Identification of an inducible catabolic system for sialic acids (nan) in Escherichia coli.

Escherichia coli K-12 and K-12 hybrid strains constructed to express a polysialic acid capsule, the K1 antigen, were able to efficiently use sialic acid as a sole carbon source. This ability was dependent on induction of at least two activities: a sialic acid-specific transport activity, and an aldolase activity specific for cleaving sialic acids. Induction over basal levels required sialic acid as the apparent inducer, and induction of both activities was repressed by glucose. Induction also required the intracellular accumulation of sialic acid, which could be either added exogenously to the medium or accumulated intracellularly through biosynthesis. Exogenous sialic acid appeared to be transported by an active mechanism that did not involve covalent modification of the sugar. Mutations affecting either the transport or degradation of sialic acid prevented its use as a carbon source and have been designated nanT and nanA, respectively. These mutations were located by transduction near min 69 on the E. coli K-12 genetic map, between argG and glnF. In addition to being unable to use sialic acid as a carbon source, aldolase-negative mutants were growth-inhibited by this sugar. Therefore, the intracellularly accumulated sialic acid was toxic in aldolase-deficient E. coli strains. The dual role of aldolase in dissimilating and detoxifying sialic acids is consistent with the apparent multiple controls on expression of this enzyme.     

Redirection of sialic acid metabolism in genetically engineered Escherichia coli.

Most microorganisms do not produce sialic acid (sialate), and those that do appear to use a biosynthetic mechanism distinct from mammals. Genetic hybrids of nonpathogenic, sialate-negative laboratory Escherichia coli K-12 strains designed for the de novo synthesis of the polysialic acid capsule from E. coli K1 proved useful in elucidating the genetics and biochemistry of capsule biosynthesis. In this article we propose a dynamic model of sialometabolism to investigate the effects of biosynthetic neu (N-acetylneuraminic acid) and catabolic nan (N-acylneuraminate) mutations on the flux of intermediates through the sialate synthetic pathway. Intracellular sialate concentrations were determined by high pH anion exchange chromatography with pulsed amperometric detection. The results indicated that a strain carrying a null defect in the gene encoding polysialyltransferase (neuS) accumulated > 50 times more CMP-sialic acid than the wild type when strains were grown in a minimal medium supplemented with glucose and casamino acids. Metabolic accumulation of CMP-sialic acid depended on a functional sialic acid synthase (neuB), as shown by the inability of a strain lacking this enzyme to accumulate a detectable endogenous sialate pool. The neuB mutant concentrated trace sialate from the medium, indicating its potential value for quantitative analysis of free sialic acids in complex biological samples. The function of the sialate aldolase (encoded by nanA) in limiting intermediate flux through the synthetic pathway was determined by analyzing free sialate accumulation in neuA (CMP-sialic acid synthetase) nanA double mutants. The combined results demonstrate how E. coli avoids a futile cycle in which biosynthetic sialate induces the system for its own degradation and indicate the feasibility of generating sialooligosaccharide precursors through targeted manipulation of sialate metabolism.     

Biochemical characterization of a paraquat-tolerant mutant of Escherichia coli

The biochemical basis for paraquat tolerance was investigated using one of the paraquat-resistant Escherichia coli mutants previously isolated. When grown in the absence of paraquat (PQ2+), the specific activities of glucose-6-phosphate dehydrogenase and NADPH:PQ2+-diaphorase, both required for the expression of PQ2+ toxicity, were comparable in the wild type and the mutant. However, growth in the presence of 1 mM PQ2+ resulted in greater induction of these two enzymes in the wild type than in the mutant. Nevertheless, when the mutant was grown in 50 mM PQ2+, the activities of these two enzymes were comparable to those of the wild type grown in the presence of 1 mM PQ2+. Measurement of cyanide-resistant respiration, an indication of intracellular superoxide generation, showed that the intracellular flux of superoxide mediated by subsaturating concentrations of paraquat was significantly lower in the mutant than in the wild type. Extracellular superoxide formation, as measured by superoxide dismutase-inhibitable cytochrome c reduction, was higher in the wild type than in the mutant whether grown in the absence or the presence of PQ2+. The mutant did not show cross-resistance toward juglone or plumbagin, compounds known to exacerbate superoxide generation. The kinetics of [14C]PQ2+ uptake showed that the wild type accumulated PQ2+ against a concentration gradient, whereas the mutant seemed to do so only by facilitated diffusion. The results indicate that the impaired paraquat uptake system in the mutant results in the physiological and biochemical differences observed between the wild type and mutant.     

Sialic acid metabolism's dual function in Haemophilus influenzae

Many bacterial commensals and pathogens use the sialic acids as carbon and nitrogen sources. In Escherichia coli, the breakdown of these sugars is catalysed by gene products of the nan (Nacylneuraminate) operon; other microorganisms may use a similar catabolic strategy. Despite the known ligand and antirecognition functions of the sialic acids, the contribution of their catabolism to infection or host colonization has never been directly investigated. We addressed these questions with Haemophilus influenzae type b, which metabolizes relatively few carbohydrates, using the infant-rat infection model. The predicted H. influenzae homologue (HI0142) of the E. coli sialic acid aldolase structural gene, nanA, was subcloned and mutagenized by insertion of a kanamycin resistance cassette. Phenotypic investigation of the resulting H. influenzae aldolase mutants showed that: (i) HI0142 is essential for sialic acid degradation; (ii) the products of the open reading frames (ORFs) flanking HI0142 (HI0140, 41, 44 and 45) are likely to have the same functions as those of their counterparts in E. coli; (iii) sialylation of the lipooligosaccharide (LOS) epitope recognized by monoclonal antibody 3F11 is dependent on an environmental source of sialic acid; (iv) a nanA mutant hypersialylates its LOS sialyl acceptor, corresponding to an apparent increased fitness of the mutant in the infant-rat model; and (v) expression of the LOS sialyl acceptor is altered in cells grown without exogenous sialic acid, indicating the direct or indirect effect of sialic acid metabolism on LOS antigenicity. Taken together the data show the dual role of sialic acid catabolism in nutrition and cell surface modulation.     

Nucleotide sequence and genetic analysis of the neuD and neuB genes in region 2 of the polysialic acid gene cluster of Escherichia coli K1.

The K1 capsular polysaccharide, a polymer of sialic acid, is an important virulence determinant of extraintestinal pathogenic Escherichia coli. The genes responsible for the synthesis and expression of the polysialic acid capsule of E. coli K1 are located on the 17-kb kps gene cluster, which is functionally divided into three regions. Central region 2 encodes proteins necessary for the synthesis, activation, and polymerization of sialic acid, while flanking regions 1 and 3 are involved in polymer transport to the cell surface. In this study, we identified two genes at the proximal end of region 2, neuD and neuB, which encode proteins with predicted sizes of 22.7 and 38.7 kDa, respectively. Several observations suggest that the neuB gene encodes sialic acid synthase. EV24, a neuB chromosomal mutant that expresses a capsule when provided exogenous sialic acid, could be complemented in trans by the cloned neuB gene. In addition, NeuB has significant sequence similarity to the product of the cpsB gene of Neisseria meningitidis group B, which is postulated to encode sialic acid synthase. We also present data indicating that neuD has an essential role in K1 polymer production. Cells harboring pSR426, which contains all of region 2 but lacks region 1 and 3 genes, produce an intracellular polymer. In contrast, no polymer accumulated in cells carrying a derivative of pSR426 lacking a functional neuD gene. Unlike strains with mutations in neuB, however, neuD mutants are not complemented by exogenous sialic acid, suggesting that NeuD is not involved in sialic acid synthesis. Additionally, cells harboring a mutation in neuD accumulated sialic acid and CMP-sialic acid. We also found no significant differences between the endogenous and exogenous sialyltransferase activities of a neuD mutant and the wild-type organism. NeuD shows significant similarity to a family of bacterial acetyltransferases, leading to the theory that NeuD is an acetyltransferase which may exert its influences through modification of other region 2 proteins.     

利用同源重组的方法提高大肠杆菌W3110天冬氨酸的积累

大肠杆菌中存在3种天冬氨酸激酶,分别为LysC,MetL,ThrA,使天冬氨酸磷酸化后分别进入Lys、Met、Thr的合成途径.因此大肠杆菌菌体中无法积累大量天冬氨酸. 以大肠杆菌W3110为出发菌株,利用Red同源重组系统分别构建了LysC、ThrA和MetL单基因缺陷株和LysC-ThrA和LysC-MetL双基因缺陷株. 采用高效液相色谱法测定L-天冬氨酸积累量. 发现除MetL单基因突变株外,其余突变株均积累了比野生型更多的L-天冬氨酸,这为代谢工程改造菌株并通过发酵法生产天冬氨酸奠定了基础.     

Role of fadR and atoC(Con) mutations in poly(3-hydroxybutyrate-co-3-hydroxyvalerate) synthesis in recombinant pha+ Escherichia coli.

Recombinant Escherichia coli fadR atoC(Con) mutants containing the polyhydroxyalkanoate (PHA) biosynthesis genes from Alcaligenes eutrophus are able to incorporate significant levels of 3-hydroxyvalerate (3HV) into the copolymer poly(3-hydroxybutyrate-co-3-hydroxyvalerate) [P(3HB-co-3HV)]. We have used E. coli fadR (FadR is a negative regulator of fatty acid oxidation) and E. coli atoC(Con) (AtoC is a positive regulator of fatty acid uptake) mutants to demonstrate that either one of these mutations alone can facilitate copolymer synthesis but that 3HV levels in single mutant strains are much lower than in the fadR atoC(Con) strain. E. coli atoC(Con) mutants were used alone and in conjunction with atoA and atoD mutants to determine that the function of the atoC(Con) mutation is to increase the uptake of propionate and that this uptake is mediated, at least in part, by atoD+. Similarly, E. coli fadR mutants were used alone and in conjunction with fadA, fadB, and fadL mutants to show that the effect of the fadR mutation is dependent on fadB+ and fadA+ gene products. Strains that were mutant in the fadB or fadA locus were unable to complement a PHA biosynthesis pathway that was mutant at the phaA locus (thiolase), but a strain containing a fadR mutation and which was fadA+ fadB+ was able to complement the phaA mutation and incorporated 3HV into P(3HB-co-3HV) to a level of 29 mol%.     

Engineering of Saccharomyces cerevisiae for the production of L-glycerol 3-phosphate

L-glycerol 3-phosphate (L-G3P) was accumulated in Saccharomyces cerevisiae by pathway engineering. Intracellular concentration of this metabolic intermediate could be increased more than 20 times compared to the wild type by overexpressing GPD1 encoding the glycerol 3-phosphate dehydrogenase in a gpp1 Delta gpp2 Delta mutant which lacks both isoenzymes of glycerol 3-phosphatase. Investigation of cellular pattern of triacylglycerols and glycerophospholipids did not reveal considerable changes due to accumulation of their precursor L-G3P. Hyperosmotic stress did not affect the L-G3P pool in the gpp1 Delta gpp2 Delta mutant overexpressing GPD1 despite an about 4-fold increase of specific GPD activity. In contrast, oxygen limitation improved intracellular L-G3P concentration by enhancing the availability of cytosolic NADH. The reduction of pyruvate decarboxylase activity by deleting PDC2 led to an additional increase. In fact, the triple mutant gpp1 Delta gpp2 Delta pdc2 Delta overexpressing GPD1 accumulated 17 mg L-G3P/g dry weight during glucose batch fermentation under oxygen limitation. This value corresponds to an about 100-fold increase compared to that found in the wild type.     

A metal-accumulator mutant of Arabidopsis thaliana.

A mutation designated man1 (for manganese accumulator) was found to cause Arabidopsis thaliana seedlings to accumulate a range of metals. The man1 mutation segregated as a single recessive locus located on chromosome 3. When grown on soil, mutant seedlings accumulated Mn (7.5 times greater than wild type), Cu (4.6 times greater than wild type), Zn (2.8 times greater than wild type), and Mg (1.8 times greater than wild type) in leaves. In addition to these metals, the man1 mutant accumulated 2.7-fold more S in leaves, primarily in the oxidized form, than wild-type seedlings. Analysis of seedlings grown by hydroponic culture showed a similar accumulation of metals in leaves of man1 mutants. Roots of man1 mutants also accumulated metals, but unlike leaves they accumulated 10-fold more total Fe (symplasmic and apoplasmic combined) than wild-type roots. Roots of man1 mutants possessed greater (from 1.8- to 20-fold) ferric-chelate reductase activity than wild-type seedings, and this activity was not responsive to changes of Mn nutrition in either genotype. Taken together, these results suggest that the man1 mutation disrupts the regulation of metal-ion uptake or homeostasis in Arabidopsis.     

Kinetic characterization in batch and continuous culture of Escherichia coli mutants affected in phosphoenolpyruvate metabolism: differences in acetic acid production

The growth kinetics of an Escherichia coli wild type strain and two derivative mutants were examined in batch cultures and in glucose-limited chemostats. One mutant (PB12) had an inactive phosphotranferase transport system and the other (PB25) had interrupted pykA and pykF genes that code for the two pyruvate kinase isoenzymes. In both batch and continuous culture, important differences in acetic acid accumulation and other metabolic activities were found. Compared to the wild type strain, we observed a reduction in acetic acid accumulation of 25 and 80% in PB25 and PB12 strains respectively, in batch culture. Continuous culture experiments revealed that compared to the other two strains, PB25 accumulated less acetic acid as a function of dilution rate. In continuous cultures, oxidoreductase metabolic activities were substantially affected in the two mutant strains. These changes in turn were reflected in different levels of biomass and CO₂ production, and in oxygen consumption.     

Mutations in Escherichia coli that relieve catabolite repression of tryptophanase synthesis. Mutations distant from the tryptophanase gene.

Two mutants are described in which the synthesis of tryptophanase is unusually insensitive to catabolite repression. Neither mutation is linked by transduction to the tryptophane structural gene, neither mutation renders the synthesis of beta-galactosidase insensitive to catabolite repression, and the mutations do not permit tryptophanase to be synthesized in strains deficient in adenyl cyclase. During growth in glucose-minimal medium the mutants maintained a similar intracellular concentration of cyclic AMP to their wild-type parent; but since in the wild type the concentration of cyclic AMP was the same in glycerol-minimal medium as in glucose-minimal medium, it is doubtful whether catabolite repression is mediated by measurable changes in the concentration of this nucleotide.     

Characterization of a cyclic nucleotide phosphodiesterase-deficient mutant in yeast

A mutation (pde1) was detected by suppressor activity on the CYR3 mutation which caused cAMP requirement for growth at 35 degrees C by the alteration of cAMP-dependent protein kinase. The pde1 mutant produced a significantly reduced level of cyclic nucleotide phosphodiesterase activity when assayed with 500 microM cAMP. Two cyclic nucleotide phosphodiesterases, I and II, were identified. Approximate molecular weights of these enzymes were 60,000 and 110,000, and the apparent Km values were 100 and 0.4 microM, respectively. The pde1 mutant was deficient in phosphodiesterase I activity. The cells carrying the pde1 mutation accumulated several times over the intracellular cAMP found in wild type cells. Phosphodiesterase I was not essential for growth of yeast cells, but controlled the intracellular cAMP levels in wild type and various mutant strains.     

Evidence for demand-regulation of ribosome accumulation in E coli

We have determined the relative concentrations of ribosomes accumulated under different growth conditions for a number of translational mutants as well as for some natural isolates of Escherichia coli. The mutants are a tRNA modification mutant (miaA), a streptomycin resistant (SmR) and a streptomycin pseudodependent (SmP) mutant as well as two ribosome ambiguity (ram) mutants. The natural isolates used in this study are known to function with submaximal ribosome kinetics. The data show that for all the ribosome mutants the concentration of ribosomes relative to that in wild type bacteria increases when the growth rate decreases. A small increase is also seen in the natural isolates. In contrast, the miaA mutant shows no increase in ribosome concentration under the same slow growth conditions. The results suggest that bacteria with kinetically impaired ribosomes can to some extent increase the number of ribosomes accumulated under poor growth conditions in order to compensate for their slower function. We use this observation to explain in part how bacteria growing in natural environments can escape the strong selection for maximized growth rates and for optimized ribosomes that are characteristic of laboratory strains.     

枯草芽孢杆菌抗脯氨酸结构类似物突变株的筛选及突变株proBA基因的克隆和序列分析

利用亚硝基胍对枯草芽孢杆菌93151进行诱变处理,获得了耐NaCl浓度达14％的突变株,同时发现该突变株也是一个抗脯氨酸反馈抑制突变菌株,其胞内自由脯氨酸的含量随着盐浓度的提高显著增加,说明其对渗透压的耐受能力与胞内自由脯氨酸的含量紧密相关。利用PCR方法克隆突变株的proBA基因,得到一个约2.3kb的DNA片段,序列分析表明该片段含有一完整的proB基因和部分proA基因,与野生菌株的proB基因相比,突变株proB基因中有3个碱基发生了改变,其中一个碱基的变化（从起始密码子开始第781位由T→A）导致了一个氨基酸发生改变（Ser→Thr),另外两个碱基变化为沉默位点突变。将该proB基因转入大肠杆菌脯氨酸营养缺陷型菌株,能够与其功能互补。同时对部分proA基因序列分析发现,其与proB基因头尾重叠。在proA基因起始密码子上游第14个碱基处有一个类似于SD的序列,其所编码的氨基酸序列与枯草芽孢杆菌168的同源性为77％。     

Selective synthesis and labeling of the polysialic acid capsule in Escherichia coli K1 strains with mutations in nanA and neuB.

The enzymes required for polysialic acid capsule synthesis in Escherichia coli K1 are encoded by region 2 neu genes of the multigenic kps cluster. To facilitate analysis of capsule synthesis and translocation, an E. coli K1 strain with mutations in nanA and neuB, affecting sialic acid degradation and synthesis, respectively, was constructed by transduction. The acapsular phenotype of the mutant was corrected in vivo by exogenous addition of sialic acid. By blocking sialic acid degradation, the nanA mutation allows intracellular metabolite accumulation, while the neuB mutation prevents dilution by the endogenous sialic acid pool and allows capsule synthesis to be controlled experimentally by the exogenous addition of sialic acid to the growth medium. Complementation was detected by bacteriophage K1F adsorption or infectivity assays. Polysialic acid translocation was observed within 2 min after addition of sialic acid to the growth medium, demonstrating the rapidity in vivo of sialic acid transport, activation, and polymerization and translocation of polysaccharide to the cell surface. Phage adsorption was not inhibited by chloramphenicol, demonstrating that de novo protein synthesis was not required for polysialic acid synthesis or translocation at 37 degrees C. Exogenous radiolabeled sialic acid was incorporated exclusively into capsular polysaccharide. The polymeric nature of the labeled capsular material was confirmed by gel permeation chromatography and susceptibility of sialyl polymers to K1F endo-N-acylneuraminidase. The ability to experimentally manipulate capsule expression provides new approaches for investigating polysialic acid synthesis and membrane translocation mechanisms.     

An essential proline in lambda repressor is required for resistance to intracellular proteolysis

Pro78 is a solvent-exposed residue at the N-terminal end of alpha-helix 5 in the DNA binding domain of lambda repressor. Random mutagenesis experiments have suggested that Pro78 is essential [Reidhaar-Olson, J.F., & Sauer, R.T. (1990) Proteins: Struct., Funct., Genet. (in press)]. To investigate the requirement for proline at this position, we constructed and studied the properties of a set of ten position 78 mutant proteins. All of these mutants have decreased intracellular activities and are expressed at significantly lower levels than wild type. Pulse-chase experiments show that the mutant proteins are rapidly degraded in the cell; the mutants examined had half-lives of 11-35 min, whereas the wild-type protein has a half-life of greater than 10 h. The rapid degradation of position 78 mutants is not suppressed by mutations that affect known Escherichia coli proteases. The Pro78----Ala mutant could be overexpressed in a dnaJ- strain and was purified. This mutant has full DNA binding activity in vitro, suggesting that its folded structure and ability to form active dimers are similar to those of wild type. The PA78 mutant (Tm = 48 degrees C) is less thermally stable than wild type (Tm = 55 degrees C). Double-mutant studies show that this instability contributes to but is not the main cause of its rapid intracellular degradation and also suggest that proteolysis proceeds from the denatured forms of proteins containing the PA78 substitution. The PA78 mutation does not appear to introduce a new cleavage site for cellular proteases, nor does the mutation enhance susceptibility to proteases such as thermolysin and trypsin in vitro.(ABSTRACT TRUNCATED AT 250 WORDS)     

High free-methionine and decreased lignin content result from a mutation in the Arabidopsis S-adenosyl-L-methionine synthetase 3 gene

As an approach to understand the regulation of methionine (Met) metabolism, Arabidopsis Met over-accumulating mutants were isolated based on their resistance to selection by ethionine. One mutant, mto3, accumulated remarkably high levels of free Met - more than 200-fold that observed for wild type - yet showed little or no difference in the concentrations of other protein amino-acids, such as aspartate, threonine and lysine. Mutant plants did not show any visible growth differences compared with wild type, except a slight delay in germination. Genetic analysis indicated that the mto3 phenotype was caused by a single, recessive mutation. Positional cloning of this gene revealed that it was a novel S-adenosylmethionine synthetase, SAMS3. A point mutation resulting in a single amino-acid change in the ATP binding domain of SAMS3 was determined to be responsible for the mto3 phenotype. SAMS3 gene expression and total SAMS protein were not changed in mto3; however, both total SAMS activity and S-adenosylmethionine (SAM) concentration were decreased in mto3 compared with wild type. Lignin, a major metabolic sink for SAM, was decreased by 22% in mto3 compared with wild type, presumably due to the reduced supply of SAM. These results suggest that SAMS3 has a different function(s) in one carbon metabolism relative to the other members of the SAMS gene family.     

Metabolic flux analysis for a ppc mutant Escherichia coli based on 13C-labelling experiments together with enzyme activity assays and intracellular metabolite measurements

The physiology and central metabolism of a ppc mutant Escherichia coli were investigated based on the metabolic flux distribution obtained by (13)C-labelling experiments using gas chromatography-mass spectrometry (GC-MS) and 2-dimensional nuclear magnetic resonance (2D NMR) strategies together with enzyme activity assays and intracellular metabolite concentration measurements. Compared to the wild type, its ppc mutant excreted little acetate and produced less carbon dioxide at the expense of a slower growth rate and a lower glucose uptake rate. Consequently, an improvement of the biomass yield on glucose was observed in the ppc mutant. Enzyme activity measurements revealed that isocitrate lyase activity increased by more than 3-fold in the ppc mutant. Some TCA cycle enzymes such as citrate synthase, aconitase and malate dehydrogenase were also upregulated, but enzymes of glycolysis and the pentose phosphate pathway were downregulated. The intracellular intermediates in the glycolysis and the pentose phosphate pathway, therefore, accumulated, while acetyl coenzyme A and oxaloacetate concentrations decreased in the ppc mutant. The intracellular metabolic flux analysis uncovered that deletion of ppc resulted in the appearance of the glyoxylate shunt, with 18.9% of the carbon flux being channeled via the glyoxylate shunt. However, the flux of the pentose phosphate pathway significantly decreased in the ppc mutant.     

Role of the conserved arginine 274 and histidine 224 and 228 residues in the NuoCD subunit of complex I from Escherichia coli

The conserved arginine 274 and histidine 224 and 228 residues in subunit NuoCD of complex I from Escherichia coli were substituted for alanine. The wild-type and mutated NuoCD subunit was expressed on a plasmid in an E. coli strain bearing a nuoCD deletion. Complex I was fully expressed in the H224A and H228A mutants, whereas the R274A mutation yielded approximately 50% expression. Ubiquinone reductase activity of complex I was studied in membranes and with purified enzyme and was 50% and 30% of the wild-type activity in the H224A and H228A mutants, respectively. The activity of R274A was less than 5% of the wild type in membranes but 20% in purified complex I. Rolliniastatin inhibited quinone reductase activity in the mutants with similar affinity as in the wild type, indicating that the quinone-binding site was not significantly altered by the mutations. Ubiquinone-dependent superoxide production by complex I was similar to the wild type in the R274A mutant but slightly higher in the H224A and H228A mutants. The EPR spectra of purified complex I from the H224A and H228A mutants did not differ from the wild type. In contrast, the signals of the N2 cluster and another fast-relaxing [4Fe-4S] cluster, tentatively assigned as N6b, were drastically decreased in the NADH-reduced R274A mutant enzyme but reappeared on further reduction with dithionite. These findings show that the redox potential of the N2 and N6b centers is shifted to more negative values by the R274A mutation. Purified complex I was reconstituted into liposomes, and electric potential was generated across the membrane upon NADH addition in all three mutant enzymes, suggesting that none of the mutations directly affect the proton-pumping machinery.