ANALYSIS OF THE STABILIZATION SYSTEM OF PSM19035-DERIVED PLASMID PBT233 IN BACILLUS-SUBTILIS
ANALYSIS OF THE STABILIZATION SYSTEM OF PSM19035-DERIVED PLASMID PBT233 IN BACILLUS-SUBTILIS
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DOI：
10.1016/0378-1119(93)90441-5
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发表时间：
1993-12-22
期刊：
影响因子：
3.5
通讯作者：
ALONSO, JC
中科院分区：
文献类型：
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作者：
CEGLOWSKI, P;BOITSOV, A;ALONSO, JC
低拷贝数、9.0 kb pSM 19035衍生质粒pBT 233在枯草芽孢杆菌中稳定遗传。已测定pBT 233的完整核苷酸（nt）序列。nt序列分析揭示了9个主要的开放阅读框架（orfs）。repS、erm 1和erm 2基因被分配给其中三个orf，并给出基因顺序：repS-orf alpha-orf beta-orf gamma-orf delta-orf epsilon-orf zeta-erm 2-erm 1。repS-orf gamma区域的基因组织类似于pAM beta 1的repE-orfI区域的基因组织。通过北方印迹检测分子量对应于repS或orf alpha+orf beta、orf gamma、orf delta和orf delta +orf zeta的信使RNA种类。23.8、81.3、34.4、10.7和32.4kDa的蛋白质分别对应于Orfs β、γ、δ、ε和ζ。使用T7启动子表达系统检测到25、81、34、10和32 kDa的放射性蛋白条带。orf beta和orf gamma分别编码与位点特异性重组酶和I型拓扑异构酶具有同源性的蛋白质。orfs，delta，zeta，编码具有未知活性的蛋白质。删除一个1.5 kb的片段（nt 2999-4552）与编码能力的orf β，orf γ和orf δ似乎不影响质粒的维护。去除3.0 kb片段（nt 4598-7689）具有编码orf orfy zeta的能力，降低了质粒的分离稳定性，但缺失了一个5.2kb的DNA片段，（nt 2546-7826）废除了它。基于这些观察，我们得出结论，pBT 233稳定依赖于一个复杂的系统，涉及质粒寡聚体和其它未知组分的解析。
The low-copy-number, 9.0-kb pSM19035-derived plasmid pBT233, is stably inherited in Bacillus subtilis. The complete nucleotide (nt) sequence of pBT233 has been determined. Analysis of the nt sequence revealed nine major open reading frames (orfs). The repS, erm1 and erm2 genes have been assigned to three of these orfs, and given the gene order, repS-orf alpha-orf beta-orf gamma-orf delta-orf epsilon-orf zeta-erm2-erm1. The organization of genes of the repS-orf gamma region resembles the organization of genes in the repE-orfI region of pAM beta 1. Messenger RNA species of molecular weights corresponding to repS, or orf alpha+orf beta, orf gamma, orf delta, and orf epsilon+orf zeta were detected by Northern blotting. Proteins of 23.8, 81.3, 34.4, 10.7 and 32.4 kDa correspond to Orfs beta, gamma, delta, epsilon and zeta, respectively. Bands of radioactive proteins of 25, 81, 34, 10 and 32 kDa were detected using the T7 promoter-expression system. The orf beta and orf gamma encode proteins that share homology to site-specific recombinases and type-I topoisomerases, respectively. The orfs, delta, epsilon and zeta, encode proteins with unknown activity. Deletion of a 1.5-kb segment (nt 2999-4552) with coding capacity for orf beta, orf gamma and orf delta does not seem to affect plasmid maintenance. Removal of a 3.0-kb fragment (nt 4598-7689) with coding capacity for orf epsilon orfy zeta reduced plasmid segregational stability, but deletion of a 5.2-kb DNA segment (nt 2546-7826) abolished it. On the basis of these observations, we conclude that pBT233 stabilization relies on a complex system, involving the resolution of plasmid oligomers and additional unknown component(s).