Identification and characterization of the tktB gene encoding a second transketolase in Escherichia coli K-12.

We isolated a transposon Tn10 insertion mutant of Escherichia coli K-12 which could not grow on MacConkey plates containing D-ribose. Characterization of the mutant revealed that the level of the transketolase activity was reduced to one-third of that of the wild type. The mutation was mapped at 63.5 min on the E. coli genetic map, in which the transketolase gene (tkt) had been mapped. A multicopy suppressor gene which complemented the tkt mutation was cloned on a 7.8-kb PstI fragment. The cloned gene was located at 53 min on the chromosome. Subcloning and sequencing of a 2.7-kb fragment containing the suppressor gene identified an open reading frame encoding a polypeptide of 667 amino acids with a calculated molecular weight of 72,973. Overexpression of the protein and determination of its N-terminal amino acid sequence defined unambiguously the translational start site of the gene. The deduced amino acid sequence showed similarity to sequences of transketolases from Saccharomyces cerevisiae and Rhodobacter sphaeroides. In addition, the level of the transketolase activity increased in strains carrying the gene in multicopy. Therefore, the gene encoding this transketolase was designated tktB and the gene formerly called tkt was renamed tktA. Analysis of the phenotypes of the strains containing tktA, tktB, or tktA tktB mutations indicated that tktA and tktB were responsible for major and minor activities, respectively, of transketolase in E. coli.     

Nucleotide sequence of gene pfkB encoding the minor phosphofructokinase of Escherichia coli K-12

The nucleotide sequence of a 1.3-kb DNA fragment containing the entire pfkB gene which codes for Pfk-2 of Escherichia coli, a minor phosphofructokinase (Pfk) enzyme, is reported. The Pfk-2 protein subunit is encoded by 924 bp, has 308 amino acids and an Mr of 33 000. Like other weakly expressed E. coli genes the codon usage in the pfkB gene is random; there is no strong bias for the usage of major tRNA isoaccepting species, and the codon preference rules of Grosjean and Fiers [Gene, 18 (1982) 199-209] are followed. This is the first report of the complete gene sequence of a phosphofructokinase.     

6-phosphogluconolactonase mutants of Escherichia coli and a maltose blue gene

Mutants lacking an enzyme of the oxidative branch of the hexose monophosphate shunt, 6-phosphogluconolactonase (pgl), have been selected as a new class of glucose-negative derivatives of a phosphoglucose isomerase (pgi) mutant. Glucose negativity is not as complete as in mutants lacking phosphoglucose isomerase and glucose-6-phosphate dehydrogenase. Pgi(+), pgl(-) strains have been constructed by transduction and grow almost normally on glucose. Genetic mapping shows that pgl lies between chlD and att-lambda, in the same position as and identical with a blu gene described by Adhya and Schwartz. These blu mutants grown on maltose were recognized by their property to turn blue after treatment with iodine. It is not known how phosphogluconolactonase deficiency causes this reaction; it might be related to accumulation of 6-phosphogluconolactone.     

Identification of the rhaA, rhaB and rhaD gene products from Escherichia coli K-12

Washed cells of Peptostreptococcus products (strain Marburg), which were incubated in the presence of CO/CO2/N2 (50%/17%/33%; 200 kPa) catalyzed the synthesis of acetate from carbon monoxide. The rate of acetate formation from CO was stimulated more than threefold by the addition of sodium (10 mM); potassium did not effect acetate synthesis. The degree of stimulation was dependent on the sodium concentration; the dependence followed simple Michaelis-Menten kinetics. The apparent Km for sodium was determined to be about 2 mmol/l. Sodium also stimulated acetate synthesis from H2 plus CO2. In the absence of added sodium the formation of formate as an intermediate in methyl group synthesis was stimulated. It is suggested that the sodium dependent reaction(s) is one (or more) of the reactions involved in methyl group synthesis from CO2.     

Insertion mutagenesis of the gene encoding the ferrichrome-iron receptor of Escherichia coli K-12.

The ferrichrome-iron receptor of Escherichia coli K-12 encoded by the fhuA gene is a multifunctional outer membrane receptor with an Mr of 78,000. It is required for the binding and uptake of ferrichrome and is the receptor for bacteriophages T5, T1, phi 80, and UC-1 as well as for colicin M. The fhuA gene was cloned into pBR322, and the recombinant plasmid pGC01 was mutagenized by the insertion of 6-base-pair TAB (two amino acid Barany) linkers into CfoI and HpaII restriction sites distributed throughout the coding region. A library of 18 TAB linker insertions in fhuA was generated; 8 of the mutations were at CfoI sites and 10 were at HpaII sites. All mutations inserted a hexamer that encoded a unique SacI site. A large deletion in fhuA was also isolated by TAB linker mutagenesis. Except for the deletion mutant, all of the linker insertion mutant FhuA proteins were found in the outer membrane in amounts similar to those found in the wild type. Five of the linker insertion mutants were susceptible to cleavage by endogenous proteolytic activity: a second FhuA-related band that migrated at approximately 72 kilodaltons could be detected on Coomassie blue-stained gels and on Western blots (immunoblots) by using a carboxy terminus-specific anti-peptide antibody. Receptor functions were measured with the mutated genes present in a single copy on the chromosome. Some of the receptors conferred wild-type phenotypes: they demonstrated growth promotion by ferrichrome and the same efficiency of plating as that of wild-type FhuA; killing by colicin M was also unaffected. Several mutants were altered in their sensitivities to the lethal agents. TAB linker insertions after amino acids 69 and 128 abolished all receptor functions. Phage T5 id not bind to these mutant FhuA proteins in detergent extracts. The deletion mutant was also defective in all FhuA functions. Sensitivity to the lethal agents of cellsl that expressed mutant FhuAs with insertions after amino acids 59 and 135 was reduced by several orders of magnitude. Insertion at other selected sites decreased some or all receptor functions only slightly. An insertion after amino acid 321 selectively eliminated ferrichrome growth promotion. Finally, a strain carrying a mutant fhuA gene on the chromosome in which the linker insertion occurred after amino acid 82 showed a tonB phenotype. These subtle perturbations that were introduced into the FhuA protein resulted in changes in its stability and in the binding and uptake of its cognate ligands.     

Cloning of the Escherichia coli K-12 hemB gene.

An Escherichia coli heme-requiring, heme-permeable mutant had no detectable 5-aminolevulinate dehydratase or porphobilinogen deaminase activities. The gene which complemented this mutation was cloned to a high-copy-number plasmid, and porphobilinogen deaminase activity was restored to normal levels, but the synthesis of 5-aminolevulinate dehydratase increased 20- to 30-fold. A maxicell procedure confirmed that the gene cloned was hemB.     

rhs gene family of Escherichia coli K-12.

Two additional members of a novel Escherichia coli gene family, the rhs genes, have been cloned and characterized. The structures of these loci, rhsC and rhsD, have been compared with those of rhsA and rhsB. All four loci contain a homologous 3.7-kilobase-pair core. Sequence comparison of the first 300 nucleotides of the cores showed that rhsA, rhsB, and rhsC are closely related, with only 1 to 2% sequence divergence, whereas rhsD is 18% divergent from the others. The beginning of the core coincides with the initiation of an open reading frame that extends beyond the 300 nucleotides compared. Whether a protein product is produced from this open reading frame has not been established. However, nucleotide substitutions which differentiate the cores have highly conservative effects on the predicted protein products; this suggests that products are made from the open reading frame and are under severe selection. The four rhs loci have been placed on both the genetic and restriction maps of E. coli K-12. A fifth rhs locus remains to be characterized. In terms of size, number, and sequence conservation, the rhs genes make up one of the most significant repetitions in E. coli, comparable to the rRNA operons.     

Identification of the phoM gene product and its regulation in Escherichia coli K-12.

Plasmids containing the chromosome region of Escherichia coli encoding phoM, whose product is a positive regulator of alkaline phosphatase expression, were isolated from the Clarke and Carbon plasmid bank. A 9.9-kilobase EcoRI fragment of plasmid pLC17-39 (subcloned into pBR322) was able to complement both phoM and thrB mutations. Restriction endonuclease analysis and in vitro mutagenesis of the hybird plasmids enabled the localization of the phoM gene locus to 3 kilobases of the cloned chromosomal fragment. The phoM gene product was identified, with maxicell techniques, as a protein with an approximate molecular weight of 55,000. A phoM-lacZ protein fusion was constructed by using a plasmid carrying the phoM gene and a derivative of phage lambda, lambda plac Mu2. Restriction endonuclease analysis of the plasmid carrying the fusion indicated that phoM is transcribed in a clockwise direction on the circular E. coli chromosome. Analysis of strains bearing the fusion on a multiple-copy plasmid or integrated at the lambda attachment site of the chromosome indicated that the synthesis of the phoM gene product was unaffected by phosphate limitation of growth. The expression of the phoM gene was studied in strains with mutations in genes encoding effectors of the pho regulon. A threefold increase in phoM expression was seen in a phoU strain in comparison with the wild-type strain.     

Identification of yacE (coaE) as the structural gene for dephosphocoenzyme A kinase in Escherichia coli K-12

Dephosphocoenzyme A (dephospho-CoA) kinase catalyzes the final step in coenzyme A biosynthesis, the phosphorylation of the 3'-hydroxy group of the ribose sugar moiety. Wild-type dephospho-CoA kinase from Corynebacterium ammoniagenes was purified to homogeneity and subjected to N-terminal sequence analysis. A BLAST search identified a gene from Escherichia coli previously designated yacE encoding a highly homologous protein. Amplification of the gene and overexpression yielded recombinant dephospho-CoA kinase as a 22.6-kDa monomer. Enzyme assay and nuclear magnetic resonance analyses of the product demonstrated that the recombinant enzyme is indeed dephospho-CoA kinase. The activities with adenosine, AMP, and adenosine phosphosulfate were 4 to 8% of the activity with dephospho-CoA. Homologues of the E. coli dephospho-CoA kinase were identified in a diverse range of organisms.     

Insertion mutations in the dam gene of Escherichia coli K-12

The dam gene of E. coli can be inactivated by insertion of Tn9 or Mud phage. Strains bearing these mutations are viable indicating that the dam gene product is dispensable.     

Molecular cloning and DNA sequencing of the Escherichia coli K-12 ald gene encoding aldehyde dehydrogenase.

The gene ald, encoding aldehyde dehydrogenase, has been cloned from a genomic library of Escherichia coli K-12 constructed with plasmid pBR322 by complementing an aldehyde dehydrogenase-deficient mutant. The ald region was sequenced, and a single open reading frame of 479 codons specifying the subunit of the aldehyde dehydrogenase enzyme complex was identified. Determination of the N-terminal amino acid sequence of the enzyme protein unambiguously established the identity and the start codon of the ald gene. Analysis of the 5'- and 3'-flanking sequences indicated that the ald gene is an operon. The deduced amino acid sequence of the ald gene displayed homology with sequences of several aldehyde dehydrogenases of eukaryotic origin but not with microbial glyceraldehyde-3-phosphate dehydrogenase.     

nfi, the gene for endonuclease V in Escherichia coli K-12.

Endonuclease V is specific for single-stranded DNA or for duplex DNA that contains uracil or that is damaged by a variety of agents (B. Demple and S. Linn, J. Biol. Chem. 257:2848-2855, 1982). Thus, it may be a versatile DNA repair enzyme. The protein was purified to apparent homogeneity, and from its N-terminal sequence, its gene, nfi, was identified. nfi is immediately downstream of hemE, at kb 4208 (90.4 min) on the current chromosomal map of Escherichia coli K-12. This region was cloned, and plasmid insertion and deletion mutants were used to study its molecular organization. Although nfi is the third of four closely spaced, codirectional genes, it is expressed independently.     

Cloning of the adenylate cyclase gene of Escherichia coli K-12

The adenylate cyclase gene of Escherichia coli has been cloned on the plasmid vector pBR325. The hybrid plasmid pTH4 obtained has a molecular weight of 6,4 megadalton and represents pBR325 plasmid with the insertion of 2,8 megadalton in the Pst1 site. The cya mutant bacteria carrying pTH4 recover their ability to utilize mannitol, lactose and other carbohydrates as carbon sources, and lose this ability again in the case of rare spontaneous excision of the DNA insert from the Pst1 site. The phenotypical effect of pTH4 in cya mutants can be only seen in the crp+ genome. The strains carrying pTH4 are also characterized by the ability of beta-galactosidase induction under conditions of catabolite repression. Besides, the bacteria containing cya+ allele on the plasmid do not grow on glycerol, which seems to be caused by toxic concentrations of methylglyoxal formed as a result of the increased intracellular level of cyclic adenosine monophosphate.     

Molecular cloning of the fdhF gene of Escherichia coli K-12

Abstract The fdhF gene of Escherichia coli , coding for at least one component of benzyl viologen-linked formate dehydrogenase (FDH-BV) activity, was isolated on a ColE1- fdhF hybrid plasmid from the Clarke and Carbon colony bank.
Endonuclease restriction maps of this plasmid and its pBR322-subcloned derivative, pLW06, were constructed. Various hybrid plasmids were further obtained by deletion of endonuclease-cleaved fragments from pLW06 DNA. Their complementation pattern was analyzed after introduction into different fdhF mutant strains. The fdhF gene was shown to be located on a 5.5 kb Bam HI- Pvu II-DNA fragment, which restored FDH-BV activity to the wild-type level.