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We report a study to determine the role of pyruvate oxidase among Escherichia coli isogenic strains with active and inactive phosphoenolpyruvate:carbohydrate phosphotransferase system (PTS). Strain PB11, displaying a specific growth rate (µ) in glucose minimal medium of 0.1 h^–1 is a ptsHI, crr operon deletion derivative of wild-type JM101 (displaying a µ of 0.70 h^–1). Strain PB12 is a spontaneous mutant obtained from PB11 after selection for its capacity to grow on glucose with a µ of 0.40 h^–1. In minimal medium cultures supplemented with glucose plus acetate, strain JM101 displayed preferential consumption of glucose, whereas strains PB11 and PB12 did not display glucose catabolic repression of acetate consumption. Inactivation of poxB caused a severe reduction in growth rate in strain PB11 when grown in the fermentor with medium containing glucose or glucose plus acetate, whereas under the same conditions poxB^–derivative strains of JM101 and PB12 were not affected. Relative transcript levels for 29 genes related to poxB transcriptional regulation and central metabolism were determined using RT-PCR. This analysis revealed 2-fold lower transcript levels for genes encoding subunits of the pyruvate dehydrogenase complex (Pdh) in strain PB11 and 4- to 6-fold higher transcript levels for poxB in strains PB11 and PB12, when compared to JM101. In addition, in the PTS^– strains, upregulation of the poxB transcription factors rpoS, soxS and marA, was detected. The results presented here strongly suggest that AcCoA is mainly synthesized from acetate produced by pyruvate oxidase in strain PB11, whereas in strains JM101 and PB12, AcCoA is synthesized preferentially from pyruvate by Pdh.

Expression of plasmid-encoded genes in bacteria is the most common strategy for the production of specific proteins in biotechnological processes. However, the synthesis of plasmid-encoded proteins and plasmid-DNA replication often places a metabolic load (metabolic burden) into the cell's biochemical capacities that usually reduces the growth rate of the producing culture (Glick BR. Biotechnol Adv 1995;13:247 -261). This metabolic burden may be related to a limited capacity of the cell to supply the extra demand of building blocks and energy required to replicate plasmid DNA and express foreign multicopy genes. Some of these required blocks are intermediaries of the pentose phosphate (PP) pathway, e.g., ribose-5-phosphate, erythrose-4-phosphate. Due to the important impact of metabolic burden on biotechnological processes, several groups have worked on developing strategies to overcome this problem, like reduction of plasmid copy number (Seo JH, Bailey JE. Biotechnol Bioeng 1985; 27:1668 -1674; Jones KL, Kim S, Keasling JD. Metab Eng 2000;3:328 -338), chromosomal insertion of the gene which product is desired, or changing the plasmidcoded antibiotic resistance gene (Hong Y, Pasternak JJ, Glick BR. Can J Microbiol 1995;41:624 -628). However, few efforts have been attempted to overcome the reduction of growth rate due to protein over-expression, by modifying central metabolic pathways (Chou C-H, Bennett GN, San KY. Biotechnol Bioeng 1994;44:952 -960). We constructed a high-copy number plasmid carrying the gene for glucose-6-phosphate dehydrogenase, zwf, under the control of an inducible trc promoter (pTRzwf04 plasmid). By transforming a wild-type strain and inducing with IPTG, it was possible to recover growth-rate from 0.46 h À1 (uninduced) to 0.64 h À1 (induced). The same transformation in an Escherichia coli zwf À , allows a growth-rate recovery from 0.43 h À1 (uninduced) to 0.62 h À1 (induced). We also studied this effect as part of a laboratory-scale biotechnology process: production of a recombinant insulin peptide by co-transforming E. coli JM101 strain with pTRzwf07, a low-copy-number plasmid that carries the same inducible construction as pTRzwf04, and with the pTEXP-MMRPI vector that carries a TrpLE-proinsulin hybrid gene. In this system, production of TrpLE-proinsulin strongly reduces growth rate; however, overexpression of zwf gene recovers with a growth rate from 0.1 h À1 in the TrpLE-proinsulin induced strain, to 0.37 h À1 when both zwf and TrpLE-proinsulin genes were induced. In this paper, we show that the engineering of the pentose phosphate pathway by modulation of the zwf gene expression level partially overcomes the possible bottleneck for the supply of building blocks and reducing power synthesized through the PP pathway, that are required for plasmid replication and plasmid-encoded protein expression. B 2004 Wiley Periodicals, Inc.

The ptsHIcrr operon was deleted from Escherichia coli wild-type JM101 to generate strain PB11 (PTS^–). In a mutant derived from PB11 that partially recovered its growth capacity on glucose by an adaptive evolution process (PB12, PTS^–Glc⁺), part of the phosphoenolpyruvate not used in glucose transport has been utilized for the synthesis of aromatic compounds. In this report, it is shown that on acetate as a carbon source, PB11 displayed a specific growth rate (μ) higher than PB12 (0.21 and 0.13 h^–1, respectively) while JM101 had a μ of 0.28 h^–1. To understand these growth differences on acetate, we compared the expression profiles of central metabolic genes by RT-PCR analysis. Obtained data revealed that some gluconeogenic genes were downregulated in both PTS^– strains as compared to JM101, while most glycolytic genes were upregulated in PB12 in contrast to PB11 and JM101. Furthermore, inactivation of gluconeogenic genes, like ppsA, sfcA, and maeB,and poxB gene that codes for pyruvate oxidase, has differential impacts in the acetate metabolism of these strains. Results indicate that growth differences on acetate in the PTS^– derivatives are due to potential carbon recycling strategies, mainly in PB11, and futile carbon cycles, especially in PB12.

The physiological role of the phosphoenolpyruvate:carbohydrate phosphotransferase system (PTS) has been studied in Escherichia coli. It has been shown that it directly or indirectly regulates the activity of most catabolic genes involved in carbohydrate transport. Accordingly, strains lacking PTS have pleiotropic phenotypes and are impaired in their capacity to grow on glucose and other PTS sugars. We have previously reported the characterization of a mutant harboring a pts operon deletion (PB11) which, as expected, showed a severe reduction of its growth capacity when incubated on glucose as carbon source, as compared to that of the isogenic wild-type strain. These observations corroborate that PTS is the main determinant of the capacity to grow on glucose and confirm the existence of other systems that allow glucose utilization although at a reduced level. To explore the physiological state and the metabolic pathways involved in glucose utilization in a pts^– background, we analyzed the global transcriptional response of the PB11 mutant when growing in minimal medium with glucose as carbon source. Genome-wide transcriptional analysis using microarrays revealed that, under this condition, expression of several genes related to carbon transport and metabolism was upregulated, as well as that of genes encoding transporters for certain nucleotides, nitrogen, phosphorus and sulfur sources. In addition, upregulation of rpoS and several genes transcribed by this sigma subunit was detected. These results indicate that the reduced capacity of glucose utilization present in the PB11 strain induces a general nutrient-scavenging response and this behavior is not dependent on a functional PTS. This condition is responsible of the utilization of secondary carbon sources in the presence of glucose.

Some school leaders have viewed programs such as Advanced Placement (AP) as an attractive option to resolve the ongoing achievement gap problem. However, the ongoing debate in the field about maintaining the ostensible purity of the AP program versus diluting it with program expansion has hindered the full utilization of AP classes to close the achievement gap among student populations. School staff must stop buying into the notion that AP is only for the top students. The paradigm shift to opening up greater access to AP courses can only occur if school leaders support the active expansion of the AP program by creating the needed infrastructure to prepare students for AP rigor. In addition to presenting the authors’ reasons, the barriers they faced, the best practices that they have developed, and the schoolwide increased student achievement based on state measures, the authors present how AP expansion provides equitable and challenging educational opportunities for all students.

High school students continue to struggle in algebra, especially in large inner-city schools with underprivileged students. Students are not the only ones skirmishing with algebra. Teachers and school leaders are just as frustrated as they search for answers to complex teaching and learning problems. Two high school principals report the results of action research they gleaned from visitations to successful high schools similar to their own in demographics in order to raise proficiency levels in mathematics in their own schools. Among the results include the focused and empowered leadership of department chairs and specific instructional pedagogies that are fostered and shared among teachers and mathematics coaches.