Division of Plant Microbe Interactions, National Botanical Research Institute, Rana Pratap Marg, Lucknow 226 001, India.
Lett Appl Microbiol. 2010 Feb;50(2):205-10. doi: 10.1111/j.1472-765X.2009.02779.x. Epub 2009 Nov 23.
To isolate the rhizosphere competence-defective transposon Tn5 mutant of Pantoea agglomerans NBRISRM (SRM) and to identify the gene causing defect in its root colonization ability.
From over 5000 clones containing Tn5, one mutant P. agglomerans NBRISRMT (SRMT) showing 6 log units less colonization when compared with SRM, after 30 days in sand-nonsterilized soil assay system was selected for further work to determine the effects of the mutation on rhizosphere competence. Southern hybridization analysis of restricted genomic DNA of SRMT demonstrated that the mutant had a single Tn5 insert. SRM increased in titre to about 2 x 10(8) CFU g(-1) root, compared with the indigenous bacterial population of heterotrophs of about 5 x 10(7) CFU g(-1) root. In contrast, 30 days later, the titre value of SRMT was almost undetectable at 1 x 10(2) CFU g(-1) root, demonstrating its inability to survive and colonize the rhizosphere. Sequencing of the flanking region of the Tn5 mutant revealed that Tn5 disrupted the purB gene.
A defect in the colonization phenotype of the SRMT was attributed to the disruption in adenylosuccinate lyase (EC 4.3.2.2) which is encoded by the pur B gene and is required for rhizosphere colonization in P. agglomerans. Significantly less exopolysaccharide and biofilm was formed by SRMT when compared to SRM, because of the disruption of the purB gene.
This work provides the first evidence for a functional role of purB gene in rhizosphere competence and root colonization by any rhizobacteria.
分离成团泛菌（Pantoea agglomerans）NBRISRM（SRM）的根际竞争缺陷型转座子 Tn5 突变体，并鉴定导致其根际定殖能力缺陷的基因。
从含有 Tn5 的 5000 多个克隆中，选择了一株在沙-未灭菌土壤模拟系统中定殖能力比 SRM 低 6 个对数单位的突变体 P. agglomerans NBRISRMT（SRMT）进行进一步研究，以确定突变对根际竞争的影响。SRMT 的限制性基因组 DNA 的 Southern 杂交分析表明，突变体有一个单一的 Tn5 插入。与土著异养菌的种群约 5 x 10(7) CFU g(-1)根相比，SRM 的浓度增加到约 2 x 10(8) CFU g(-1)根。相比之下，30 天后，SRMT 的浓度值几乎无法检测到 1 x 10(2) CFU g(-1)根，表明其无法在根际中生存和定殖。Tn5 突变体侧翼区的测序表明，Tn5 破坏了 purB 基因。
SRMT 的定殖表型缺陷归因于其对腺苷酸琥珀酸裂解酶（EC 4.3.2.2）的破坏，该酶由 purB 基因编码，是成团泛菌根际定殖所必需的。与 SRM 相比，SRMT 形成的胞外多糖和生物膜明显减少，因为 purB 基因被破坏。
这项工作首次提供了 purB 基因在任何根际细菌的根际竞争力和根际定殖中的功能作用的证据。
