The CafA protein required for the 5'-maturation of 16 S rRNA is a 5'-end-dependent ribonuclease that has context-dependent broad sequence specificity

The CafA protein, which was initially described as having a role in either Escherichia coli cell division or chromosomal segregation, has recently been shown to be required for the maturation of the 5'-end of 16 S rRNA. The sequence of CafA is similar to that of the N-terminal ribonucleolytic half of RNase E, an essential E. coli enzyme that has a central role in the processing of rRNA and the decay of mRNA and RNAI, the antisense regulator of ColE1-type plasmids. We show here that a highly purified preparation of CafA is sufficient in vitro for RNA cutting. We detected CafA cleavage of RNAI and a structured region from the 5'-untranslated region of ompA mRNA within segments cleavable by RNaseE, but not CafA cleavage of 9 S RNA at its "a" RNase E site. The latter is consistent with the finding that the generation of 5 S rRNA from its 9 S precursor can be blocked by inactivation of RNase E in cells that are wild type for CafA. Interestingly, however, a decanucleotide corresponding in sequence to the a site of 9 S RNA was cut efficiently indicating that cleavage by CafA is regulated by the context of sites within structured RNAs. Consistent with this notion is our finding that although 23 S rRNA is stable in vivo, a segment from this RNA is cut efficient by CafA at multiple sites in vitro. We also show that, like RNase E cleavage, the efficiency of cleavage by CafA is dependent on the presence of a monophosphate group on the 5'-end of the RNA. This finding raises the possibility that the context dependence of cleavage by CafA may be due at least in part to the separation of a cleavable sequence from the 5'-end of an RNA. Comparison of the sites surrounding points of CafA cleavage suggests that this enzyme has broad sequence specificity. Together with the knowledge that CafA can cut RNAI and ompA mRNA in vitro within segments whose cleavage in vivo initiates the decay of these RNAs, this finding suggests that CafA may contribute at some point during the decay of many RNAs in E. coli.     

Regions of RNase E important for 5'-end-dependent RNA cleavage and autoregulated synthesis

RNase E is an important regulatory enzyme that plays a key role in RNA processing and degradation in Escherichia coli. Internal cleavage by this endonuclease is accelerated by the presence of a monophosphate at the RNA 5' end. Here we show that the preference of E. coli RNase E for 5'-monophosphorylated substrates is an intrinsic property of the catalytically active amino-terminal half of the enzyme and does not require the carboxy-terminal region. This property is shared by the related E. coli ribonuclease CafA (RNase G) and by a cyanobacterial RNase E homolog derived from Synechocystis, indicating that the 5'-end dependence of RNase E is a general characteristic of members of this ribonuclease family, including those from evolutionarily distant species. Although it is dispensable for 5'-end-dependent RNA cleavage, the carboxy-terminal half of RNase E significantly enhances the ability of this ribonuclease to autoregulate its synthesis in E. coli. Despite similarities in amino acid sequence and substrate specificity, CafA is unable to replace RNase E in sustaining E. coli cell growth or in regulating RNase E production, even when overproduced sixfold relative to wild-type RNase E levels.     

Substrate preferences of caffeic acid/5-hydroxyferulic acid 3/5-O-methyltransferases in developing stems of alfalfa (Medicago sativa L.)

Caffeic acid/5-hydroxyferulic acid 3/5-O-methyltransferase (COMT, EC 1.2.1.68) catalyzes at least two reactions in lignin biosynthesis. Of its two supposed substrates in the lignin pathway, COMT from most sources methylates 5-hydroxyferulic acid (5HFA) with two to three times higher activity than caffeic acid (CafA). The ratio of activity for 5HFA compared with CafA increases with the developmental age of alfalfa (Medicago sativa L.) stem internodes, from approximately 1:1 in young (third and fourth) internodes to 2:1 in mature (seventh and eighth) internodes. This observation, together with immunoblot analysis using antiserum raised against recombinant alfalfa COMT, suggests the presence of a different form of COMT, having preference for CafA compared with 5HFA, in young internodes. This apparently new O-methyltransferase (COMT II) was separated from the previously characterized COMT (COMT I) by anion exchange and hydrophobic interaction chromatography. COMT I, but not COMT II, was found in mature internodes. COMT II was not recognized by anti-(COMT I) serum. Furthermore, in addition to substrate preference, COMT II differed from COMT I in native relative molecular mass, pH optimum, and its very low K(m) for CafA. The possible physiological role of COMT II is discussed.     

Immunocytochemical localization of the elongation factor Tu in E. coli cells

The localization of the elongation factor Tu (EF-Tu) in ultrathin cryosections of E. coli cells was determined with the electron microscope using a highly specific immunological labelling technique. EF-Tu is distributed almost homogeneously throughout the cytoplasm. Although it has often been suggested that EF-Tu could be part of a putative prokaryotic cytoskeleton, we did not find any evidence for supramolecular assemblies, such as fibres or filaments, containing a large amount of EF-Tu. EF-Tu was not observed in association with the outer cell membrane and periplasmic space. A topological relationship with the inner membrane is not apparent in our micrographs. In cells in which the EF-Tu level is raised significantly, the protein piles up in discrete cell regions.     

Confocal scanning light microscopy of the Escherichia coli nucleoid: comparison with phase-contrast and electron microscope images.

The nucleoid of living and OsO4- or glutaraldehyde-fixed cells of Escherichia coli strains was studied with a phase-contrast microscope, a confocal scanning light microscope, and an electron microscope. The trustworthiness of the images obtained with the confocal scanning light microscope was investigated by comparison with phase-contrast micrographs and reconstructions based on serially sectioned material of DNA-containing and DNA-less cells. This comparison showed higher resolution of the confocal scanning light microscope as compared with the phase-contrast microscope, and agreement with results obtained with the electron microscope. The effects of fixation on the structure of the nucleoid were studied in E. coli B/r H266. Confocal scanning light micrographs and electron microscopic reconstructions showed that the shape of the nucleoid remained similar after OsO4 or glutaraldehyde fixation; however, the OsO4 nucleoid appeared to be somewhat smaller and more centralized within the cell.     

Human NAD(+)-dependent mitochondrial malic enzyme. cDNA cloning, primary structure, and expression in Escherichia coli

Mitochondrial NAD(+)-dependent malic enzyme (EC 1.1.1.40) is expressed in rapidly proliferating cells and tumor cells, where it is probably linked to the conversion of amino acid carbon to pyruvate. In this paper, we report the cDNA cloning, amino acid sequence, and expression in Escherichia coli of functional human NAD(+)-dependent mitochondrial malic enzyme. The cDNA is 1,923 base pairs long and contains an open reading frame coding for a 584-amino acid protein. The molecular mass is 65.4 kDa for the unprocessed precursor protein. Comparison of the amino acid sequence of the human protein with the published NADP(+)-dependent mammalian cytosolic or plant chloroplast malic enzymes reveals highly conserved regions interrupted with long stretches of amino acids without significant homology. Expression of the processed protein in E. coli yielded an enzyme with the same kinetic and allosteric properties as malic enzyme purified from human cells.     

Nutritional intervention with omega-3 fatty acids enhances tumor response to anti-neoplastic agents

Nutritional intervention with specific fatty acids depresses tumor growth and enhances tumor responsiveness to chemotherapy. Supplementation of tumors with long chained omega-3 polyunsaturated fatty acids results in enrichment of tumor phospholipid fractions with omega-3 fatty acids resulting in an altered membrane composition and function. Tumors enriched with long chained omega-3 polyunsaturated fatty acids possess membranes with increased fluidity, an elevated unsaturation index, enhanced transport capabilities that results in accumulation of selective anti-cancer agents, increased activity of selected drug activating enzymes, and alteration of signaling pathways important for cancer progression. These nutritionally induced changes in tumor fatty acid composition result in increased sensitivity to chemotherapy, especially in tumor lines that are resistant to chemotherapy and cause specific enhancement of cytotoxicity to tumor cells and protection of normal cells. Pre-disposing tumors to increased chemo-sensitivity through nutritional intervention with specific fatty acids has the potential to improve patient response to chemotherapy with fewer untoward side effects if these pre-clinical findings carry over into a clinical setting.     

Various morphological aspects of Escherichia coli lysis by RNA bacteriophage MS2 observed by transmission and scanning electron microscopes

Escherichia coli cell lysis profiles induced by the cloned lysis gene from RNA bacteriophages MS2 were presented. Transmission electron micrographs showed that ballooning structures appeared on the cell surfaces and the others were leaking materials through the cell wall. Harsh rupture of the host cell was also observed. Scanning electron micrographs revealed many extruded structures from the cells. For the scanning electron microscopy, plastic sheets coated with calf dermis collagen and SEMPORE filters were successfully used to collect the samples and for various chemical treatments. The lysing cells by bacteriophage MS2 lysis gene observed in transmission and scanning electron micrographs showed various morphological aspects of the intermediates in the lysing process.     

Filamentous Cells of Escherichia coli Formed in the Presence of Mitomycin

The effects of mitomycin C on cell elongation of Escherichia coli B were studied. Filament formation was most marked in cultures treated with a moderate level (1 mug/ml) of the antibiotic, becoming less obvious at higher levels (10 mug/ml). Cells treated with a bacteriostatic concentration (0.1 mug/ml or less) of mitomycin C were also significantly elongated. The filamentous or elongated cells appeared to lack septa, since their spheroplasts were considerably larger than those formed from normal cells. The appearance of empty spheres also indicated some defects in the surfaces of the filamentous cells. Electron micrographs of the filaments revealed a characteristic difference in the arrangement of the nuclei in the filaments formed in the presence of low (0.1 mug/ml) and high (5 mug/ml) concentrations of mitomycin C. The filaments formed by the low level of mitomycin C had normal well-defined nuclear bodies distributed along the long axis, whereas those formed by the elevated level of the antibiotic contained smaller nuclei. The latter were characteristically confined to the center of the cells and did not extend out to the tips of the filaments.     

Purification and DNA-binding properties of human papillomavirus type 16 E6 protein expressed in Escherichia coli

Unfused human papillomavirus type 16 (HPV 16) E6 protein was expressed in Escherichia coli using a lambda PL promoter system. The protein was isolated from the cells as inclusion bodies, extracted by 6 M guanidine-HCl, and purified by chromatography. The purified protein had high affinity to DNA and was demonstrated for the first time to bind to a specific sequence within the long control region of HPV 16.     

Survival of Legionella pneumophila in the cold-water ciliate Tetrahymena vorax.

The processing of phagosomes containing Legionella pneumophila and Escherichia coli were compared in Tetrahymena vorax, a hymenostome ciliated protozoan that prefers lower temperatures. L. pneumophila did not multiply in the ciliate when incubated at 20 to 22 degrees C, but vacuoles containing L. pneumophila were retained in the cells for a substantially longer time than vacuoles with E. coli. Electron micrographs showed no evidence of degradation of L. pneumophila cells through 12 h, while E. coli cells in the process of being digested were observed in vacuoles 75 min after the addition of the bacterium. T. vorax ingested L. pneumophila normally, but by 10 to 15 min, the vacuolar membrane appeared denser than that surrounding nascent or newly formed phagosomes. In older vacuoles, electron-dense particles lined portions of the membrane. Acidification of the phagosomes indicated by the accumulation of neutral red was similar in T. vorax containing L. pneumophila or E. coli. This ciliate could provide a model for the analysis of virulence-associated intracellular events independent of the replication of L. pneumophila.     

sn-Glycerol-3-phosphate transport in Salmonella typhimurium

Salmonella typhimurium contains a transport system for sn-glycerol-3-phosphate that is inducible by growth on glycerol and sn-glycerol-3-phosphate. In fully induced cells, the system exhibited an apparent Km of 50 microM and a Vmax of 2.2 nmol/min . 10(8) cells. The corresponding system in Escherichia coli exhibits, under comparable conditions, a Km of 14 microM and a Vmax of 2.2 nmol/min . 10(8) cells. Transport-defective mutants were isolated by selecting for resistance against the antibiotic fosfomycin. They mapped in glpT at 47 min in the S. typhimurium linkage map, 37% cotransducible with gyrA. In addition to the glpT-dependent system, S. typhimurium LT2 contains, like E. coli, a second, ugp-dependent transport system for sn-glycerol-3-phosphate that was derepressed by phosphate starvation. A S. typhimurium DNA bank containing EcoRI restriction fragments in phage lambda gt7 was used to clone the glpT gene in E. coli. Lysogens that were fully active in the transport of sn-glycerol-3-phosphate with a Km of 33 microM and a Vmax of 2.0 nmol/min . 10(8) cells were isolated in a delta glpT mutant of E. coli. The EcoRI fragment harboring glpT was 3.5 kilobases long and carried only part of glpQ, a gene distal to glpT but on the same operon. The fragment was subcloned in multicopy plasmid pACYC184. Strains carrying this hybrid plasmid produced large amounts of cytoplasmic membrane protein with an apparent molecular weight of 33,000, which was identified as the sn-glycerol-3-phosphate permease. Its properties were similar to the corresponding E. coli permease. The presence of the multicopy glpT hybrid plasmid had a strong influence on the synthesis or assembly of other cell envelope proteins of E. coli. For instance, the periplasmic ribose-binding protein was nearly absent. On the other hand, the quantity of an unidentified E. coli outer membrane protein usually present only in small amounts increased.     

Survival of Legionella pneumophila in the cold-water ciliate Tetrahymena vorax.

The processing of phagosomes containing Legionella pneumophila and Escherichia coli were compared in Tetrahymena vorax, a hymenostome ciliated protozoan that prefers lower temperatures. L. pneumophila did not multiply in the ciliate when incubated at 20 to 22 degrees C, but vacuoles containing L. pneumophila were retained in the cells for a substantially longer time than vacuoles with E. coli. Electron micrographs showed no evidence of degradation of L. pneumophila cells through 12 h, while E. coli cells in the process of being digested were observed in vacuoles 75 min after the addition of the bacterium. T. vorax ingested L. pneumophila normally, but by 10 to 15 min, the vacuolar membrane appeared denser than that surrounding nascent or newly formed phagosomes. In older vacuoles, electron-dense particles lined portions of the membrane. Acidification of the phagosomes indicated by the accumulation of neutral red was similar in T. vorax containing L. pneumophila or E. coli. This ciliate could provide a model for the analysis of virulence-associated intracellular events independent of the replication of L. pneumophila.     

Phase separation between nucleoid and cytoplasm in Escherichia coli as defined by immersive refractometry.

The refractive indices of nucleoid and cytoplasm in Escherichia coli were derived theoretically and experimentally. For the theoretical estimates, we made use of the known macromolecular composition of E. coli B/r (G. Churchward and H. Bremer, J. Theor. Biol. 94:651-670, 1982) and of estimates of cell and nucleoid volumes. These were obtained from micrographs of living bacteria made with a confocal scanning light microscope. The theoretical values were calculated, assuming that all DNA occurred in the nucleoid and that all protein and RNA occurred in the cytoplasm. Comparison with experimental refractive index values directly obtained by immersive refractometry showed that, besides its DNA, the nucleoid must contain an additional amount of solids equivalent to 8.6% (wt/vol) protein. With the nucleoid containing 6.8% (wt/vol) DNA and 8.6% (wt/vol) protein and the cytoplasm containing 21% (wt/vol) protein and 4% (wt/vol) RNA, a mass difference is obtained, which accounts for the phase separation observed between the nucleoid and cytoplasm in living cells by phase-contrast microscopy. The decrease in the refractive index of the nucleoid relative to that of the cytoplasm observed upon, for instance, OsO4 fixation was interpreted as being indicative of the loss of protein content in the nucleoid.     

Suppressive effect of l-phenylalanine on lignin peroxidase in the white-rot fungus Phanerochaete chrysosporium

Abstract Guanosine-5'-diphosphate-3'-diphosphate (ppGpp), an effector for many metabolic pathways, is synthesized by the relA gene product after amino acid limitation. Studies of stringent controlled Escherichia coli CP78 (relA⁺) and relaxed controlled E. coli CP79 (relA⁻) were carried out to test whether these strains differ in the appearance of their cytoplasmic membranes after induction of stringent and relaxed response. Cytoplasmic membrane structures of the cells were investigated by freeze-fracture electron microscopy after cooling the cells. The obtained micrographs showed a net-like distribution of the particles in the cytoplasmic membranes of relaxed controlled cells whereas such a pattern was not detectable in the stringent controlled counterparts.     

Synthesis of a Bacillus subtilis small, acid-soluble spore protein in Escherichia coli causes cell DNA to assume some characteristics of spore DNA.

Small, acid-soluble proteins (SASP) of the alpha/beta-type are associated with DNA in spores of Bacillus subtilis. Induction of synthesis of alpha/beta-type SASP in Escherichia coli resulted in rapid cessation of DNA synthesis, followed by a halt in RNA and then protein accumulation, although significant mRNA and protein synthesis continued. There was a significant loss in viability associated with SASP synthesis in E. coli: recA+ cells became extremely long filaments, whereas recA mutant cells became less filamentous. The nucleoids of cells with alpha/beta-type SASP were extremely condensed, as viewed in both light and electron microscopes, and immunoelectron microscopy showed that the alpha/beta-type SASP were associated with the cell DNA. Induction of alpha/beta-type SASP synthesis in E. coli increased the negative superhelical density of plasmid DNA by approximately 20%; UV irradiation of E. coli with alpha/beta-type SASP gave reduced yields of thymine dimers but significant amounts of the spore photoproduct. These changes in E. coli DNA topology and photochemistry due to alpha/beta-type SASP are similar to the effects of alpha/beta-type SASP on the DNA in Bacillus spores, further suggesting that alpha/beta-type SASP are a major factor determining DNA properties in bacterial spores.     

Structure of helical RecA-DNA complexes. III. The structural polarity of RecA filaments and functional polarity in the RecA-mediated strand exchange reaction

The RecA protein of Escherichia coli has been used in vitro to mediate a strand-exchange reaction between homologous DNA molecules. A three-dimensional reconstruction of a RecA filament on double-stranded DNA has been previously determined from electron micrographs, and the reconstruction displays a clear axial polarity. The RecA-mediated strand-exchange reaction between a double-stranded DNA and a homologous single-stranded DNA that is complexed with a RecA helical polymer proceeds with a known polarity. Using image analysis of electron micrographs, we have determined the relation between the structural polarity of RecA filaments and the 3' and 5' polarity of single-stranded DNA. Thus, the structural polarity of RecA filaments can now be related to the direction in which the RecA-mediated strand-exchange reaction advances along the complexed single-stranded DNA.