Effects of edd and pgi Disruptions on Inosine Accumulation in Escherichia coli

Using an inosine-producing mutant of Escherichia coli, the contributions of the central carbon metabolism for overproducing inosine were investigated. Sodium gluconate instead of glucose was tested as a carbon source to increase the supply of ribose-5-phosphate through the oxidative pentose phosphate pathway. The edd (6-phosphogluconate dehydrase gene)-disrupted mutant accumulated 2.5 g/l of inosine from 48 g/l of sodium gluconate, compared with 1.4 g/l of inosine in the edd wild strain. The rpe (ribulose phosphate 3-epimerase gene)-disrupted mutant resulted in low cell growth and low inosine production on glucose and on gluconate. The disruption of pgi (glucose-6-phosphate isomerase gene) was effective for increasing the accumulation of inosine from glucose but resulted in low cell growth. The pgi-disrupted mutant accumulated 3.7 g/l of inosine from 40 g/l of glucose when 8 g/l of yeast extract was added to the medium. Furthermore, to improve effective utilization of adenine, the yicP (adenine deaminase gene)-disrupted mutant was evaluated. It showed higher inosine accumulation, of 3.7 g/l, than that of 2.8 g/l in the yicP wild strain when 4 g/l of yeast extract was added to the medium.     

Effects of xapA and guaA disruption on inosine accumulation in Escherichia coli

A xapA-disrupted mutant was studied to minimize hypoxanthine production and to improve inosine productivity in mutants of Escherichia coli. The xapA-disrupted mutant accumulated 5.6 g/l of inosine from 40 g/l of glucose, while the parent strain accumulated 4.6 g/l. This result indicates that xapA is activated in xapA-positive inosine-producers and that xapA disruption might be useful for improving inosine productivity.     

Investigation of various genotype characteristics for inosine accumulation in Escherichia coli W3110

For the derivation of an inosine-overproducing strain from the wild type microorganism, it is known that the addition of an adenine requirement, removal of purine nucleoside hydrolyzing activity, removal of the feedback inhibition, and repression of key enzymes in the purine nucleotides biosynthetic pathway are essential. Thus, the disruption of purA (adenine requirement), deoD (removal of purine nucleosides phosphorylase activity), purR (derepression of the regulation of purine nucleotides biosynthetic pathway), and the insensitivity of the feedback inhibition of phosphoribosylpyrophosphate (PRPP) amidotransferase by adenosine 5'-monophosphate (AMP) and guanosine 5'-monophosphate (GMP) were done in the Escherichia coli strain W3110, and then the inosine productivity was estimated. In the case of using a plasmid harboring the PRPP amidotransferase gene (purF) that encoded a desensitized PRPP amidotransferase, purF disrupted mutants were used as the host strains. It was found that the innovation of the four genotypes brought about a small amount of inosine accumulation. Furthermore, an adenine auxotrophic mutant of E. coli showed inappropriate adenine use because its growth could not respond efficiently to the concentration of adenine added. As the presence of adenosine deaminase is well known in E. coli and it is thought to be involved in adenine use, a mutant disrupted adenosine deaminase gene (add) was constructed and tested. The mutant, which is deficient in purF, purA, deoD, purR, and add genes, and harboring the desensitized purF as a plasmid, accumulated about 1 g of inosine per liter. Although we investigated the effects of purR disruption and purF gene improvement, unexpectedly an increase in the inosine productivity could not be found with this mutant.     

Deletion of pgi alters tryptophan biosynthesis in a genetically engineered strain of Escherichia coli.

Deletion of the structural gene for phosphoglucose isomerase (pgi) of Escherichia coli dramatically alters the path of glucose catabolism by diverting carbon into the hexose monophosphate shunt. The effect of this genetic alteration on the conversion of glucose to tryptophan by strains optimized for the biosynthesis of this amino acid was determined by using 13C-nuclear magnetic resonance spectroscopy in vivo. Pgi- strains converted glucose to tryptophan almost twice as efficiently as did their Pgi+ counterparts.     

Deletion of pgi alters tryptophan biosynthesis in a genetically engineered strain of Escherichia coli.

Deletion of the structural gene for phosphoglucose isomerase (pgi) of Escherichia coli dramatically alters the path of glucose catabolism by diverting carbon into the hexose monophosphate shunt. The effect of this genetic alteration on the conversion of glucose to tryptophan by strains optimized for the biosynthesis of this amino acid was determined by using 13C-nuclear magnetic resonance spectroscopy in vivo. Pgi- strains converted glucose to tryptophan almost twice as efficiently as did their Pgi+ counterparts.     

Monovalent cation activation in Escherichia coli inosine 5'-monophosphate dehydrogenase

Inosine 5'-monophosphate dehydrogenase (IMPDH) catalyzes the oxidation of inosine 5'-monophosphate (IMP) to xanthosine 5'-monophosphate with the concomitant reduction of NAD to NADH. Escherichia coli IMPDH is activated by K(+), Rb(+), NH(+)(4), and Cs(+). K(+) activation is inhibited by Li(+), Na(+), Ca(2+), and Mg(2+). This inhibition is competitive versus K(+) at high K(+) concentrations, noncompetitive versus IMP, and competitive versus NAD. Thus monovalent cation activation is linked to the NAD site. K(+) increases the rate constant for the pre-steady-state burst of NADH production, possibly by increasing the affinity of NAD. Three mutant IMPDHs have been identified which increase the value of K(m) for K(+): Asp13Ala, Asp50Ala, and Glu469Ala. In contrast to wild type, both Asp13Ala and Glu469Ala are activated by all cations tested. Thus these mutations eliminate cation selectivity. Both Asp13 and Glu469 appear to interact with the K(+) binding site identified in Chinese hamster IMPDH. Like wild-type IMPDH, K(+) activation of Asp50Ala is inhibited by Li(+), Na(+), Ca(2+), and Mg(2+). However, this inhibition is noncompetitive with respect to K(+) and competitive with respect to both IMP and NAD. Asp50 interacts with residues that form a rigid wall in the IMP site; disruption of this wall would be expected to decrease IMP binding, and the defect could propagate to the proposed K(+) site. Alternatively, this mutation could uncover a second monovalent cation binding site.     

Studies on the accumulation of putrescine and spermidine in Escherichia coli

The rate of accumulation of the polyamines spermidine and putrescine by E. coli depended on growth rate. Spermidine accumulation was faster in chemostat cultures with high dilution rates than in those with low dilution rates and was slower in bacteria that had been grown for several generations with either putrescine or spermidine, suggesting that the spermidine-uptake system was repressed by exogenous polyamines. The uptake of spermidine required metabolic energy. Thus accumulation occurred in an energy-starved unc strain only upon addition of glucose (or D-lactate to a smaller extent). With glucose present accumulation occurred in an unc, frd strain under anaerobic conditions, suggesting that ATP drives uptake. However, accumulation was generally sensitive to carbonylcyanide m-chlorophenylhydrazone (CCCP), indicating that the proton motive force was involved in uptake. Unlike spermidine, putrescine accumulation was faster in slow-growing than in fast-growing cultures. This may have been due to greater efflux of putrescine at faster growth rates. Accumulation of putrescine was faster following prolonged growth with either putrescine or spermidine, suggesting induction of the putrescine-uptake system by exogenous polyamines. Like spermidine accumulation, putrescine accumulation required metabolic energy. Accumulation was insensitive to CCCP and occurred only when glucose was added to energy-starved unc bacteria, suggesting that high-energy bonds may drive the uptake of putrescine.     

The copper-dependent accumulation of magnesium in Escherichia coli

Cu2(+)-induced accumulation of Mg ions by E. coli cells has been studied. The accumulation was demonstrated to take place only when the cell had endogenous energetic resources. The data obtained and their correlation with the data on Cu2+ binding by bacterial cells and Cu2(+)-dependent streptomycin accumulation allowed to conclude that copper induced nonspecific potential-dependent influx of cations into cells.     

Silver accumulation and resistance in Escherichia coli R1

E. coli R1 contains at least 2 large plasmids (83 and 77 kb) while E. coli S1 was previously cured of the 83 kb plasmid. Transformation using artificial competence, high-voltage electroporation, and plasmid mobilization experiments with the mobilizing plasmid RP4, failed to ascertain the 83 kb plasmid was responsible for Ag(+)-resistance. Silver accumulation by an Ag(+)-sensitive E. coli S1 strain was 5-fold higher than an Ag(+)-resistant E. coli R1 strain. The Ag(+)-resistant E. coli R1 strain produced 33% more H2S and 32% more intracellular acid labile SH than the Ag(+)-sensitive E. coli R1 strain when grown in the absence of AgNO3. Hydrophobic interaction chromatography revealed E. coli R1 displayed higher cell surface hydrophobicity than E. coli S1. HPLC protein analysis of cell-free extracts also revealed differences between protein fractions in E. coli R1 and S1 strains.     

Genetic basis of growth adaptation of Escherichia coli after deletion of pgi, a major metabolic gene

Bacterial survival requires adaptation to different environmental perturbations such as exposure to antibiotics, changes in temperature or oxygen levels, DNA damage, and alternative nutrient sources. During adaptation, bacteria often develop beneficial mutations that confer increased fitness in the new environment. Adaptation to the loss of a major non-essential gene product that cripples growth, however, has not been studied at the whole-genome level. We investigated the ability of Escherichia coli K-12 MG1655 to overcome the loss of phosphoglucose isomerase (pgi) by adaptively evolving ten replicates of E. coli lacking pgi for 50 days in glucose M9 minimal medium and by characterizing endpoint clones through whole-genome re-sequencing and phenotype profiling. We found that 1) the growth rates for all ten endpoint clones increased approximately 3-fold over the 50-day period; 2) two to five mutations arose during adaptation, most frequently in the NADH/NADPH transhydrogenases udhA and pntAB and in the stress-associated sigma factor rpoS; and 3) despite similar growth rates, at least three distinct endpoint phenotypes developed as defined by different rates of acetate and formate secretion. These results demonstrate that E. coli can adapt to the loss of a major metabolic gene product with only a handful of mutations and that adaptation can result in multiple, alternative phenotypes.     

Active accumulation of tetracycline by Escherichia coli

1. At low concentrations of tetracycline (10mug/ml) net accumulation of the drug by Escherichia coli cells ceased after 7-10min. 2. At higher concentrations of tetracycline (>30mug/ml) the period of net accumulation of the drug was significantly extended. 3. The efflux of tetracycline from E. coli cells transferred from medium containing 10mug of tetracycline/ml to drug-free medium was a rapid temperature-dependent process and was accelerated by 2,4-dinitrophenol. 4. As the concentration of tetracycline in the preloading phase was increased, the rate of subsequent efflux of the drug progressively declined. The efflux of drug from cells preloaded in medium containing 200mug of tetracycline/ml was negligible, although efflux was readily provoked by 2,4-dinitrophenol, by N-ethylmaleimide or by omission of glucose from the medium. 5. The initial rate of uptake of tetracycline by E. coli cells was linearly proportional to the concentration of tetracycline in the medium up to the maximum concentration of drug obtainable under the experimental conditions used (400mug/ml, 0.83mm). 6. Although N-ethylmaleimide strongly inhibited the accumulation of tetracycline by E. coli, no evidence was obtained for the direct involvement of thiol groups in the transport process. It was concluded that N-ethylmaleimide inhibited accumulation by interruption of the energy supply of the cells. 7. Osmotic shock of E. coli cells did not significantly affect the influx of tetracycline, but promoted both efflux of tetracycline and cell lysis in cells treated with a high concentration of tetracycline. 8. A study of the distribution of tetracycline among the subcellular fractions of penicillin-induced spheroplasts preincubated with various concentrations of tetracycline indicated that 60-70% of the accumulated tetracycline was in the high-speed supernatant fraction. Sephadex chromatography showed that the tetracycline of this fraction was present as the free drug. Sephadex chromatography of a detergent extract of the membrane fraction, however, indicated that a significant proportion of the tetracycline radioactivity of this fraction was apparently bound to some macromolecular component. 9. Cellulose phosphate paper chromatography of cold-acid extracts of spheroplasts preloaded with tetracycline indicated that the accumulated drug was chemically unchanged. 10. Membrane preparations isolated from osmotically lysed penicillin-induced spheroplasts showed a temperature-dependent binding of tetracycline that was not energy-dependent and was not inhibited by N-ethylmaleimide. The binding process was stimulated by omitting Mg(2+) from the medium, but conversely was profoundly inhibited by EDTA. 11. The relevance of these findings to the probable mechanism of active tetracycline accumulation by E. coli is discussed.     

The binding of inosine monophosphate to Escherichia coli carbamoyl phosphate synthetase.

Carbamoyl phosphate synthetase (CPS) from Escherichia coli catalyzes the formation of carbamoyl phosphate, which is subsequently employed in both the pyrimidine and arginine biosynthetic pathways. The reaction mechanism is known to proceed through at least three highly reactive intermediates: ammonia, carboxyphosphate, and carbamate. In keeping with the fact that the product of CPS is utilized in two competing metabolic pathways, the enzyme is highly regulated by a variety of effector molecules including potassium and ornithine, which function as activators, and UMP, which acts as an inhibitor. IMP is also known to bind to CPS but the actual effect of this ligand on the activity of the enzyme is dependent upon both temperature and assay conditions. Here we describe the three-dimensional architecture of CPS with bound IMP determined and refined to 2.1 A resolution. The nucleotide is situated at the C-terminal portion of a five-stranded parallel beta-sheet in the allosteric domain formed by Ser(937) to Lys(1073). Those amino acid side chains responsible for anchoring the nucleotide to the polypeptide chain include Lys(954), Thr(974), Thr(977), Lys(993), Asn(1015), and Thr(1017). A series of hydrogen bonds connect the IMP-binding pocket to the active site of the large subunit known to function in the phosphorylation of the unstable intermediate, carbamate. This structural analysis reveals, for the first time, the detailed manner in which CPS accommodates nucleotide monophosphate effector molecules within the allosteric domain.     

Improvement in organic solvent tolerance by double disruptions of proV and marR genes in Escherichia coli

Aims: To investigate the involvement of osmoprotectant transporters in organic solvent tolerance in Escherichia coli and to construct an E. coli strain with high organic solvent tolerance. Methods and Results: The organic solvent tolerance of ΔbetT, ΔproV, ΔproP or ΔputP single‐gene knockout mutants of E. coli K‐12 strain was examined. Among these mutants, the organic solvent tolerance of the ΔproV mutant remarkably increased compared with that of the parent strain. It has been known that a marR mutation confers tolerance on E. coli to organic solvents. A ΔproV and ΔmarR double‐gene mutant was more tolerant to organic solvents than the ΔproV or ΔmarR single‐gene mutant. The n‐hexane amount accumulated in E. coli cells was examined after incubation in an n‐hexane‐aqueous medium two‐phase system. The intracellular n‐hexane level in the ΔproV and ΔmarR double‐gene mutant was kept lower than those of the parent strain, ΔproV mutant and ΔmarR mutant. Conclusions: The organic solvent tolerance level in E. coli highly increased by dual disruption of proV and marR. Significance and Impact of the Study: This study suggests a new strategy for increasing the organic solvent tolerance level in E. coli to improve the usability of the whole‐cell biocatalysts in two‐phase systems employing organic solvents.     

Molybdenum accumulation in chlD mutants of Escherichia coli.

The content of molybdenum in wild-type and chlD cells was measured under a variety of growth conditions to determine if cells with a defective chlD gene were able to accumulate molybdenum. The chlD cells accumulated less molybdenum than wild-type cells did but concentrated molybdenum to a level at least 20-fold higher than the concentration in the culture medium. Molybdenum was present within spheroplasts of chlD cells and was not dialyzable. The chlD cells accumulated as much molybdenum as wild-type cells did when grown in medium containing 0.1 mM molybdate; thus, the capability of incorporation of molybdenum into cellular component(s) was equivalent to that of the wild type under these conditions.