Regulation of Escherichia coli K-12 hexuronate system genes: exu regulon.

Two types of Escherichia coli K-12 regulatory mutants, partially or totally negative for the induction of the five catabolic enzymes (uronic isomerase, uxaC; altronate oxidized nicotinamide adenine dinucleotide: uxaB; mannonate hydrolyase, uxuA) and the transport system (exuT) of the hexuronate-inducible pathway, were isolated and analyzed enzymatically. Hexuronate-catabolizing revertants of the negative mutants showed a constitutive synthesis for some or all of these enzymes. Negative and constitutive mutations were localized in the same genetic locus, called exuR, and the following order for the markers situated between the min 65 and 68 was determined: argG--exuR--exuT--uxaC--uxaA--tolC. The enzymatic characterization of the pleiotropic negative and constitutive mutants of the exuR gene suggests that the exuR regulatory gene product exerts a specific and total control on the three exuT, uszB, and uxaC-uxaA operons of the galacturonate pathway and a partial control on the uxuA-uxuB operon of the glucuronate pathway. The analysis of diploid strains conatining both the wild type and a negative or constitutive allele of the exuR gene, as well as the analysis of thermosensitive mutants of the exuR gene, was in agreement with a negative regulatory mechanism for the control of the hexuronate system.     

Hydroxylamine reductase activity of the hybrid cluster protein from Escherichia coli

The hybrid cluster protein (HCP; formerly termed the prismane protein) has been extensively studied due to its unique spectroscopic properties. Although the structural and spectroscopic characteristics are well defined, its enzymatic function, up to this point, has remained unidentified. While it was proposed that HCP acts in some step of nitrogen metabolism, a specific role for this enzyme remained unknown. Recent studies of HCP purified from Escherichia coli have identified a novel hydroxylamine reductase activity. These data reveal the ability of HCP to reduce hydroxylamine in vitro to form NH(3) and H(2)O. Further biochemical analyses were completed in order to determine the effects of various electron donors, different pH levels, and the presence of CN(-) on in vitro hydroxylamine reduction.     

Cloning of the 3'-phosphoadenylyl sulfate reductase and sulfite reductase genes from Escherichia coli K-12

The structural genes for 3'-phosphoadenylyl sulfate (PAPS) reductase (cysH) and sulfite reductase (alpha and beta subunits; EC 1.8.1.2)(cysI and cysJ) of Escherichia coli K-12 have been cloned by complementation. pCYSI contains two PstI fragments (18.3 and 2.9 kb) which complement cysH-, cysI-, and cysJ- mutants. Subcloning showed that the cysH gene is located on a 1.6-kb ClaI subfragment (pCYSI-3) whereas cysI and most of cysJ are carried on a 3.7-kb ClaI subfragment (pCYSI-5). The PAPS reductase gene is closely linked to the sulfite reductase genes, but its expression is regulated by a unique promoter. The cysI and cysJ genes, on the other hand, are transcribed as an operon and the promoter precedes the cysI gene. Maxicell analysis demonstrated that pCYSI encodes three polypeptides of Mr 27,000, 57,000, and 60,000, in addition to the tetracycline-resistance determinant. The 60- and 57-kDa proteins are most likely the alpha and beta subunits, respectively, of E. coli sulfite reductase while the 27-kDa protein is putatively identified as PAPS reductase. Preliminary data suggest that the alpha and beta subunits of sulfite reductase are encoded by cysI and cysJ, respectively.     

Structure and spectroscopy of the periplasmic cytochrome c nitrite reductase from Escherichia coli

The crystal structure and spectroscopic properties of the periplasmic penta-heme cytochrome c nitrite reductase (NrfA) of Escherichia coli are presented. The structure is the first for a member of the NrfA subgroup that utilize a soluble penta-heme cytochrome, NrfB, as a redox partner. Comparison to the structures of Wolinella succinogenes NrfA and Sulfospirillum deleyianum NrfA, which accept electrons from a membrane-anchored tetra-heme cytochrome (NrfH), reveals notable differences in the protein surface around heme 2, which may be the docking site for the redox partner. The structure shows that four of the NrfA hemes (hemes 2-5) have bis-histidine axial heme-Fe ligation. The catalytic heme-Fe (heme 1) has a lysine distal ligand and an oxygen atom proximal ligand. Analysis of NrfA in solution by magnetic circular dichroism (MCD) suggested that the oxygen ligand arose from water. Electron paramagnetic resonance (EPR) spectra were collected from electrochemically poised NrfA samples. Broad perpendicular mode signals at g similar 10.8 and 3.5, characteristic of weakly spin-coupled S = 5/2, S = 1/2 paramagnets, titrated with E(m) = -107 mV. A possible origin for these are the active site Lys-OH(2) coordinated heme (heme 1) and a nearby bis-His coordinated heme (heme 3). A rhombic heme Fe(III) EPR signal at g(z) = 2.91, g(y) = 2.3, g(x) = 1.5 titrated with E(m) = -37 mV and is likely to arise from bis-His coordinated heme (heme 2) in which the interplanar angle of the imidazole rings is 21.2. The final two bis-His coordinated hemes (hemes 4 and 5) have imidazole interplanar angles of 64.4 and 71.8. Either, or both, of these hemes could give rise to a "Large g max" EPR signal at g(z)() = 3.17 that titrated at potentials between -250 and -400 mV. Previous spectroscopic studies on NrfA from a number of bacterial species are considered in the light of the structure-based spectro-potentiometric analysis presented for the E. coli NrfA.     

Functional characterization of the Escherichia coli K-12 yiaMNO transport protein genes

The yiaMNO genes of Escherichia coli K-12 encode a binding protein-dependent secondary, or tri-partite ATP-independent periplasmic (TRAP), transporter. Since only a few members of this family have been functionally characterized to date, we aimed to identify the substrate for this transporter. Cells that constitutively express the yiaK-S gene cluster metabolized the rare pentose L-xylulose, while deletion of the yiaMNO transporter genes reduced L-xylulose metabolism. The periplasmic substrate-binding protein YiaO was found to bind L-xylulose, and stimulated L-xylulose uptake by spheroplasts. These date indicate that the yiaMNO transporter mediates uptake of this rare pentose.     

Purification and properties of glutamate binding protein from the periplasmic space of Escherichia coli K-12.

Glutamate binding protein released from the periplasmic space of Escherichia coli K-12 by lysozyme-EDTA treatment was purified to homogeneity and its physical and chemical properties were studied. It is a basic protein with a pI of 9.1. Its molecular weight, determined in an analytical ultracentrifuge, and by gel filtration on Sephadex G-100 and dodecylsulphate acrylamide is 29 700, 27 800 and 32 000, respectively. The KD value for glutamate was 6.7 - 10- minus 6 M. L-Aspartate, reduced glutathione, G-glutamate-gamma-benzylester and L-glutamate-gamma-ethylester competitively inhibited glutamate binding with K-i; values of 7.8 - 10- minus 5, 1.1 - 10- minus 5, 1.0 - 10- minus 5 and 1.0 - 10- minus 5 M, respectively. Spheroplasts retained 40% of glutamate transport as compared to intact cells. The glutamate binding activity of a glutamate-utilizing strain (CS7), was 1.6 times as high as that of the glutamate non-utilizing parent strain (CS101). Similarly, the glutamate binding activity of a temperature conditional glutamate-utilizing mutant (CS2-TC) was 1.9 times higher when grown at the permissive temperature (42 degrees C) than when grown at the restrictive temperature (30 degrees C).     

On the functional organization of the arg ECBH cluster of genes in Escherichia coli K-12

Summary Among the four seemingly adjacent loci of the argECBH cluster of E. coli K-12, the last three are shown to belong to the same unit of coordinated expression; the latter exhibits a clockwise polarity in contrast to all other known E. coli operons, except the cluster governing the synthesis of the pyruvate dehydrogenase complex.The analysis of several deletion and nonsense mutants suggests that argE (the expression of which is not strictly correlated with the functioning of the argCBH group) has the same polarity but is not integrated with the three other genes into one operon.Between polar argC B and B mutants the coefficient of repressibility of enzyme H synthesis varies widely. This feature resembles the reduced repressibility of distal gene activity found in polar mutants in the tryptophan operons of E. coli and S. typhimurium but not in the lac, gal (E. coli) and his (S. typhimurium) operons.Possible implications of the present results and some relevant data that have appeared in the recent literature are discussed.Research fellow of the Institute for the Encouragement of Scientific Research in Industry and Agriculture, Belgium.Research fellow of the National Fund of Scientific Research, Belgium.     

Purification of dihydrofolate reductase amplified in Escherichia coli K-12

The Escherichia coli strain carrying pTP 6-10 which was constructed in our previous work (Iwakura, M., et al. (1983) J. Biochem. 93, 927-930) produces more than 400-fold dihydrofolate reductase as compared with the strain without the plasmid. Dihydrofolate reductase was highly purified from the cell-free extract of the plasmid strain simply by two steps; ammonium sulfate fractionation and ion-exchange chromatography. By 10-fold purification, the enzyme was essentially homogeneous as judged by polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate. The restriction map of pTP 6-10 was also determined and the plasmid was shown to have an Ava I, an EcoR I, a Pst I, a Pvu I, and a Pvu II site. Our results indicate that the plasmid strain is suitable as a source of the enzyme and that plasmid pTP 6-10 is promising as a versatile plasmid vector for efficiently yielding the product of the cloned gene.     

The complete nucleotide sequence of the ilvGMEDA cluster of Escherichia coli K-12

The ilvGMEDA gene cluster of Escherichia coli K-12 has been the focus of intensive genetic and biochemical analysis for the past 30 years. Genetic regulation of the ilvGMEDA cluster involves attenuation, internal promoters, internal Rho-dependent termination sites, a site of polarity in the ilvG pseudogene of the wild-type organism, and autoregulation by the ilvA gene product, the biosynthetic L-threonine deaminase. We have now completed the nucleotide sequence of the 6600-bp cluster and have analyzed it, along with the ilvYC, ilvBN, and ilvIH genes, for codon frequencies and possible evolutionary relationships. The isoleucine content of each of the gene products of the ilvGMEDA cluster is quite similar (less than a two-fold variation), thus excluding one possible interpretation of the isoleucine-specific downstream amplification phenomenon. There is no evidence for retrograde evolution in the cluster since no significant homologies are detectable among genes that catalyze sequential reactions of the pathway. A highly significant homology does exist, however, between the threonine deaminases of yeast mitochondria and E. coli. The sequence at the boundary of the ilvA and ilvD genes is TAATAATG, so that the second TAA stop codon of ilvD overlaps the ATG initiation codon of ilvA.     

Molecular cloning of the Escherichia coli K-12 entACGBE genes

Abstract A 7-kb piece of Escherichia coli DNA that contains five genes ( entA, C, G, B and E ) required for the biosynthesis of the iron transport molecule enterochelin was isolated. A restriction map was constructed and proteins specified by the E. coli DNA were identified in mini- and maxicell systems. Plasmids containing portions of the entACGBE DNA generated by BAL31 digestion or restriction enzyme treatment were constructed; complementation studies done with these indicated that the five genes constitute an operon. The approximate site of the promoter was determined and the product of entE was tentatively identified as an M _r 63000 polypeptide.