Genetic and physiological characterization of a Rhizobium etli mutant strain unable to synthesize poly-beta-hydroxybutyrate
Genetic and physiological characterization of a Rhizobium etli mutant strain unable to synthesize poly-beta-hydroxybutyrate
复制标题
DOI：
10.1128/jb.178.6.1646-1654.1996
复制
发表时间：
1996-03-01
影响因子：
3.2
通讯作者：
Mora, J
中科院分区：
文献类型：
--
作者：
Cevallos, MA;Encarnacion, S;Mora, J
根瘤菌等在共生和自由生活中积累聚-β-羟基丁酸酯(PHB)。PHB是一种储备材料，当不满足最佳生长条件时，它充当碳和/或电子吸收。有人提出，在共生中，PHB可以将固氮延长到种子发育的最后阶段，但到目前为止还没有实验来检验这一命题。为了直接解决这些问题，我们通过在PHB合成酶结构基因(PHAC)中插入一个Omega-KM插入子，构建了一个R.etli PHB阴性突变体。文中还报道了R.etli phac基因的鉴定和序列测定。生理研究表明，PHB阴性突变株不能合成PHB，分泌的乳酸、乙酸盐、丙酮酸、β-羟丁酸、富马酸和苹果酸均高于野生型菌株。突变菌株的NAD(+)/NADH比值低于亲本菌株。PHB阴性突变体的氧化能力降低。因此，突变菌株在添加葡萄糖或丙酮酸的最低限度的培养基中生长的能力严重减弱。我们提出，在自由生命中，PHB的合成隔离了还原能力，允许三元酸循环在氧气为限制因素的条件下进行。在与普通菜豆的共生中，PHB阴性突变体诱导的根瘤延长了固氮能力。
Rhizobium etli accumulates poly-beta-hydroxybutyrate (PHB) in symbiosis and in free life. PHB is a reserve material that serves as a carbon and/or electron sink when optimal growth conditions are not met. It has been suggested that in symbiosis PHB can prolong nitrogen fixation until the last stages of seed development, but experiments to test this proposition have not been done until now. To address these questions in a direct way, we constructed an R. etli PHB-negative mutant by the insertion of an Omega-Km interposon within the PHB synthase structural gene (phaC). The identification and sequence of the R. etli phaC gene are also reported here. Physiological studies showed that the PHB-negative mutant strain was unable to synthesize PHB and excreted more lactate, acetate, pyruvate, beta-hydroxybutyrate, fumarate, and malate than the wild-type strain. The NAD(+)/NADH ratio in the mutant strain was lower than that in the parent strain. The oxidative capacity of the PHB-negative mutant was reduced. Accordingly, the ability to grow in minimal medium supplemented with glucose or pyruvate was severely diminished in the mutant strain. We propose that in free life PHB synthesis sequesters reductive power, allowing the tricarboxylic acid cycle to proceed under conditions in which oxygen is a limiting factor. In symbiosis with Phaseolus vulgaris, the PHB-negative mutant induced nodules that prolonged the capacity to fix nitrogen.