A Gram-Negative Anaerobic Bacterium That Utilizes O-Methyl Substituents of Aromatic Acids

A novel strain of gram-negative anaerobic rods which utilized O-methyl substituents of monoaromatic acids as a sole organic source of carbon was isolated from municipal sewage sludge. Energy for growth seemed to be generated by an acetate formation pathway. The growth yield in defined medium was 7.9 g (dry weight) of cells per mol of ferulate utilized. This isolate and other O-demethylating anaerobes may play a role in the turnover of acetate and the metabolism of highly methoxylated lignaceous materials in anaerobic environments.     

Identification of Novel Cytotoxic Peptide KENPVLSLVNGMF from Marine Sponge <Emphasis Type="Italic">Xestospongia testudinaria</Emphasis>, with Characterization of Stability in Human Serum

Resistance and side effects are common problems for anticancer drugs used in chemotherapy. Thus, continued research to discover novel and specific anticancer drugs is obligatory. Marine sponges hold great promise as a source of potent cytotoxic peptides with future applications in cancer treatments. This study aimed to purify and identify cytotoxic peptides from the protein hydrolysates of the giant barrel sponge Xestospongia testudinaria, guided by a cytotoxicity assay based on the human cervical cancer cell line (HeLa). Comparison among trypsin, chymotrypsin, papain and alcalase hydrolysates of X. testudinaria revealed papain hydrolysate (PH) to be the most active. PH was purified consecutively by membrane ultrafiltration, gel filtration chromatography, and reversed-phase high performance liquid chromatography (RP-HPLC). Following liquid chromatography-tandem mass spectrometric analysis, two peptides were identified from the most cytotoxic RP-HPLC fraction: KENPVLSLVNGMF and LLATIPKVGVFSILV. Between the two, only the synthetic peptide KENPVLSLVNGMF showed cytotoxicity toward HeLa cells in a dose-dependent manner. KENPVLSLVNGMF (EC₅₀ 0.67 mM) was 3.8-fold more cytotoxic compared with anticancer drug 5-fluorouracil (EC₅₀ 2.56 mM). Furthermore, KENPVLSLVNGMF show only marginal 5% cytotoxicity to Hek293, a non-cancerous, human embryonic kidney cell line, when tested at 0.67 mM. The half-life of the peptide was 3.2?±?0.5 h in human serum in vitro, as revealed by RP-HPLC analyses. These results suggest that KENPVLSLVNGMF identified from X. testudinaria papain hydrolysate has potential applications as peptide lead in future development of potent and specific anticancer drugs.     

Hydrophobic mannosides act as acceptors for trypanosome {alpha}-mannosyltransferases

A series of hydrophobic mannosides were synthesized and testedfor their ability to act as acceptor substrates for mannosyltransferasesin a Trypanosoma brucei cell-free system. The thiooctyl -mannosidesand octyl -mannosides all accepted single mannose residues in-linkage, as judged by thin layer chromatography of the productsbefore and after jack bean -mannosidase digestion. The mannosylationreactions were inhibited by amphomycin, suggesting that theimmediate donor was dolicholphosphate-mannose (Dol-P-Man) inall cases. The transferred -mannose residues were shown to beboth 1-2 and 1-6 linked by Aspergillus phoenicis -mannosidaseand acetolysis treatments, respectively. These data suggestthat the compounds can act as acceptor substrates for the Dol-P-Mandependent 1-2 and 1-6 mannosyltransferases of the GPI biosyntheticpathway and/or the dolichol-cycle of protein N-glycosylation.One of the compounds, Man1-6Man1-O-(CH₂)₇CH₃, inhibited endogenousGPI biosynthesis in the cell-free system, suggesting that itcould be a substrate for the trypanosome Dol-P-Man:Man₂GlcN-Pl1-2 mannosyltransferase. dolichol glycosylphosphatidylinositol mannosyltransferase trypanosome     

Functional metagenomic selection of ribulose 1, 5‐bisphosphate carboxylase/oxygenase from uncultivated bacteria

Ribulose 1,5‐bisphosphate carboxylase/oxygenase (RubisCO) is a critical yet severely inefficient enzyme that catalyses the fixation of virtually all of the carbon found on Earth. Here, we report a functional metagenomic selection that recovers physiologically active RubisCO molecules directly from uncultivated and largely unknown members of natural microbial communities. Selection is based on CO₂‐dependent growth in a host strain capable of expressing environmental deoxyribonucleic acid (DNA), precluding the need for pure cultures or screening of recombinant clones for enzymatic activity. Seventeen functional RubisCO‐encoded sequences were selected using DNA extracted from soil and river autotrophic enrichments, a photosynthetic biofilm and a subsurface groundwater aquifer. Notably, three related form II RubisCOs were recovered which share high sequence similarity with metagenomic scaffolds from uncultivated members of the Gallionellaceae family. One of the Gallionellaceae RubisCOs was purified and shown to possess CO₂/O₂ specificity typical of form II enzymes. X‐ray crystallography determined that this enzyme is a hexamer, only the second form II multimer ever solved and the first RubisCO structure obtained from an uncultivated bacterium. Functional metagenomic selection leverages natural biological diversity and billions of years of evolution inherent in environmental communities, providing a new window into the discovery of CO₂‐fixing enzymes not previously characterized.     

Purification, crystallisation and preliminary X-ray diffraction characterisation of methanol dehydrogenase from Methylosinus trichosporium OB3b

Methanol dehydrogenase was purified from the obligate methanotroph, Methylosinus trichosporium OB3b, in two steps from disrupted biomass by aqueous two-phase partition and ion-exchange chromatography. Copartitioning of a cytochrome c was dependent upon the pH at which aqueous partition was carried out. The native enzyme has a Mr of 120,000, as determined by gel filtration chromatography, and consists of two identical subunits. The purified enzyme contained four electrophoretically distinct isoenzymes, with pI values of 6.3, 6.58, 6.63 and 6.88. The native enzyme has been crystallised in a form suitable for high-resolution X-ray crystallographic studies. The crystals diffract to better than 0.19 nm spacing and are relatively stable to irradiation with X-rays. The space group is P6(1)22 (or P6(5)22) with cell dimensions a = b = 10.21 nm, c = 29.32 nm and the crystal probably contains a single monomer in the asymmetric unit.     

Style proteins of a wild tomato (Lycopersicon peruvianum) associated with expression of self-incompatibility

The identification, isolation and aminoterminal sequencing of two S-genotype-associated proteins from style extracts of Lycopersicon peruvianum Mill. is reported. There is a high level of homology between these two sequences and with the amino-terminal sequences of other S-allele-associated glycoproteins isolated from Nicotiana alata Link et Otto. These sequences were obtained by a new high-sensitivity method of selected twodimensional gel analysis followed by electroelution and purification of proteins by inverse-gradient high-performance liquid chromatography before sequencing.Abbreviations HPLC high-performance liquid chromatography - Mr relative molecular mass - PTH phenylthiohydrantoin - SDS sodium dodecyl sulphate