Studies on the Triangular-shaped Extremely Halophilic Archaeon

日本盐节菌（Haloarcula japonica）TR-1菌株是一株三角盘状嗜盐古菌，对其分类学特征、细胞分裂方式和细胞被膜超微结构进行了研究。然而，维持三角形形状的机制仍然不清楚。我们证明发生在其细胞表面的糖蛋白（细胞表面糖蛋白：CSG），这似乎是重要的，在维护的特征形状的Ha. japonica。对日本血吸虫CSG进行了纯化和鉴定。十二烷基硫酸钠-聚丙烯酰胺凝胶电泳（SDS-PAGE）表明，其表观分子量为170 kDa，糖含量约为12%（w/w）。多肽部分含有大比例的酸性氨基酸。合成了针对盐生盐杆菌和火山盐铁菌的CSG之间保守区域的寡核苷酸并用作PCR引物。从染色体上扩增出与CSG相关的序列 关于我们 通过PCR方法扩增了日本血吸虫CSG基因的一个片段，该片段大小约为890 bp，在大肠杆菌中进行了克隆和序列测定。根据预测的氨基酸序列，日本血吸虫的CSG与Hb、盐生菌和火山血吸虫的CSG具有明显的同源性。以PCR产物为探针，通过菌落杂交筛选转化子。发现阳性克隆含有具有相同的3.5 kb EcoRI插入片段的重组质粒。部分核苷酸序列分析表明，CSG基因的5 '端及上游区域为一个保守区。因此，通过基因测序，从另一个日本血吸虫基因组文库中克隆了2.5kb的Kpn I片段。3.5kb和2.5kb片段重叠1.4kb。将上述3.5-kb和2.5-kb片段在同一SacI位点重组，得到4.6-kb KpnI-EcoRI片段，然后插入pUC 119载体，构建质粒pJAG 16。少

Haloarcula japonica strain TR-1 is a predominantly triangular disc-shaped halophilic archaeon.Taxonomic characteristics, mode of cell division and ultrastructure of cell envelope have been studied for the strain to date. However, the mechanisms maintaing the triangular shape have been still unclear. We demonstrated occurrence of a glycoprotein on its cell surface (cell surface glycoprotein : CSG), which seemed to be important in maintenance of the characteristic shape of Ha.japonica. The Ha.japonica CSG was purified and characterized. It had an apparent molecular mass of 170 kDa in sodium dodecyl sulfate-polyacrylamide gel electrophoresis(SDS-PAGE)and the carbohydrate content was about 12%(w/w). The polypeptide portion contained a large proportion of acidic amino acids. Oligonucleotides, directed against conserved regions between the CSGs from Halobacterium halobium and Haloferax volcanii, were synthesized and used as PCR primers. CSG-related sequences were amplified from the chromosom … More al DNA of Ha.japonica by PCR.The PCR-generated DNA fragment of about 890 bp, presumed to be a part of the CSG gene of Ha.japonica, was cloned in Escherichia coli and sequenced. According to the predicted amino acid sequence, the CSG from Ha.japonica showed apparent homology with those from Hb, halobium and Hf.volcanii.Chromosomal DNA from Ha.japonica was digested with EcoRI,ligated to a vactor plasmid, and then transformed into E.coli to construct a genomic library. Transformants were screened by colony hybridization using the PCR product as a probe. Positive clones were found to contain recombinant plasmids with an identical 3.5-kb EcoRI insert. The partial nucleotide sequence indicated that the 5'end of the CSG gene and upstream region. Therefore, the KpnI fragment of 2.5 kb was cloned from another genomic library of Ha.japonica by gene walding. The 3.5-kb and 2.5-kb fragments overlapped by 1.4 kb. The 4.6-kb KpnI-EcoRI fragment was obtained by recombining the above 3.5-kb and 2.5-kb fragments at a common SacI site, and then inserted in pUC119 vector to construct plasmid pJAG16.SDS-PAGE of cell extract of E.coli harboring pJAG16 revealed that the Ha.japonica CSG gene was expressid in E.coli. Less