Synthetic Lethality of the lytE cwlO Genotype in Bacillus subtilis Is Caused by Lack of D,L-Endopeptidase Activity at the Lateral Cell Wall

Bacterial peptidoglycan acts as an exoskeleton to protect the bacterial cell. Although peptidoglycan biosynthesis by penicillin-binding proteins is well studied, few studies have described peptidoglycan disassembly, which is necessary for a dynamic structure that allows cell growth. In Bacillus subtilis, more than 35 genes encoding cell wall lytic enzymes have been identified; however, only two D,L-endopeptidases (lytE and cwlO) are involved in cell proliferation. In this study, we demonstrated that the D,L-endopeptidase activity at the lateral cell wall is essential for cell proliferation. Inactivation of LytE and CwlO by point mutation of the catalytic residues caused cell growth defects. However, the forced expression of LytF or CwlS, which are paralogs of LytE, did not suppress lytE cwlO synthetic lethality. Subcellular localization studies of these D,L-endopeptidases showed LytE and CwlS at the septa and poles, CwlO at the cylindrical part of the cell, and LytE at the septa and poles as well as the cylindrical part. Furthermore, construction of N-terminal and C-terminal domain-swapped enzymes of LytE, LytF, CwlS, and CwlO revealed that localization was dependent on the N-terminal domains. Only the chimeric proteins that were enzymatically active and localized to the sidewall were able to suppress the synthetic lethality, suggesting that the lack of D,L-endopeptidase activity at the cylindrical part of the cell leads to a growth defect. The functions of LytE and CwlO in cell morphogenesis were discussed.ArticleJOURNAL OF BACTERIOLOGY. 194(4):796-803 (2012)journal articl

Bacillus subtilis Cw1Q (previous YjbJ) is a bifunctional enzyme exhibiting muramidase and soluble-lytic transglycosylase activities

CwIQ (previous YjbJ) is one of the putative cell wall hydrolases in Bacillus subtilis. Its domain has an amino acid sequence similar to the soluble-lytic transglycosylase (SLT) of Escherichia coli Slt70 and also goose lysozyme (muramidase). To characterize the enzyme, the domain of CwIQ was cloned and expressed in E. coil. The purified CwIQ protein exhibited cell wall hydrolytic activity. Surprisingly, RP-HPLC, mass spectrometry (MS), and MS/MS analyses showed that CwIQ produces two products, 1,6-anhydro-N-acetylmuramic acid and N-acetylmuramic acid, thus indicating that CwIQ is a bifunctional enzyme. The site-directed mutagenesis revealed that glutamic acid 85 (Glu-85) is an amino acid residue essential to both activities. (C) 2010 Elsevier Inc. All rights reserved.ArticleBIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS. 398(3):606-612 (2010)journal articl

Thermal Infrared and Optical Photometry of Asteroidal Comet C/2002 CE10_{10}

C/2002 CE$_{10}$ is an object in a retrograde elliptical orbit with Tisserand parameter $-0.853$ indicating a likely origin in the Oort Cloud. It appears to be a rather inactive comet since no coma and only a very weak tail was detected during the past perihelion passage. We present multi-color optical photometry, lightcurve and thermal mid-IR observations of the asteroidal comet. \textcolor{blue}{ With the photometric analysis in $BVRI$, the surface color is found to be redder than asteroids, corresponding to cometary nuclei and TNOs/Centaurs. The time-resolved differential photometry supports a rotation period of 8.19$\pm$0.05 h. The effective diameter and the geometric albedo are 17.9$\pm$0.9 km and 0.03$\pm$0.01, respectively, indicating a very dark reflectance of the surface. The dark and redder surface color of C/2002 CE$_{10}$ may be attribute to devolatilized material by surface aging suffered from the irradiation by cosmic rays or from impact by dust particles in the Oort Cloud. Alternatively, C/2002 CE$_{10}$ was formed of very dark refractory material originally like a rocky planetesimal. In both cases, this object lacks ices (on the surface at least). The dynamical and known physical characteristics of C/2002 CE$_{10}$ are best compatible with those of the Damocloids population in the Solar System, that appear to be exhaust cometary nucleus in Halley-type orbits. The study of physical properties of rocky Oort cloud objects may give us a key for the formation of the Oort cloud and the solar system.Comment: 9 pages, 4 figures accepted to Icaru

Solution Structure of IseA, an Inhibitor Protein of DL-Endopeptidases from Bacillus subtilis, Reveals a Novel Fold with a Characteristic Inhibitory Loop

In Bacillus subtilis, LytE, LytF, CwlS, and CwlO are vegetative autolysins, DL-endopeptidases in the NlpC/P60 family, and play essential roles in cell growth and separation. IseA (YoeB) is a proteinaceous inhibitor against the DL-endopeptidases, peptidoglycan hydrolases. Overexpression of IseA caused significantly long chained cell morphology, because IseA inhibits the cell separation DL-endopeptidases post-translationally. Here, we report the first three-dimensional structure of IseA, determined by NMR spectroscopy. The structure includes a single domain consisting of three alpha-helices, one 3(10)-helix, and eight beta-strands, which is a novel fold like a "hacksaw." Noteworthy is a dynamic loop between beta 4 and the 3(10)-helix, which resembles a "blade." The electrostatic potential distribution shows that most of the surface is positively charged, but the region around the loop is negatively charged. In contrast, the LytF active-site cleft is expected to be positively charged. NMR chemical shift perturbation of IseA interacting with LytF indicated that potential interaction sites are located around the loop. Furthermore, the IseA mutants D100K/D102K and G99P/G101P at the loop showed dramatic loss of inhibition activity against LytF, compared with wild-type IseA, indicating that the beta 4-3(10) loop plays an important role in inhibition. Moreover, we built a complex structure model of IseA-LytF by docking simulation, suggesting that the beta 4-3(10) loop of IseA gets stuck deep in the cleft of LytF, and the active site is occluded. These results suggest a novel inhibition mechanism of the hacksaw-like structure, which is different from known inhibitor proteins, through interactions around the characteristic loop regions with the active-site cleft of enzymes.ArticleJOURNAL OF BIOLOGICAL CHEMISTRY. 287(53):44736-44748 (2012)journal articl

Mechanistic basis for the recognition of laminin-511 by α6β1 integrin

Mamoru Takizawa, Takao Arimori, Yukimasa Taniguchi, Yu Kitago, Erika Yamashita, Junichi Takagi and Kiyotoshi Sekiguchi, "Mechanistic basis for the recognition of laminin-511 by α6β1 integrin", Science Advances, Vol. 3, No. 9, e1701497, AAAS, 201

Identification and Characterization of a Novel Polysaccharide Deacetylase C (PdaC) from Bacillus subtilis

Cell wall metabolism and cell wall modification are very important processes that bacteria use to adjust to various environmental conditions. One of the main modifications is deacetylation of peptidoglycan. The polysaccharide deacetylase homologue, Bacillus subtilis YjeA (renamed PdaC), was characterized and found to be a unique deacetylase. The pdaC deletion mutant was sensitive to lysozyme treatment, indicating that PdaC acts as a deacetylase. The purified recombinant and truncated PdaC from Escherichia coli deacetylated B. subtilis peptidoglycan and its polymer, (-GlcNAc-MurNAc[-L-Ala-D-Glu]-)(n). Surprisingly, RP-HPLC and ESI-MS/MS analyses showed that the enzyme deacetylates N-acetylmuramic acid (MurNAc) not GlcNAc from the polymer. Contrary to Streptococcus pneumoniae PgdA, which shows high amino acid sequence similarity with PdaC and is a zinc-dependent GlcNAc deacetylase toward peptidoglycan, there was less dependence on zinc ion for deacetylation of peptidoglycan by PdaC than other metal ions (Mn2+, Mg2+, Ca2+). The kinetic values of the activity toward B. subtilis peptidoglycan were K-m = 4.8 mM and k(cat) = 0.32 s(-1). PdaC also deacetylated N-acetylglucosamine (GlcNAc) oligomers with a K-m = 12.3 mM and k(cat) = 0.24 s(-1) toward GlcNAc(4). Therefore, PdaC has GlcNAc deacetylase activity toward GlcNAc oligomers and MurNAc deacetylase activity toward B. subtilis peptidoglycan.ArticleJOURNAL OF BIOLOGICAL CHEMISTRY. 287(13):9765-9776 (2012)journal articl

Suppression of Propionibacterium acnes

Purpose. Macrophages serve as sweepers of microbes and inflammation-derived wastes and regulators of inflammation. Some traditional Japanese medicines are reported to have adjuvant effects by modifying macrophages. Our aim was to characterize the actions of jumihaidokuto (JHT) for treatment of skin inflammations including acne vulgaris, in which Propionibacterium acnes has pathogenic roles. Methods. Dermatitis was induced in rat ears by intradermal injection of P. acnes. JHT or prednisolone (PDN) was given orally, and ear thickness and histology were evaluated. The effects of constituents and metabolites of JHT on monocytes were tested by cell-based assays using the human monocytic THP-1 cell. Results. JHT and PDN suppressed the ear thickness induced by P. acnes injection. Histological examinations revealed that JHT, but not PDN, promoted macrophage accumulation at 24 h after the injection. PDN suppressed the macrophage chemokine MCP-1 in the inflamed ears, while JHT did not affect it. The JHT constituents liquiritigenin and isoliquiritin increased expression of CD86 (type-1 macrophage marker) and CD192 (MCP-1 receptor) and enhanced phagocytosis by THP-1. Conclusions. JHT suppressed dermatitis, probably by enhancing type-1 macrophage functions, with an action different from PDN. JHT may be a beneficial drug in treatment of skin inflammation induced by P. acnes

Inhibition of Hepatitis C Virus Replication and Viral Helicase by Ethyl Acetate Extract of the Marine Feather Star Alloeocomatella polycladia

Hepatitis C virus (HCV) is a causative agent of acute and chronic hepatitis, leading to the development of hepatic cirrhosis and hepatocellular carcinoma. We prepared extracts from 61 marine organisms and screened them by an in vitro fluorescence assay targeting the viral helicase (NS3), which plays an important role in HCV replication, to identify effective candidates for anti-HCV agents. An ethyl acetate-soluble fraction of the feather star Alloeocomatella polycladia exhibited the strongest inhibition of NS3 helicase activity, with an IC50 of 11.7 µg/mL. The extract of A. polycladia inhibited interaction between NS3 and RNA but not ATPase of NS3. Furthermore, the replication of the replicons derived from three HCV strains of genotype 1b in cultured cells was suppressed by the extract with an EC50 value of 23 to 44 µg/mL, which is similar to the IC50 value of the NS3 helicase assay. The extract did not induce interferon or inhibit cell growth. These results suggest that the unknown compound(s) included in A. polycladia can inhibit HCV replication by suppressing the helicase activity of HCV NS3. This study may present a new approach toward the development of a novel therapy for chronic hepatitis C