Simou Olga M, Pantazaki Anastasia A
Laboratory of Biochemistry, Dept. of Chemistry, Aristotle University of Thessaloniki, 54124, Thessaloniki, Greece.
Appl Microbiol Biotechnol. 2014 Feb;98(3):1205-21. doi: 10.1007/s00253-013-4980-0. Epub 2013 May 18.
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.
嗜热栖热菌HB8会积累聚羟基脂肪酸酯（PHA）作为细胞内颗粒，细胞将其用作碳和能量储存化合物。PHA颗粒是从以葡萄糖酸钠（1.5% w/v）作为碳源生长的细胞中分离出来的。通过各种方法证明，裂解活性与PHA颗粒密切相关并作用于PHA颗粒。专门的裂解转糖基酶（LTG）是能够局部降解革兰氏阴性菌肽聚糖（PG）网络的溶菌酶。这些酶切割PG中N-乙酰胞壁酸（MurNAc）和N-乙酰葡糖胺（GlcNAc）残基之间的β-1,4-糖苷键。使用溶菌平板试验检测类似溶菌酶的活性。通过检测N-乙酰葡糖胺糖苷酶（NAG）（E.C.3.2.1.5.52）水解合成底物对硝基苯基-N-乙酰-β-D-葡糖胺苷（pNP-GlcNAc）释放对硝基苯酚（pNP）和GlcNAc来检测几丁质酶活性。使用酶谱分析揭示了两种丰富的LTG，它们在SDS-PAGE中水解溶壁微球菌的细胞壁或掺入的纯化PG作为天然底物，然后复性。这些蛋白质在SDS-PAGE和考马斯亮蓝染色凝胶中的分子量分别为110 kDa和32 kDa，通过基质辅助激光解吸/电离质谱（MALDI-MS）进行分析。110 kDa的蛋白质被鉴定为含有S层结构域的蛋白质[gi|336233805]，而32 kDa的蛋白质与假定蛋白VDG1235_2196（gi/254443957）相似。总体而言，PG水解酶在PHA颗粒中的定位似乎涉及其从膜的生物发生过程，并且可能促进隔膜PG的分裂和子细胞的分离。
