title: Evidence for lytic transglycosylase and β-N-acetylglucosaminidase activities located at the polyhydroxyalkanoates (PHAs) granules of Thermus thermophilus HB8.
abstract: The thermophilic bacterium Thermus thermophilus HB8 accumulates polyhydroxyalkanoates (PHAs) as intracellular granules used by cells as carbon and energy storage compounds. PHAs granules were isolated from cells grown in sodium gluconate (1.5 % w/v) as carbon source. Lytic activities are strongly associated and act to the PHAs granules proved with various methods. Specialized lytic trasglycosylases (LTGs) are muramidases capable of locally degrading the peptidoglycan (PG) meshwork of Gram negative bacteria. These enzymes cleave the β-1,4-glycosidic linkages between the N-acetylmuramic acid (MurNAc) and N-acetylglucosamine (GlcNAc) residues of PG. Lysozyme-like activity/-ies were detected using lysoplate assay. Chitinolytic activity/-ies, were detected as N-acetyl glucosaminidases (NAG) (E.C.3.2.1.5.52) hydrolyzing the synthetic substrate p-nitrophenyl-N-acetyl-β-D-glucosaminide (pNP-GlcNAc) releasing pNP and GlcNAc. Using zymogram analysis two abundant LTGs were revealed hydrolyzing cell wall of Micrococcus lysodeikticus or purified PG incorporated as natural substrates, in SDS-PAGE and then renaturation. These proteins corresponded in a SDS-PAGE and Coomassie-stained gel in molecular mass of 110 and 32 kDa respectively, were analyzed by MALDI-MS (Matrix-assisted laser desorption/ionization-Mass Spectrometry). The 110 kDa protein was identified as an S-layer domain-containing protein [gi|336233805], while the 32 kDa similar to the hypothetical protein VDG1235_2196 (gi/254443957). Overall, the localization of PG hydrolases in PHAs granules appears to be involved to their biogenesis from membranes, and probably promoting septal PG splitting and daughter cell separation.
authors.affiliation: Laboratory of Biochemistry, Dept. of Chemistry, Aristotle University of Thessaloniki, 54124, Thessaloniki, Greece.
authors.forename: Olga M
authors.initials: OM
authors.lastname: Simou
authors.forename: Anastasia A
authors.initials: AA
authors.lastname: Pantazaki
mesh_terms: D000118:Acetylglucosaminidase / Q000737:chemistry / Q000302:isolation & purification* / Q000378:metabolism*; D002473:Cell Wall / Q000378:metabolism; D004591:Electrophoresis, Polyacrylamide Gel; D006868:Hydrolysis; D008837:Micrococcus / Q000378:metabolism; D008970:Molecular Weight; D010457:Peptidoglycan / Q000378:metabolism; D046928:Peptidoglycan Glycosyltransferase / Q000737:chemistry / Q000302:isolation & purification* / Q000378:metabolism*; D054813:Polyhydroxyalkanoates / Q000378:metabolism*; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization; D016963:Thermus thermophilus / Q000201:enzymology* / Q000378:metabolism*
chemical_list: D010457:Peptidoglycan; D054813:Polyhydroxyalkanoates; D046928:Peptidoglycan Glycosyltransferase; D000118:Acetylglucosaminidase