Characterization of the RcsC→YojN→RcsB Phosphorelay Signaling Pathway Involved in Capsular Synthesis in Escherichia coli

Escherichia coli and other enteric microorganisms produce an extracellular polysaccharide capsule, called colanic acid, under certain environmental conditions. This capsular synthesis is regulated by the RcsC (sensor kinase)→YojN (phosphotransfer intermediate)→RcsB (response regulator) phosphorelay signal transduction under certain growth conditions. Nonetheless, little is known about signals that exaggerate the Rcs-system. To gain insight into signals that activate the Rcs-system, here we searched for genes that activate the Rcs-system, provided that those on a multicopy plasmid were introduced into E. coli. We identified several such genes, namely, rcsB, rcsA, djlA, lolA, and ompG. The DjlA, LolA, and OmpG proteins are particularly interesting in that they are all located on the cell surface, where the primary sensor RcsC histidine-kinase is localized. Implications of these findings are discussed with special reference to the mechanism by which RcsC perceives external signals.     

Engineered Escherichia coli for Short-Chain-Length Medium-Chain-Length Polyhydroxyalkanoate Copolymer Biosynthesis from Glycerol and Dodecanoate

Short-chain-length medium-chain-length polyhydroxyalkanoate (SCL-MCL PHA) copolymers are promising as bio-plastics with properties ranging from thermoplastics to elastomers. In this study, the hybrid pathway for the biosynthesis of SCL-MCL PHA copolymers was established in recombinant Escherichia coli by co-expression of β-ketothiolase (PhaA _Re ) and NADPH-dependent acetoacetyl-CoA reductase (PhaB _Re ) from Ralstonia eutropha together with PHA synthases from R. eutropha (PhaC _Re ), Aeromonas hydrophila (PhaC _Ah ), and Pseudomonas putida (PhaC2 _Pp ) and with (R)-specific enoyl-CoA hydratases from P. putida (PhaJ1 _Pp and PhaJ4 _Pp ), and A. hydrophila (PhaJ _Ah ). When glycerol supplemented with dodecanoate was used as primary carbon source, E. coli harboring various combinations of PhaABCJ produced SCL-MCL PHA copolymers of various monomer compositions varying from C₄ to C₁₀. In addition, polymer property analysis suggested that the copolymers produced from this recombinant source have thermal properties (lower glass transition and melting temperatures) superior to polyhydroxybutyrate homopolymer.     

Production of Hydroxlated Flavonoids with Cytochrome P450 BM3 Variant F87V and Their Antioxidative Activities

A variant of P450 BM3 with an F87V substitution [P450 BM3 (F87V)] is a substrate-promiscuous cytochrome P450 monooxygenase. We investigated the bioconversion of various flavonoids (favanones, chalcone, and isoflavone) by using recombinant Escherichia coli cells, which expressed the gene coding for P450 BM3 (F87V), to give their corresponding hydroxylated products. Potent antioxidative activities were observed in some of the products.     

Phytotoxic Metabolites of Phyllosticta sp.

The additional new metabolites, named phyllostine (II) and 3-chloro-2,5-dihydroxybenzyl alcohol (III) from the cultural filtrates of Phyllosticta sp. have been isolated. The chemical structure of II have been established by spectral studies and chemical conversion from the known I, and the chlorine-containing metabolite (III) by spectral and synthetic studies. The metabolite (II) exhibits the similar phytotoxic effects as I, but the metabolite III does less phytotoxicity than I and II on the leaf test.     

Effect of 1-n-Decylimidazole on Gibberellin Biosynthesis in Tan-ginbozu,a Dwarf Variety of Rice

Previous structural studies of less-polar dimers in autoxidized methyl linoleate (ML) have been extended to polar dimers. After isolation by successive silicic acid and gel permeation chromatography, the dimeric fraction of linoleate was separated into two major fractions, A₁ and A₂, according to their polarities. The polar dimers (A₁) were further fractionated by HPLC either directly or after reduction with triphenyl phosphine on a micro silica column. Isolated subfractions were characterized by UV, IR, GC-MS and FD-MS after suitable derivatizations. FD-MS of all these dimers showed a molecular ion peak which corresponds to 2 × ML + 6 × O and the reduction of each subfraction with stannous chloride gave equimolar amounts of 9 and 13-hydroxy octadecadienoate, and 9, 10, 13 and/or 9, 12, 13-trihydroxy octadecenoate. These results combined with others show that the A₁ dimers are composed of isomeric mixtures containing a peroxide bridge linking a methyl octadecadienoate and a 9, 12 and/or 10, 13-dihydroperoxy octadecenoate across C-9 and/or 13 on each of them.     

Isolation and Sequences of Peptic Peptides,and the Complete Sequence of Ile Chain of Ricin D

Sixteen peptic peptides, which contain arginine(s) or lysine, were isolated from cyanogen bromide fragments CB I and CB II of Ile-chain. Sequence determination has been performed on most of these peptides to provide overlaps for the tryptic peptides. Thus, complete amino acid sequence of Ile-chain consisting of 265 amino acid residues was determined. Some structural characteristics of the protein are also discussed.     

Synthesis of Polyoxamic Acid (2-Amino-2-deoxy-l-xylonic Acid

The rate of precipitation of the retrograded amylose product from a dil. amylose solution was determined by the centrifugal method. The results showed that the relation of the quantity of precipitate vs. time did not fit the typical second order reaction for the coalescence of colloidal particles but fitted the crystallization formula, in appearance.

The rate of precipitation was in proportion to (c-c_a)^1.5, where c is the amylose concentration and c_a the concentration of the dil. solution phase in the phase-separated solution. When the temperature dependence of the rate was treated according to the crystallization of polymers, it was found that the rate was in proportion to T_m²/T(ΔT)², where T_m is the melting point of the polymer in solution and ΔT is (T_m?T). The T_m thus obtained was 120°C for an amylose solution. These results suggested a certain correlation between the amylose retrogradation and the crystallization.     

Identification and Classification of Two-component Systems That Affect rpoS Expression in Escherichia coli

The rpoS-encoded σ^S subunit of RNA polymerase regulates the expression of stationary phase and stress response genes in Escherichia coli. Recent study of our DNA microarray analysis suggested that the rpoS expression is affected by multiple two-component systems. In this study, we identified two-component-system mutants in which the rpoS expression increased. The regulatory manner of the systems on rpoS expression is suggested.     

Escherichia coli tRNAs Are Resistant to the Hyperprocessing Reaction of Homologous E. coli Ribonuclease P Ribozyme

Bacterial ribonuclease P RNA ribozyme can do the hyperprocessing reaction, the internal cleavage reaction of some floppy eukaryotic tRNAs. The hyperprocessing reaction can be used as a detection tool to examine the stability of the cloverleaf shape of tRNA. Until now, the hyperprocessing reaction has been observed in the heterologous combination of eukaryotic tRNAs and bacterial RNase P enzymes. In this paper, we examined the hyperprocessing reaction of Escherichia coli tRNAs by homologous E. coli RNase P, to find that these homologous tRNAs were resistant to the toxic hyperprocessing reaction. Our results display the evidence for molecular co-evolution between homologous tRNAs and RNase P in the bacterium E. coli.     

A Simple Method for Multiple Modification of the Escherichia coli K-12 Chromosome

We developed a simple method of generating markerless deletions in the Escherichia coli chromosome. The method consists of two recombination events stimulated by λ Red recombinase. The first recombination replaced a target region with a marker cassette and the second then eliminated the marker cassette. The marker cassette included an antibiotic resistant gene and a negative selection marker (Bacillus subtilis sacB). Since sacB makes E. coli sensitive to sucrose, a markerless deletion strain was successfully selected using its sucrose-resistant phenotype. To stimulate these recombination events, 1-kbp homologous sequences adjacent to the target region were connected to both ends of the marker cassette or connected to each other by PCR. The average efficiency of the recombinations was 24% and 93% respectively. Eliminating the marker cassette with a fragment including an additional sequence, insertion was also possible. This markerless deletion method should be useful in creating a highly modified E. coli chromosome.