Microbial Gutta-Percha Degradation Shares Common Steps with Rubber Degradation by Nocardia nova SH22a
Microbial Gutta-Percha Degradation Shares Common Steps with Rubber Degradation by Nocardia nova SH22a
复制标题
DOI：
10.1128/aem.03016-12
复制
发表时间：
2013-02-01
影响因子：
4.4
通讯作者：
Steinbuechel, Alexander
中科院分区：
文献类型：
--
作者：
Luo, Quan;Hiessl, Sebastian;Steinbuechel, Alexander
以一株能降解杜仲胶（GP）和天然橡胶（NR）的细菌Nocardia nova SH 22 a为试材，研究了GP和NR降解途径的关系及GP降解机理。对于该菌株，系统地优化了电穿孔方案，并且实现了高达4.3 × 10（7）CFU/μ g质粒DNA的效率。将该优化方案应用于N. nova SH 22 a，构建了该细菌的基于Tn 5096的转座子诱变文库。在约12，000个安普霉素抗性转化体中，我们鉴定了76个GP或NR利用缺陷的稳定突变体。而10个突变体是专门缺陷的GP利用，其他66个突变体的生长受到影响的GP和NR。这表明这两种降解途径非常相似，并且有许多共同的步骤。GP降解缺陷突变体的数量较多可以用以下两种方式之一来解释：（i）GP途径更复杂，包含更具体的步骤，或（ii）两种途径的步骤几乎相同，但在GP降解的情况下，每个步骤中涉及的酶较少。转座位点的分析和对感兴趣基因的遗传研究证实了α-甲酰基辅酶A消旋酶在GP和NR的降解中的关键作用。我们还证明了参与氧化还原反应，β-氧化，和复杂的细胞包膜脂质的合成GP的降解酶的可能参与。
Nocardia nova SH22a, a bacterium capable of degrading gutta-percha (GP) and natural rubber (NR), was used to investigate the GP degradation mechanism and the relations between the GP and NR degradation pathways. For this strain, a protocol of electroporation was systematically optimized, and an efficiency of up to 4.3 x 10(7) CFU per mu g of plasmid DNA was achieved. By applying this optimized protocol to N. nova SH22a, a Tn5096-based transposon mutagenesis library of this bacterium was constructed. Among about 12,000 apramycin-resistant transformants, we identified 76 stable mutants defective in GP or NR utilization. Whereas 10 mutants were specifically defective in GP utilization, the growth of the other 66 mutants was affected on both GP and NR. This indicated that the two degradation pathways are quite similar and share many common steps. The larger number of GP-degrading defective mutants could be explained in one of two ways: either (i) the GP pathway is more complex and harbors more specific steps or (ii) the steps for both pathways are almost identical, but in the case of GP degradation there are fewer enzymes involved in each step. The analysis of transposition loci and genetic studies on interesting genes confirmed the crucial role of an alpha-methylacyl-coenzyme A racemase in the degradation of both GP and NR. We also demonstrated the probable involvement of enzymes participating in oxidoreduction reactions, beta-oxidation, and the synthesis of complex cell envelope lipids in the degradation of GP.