Oxygen regulation of L-1,2-propanediol oxidoreductase activity in Escherichia coli.

Regardless of the respiratory conditions of the culture, Escherichia coli synthesizes an active propanediol oxidoreductase. Under anaerobic conditions, the enzyme remained fully active and accomplished its physiological role, while under aerobic conditions, it was inactivated in a process that did not depend on protein synthesis or on the presence of a carbon source.     

Crystal structure of an iron-dependent group III dehydrogenase that interconverts L-lactaldehyde and L-1,2-propanediol in Escherichia coli

The FucO protein, a member of the group III "iron-activated" dehydrogenases, catalyzes the interconversion between L-lactaldehyde and L-1,2-propanediol in Escherichia coli. The three-dimensional structure of FucO in a complex with NAD(+) was solved, and the presence of iron in the crystals was confirmed by X-ray fluorescence. The FucO structure presented here is the first structure for a member of the group III bacterial dehydrogenases shown experimentally to contain iron. FucO forms a dimer, in which each monomer folds into an alpha/beta dinucleotide-binding N-terminal domain and an all-alpha-helix C-terminal domain that are separated by a deep cleft. The dimer is formed by the swapping (between monomers) of the first chain of the beta-sheet. The binding site for Fe(2+) is located at the face of the cleft formed by the C-terminal domain, where the metal ion is tetrahedrally coordinated by three histidine residues (His200, His263, and His277) and an aspartate residue (Asp196). The glycine-rich turn formed by residues 96 to 98 and the following alpha-helix is part of the NAD(+) recognition locus common in dehydrogenases. Site-directed mutagenesis and enzyme kinetic assays were performed to assess the role of different residues in metal, cofactor, and substrate binding. In contrast to previous assumptions, the essential His267 residue does not interact with the metal ion. Asp39 appears to be the key residue for discriminating against NADP(+). Modeling L-1,2-propanediol in the active center resulted in a close approach of the C-1 hydroxyl of the substrate to C-4 of the nicotinamide ring, implying that there is a typical metal-dependent dehydrogenation catalytic mechanism.     

Mapping of insertion element IS5 in the Escherichia coli K-12 chromosome. Chromosomal rearrangements mediated by IS5

We identified phage clones containing insertion element IS5 in a set of 476 lambda phage clones carrying chromosomal segments that cover almost the entire chromosome of Escherichia coli K-12 W3110. Precise locations and orientations of IS5 were then determined by cleavage analysis of phage DNAs containing them. We mapped 23 copies of IS5 (named is5A to is5W) on the W3110 chromosome. Among them, ten were identified as the common elements present at the same locations in both chromosomes of W3110 and another E. coli K-12 strain, JE5519. While most of the mapped IS5 elements were scattered over the W3110 chromosome, four copies of IS5 (designated is5L, is5M, is5N and is5O) were in a region representing tandem duplication of a DNA segment flanked by two copies of IS5. Interestingly, one unit of this DNA segment as well as a portion of it was seen also in a tandem array in a different region where two copies of IS5 (designated is5P and is5Q) were present. In particular two pairs of the mapped IS5 elements may have been involved in inversion of the chromosomal segments in two of the E. coli K-12 derivatives.     

Constitutive activation of L-fucose genes by an unlinked mutation in Escherichia coli.

Wild-type Escherichia coli cannot grow on L-1,2-propanediol; mutants that can do so have increased basal activity of an NAD-linked L-1,2-propanediol oxidoreductase. This enzyme belongs to the L-fucose system and functions normally as L-lactaldehyde reductase during fermentation of the methylpentose. In wild-type cells, the activity of this enzyme is fully induced only anaerobically. Continued aerobic selection for mutants with an improved growth rate on L-1,2-propanediol inevitably leads to full constitutive expression of the oxidoreductase activity. When this occurs, L-fuculose 1-phosphate aldolase concomitantly becomes constitutive, whereas L-fucose permease, L-fucose isomerase, and L-fuculose kinase become noninducible. It is shown in this study that the noninducibility of the three proteins can be changed by two different kinds of suppressor mutations: one mapping external to and the other within the fuc gene cluster. Both mutations result in constitutive synthesis of the permease, the isomerase, and the kinase, without affecting synthesis of the oxidoreductase and the aldolase. Since expression of the fuc structural genes is activated by a protein specified by the regulator gene fucR, and since all the known genes of the fuc system are clustered at minute 60.2 of the chromosome, the external gene in which the suppressor mutation can occur probably has an unrelated function in the wild-type strain. The internal suppressor mutation might be either in fucR or in the promoter region of the genes encoding the permease, the isomerase, and the kinase, if these genes belong to the same operon.     

Suppression of hypersensitivity of Escherichia coli acrB mutant to organic solvents by integrational activation of the acrEF operon with the IS1 or IS2 element

The AcrAB-TolC efflux pump plays an intrinsic role in resistance to hydrophobic solvents in Escherichia coli. E. coli OST5500 is hypersensitive to solvents due to inactivation of the acrB gene by insertion of IS30. Suppressor mutants showing high solvent resistance were isolated from OST5500. These mutants produced high levels of AcrE and AcrF proteins, which were not produced in OST5500, and in each mutant an insertion sequence (IS1 or IS2) was found integrated upstream of the acrEF operon, coding for the two proteins. The suppressor mutants lost solvent resistance on inactivation of the acrEF operon. The solvent hypersensitivity of OST5500 was suppressed by introduction of the acrEF operon with IS1 or IS2 integrated upstream but not by introduction of the operon lacking the integrated IS. It was concluded that IS integration activated acrEF, resulting in functional complementation of the acrB mutation. The acrB mutation was also complemented by a plasmid containing acrF or acrEF under the control of Plac. The wild-type tolC gene was found to be essential for complementation of the acrB mutation by acrEF. Thus, it is concluded that in these cells a combination of the proteins AcrA, AcrF, and TolC or the proteins AcrE, AcrF, and TolC is functional in solvent efflux instead of the AcrAB-TolC efflux pump.     

Expression of the histidine operon in rho mutants of Escherichia coli.

Expression of the Escherichia coli histidine operon was measured in four independently isolated sets of strains carrying ten different rho mutations. Rho factor does not act as a major regulatory element of histidine operon attenuation.     

Operator-constitutive mutants in the threonine operon of Escherichia coli K-12.

Three Escherichia coli K-12 mutant strains resistant to DL-alpha-amino-beta-hydroxyvaleric acid were isolated in which the expression of the thr operon is constitutive. The localization and dominance properties of the mutations involved, called thrO, are those of operator mutations. The gene sequence is OABC as suggested by earlier studies.     

Regulatory mutants of the aroF-tyrA operon of Escherichia coli K-12.

The regulatory region of the aroF-tyrA operon was fused to the chloramphenicol acetyltransferase (cat) gene on a plasmid vector. Expression of the cat gene was subject to repression by tyrR+. This fusion was used to isolate regulatory mutants with increased expression of the cat gene in which repression by tyrR+ was affected. Nucleotide sequencing of these mutants has led to the identification of three sites involved in the repression of aroF by tyrR+. The existence of a functional promoter divergently transcribing from the aroF regulatory region was also demonstrated by using the cat fusion vector. The expression of this promoter is also regulated by tyrR+.     

Metabolic engineering of Escherichia coli for the production of 1,2-propanediol from glycerol

Due to its availability, low‐price, and high degree of reduction, glycerol has become an attractive carbon source for the production of fuels and reduced chemicals. Using the platform we have established from the identification of key pathways mediating fermentative metabolism of glycerol, this work reports the engineering of Escherichia coli for the conversion of glycerol into 1,2‐propanediol (1,2‐PDO). A functional 1,2‐PDO pathway was engineered through a combination of overexpression of genes involved in its synthesis from the key intermediate dihydroxyacetone phosphate (DHAP) and the manipulation of the fermentative glycerol utilization pathway. The former included the overexpression of methylglyoxal synthase (mgsA), glycerol dehydrogenase (gldA), and aldehyde oxidoreductase (yqhD). Manipulation of the glycerol utilization pathway through the replacement of the native E. coli PEP‐dependent dihydroxyacetone kinase (DHAK) with an ATP‐dependent DHAK from C. freundii increased the availability of DHAP allowing for higher 1,2‐PDO production. Analysis of the major fermentative pathways indentified ethanol as a required co‐product while increases in 1,2‐PDO titer and yield were achieved through the disruption of the pathways for acetate and lactate production. Combination of these key metabolic manipulations resulted in an engineered E. coli strain capable of producing 5.6 g/L 1,2‐PDO, at a yield of 21.3% (w/w). This strain also performed well when crude glycerol, a by‐product of biodiesel production, was used as the substrate. The titer and yield achieved in this study were favorable to those obtained with the use of E. coli for the production of 1,2‐PDO from common sugars. Biotechnol. Bioeng. 2011; 108:867–879. © 2010 Wiley Periodicals, Inc.     

Evidence for an internal promoter in the Escherichia coli threonine operon.

Auxotrophic mutants of Halobacterium volcanii generated by chemical mutagenesis were used to demonstrate a native genetic transfer system in this extremely halophilic member of the class Archaeobacteria.