Characterisation of mutants of Escherichia coli K12, selected by resistance to streptozotocin

Summary From cultures of sensitive bacteria, treated with the antibiotic streptozotocin, two classes of resistant mutants can be isolated: 1) mutants, resistant under all the conditions tested to even the highest doses of the antibiotic. These are either pleiotropicdefective, pts-mutants, or more frequently, mutants lacking a transport system (enzyme II^Nag-complex of the PEP-dependent phosphotransferase system) encoded by the gene nagE. This gene is inducible by N-acetyl-glucosamine and seems to be part of the nag operon. The transport system in question is responsible for the uptake of N-acetyl-glucosamine, of D-glucosamine and of streptozotocin; 2) conditional resistant mutants which are unable to energize or to synthesize the streptozotocin transport system under certain growth conditions but do have the transport activity under other conditions. These include a) mutants auxotrophic for amino acids, vitamins, or nucleotides, b) mutants negative or sensitive to carbohydrates in the medium, and c) mutants with defects in energy metabolism such as PEP synthesis.     

Effects of monovalent cations on derepression of phosphate transport in yeast

The effect of monovalent cations on derepression of phosphate transport was studied. It was found that ammonium, K⁺ and Rb⁺ accelerate the derepression of phosphate transport produced by glucose in yeast (Saccharomyces cerevisiae). Na⁺ and Li⁺ were ineffective in accelerating derepression; Cs⁺ produced only a minor stimulation. The concentration range of both K⁺ and NH₄⁺ that accelerated derepression was similar to that required for transport to occur. In the case of ammonium, the effects seem to depend exclusively on the so-called low-affinity transport system. The effect was strongly dependent on pH, with an optimum around 6; however, the increase in the pH of the medium did not produce in itself a high increase of the depression. Derepression was dependent on the presence of glucose, and it was very low with ethanol as substrate. The mechanism seems to depend on the ability that both K⁺ and NH₄⁺ have to decrease the membrane potential of the cell while transported, and not on the capacity to produce the alkalinization of the cell interior. In addition, the phenomenon depends on the presence of glucose as substrate, which indicates the involvement of some product of glucose metabolism in the mechanism, and possibly some relation to catabolic repression.     

Comparison of the sequences of the nagE operons from Klebsiella pneumoniae and Escherichia coli K12: enhanced variability of the enzyme IIN-acetylglucosamine in regions connecting functional domains.

Summary The nagE operon, encoding the enzyme II specific for N-acetylglucosamine (EII^Nag), and adjacent DNA from the chromosome of Klebsiella pneumoniae were sequenced and compared with the corresponding sequence from Escherichia colt K12. The deduced EII^Nag sequences differ in 72 out of 651 amino acids, the K. pneumoniae sequence being three residues longer. The amino acid differences were distributed unevenly, and were most frequent in regions connecting the three functional domains of the protein. In the nagE-nagB intergenic region, two promoter, two operator, and one CAP consensus sequence with regulatory functions were highly conserved. The nag structural genes from both species were very similar (83% DNA similarity; 89% amino acid similarity) except for frequent AT to GC exchanges in the wobble base of codons in K. pneumoniae DNA relative to the E. coli DNA.     

Analysis of the nag regulon from Escherichia coli K12 and Klebsiella pneumoniae and of its regulation

Summary Four genes, nagR, A, B and E, clustered in the nag locus of Escherichia coli K12 and Klebsiella pneumoniae, were cloned and physically mapped, and the corresponding gene products involved in amino sugar metabolism identified. Expression of the nag genes was also analysed using a series of lacZ fusions. In both bacteria, the genes are arranged in two divergent operons and controlled by a common NagR repressor. The corresponding gene nagR was found to map in the first operon together with the promoter proximal gene nagB, encoding the enzyme d-glucosamine isomerase (deaminase) (NagB) and the middle gene nagA, coding for N-acetyl-glucosamine deacetylase (NagA). Polar mutations in nagB and nagA prevent the efficient expression of nagR and cause constitutive expression of all nag genes. This includes the gene nagE encoding Enzyme II^Nag of the phosphoenolpyruvate-dependent carbohydrate phosphotransferase system (PTS), encoded in the second divergently transcribed operon. No further gene is found in this operon which in both organisms is directly adjacent to the gene glnS. It is interesting that the NagR repressor also affects the mannose PTS (genes manX, Y, Z), the second transport system involved in amino sugar uptake and phosphorylation.     

Glucosamine as carbon source for amino acid-producing Corynebacterium glutamicum

Corynebacterium glutamicum grows with a variety of carbohydrates and carbohydrate derivatives as sole carbon sources; however, growth with glucosamine has not yet been reported. We isolated a spontaneous mutant (M4) which is able to grow as fast with glucosamine as with glucose as sole carbon source. Glucosamine also served as a combined source of carbon, energy and nitrogen for the mutant strain. Characterisation of the M4 mutant revealed a significantly increased expression of the nagB gene encoding the glucosamine-6P deaminase NagB involved in degradation of glucosamine, as a consequence of a single mutation in the promoter region of the nagAB-scrB operon. Ectopic nagB overexpression verified that the activity of the NagB enzyme is in fact the growth limiting factor under these conditions. In addition, glucosamine uptake was studied, which proved to be unchanged in the wild-type and M4 mutant strains. Using specific deletion strains, we identified the PTS^Glc transport system to be responsible for glucosamine uptake in C. glutamicum. The affinity of this uptake system for glucosamine was about 40-fold lower than that for its major substrate glucose. Because of this difference in affinity, glucosamine is efficiently taken up only if external glucose is absent or present at low concentrations. C. glutamicum was also examined for its suitability to use glucosamine as substrate for biotechnological purposes. Upon overexpression of the nagB gene in suitable C. glutamicum producer strains, efficient production of both the amino acid l-lysine and the diamine putrescine from glucosamine was demonstrated.     

Metabolic Engineering of Escherichia coli for Production of Enantiomerically Pure (R)-(−)-Hydroxycarboxylic Acids

A heterologous metabolism of polyhydroxyalkanoate (PHA) biosynthesis and degradation was established in Escherichia coli by introducing the Ralstonia eutropha PHA biosynthesis operon along with the R. eutropha intracellular PHA depolymerase gene. By with this metabolically engineered E. coli, enantiomerically pure (R)-3-hydroxybutyric acid (R3HB) could be efficiently produced from glucose. By employing a two-plasmid system, developed as the PHA biosynthesis operon on a medium-copy-number plasmid and the PHA depolymerase gene on a high-copy-number plasmid, R3HB could be produced with a yield of 49.5% (85.6% of the maximum theoretical yield) from glucose. By integration of the PHA biosynthesis genes into the chromosome of E. coli and by introducing a plasmid containing the PHA depolymerase gene, R3HB could be produced without plasmid instability in the absence of antibiotics. This strategy can be used for the production of various enantiomerically pure (R)-hydroxycarboxylic acids from renewable resources.     

Investigation of the NADH/NAD+ ratio in Ralstonia eutropha using the fluorescence reporter protein Peredox

Ralstonia eutropha is a hydrogen-oxidizing (“Knallgas”) bacterium that can easily switch between heterotrophic and autotrophic metabolism to thrive in aerobic and anaerobic environments. Its versatile metabolism makes R. eutropha an attractive host for biotechnological applications, including H₂-driven production of biodegradable polymers and hydrocarbons. H₂ oxidation by R. eutropha takes place in the presence of O₂ and is mediated by four hydrogenases, which represent ideal model systems for both biohydrogen production and H₂ utilization. The so-called soluble hydrogenase (SH) couples reversibly H₂ oxidation with the reduction of NAD⁺ to NADH and has already been applied successfully in vitro and in vivo for cofactor regeneration. Thus, the interaction of the SH with the cellular NADH/NAD⁺ pool is of major interest. In this work, we applied the fluorescent biosensor Peredox to measure the [NADH]:[NAD⁺] ratio in R. eutropha cells under different metabolic conditions. The results suggest that the sensor operates close to saturation level, indicating a rather high [NADH]:[NAD⁺] ratio in aerobically grown R. eutropha cells. Furthermore, we demonstrate that multicomponent analysis of spectrally-resolved fluorescence lifetime data of the Peredox sensor response to different [NADH]:[NAD⁺] ratios represents a novel and sensitive tool to determine the redox state of cells.     

Physiological and Biochemical Characteristics and Capacity for Polyhydroxyalkanoates Synthesis in a Glucose-Utilizing Strain of Hydrogen-Oxidizing Bacteria, Ralstonia eutropha B8562

The physiological, biochemical, genetic, and cultural characteristics of the glucose-utilizing mutant strain Ralstonia eutropha B8562 were investigated in comparison with the parent strain R. eutropha B5786. The morphological, cultural, and biochemical characteristics of strain R. eutropha B8562 were similar to those of strain R. eutropha B5786. Genetic analysis revealed differences between the 16S rRNA gene sequences of these strains. The growth characteristics of the mutant using glucose as the sole carbon and energy source were comparable with those of the parent strain grown on fructose. Strain B8562 was characterized by high yields of polyhydroxyalkanoate (PHA) from different carbon sources (CO₂, fructose, and glucose). In batch culture with glucose under nitrogen limitation, PHA accumulation reached 90% of dry weight. In PHA, β-hydroxybutyrate was predominant (over 99 mol %); β-hydroxyvalerate (0.25–0.72 mol %) and β-hydroxyhexanoate (0.008–1.5 mol %) were present as minor components. The strain has prospects as a PHA producer on glucose-containing media.     

Cataracts and Microphthalmia Caused by a Gja8 Mutation in Extracellular Loop 2

The mouse semi-dominant Nm2249 mutation displays variable cataracts in heterozygous mice and smaller lenses with severe cataracts in homozygous mice. This mutation is caused by a Gja8^R205G point mutation in the second extracellular loop of the Cx50 (or α8 connexin) protein. Immunohistological data reveal that Cx50-R205G mutant proteins and endogenous wild-type Cx46 (or α3 connexin) proteins form diffuse tiny spots rather than typical punctate signals of normal gap junctions in the lens. The level of phosphorylated Cx46 proteins is decreased in Gja8^R205G/R205G mutant lenses. Genetic analysis reveals that the Cx50-R205G mutation needs the presence of wild-type Cx46 to disrupt lens peripheral fibers and epithelial cells. Electrophysiological data in Xenopus oocytes reveal that Cx50-R205G mutant proteins block channel function of gap junctions composed of wild-type Cx50, but only affect the gating of wild-type Cx46 channels. Both genetic and electrophysiological results suggest that Cx50-R205G mutant proteins alone are unable to form functional channels. These findings imply that the Gja8^R205G mutation differentially impairs the functions of Cx50 and Cx46 to cause cataracts, small lenses and microphthalmia. The Gja8^R205G mutation occurs at the same conserved residue as the human GJA8^R198W mutation. This work provides molecular insights to understand the cataract and microphthalmia/microcornea phenotype caused by Gja8 mutations in mice and humans.     

Metabolite profiles of polyhydroxyalkanoate-producing Ralstonia eutropha H16

This study describes metabolite profiles of Ralstonia eutropha H16 focusing on biosynthesis of polyhydroxyalkanoates (PHAs), bacterial polyesters attracted as biodegradable bio-based plastics. As CoA-thioesters are important intermediates in PHA biosynthesis, four kinds of acyl-CoAs with medium chain length were prepared and used to establish analytical conditions for capillary electrophoresis-electron spray ionization-tandem mass spectrometry (CE–ESI-MS/MS). Metabolites were extracted from R. eutropha cells in growth, PHA production, and stationary phases on fructose and PHA production phase on octanoate, and subjected to stable isotope dilution-based comparative quantification by multiple reaction monitoring using CE–ESI-MS/MS and ¹³C-labeled metabolites prepared by extraction from R. eutropha mutant grown on U-¹³C₆-glucose. This procedure allowed to quantify relative changes of 94 ionic metabolites including CoA-thioesters. Hexose-phosphates except for glucose 1-phosphate were decreased in the PHA production phase than in the growth phase, suggesting reduced flux of sugar degradation after the cell growth. Several intermediates in TCA cycle and gluconeogenesis were increased in the PHA production phase on octanoate. Interestingly, ribulose 1,5-bisphosphate were detected in all the samples examined, raising possibilities of CO₂ fixation by Calvin–Benson–Bassham cycle in this bacterium even under heterotrophic growth conditions. Turnover of acyl moieties through β-oxidation was suggested to be active on fructose, as CoA-thioesters of C₆ and C₈ were detected in the fructose-grown cells. In addition, major metabolic pools in R. eutropha cells were estimated from the signal intensities. The results of the present study provided new insights into global metabolisms in PHA-producing R. eutropha.     

Different UmuC requirements for generation of different kinds of UV-induced mutations in Escherichia coli

An Escherichia coli strain bearing the dnaQ49 mutation, which results in a defective s subunit of DNA polymerase III, and carrying the lexA71 mutation, which causes derepression of the SOS regulon, is totally unable to maintain high-copy-number plasmids containing the umuDC operon. The strain is also unable to maintain the pAN4 plasmid containing a partial deletion of the umuD gene but retaining the wild-type umuC gene. These results suggest that a high cellular level of UmuC is exceptionally harmful to the defective DNA polymerase III of the dnaQ49 mutant. We have used this finding as a basis for selection of new plasmid umuC mutants. The properties of two such mutants, bearing the umuC61 or umuC95 mutation, are described in detail. In the umuC122:: Tn 5 strain harbouring the mutant plasmids, UV-induced mutagenesis is severely decreased compared to that observed with the parental umuDC ⁺ plasmid. Interestingly, while the frequency of UV-induced GC → AT transitions is greatly reduced, the frequency of AT → TA transversions is not affected. Both mutant plasmids bear frameshift mutations within the same run of seven A residues present in umuC ⁺; in umuC61 the run is shortened to six A whereas in umuC95 is lengthened to eight A. We have found in both umuC61 and umuC95 that translation is partially restored to the proper reading frame. We propose that under conditions of limiting amounts of UmuC, the protein preferentially facilitates processing of only some kinds of UV-induced lesions.     

The rectification control and physiological relevance of potassium channel OsAKT2

AKT2 potassium (K⁺) channels are members of the plant Shaker family which mediate dual-directional K⁺ transport with weak voltage-dependency. Here we show that OsAKT2 of rice (Oryza sativa) functions mainly as an inward rectifier with strong voltage-dependency and acutely suppressed outward activity. This is attributed to the presence of a unique K191 residue in the S4 domain. The typical bi-directional leak-like property was restored by a single K191R mutation, indicating that this functional distinction is an intrinsic characteristic of OsAKT2. Furthermore, the opposite R195K mutation of AtAKT2 changed the channel to an inward-rectifier similar to OsAKT2. OsAKT2 was modulated by OsCBL1/OsCIPK23, evoking the outward activity and diminishing the inward current. The physiological relevance in relation to the rectification diversity of OsAKT2 was addressed by functional assembly in the Arabidopsis (Arabidopsis thaliana) akt2 mutant. Overexpression (OE) of OsAKT2 complemented the K⁺ deficiency in the phloem sap and leaves of the mutant plants but did not significantly contribute to the transport of sugars. However, the expression of OsAKT2-K191R overcame both the shortage of phloem K⁺ and sucrose of the akt2 mutant, which was comparable to the effects of the OE of AtAKT2, while the expression of the inward mutation AtAKT2-R195K resembled the effects of OsAKT2. Additionally, OE of OsAKT2 ameliorated the salt tolerance of Arabidopsis.

The presence of a unique K191 residue retains the activity of rice potassium channel OsAKT2 mainly as an inward rectifier (Mode I) that emphasizes its in planta role of phloem K⁺ translocation.     

Genetic Analysis Reveals the Essential Role of Nitrogen Phosphotransferase System Components in Sinorhizobium fredii CCBAU 45436 Symbioses with Soybean and Pigeonpea Plants

The nitrogen phosphotransferase system (PTS^Ntr) consists of EI^Ntr, NPr, and EIIA^Ntr. The active phosphate moiety derived from phosphoenolpyruvate is transferred through EI^Ntr and NPr to EIIA^Ntr. Sinorhizobium fredii can establish a nitrogen-fixing symbiosis with the legume crops soybean (as determinate nodules) and pigeonpea (as indeterminate nodules). In this study, S. fredii strains with mutations in ptsP and ptsO (encoding EI^Ntr and NPr, respectively) formed ineffective nodules on soybeans, while a strain with a ptsN mutation (encoding EIIA^Ntr) was not defective in symbiosis with soybeans. Notable reductions in the numbers of bacteroids within each symbiosome and of poly-β-hydroxybutyrate granules in bacteroids were observed in nodules infected by the ptsP or ptsO mutant strains but not in those infected with the ptsN mutant strain. However, these defects of the ptsP and ptsO mutant strains were recovered in ptsP ptsN and ptsO ptsN double-mutant strains, implying a negative role of unphosphorylated EIIA^Ntr in symbiosis. Moreover, the symbiotic defect of the ptsP mutant was also recovered by expressing EI^Ntr with or without the GAF domain, indicating that the putative glutamine-sensing domain GAF is dispensable in symbiotic interactions. The critical role of PTS^Ntr in symbiosis was also observed when related PTS^Ntr mutant strains of S. fredii were inoculated on pigeonpea plants. Furthermore, nodule occupancy and carbon utilization tests suggested that multiple outputs could be derived from components of PTS^Ntr in addition to the negative role of unphosphorylated EIIA^Ntr.     

A direct comparison of approaches for increasing carbon flow to aromatic biosynthesis inEscherichia coli

Different approaches to increasing carbon commitment to aromatic amino acid biosynthesis were compared in isogenic strains ofEscherichia coli. In a strain having a wild-type PEP: glucose phosphotransferase (PTS) system, inactivation of the genes encoding pyruvate kinase (pykA andpykF) resulted in a 3.4-fold increase in carbon flow to aromatic biosynthesis. In a strain already having increased carbon flow to aromatics by virtue of overexpression of thetktA gene (encoding transketolase), thepykA and/orpykF mutations had no effect. A PTS glucose⁺ mutant showed a 1.6-fold increase in carbon flow to aromatics compared to the PTS⁺ control strain. In the PTS^– glucose⁺ host background, overexpression oftktA caused a further 3.7-fold increase in carbon flow, while inactivation ofpykA andpykF caused a 5.8-fold increase. When all of the variables tested (PTS^– glucose⁺,pykA, pykF, and overexpressedtktA) were combined in a single strain, a 19.9-fold increase in carbon commitment to aromatic biosynthesis was achieved.     

A new shuttle vector for gene expression in biopolymer-producing Ralstonia eutropha

Ralstonia eutropha (formerly Alcaligenes eutrophus) is a fascinating microorganism with a great scientific importance and an immense commercial potential. A new genetic transformation system for the organism would greatly facilitate the biological study and molecular engineering of this organism. We report here a versatile gene expression method for the genetic engineering of R. eutropha. This method, based on a simplified electroporation protocol, uses a recombinant plasmid, pBS29-P2, containing a Pseudomonas syringae promoter (P2) and two antibiotic-resistance markers (i.e., genes coding for kanamycin (Km)- and tetracycline (Tc)-resistance). Using this method, we successfully achieved transformation of wild-type R. eutropha and its poly(hydroxyalkanoate)-negative mutant, R. eutropha PHB⁻4, with various pBS29-P2-based recombinants. A transformation frequency as high as 4 × 10³ Km-resistance colonies/μg DNA was obtained per electroporation experiment. We further demonstrated the successful expression of a heterologous gene coding for green-fluorescent-protein by fluorescence measurement. In addition, our results indicated the expression of a truncated but active Streptomyces coelicolor α-galactosidase in R. eutropha.