Glucitol-specific enzymes of the phosphotransferase system in Escherichia coli. Nucleotide sequence of the gut operon

The complete nucleotide sequence of the glucitol (gut) operon in Escherichia coli has been determined. The glucitol-specific Enzyme II and Enzyme III of the phosphoenolpyruvate:sugar phosphotransferase system as well as glucitol-6-phosphate dehydrogenase which are encoded by the gutA, gutB, and gutD genes of the gut operon, respectively, are predicted to consist of 506 (Mr = 54,018), 123 (Mr = 13,306), and 259 (Mr = 27,866) amino acyl residues, respectively. The hydropathic profile of the Enzyme IIgut revealed 7 or 8 long hydrophobic segments which may traverse the cell membrane as alpha-helices as well as 2 or 4 short strongly hydrophobic stretches which may traverse the membrane as beta-structure. The number of amino acyl residues in the sum of the molecular weights of the glucitol Enzyme II-III pair are nearly the same as those of the mannitol Enzyme II. The ratio of hydrophobic to hydrophilic amino acyl residues and the numbers of the hydrophobic segments are also nearly the same for both transport systems. However, no significant homology was found in the nucleotide or amino acyl sequences of the two systems. Glucitol-6-phosphate dehydrogenase was found to exhibit sequence homology with ribitol dehydrogenase. A repetitive extragenic palindromic sequence was found in the 3'-flanking region of the gutD gene, suggesting the presence of a gene downstream from the gutD gene.     

Regulation of gluconeogenesis by the glucitol enzyme III of the phosphotransferase system in Escherichia coli.

The gut operon was subcloned into various plasmid vectors (M. Yamada and M. H. Saier, Jr., J. Bacteriol. 169:2990-2994, 1987). Constitutive expression of the plasmid-encoded operon prevented utilization of alanine and Krebs cycle intermediates when they were provided as sole sources of carbon for growth. Expression of the gutB gene alone (encoding the glucitol enzyme III), subcloned downstream from either the lactose promoter or the tetracycline resistance promoter, inhibited utilization of the same compounds. On the other hand, overexpression of the gutA gene (encoding the glucitol enzyme II) inhibited the utilization of a variety of sugars as well as alanine and Krebs cycle intermediates by an apparently distinct mechanism. Phosphoenolpyruvate carboxykinase activity was greatly reduced in cells expressing high levels of the cloned gutB gene but was nearly normal in cells expressing high levels of the gutA gene. A chromosomal mutation in the gutR gene, which gave rise to constitutive expression of the chromosomal gut operon, also gave rise to growth inhibition on gluconeogenic substrates as well as reduced phosphoenolpyruvate carboxykinase activity. Phosphoenolpyruvate synthase activity in general varied in parallel with that of phosphoenolpyruvate carboxykinase. These results suggest that high-level expression of the glucitol enzyme III of the phosphotransferase system can negatively regulate gluconeogenesis by repression or inhibition of the two key gluconeogenic enzymes, phosphoenolpyruvate carboxykinase and phosphoenolpyruvate synthase.     

Genetic evidence for glucitol-specific enzyme III, an essential phosphocarrier protein of the Salmonella typhimurium glucitol phosphotransferase system.

Positive selection procedures were developed for the isolation of mutants defective in components of the glucitol-specific catabolic enzyme system in Salmonella typhimurium. gutA (enzyme IIgut-negative), gutB (enzyme IIIgut-negative), and gutC (constitutive for the glucitol operon) mutants were isolated and characterized biochemically and genetically. The gene order was shown to be gutCAB.     

Chromosomal localization of gut, fruC, and pfk mutations affecting genes involved in Bacillus subtilis D-glucitol catabolism.

Mutations affecting the genes involved in B. subtilis D-glucitol catabolism were mapped either by PBS1-mediated transduction or DNA-mediated transformation. It was shown that the genes gutA and gutB coding for the D-glucitol permease and the D-glucitol dehydrogenase, respectively, and regulatory locus gutR are clustered in a gut operon localized between purB and dal close to the pha marker. A mutation affecting fructokinase activity (fruC) was mapped near the gut markers. The fruC gene does not belong to the operon. A mutation affecting phosphofructokinase activity (pfk) was mapped between the leuA and aroG markers.     

Cloning,sequencing and partial characterisation of sorbitol transporter (srlT) gene encoding phosphotransferase system,glucitol/sorbitol-specific IIBC components of Erwinia herbicola ATCC 21998 – Cloning,sequencing and partial characterisation of sorbitol specific transporter (srlT) gene of Erwinia herbicola ATCC 21998

A DNA fragment of approximately 1500 bp, harbouring the sorbitol transport gene (srlT), was amplified from the chromosomal DNA of Erwinia herbicola ATCC 21998 by PCR and cloned in Escherichia coli JM109. Degenerate oligonucleotide primers used were designed based on the conserved regions in the gene sequences within the gut operon of E. coli (Gene Bank accession no. J02708) and the srl operon of Erwinia amylovora (Gene Bank accession no. Y14603). The cloned DNA fragment was sequenced and found to contain an open reading frame of 1473 nucleotides coding for a protein of 491 amino acids, corresponding to a mass of 52410 Da. The nucleotide sequence of this ORF was highly homologous to that of the gutA gene of Escherichia coli gut operon, the srlE gene of Shigella flexrni and the sorbitol transporter gene sequence of Escherichia coli K12 (Gene Bank accession nos. J02708, AE016987 and D90892 respectively). The protein sequence showed significant homology to that of the phosphotransferase system i.e. the glucitol/sorbitol-specific IIBC components of Escherichia coli and Erwinia amylovora (P56580, O32522). The cloned DNA fragment was introduced into a pRA90 vector and the recombinant was used for developing srlT mutants of Erwinia herbicola, by homologous recombination. Mutants obtained were unable to grow on minimal medium with sorbitol. The insertion of the pRA90 vector inside the srlT gene sequence of the mutants was confirmed by DNA-DNA hybridisation.     

Biochemical and genetic study of D-glucitol transport and catabolism in Bacillus subtilis.

The catabolic pathway of D-glucitol (sorbitol) in Bacillus subtilis Marburg 168M is characterized. It includes (i) a transport step catalyzed by a D-glucitol permease which is affected by the gutA mutations, (ii) an oxidation step of the intracellular D-glucitol catalyzed by a D-glucitol dehydrogenase, generating intracellular fructose, affected by gutB mutations, and (iii) phosphorylation of the intracellular fructose either at the C1 site or at the C6 site as described previously (A. Delobbe et al., Eur. J. Biochem., 66:485-491, 1976; A. Delobbe et al., EUR. J. Biochem. 51:503-510, 1975). Additional data are given concerning the phosphorylation of fructose by a fructokinase (fructose ATP 6-phosphotransferase), which is affected by the fruC mutation. The isolation of regulatory mutants affected in gutR that synthesize constitutively both the permease and the dehydrogenase indicates the existence of a D-glucitol operon in B. subtilis. Unlike the wild-type strain, these mutants are able to utilize D-xylitol as sole carbon source.     

Molecular population genetics of Escherichia coli: DNA sequence diversity at the celC, crr, and gutB loci of natural isolates.

The DNA sequences of three genes--celC, crr, and gutB--have been determined for each of 11 or 12 natural isolates of Escherichia coli from the ECOR collection. These genes encode the phosphoenolpyruvate-dependent phosphotransferase-system enzyme III proteins specific for beta-glucoside sugars (celC), glucose (crr), and glucitol (gutB), respectively. There is little evidence of recombination at or among these loci; among these strains, relationships inferred from each gene are largely consistent with each other and with the relationship inferred from multilocus enzyme electrophoresis. DNA sequence diversity is similar for all three genes, particularly when silent (synonymous) sites only are considered. This is surprising because there is much stronger codon usage bias at crr than at celC or gutB. The extent of divergence in the protein sequences encoded by these three genes varies considerably. The constitutively expressed glucose-specific enzyme is completely conserved. It is surprising that the inducible glucitol-specific enzyme, which is functional, is more variable than the cellobiose-specific enzyme, which is cryptic; the latter might be expected to be under less (if any) purifying selection.     

Bacterial metabolism of naphthalene: construction and use of recombinant bacteria to study ring cleavage of 1,2-dihydroxynaphthalene and subsequent reactions.

The reactions involved in the bacterial metabolism of naphthalene to salicylate have been reinvestigated by using recombinant bacteria carrying genes cloned from plasmid NAH7. When intact cells of Pseudomonas aeruginosa PAO1 carrying DNA fragments encoding the first three enzymes of the pathway were incubated with naphthalene, they formed products of the dioxygenase-catalyzed ring cleavage of 1,2-dihydroxynaphthalene. These products were separated by chromatography on Sephadex G-25 and were identified by 1H and 13C nuclear magnetic resonance spectroscopy and gas chromatography-mass spectrometry as 2-hydroxychromene-2-carboxylate (HCCA) and trans-o-hydroxybenzylidenepyruvate (tHBPA). HCCA was detected as the first reaction product in these incubation mixtures by its characteristic UV spectrum, which slowly changed to a spectrum indicative of an equilibrium mixture of HCCA and tHBPA. Isomerization of either purified product occurred slowly and spontaneously to give an equilibrium mixture of essentially the same composition. tHBPA is also formed from HCCA by the action of an isomerase enzyme encoded by plasmid NAH7. The gene encoding this enzyme, nahD, was cloned on a 1.95-kb KpnI-BglII fragment. Extracts of Escherichia coli JM109 carrying this fragment catalyzed the rapid equilibration of HCCA and tHBPA. Metabolism of tHBPA to salicylaldehyde by hydration and aldol cleavage is catalyzed by a single enzyme encoded by a 1-kb MluI-StuI restriction fragment. A mechanism for the hydratase-aldolase-catalyzed reaction is proposed. The salicylaldehyde dehydrogenase gene, nahF, was cloned on a 2.75-kb BamHI fragment which also carries the naphthalene dihydrodiol dehydrogenase gene, nahB. On the basis of the identification of the enzymes encoded by various clones, the gene order for the nah operon was shown to be p, A, B, F, C, E, D.     

Genes involved in the anaerobic degradation of ethylbenzene in a denitrifying bacterium,strain EbN1

Genes involved in anaerobic degradation of the petroleum hydrocarbon ethylbenzene in the denitrifying Azoarcus-like strain EbN1 were identified on a 56-kb DNA contig obtained from shotgun sequencing. Ethylbenzene is first oxidized via ethylbenzene dehydrogenase to (S)-1-phenylethanol; this is converted by (S)-1-phenylethanol dehydrogenase to acetophenone. Further degradation probably involves acetophenone carboxylase forming benzoylacetate, a ligase forming benzoylacetyl-CoA, and a thiolase forming acetyl-CoA and benzoyl-CoA. Genes of this pathway were identified via N-terminal sequences of proteins isolated from strain EbN1 and by sequence similarities to proteins from other bacteria. Ethylbenzene dehydrogenase is encoded by three genes (ebdABC), in accordance with the heterotrimeric enzyme structure. Binding domains for a molybdenum cofactor (in subunit EbdA) and iron/sulfur-clusters (in subunits EbdA and EbdB) were identified. The previously observed periplasmic location of the enzyme was corroborated by the presence of a twin-arginine leader peptide characteristic of the Tat system for protein export. A fourth gene (ebdD) was identified, the product of which may act as an enzyme-specific chaperone in the maturation of the molybdenum-containing subunit. A distinct gene (ped) coding for (S)-1-phenylethanol dehydrogenase apparently forms an operon with the ebdABCD genes. The ped gene product with its characteristic NAD(P)-binding motif in the N-terminal domain belongs to the short-chain dehydrogenase/reductase (SDR) superfamily. A further operon apparently contains five genes (apc1-5) suggested to code for subunits of acetophenone carboxylase. Four of the five gene products are similar to subunits of acetone carboxylase from Xanthobacter autotrophicus. Upstream of the apc genes, a single gene (bal) was identified which possibly codes for a benzoylacetate CoA-ligase and which is co-transcribed with the apc genes. In addition, an apparent operon containing almost all genes required for beta-oxidation of fatty acids was detected; one of the gene products may be involved in thiolytic cleavage of benzoylacetyl-CoA. The DNA fragment also included genes for regulatory systems; these were two sets of two-component systems, two LysR homologs, and a TetR homolog. Some of these proteins may be involved in ethylbenzene-dependent gene expression.     

Multiple regulatory elements for the glpA operon encoding anaerobic glycerol-3-phosphate dehydrogenase and the glpD operon encoding aerobic glycerol-3-phosphate dehydrogenase in Escherichia coli: further characterization of respiratory control.

In Escherichia coli, sn-glycerol-3-phosphate can be oxidized by two different flavo-dehydrogenases, an anaerobic enzyme encoded by the glpACB operon and an aerobic enzyme encoded by the glpD operon. These two operons belong to the glp regulon specifying the utilization of glycerol, sn-glycerol-3-phosphate, and glycerophosphodiesters. In glpR mutant cells grown under conditions of low catabolite repression, the glpA operon is best expressed anaerobically with fumarate as the exogenous electron acceptor, whereas the glpD operon is best expressed aerobically. Increased anaerobic expression of glpA is dependent on the fnr product, a pleiotropic activator of genes involved in anaerobic respiration. In this study we found that the expression of a glpA1(Oxr) (oxygen-resistant) mutant operon, selected for increased aerobic expression, became less dependent on the FNR protein but more dependent on the cyclic AMP-catabolite gene activator protein complex mediating catabolite repression. Despite the increased aerobic expression of glpA1(Oxr), a twofold aerobic repressibility persisted. Moreover, anaerobic repression by nitrate respiration remained normal. Thus, there seems to exist a redox control apart from the FNR-mediated one. We also showed that the anaerobic repression of the glpD operon was fully relieved by mutations in either arcA (encoding a presumptive DNA recognition protein) or arcB (encoding a presumptive redox sensor protein). The arc system is known to mediate pleiotropic control of genes of aerobic function.     

Transcriptional analysis of the Azospirillum brasilense indole-3-pyruvate decarboxylase gene and identification of a cis-acting sequence involved in auxin responsive expression

Expression of the Azospirillum brasilense ipdC gene, encoding an indole-3-pyruvate decarboxylase, a key enzyme in the production of indole-3-acetic acid (IAA) in this bacterium, is upregulated by IAA. Here, we demonstrate that the ipdC gene is the promoter proximal gene in a bicistronic operon. Database searches revealed that the second gene of this operon, named iaaC, is well conserved evolutionarily and that the encoded protein is homologous to the Escherichia coli protein SCRP-27A, the zebrafish protein ES1, and the human protein KNP-I/GT335 (HES1), all of unknown function and belonging to the DJ-1/PfpI superfamily. In addition to this operon structure, iaaC is also transcribed monocistronically. Mutation analysis of the latter gene indicated that the encoded protein is involved in controlling IAA biosynthesis but not ipdC expression. Besides being upregulated by IAA, expression of the ipdC-iaaC operon is pH dependent and maximal at acidic pH. The ipdC promoter was studied using a combination of deletion analyses and site-directed mutagenesis. A dyadic sequence (ATTGTTTC(GAAT)GAAACAAT), centered at -48 was demonstrated to be responsible for the IAA inducibility. This bacterial auxin-responsive element does not control the pH-dependent expression of ipdC-iaaC.     

Molecular characterization of Lactobacillus plantarum genes for beta-ketoacyl-acyl carrier protein synthase III (fabH) and acetyl coenzyme A carboxylase (accBCDA), which are essential for fatty acid biosynthesis

Genes for subunits of acetyl coenzyme A carboxylase (ACC), which is the enzyme that catalyzes the first step in the synthesis of fatty acids in Lactobacillus plantarum L137, were cloned and characterized. We identified six potential open reading frames, namely, manB, fabH, accB, accC, accD, and accA, in that order. Nucleotide sequence analysis suggested that fabH encoded beta-ketoacyl-acyl carrier protein synthase III, that the accB, accC, accD, and accA genes encoded biotin carboxyl carrier protein, biotin carboxylase, and the beta and alpha subunits of carboxyltransferase, respectively, and that these genes were clustered. The organization of acc genes was different from that reported for Escherichia coli, for Bacillus subtilis, and for Pseudomonas aeruginosa. E. coli accB and accD mutations were complemented by the L. plantarum accB and accD genes, respectively. The predicted products of all five genes were confirmed by using the T7 expression system in E. coli. The gene product of accB was biotinylated in E. coli. Northern and primer extension analyses demonstrated that the five genes in L. plantarum were regulated polycistronically in an acc operon.