Cloning of genes specifying carbohydrate catabolism in Pseudomonas aeruginosa and Pseudomonas putida.

A 6.0-kilobase EcoRI fragment of the Pseudomonas aeruginosa PAO chromosome containing a cluster of genes specifying carbohydrate catabolism was cloned into the multicopy plasmid pRO1769. The vector contains a unique EcoRI site for cloning within a streptomycin resistance determinant and a selectable gene encoding gentamicin resistance. Mutants of P. aeruginosa PAO transformed with the chimeric plasmid pRO1816 regained the ability to grow on glucose, and the following deficiencies in enzyme or transport activities corresponding to the specific mutations were complemented: glcT1, glucose transport and periplasmic glucose-binding protein; glcK1, glucokinase; and edd-1, 6-phosphogluconate dehydratase. Two other carbohydrate catabolic markers that are cotransducible with glcT1 and edd-1 were not complemented by plasmid pRO1816: zwf-1, glucose-6-phosphate dehydrogenase; and eda-9001, 2-keto-3-deoxy-6-phosphogluconate aldolase. However, all five of these normally inducible activities were expressed at markedly elevated basal levels when transformed cells of prototrophic strain PAO1 were grown without carbohydrate inducer. Vector plasmid pRO1769 had no effect on the expression of these activities in transformed mutant or wild-type cells. Thus, the chromosomal insert in pRO1816 contains the edd and glcK structural genes, at least one gene (glcT) that is essential for expression of the glucose active transport system, and other loci that regulate the expression of the five clustered carbohydrate catabolic genes. The insert in pRO1816 also complemented the edd-1 mutation in a glucose-negative Pseudomonas putida mutant but not the eda-1 defect in another mutant. Moreover, pRO1816 caused the expression of high specific activities of glucokinase, an enzyme that is naturally lacking in these strains of Pseudomonas putida.     

Pseudomonas aeruginosa mutants affected in anaerobic growth on arginine: evidence for a four-gene cluster encoding the arginine deiminase pathway.

Pseudomonas aeruginosa PAO was able to grow in the absence of exogenous terminal electron acceptors, provided that the medium contained 30 to 40 mM L-arginine and 0.4% yeast extract. Under strictly anaerobic conditions (O2 at less than 1 ppm), growth could be measured as an increase in protein and proceeded in a non-exponential way; arginine was largely converted to ornithine but not entirely consumed at the end of growth. In the GasPak anaerobic jar (Becton Dickinson and Co.), the wild-type strain PAO1 grew on arginine-yeast extract medium in 3 to 5 days; mutants could be isolated that were unable to grow under these conditions. All mutants (except one) were defective in at least one of the three enzymes of the arginine deiminase pathway (arcA, arcB, and arcC mutants) or in a novel function that might be involved in anaerobic arginine uptake (arcD mutants). The mutations arcA (arginine deiminase), arcB (catabolic ornithine carbamoyltransferase), arcC (carbamate kinase), and arcD were highly cotransducible and mapped in the 17-min chromosome region. Some mutations in the arc cluster led to low, noninducible levels of all three arginine deiminase pathway enzymes and thus may affect control elements required for induction of the postulated arc operon. Two fluorescent pseudomonads (P. putida and P. fluorescens) and P. mendocina, as well as one PAO mutant, possessed an inducible arginine deiminase pathway and yet were unable to grow fermentatively on arginine. The ability to use arginine-derived ATP for growth may provide P. aeruginosa with a selective advantage when oxygen and nitrate are scarce.     

Characterization and Genetic Mapping of Phosphoglucoisomerase Mutations in Mannitol-Negative Mutants of Pseudomonas aeruginosa

Phosphoglucoisomerase (pgi) mutations in a number of independently isolated mannitol-negative mutants of Pseudomonas aeruginosa PAO1 were mapped on the chromosome by plasmid FP5-mediated conjugation and by cotransduction with the generalized transducing phages G101 and F116L. Mutant allele pgi-9001 exhibited linkage to ilvB, C-9059 (46–85%), car-9003 (93–100%), and pur-9047 (70%), but not with met-9011, in FP5-mediated conjugational crosses. All known pgi mutations and several previously uncharacterized mannitol-negative mutations exhibited transductional linkage to two independent car mutations at frequencies ranging from 13% to 42% and 53% to 99%, in transductional crosses mediated by phages G101 and F116L respectively. These pgi and mannitol-negative mutations also were cotransducible at very low frequencies (<1%) with two independent ilv mutations. Cotransduction of the car and ilv loci could not be detected. These data suggest the location of pgi within the first minute of the P. aeruginosa chromosome closely linked to the car marker and probably between the ilv and car loci. All of the mannitol-negative mutations that exhibited linkage to the car and ilv loci were characterized as pgi mutations by enzyme assays. A phenotypically similar, mannitol-negative mutatant was shown to contain a mutation in glucose-6-phosphate dehydrogenase (zwf-9012) that maps to a different region on the chromosome. Received: 26 April 1996 / Accepted: 10 June 1996     

Menaquinone biosynthesis: mutants of Escherichia coli K-12 requiring 2-succinylbenzoate.

Two independent mutants of Escherichia coli K-12, selected for their inability to grow anaerobically with fumarate as the terminal electron acceptor, were shown to be deficient in menaquinone biosynthesis. In both cases, exogenously supplied 2-succinylbenzoate promoted normal anaerobic growth on a lactate plus fumarate medium. Anaerobic growth of the mutants on glucose minimal medium was impaired but could be restored to normal by adding either uracil or 2-succinylbenzoate. The addition of 2-succinylbenzoate (but not uracil) permitted the synthesis of menaquinone and demethylmenaquinone by both mutants. The menaquinone content of the parental strain grown on lactate plus fumarate was three times greater than observed after growth on glucose. Transduction studies with phage P1 showed that the two mutations are very closely linked and probably affect the same gene, menC, which is cotransducible with nalA (23%), glpT (51%), and purF (8 to 14%). The gene order nalA-nrdA-glpTA-menC-purF was indicated. The results were consistent with 2-succinylbenzoate being an intermediate in menaquinone biosynthesis and show that the gene designated menC (located at 48.65 min of the E. coli chromosome) is involved in the conversion of chorismate to 2-succinylbenzoate. It was also concluded that menaquinone is essential for electron transport to fumarate in E. coli.     

Transport of glucose, gluconate, and methyl alpha-D-glucoside by Pseudomonas aeruginosa

Glucose transport by Pseudomonas aeruginosa was studied. These studies were enhanced by the use of a mutant, strain PAO 57, which was unable to grow on glucose but which formed the inducible glucose transport system when grown in media containing glucose or other inducers such as 2-deoxy-d-glucose. Both PAO 57 and parental strain PAO transported glucose with an apparent K(m) of 7 muM. Free glucose was concentrated intracellularly by P. aeruginosa PAO 57 over 200-fold above the external level. These data constitute direct evidence that glucose is transported via active transport by P. aeruginosa. Various experimental data clearly indicated that P. aeruginosa PAO transported methyl alpha-d-glucose (alpha-MeGlc) via the glucose transport system. The apparent K(m) of alpha-MeGlc transport was 7 mM which indicated a 1,000-fold lower affinity of the glucose transport system for alpha-MeGlc than for glucose. While only unchanged alpha-MeGlc was detected intracellularly in P. aeruginosa, alpha-MeGlc was actually concentrated intracellularly less than 2-fold over the external level. Membrane vesicles of P. aeruginosa PAO retained transport activity for gluconate. This solute was concentrated intravesicularly several-fold over the external level. A component of the glucose transport system is believed to have been lost during vesicle preparation since glucose per se was not transported. Instead; glucose was converted to gluconate by membrane-associated glucose dehydrogenase and gluconate was then transported into the vesicles. Although this may constitute an alternate system for glucose transport, it is not a necessary prerequisite for glucose transport by intact cells since P. aeruginosa PAO 57, which lacks glucose dehydrogenase, was able to transport glucose at a rate equal to the parental strain.     

Cloning and nucleotide sequence of the glpD gene encoding sn-glycerol-3-phosphate dehydrogenase of Pseudomonas aeruginosa.

Nitrosoguanidine-induced Pseudomonas aeruginosa mutants which were unable to utilize glycerol as a carbon source were isolated. By utilizing PAO104, a mutant defective in glycerol transport and sn-glycerol-3-phosphate dehydrogenase (glpD), the glpD gene was cloned by a phage mini-D3112-based in vivo cloning method. The cloned gene was able to complement an Escherichia coli glpD mutant. Restriction analysis and recloning of DNA fragments located the glpD gene to a 1.6-kb EcoRI-SphI DNA fragment. In E. coli, a single 56,000-Da protein was expressed from the cloned DNA fragments. An in-frame glpD'-'lacZ translational fusion was isolated and used to determine the reading frame of glpD by sequencing across the fusion junction. The nucleotide sequence of a 1,792-bp fragment containing the glpD region was determined. The glpD gene encodes a protein containing 510 amino acids and with a predicted molecular weight of 56,150. Compared with the aerobic sn-glycerol-3-phosphate dehydrogenase from E. coli, P. aeruginosa GlpD is 56% identical and 69% similar. A similar comparison with GlpD from Bacillus subtilis reveals 21% identity and 40% similarity. A flavin-binding domain near the amino terminus which shared the consensus sequence reported for other bacterial flavoproteins was identified.     

Isolation and characterization of a Pseudomonas aeruginosa PAO mutant defective in the structural gene for the LIVAT-binding protein.

A mutant of Pseudomonas aeruginosa PAO which has a defect in the structural gene for a binding protein for leucine, isoleucine, valine, alanine, and threonine (LIVAT-binding protein) was isolated and characterized. DL-4-azaleucine was taken up via the high-affinity branched-chain amino acid transport system (LIV-I), but not via the low affinity system (LIV-II), and then inhibited the growth of P. aeruginosa cells. This finding enabled us to select mutants defective in the LIV-I transport system alone. Among such mutants, strain PAO3530 was found to produce an altered LIVAT-binding protein. The shock fluid of this strain contained a normal level of the protein which corresponded to the wild-type LIVAT-binding protein as judged by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and by an immunological test. However, the shock fluid showed almost no binding activity for branched-chain amino acids, suggesting that strain PAO3530 has a defect in the structural gene for the LIVAT-binding protein. The mutation locus (bra-310) was mapped in a region between cnu-9001 and oru-325 on the chromosome of P. aeruginosa PAO by conjugation mediated by plasmid FP5 or R68.45.     

Mapping and characterization of two mutations to antibiotic supersusceptibility in Pseudomonas aeruginosa

Two mutations associated with antibiotic supersusceptibility in Pseudomonas aeruginosa strain Z61 were transferred separately into strain PAO222, using R68.45-mediated conjugation and phage F116L transduction. One mutation (absA) was 40% contransducible with pro-82 at 26 min on the P. aeruginosa chromosome and was associated with increased susceptibility to beta-lactams, gentamicin and hydrophobic agents. Strains carrying the absA mutation also displayed enhanced uptake of a hydrophobic fluorescent probe, 1-N-phenylnaphthylamine, and were found, by SDS-PAGE, to be altered in the pattern of lipopolysaccharide O-antigen distribution. The other mutation (absB), associated with increased susceptibility to beta-lactams and gentamicin but not to hydrophobic agents, was cotransducible with met-28 and proC at 20 min on the chromosome. The absB mutation caused a structurally undefined alteration in the physical interaction of EDTA and gentamicin with the outer membrane.     

Locus of the Pseudomonas aeruginosa toxin A gene.

The gene for Pseudomonas aeruginosa toxin A has been mapped in the late region of the chromosome of strain PAO. Strain PAO-PR1, which produces parental levels of toxin A antigen that is enzymatically inactive and nontoxic, was used as the donor for R68.45 plasmid-mediated genetic exchange. Strain PAO-PR1 (toxA1) was mated with toxin A-producing strains, and exconjugates for selected prototrophic markers were tested for the transfer of toxA1. The toxA1 gene was located between cnu-9001 and pur-67 at approximately 85 min on the PAO chromosome.     

Formation of aromatic amino acid pools in Escherichia coli K-12

Phenylalanine, tyrosine, and tryptophan were taken up into cells of Escherichia coli K-12 by a general aromatic transport system. Apparent Michaelis constants for the three amino acids were 4.7 x 10(-7), 5.7 x 10(-7), and 4.0 x 10(-7)m, respectively. High concentrations (> 0.1 mm) of histidine, leucine, methionine, alanine, cysteine, and aspartic acid also had an affinity for this system. Mutants lacking the general aromatic transport system were resistant to p-fluorophenylalanine, beta-2-thienylalanine, and 5-methyltryptophan. They mapped at a locus, aroP, between leu and pan on the chromosome, being 30% cotransducible with leu and 43% cotransducible with pan. Phenylalanine, tyrosine, and tryptophan were also transported by three specific transport systems. The apparent Michaelis constants of these systems were 2.0 x 10(-6), 2.2 x 10(-6), and 3.0 x 10(-6)m, respectively. An external energy source, such as glucose, was not required for activity of either general or specific aromatic transport systems. Azide and 2,4-dinitrophenol, however, inhibited all aromatic transport, indicating that energy production is necessary. Between 80 and 90% of the trichloroacetic acid-soluble pool formed from a particular exogenous aromatic amino acid was generated by the general aromatic transport system. This contribution was abolished when uptake was inhibited by competition by the other aromatic amino acids or by mutation in aroP. Incorporation of the former amino acid into protein was not affected by the reduction in its pool size, indicating that the general aromatic transport system is not essential for the supply of external aromatic amino acids to protein synthesis.     

Isolation and characterization of catabolite repression control mutants of Pseudomonas aeruginosa PAO.

Independently controlled, inducible, catabolic genes in Pseudomonas aeruginosa are subject to strong catabolite repression control by intermediates of the tricarboxylic acid cycle. Mutants which exhibited a pleiotropic loss of catabolite repression control of multiple pathways were isolated. The mutations mapped in the 11-min region of the P. aeruginosa chromosome near argB and pyrE and were designated crc. Crc- mutants no longer showed repression of mannitol and glucose transport, glucose-6-phosphate dehydrogenase, glucokinase, Entner-Doudoroff dehydratase and aldolase, and amidase when grown in the presence of succinate plus an inducer. These activities were not expressed constitutively in Crc- mutants but exhibited wild-type inducible expression.     

Mutants of Salmonella typhimurium That Are Insensitive to Catabolite Repression of Proline Degradation

In Salmonella typhimurium the two enzymes of proline catabolism, proline oxidase and Delta(1)-pyrroline-5-carboxylic acid dehydrogenase, are subject to catabolite repression when the cells are grown in the presence of glucose. Mutants partially relieved of catabolite repression (PutR) for the proline catabolic enzymes have been isolated by selection on agar plates containing glucose and proline. The specificity of the catabolite repression-insensitive character for the enzymes of proline utilization has been confirmed by an analysis of other unrelated catabolic enzymes. Histidase and amylomaltase of the mutant strains are equally as sensitive to glucose repression as are the enzymes from the wild type. All four PutR mutants exhibit higher induced and higher basal levels of proline oxidase as compared with the corresponding wild-type levels. The mutations of three strains tested are cotransducible with constitutive, pleiotrophic-negative and structural gene mutations of the put region. Three-factor crosses indicate that two putR mutations are located at one end of the cluster of put mutations.     

Location on the Salmonella typhimurium Chromosome of the Gene Encoding Nucleoside Diphosphokinase (ndk)

The gene encoding nucleosidediphosphate kinase (ndk) was located at 55 units on the Salmonella typhimurium chromosome. The ndk locus was 83% cotransducible with hisS and 2% cotransducible with glyA in phage P22-mediated crosses. A nucleosidediphosphate kinase mutant that produced only 10% of the wild-type enzyme activity (ndk-1) grew normally and produced a heat-labile enzyme.     

Phenylarsine oxide stimulates hexose transport in 3T3-L1 adipocytes by a mechanism other than an increase in surface transporters

Phenylarsine oxide (PAO) has been shown to exert a biphasic effect on glucose transport in 3T3-L1 adipocytes. At 10 microM, PAO activates transport threefold, but at higher concentrations an inhibition of transport is observed. In this paper we report a procedure for the subcellular fractionation of these cells which we use to examine the distribution of glucose transporters following PAO challenge. Quantitative immunoblotting showed that the glucose transporter content of the plasma membrane fraction increased with increasing PAO concentrations; a parallel increase in another insulin-responsive protein, the transferrin receptor, also occurred. However, cell-surface labeling procedures for the glucose transporter and transferrin receptor showed that PAO actually decreased the cell-surface concentrations of these proteins; the basis of this discrepancy may be that in the presence of PAO, intracellular vesicles containing these proteins associate with the plasma membrane, but do not fuse with it. The possibility that PAO modulated transport by direct interaction with the glucose transporter was investigated by examining the effects of PAO on transport in both erythrocytes and a reconstituted system of purified erythrocyte transporter in lipid vesicles. PAO was without effect on the rate of transport in these systems. The hypothesis that the stimulatory effect of PAO on transport might be due to the activation of the insulin receptor kinase activity was examined by assessing the phosphotyrosine content of the receptor and other proteins using anti-phosphotyrosine antibodies. PAO alone caused no detectable increase in receptor phosphotyrosine content. However, the combination of PAO and insulin led to the tyrosine phosphorylation of two proteins of Mr 68,000 and 57,000 which were not detected in cells treated with either PAO or insulin, and an increased phosphotyrosine content of proteins of Mr 95,000 and 165,000 when compared to cells treated with insulin alone.     

Phenylarsine oxide inhibits insulin-dependent glucose transport activity in rat soleus muscles

Phenylarsine oxide (PAO) is known to block insulin-stimulated glucose transport activity in 3T3 L1 adipocytes at a post-receptor step. Herein, we demonstrate that, at right concentration, PAO also inhibits insulin activation of glucose uptake in rat soleus muscles but does not affect basal level of uptake. In control experiments, insulin stimulation of 2-deoxy-D-glucose uptake is about 400% of that of the control level. After PAO treatment, the stimulation reduces to 150% of the control. Since the intracellular level of ATP remains unchanged after PAO treatment, when measured by phosphorus-31 nmr spectroscopy, this inhibition is not due to depletion of ATP pool size. Moreover, PAO does not affect autophosphorylation of the insulin receptors purified from rat soleus muscles, implying that the PAO blockage of insulin-dependent glucose uptake in soleus muscles also may be post-receptor.     

Phenylarsine oxide stimulates a cytosolic tyrosine kinase activity and glucose transport in mouse fibroblasts

In the present report we further approach the mechanism by which insulin and phenylarsine oxide (PAO), a trivalent arsenical compound, regulate glucose transport in mouse fibroblasts (NIH3T3). First, we show that PAO is a powerful stimulatory agent on glucose transport. Second, at least three series of observations indicate that this action of PAO is not mediated through the insulin receptor: (i) the same effect of PAO is observed in NIH3T3 and in transfected cells expressing 6 x 10(6) insulin receptors, while the effect of insulin is markedly increased in the transfected cells; (ii) PAO does not affect the tyrosine phosphorylation of the insulin receptor; (iii) the tyrosine kinase activity of the insulin receptor toward exogenous substrates is not increased by PAO. Since PAO appears to act on glucose transport by a different mechanism than insulin, we have compared the effect of PAO and insulin on tyrosine phosphorylation of cellular proteins. Using Western blot analysis we did not detect common substrates in PAO- and insulin-treated cells. However, we found in cell extracts from both PAO- and insulin-treated cells a 50-kDa protein that is immunoprecipitated by antiphosphotyrosine antibody. In addition, PAO activates a cytosolic tyrosine kinase capable of poly(Glu/Tyr) phosphorylation. As a whole, our data suggest that the 50-kDa protein found in cells incubated with PAO and insulin could be the convergence point of the insulin and PAO signaling pathways.     

Regulation of the Escherichia coli methylgalactoside transport system by gene mglD.

Constitutive activity of the methylgalactoside transport system of Escherichia coli K-12 is shown to result from mutation of a genetic locus distinct from the two previously described regulatory loci for this permease. Employing an autoradiographic procedure whereby constitutive and inducible cells can be differentiated, it is demonstrated that this locus, termed mglD, is 20% cotransducible with ptsF by bacteriophage P1. Selection for constitutive mutants among an inducible population yielded cells who mutations mapped in mglD. Cotransduction of mglD with mglB, minus C, and minus A, three genes required for activity of the methylgalactoside transport system, is 95, 88, and 81%, respectively. The results of recombination studies employing three and four factors indicate that the order of genes in this region is ptsF, mglD, B, C, A.     

On the regulation of adenosine 3', 5'-monophosphate synthesis in bacteria. I. Effect of carbon source variation on cyclic AMP synthesis in Escherichia coli B/r.

1. The effect of carbon source variation in bacterial growth media on their growth rate, inducible enzyme and cyclic AMP synthesis was examined: an inverse relationship between the culture's growth rate and its differential rate of inducible enzyme (tryptophanase and beta-galactosidase), and cyclic AMP synthesis was found. 2. The effect of the culture's growth phase on its sensitivity or resistance to glucose catabolite repression was determined in the wild type and a catabolite insensitive mutant (ABDROI): the wild type's sensitivity to glucose repression was not affected, whereas the insensitivity of the mutant was found to be limited to its early logarithmic phase of growth. At late log, or stationary phase, the mutant was found to be sensitive to glucose repression. 3. Examination of the kinetics of glucose uptake by the mutant, using alpha-[1 4-C] methyl-glucoside showed evidence for two transport systems each with a different affinity to glucose. A low affinity transport system (apparent Km of 3.4-10-minus 5 M) which appears mostly at the early logarithmic phase of growth. A high affinity transport system (apparent Km of 1.2-10-minus 5 M) which appears mostly at the late log and stationary phases of growth. 4. The effect of the culture density variation on its sensitivity to glucose repression showed that sensitivity to glucose catabolic repression is primarily a reflection of the formation of an allosteric effector molecule between glucose and its specific transport molecule which in turn regulates the activity of the adenylate cyclase.     

Evidence for an Altered Operator Specificity: Catabolite Repression Control of the Leucine Operon in Salmonella typhimurium

A mutation, GD-1, in the leucine operon imposed unusual growth characteristics upon a leucine auxotrophic strain bearing the leucine operator mutation, leu-500. The strain with the GD-1 mutation was able to grow on a minimal salts medium when citrate was the sole carbon source, but required leucine when glucose was present. Tests with a large number of carbohydrates suggest that in the strain bearing the GD-1 mutation the leucine biosynthetic enzymes are under catabolite repressor control. Recombination studies indicate that the GD-1 mutation is a secondary alteration of the leucine operator at or very close to the site of the leu-500 mutation. Mutations at the supX locus (previously termed su leu 500 and located on the chromosome between the cysteine B and tryptophan gene clusters) result in elimination of the catabolite repression effect. The data are interpreted as an indication that the GD-1 and leu-500 mutations alter the leucine operator with respect to its specificity of response to repressors.     

The regulation of transport of glucose, gluconate and 2-oxogluconate and of glucose catabolism in Pseudomonas aeruginosa.

1. The induction by glucose and gluconate of the transport systems and catabolic enzymes for glucose, gluconate and 2-oxogluconate was studied with Pseudomonas aeruginosa PAO1 growing in a chemostat under conditions of nitrogen limitation with citrate as the major carbon source. 2. In the presence of a residual concentration of 30mM-citrate an inflowing glucose concentration of 6-8 mM was required to induce the glucose-transport system and associated catabolic enzymes. When the glucose concentration was raised to 20mM the glucose-transport system was repressed, but the transport system for gluconate, and at higher glucose concentrations, that for 2-oxogluconate, were induced. No repression of the glucose-catabolizing enzymes occurred at the higher inflowing glucose concentrations. 3. In the presence of 30mM-citrate no marked threshold concentration was required for the induction of the gluconate-transport system by added gluconate. 4. In the presence of 30mM-citrate and various concentrations of added glucose and gluconate, the activity of the glucose-transport system accorded with the proposal that a major factor concerned in the repression of this system was the concentration of gluconate, produced extracellularly by glucose dehydrogenase. 5. This proposal was supported by chemostat experiments with mutants defective in glucose dehydrogenase. Such mutants showed no repression of the glucose-transport system by high inflowing concentrations, but with a mutant apparently defective only in glucose dehydrogenase, the addition of gluconate caused repression of the glucose-transport system. 6. Studies with the mutants showed that both glucose and gluconate can induce the enzymes of the Entner-Doudoroff system, whereas for the induction of the gluconate-transport system glucose must be converted into gluconate.