the fusion whose fusion joint was at codon 48. The level of beta-galactosidase in the other seven fusion strains whose fusion joints were located further downstream in the 6PGD-coding sequence showed the same dependence on growth rate as 6PGD in a normal strain. beta-Galactosidase levels were not affected by the presence of a gnd+ gene in trans to any of the fusions. The results suggest that all sites necessary for growth rate-dependent regulation of 6PGD level lie in gnd upstream from codon 118 and that an essential site of negative control lies within the coding sequence, between codons 48 and 118.