Insertion mutation of the form I cbbL gene encoding ribulose bisphosphate carboxylase/oxygenase (RuBisCO) in Thiobacillus neapolitanus results in expression of form II RuBisCO, loss of carboxysomes, and an increased CO2 requirement for growth
Insertion mutation of the form I cbbL gene encoding ribulose bisphosphate carboxylase/oxygenase (RuBisCO) in Thiobacillus neapolitanus results in expression of form II RuBisCO, loss of carboxysomes, and an increased CO2 requirement for growth
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DOI：
10.1128/jb.180.16.4133-4139.1998
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发表时间：
1998-08-01
影响因子：
3.2
通讯作者：
Shively, JM
中科院分区：
文献类型：
--
作者：
Baker, SH;Jin, SM;Shively, JM
先前已经确定，那不勒斯硫杆菌通过使用 I 型核酮糖二磷酸羧化酶/加氧酶 (RuBisCO) 来固定 CO2，大部分酶被隔离在羧基体中，并且该酶的基因 cbbL 和 cbbS 是假定的羧基体操纵子的一部分。在本研究中，对 cbbL 和 cbbS 进行了克隆和测序。 RNA 分析表明，cbbL 和 cbbS 共同转录大约 2,000 个核苷酸大小的信息。将卡那霉素抗性盒插入 cbbL 会导致转录过早终止；产生了极性突变体。该突变体能够固定二氧化碳，但需要补充二氧化碳才能生长。在蔗糖梯度上从野生型和突变体中分离细胞蛋白，并对收集的级分中的 RuBisCO 活性进行随后的分析表明，突变体通过使用 II 型 RuBisCO 同化 CO2。使用针对 I 型和 II 型 RuBisCO 产生的抗体进行免疫印迹分析证实了这一点。该突变体不具有羧基体。存在较小的空内含物，但生化分析表明，如果它们与羧基相关，则它们没有功能，即不包含 RuBisCO。 Northern分析表明，产生了羧基体的一些壳成分，这可以解释突变体中这些内含物的存在。
It has been previously established that Thiobacillus neapolitanus fixes CO2 by using a form I ribulose bisphosphate carboxylase/oxygenase (RuBisCO), that much of the enzyme is sequestered into carboxysomes, and that the genes for the enzyme, cbbL and cbbS, are part of a putative carboxysome operon. In the present study, cbbL and cbbS were cloned and sequenced. Analysis of RNA showed that cbbL and cbbS are cotranscribed on a message approximately 2,000 nucleotides in size. The insertion of a kanamycin resistance cartridge into cbbL resulted in a premature termination of transcription; a polar mutant was generated. The mutant is able to fix CO2, but requires a CO2 supplement for growth. Separation of cellular proteins from both the wild type and the mutant on sucrose gradients and subsequent analysis of the RuBisCO activity in the collected fractions showed that the mutant assimilates CO2 by using a form II RuBisCO. This was confirmed by immunoblot analysis using antibodies raised against form I and form II RuBisCOs. The mutant does not possess carboxysomes. Smaller, empty inclusions are present, but biochemical analysis indicates that if they are carboxysome related, they are not functional, i.e., do not contain RuBisCO. Northern analysis showed that some of the shell components of the carboxysome are produced, which may explain the presence of these inclusions in the mutant.