Reed J W, Walker G C
Department of Biology, Massachusetts Institute of Technology, Cambridge 02139.
Genes Dev. 1991 Dec;5(12A):2274-87. doi: 10.1101/gad.5.12a.2274.
Rhizobium meliloti exoD mutants are deficient in invasion of alfalfa nodules and, as a consequence, the nodules that exoD strains induce fail to fix nitrogen. These nodules appear to be arrested at the same stage as nodules induced by other exo mutants, which do not make an acidic exopolysaccharide called EPS I, or by ndv mutants, which do not produce a periplasmic cyclic beta(1,2) glucan. However previous genetic and biochemical evidence suggested that the nodule invasion defect of exoD mutants arose from a biochemical deficiency distinct from those of both EPS I-deficient exo mutants and ndv mutants. In this study, we characterize mutant phenotypes of exoD strains in both free-living and symbiotic states. Nodules induced by exoD mutants are generally small and empty of bacteria, and exhibit the same structural features as nodules induced by other invasion-deficient mutants. Putative incipient infection threads were visible in outer cortical cells of these nodules but not in the plant cells in the interior of the nodule. We show that exoD mutants are sensitive to alkaline conditions, ceasing to grow at elevated pH in liquid yeast extract cultures and exhibiting decreased viability in alkaline medium. Interestingly, we find that buffering the plant growth medium at slightly acidic pH (6.0-6.5) restores the ability of exoD mutants to invade alfalfa nodules. exoD mutants are thus alkali sensitive for both free-living and symbiotic phenotypes. This result implies that the nodule invasion defect of exoD mutants arises from their sensitivity to alkaline conditions and, furthermore, that alkaline conditions may obtain in the developing infection thread. The deduced amino acid sequence of ExoD is extremely hydrophobic, suggesting that the protein is membrane associated. We propose models whereby absence of a putative membrane protein might lead to sensitivity to alkaline conditions and consequent arrest of nodule invasion.
苜蓿中华根瘤菌exoD突变体在侵染苜蓿根瘤方面存在缺陷，因此，exoD菌株诱导形成的根瘤无法固氮。这些根瘤似乎停滞在与其他exo突变体（不能产生一种名为EPS I的酸性胞外多糖）或ndv突变体（不能产生周质环状β(1,2)葡聚糖）诱导形成的根瘤相同的阶段。然而，先前的遗传学和生物化学证据表明，exoD突变体的根瘤侵染缺陷源于一种与EPS I缺陷型exo突变体和ndv突变体不同的生化缺陷。在本研究中，我们对exoD菌株在自由生活和共生状态下的突变体表型进行了表征。exoD突变体诱导形成的根瘤通常较小且无细菌，并且表现出与其他侵染缺陷型突变体诱导形成的根瘤相同的结构特征。在这些根瘤的外皮层细胞中可见假定的初期感染丝，但在根瘤内部的植物细胞中则不可见。我们发现exoD突变体对碱性条件敏感，在液体酵母提取物培养基中，pH升高时停止生长，并且在碱性培养基中活力下降。有趣的是，我们发现将植物生长培养基缓冲至微酸性pH（6.0 - 6.5）可恢复exoD突变体侵染苜蓿根瘤的能力。因此，exoD突变体在自由生活和共生表型方面均对碱敏感。这一结果表明，exoD突变体的根瘤侵染缺陷源于它们对碱性条件的敏感性，此外，在发育中的感染丝中可能存在碱性条件。ExoD推导的氨基酸序列具有极强的疏水性，表明该蛋白与膜相关。我们提出了一些模型，据此推测膜蛋白的缺失可能导致对碱性条件敏感，进而导致根瘤侵染停滞。
