Yadav Kavita, Kumar Chanchal, Archana G, Naresh Kumar G
Molecular Microbial Biochemistry Laboratory, Department of Biochemistry, Faculty of Science, The Maharaja Sayajirao University of Baroda, Vadodara, India.
Department of Microbiology and Biotechnology Centre, Faculty of Science, The Maharaja Sayajirao University of Baroda, Vadodara, India.
PLoS One. 2014 Apr 4;9(4):e92400. doi: 10.1371/journal.pone.0092400. eCollection 2014.
Oxalate secretion was achieved in Pseudomonas fluorescens ATCC 13525 by incorporation of genes encoding Aspergillus niger oxaloacetate acetyl hydrolase (oah), Fomitopsis plaustris oxalate transporter (FpOAR) and Vitreoscilla hemoglobin (vgb) in various combinations. Pf (pKCN2) transformant containing oah alone accumulated 19 mM oxalic acid intracellularly but secreted 1.2 mM. However, in the presence of an artificial oxalate operon containing oah and FpOAR genes in plasmid pKCN4, Pf (pKCN4) secreted 13.6 mM oxalate in the medium while 3.6 mM remained inside. This transformant solubilized 509 μM of phosphorus from rock phosphate in alfisol which is 4.5 fold higher than the Pf (pKCN2) transformant. Genomic integrants of P. fluorescens (Pf int1 and Pf int2) containing artificial oxalate operon (plac-FpOAR-oah) and artificial oxalate gene cluster (plac-FpOAR-oah, vgb, egfp) secreted 4.8 mM and 5.4 mM oxalic acid, released 329 μM and 351 μM P, respectively, in alfisol. The integrants showed enhanced root colonization, improved growth and increased P content of Vigna radiata plants. This study demonstrates oxalic acid secretion in P. fluorescens by incorporation of an artificial operon constituted of genes for oxalate synthesis and transport, which imparts mineral phosphate solubilizing ability to the organism leading to enhanced growth and P content of V. radiata in alfisol soil.
通过以各种组合方式导入编码黑曲霉草酰乙酸乙酰水解酶（oah）、拟层孔菌草酸转运蛋白（FpOAR）和透明颤菌血红蛋白（vgb）的基因，在荧光假单胞菌ATCC 13525中实现了草酸盐分泌。仅含有oah的Pf（pKCN2）转化体在细胞内积累了19 mM草酸，但分泌了1.2 mM。然而，在质粒pKCN4中存在包含oah和FpOAR基因的人工草酸操纵子时，Pf（pKCN4）在培养基中分泌了13.6 mM草酸盐，而细胞内还残留3.6 mM。该转化体从淋溶土中的磷矿粉中溶解了509 μM的磷，这比Pf（pKCN2）转化体高4.5倍。含有人工草酸操纵子（plac-FpOAR-oah）和人工草酸基因簇（plac-FpOAR-oah、vgb、egfp）的荧光假单胞菌基因组整合体（Pf int1和Pf int2）在淋溶土中分别分泌了4.8 mM和5.4 mM草酸，释放了329 μM和351 μM的磷。这些整合体表现出增强的根部定殖能力、促进了豇豆植株的生长并增加了其磷含量。本研究表明，通过导入由草酸合成和转运基因组成的人工操纵子，荧光假单胞菌能够分泌草酸，这赋予了该生物体溶解矿物磷酸盐的能力，从而促进了淋溶土中豇豆的生长并增加了其磷含量。
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