Lagares A, Caetano-Anollés G, Niehaus K, Lorenzen J, Ljunggren H D, Pühler A, Favelukes G
Cátedra de Química Biológica I, Facultad de Ciencias Exactas, Universidad Nacional de La Plata, Argentina.
J Bacteriol. 1992 Sep;174(18):5941-52. doi: 10.1128/jb.174.18.5941-5952.1992.
A transposon Tn5-induced mutant of Rhizobium meliloti Rm2011, designated Rm6963, showed a rough colony morphology on rich and minimal media and an altered lipopolysaccharide (LPS). Major differences from the wild-type LPS were observed in (i) hexose and 2-keto-3-deoxyoctonate elution profiles of crude phenol extracts chromatographed in Sepharose CL-4B, (ii) silver-stained sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis patterns of crude and purified LPS fractions, and (iii) immunoreactivities otherwise present in purified LPS of the parental strain Rm2011. In addition, Rm6963 lost the ability to grow in Luria-Bertani medium containing the hydrophobic compounds sodium deoxycholate or SDS and showed a decrease in survival in TY medium supplemented with high calcium concentrations. The mutant also had altered symbiotic properties. Rm6963 formed nodules that fixed nitrogen but showed a delayed or even reduced ability to nodulate the primary root of alfalfa without showing changes in the position of nodule distribution profiles along the roots. Furthermore, 2 to 3 weeks after inoculation, plants nodulated by Rm6963 were smaller than control plants inoculated with wild-type bacteria in correlation with a transient decrease in nitrogen fixation. In most experiments, the plants recovered later by expressing a full nitrogen-fixing phenotype and developing an abnormally high number of small nodules in lateral roots after 1 month. Rm6963 was also deficient in the ability to compete for nodulation. In coinoculation experiments with equal bacterial numbers of both mutant and wild-type rhizobia, only the parent was recovered from the uppermost root nodules. A strain ratio of approximately 100 to 1 favoring the mutant was necessary to obtain an equal ratio (1:1) of nodule occupancy. These results show that alterations in Rm6963 which include LPS changes lead to an altered symbiotic phenotype during the association with alfalfa that affects the timing of nodule emergence, the progress of nitrogen fixation, and the strain competitiveness for nodulation.
苜蓿根瘤菌Rm2011的一个转座子Tn5诱导突变体，命名为Rm6963，在丰富培养基和基本培养基上呈现粗糙的菌落形态，且脂多糖（LPS）发生改变。在以下方面观察到与野生型LPS的主要差异：（i）在琼脂糖CL-4B上进行层析的粗酚提取物的己糖和2-酮-3-脱氧辛酸洗脱图谱；（ii）粗LPS组分和纯化LPS组分的银染十二烷基硫酸钠（SDS）-聚丙烯酰胺凝胶电泳图谱；（iii）亲本菌株Rm2011纯化LPS中原本存在的免疫反应性。此外，Rm6963失去了在含有疏水化合物脱氧胆酸钠或SDS的Luria-Bertani培养基中生长的能力，并且在补充高钙浓度的TY培养基中的存活率降低。该突变体的共生特性也发生了改变。Rm6963形成了能固氮的根瘤，但在结瘤苜蓿主根时能力延迟甚至降低，且沿根的根瘤分布轮廓位置未发生变化。此外，接种后2至3周，被Rm6963结瘤的植株比接种野生型细菌的对照植株小，这与固氮的短暂下降相关。在大多数实验中，植株后来恢复，1个月后表现出完全的固氮表型，并在侧根上形成异常多的小根瘤。Rm6963在竞争结瘤能力方面也存在缺陷。在突变型和野生型根瘤菌数量相等的共接种实验中，仅在最上部的根瘤中回收了亲本。需要大约100比1的菌株比例有利于突变体才能获得相等的（1:1）根瘤占有率。这些结果表明，Rm6963中的改变包括LPS变化，导致与苜蓿共生期间共生表型改变，影响根瘤出现的时间、固氮进程以及菌株的结瘤竞争力。
