Clustering of genes for L-fucose dissimilation by Escherichia coli.

Aerobic and anaerobic L-fucose utilization by Escherichia coli involves an inducible trunk pathway mediated by a permease, an isomerase, a kinase, and an aldolase. Tn5 insertion mutants of a parental strain expressing this pathway constitutively were used to map the positions of the structural genes by transduction. Results from this and previous studies show that all of the structural genes of the L-fucose trunk pathway map between eno and argA at minute 60.2 of the chromosome.     

Proton-linked L-fucose transport in Escherichia coli.

1. Addition of L-fucose to energy-depleted anaerobic suspensions of Escherichia coli elicited an uncoupler-sensitive alkaline pH change diagnostic of L-fucose/H+ symport activity. 2. L-Galactose or D-arabinose were also substrates, but not inducers, for the L-fucose/H+ symporter. 3. L-Fucose transport into subcellular vesicles was dependent upon respiration, displayed a pH optimum of about 5.5, and was inhibited by protonophores and ionophores. 4. These results showed that L-fucose transport into E. coli was energized by the transmembrane electrochemical gradient of protons. 5. Neither steady state kinetic measurements nor assays of L-fucose binding to periplasmic proteins revealed the existence of a second L-fucose transport system.     

Cross-induction of the L-fucose system by L-rhamnose in Escherichia coli.

Dissimilation of L-fucose as a carbon and energy source by Escherichia coli involves a permease, an isomerase, a kinase, and an aldolase encoded by the fuc regulon at minute 60.2. Utilization of L-rhamnose involves a similar set of proteins encoded by the rha operon at minute 87.7. Both pathways lead to the formation of L-lactaldehyde and dihydroxyacetone phosphate. A common NAD-linked oxidoreductase encoded by fucO serves to reduce L-lactaldehyde to L-1,2-propanediol under anaerobic growth conditions, irrespective of whether the aldehyde is derived from fucose or rhamnose. In this study it was shown that anaerobic growth on rhamnose induces expression of not only the fucO gene but also the entire fuc regulon. Rhamnose is unable to induce the fuc genes in mutants defective in rhaA (encoding L-rhamnose isomerase), rhaB (encoding L-rhamnulose kinase), rhaD (encoding L-rhamnulose 1-phosphate aldolase), rhaR (encoding the positive regulator for the rha structural genes), or fucR (encoding the positive for the fuc regulon). Thus, cross-induction of the L-fucose enzymes by rhamnose requires formation of L-lactaldehyde; either the aldehyde itself or the L-fuculose 1-phosphate (known to be an effector) formed from it then interacts with the fucR-encoded protein to induce the fuc regulon.     

Mutations in two unlinked genes are required to produce asparagine auxotrophy in Escherichia coli.

Escherichia coli K-12 has two genes, asnA+ and asnB+, either one of which is able to satisfy the need of cells for asparagine. In order for a strain to have an auxotrophic requirement for asparagine, both genes must be mutationally inactivated. We obtained mutants with Tn5 inserted in asnB. asnB was mapped by conjugation and by three-factor P1 transductions at 15 min on the E coli K-12 linkage map, between ubiF and nagB. Specialized transducing phage lamba 781 supE was shown to carry asnB, as well as supE, ubiF, nagA, and nagB. asnA is the previously mapped ilv-linked asn locus, whiich is between uncB and rbs. E. coli C also has two asn genes, corresponding to asnA and asnB.     

Oligomerization-mediated activation of plasmid-borne genes in Escherichia coli

A plasmid carrying a weakly expressed neomycin phosphotransferase (neo) gene from the transposable element Tn5 was found to confer elevated levels of antibiotic resistance on its host cell when it existed in a non-monomeric state. This activation of the neo gene appeared to be a generalized effect which can be exerted on any plasmid-encoded gene, since specific sequences were not required for enhanced neo expression, and the activity of a plasmid-borne chloramphenicol acetyltransferase gene could be similarly induced by oligomerization. The potential role that multiple origins of replication present in such oligomeric plasmids play in these observed increases in gene expression is discussed.     

NAD-linked aldehyde dehydrogenase for aerobic utilization of L-fucose and L-rhamnose by Escherichia coli.

Mutant analysis revealed that complete utilization of L-fucose and L-rhamnose by Escherichia coli requires the activity of a common NAD-linked aldehyde dehydrogenase which converts L-lactaldehyde to L-lactate. Mutations affecting this activity mapped to the ald locus at min 31, well apart from the fuc genes (min 60) encoding the trunk pathway for L-fucose dissimilation (as well as L-1,2-propanediol oxidoreductase) and the rha genes (min 88) encoding the trunk pathway for L-rhamnose dissimilation. Mutants that grow on L-1,2-propanediol as a carbon and energy source also depend on the ald gene product for the conversion of L-lactaldehyde to L-lactate.     

C-shaped cells caused by expression of an ftsA mutation in Escherichia coli.

A plasmid, pDLL4, was isolated from a Tn5tac1 mutagenesis experiment with plasmid pZAQ. When pDLL4 was transformed into wild-type rod-shaped cells, it caused cells in the population to become curved (C-shaped or convoluted). The Tn5tac1 transposon was integrated within the carboxyl end of the ftsA gene in pDLL4. This mutation was designated ftsAc. Subcloning ftsAc DNA into another plasmid vector verified that the curved-cell phenotype was caused by the expression of this altered gene. DNA sequence analysis of the ftsAc mutation revealed that the transposition event changed the DNA so that the last 28 amino acids of the FtsA protein were lost and 5 new amino acids were added. A radioactive peptide band corresponding to this truncated FtsAc protein was identified by a T7 promoter-T7 polymerase protein labeling system. Observations of thin sections of these curved cells with an electron microscope revealed aggregates of striated cylindrical structures traversing the cytoplasm. The ends of these aggregates appear to be at or near the cell membrane. The linear periodicity of the cylinders was approximately 11 nm, and the diameter of a cylinder was about 15 nm. Aggregates of as many as five cylinders were arrayed diagonally to the long axis of the curved cells, a finding that suggests that some type of internal organization may be causing the curved cell shape.     

Suppressor genes of a dnaA temperature sensitive mutation in Escherichia coli

Summary Recombinant plasmids were constructed from EcoRI digests of Escherichia coli chromosomal DNA and pMB9 DNA by selecting for suppression of a dnaA–T46 temperature-sensitive mutation. Two types of plasmid capable of suppressing the dnaA mutation were isolated. They did not carry any genetic markers around dnaA and physical mapping with various restriction enzymes showed that neither of the plasmids contained the dnaA gene. One plasmid, pYT47, was characterized further and the protein responsible for the suppression was identified by two-dimensional gel electrophoresis. The molecular weight of the suppressor protein was about 68 Kdal and thus is clearly different from the dnaA gene product.     

Production of L-xylose from L-xylulose using Escherichia coli L-fucose isomerase

l-Xylulose was used as a raw material for the production of l-xylose with a recombinantly produced Escherichia colil-fucose isomerase as the catalyst. The enzyme had a very alkaline pH optimum (over 10.5) and displayed Michaelis-Menten kinetics for l-xylulose with a K_m of 41 mM and a V_max of 0.23 μmol/(mg min). The half-lives determined for the enzyme at 35 °C and at 45 °C were 6 h 50 min and 1 h 31 min, respectively. The reaction equilibrium between l-xylulose and l-xylose was 15:85 at 35 °C and thus favored the formation of l-xylose. Contrary to the l-rhamnose isomerase catalyzed reaction described previously [14]l-lyxose was not detected in the reaction mixture with l-fucose isomerase. Although xylitol acted as an inhibitor of the reaction, even at a high ratio of xylitol to l-xylulose the inhibition did not reach 50%.     

Thiol-sensitive genes of Escherichia coli.

The effect of 1-thioglycerol on the expression of genes of Escherichia coli was investigated. Pulse-labeled proteins from aerobically growing, 1-thioglycerol-treated E. coli were separated by two-dimensional gel electrophoresis, and their radioactivities were compared with those of identical proteins from nontreated cells. The first 10 min of exposure to thiol stimulated the synthesis of 10% of the observed proteins and inhibited the production of 16% of the proteins. After 30 min of growth with thiol, the synthesis of 44% of the observed proteins was inhibited and synthesis of 18% of the proteins was stimulated. In general, the expression of genes of carbohydrate metabolism, amino acid metabolism, and protein biosynthesis were inhibited, while nucleic acid synthetic and repair gene expressions showed mixed responses. Synthesis of transport proteins was not affected. Transient stimulation of oxidative-stress proteins and sustained stimulation of the expressions of trxB, ompA, and ompB genes and those of several unidentified gene products were also observed. Whether these complex responses merely reflect adjustments by cellular subsystems to a suddenly reducing environment or whether they are manifestations of a reductive-stress regulon will have to await genetic analysis of this phenomenon.     

Metabolism of L-fucose and L-rhamnose in Escherichia coli: aerobic-anaerobic regulation of L-lactaldehyde dissimilation.

L-Lactaldehyde is a branching point in the metabolic pathway of L-fucose and L-rhamnose utilization. Under aerobic conditions, L-lactaldehyde is oxidized to L-lactate by the enzyme lactaldehyde dehydrogenase, while under anaerobic conditions, L-lactaldehyde is reduced to L-1,2-propanediol by the enzyme propanediol oxidoreductase. Aerobic growth on either of the methyl pentoses induces a lactaldehyde dehydrogenase enzyme which is inhibited by NADH and is very stable under anaerobic conditions. In the absence of oxygen, the cell shifts from the oxidation of L-lactaldehyde to its reduction, owing to both the induction of propanediol oxidoreductase activity and the decrease in the NAD/NADH ratio. The oxidation of L-lactaldehyde to L-lactate is again restored upon a change to aerobic conditions. In this case, only the NAD/NADH ratio may be invoked as a regulatory mechanism, since both enzymes remain active after this change. Experimental evidence in the presence of rhamnose with mutants unable to produce L-lactaldehyde and mutants capable of producing but not further metabolizing it points toward L-lactaldehyde as the effector molecule in the induction of lactaldehyde dehydrogenase. Analysis of a temperature-sensitive mutation affecting the synthesis of lactaldehyde dehydrogenase permitted us to locate an apparently single regulator gene linked to the ald locus at 31 min and probably acting as a positive control element on the expression of the structural gene.     

Metabolism of L-fucose and L-rhamnose in Escherichia coli: differences in induction of propanediol oxidoreductase.

Escherichia coli is capable of growing on L-fucose or L-rhamnose as a sole source of carbon and energy. When grown under anaerobic conditions on either sugar, a nicotinamide adenine dinucleotide-linked L-lactaldehyde:propanediol oxidoreductase activity is induced. The functioning of this enzyme results in the regeneration of oxidized nicotinamide adenine dinucleotide. Conditions of induction of the enzyme activity were studied and were found to display different characteristics on each sugar. In the rhamnose-grown cells, the increase in enzyme activity detected under inducing conditions was accompanied by the synthesis of propanediol oxidoreductase, as measured by the appearance in the extracts of a protein that reacts with propanediol oxidoreductase antibodies. In contrast, in fucose-grown cells, the level of propanediol oxidoreductase as measured by enzyme antibody-reacting material was high under noninducing and inducing conditions. Thus, the increase in enzyme activity detected in going from noninducing to inducing conditions in fucose-grown cells did not depend on the appearance of the specific protein but on the activation of the propanediol oxidoreductase already present in the cells in an inactive form. The propanediol oxidoreductase of both homologous systems should consequently be regulated by different control mechanisms.     

Complementation of an Escherichia coli proC mutation by a gene cloned from Treponema pallidum.

Little is known concerning the biosynthetic and metabolic capabilities of the syphilis agent, Treponema pallidum, because of the inability to cultivate continuously the organism in vitro. To circumvent the problem of cultivation, researchers have used recombinant DNA technology to express treponemal protein antigens in Escherichia coli. However, with a few notable exceptions, the specific cellular roles of these cloned treponemal proteins have not been determined. In this study, a cosmid library of T. pallidum genomic DNA was constructed and amplified by repackaging infective lambda bacteriophage particles in vivo. Recombinant clones capable of complementing a null mutation in the E. coli proC gene encoding 1-pyrroline-5-carboxylate (P5C) reductase (EC 1.5.1.2) were subsequently identified. The complementing activity was eventually localized to a 2.3-kilobase BglII-HindIII fragment that hybridized to the same-size fragment of a BglII-HindIII digest of T. pallidum DNA. Two proteins of 41 and 27 kilodaltons (kDa) were encoded by this fragment, as determined by maxicell analysis. Although only the 41-kDa protein could be specifically precipitated by experimental syphilitic rabbit antisera, it was the 27-kDa protein that was responsible for the proC-complementing activity. The recombinant P5C reductase differed from the native E. coli enzyme by a number of biochemical properties. The cloning of a T. pallidum gene encoding P5C reductase strongly suggests that this pathogen has the ability to synthesize proline and possibly other amino acids.     

EcoCyc: an encyclopedia of Escherichia coli genes and metabolism.

The encyclopedia of Escherichia coli genes and metabolism (EcoCyc) is a database that combines information about the genome and the intermediary metabolism of E.coli. It describes 2034 genes, 306 enzymes encoded by these genes, 580 metabolic reactions that occur in E.coli and the organization of these reactions into 100 metabolic pathways. The EcoCyc graphical user interface allows query and exploration of the EcoCyc database using visualization tools such as genomic map browsers and automatic layouts of metabolic pathways. EcoCyc spans the space from sequence to function to allow investigation of an unusually broad range of questions. EcoCyc can be thought of as both an electronic review article, because of its copious references to the primary literature, and as an in silico model of E.coli that can be probed and analyzed through computational means.     

Influence of gyrA mutation on expression of Erwinia chrysanthemi clb genes cloned in Escherichia coli.

Erwinia chrysanthemi clb genes cloned into Nals Escherichia coli allowed growth on cellobiose, arbutin, or salicin. In contrast, Nalr isogenic strains grew only on cellobiose. It is proposed that expression of cloned E. chrysanthemi clb genes is reduced by the E. coli chromosomal gyrA (Nalr) mutation, resulting in apparent segregation of the Clb and Arb Sal characters.