Karunakaran R, Ebert K, Harvey S, Leonard M E, Ramachandran V, Poole P S
School of Biological Sciences, University of Reading, Whiteknights, P.O. Box 228, Reading RG6 6AJ, United Kingdom.
J Bacteriol. 2006 Sep;188(18):6661-8. doi: 10.1128/JB.00641-06.
In the absence of added thiamine, Rhizobium leguminosarum bv. viciae strain 3841 does not grow in liquid medium and forms only "pin" colonies on agar plates, which contrasts with the good growth of Sinorhizobium meliloti 1021, Mesorhizobium loti 303099, and Rhizobium etli CFN42. These last three organisms have thiCOGE genes, which are essential for de novo thiamine synthesis. While R. leguminosarum bv. viciae 3841 lacks thiCOGE, it does have thiMED. Mutation of thiM prevented formation of pin colonies on agar plates lacking added thiamine, suggesting thiamine intermediates are normally present. The putative functions of ThiM, ThiE, and ThiD are 4-methyl-5-(beta-hydroxyethyl) thiazole (THZ) kinase, thiamine phosphate pyrophosphorylase, and 4-amino-5-hydroxymethyl-2-methyl pyrimidine (HMP) kinase, respectively. This suggests that a salvage pathway operates in R. leguminosarum, and addition of HMP and THZ enabled growth at the same rate as that enabled by thiamine in strain 3841 but elicited no growth in the thiM mutant (RU2459). There is a putative thi box sequence immediately upstream of the thiM, and a gfp-mut3.1 fusion to it revealed the presence of a promoter that is strongly repressed by thiamine. Using fluorescent microscopy and quantitative reverse transcription-PCR, it was shown that thiM is expressed in the rhizosphere of vetch and pea plants, indicating limitation for thiamine. Pea plants infected by RU2459 were not impaired in nodulation or nitrogen fixation. However, colonization of the pea rhizosphere by the thiM mutant was impaired relative to that of the wild type. Overall, the results show that a thiamine salvage pathway operates to enable growth of Rhizobium leguminosarum in the rhizosphere, allowing its survival when thiamine is limiting.
在不添加硫胺素的情况下，豌豆根瘤菌蚕豆生物型3841菌株在液体培养基中不生长，在琼脂平板上仅形成“针状”菌落，这与苜蓿中华根瘤菌1021、百脉根中生根瘤菌303099和菜豆根瘤菌CFN42的良好生长形成对比。后三种微生物具有thiCOGE基因，这对于硫胺素的从头合成至关重要。虽然豌豆根瘤菌蚕豆生物型3841缺乏thiCOGE，但它确实有thiMED。thiM的突变阻止了在缺乏添加硫胺素的琼脂平板上形成针状菌落，这表明硫胺素中间体通常存在。ThiM、ThiE和ThiD的推定功能分别是4-甲基-5-（β-羟乙基）噻唑（THZ）激酶、硫胺素磷酸焦磷酸化酶和4-氨基-5-羟甲基-2-甲基嘧啶（HMP）激酶。这表明在豌豆根瘤菌中存在一条补救途径，添加HMP和THZ能使菌株3841以与硫胺素相同的速率生长，但在thiM突变体（RU2459）中却无法促进生长。在thiM的紧邻上游有一个推定的thi框序列，与gfp-mut3.1融合表明存在一个被硫胺素强烈抑制的启动子。使用荧光显微镜和定量逆转录PCR表明，thiM在巢菜和豌豆植物的根际中表达，表明硫胺素存在限制。被RU2459感染的豌豆植物在结瘤或固氮方面没有受到损害。然而，相对于野生型，thiM突变体在豌豆根际的定殖受到损害。总体而言，结果表明硫胺素补救途径发挥作用，使豌豆根瘤菌能够在根际生长，从而在硫胺素有限时得以存活。
