Escherichia coli K-12 Mutants Altered in the Transport Systems for Oligo- and Dipeptides

Two mutants of Escherichia coli K-12, defective in the oligopeptide and dipeptide transport system, are described. A mutant defective in the oligopeptide transport system (opp-1) was isolated as resistant to the inhibitory action of triornithine; this mutant is also resistant to glycylglycylvaline and does not concentrate (14)C-glycylglycylglycine, although it is still as sensitive as the parental strain to glycylvaline and valine. Starting from the opp-1 strain, a mutant defective also in the dipeptide transport system (dpp-1) was isolated; this mutant is resistant to the inhibitory action of glycylvaline, valylleucine, and leucylvaline and does not concentrate (14)C-glycylglycine, although it is still as sensitive as the parental strain to valine. The apparent kinetic constants for oligopeptide and dipeptide transport were measured. The opp marker is co-transducible with trp at 27 min on the E. coli genetic map. The dpp locus is separated from opp and is located between proC (10 min) and opp.     

Identification of a new gene, molR, essential for utilization of molybdate by Escherichia coli.

A mutation in a new gene, molR, prevented the synthesis in Escherichia coli of molybdoenzymes, including the two formate dehydrogenase isoenzymes, nitrate reductase and trimethylamine-N-oxide reductase. This phenotype was suppressed by supplementing the media with molybdate. Thus, the molR mutant was phenotypically similar to previously described chlD mutants, thought to be defective in molybdate transport. The molR gene is located at 65.3 min in the E. coli chromosome, in contrast to the chlD gene, which maps at 17 min and thus can be readily distinguished. The molR gene is also cotransducible with a hitherto unidentified gene essential for the production of 2-oxoglutarate from isocitrate, designated icdB (located at 66 min). The molR mutant strain SE1100 also failed to produce the hydrogenase component of formate hydrogenlyase (HYD3) in molybdate-unsupplemented media. The amount of molybdate required by strain SE1100 for the production of parental levels of formate hydrogenlyase activity was dependent on the growth medium. In Luria-Bertani medium, this value was about 100 microM, and in glucose-minimal medium, 1.0 microM was sufficient. In low-sulfur medium, this value decreased to about 50 nM. The addition of sulfate or selenite increased the amount of molybdate needed for the production of formate hydrogenlyase activity. These data suggest that in the absence of the high-affinity molybdate transport system, E. coli utilizes sulfate and selenite transport systems for transporting molybdate, preferring sulfate transport over the selenite transport system.     

Neutral amino acid transport in Pseudomonas fluorescens

Membrane transport of beta-alanine, l-alanine, and l-proline was studied in a beta-alanine transaminaseless mutant (strain 67) of Pseudomonas fluorescens. In this mutant beta-alanine is metabolically inert, and it was therefore possible to demonstrate active transport of this substrate in the absence of intracellular catabolism. The permease which catalyzes the uptake of beta-alanine also transports l-proline and l-alanine. This common transport system was distinguished from permeases which transport only l-alanine and only l-proline by competition studies in strain 67 and by studies of transport specificity in a permeaseless mutant (strain 67/4MTR).     

Genetic and Metabolic Controls for Sulfate Metabolism in Neurospora crassa: Isolation and Study of Chromate-Resistant and Sulfate Transport-Negative Mutants

Mutants of Neurospora resistant to chromate were selected and all were found to map at a single genetic locus designated as cys-13. The chromate-resistant mutants grow at a wild-type rate on minimal media but are partially deficient in the transport of inorganic sulfate, especially during the conidial stage. An unlinked mutant, cys-14, is sensitive to chromate but transports sulfate during the mycelial stage at only 25% of the wild-type rate; cys-14 also grows at a fully wild-type rate on minimal media. The double-mutant strain, cys-13;cys-14, cannot utilize inorganic sulfate for growth and completely lacks the capacity to transport this anion. The only biochemical lesion that has been detected for the double-mutant strain is its loss in capacity for sulfate transport. Neurospora appears to possess two distinct sulfate permease species encoded by separate genetic loci. The transport system (permease I) encoded by cys-13 predominates in the conidial stage and is replaced by sulfate permease II, encoded by the cys-14 locus, during outgrowth into the mycelial phase. The relationship of these new mutants to cys-3, a regulatory gene that appears to control their expression, is discussed.     

Basic Amino Acid Transport in Escherichia coli: Properties of Canavanine-Resistant Mutants

A mutant of Escherichia coli strain CanR 22 has been isolated which is resistant to growth inhibition by canavanine, an analogue of arginine. The properties of this strain and of another canavanine-resistant mutant, JC182-5 (isolated by Celis et al. [5]), were studied. The mutation is pleiotropic in that it results in a reduction in the activity of two distinct permeases, the arginine-specific and lysine-arginine-ornithine transport systems. The lesion maps at min 56 of the E. coli linkage map, at or near the argP locus. Although strain CanR 22 excretes arginine, this excretion appears to result from reduced ability to concentrate arginine, rather than the loss of transport ability being the result of excretion. This conclusion is based on findings with a canavanine-resistant strain auxotrophic for arginine, which exhibits transport properties similar to those of the prototrophic strains. Additionally, growth in the presence of arginine or ornithine results in a repression of the activity of the two basic amino acid transport systems. Neither the arginine-specific nor the lysine-arginine-ornithine binding proteins of the mutant cells show significant alterations in terms of amount, physical properties, or kinetic parameters. These observations lead to the proposal of a model for the two basic amino acid transport systems in which two carrier proteins with different specificities interact with a common energy coupling mechanism. A lesion in the gene (or one of the genes) for this coupling mechanism can confer canavanine resistance.     

Biochemical characterization of a thialysine-resistant clone of CHO cells

The intracellular transport and the activation of lysine, thialysine and selenalysine have been investigated in a thialysine-resistant CHO cell mutant strain in comparison with the parental strain. The cationic amino acid transport system responsible for the transport of these 3 amino acids shows no differences between the 2 strains as regards its affinity for each of these amino acids. On the other hand the Vmax of the transport system in the mutant is about double that in the parental strain. The lysyl-tRNA synthetase, assayed both as ATP = PPi exchange reaction and lysyl-tRNA synthesis, shows a lower affinity for thialysine and selenalysine than for lysine in both strains; in the mutant, however, the difference is even greater. Thus the thialysine resistance of the mutant is mainly due to the properties of its lysyl-tRNA synthetase, which shows a greater difference of the affinities for lysine and thialysine with respect to the parental strain.     

The pyocin Sa receptor of Pseudomonas aeruginosa is associated with ferripyoverdin uptake.

We have used Tn5 mutagenesis to obtain a mutant resistant to pyocin Sa. When grown in iron-deficient succinate medium this mutant lacked an 85-kDa iron-regulated outer membrane protein (IROMP), and expression of a 75-kDa IROMP was increased compared with that in the parent strain. The mutant was deficient in pyoverdin biosynthesis and showed a 95% decrease in transport of ferripyoverdin purified from the parent strain, suggesting that the 85-kDa IROMP is the specific receptor for ferripyoverdin and pyocin Sa. The mutant compensated for the deficiency in pyoverdin biosynthesis and transport by exhibiting a fourfold increase in ferripyochelin transport. The low-level transport of ferripyoverdin in the Sa-resistant mutant, which extended to heterologous pyoverdins from other strains, suggests that Pseudomonas aeruginosa has a second ferripyoverdin uptake system of lower affinity and broader specificity.     

Inactivation of the Escherichia coli K-12 twin-arginine translocation system promotes increased hydrogen production

The effect of deleting the genes encoding the twin-arginine translocation (Tat) system on H2 production by Escherichia coli strain MC4100 and its formate hydrogenlyase upregulated mutant (DeltahycA) was investigated. H2 evolution tests using two mutant strains defective in Tat transport (DeltatatC and DeltatatA-E) showed that the rate doubled from 0.88+/-0.28 mL H2 mg dry weight-1 L culture-1 in the parental strain, to 1.70+/-0.15 and 1.75+/-0.18 mL H2 mg dry weight-1 L culture-1, respectively, in the DeltatatC and DeltatatA-E strains. This increase was comparable to that of a previously characterized hydrogen over-producing E. coli strain carrying a DeltahycA allele. Construction of a tatC, DeltahycA double deletion strain did not increase hydrogen production further. Inactivation of the Tat system prevents correct assembly of the uptake hydrogenases and formate dehydrogenases in the cytoplasmic membrane and it is postulated that the subsequent loss of basal levels of respiratory-linked hydrogen and formate oxidation accounts for the observed increases in formate-dependent hydrogen evolution.     

Sugar uptake and sensitivity to carbon catabolite regulation in Streptomyces peucetius var. caesius

Streptomyces peucetius var. caesius produces a family of secondary metabolites called anthracyclines. Production of these compounds is negatively affected in the presence of glucose, galactose, and lactose, but the greatest effect is observed under conditions of excess glucose. Other carbon sources, such as arabinose or glutamate, show either no effect or stimulate production. Among the carbon sources that negatively affect anthracycline production, glucose is consumed in greater concentrations. We determined glucose and galactose transport in S. peucetius var. caesius and in a mutant of this strain whose anthracycline production is insensitive to carbon catabolite repression (CCR). In the original strain, incorporation of glucose and galactose was stimulated when the microorganism was grown in media containing these sugars, although we also observed basal galactose incorporation. Both the induced and the basal incorporation of galactose were suppressed when the microorganism was grown in the presence of glucose. Furthermore, adding glucose directly during the transport assay also inhibited galactose incorporation. In the mutant strain, we observed a reduction in both glucose (48%) and galactose (81%) incorporation compared to the original. Galactose transport in this mutant showed reduced sensitivity to the negative effect of glucose; however, it was still sensitive to inhibition. The deficient transport of these sugars, as well as CCR sensitivity to glucose in this mutant was corrected when the mutant was transformed with the SCO2127 region of the Streptomyces coelicolor genome. Our results support a role for glucose as the most easily utilized carbon source capable of exerting the greatest repression on anthracycline biosynthesis. In consequence, glucose also prevented the repressive effect of galactose by suppressing its incorporation. This suggests the participation of an integral regulatory system, which is initiated by an increase in incorporation of repressive sugars and their metabolism as a prerequisite for establishing the phenomenon of CCR in S. peucetius var. caesius.     

Isolation of a fosfomycin-hypersensitive mutant and production of α-d-glucose-l-phosphate from Bacillus sp. BA-3796 screened by its use

The development of a very sensitive and highly specific screening method for detection of new cell wall inhibitors of the fosfomycin type is described. A fosfomycin-hypersensitive mutant, f-360, was isolated from Staphylococcus aureus Newman by selection with fosfomycin, an antibiotic that inhibits synthesis of the bacterial cell wall. The mutant f-360 was 50-fold more sensitive than the parent strain to fosfomycin. The mutant was constitutive for the hexose phosphate transport system. Using the organism in screening, BA-3796, which had an antibacterial activity against mutant f-360 was found to be produced by a bacterium designated Bacillus sp. BA-3796. Starch and beef extract were the most suitable carbon and nitrogen sources for BA-3796 production and the amount of BA-3796 reached 3 g/l at a maximum level. The purified BA-3796 was identified as α-d-glucose-l-phosphate by its various physiochemical properties. α-d-Glucose-1-phosphate showes an antibacterial activity against Staphylococci in the presence of a slight amount pf α-d-glucose-6-phosphate.     

Molybdate transport and its effect on nitrogen utilization in the cyanobacterium Anabaena variabilis ATCC 29413

Molybdenum is an essential component of the cofactors of many metalloenzymes including nitrate reductase and Mo-nitrogenase. The cyanobacterium Anabaena variabilis ATCC 29413 uses nitrate and atmospheric N2 as sources of nitrogen for growth. Two of the three nitrogenases in this strain are Mo-dependent enzymes, as is nitrate reductase; thus, transport of molybdate is important for growth of this strain. High-affinity transport of molybdate in A. variabilis was mediated by an ABC-type transport system encoded by the products of modA and modBC. The modBC gene comprised a fused orf including components corresponding to modB and modC of Escherichia coli. The deduced ModC part of the fused gene lacked a recognizable molybdate-binding domain. Expression of modA and modBC was induced by starvation for molybdate. Mutants in modA or modBC were unable to grow using nitrate or Mo-nitrogenase. Growth using the alternative V-nitrogenase was not impaired in the mutants. A high concentration of molybdate (10 microM) supported normal growth of the modBC mutant using the Nif1 Mo-nitrogenase, indicating that there was a low-affinity molybdate transport system in this strain. The modBC mutant did not detectably transport low concentrations of 99Mo (molybdate), but did transport high concentrations. However, such transport was observed only after cells were starved for sulphate, suggesting that an inducible sulphate transport system might also serve as a low-affinity molybdate transport system in this strain.     

Engineering Lactococcus lactis for production of mannitol: high yields from food-grade strains deficient in lactate dehydrogenase and the mannitol transport system

Mannitol is a sugar polyol claimed to have health-promoting properties. A mannitol-producing strain of Lactococcus lactis was obtained by disruption of two genes of the phosphoenolpyruvate (PEP)-mannitol phosphotransferase system (PTS(Mtl)). Genes mtlA and mtlF were independently deleted by double-crossover recombination in strain L. lactis FI9630 (a food-grade lactate dehydrogenase-deficient strain derived from MG1363), yielding two mutant (Delta ldh Delta mtlA and Delta ldh Delta mtlF) strains. The new strains, FI10091 and FI10089, respectively, do not possess any selection marker and are suitable for use in the food industry. The metabolism of glucose in nongrowing cell suspensions of the mutant strains was characterized by in vivo (13)C-nuclear magnetic resonance. The intermediate metabolite, mannitol-1-phosphate, accumulated intracellularly to high levels (up to 76 mM). Mannitol was a major end product, one-third of glucose being converted to this hexitol. The double mutants, in contrast to the parent strain, were unable to utilize mannitol even after glucose depletion, showing that mannitol was taken up exclusively by PEP-PTS(Mtl). Disruption of this system completely blocked mannitol transport in L. lactis, as intended. In addition to mannitol, approximately equimolar amounts of ethanol, 2,3-butanediol, and lactate were produced. A mixed-acid fermentation (formate, ethanol, and acetate) was also observed during growth under controlled conditions of pH and temperature, but mannitol production was low. The reasons for the alteration in the pattern of end products under nongrowing and growing conditions are discussed, and strategies to improve mannitol production during growth are proposed.     

A mutation affecting a second component of the F0 portion of the magnesium ion-stimulated adenosine triphosphatase of Escherichia coli K12. The uncC424 allele.

A new mutant strain of Escherichia coli in which phosphorylation is uncoupled from electron transport was isolated. The new mutant strain has a similar phenotype to the uncB mutant described previously; results from reconstitution experiments in vitro indicate that the new mutation also affects a component of the F0 portion of the Mg2+-stimulated adenosine triphosphatase. A method was developed to incorporate mutant unc alleles into plasmids. Partial diploid strains were prepared in which the uncB402 allele was incorporated into the plasmid and the new unc mutation into the chromosome, or vice versa. Complementation between the mutant unc alleles was indicated by growth on succinate, growth yields on glucose, ATP-dependent transhydrogenase activities, ATP-induced atebrin-fluorescence quenching and oxidative-phosphorylation measurements. The gene in which the new mutation occurs is therefore distinct from the uncB gene, and the mutant allele was designated uncC424.     

Cyanate is transported by the nitrate permease in Azotobacter chroococcum

Abstract Azotobacter chroococcum cells exhibiting the capacity to take up nitrate actively could transport [¹⁴C]cyanate. This activity was dependent on the nitrogen source present in the culture medium, ammonium acting as a repressor and nitrate as an inducer. The uptake of cyanate required metabolic energy and was absent from A. Chroococcum TR1, a mutant strain lacking the nitrate transport system, but was present at wild-type levels in A. chroococcum E4, a mutant strain deficient in nitrate reductase. These results show that cyanate is transported by the nitrate permease in A. chroococcum and therefore [¹⁴C]cyanate may be useful as a nitrate analogue for studies on nitrate transport.     

Glutamate transport in membrane vesicles of the wild-type strain and glutamate-utilizing mutants of Escherichia coli.

A highly specific energy-dependent glutamate transport system was demonstrated in membrane vesicles of glutamate-utilizing Escherichia coli K-12 mutants. The glutamate transport activity of membranes from the parent strain, unable to grow on glutamate, was very low. With ascorbate-phenazine methosulfate as the electron donor, mutant preparations displayed 17 to 20 times higher activity than did the wild type. However, the affinity of the mutant carrier for L-glutamate remained the same as in the parent strain. Comparative inhibition analysis of glutamate transport in whole cells and membrane vesicles and of in vitro binding of glutamate to a specific periplasmic-binding protein suggests that under certain conditions the latter may be a component of the E. coli K-12 glutamate transport system.     

Astaxanthin production by a highly photosensitive Haematococcus mutant

Haematococcus pluvialis synthesizes a high yield of astaxanthin using CO₂ in a photoautotrophic culture without contaminant heterotrophs; however, it takes too long to induce astaxanthin production. In this study, a highly photosensitive mutant strain was attained by conventional random mutagenesis and an efficient isolation method to shorten induction time. Sensitivity to photoinhibition in this mutant was raised by a partial lesion in the photosystem II (PSII) of photosynthesis, thereby prompting a change in cellular morphology as well as stimulating carotenogenesis (astaxanthin production). As a result, the concentrations of cell biomass and astaxanthin were dramatically increased by 27% and 62% under strong light and 79% and 153% under moderate light, respectively. This Haematococcus mutant would be useful for the economical astaxanthin production capable of reducing the light energy cost in a photoautotrophic culture system, even in areas with insufficient sunlight.     

Depression of uracil uptake by ammonium in Neurospora crassa.

The mechanism of uracil uptake and one aspect of its regulation were studied in germinated conidia of Neurospora crassa. Uracil was found to be taken up by a transport mechanism that did not exhibit Michaelis-Menten kinetics. Rather, the kinetic patterns indicated two separate systems or a single transport mechanism with negative cooperativity. Cytosine and thymine inhibited uracil uptake, but uridine did not. The mutant strain uc-5-pyr-1, which failed to transport uracil, was used in reversion studies and to map the uc-5 locus. Spontaneous reversion rates at the uc-5 locus were found to be approximately 2 x 10(-8), indicating that the uc-5 lesion results from a single mutation. Loss of the uracil transport function through a single mutation favors the model of a single transport mechanism with negative cooperativity. Uracil uptake was significantly decreased in the presence of NH 4+, and evidence is presented for repression by NH4+ of a uracil transport system. Growth rates of pyrimidine-requiring and wild-type strains measured in the presence and absence of NH4+, with uracil as the pyrimidine supplement, showed that NH4+ decreased the growth rates of the pyrimidine-requiring strains significantly, while having no effect on wild-type growth rates.