Chloroplast PNPase exists as a homo-multimer enzyme complex that is distinct from the Escherichia coli degradosome.

In Escherichia coli, the exoribonuclease polynucleotide phosphorylase (PNPase), the endoribonuclease RNase E, a DEAD-RNA helicase and the glycolytic enzyme enolase are associated with a high molecular weight complex, the degradosome. This complex has an important role in processing and degradation of RNA. Chloroplasts contain an exoribonuclease homologous to E. coli PNPase. Size exclusion chromatography revealed that chloroplast PNPase elutes as a 580-600 kDa complex, suggesting that it can form an enzyme complex similar to the E. coli degradosome. Biochemical and mass-spectrometric analysis showed, however, that PNPase is the only protein associated with the 580-600 kDa complex. Similarly, a purified recombinant chloroplast PNPase also eluted as a 580-600 kDa complex after gel filtration chromatography. These results suggest that chloroplast PNPase exists as a homo-multimer complex. No other chloroplast proteins were found to associate with chloroplast PNPase during affinity chromatography. Database analysis of proteins homologous to E. coli RNase E revealed that chloroplast and cyanobacterial proteins lack the C-terminal domain of the E. coli protein that is involved in assembly of the degradosome. Together, our results suggest that PNPase does not form a degradosome-like complex in the chloroplast. Thus, RNA processing and degradation in this organelle differ in several respects from those in E. coli.     

Homologous pairing of single-stranded DNA and superhelical double-stranded DNA catalyzed by RecO protein from Escherichia coli.

The recO gene product is required for DNA repair and some types of homologous recombination in wild-type Escherichia coli cells. RecO protein has been previously purified and shown to bind to single- and double-stranded DNA and to promote the renaturation of complementary single-stranded DNA molecules. In this study, purified RecO protein was shown to catalyze the assimilation of single-stranded DNA into homologous superhelical double-stranded DNA, an activity also associated with RecA protein. The RecO protein-promoted strand assimilation reaction requires Mg2+ and is ATP independent. Because of the biochemical similarities between RecO and RecA proteins, the ability of RecO protein to substitute for RecA protein in DNA repair in vivo was also assessed in this study. The results show that overexpression of RecO protein partially suppressed the UV repair deficiency of a recA null mutant and support the hypothesis that RecO and RecA proteins are functionally similar with respect to strand assimilation and the ability to enhance UV survival. These results suggest that RecO and RecA proteins may have common functional properties.     

Escherichia coli is able to grow with negligible sodium ion extrusion activity at alkaline pH.

The Escherichia coli mutant NM81, which is deficient in the nhaA gene for the sodium/proton antiporter, still has a sodium ion extrusion activity because of a second antiporter encoded by nhaB (E. Padan, N. Maisler, D. Taglicht, R. Karpel, and S. Schuldiner, J. Biol. Chem. 264:20297-20302, 1989). By chance, we have found that E. coli pop6810 already contains a mutation affecting the sodium ion circulation, probably in or near nhaB, and that its delta nhaA mutant, designated RS1, has no sodium ion extrusion activity at alkaline pH. The growth of RS1 was inhibited completely by 0.1 M sodium, whereas growth inhibition of NM81 was observed only at sodium concentrations greater than 0.2 M. RS1 grew at a normal rate in an alkaline medium containing a low sodium concentration. Furthermore, RS1 grew with a negligible proton motive force in the alkaline medium containing carbonyl cyanide m-chlorophenylhydrazone. The transport activities for proline and serine were not impaired in RS1, suggesting that these transport systems could be driven by the proton motive force at alkaline pH. These findings led us to conclude that the operation of the sodium/proton antiporter is not essential for growth at alkaline pH but that the antiporter is required for maintaining a low internal sodium concentration when the growth medium contains a high concentration of these ions.     

Escherichia coli DNA helicase II is active as a monomer

Helicases are thought to function as oligomers (generally dimers or hexamers). Here we demonstrate that although Escherichia coli DNA helicase II (UvrD) is capable of dimerization as evidenced by a positive interaction in the yeast two-hybrid system, gel filtration chromatography, and equilibrium sedimentation ultracentrifugation (Kd = 3.4 microM), the protein is active in vivo and in vitro as a monomer. A mutant lacking the C-terminal 40 amino acids (UvrDDelta40C) failed to dimerize and yet was as active as the wild-type protein in ATP hydrolysis and helicase assays. In addition, the uvrDDelta40C allele fully complemented the loss of helicase II in both methyl-directed mismatch repair and excision repair of pyrimidine dimers. Biochemical inhibition experiments using wild-type UvrD and inactive UvrD point mutants provided further evidence for a functional monomer. This investigation provides the first direct demonstration of an active monomeric helicase, and a model for DNA unwinding by a monomer is presented.     

Genetic transformation in freshwater: Escherichia coli is able to develop natural competence.

Until now, Escherichia coli was thought to be unable to develop natural competence, i.e., genetic transformation could be achieved only artificially with the aid of nonphysiological concentrations of calcium ions or by other treatments. We have tested the competence development of E. coli through transformation under natural conditions in river water, springwater, and mineral water which contained between 0 and 11 mM Ca2+, using pUC18 DNA. The presence of calcium ions at concentrations as low as 1 to 2 mM was sufficient to obtain transformants. Variations in the temperature of incubation were not required for competence development but had an influence on the transformation frequency. Using water from mineral springs originating from calcareous regions, we have obtained transformation frequencies with laboratory strains of E. coli similar to those reported for other gram-negative bacteria known to develop natural competence. The competence development of E. coli is most probably internally regulated (as for the other gram-negative bacteria), and inadequate conditions chosen for the transformation tests in the laboratory might impair the detection of higher natural transformation frequencies. The results will enhance our knowledge about the fate of laboratory or production strains of E. coli cells reaching natural aquatic ecosystems.     

Synthesis of complex forms of bacteriophage phiX174 double-stranded DNA in a temperature-sensitive dnaC mutant of Escherichia coli C.

Fast-sedimenting forms of bacteriophage phiX174 double-stranded replicative-form DNA observed in normal infections continued to accumulate at the nonpermissive temperature in a temperature-sensitive dnaC mutant of Escherichia coli. These complex molecules accounted for up to half of the DNA synthesized during short pulses at the nonpermissive temperature. They were the dead-end products of DNA synthesis, not intermediates in normal replicative-form replication. The data suggest that these higher-than-normal-molecular-weight DNA molecules result from abnormal initiation of phiX174 replicative-form DNA replication.     

Escherichia coli DNA photolyase is a flavoprotein

Escherichia coli DNA photolyase (photoreactivating enzyme) was purified to homogeneity from a strain that greatly overproduces the protein. The purified enzyme has absorption peaks at 280 and 380 nm, a fluorescence emission peak at 480 nm and, upon denaturation, releases a chromophore that has the spectroscopic properties of flavin adenine dinucleotide (FAD), indicating that FAD is an intrinsic chromophore of the enzyme.     

FtsH exists as an exceptionally large complex containing HflKC in the plasma membrane of Escherichia coli

FtsH is an ATP-dependent and membrane-associated protease, which exerts processive proteolysis against membrane-embedded and soluble substrate proteins. Although previous studies suggested that it functions as a homo-oligomer and it also interacts with HflK-HflC membrane protein complex (HflKC), it is still important to address the question of what kind of supramolecular assembly FtsH forms in wild-type cells. Now we show that FtsH in wild-type Escherichia coli cells exists exclusively as a large complex, termed FtsH holo-enzyme, which can be separated from bulk of membrane proteins after detergent solubilization and velocity sedimentation. This complex appears to have molecular mass of around 1000 kDa. A tentative model is presented that it is composed of hexamers of FtsH and of HflKC, with an ability to bind one or a few substrate molecules.     

DNA cleavage and degradation by the SbcCD protein complex from Escherichia coli.

The SbcCD protein is a member of a group of nucleases found in bacteriophage T4 and T5, eubacteria, archaebacteria, yeast, Drosophila, mouse and man. Evidence from electron microscopy has revealed a distinctive structure consisting of two globular domains linked by a long region of coiled coil, similar to that predicted for the members of the SMC family. That a nuclease should have such an unusual structure suggests that its mode of action may be complex. Here we show that the protein degrades duplex DNA in a 3'-->5' direction. This degradation releases products half the length of the original duplex suggesting simultaneous degradation from two duplex ends. This may provide a link to the unusual structure of the protein since our data are consistent with recognition and cleavage of DNA ends followed by 3'-->5' nicking by two nucleolytic centres within a single nuclease molecule that releases a half length limit product. We also show that cleavage is not simply at the point of a single-strand/double-stand transition and that despite the dominant 3'-->5' polarity of degradation, a 5' single-strand can be cleaved when attached to duplex DNA. The implications of this mechanism for the processing of hairpins formed during DNA replication are discussed.     

Polyribosomes of Escherichia coli. I. Isolation of polysomes from a complex of DNA and membrane

Summary The distribution of pulse labeled RNA, pulse labeled protein, soluble enzyme (-galactosidase) and polyribosomes between low speed (10 min 20000xg) supernatant and pellet of E. coli lysates was examined. This distribution was greatly changed by addition of deoxyribonuclease to the lysing medium. Large amounts of polysomes sedimented with DNA at low centrifugal forces. A complex of membrane, DNA, polyribosomes and RNA polymerase could be separated from unlysed cells by surcrose gradient centrifugation. The polysomes present in this complex (Polysomes II) were separated from the polysomes which were found in the cytoplasma (Polysomes I). Polysomes II contain very few ribosomal subunits and 70S ribosomes.     

Evidence for ATP binding and double-stranded DNA binding by Escherichia coli RecF protein.

RecF protein is one of the important proteins involved in DNA recombination and repair. RecF protein has been shown to bind single-stranded DNA (ssDNA) in the absence of ATP (T. J. Griffin IV and R. D. Kolodner, J. Bacteriol. 172:6291-6299, 1990; M. V. V. S. Madiraju and A. J. Clark, Nucleic Acids Res. 19:6295-6300, 1991). In the present study, using 8-azido-ATP, a photo-affinity analog of ATP, we show that RecF protein binds ATP and that the binding is specific in the presence of DNA. 8-Azido-ATP photo-cross-linking is stimulated in the presence of DNA (both ssDNA and double-stranded DNA [dsDNA]), suggesting that DNA enhances the affinity of RecF protein for ATP. These data suggest that RecF protein possesses independent ATP- and DNA-binding sites. Further, we find that stable RecF protein-dsDNA complexes are obtained in the presence of ATP or ATP-gamma-S [adenosine-5'-O-(3-thio-triphosphate)]. No other nucleoside triphosphates served as necessary cofactors for dsDNA binding, indicating that RecF is an ATP-dependent dsDNA-binding protein. Since a mutation in a putative phosphate-binding motif of RecF protein results in a recF mutant phenotype (S. J. Sandler, B. Chackerian, J. T. Li, and A. J. Clark, Nucleic Acids Res. 20:839-845, 1992), we suggest on the basis of our data that the interactions of RecF protein with ATP, with dsDNA, or with both are physiologically important for understanding RecF protein function in vivo.     

Engineering nonphosphorylative metabolism to synthesize mesaconate from lignocellulosic sugars in Escherichia coli

Dicarboxylic acids are attractive biosynthetic targets due to their broad applications and their challenging manufacturing process from fossil fuel feedstock. Mesaconate is a branched, unsaturated dicarboxylic acid that can be used as a co-monomer to produce hydrogels and fire-retardant materials. In this study, we engineered nonphosphorylative metabolism to produce mesaconate from d-xylose and l-arabinose. This nonphosphorylative metabolism is orthogonal to the intrinsic pentose metabolism in Escherichia coli and has fewer enzymatic steps and a higher theoretical yield to TCA cycle intermediates than the pentose phosphate pathway. Here mesaconate production was enabled from the d-xylose pathway and the l-arabinose pathway. To enhance the transportation of d-xylose and l-arabinose, pentose transporters were examined. We identified the pentose/proton symporter, AraE, as the most effective transporter for both d-xylose and l-arabinose in mesaconate production process. Further production optimization was achieved by operon screening and metabolic engineering. These efforts led to the engineered strains that produced 12.5 g/l and 13.2 g/l mesaconate after 48 h from 20 g/l of d-xylose and l-arabinose, respectively. Finally, the engineered strain overexpressing both l-arabinose and d-xylose operons produced 14.7 g/l mesaconate from a 1:1 d-xylose and l-arabinose mixture with a yield of 85% of the theoretical maximum. (0.87 g/g). This work demonstrates an effective system that converts pentoses into a value-added chemical, mesaconate, with promising titer, rate, and yield.