Regulation of the divergent guaBA and xseA promoters of Escherichia coli by the cyclic AMP receptor protein.

The gua promoter (guaP) of Escherichia coli resembles those for ribosomal RNA (rrn) operons and lies in a close back-to-back arrangement with the promoter for xseA (xseP). Transcription from guaP is subject to stringent control and growth-rate-dependent regulation, and to repression by DnaA and PurR. In addition, transcription from guaP is regulated by the cyclic AMP receptor protein (CRP).

The purB gene of Escherichia coli K-12 is located in an operon.

The structural gene (purB) for succinyl-AMP (S-AMP) lyase and three additional ORFs are on the same DNA strand of the chromosome of Escherichia coli. Cassette mutagenesis and primer extension mapping demonstrated that purB is co-transcribed with an upstream gene (ORF23, or ycfC) encoding a 22.9 kDa membrane-associated protein of non-essential, but unknown, function unrelated to purine biosynthesis.

Specific binding of DnaA protein to a DnaA box in the guaB gene of Escherichia coli K12.

Expression of the guaBA operon of Escherichia coli is regulated by the DNA replication-initiating protein, DnaA. Two DnaA boxes, which are potential binding sites for DnaA, are present in the gua operon. One box (with 8/9 match to the DnaA box consensus sequence) is at the gua promoter; the other box, which has a consensus sequence, is on the non-transcribed strand within the guaB coding region approximately 200 bp downstream of the initiation codon.

Stringent and growth-rate-dependent control of the gua operon of Escherichia coli K-12.

The promoter of the gua operon has been located by transcript mapping using primer extension with reverse transcriptase. The surrounding nucleotide sequence has features characteristic of promoters under stringent and growth-rate-dependent regulation, namely a GC-rich discriminator next to the -10 hexamer, an upstream AT-rich sequence (the UP element) and potential FIS-binding sites. Transcriptional activity of the gua promoter was examined using transcriptional fusions to lacZ placed at a single chromosomal location.

Regulation of the gua operon of Escherichia coli by the DnaA protein.

The guaBA operon determines production of the two enzymes required to convert hypoxanthine to guanine at the nucleotide level during guanine nucleotide biosynthesis. Two DnaA boxes, binding sites for the DNA replication-initiating DnaA protein, are present in the gua operon, one at the gua promoter (guaP) and the other within the guaB coding sequence. Regulation of the guaBA operon by DnaA protein was studied using strains carrying chromosomal gua-lacZ fusions.

Secondary attachment site for bacteriophage lambda in the guaB gene of Escherichia coli.

lambda gua transducing bacteriophages were used to identify and sequence the secondary attachment site for lambda in the guaB gene of Escherichia coli. The sequence matched the primary core sequence at nine positions, and a putative integrase binding-site overlapped the left core-arm junction. Recombinational crossover occurred between nucleotides -3 and +2 of the core region.

Nucleotide sequence and organisation of the gua promoter region of Escherichia coli.

Overlapping restriction fragments of DNA carrying the gua promoter region of Escherichia coli have been cloned using promoter-probe plasmids. Antibiotic resistance conferred by the constructed plasmids is repressed by guanine and enhanced by adenine, two features characteristic of expression of the gua operon. The nucleotide sequence of these fragments reveals the gua promoter 43 bp upstream of the translational start codon for inosine 5'-monophosphate (IMP) dehydrogenase.

Molecular cloning and characterisation of the gua regulatory region of Escherichia coli K12.

The regulatory region of the gua operon of Escherichia coli is contained within a 2.1 kb EcoR1 restriction fragment isolated from a lambda pgua transducing phage. This DNA fragment was inserted into pPV33-II, a promoter-cloning vector, where it activated the gene(s) for tetracycline resistance.

Citrate synthase activity in Escherichia coli harbouring hybrid plasmids containing the gltA gene.

A hybrid plasmid, pDB2, was constructed by ligating a 3.24 kb EcoRI/HindIII fragment of the Escherichia coli chromosome into pBR322. This was used to transform a gltA mutant which was devoid of citrate synthase activity.

Construction and characterization of guaB-lacZ fusions in Escherichia coli K12.

GuaB-lacZ fusion strains of Escherichia coli K12 have been constructed using the Casadaban (1976) gene-fusion technique. The major modification to the procedure was the removal of the guanine requirement of Mu-induced guaA auxotrophs by introduction of a ColEl-gua+ plasmid prior to selection of the fusion. Conversion to prototrophy was necessary to overcome problems associated with repression of the gua operon by guanine added to selection media.

Dual control of the gua operon of Escherichia coli K12 by adenine and guanine nucleotides.

The gua operon of Escherichia coli K12 comprises structural genes for the two enzymes, IMP dehydrogenase and GMP synthetase, required for the biosynthesis of GMP from IMP. The specific activities of these enzymes were measured in various purine auxotrophs. GuaA and guaB mutants (guanine-specific) were depressed under conditions of growth limitation by guanine but were repressed by excess guanine.

Active-site modification of native and mutant forms of inosine 5'-monophosphate dehydrogenase from Escherichia coli K12.

IMP dehydrogenase of Escherichia coli was irreversibly inactivated by Cl-IMP (6-chloro-9-beta-d-ribofuranosylpurine 5'-phosphate, 6-chloropurine ribotide). The inactivation reaction showed saturation kinetics. 6-Chloropurine riboside did not inactivate the enzyme.

Complementation in vitro between guaB mutants of Escherichia coli K12.

Guanine auxotrophs of Escherichia coli were isolated following mutagenesis by N-methyl-N'-nitro-N-nitrosoguanidine or ethyl methanesulphonate. The mutants were classified according to growth properties and absence of IMP dehydrogenase or GMP synthetase activity. Mutations in guaB (IMP dehydrogenase-less) were analysed by reversion and suppression tests; all were of the base substitution missense type except for one possible frameshift and one polar nonsense mutation.

Inosine 5'-monophosphate dehydrogenase of Escherichia coli. Purification by affinity chromatography, subunit structure and inhibition by guanosine 5'-monophosphate.

Escherichia coli IMP dehydrogenase (EC 1.2.1.

The gua operon of Escherichia coli K-12: evidence for polarity from guaB to guaA.

Guanine auxotrophs of Escherichia coli K-12 were isolated after mutagenesis with N-methyl-N'-nitro-N-nitrosoguanidine, ethyl methane sulfonate, or the acridine mustard ICR 372. guaA (xanthosine 5'-monophosphate [XMP] aminase-less) mutants were distinguished from guaB (inosine 5'-monophosphate [IMP] dehydrogenase-less) mutants by their growth response to xanthine and by enzyme assay. Mutations were classified as base substitutions or frameshift on the basis of mutagen-induced reversion patterns.

Inosine 5'-monophosphate dehydrogenase of Escherichia coli K12: the nature of the inhibition by guanosine 5'-monophosphate.

When IMP is the variable substrate, IMP dehydrogenase (EC 1.2.1.

Microbial metabolism of C 1 and C 2 compounds. The involvement of glycollate in the metabolism of ethanol and of acetate by Pseudomonas AM1.

Pseudomonas AM1 grows on ethanol with a mean generation time of about 10h. A single alcohol dehydrogenase is responsible for oxidation of both methanol and ethanol. It is proposed that the glyoxylate bypass does not operate in Pseudomonas AM1 during growth on ethanol.

Microbial metabolism of C 1 and C 2 compounds. The role of glyoxylate, glycollate and acetate in the growth of Pseudomonas AM1 on ethanol and on C 1 compounds.

Succinate (or a product of succinate metabolism) is a catabolite repressor of some enzymes of the serine pathway (hydroxypyruvate reductase, serine-glyoxylate aminotransferase and glycerate kinase) but not of methanol dehydrogenase nor methylamine dehydrogenase. A mutant (PCT64) of Pseudomonas AM1, which is unable to grow on C(1) compounds, lacks glycerate kinase, showing that this enzyme is essential for the operation of the serine pathway. Mutant PCT48, unable to convert acetate into glycollate, has lost the ability to grow both on C(1) compounds and on ethanol.