title: Thiamine is synthesized by a salvage pathway in Rhizobium leguminosarum bv. viciae strain 3841.
abstract: 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.
authors.affiliation: School of Biological Sciences, University of Reading, Whiteknights, P.O. Box 228, Reading RG6 6AJ, United Kingdom.
authors.forename: R
authors.initials: R
authors.lastname: Karunakaran
authors.forename: K
authors.initials: K
authors.lastname: Ebert
authors.forename: S
authors.initials: S
authors.lastname: Harvey
authors.forename: M E
authors.initials: ME
authors.lastname: Leonard
authors.forename: V
authors.initials: V
authors.lastname: Ramachandran
authors.forename: P S
authors.initials: PS
authors.lastname: Poole
mesh_terms: D019883:Alkyl and Aryl Transferases / Q000502:physiology; D019677:Artificial Gene Fusion; D001665:Binding Sites; D015169:Colony Count, Microbial; D017353:Gene Deletion; D015964:Gene Expression Regulation, Bacterial; D005798:Genes, Bacterial; D049452:Green Fluorescent Proteins / Q000096:biosynthesis / Q000235:genetics; D008856:Microscopy, Fluorescence; D009586:Nitrogen Fixation; D018532:Pisum sativum / Q000382:microbiology; D017853:Phosphotransferases (Alcohol Group Acceptor) / Q000378:metabolism; D017856:Phosphotransferases (Phosphate Group Acceptor) / Q000378:metabolism; D018517:Plant Roots / Q000382:microbiology; D011401:Promoter Regions, Genetic; D011743:Pyrimidines / Q000378:metabolism; D012329:RNA, Bacterial / Q000032:analysis; D012333:RNA, Messenger / Q000032:analysis; D020133:Reverse Transcriptase Polymerase Chain Reaction; D016961:Rhizobium leguminosarum / Q000235:genetics* / Q000254:growth & development / Q000378:metabolism*; D013831:Thiamine / Q000096:biosynthesis*; D013844:Thiazoles / Q000378:metabolism; D031306:Vicia / Q000382:microbiology
chemical_list: C101901:4-amino-5-hydroxymethyl-2-methylpyrimidine; D011743:Pyrimidines; D012329:RNA, Bacterial; D012333:RNA, Messenger; D013844:Thiazoles; D049452:Green Fluorescent Proteins; D019883:Alkyl and Aryl Transferases; C435495:thiamin phosphate synthase; C108435:4-methyl-5-(beta-hydroxyethyl)thiazole kinase; D017853:Phosphotransferases (Alcohol Group Acceptor); C108436:4-amino-5-(hydroxymethyl)-2-methylpyrimidine phosphate kinase; D017856:Phosphotransferases (Phosphate Group Acceptor); D013831:Thiamine