Metabolite Profiling Uncovers Plasmid-Induced Cobalt Limitation under Methylotrophic Growth Conditions
Metabolite Profiling Uncovers Plasmid-Induced Cobalt Limitation under Methylotrophic Growth Conditions
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DOI：
10.1371/journal.pone.0007831
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发表时间：
2009-11-13
期刊：
影响因子：
3.7
通讯作者：
Vorholt, Julia A.
中科院分区：
文献类型：
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作者：
Kiefer, Patrick;Buchhaupt, Markus;Vorholt, Julia A.
背景：质粒在细胞中的引入和维持通常与生长速率的降低有关。这种增长减少的原因是不清楚的，在许多cases.Methodology/主要调查结果：我们观察到一个令人惊讶的大减少约50%的扭脱甲基杆菌AM1在甲基营养型生长过程中的质粒，pCM80表达tetA基因的存在下，相对于野生型的增长率。在非甲基营养生长条件下生长期间观察到不太明显的生长延迟;这表明抑制一碳代谢，而不是一般的生长抑制或代谢负担。代谢物组分析显示，乙基丙二酰辅酶A和甲基丙二酰辅酶A的库大小分别增加了6倍和35倍以上，相对于野生型，这表明这些中心中间体的转化强烈减少，这是该模型甲基营养型中乙醛酸再生所必需的。对M.赋予卡那霉素抗性的extorquens AM1 pCM 160。乙基丙二酰辅酶A途径的这些中间体的共同点是通过辅酶B-12依赖性突变酶进行转化，所述辅酶B-12依赖性突变酶具有钴作为中心配体。通过提供更高的钴浓度，异柠檬酸裂解酶的异源表达作为乙醛酸再生的替代途径，或通过鉴定和过量生产的蛋白质参与钴import.Conclusions/Significance：这项研究表明，引入的质粒导致明显的抑制钴依赖性酶的乙基丙二酰辅酶A途径。可能的解释，并指出有限的钴浓度在细胞中的抗生素压力的结果。
Background: The introduction and maintenance of plasmids in cells is often associated with a reduction of growth rate. The reason for this growth reduction is unclear in many cases.Methodology/Principal Findings: We observed a surprisingly large reduction in growth rate of about 50% of Methylobacterium extorquens AM1 during methylotrophic growth in the presence of a plasmid, pCM80 expressing the tetA gene, relative to the wild-type. A less pronounced growth delay during growth under non-methylotrophic growth conditions was observed; this suggested an inhibition of one-carbon metabolism rather than a general growth inhibition or metabolic burden. Metabolome analyses revealed an increase in pool sizes of ethylmalonyl-CoA and methylmalonyl-CoA of more than 6- and 35-fold, respectively, relative to wild type, suggesting a strongly reduced conversion of these central intermediates, which are essential for glyoxylate regeneration in this model methylotroph. Similar results were found for M. extorquens AM1 pCM160 which confers kanamycin resistance. These intermediates of the ethylmalonyl-CoA pathway have in common their conversion by coenzyme B-12-dependent mutases, which have cobalt as a central ligand. The one-carbon metabolism-related growth delay was restored by providing higher cobalt concentrations, by heterologous expression of isocitrate lyase as an alternative path for glyoxylate regeneration, or by identification and overproduction of proteins involved in cobalt import.Conclusions/Significance: This study demonstrates that the introduction of the plasmids leads to an apparent inhibition of the cobalt-dependent enzymes of the ethylmalonyl-CoA pathway. Possible explanations are presented and point to a limited cobalt concentration in the cell as a consequence of the antibiotic stress.