Studies on the enzymatic synthesis of enterochelin in Escherichia coli K-12, Salmonella typhimurium and Klebsiella pneumoniae. Physical association of enterochelin synthetase components in vitro

Further evidence is presented in support of the proposal made previously (Greenwood, K.T. and Luke, R.K.J. (1976) Biochim. Biophys. Acta 454, 285-297) that components of the Escherichia coli enterochelin synthetase system physicaloly associate to form enzyme complexes. Evidence for the existence of three enzyme complexes, designated in order of increasing stability G-D < F-D < F-D-G, has been obtained following gel filtration and chromatography on DEAE-Sephadex. Persistence of the F-D and G-D complexes during chromatography appears to depend on the flow rate of the column. On the basis of complementation with appropriate ent mutants of E. coli, activities corresponding to those of the D, E, F and G components of enterochelin synthetase in E. coli have been detected in cell-extracts of both Salmonella typhimurium and Klebsiella pneumoniae (formerly Aerobacter aerogenes) strains. These are designated D', E', F' and G' activities. Components E' and G' are eluted from Sephadex G-100 in similar fashion to their E. coli counterparts. Peaks of F' and D' activities however, are eluted together at a position corresponding to that of the E. coli F component. We suggest that in S. typhimurium and K. pneumoniae, either a single polypeptide combines the functions of the E. coli F and D components, or that separate F' and D' components form a stable complex and that activity of uncomplexed D' and component was not detected under the conditions used during chromatography and assay.     

Regulation of Glyoxylate Metabolism in Escherichia coli K-12

The relative contributions of the dicarboxylic acid and the tricarboxylic acid cycles to the oxidative catabolism of glyoxylate in Escherichia coli K-12 were deduced by analysis of mutant strains that were blocked in the formation of glyoxylate carboligase and of malate synthase G (the "glycolate form" of malate synthase). Mutant strains unable to form malate synthase G were unimpaired in their ability to oxidize glyoxylate. Hence, the dicarboxylic acid cycle does not appear to play an essential role in this process. Organisms blocked in the synthesis of glyoxylate carboligase did not oxidize glyoxylate at a detectable rate, indicating that wild-type organisms convert glyoxylate to acetyl-coenzyme A and oxidize it via the tricarboxylic acid cycle. The foregoing evidence indicates that malate synthase G plays an anaplerotic role during growth with glycolate or acetate as the carbon source. The in vivo activity of malate synthase G was not detectable when the intracellular concentration of acetyl-coenzyme A was low, suggesting that this substrate or a closely related metabolite exerts a sensitive positive control over the enzyme. The synthesis of malate synthase G appears to be induced directly by glycolate which may be formed by a constitutive reduced nicotinamide adenine dinucleotide phosphate-dependent glyoxylate reductase in glyoxylate- or acetate-grown cells.     

Map location of arginyl-tRNA synthetase mutations in Escherichia coli K-12

Summary Mutants of Escherichia coli K-12 previously isolated in the authors' laboratory have reduced arginyl-tRNA synthetase activity. The mutants fall into two classes. All mutants grow slowly on arginine-free medium. On arginine-supplemented medium some mutants grow at a normal rate (Class I) while others still grow slowly (Class II). Matings were performed to located a Class I and a Class II mutation on the E. coli chromosome map, and on the basis of our results we have assigned both to one locus, argS.     

Regulation of the Escherichia coli K-12 uvrB operon

The UV light inducibility of the uvrB operon of Escherichia coli K-12 was previously demonstrated by exploiting a strain in which the gene for the enzyme beta-galactosidase was inserted into the uvrB operon. This insert is now shown to be located within the structural gene for the uvrB enzyme, leaving the regulatory sequences of the operon intact. Analyses to quantitate the induction of this system show that derepression of the operon is first detectable 5 min after UV exposure, with the rate of synthesis increasing to four to six times the uninduced rate during the subsequent 30 min. Induction is unaffected by mutations in other components of nucleotide excision repair. The control of uvrB was found to result from direct repression by the lexA gene product, with the recA gene product playing an indirect role. Nucleotide excision repair thus seems to be part of the SOS response.     

Regulation of lysine decarboxylase activity in Escherichia coli K-12

The biodegradative lysine decarboxylase of E. coli has been reported to attain a higher specific activity when grown to saturation in the presence of excess lysine under conditions of low pH and absence of aeration. In order to examine possible sources of the pH and anaerobic regulation, a series of isogenic strains of E. coli K-12 were constructed. The effects of cadR^-, fnr ^-, cya ^-, crp ^-and pgl ^-mutations on lysine decarboxylase expression were examined. Cultures were grown in a lysine supplemented rich medium at pH 5.5, pH 6.8, and pH 8.0 with and without aeration and the enzyme was assayed from log phase cultures. The results suggested that the pH and air responses were independent and that these known regulatory processes are not responsible for this regulation of the biodegradative lysine decarboxylase.     

Regulation of D-arabinose utilization in Escherichia coli K-12.

Studies involving lambda phage transduction of the D-arabinose utilization gene (dar+) in Escherichia coli K-12 indicated the product of this gene to be a transdominant activator. An apparent anomaly regarding this hypothesis exists in that a diploid recessive lysogen (lambda dar-/dar-) can spontaneously become capable of growth on D-arabinose.     

Iron uptake in pseudorevertants of Escherichia coli K-12 mutants with multiple defects in the enterochelin system

Iron uptake in pseudorevertants of Escherichia coli K-12 strains which lack the ability to synthesize enterochelin, 2,3-dihydroxybenzoate, and the ferrienterochelin receptor protein was characterized. In four independent pseudorevertants, the suppressor mutations which permitted growth in iron-poor environments appeared to be located in ompB, the regulatory locus for the porin proteins. Unlike wild-type cells, the pseudorevertants were unable to utilize ferrienterochelin and could acquire iron from citrate without induction by prior growth in citrate. The energy requirements of the pseudorevertant system appeared to be identical to those of the enterochelin system. Evidence that loss of the porin proteins results in the secretion by the pseudorevertants of a molecule with siderophore activity is presented; this siderophore is able to remove iron from the non-biological iron chelators nitrilotriacetic acid and , -dipyridyl but not from the siderophores ferrichrome and enterochelin.     

Evidence for cAMP-mediated control of isoleucyl-tRNA synthetase formation in Escherichia coli K-12

The ability of cAMP to inhibit isoleucyl-tRNA synthetase (IRS) formation has been demonstrated in wild type K-12 Escherichia coli and two adenyl-cyclase (cya) mutants. cAMP appeared not to have any effect on either the valyl- or arginyl-tRNA synthetase (VRS and ARS respectively). Addition of cAMP led to a reduction in rate of IRS synthesis but not VRS or ARS. Furthermore, derepression of IRS and VRS by isoleucine limitation was completely prevented by cAMP.Abbreviations IRS isoleucyl-tRNA synthetase - VRS valyl-tRNA synthetase - ARS arginyl-tRNA synthetase - cAMP cyclic adenosine-3,5-monophosphate - Cya adenyl cyclase Gene - CRP cAMP receptor protein - O.D. optical density     

Location of three genes concerned with the conversion of 2,3-dihydroxybenzoate into enterochelin in Escherichia coli K-12

Mutants of Escherichia coli K-12 unable to synthesize the iron-sequestering compound, enterochelin, from 2,3-dihydroxybenzoate have been isolated and divided into three classes on the basis of tests for enzymatic complementation. The genes affected (designated entD, entE, and entF) have been mapped by cotransduction and are located at about minute 14 on the E. coli genome. They were found to be closely linked to other genes (entA, entB, and entC) concerned with enterochelin biosynthesis and a gene (fep) concerned with the uptake of the iron-enterochelin complex. No detectable diffusible intermediate in the formation of enterochelin from 2,3-dihydroxybenzoate was formed by cell extracts of mutants carrying mutations in the entD, entE, or entF genes.     

Escherichia coli K-12 lysyl-tRNA synthetase mutant with a novel reversion pattern

Fast-growing revertants have been selected from a slow-growing lysyl-tRNA synthetase mutant. All of the revertants had increased lysyl-tRNA synthetase activity compared with the mutant (5- to 85-fold), and in some revertants this amounted to two to three times the wild-type synthetase activity. Two-dimensional gel electrophoresis of a whole-cell extract of revertant IH2018 (1.5- to 2-fold wild-type synthetase activity) showed that the increase in synthetase activity is due to the induction of cryptic lysyl-tRNA synthetase forms and not to a change in the constitutive lysyl-tRNA synthetase. Genetic studies have shown that a locus termed rlu (for regulation of lysU ) which is cotransducible with purF at 49.5 min influences the amount of the cryptic lysyl-tRNA synthetase.     

Studies on the enzymatic synthesis of enterochelin in Escherichia coli K-12. Four polypeptides involved in the conversion of 2,3-dihydroxybenzoate to enterochelin.

Four gene products involved in the enzymatic synthesis of enterochelin from 2,3-dihydroxybenzoate, L-serine and ATP (Luke, R.K.L. and Gibson, F. (1971) J. Bacteriol. 107,557-562; Woodrow, G.C., Young, I.G. and Gibson, F. (1975) J. Bacteriol. 124, 1-6) have been partially purified using a previously reported fractionation procedure (Bryce, G.F. and Brot, N. (1972) Biochemistry 11, 1708-1715). The products of genes E, F and G have been separated from each other and correspond to the E1, E2 and E3 activities described by Bryce and Brot. These three gene products were not completely separated from the product of gene D. We refer to these gene products as components E, F, G and D of the enzymic apparatus for biosynthesis of enterochelin. Certain properties and functions of the four semi-purified components have been investigated. The E component is involved in the activation of 2,3-dihydroxybenzoate and the F component in the activation of L-serine. The D component physically associates with the F and G components during gel filtration and chromatography on DEAE Sephadex. It is proposed that the synthesis of enterochelin from L-serine and 2,3-dihydroxybenzoic acid is catalysed in vivo by a multienzyme complex, enterochelin synthetase.     

Regulation of the Pool Size of Valine in Escherichia coli K-12

Three mutations (ilvH611, ilvH612, and ilvH613) are described which make Escherichia coli K-12 resistant to valine inhibition and are located near leu. The expression of the ilv genes appears to be normal in these mutants since the isoleucine-valine biosynthetic enzymes are not derepressed relative to the wild type. The intracellular concentration of valine is, however, higher in the mutants than in the isogenic ilvH(+) strain. These mutants also excrete valine, probably because of the high intracellular concentration of this amino acid. The pool size of valine is regulated independently from that of isoleucine and leucine. The increased intracellular concentration of valine is due to a decreased feedback inhibition that valine exerts on its own biosynthetic pathway. In fact, acetolactate synthase activity assayed in extracts of ilvH612 and ilvH613 mutants is more resistant to valine inhibition than the activity assayed in the ilvH(+) isogenic strain. Two forms of acetolactate synthase activity can be separated from these extracts by adsorption and elution on hydroxylapatite. One of them is as sensitive to valine inhibition as that of the wild type, the other is more resistant to valine inhibition.     

Mapping of the constitutive lysyl-tRNA synthetase gene of Escherichia coli K-12.

The constitutive lysyl-tRNA synthetase gene (lysS) was mapped at 62.1 min on the Escherichia coli chromosome by a combination of conjugation and transduction, with physical confirmation by two-dimensional gel electrophoresis. Revertant analysis suggests that the altered isoelectric point and the low amount of the mutant LysS protein may be due to a single mutational event.     

Enzymatic hydrolysis of enterochelin and its iron complex in Escherichia Coli K-12. Properties of enterochelin esterase.

Properties of the enzyme which hydrolyses enterochelin (a cyclic trimer of 2,3-dihydroxy-N-benzoyl-L-serine) to 2,3-dihydroxybenzoylserine have been investigated with a view to resolving discrepancies between earlier reports. Enterochelin esterase, previously reported to consists of two components (O'Brien, I.G., Cox, G.B. and Gibson, F. (1971) Biochim. Biophys. Acta 237, 537-549), has been shown to be fully active in the absence of the so-called A component. The hydrolase described previously (Bryce, G.F. and Brot, N. (1972) Biochemistry 11, 1708-1715) as being able to break down enterochelin but not its iron complex, ferric-enterochelin, appears to be identical with the B component of enterochelin esterase. The single component enterochelin esterase corresponding to what was previously described as component B, hydrolyses both enterochelin and ferric-enterochelin. Under the assay conditions used, enterochelin is hydrolysed 2.5 times faster than the complex. Enzymatic activity is inhibited by N-ethylmaleimide and is lost rapidly at 37 degrees C. Activity is stabilized in the presence of ferric-enterochelin, enterochelin, dithiothreitol or certain protein fractions.     

Iron transport in Escherichia coli K-12

The study of iron uptake promoted by 2,3-dihydroxybenzoate (DHB) into Escherichia coli K-12 aroB mutants allowed some dissection of outer and cytoplasmic membrane functions. These strains are unable to produce the iron-transporting chelate enterochelin, unless fed with a precursor such as DHB. When added to the medium, enterochelin and its natural breakdown products, the linear dimer and trimer of 2,3-dihydroxybenzoylserine (DBS), efficiently transported iron via the feuB, tonB and fep gene products. Thus mutants in these genes were defective in transport of the above chelates. However, feuB and tonB mutants were able to take up iron when DHB was added to the medium. Thus DHB-promoted iron uptake bypassed two functions required for the transport of ferric-enterochelin from the medium. One of these functions, feuB, has been shown to be an outer membrane protein. In contrast to three other iron transport systems including ferric-enterochelin uptake, DHB-promoted iron uptake was little affected by the uncoupler 2,4-dinitrophenol. Dissipation of the energized state of the cytoplasmic membrane apparently only affects those iron transport systems which require an outer membrane protein. Since DHB-promoted iron uptake bypasses the feuB outer membrane protein and the tonB function, it is concluded that, in ferricenterochelin transport, the tonB gene may function in coupling the energized state of the cytoplasmic membrane to the protein-dependent outer membrane permeability. DHB-promoted iron uptake required the synthesis and enzymatic breakdown of enterochelin as judged by the effects of the entF and fesB mutations. A fep mutant was not only deficient in the transport of the ferric chelates of enterochelin and its breakdown products, but was also deficient in DHB-promoted iron uptake. A scheme is presented in which iron diffuses as DHB-complex through the outer membrane, and is subsequently captured by enterochelin or DBS dimer or trimer and translocated across the cytoplasmic membrane.List of Abbreviations DHB 2,3-dihydroxybenzoate - DBS 2,3-dihydroxybenzoylserine - NTA nitrilotriacetate - DNP 2,4-dinitrophenol