Involvement of lactaldehyde dehydrogenase in several metabolic pathways of Escherichia coli K12

Lactaldehyde dehydrogenase (E.C. 1.2.1.22) of Escherichia coli has been purified to homogeneity. It has four apparently equal subunits (molecular weight 55,000 each) and four NAD binding sites per molecule of native enzyme. The enzyme is inducible, only under aerobic conditions, by at least three different types of molecules, the sugars fucose and rhamnose, the diol ethylene glycol and the amino acid glutamate. The enzyme catalyzes the irreversible oxidation of several aldehydes with a Km in the micromolar range for alpha-hydroxyaldehydes (lactaldehyde, glyceraldehyde, or glycolaldehyde) and a higher Km, in the millimolar range, for the alpha-ketoaldehyde methylglyoxal. It displays substrate inhibition with all these substrates. NAD is the preferential cofactor. The functional and structural features of the enzyme indicate that it is not an isozyme of other E. coli aldehyde dehydrogenases such as glyceraldehyde phosphate dehydrogenase, glycolaldehyde dehydrogenase, or acetaldehyde dehydrogenase. The enzyme, previously described as specific for lactaldehyde, is thus identified as a dehydrogenase with a fairly general role in aldehyde oxidation, and it is probably involved in several metabolic pathways.     

Identification of Escherichia coli K12 YdcW protein as a gamma-aminobutyraldehyde dehydrogenase

Gamma-aminobutyraldehyde dehydrogenase (ABALDH) from wild-type E. coli K12 was purified to apparent homogeneity and identified as YdcW by MS-analysis. YdcW exists as a tetramer of 202+/-29 kDa in the native state, a molecular mass of one subunit was determined as 51+/-3 kDa. Km parameters of YdcW for gamma-aminobutyraldehyde, NAD+ and NADP+ were 41+/-7, 54+/-10 and 484+/-72 microM, respectively. YdcW is the unique ABALDH in E. coli K12. A coupling action of E. coli YgjG putrescine transaminase and YdcW dehydrogenase in vitro resulted in conversion of putrescine into gamma-aminobutyric acid.     

Crystal structure of lactaldehyde dehydrogenase from Escherichia coli and inferences regarding substrate and cofactor specificity

Aldehyde dehydrogenases catalyze the oxidation of aldehyde substrates to the corresponding carboxylic acids. Lactaldehyde dehydrogenase from Escherichia coli (aldA gene product, P25553) is an NAD(+)-dependent enzyme implicated in the metabolism of l-fucose and l-rhamnose. During the heterologous expression and purification of taxadiene synthase from the Pacific yew, lactaldehyde dehydrogenase from E. coli was identified as a minor (相似文献   

Pyridoxine-requiring mutants of Escherichia coli: glycolaldehyde dehydrogenase is not coded for by the pdxB gene.

Twenty-seven independent pyridoxineless mutants belonging to genetic linkage group I were assayed for glycolaldehyde dehydrogenase. Some mutants lacked enzyme activity entirely, and others showed activity ranging from very low to wild-type levels. Reversion to pyridoxine independence usually had no effect upon this activity. Transfer of the pyridoxine genes to a common host that had wild-type levels of enzyme activity made the recipient pyridoxineless without affecting the activity. These results negate the idea of an obligatory role for glycolaldehyde dehydrogenase in pyridoxine biosynthesis.     

Induction of the soxRS regulon of Escherichia coli by glycolaldehyde

The ability of short-chain sugars to cause oxidative stress has been examined using glycolaldehyde as the simplest sugar. Short-chain sugars autoxidize in air, producing superoxide and alpha,beta-dicarbonyls. In Escherichia coli the soxRS regulon mediates an oxidative stress response, which protects the cell against both superoxide-generating agents and nitric oxide. In superoxide dismutase-deficient E. coli mutants, glycolaldehyde induces fumarase C and nitroreductase A, which are regulated as members of the soxRS regulon. A mutational defect in soxRS eliminates that induction. This establishes that glycolaldehyde can cause induction of this defensive regulon. This effect of glycolaldehyde was oxygen-dependent, was not shown by glyoxal, and was not seen in the superoxide dismutase-replete parental strain, and it was abolished by a cell-permeable SOD mimetic. All of these suggest that superoxide radicals produced by the oxidation of glycolaldehyde played a key role in the induction.     

Green fluorescent protein functions as a reporter for protein localization in Escherichia coli

The use of green fluorescent protein (GFP) as a reporter for protein localization in Escherichia coli was explored by creating gene fusions between malE (encoding maltose-binding protein [MBP]) and a variant of gfp optimized for fluorescence in bacteria (GFPuv). These constructs encode hybrid proteins composed of GFP fused to the carboxy-terminal end of MBP. Fluorescence was not detected when the hybrid protein was synthesized with the MBP signal sequence. In contrast, when the MBP signal sequence was deleted, fluorescence was observed. Cell fractionation studies showed that the fluorescent MBP-GFP hybrid protein was localized in the cytoplasm, whereas the nonfluorescent version was localized to the periplasmic space. Smaller MBP-GFP hybrid proteins, however, exhibited abnormal fractionation. Expression of the gene fusions in different sec mutants, as well as signal sequence processing assays, confirmed that the periplasmically localized hybrid proteins were exported by the sec-dependent pathway. The distinction between fluorescent and nonfluorescent colonies was exploited as a scorable phenotype to isolate malE signal sequence mutations. While expression of hybrid proteins comprised of full-length MBP did not result in overproduction lethality characteristic of some exported beta-galactosidase hybrid proteins, synthesis of shorter, exported hybrid proteins was toxic to the cells. Purification of MBP-GFP hybrid protein from the different cellular compartments indicated that GFP is improperly folded when localized outside of the cytoplasm. These results suggest that GFP could serve as a useful reporter for genetic analysis of bacterial protein export and of protein folding.     

Identification of a segment of the Escherichia coli Tsx protein that functions as a bacteriophage receptor area.

The Escherichia coli outer membrane protein Tsx functions as a nucleoside-specific channel and serves as the receptor for colicin K and a number of T-even-type bacteriophages, including phage T6. To identify those segments of the Tsx protein that are important for its phage receptor function, we devised a selection and screening procedure which allowed us to isolate phage-resistant strains synthesizing normal amounts of Tsx. Three different Tsx-specific phages (T6, Ox1, and H3) were employed for the selection of phage-resistant derivatives of a strain expressing a tsx(+)-lacZ+ operon fusion, and 28 tsx mutants with impaired phage receptor function were characterized. Regardless of the Tsx-specific phage used for the initial mutant selection, cross-resistance against a set of six different Tsx phages invariably occurred. With one exception, these mutant Tsx proteins could still serve as a colicin K receptor. DNA sequence analysis of 10 mutant tsx genes revealed the presence of four distinct tsx alleles: two point mutations, an 18-bp deletion, and a 27-bp tandem duplication. In three isolates, Asn-249 was replaced by a Lys residue (tsx-504), and in four others, residue Asn-254 was replaced by Lys (tsx-505). The deletion (tsx-506; one isolate) removed six amino acids (residue 239 to residue 244) from the 272-residue Tsx polypeptide chain, and the DNA duplication (tsx-507; two isolates) resulted in the addition of nine extra amino acids (residue 229 to residue 237) to the Tsx protein. In contrast to the wild-type Tsx protein and the other mutant Tsx proteins the Tsx-507 protein was cleaved by trypsin when intact cells were treated with this protease. The Tsx proteins encoded by the four tsx alleles still functioned in deoxyadenosine uptake in vivo, demonstrating that their nucleoside-specific channel activity was not affected by the alterations that caused the loss of their phage receptor function. HTe changes in the Tsx polypeptide that confer resistance against the Tsx-specific phages are clustered in a small region near the carboxy terminus of Tsx. Our results are discussed in terms of a model for the topological organization of the carboxy-terminal end of the Tsx protein within the outer membrane.     

Pilus formation and protein secretion by the same machinery in Escherichia coli

The secreton (type II secretion) and type IV pilus biogenesis branches of the general secretory pathway in Gram-negative bacteria share many features that suggest a common evolutionary origin. Five components of the secreton, the pseudopilins, are similar to subunits of type IV pili. Here, we report that when the 15 genes encoding the pullulanase secreton of Klebsiella oxytoca were expressed on a high copy number plasmid in Escherichia coli, one pseudopilin, PulG, was assembled into pilus-like bundles. Assembly of the 'secreton pilus' required most but not all of the secreton components that are essential for pullulanase secretion, including some with no known homologues in type IV piliation machineries. Two other pseudopilins, pullulanase and two outer membrane-associated secreton components were not associated with pili. Thus, PulG is probably the major component of the pilus. Expression of a type IV pilin gene, the E.coli K-12 gene ppdD, led to secreton-dependent incorporation of PpdD pilin into pili without diminishing pullulanase secretion. This is the first demonstration that pseudopilins can be assembled into pilus-like structures.     

Identification of a trans-dominant mutation affecting proline dehydrogenase in Escherichia coli

L-Proline dehydrogenase catalyzes the oxidation of L-proline to delta 1-pyrroline-5-carboxylate, a reaction that is an important step in the utilization of proline as a carbon or nitrogen source by bacteria. A mutant of Escherichia coli K-12 lacking L-leucyl-tRNA:protein transferase had been found previously to contain about five times as much proline dehydrogenase activity as its parent strain. This difference has now been shown to be due to the presence in the parent strain of a previously unrecognized mutation. This mutation, which has been designated put-4977, specifically affects proline dehydrogenase rather than proline uptake. Although proline dehydrogenase remains inducible by L-proline in strains carrying the mutation, there is a premature cessation of differential synthesis during induction that results in a lower specific activity. The mutation shows about 50% P1-mediated cotransduction with pyrC and is therefore located at about 22 min on the E. coli chromosome. Merodiploids containing a normal F' factor still exhibit decreased enzyme activity, indicating that the put-4977 mutation is trans-dominant. The mutation cannot be detected in present stocks of the transferase-deficient mutant, suggesting that this mutant is a revertant for put-4977.     

Identification of the heat-inducible protein C15.4 as the groES gene product in Escherichia coli

The product of the Escherichia coli morphogenetic gene groES (mopB) was identified as the heat-inducible protein C15.4 by two-dimensional gel analysis of the products of wild-type and mutant alleles carried on the bacterial chromosome, on a hybrid plasmid, and on a transducing phage.     

Identification of the dITP- and XTP-hydrolyzing protein from Escherichia coli

A hypothetical 21.0 kDa protein (ORF O197) from Escherichia coli K-12 was cloned, purified, and characterized. The protein sequence of ORF O197 (termed EcO197) shares a 33.5% identity with that of a novel NTPase from Methanococcus jannaschii. The EcO197 protein was purified using Ni-NTA affinity chromatography, protease digestion, and gel filtration column. It hydrolyzed nucleoside triphosphates with an O6 atom-containing purine base to nucleoside monophosphate and pyrophosphate. The EcO197 protein had a strong preference for deoxyinosine triphosphate (dITP) and xanthosine triphosphate (XTP), while it had little activity in the standard nucleoside triphosphates (dATP, dCTP, dGTP, and dTTP). These aberrant nucleotides can be produced by oxidative deamination from purine nucleotides in cells; they are potentially mutagenic. The mutation protection mechanisms are caused by the incorporation into DNA of unwelcome nucleotides that are formed spontaneously. The EcO197 protein may function to eliminate specifically damaged purine nucleotide that contains the 6-keto group. This protein appears to be the first eubacterial dITP- and XTPhydrolyzing enzyme that has been identified.     

Evidence that TET protein functions as a multimer in the inner membrane of Escherichia coli.

The inner membrane TET (TetA) protein, which is involved in Tn10-mediated microbial tetracycline resistance, consists of two domains, alpha and beta, both of which are needed for tetracycline resistance and efflux (M.S. Curiale, L.M. McMurry, and S.B. Levy, J. Bacteriol. 157:211-217, 1984). Since tetracycline-sensitive mutants in one domain can partially complement sensitive mutants in the other domain and since some sensitive mutants show dominance over the wild type, a multimeric structure for TET in the membrane had been suggested. We have studied this possibility by using tetA-phoA gene fusions. We fused all but the last 40 base pairs of the tetA gene with the carboxy terminus of the phoA gene for alkaline phosphatase (PhoA), whose activity requires its dimerization in the periplasm. The tetA-phoA fusion protein was under control of the tetracycline-inducible regulatory system for the tetA gene. Induction led to the synthesis of a 78,000-dalton inner membrane protein. Tetracycline resistance was expressed at reduced levels, consistent with the terminal beta domain deletion. Alkaline phosphatase activity was also present, but at low levels, suggesting that some, but not all, of the fusion proteins had their carboxy-terminal ends in the periplasm. When wild-type or mutant TET proteins were present in the same cell with the fusion protein, the tetracycline resistance level was affected (raised or lowered); however, phosphatase activity was reduced only when TET proteins with intact or near-intact beta domains were present. These findings suggest that TET functions as a multimer and that intact beta domains, on TET molecules in the heterologous multimer, either allow fewer PhoA moieties to project into the periplasm or sterically hinder PhoA moieties from dimerizing.     

Identification of the Escherichia coli recN gene product as a major SOS protein.

The recA+ lexA+-dependent induction of four Escherichia coli SOS proteins was readily observed by two-dimensional gel analysis. In addition to the 38-kilodalton (kDa) RecA protein, which was induced in the greatest amounts and was readily identified, three other proteins of 115, 62, and 12 kDa were seen. The 115-kDa protein is the product of the uvrA gene, which is required for nucleotide excision repair and has previously been shown to be induced in the SOS response. The 62-kDa protein, which was induced to high intracellular levels, is the product of recN, a gene required for recBC-independent recombination. The recA and recN genes were partially derepressed in a recBC sbcB genetic background, a phenomenon which might account for the recombination proficiency of such strains. The 12-kDa protein has yet to be identified.     

Identification of functional domains of the Escherichia coli SeqA protein

The Escherichia coli SeqA protein, a negative regulator of chromosomal DNA replication, prevents the overinitiation of replication within one cell cycle by binding to hemimethylated G-mA-T-C sequences in the replication origin, oriC. In addition to the hemimethylated DNA-binding activity, the SeqA protein has a self-association activity, which is also considered to be essential for its regulatory function in replication initiation. To study the functional domains responsible for the DNA-binding and self-association activities, we performed a deletion analysis of the SeqA protein and found that the N-terminal (amino acid residues 1-59) and the C-terminal (amino acid residues 71-181) regions form structurally distinct domains. The N-terminal domain, which is not involved in DNA binding, has the self-association activity. In contrast, the C-terminal domain, which lacks the self-association activity, specifically binds to the hemimethylated G-mA-T-C sequence. Therefore, two essential SeqA activities, self-association and DNA-binding, are independently performed by the structurally distinct N-terminal and C-terminal domains, respectively.     

Identification of the AraE transport protein of Escherichia coli.

1. Two arabinose-inducible proteins are detected in membrane preparations from strains of Escherichia coli containing arabinose-H+ (or fucose-H+) transport activity; one protein has an apparent subunit relative molecular mass (Mr) of 36 000-37 000 and the other has Mr 27 000. 2. An araE deletion mutant was isolated and characterized; it has lost arabinose-H+ symport activity and the arabinose-inducible protein of Mr 36 000, but not the protein of Mr 27 000. 3. An araE+ specialized transducing phage was characterized and used to re-introduce the araE+ gene into the deletion strain, a procedure that restores both arabinose-H+ symport activity and the protein of Mr 36,000. 4. N-Ethylmaleimide inhibits arabinose transport and partially inhibits arabinose-H+ symport activity. 5. N-Ethylmaleimide modifies an arabinose-inducible protein of Mr 36 000-38 000, and arabinose protects the protein against the reagent. 6. These observations identify an arabinose-transport protein of Escherichia coli as the product of the araE+ gene. 7. The protein was recognized as a single spot staining with Coomassie Blue after two-dimensional gel electrophoresis.