Peptidoglycan hydrolase, LytF (CwlE), was determined to be identical to YhdD (deduced cell wall binding protein) by zymography after insertional inactivation of the yhdD gene. YhdD exhibits high sequence similarity with CwlF (PapQ, LytE) and p60 ofListeria monocytogenes. The N-terminal region of YhdD has a signal sequence followed by five tandem repeated regions containing polyserine residues. The C-terminal region corresponds to the catalytic domain, because a truncated protein without the N-terminal region retained cell wall hydrolase activity. The histidine-tagged LytF protein produced in Escherichia coli cells hydrolyzed the linkage of d-γ-glutamyl-meso-diaminopimelic acid in murein peptides, indicating that it is ad,l-endopeptidase. Northern hybridization and primer extension analyses indicated that the lytF gene was transcribed by EςD RNA polymerase. Disruption oflytF led to slightly filamentous cells, and a lytF cwlF double mutant exhibited extraordinary microfiber formation, which is similar to the cell morphology of the cwlF sigDmutant.

Bacillus subtilis produces a 35-kDa cell wall hydrolase, CwlF, during vegetative growth. The CwlF protein was extracted from B. subtilis cwlB sigD mutant cells and separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. N-terminal amino acid sequencing revealed that its sequence is completely identical to that of the internal region of thepapQ gene product. Disruption of the papQ gene in the B. subtilis chromosome led to the complete loss of CwlF, indicating that papQ is identical tocwlF. CwlF exhibits high sequence similarity to the p60 proteins of Listeria species, NlpC proteins ofEscherichia coli and Haemophilus influenzae, and Enp2 protein of Bacillus sphaericus. The β-galactosidase activity of the cwlF-lacZ transcriptional fusion and Northern blot analysis of the cwlF gene indicated that the gene is expressed as a monocistronic operon during the exponential growth phase, and primer extension analysis suggested that the cwlF gene is transcribed mainly by EςA RNA polymerase and weakly by EςH RNA polymerase. While the cells of the cwlF-deficient mutant were about twice as long as those of the wild-type strain, the cwlF sigD double mutant cells exhibited extraordinary microfiber formation, in contrast to the filamentation of the sigD mutant. The CwlF production was not affected by the pleiotropic mutationsflaD1 and degU32(Hy), which endow cells with the ability of extensive filamentation.

No abstract

Photoinduced morphological changes in phosphatidylcholine vesicles are triggered by a Malachite Green leuconitrile derivative dissolved in the lipidic membrane, and are observed at Malachite Green derivative/lipid ratios <5 mol %. This Malachite Green derivative is a photoresponsive compound that undergoes ionization to afford a positive charge on the molecule by UV irradiation. The Malachite Green derivative exhibits amphiphilicity when ionized photochemically, whereas it behaves as a lipophilic compound under dark conditions. Cryo-transmission electron microscopy was used to determine vesicle morphology. The effects of the Malachite Green derivative on vesicles were studied by dynamic light scattering and fluorescence resonance energy transfer. Irradiation of vesicles containing the Malachite Green derivative induces nonspherical vesicle morphology, fusion of vesicles, and membrane solubilization, depending on conditions. Furthermore, irradiation of the Malachite Green derivative induces the release of a vesicle-encapsulated compound.

Recently, we synthesized pyrimidine derivatives of the 2′-O,4′-C-methylenoxymethylene-bridged nucleic-acid (2′,4′-BNACOC) monomer, the sugar conformation of which is restricted in N-type conformation by a seven-membered bridged structure. Oligonucleotides (BNACOC) containing this monomer show high affinity with complementary single-stranded RNA and significant resistance to nuclease degradation. Here, BNACOC consisting of 2′,4′-BNACOC monomers bearing all four bases, namely thymine, 5-methylcytosine, adenine and guanine was efficiently synthesized and properties of duplexes containing the 2′,4′-BNACOC monomers were investigated by UV melting experiments and circular dichroism (CD) spectroscopy. The UV melting curve analyses showed that the BNACOC/BNACOC duplex possessed excellent thermal stability and that the BNACOC increased thermal stability with a complementary RNA strand. On the other hand, BNACOC/DNA heteroduplexes showed almost the same thermal stability as RNA/DNA heteroduplexes. Furthermore, mismatched sequence studies showed that BNACOC generally improved the sequence selectivity with Watson–Crick base-pairing compared to the corresponding natural DNA and RNA. A CD spectroscopic analysis indicated that the BNACOC formed duplexes with complementary DNA and RNA in a manner similar to natural RNA.