Expression of thiamin biosynthetic genes (thiCOGE) and production of symbiotic terminal oxidase cbb3 in Rhizobium etli

Abstract

In this paper we report the cloning and sequence analysis of four genes, located on plasmid pb, which are involved in the synthesis of thiamin in Rhizobium etli (thiC, thiO, thiG, and thiE). Two precursors, 4-methyl-5-(␤-hydroxyethyl)thiazole monophosphate and 4-amino-5-hydroxymethylpyrimidine pyrophosphate, are coupled to form thiamin monophosphate, which is then phosphorylated to make thiamin pyrophosphate. The first open reading frame (ORF) product, of 610 residues, has significant homology (69% identity) with the product of thiC from Escherichia coli, which is involved in the synthesis of hydroxymethylpyrimidine. The second ORF product, of 327 residues, is the product of a novel gene denoted thiO. A protein motif involved in flavin adenine dinucleotide binding was found in the amino-terminal part of ThiO; also, residues involved in the catalytic site of D-amino acid oxidases are conserved in ThiO, suggesting that it catalyzes the oxidative deamination of some intermediate of thiamin biosynthesis. The third ORF product, of 323 residues, has significant homology (38% identity) with ThiG from E. coli, which is involved in the synthesis of the thiazole. The fourth ORF product, of 204 residues, has significant homology (47% identity) with the product of thiE from E. coli, which is involved in the condensation of hydroxymethylpyrimidine and thiazole. Strain CFN037 is an R. etli mutant induced by a single Tn5mob insertion in the promoter region of the thiCOGE gene cluster. The Tn5mob insertion in CFN037 occurred within a 39-bp region which is highly conserved in all of the thiC promoters analyzed and promotes constitutive expression of thiC. Primer extension analysis showed that thiC transcription in strain CFN037 originates within the Tn5 element. Analysis of c-type protein content and expression of the fixNOQP operon, which codes for the symbiotic terminal oxidase cbb 3 , revealed that CFN037 produces the cbb 3 terminal oxidase. These data show a direct relationship between expression of thiC and production of the cbb 3 terminal oxidase. This is consistent with the proposition that a purine-related metabolite, 5-aminoimidazole-4-carboxamide ribonucleotide, is a negative effector of the production of the symbiotic terminal oxidase cbb 3 in R. etli.

Structure-Function Correlation in Glycine Oxidase from Bacillus subtilis

“…On the other hand, a different role of GO in the formation of the thiazole moiety of thiamine was recently proposed (8); this role requires that the imine product of dehydrogenation of glycine is trapped at the active site of GO with the thiocarboxylate produced by the following enzyme of the metabolic pathway, ThiS. This proposal is also supported by the sequence homology between GO and ThiO, a protein that has been suggested to catalyze this reaction in R. etli (31). This nucleophilic addition may occur before hydrolysis of the imine, which, when it is released from the enzyme, takes place immediately.…”

“…Furthermore, the primary sequence of GO shows a high degree of conservation with the product of thiO gene of Rhizobium etli (23.0% of sequence identity). thiO is the second open reading frame of four genes (thiC, thiO, thiG, and thiE) located on plasmid pb, which are involved in the synthesis of thiamine in R. etli (31). R. etli ThiO protein (a 327-amino acid protein) contains at its N terminus a flavin adenine dinucleotide-binding motif and shares many of the residues involved in the catalytic site of DAAO; it has been also suggested that ThiO may have amino acid oxidase activity (31).…”

“…thiO is the second open reading frame of four genes (thiC, thiO, thiG, and thiE) located on plasmid pb, which are involved in the synthesis of thiamine in R. etli (31). R. etli ThiO protein (a 327-amino acid protein) contains at its N terminus a flavin adenine dinucleotide-binding motif and shares many of the residues involved in the catalytic site of DAAO; it has been also suggested that ThiO may have amino acid oxidase activity (31). In E. coli five genes (thiC, thiE, thiF, thiG, and thiH), proposed to be a single transcription unit, are involved in thiamine biosynthesis (32).…”

A conserved RNA structure ( thi box) is involved in regulation of thiamin biosynthetic gene expression in bacteria

“…The transcription start site (tss) of the thiC gene lies 211 bases upstream of thiC start codon. The RNA untranslated leader contains a 38-base sequence, named thi box, that is highly conserved in the upstream region of thi genes of organisms as diverse as E. coli, Synechocystis, Bacillus subtilis, and Mycobacterium tuberculosis (14).In this work, we found by functional analysis of the untranslated RNA leader that repression by thiamin is a posttranscriptional event and that the thi box is an element indispensable for the expression of thiCOGE. We also show that the region surrounding the Shine-Dalgarno (S-D) sequence and the first 443 nt of the coding region participate in the regulation of thiCOGE expression by thiamin.…”

Mutational Analysis of ThiH, a Member of the Radical S-Adenosylmethionine (AdoMet) Protein Superfamily

“…Organisms that are able to synthesize thiamine yet lack thiH (e.g. B. subtilis and Rizobium etli) have thiO, which encodes a glycine oxidase (11,21). The remaining genes involved in thiamine synthesis are more widely conserved (22,23).…”