Cary J W, Petersen D J, Papoutsakis E T, Bennett G N
Department of Biochemistry, Rice University, Houston, Texas 77251.
J Bacteriol. 1988 Oct;170(10):4613-8. doi: 10.1128/jb.170.10.4613-4618.1988.
A 13.6-kilobase (kb) Sau3AI restriction endonuclease fragment of Clostridium acetobutylicum DNA cloned into pBR322 enabled Escherichia coli ato mutants to grow on butyrate as a sole carbon source (But+). Complementation of the ato defect by the recombinant plasmid pJC6 was due to expression of the genes for phosphotransbutyrylase (PTB) and butyrate kinase (BK). Both genes were efficiently expressed in E. coli, as their products were readily detected by sodium dodecyl sulfate-polyacrylamide gel electrophoresis of whole-cell extracts. PTB was found to have a polypeptide subunit molecular weight of approximately 31,000, while that of BK was approximately 39,000. Deletion analysis and Tn5 mutagenesis of plasmid pJC7 (a But+ subclone containing a 4.4-kb BamHI fragment from the insert of pJC6) localized the PTB and BK genes within a region spanning approximately 2.9 kb. Preliminary evidence suggests that the two genes may form an operon that is transcribed as a single unit from a promoter of clostridial origin within the 4.4-kb insert of pJC7.
将丙酮丁醇梭菌DNA的一个13.6千碱基（kb）的Sau3AI限制性内切酶片段克隆到pBR322中，使大肠杆菌ato突变体能够以丁酸盐作为唯一碳源生长（But+）。重组质粒pJC6对ato缺陷的互补作用是由于磷酸转丁酰酶（PTB）和丁酸盐激酶（BK）基因的表达。这两个基因在大肠杆菌中均有效表达，因为通过全细胞提取物的十二烷基硫酸钠-聚丙烯酰胺凝胶电泳可轻易检测到它们的产物。发现PTB的多肽亚基分子量约为31,000，而BK的约为39,000。对质粒pJC7（一个But+亚克隆，包含来自pJC6插入片段的4.4-kb BamHI片段）进行缺失分析和Tn5诱变，将PTB和BK基因定位在一个约2.9 kb的区域内。初步证据表明，这两个基因可能形成一个操纵子，从pJC7的4.4-kb插入片段内的梭菌来源启动子作为一个单一单元进行转录。
