A Moonlighting Enzyme Links Escherichia coli Cell Size with Central Metabolism

Growth rate and nutrient availability are the primary determinants of size in single-celled organisms: rapidly growing Escherichia coli cells are more than twice as large as their slow growing counterparts. Here we report the identification of the glucosyltransferase OpgH as a nutrient-dependent regulator of E. coli cell size. During growth under nutrient-rich conditions, OpgH localizes to the nascent septal site, where it antagonizes assembly of the tubulin-like cell division protein FtsZ, delaying division and increasing cell size. Biochemical analysis is consistent with OpgH sequestering FtsZ from growing polymers. OpgH is functionally analogous to UgtP, a Bacillus subtilis glucosyltransferase that inhibits cell division in a growth rate-dependent fashion. In a striking example of convergent evolution, OpgH and UgtP share no homology, have distinct enzymatic activities, and appear to inhibit FtsZ assembly through different mechanisms. Comparative analysis of E. coli and B. subtilis reveals conserved aspects of growth rate regulation and cell size control that are likely to be broadly applicable. These include the conservation of uridine diphosphate glucose as a proxy for nutrient status and the use of moonlighting enzymes to couple growth rate-dependent phenomena to central metabolism.     

Clustered Genes for Galactose Metabolism from Streptococcus mutans Cloned in Escherichia coli

DNA cloned into Escherichia coli from a serotype c strain of Streptococcus mutans allowed a galKTE mutant to utilize galactose for growth. However, the DNA does not appear to encode enzymes of the Leloir pathway used by E. coli, but rather appears to encode enzymes of the tagatose phosphate pathway.     

Metabolic Flux Ratio Analysis of Genetic and Environmental Modulations of Escherichia coli Central Carbon Metabolism

The response of Escherichia coli central carbon metabolism to genetic and environmental manipulation has been studied by use of a recently developed methodology for metabolic flux ratio (METAFoR) analysis; this methodology can also directly reveal active metabolic pathways. Generation of fluxome data arrays by use of the METAFoR approach is based on two-dimensional (13)C-(1)H correlation nuclear magnetic resonance spectroscopy with fractionally labeled biomass and, in contrast to metabolic flux analysis, does not require measurements of extracellular substrate and metabolite concentrations. METAFoR analyses of E. coli strains that moderately overexpress phosphofructokinase, pyruvate kinase, pyruvate decarboxylase, or alcohol dehydrogenase revealed that only a few flux ratios change in concert with the overexpression of these enzymes. Disruption of both pyruvate kinase isoenzymes resulted in altered flux ratios for reactions connecting the phosphoenolpyruvate (PEP) and pyruvate pools but did not significantly alter central metabolism. These data indicate remarkable robustness and rigidity in central carbon metabolism in the presence of genetic variation. More significant physiological changes and flux ratio differences were seen in response to altered environmental conditions. For example, in ammonia-limited chemostat cultures, compared to glucose-limited chemostat cultures, a reduced fraction of PEP molecules was derived through at least one transketolase reaction, and there was a higher relative contribution of anaplerotic PEP carboxylation than of the tricarboxylic acid (TCA) cycle for oxaloacetate synthesis. These two parameters also showed significant variation between aerobic and anaerobic batch cultures. Finally, two reactions catalyzed by PEP carboxykinase and malic enzyme were identified by METAFoR analysis; these had previously been considered absent in E. coli cells grown in glucose-containing media. Backward flux from the TCA cycle to glycolysis, as indicated by significant activity of PEP carboxykinase, was found only in glucose-limited chemostat culture, demonstrating that control of this futile cycle activity is relaxed under severe glucose limitation.     

Small RNAs in Escherichia coli

Bacterial cells contain several small RNAs (sRNAs) that are not translated. These stable, abundant RNAs act by multiple mechanisms, such as RNA-RNA basepairing, RNA-protein interactions and intrinsic RNA activity, and regulate diverse cellular functions, including RNA processing, mRNA stability, translation, protein stability and secretion.     

EcoCyc: Enyclopedia of Escherichia coli Genes and Metabolism.

The Encyclopedia of Genes and Metabolism (EcoCyc) is a database that combines information about the genome and the intermediary metabolism of Escherichia coli. It describes 2970 genes of E.coli, 547 enzymes encoded by these genes, 702 metabolic reactions that occur in E.coli and the organization of these reactions into 107 metabolic pathways. The EcoCyc graphical user interface allows scientists to query and explore the EcoCyc database using visualization tools such as genomic-map browsers and automatic layouts of metabolic pathways. EcoCyc spans the space from sequence to function to allow scientists to investigate an unusually broad range of questions. EcoCyc can be thought of as both an electronic review article because of its copious references to the primary literature, and as an in silicio model of E.coli metabolism that can be probed and analyzed through computational means.     

Silencing of NADPH-Dependent Oxidoreductase Genes (yqhD and dkgA) in Furfural-Resistant Ethanologenic Escherichia coli

Low concentrations of furfural are formed as a side product during the dilute acid hydrolysis of hemicellulose. Growth is inhibited by exposure to furfural but resumes after the complete reduction of furfural to the less toxic furfuryl alcohol. Growth-based selection was used to isolate a furfural-resistant mutant of ethanologenic Escherichia coli LY180, designated strain EMFR9. Based on mRNA expression levels in the parent and mutant in response to furfural challenge, genes encoding 12 oxidoreductases were found to vary by more than twofold (eight were higher in EMFR9; four were higher in the parent). All 12 genes were cloned. When expressed from plasmids, none of the eight genes in the first group increased furfural tolerance in the parent (LY180). Expression of three of the silenced genes (yqhD, dkgA, and yqfA) in EMFR9 was found to decrease furfural tolerance compared to that in the parent. Purified enzymes encoded by yqhD and dkgA were shown to have NADPH-dependent furfural reductase activity. Both exhibited low K_m values for NADPH (8 μM and 23 μM, respectively), similar to those of biosynthetic reactions. Furfural reductase activity was not associated with yqfA. Deleting yqhD and dkgA in the parent (LY180) increased furfural tolerance, but not to the same extent observed in the mutant EMFR9. Together, these results suggest that the process of reducing furfural by using an enzyme with a low K_m for NADPH rather than a direct inhibitory action is the primary cause for growth inhibition by low concentrations of furfural.A wide variety of fermentation products can be made using sugars from lignocellulosic biomass as a substrate (9, 13, 16, 39). Prior to fermentation, however, the carbohydrate polymers cellulose and hemicellulose must be converted to soluble sugars, using a combination of chemical and enzymatic processes (40, 42, 43). Chemical processes are accompanied by side reactions that produce a mixture of minor products such as alcohols, acids, and aldehydes that have a negative effect on the metabolism of microbial biocatalysts. Alcohols (catechol, syringol, etc.) have been shown to act by permeabilizing the cell membrane and toxicity correlated well with the hydrophobicity of the molecule (47). Organic acids (acetate, formate, etc.) are thought to cross the membrane in neutral form and ionize within the cytoplasm, inhibiting growth by acidifying the cytoplasm and collapsing the proton motive force (32, 48). The inhibitory mechanisms of aldehydes are more complex. Aldehydes can react to form products with many cellular constituents, in addition to direct physical and metabolic effects (27, 36). In aggregate, these side products from chemical pretreatments can retard cell growth and slow the fermentation of biomass-derived sugars (10, 31).Furfural (a dehydration product of pentose sugars) is of particular importance. Furfural content in dilute acid hydrolysates of hemicellulose has been correlated with toxicity (23). Removal of furfural by lime addition (pH 10) rendered hydrolysates readily fermentable while readdition of furfural restored toxicity (22). Furfural has also been shown to potentiate the toxicity of other compounds known to be present in acid hydrolysates of hemicellulose (47-49). Furfural has been reported to alter DNA structure and sequence (3, 17), inhibit glycolytic enzymes (6), and slow sugar metabolism (11).The ability of fermenting organisms to function in the presence of these inhibitors has been researched extensively. Encapsulation of Saccharomyces cerevisiae in alginate has been shown to be protective and improve fermentation in acid hydrolysates of hemicellulose (38). Strains of S. cerevisiae have been previously described with improved resistance to hydrolysate inhibitors (1, 20, 29). Escherichia coli (7), S. cerevisiae (2, 19), and other microorganisms (4) have been shown to contain enzymes that catalyze the reduction of furfural to the less toxic product, furfuryl alcohol (49). In E. coli, furfural reductase activity appears to be NADPH-dependent (7). An NADPH-dependent furfural reductase was purified from E. coli although others may also be present. Two NADPH-dependent enzymes capable of reducing 5-hydroxymethyl furfural (a dehydration product of hexose sugars) have been characterized in S. cerevisiae and identified as the ADH6 gene product (2, 34) and a mutant form of ADH1 (2).In this paper, we describe the isolation of a furfural-resistant E. coli mutant (EMFR9) in which furfural reductase activity is lower than that of the parent (LY180) due to decreased expression of yqhD and dkgA. The reduction of furfural by these two NADPH-dependent oxidoreductases is proposed to inhibit growth by depleting the NADPH needed for biosynthesis.     

S-Methylmethionine Metabolism in Escherichia coli

Selenium-accumulating Astragalus spp. contain an enzyme which specifically transfers a methyl group from S-methylmethionine to the selenol of selenocysteine, thus converting it to a nontoxic, since nonproteinogenic, amino acid. Analysis of the amino acid sequence of this enzyme revealed that Escherichia coli possesses a protein (YagD) which shares high sequence similarity with the enzyme. The properties and physiological role of YagD were investigated. YagD is an S-methylmethionine: homocysteine methyltransferase which also accepts selenohomocysteine as a substrate. Mutants in yagD which also possess defects in metE and metH are unable to utilize S-methylmethionine for growth, whereas a metE metH double mutant still grows on S-methylmethionine. Upstream of yagD and overlapping with its reading frame is a gene (ykfD) which, when inactivated, also blocks growth on methylmethionine in a metE metH genetic background. Since it displays sequence similarities with amino acid permeases it appears to be the transporter for S-methylmethionine. Methionine but not S-methylmethionine in the medium reduces the amount of yagD protein. This and the existence of four MET box motifs upstream of yfkD indicate that the two genes are members of the methionine regulon. The physiological roles of the ykfD and yagD products appear to reside in the acquisition of S-methylmethionine, which is an abundant plant product, and its utilization for methionine biosynthesis.     

Gluconate Metabolism in Escherichia coli

On the basis of information available in the literature, gluconate dissimilation in Escherichia coli is thought to occur via the hexose monophosphate pathway. Evidence is presented in this study that gluconate is catabolized in this organism via an inducible Entner-Doudoroff pathway. This evidence is based on chromatographic examination of end products produced from (14)C-labeled gluconate or glucose, distribution of (14)C in the carbon atoms of pyruvate formed from specifically labeled (14)C-glucose and (14)C-gluconate, and the ability of cell-free extracts to produce pyruvate from 6-phosphogluconate. Degradation of gluconate by an Entner-Doudoroff pathway occurred simultaneously with a glycolytic cleavage of glucose. A relationship between gluconate-induced, Entner-Doudoroff pathway activity and catabolism of glucose in Escherichia coli and other bacterial species is discussed.     

Genes for 7S RNAs can replace the gene for 4.5S RNA in growth of Escherichia coli.

4.5S RNAs of eubacteria and 7S RNAs of archaebacteria and eukaryotes exist in a hairpin conformation. The apex of this hairpin displays structural and sequence similarities among both 4.5S and 7S RNAs. Furthermore, a hyphenated sequence of 16 nucleotides is conserved in all eubacterial 4.5S RNAs examined. In this article I report that 7S RNAs that contain this 16-nucleotide sequence are able to replace 4.5S RNAs and permit growth of Escherichia coli.     

Competition among Hfq-binding small RNAs in Escherichia coli

A major class of small bacterial RNAs (sRNAs) regulate translation and mRNA stability by pairing with target mRNAs, dependent upon the RNA chaperone Hfq. Hfq, related to the Lsm/Sm families of splicing proteins, binds the sRNAs and stabilizes them in vivo and stimulates pairing with mRNAs in vitro. Although Hfq is abundant, the sRNAs, when induced, are similarly abundant. Therefore, Hfq may be limiting for sRNA function. We find that, when overexpressed, a number of sRNAs competed with endogenous sRNAs for binding to Hfq. This correlated with lower accumulation of the sRNAs (presumably a reflection of the loss of Hfq binding), and lower activity of the sRNAs in regulating gene expression. Hfq was limiting for both positive and negative regulation by the sRNAs. In addition, deletion of the gene for an expressed and particularly effective competitor sRNA improved the regulation of genes by other sRNAs, suggesting that Hfq is limiting during normal growth conditions. These results support the existence of a hierarchy of sRNA competition for Hfq, modulating the function of some sRNAs.     

Mutations Affecting Gluconate Metabolism in Escherichia coli

A mutant of Escherichia coli K-12 that does not ferment gluconate on fermentation plates was isolated and characterized. This mutant, designated M2, shows a long lag for growth on gluconate mineral medium and somewhat reduced levels of high-affinity transport, gluconokinase, and gluconate-6-P dehydrase activities in the log phase of growth. The mutation involved is near malA. Deletion mutants in which malA region was affected were also studied. They were found to affect the function of different genes involved in gluconate metabolism.     

大肠杆菌海藻糖的代谢调控

海藻糖是一种重要的抗逆物质。大肠杆菌中otsBA操纵子编码的两种酶负责海藻糖合成。otsBA基因的表达受渗透压诱导和σs因子的调节。细胞的周质海藻糖酶(treA)将外源海藻糖分解成两个葡萄糖分子。尽管大肠杆菌中渗透压诱导合成的海藻糖并不能保护细胞抗干燥,我们将otsA单个基因通过农杆菌转入烟草时,转基因株提高了耐盐和抗干燥特性,同时在转基因烟草提取物中检测到海藻糖,证明otsA基因在烟草中表达并合成海藻糖。我们认为若将otsA基因转入其它植物,可望改善这些植物的抗干旱、耐盐碱特性和延长采摘后的保鲜期 。