Genes involved in the uptake and catabolism of gluconate by Escherichia coli.

The isolation and properties of a mutant of Escherichia coli K12 that is totally unable to take up and utilize gluconate are described. Genetical analysis shows this phenotype to be associated with two lesions. One phenotype, designated GntM-, is the result of a mutation in a gene co-transducible with malA; the other, designated GNTS-, is the result of a mutation in a gene (GntS) co-transducible with fdp. The GntS--phenotype differs little from that of wild-type cells, but GntM- GntS+ organisms grow on gluconate only after a prolonged lag and form a gluconate uptake system that is strongly repressed by pyruvate. Moreover, such GntM- mutants readily give rise to further mutants that form a gluconate uptake system, gluconate kinase and 6-phosphogluconate dehydratase consititutively; in partial diploids, this constitutivity is recessive to the inducible character. It is postulated that the GntM- phenotype is due to malfunction of a negative control gene gntR, and that gntS+ specifies the activity of a gluconate uptake system.     

ExbBD-dependent transport of maltodextrins through the novel MalA protein across the outer membrane of Caulobacter crescentus

Analysis of the genome sequence of Caulobacter crescentus predicts 67 TonB-dependent outer membrane proteins. To demonstrate that among them are proteins that transport nutrients other than chelated Fe(3+) and vitamin B(12)-the substrates hitherto known to be transported by TonB-dependent transporters-the outer membrane protein profile of cells grown on different substrates was determined by two-dimensional electrophoresis. Maltose induced the synthesis of a hitherto unknown 99.5-kDa protein, designated here as MalA, encoded by the cc2287 genomic locus. MalA mediated growth on maltodextrins and transported [(14)C]maltodextrins from [(14)C]maltose to [(14)C]maltopentaose. [(14)C]maltose transport showed biphasic kinetics, with a fast initial rate and a slower second rate. The initial transport had a K(d) of 0.2 microM, while the second transport had a K(d) of 5 microM. It is proposed that the fast rate reflects binding to MalA and the second rate reflects transport into the cells. Energy depletion of cells by 100 microM carbonyl cyanide 3-chlorophenylhydrazone abolished maltose binding and transport. Deletion of the malA gene diminished maltose transport to 1% of the wild-type malA strain and impaired transport of the larger maltodextrins. The malA mutant was unable to grow on maltodextrins larger than maltotetraose. Deletion of two C. crescentus genes homologous to the exbB exbD genes of Escherichia coli abolished [(14)C]maltodextrin binding and transport and growth on maltodextrins larger than maltotetraose. These mutants also showed impaired growth on Fe(3+)-rhodotorulate as the sole iron source, which provided evidence of energy-coupled transport. Unexpectedly, a deletion mutant of a tonB homolog transported maltose at the wild-type rate and grew on all maltodextrins tested. Since Fe(3+)-rhodotorulate served as an iron source for the tonB mutant, an additional gene encoding a protein with a TonB function is postulated. Permeation of maltose and maltotriose through the outer membrane of the C. crescentus malA mutant was slower than permeation through the outer membrane of an E. coli lamB mutant, which suggests a low porin activity in C. crescentus. The pores of the C. crescentus porins are slightly larger than those of E. coli K-12, since maltotetraose supported growth of the C. crescentus malA mutant but failed to support growth of the E. coli lamB mutant. The data are consistent with the proposal that binding of maltodextrins to MalA requires energy and MalA actively transports maltodextrins with K(d) values 1,000-fold smaller than those for the LamB porin and 100-fold larger than those for the vitamin B(12) and ferric siderophore outer membrane transporters. MalA is the first example of an outer membrane protein for which an ExbB/ExbD-dependent transport of a nutrient other than iron and vitamin B(12) has been demonstrated.     

Glucose and Gluconate Metabolism in an Escherichia coli Mutant Lacking Phosphoglucose Isomerase

A single gene mutant lacking phosphoglucose isomerase (pgi) was selected after ethyl methane sulfonate mutagenesis of Escherichia coli strain K-10. Enzyme assays revealed no pgi activity in the mutant, whereas levels of glucokinase, glucose-6-phosphate dehydrogenase, and gluconate-6-phosphate dehydrogenase were similar in parent and mutant. The amount of glucose released by acid hydrolysis of the mutant cells after growth on gluconate was less than 2% that released from parent cells; when grown in the presence of glucose, mutant and parent cells contained the same amount of glucose residues. The mutant grew on glucose one-third as fast as the parent; it also grew much slower than the parent on galactose, maltose, and lactose. On fructose, gluconate, and other carbon sources, growth was almost normal. In both parent and mutant, gluconokinase and gluconate-6-phosphate dehydrase were present during growth on gluconate but not during growth on glucose. Assay and degradation of alanine from protein hydrolysates after growth on glucose-1-(14)C and gluconate-1-(14)C showed that in the parent strain glucose was metabolized by the glycolytic path and the hexose monophosphate shunt. Gluconate was metabolized by the Entner-Doudoroff path and the hexose monophosphate shunt. The mutant used glucose chiefly by the shunt, but may also have used the Entner-Doudoroff path to a limited extent.     

On Some Genetic Aspects of Phage λ Resistance in E. COLI K12

Most mutations rendering E. coli K12 resistant to phage lambda, map in two genetic regions malA and malB.-The malB region contains a gene lamB specifically involved in the lambda receptor synthesis. Twenty-one independent lamB mutations studied by complementation belonged to a single cistron. This makes it very likely that lamB is monocistronic. Among the lamB mutants some are still sensitive to a host range mutant of phage lambda. Mutations mapping in a proximal gene essential for maltose metabolism inactivate gene lamB by polarity confirming that both genes are part of the same operon. Because cases of intracistronic complementation have been found, the active lamB product may be an oligomeric protein.-Previously all lambda resistant mutations in the malA region have been shown to map in the malT cistron. malT is believed to be a positive regulatory gene necessary for the induction of the "maltose operons" in the malA region and in the malB region of the E. coli K12 genetic map. No trans dominant malT mutation have been found. Therefore if they exist, they occur at a frequency of less than 10(-8), or strongly reduce the growth rate of the mutants.     

Utilization of gluconate by Escherichia coli. A role of adenosine 3'':5''-cyclic monophosphate in the induction of gluconate catabolism.

1. Cultures of Escherichia coli growing on gluconate use both gluconate and glucose when glucose is added. 2. Glycerol-grown cells adapt to gluconate utilization even in media containing glucose as well as gluconate. 3. The rates of gluconate utilization by cells growing on a mixture of glucose and gluconate, and the specific activities of the gluconate uptake system and of gluconate kinase, are greater if adenosine 3':5'-cyclic monophosphate (cyclic AMP) is present in the medium than in its absence. 4. Growth on media containing gluconate and cyclic AMP is accompanied by the formation of methyl glyoxal and pyruvate, and progressive inhibition of growth. 5. A mutant devoid of adenylate cyclase activity (cya) grew well on glucose in the absence of exogenous cyclic AMP but grew only poorly on gluconate; neither the gluconate uptake system nor gluconate kinase was adequately induced. The addition of cyclic AMP promoted growth on gluconate and facilitated the induction of proteins required for gluconate catabolism. 6. Phage Pl-mediated transduction of cya+ into the cya-mutant also restored the wild-type phenotype in its ability to adapt to gluconate utilization.     

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.     

Utilization of gluconate by Escherichia coli. Induction of gluconate kinase and 6-phosphogluconate dehydratase activities

1. A mutant of Escherichia coli, devoid of phosphopyruvate synthetase, glucosephosphate isomerase and 6-phosphogluconate dehydrogenase activities, grew readily on gluconate and inducibly formed an uptake system for gluconate, gluconate kinase and 6-phosphogluconate dehydratase while doing so. 2. This mutant also grew on glucose 6-phosphate and inducibly formed 6-phosphogluconate dehydratase; however, the formation of the gluconate uptake system and gluconate kinase was not induced under these conditions. 3. The use of the Entner–Doudoroff pathway for the dissimilation of 6-phosphogluconate, derived from either gluconate or glucose 6-phosphate, by this mutant was also demonstrated by the accumulation of 2-keto-3-deoxy-6-phosphogluconate (3-deoxy-6-phospho-l-glycero-2-hexulosonate) from both these substrates in a similar mutant that also lacked phospho-2-keto-3-deoxygluconate aldolase activity. 4. Glucose 6-phosphate inhibits the continued utilization of fructose by cultures of the mutants growing on fructose, as it does in wild-type E. coli. 5. The mutants do not use glucose for growth. This is shown to be due to insufficiency of phosphopyruvate, which is required for glucose uptake.     

Regulatory Mutations Affecting the Gluconate System in Escherichia coli

A spontaneously arising regulatory mutant of the gluconate system in Escherichia coli was isolated. This mutant became constitutive, probably in one step, for gluconate high-affinity transport, gluconokinase, and gluconate-6-P dehydrase. The mutation involved (gntR18) is cotransducible with asd. Pseudorevertants, derived from a mutant (M2) that shows a long lag for growth on gluconate mineral medium, were also isolated and characterized. They give constitutive levels of gluconokinase and gluconate-6-P dehydrase but lack high-affinity transport function. Genetic experiments performed with one of these pseudorevertants (M4) indicate that it carries a secondary mutation in the gntR gene. The M4 phenotype is thus the result of the interaction of expression of a constitutive mutation (gntR4) with the mutation of strain M2 (gntM2).     

The YvcK protein is required for morphogenesis via localization of PBP1 under gluconeogenic growth conditions in Bacillus subtilis

The YvcK protein was previously shown to be dispensable when B. subtilis cells are grown on glycolytic carbon sources but essential for growth and normal shape on gluconeogenic carbon sources. Here, we report that YvcK is localized as a helical-like pattern in the cell. This localization seems independent of the actin-like protein, MreB. A YvcK overproduction restores a normal morphology in an mreB mutant strain when bacteria are grown on PAB medium. Reciprocally, an additional copy of mreB restores a normal growth and morphology in a yvcK mutant strain when bacteria are grown on a gluconeogenic carbon source like gluconate. Furthermore, as already observed for the mreB mutant, the deletion of the gene encoding the penicillin-binding protein PBP1 restores growth and normal shape of a yvcK mutant on gluconeogenic carbon sources. The PBP1 is delocalized in an mreB mutant grown in the absence of magnesium and in a yvcK mutant grown on gluconate medium. Interestingly, its proper localization can be rescued by YvcK overproduction. Therefore, in gluconeogenic growth conditions, YvcK is required for the correct localization of PBP1 and hence for displaying a normal rod shape.     

Effect of phosphoglycerate mutase deficiency on heterotrophic and autotrophic carbon metabolism of Alcaligenes eutrophus.

Mutants of Alcaligenes eutrophus were isolated on the basis of their inability to grow on succinate as the sole source of carbon and energy. The mutants also failed to grow on other gluconeogenic substrates, including pyruvate, acetate, and citrate. Simultaneously, they had lost their capability for autotrophic growth. The mutants grew, but slower than the wild type, on fructose or gluconate. Growth retardation on gluconate was more pronounced. The mutants lacked phosphoglycerate mutase activity, and spontaneous revertants of normal growth phenotype had regained the activity. The physiological characteristics of the mutants indicate the role of phosphoglycerate mutase in heterotrophic and autotrophic carbon metabolism of A. eutrophus. Although the enzyme is necessary for gluconeogenesis during heterotrophic growth on three- or four-carbon substrates, its glycolytic function is not essential for the catabolism of fructose or gluconate via the Entner-Doudoroff pathway. The enzyme is required during autotrophic growth as a catalyst in the biosynthetic route leading from glycerate 3-phosphate to pyruvate. It is suggested that the mutants accomplish the complete degradation of fructose and gluconate mutase lesion. The catabolically produced triose phosphates are converted to fructose 6-phosphate which is rechanneled into the Entner-Doudoroff pathway. This carbon recycling mechanism operates less effectively in mutant cells growing on gluconate.     

Mutation causing reverse osmoregulation of synthesis of OmpF, a major outer membrane protein of Escherichia coli.

Supplementation of growth media with high concentrations of substances like sucrose results in the induction of OmpC synthesis and the suppression of OmpF synthesis. We isolated a novel mutant in which OmpF synthesis is in the opposite direction from normal osmoregulation. By transductional mapping, the mutation was localized at 75 min between malA and aroB on the Escherichia coli chromosome map where the ompR-envZ region is. The mutation was suppressed by a plasmid carrying the ompR gene but not by a plasmid carrying the envZ gene alone. The mutation also resulted in the almost complete suppression of OmpC synthesis. However, the remaining OmpC synthesis was osmoregulated normally. Based on these observations, the mechanism of osmoregulation of OmpF-OmpC synthesis is discussed.     

Glucose and Gluconate Metabolism in a Mutant of Escherichia coli Lacking Gluconate-6-phosphate Dehydrase

A mutant lacking gluconate-6-phosphate dehydrase (the first enzyme of the Entner-Doudoroff pathway) was isolated after ethyl methane sulfonate mutagenesis of Escherichia coli. Other enzymes of gluconate metabolism (gluconokinase, gluconate-6-phosphate dehydrogenase, and 2-keto-3-deoxygluconate-6-phosphate aldolase) were present in the mutant. When the mutant was grown on gluconate-1-(14)C, alanine isolated from protein was unlabeled, showing that the dehydrase was absent in vivo and that the sole pathway of gluconate metabolism in the mutant was the hexose monophosphate shunt. The mutant grew on gluconate with a doubling time of 155 min, compared with the parent strain's 56 min. On glucose and fructose it grew with normal doubling times. Thus, in E. coli, the Entner-Doudoroff pathway is used for gluconate metabolism but not for glucose metabolism.     

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.     

Participation of the Entner-Doudoroff pathway in Escherichia coli strains with an inactive phosphotransferase system (PTS- Glc+) in gluconate and glucose batch cultures

The activity of the enzymes of the central metabolic pathways has been the subject of intensive analysis; however, the Entner-Doudoroff (ED) pathway has only recently begun to attract attention. The metabolic response to edd gene knockout in Escherichia coli JM101 and PTS- Glc+ was investigated in gluconate and glucose batch cultures and compared with other pyruvate kinase and PTS mutants previously constructed. Even though the specific growth rates between the strain carrying the edd gene knockout and its parent JM101 and PTS- Glc+ edd and its parent PTS- Glc+ were very similar, reproducible changes in the specific consumption rates and biomass yields were obtained when grown on glucose. These results support the participation of the ED pathway not only on gluconate metabolism but on other metabolic and biochemical processes in E. coli. Despite that gluconate is a non-PTS carbohydrate, the PTS- Glc+ and derived strains showed important reductions in the specific growth and gluconate consumption rates. Moreover, the overall activity of the ED pathway on gluconate resulted in important increments in PTS- Glc+ and PTS- Glc+ pykF mutants. Additional results obtained with the pykA pykF mutant indicate the important contribution of the pyruvate kinase enzymes to pyruvate synthesis and energy production in both carbon sources.     

6-Phosphogluconate dehydratase deficiency in pleiotropic carbohydrate-negative mutant strains of Pseudomonas aeruginosa.

Mutants of Pseudomonas aeruginosa, strain PAO, have been isolated that are unable to grow on mannitol, glucose, gluconate, or 2-ketogluconate, cut that exhibit wild-type growth on pyruvate, lactate, citrate, succinate, or acetate. Although some of these mutants were also unable to grow on glycerol, the mutations formed a single linkage group by quantitative transductional analysis with phage F116 on glucose minimal agar medium. Cell extracts of all mutant strains were either lacking or severely deficient in 6-phosphogluconate dehydratase activity. Glu+ transductants derived from mutant strains that retained the wild-type ability for growth at the expense of glycerol also regained the ability to grow on all C-6 compounds. Although a role for the pentose phosphate pathway in the catabolism of C6 substrates was not found, a functional Entner-Doudoroff pathway appears to be essential for the catabolism of mannitol, glucose, gluconate, and 2-ketogluconate.     

Phosphoglucose isomerase mutant of Rhizobium meliloti.

A mutant strain of complex phenotype was selected in Rhizobium meliloti after nitrosoguanidine mutagenesis. It failed to grow on mannitol, sorbitol, fructose, mannose, ribose, arabitol, or xylose, but grew on glucose, maltose, gluconate, L-arabinose, and many other carbohydrates. Assay showed the enzyme lesion to be in phosphoglucose isomerase (pgi), and revertants, which were of normal growth phenotype, contained the enzyme again. Nonpermissive substrates such as fructose and xylose prevented growth on permissive ones such as L-arabinose, and in such situations there was high accumulation of fructose 6-phosphate. The mutant strain had about 20% as much exopolysaccharide as the parent. Nitrogen fixation by whole plants was low and delayed when the mutant strain was the inoculant.     

Role of the pentose phosphate pathway and the Entner–Doudoroff pathway in glucose metabolism of Gluconobacter oxydans 621H

Glucose catabolism by the obligatory aerobic acetic acid bacterium Gluconobacter oxydans 621H proceeds in two phases comprising rapid periplasmic oxidation of glucose to gluconate (phase I) and oxidation of gluconate to 2-ketogluconate or 5-ketogluconate (phase II). Only a small amount of glucose and part of the gluconate is taken up into the cells. To determine the roles of the pentose phosphate pathway (PPP) and the Entner–Doudoroff pathway (EDP) for intracellular glucose and gluconate catabolism, mutants defective in either the PPP (Δgnd, Δgnd zwf*) or the EDP (Δedd–eda) were characterized under defined conditions of pH 6 and 15 % dissolved oxygen. In the presence of yeast extract, neither of the two pathways was essential for growth with glucose. However, the PPP mutants showed a reduced growth rate in phase I and completely lacked growth in phase II. In contrast, the EDP mutant showed the same growth behavior as the reference strain. These results demonstrate that the PPP is of major importance for cytoplasmic glucose and gluconate catabolism, whereas the EDP is dispensable. Reasons for this difference are discussed.     

Transport of gluconate in Rhodotorula glutinis. Inactivation by glucose of the uptake system.

Transport of gluconate has been studied in a wild-type strain of Rhodotorula glutinis and in a mutant derived from it which has acquired the ability to grow on gluconate as the only carbon and energy source. The transport is energy dependent. It shows the same K_m for gluconate (0.1 mm) between pH 4.7 and 7, which suggests that the negatively charged gluconate is the true substrate for the transport system. The rate of gluconate uptake is much lower in the wild type than in the mutant. The mutant grown on gluconate transports gluconate much faster than if grown on other carbon sources. Glucose rapidly and irreversibly inactivates the transport system. This inactivation can also be effected by δ-gluconolactone and to a lesser extent by acetate; it is not prevented by gluconate and occurs also in the presence of cycloheximide.     

Genetic definition of the substrate selectivity of outer membrane porin protein OprD of Pseudomonas aeruginosa.

Earlier studies proved that Pseudomonas aeruginosa OprD is a specific porin for basic amino acids and imipenem. It was also considered to function as a nonspecific porin that allowed the size-dependent uptake of monosaccharides and facilitation of the uptake of quinolone and other antibiotics. In the present study, we utilized P. aeruginosa strains with genetically defined levels of OprD to characterize the in vivo substrate selectivity of this porin. An oprD::omega interposon mutant was constructed by gene replacement utilizing an in vitro mutagenized cloned oprD gene. In addition, OprD was overexpressed from the lac promoter by cloning the oprD gene into the broad-host-range plasmid pUCP19. To test the substrate selectivity, strains were grown in minimal medium with limiting concentrations of the carbon sources glucose, gluconate, or pyruvate. In minimal medium with 0.5 mM gluconate, the growth rates of the parent strain H103 and its oprD::omega mutant H729 were only 60 and 20%, respectively, of that of the OprD-overexpressing strain H103(pXH2). In contrast, no significant differences were observed in the growth rates of these three strains on glucose or pyruvate, indicating that OprD selectively facilitated the transport of gluconate. To determine the role of OprD in antibiotic uptake, nine strains representing different levels of OprD and OprF were used to determine the MICs of different antibiotics. The results clearly demonstrated that OprD could be utilized by imipenem and meropenem but that, even when substantially overexpressed, it could not be significantly utilized by other beta-lactams, quinolones, or aminoglycosides. In addition, competition experiments confirmed that imipenem had common binding sites with basic amino acids in the OprD channel, but not with gluconate or glucose.     

Behavior of Coliphage Lambda in Shigella flexneri 2a

The insensitivity of wild-type Shigella flexneri 2a to coliphage lambda is a consequence of its native genetic defect in the malA gene cluster. The "smooth" S. flexneri 2a lipopolysaccharide layer affects the efficient adsorption of lambda. Derivatives, capable of serving as functional hosts for lambda, were obtained by repairing the malA lesion, enabling the expression of the malB-lambdarcp region of S. flexneri. Introduction of a mutation into S. flexneri causing a "rough" lipopolysaccharide character resulted in more efficient adsorption of lambda. Such S. flexneri hosts can be stably lysogenized and upon induction yield gal(+)-transducing lysates. Lambda propagated on a malA(+) rough S. flexneri host was restricted by Escherichia coli K-12 and E. coli B, but not by E. coli C. This S. flexneri host did not restrict lambda grown on these E. coli strains.