A Mutant Allele of rpoD Results in Increased Conversion of Aminoimidazole Ribotide to Hydroxymethyl Pyrimidine in Salmonella enterica

Abstract

An allele of rpoD (rpoD1181) that results in increased synthesis of the pyrimidine moiety of thiamine in Salmonella enterica was identified. The S508Y substitution caused by rpoD1181 is analogous to the S506F derivative of the Escherichia coli protein. The properties of this E. coli mutant protein have been well characterized in vitro. Identification of a metabolic phenotype caused by the rpoD1181 allele of S. enterica allows past in vitro results to be incorporated in continuing efforts to understand cellular processes that are integrated with the thiamine biosynthetic pathway.The essential cofactor thiamine pyrophosphate is generated by the condensation of two independently synthesized molecules, 4-methyl-5(␤-hydroxyethyl) thiazole phosphate and 4-amino-5-hydroxymethyl-2-methylpyrimidine pyrophosphate (HMP-PP) ( Fig. 1) (3). The precursor of the pyrimidine moiety is 5-aminoimidazole ribotide (AIR), an intermediate in the purine biosynthetic pathway. HMP-P synthesis involves a complex intramolecular rearrangement and requires at least the product of the thiC gene (24, 26). Reconstitution of HMP-P synthesis in vitro has not yet been reported.Past work has identified loci other than thi biosynthetic enzymes that are required for efficient HMP-P synthesis. Characterization of these loci has implicated the oxidative pentose phosphate pathway, pantothenate (and/or coenzyme A [CoA]), and iron-sulfur cluster metabolism in the synthesis of HMP (10, 13, 14, 21, 23).Work described here was initiated to probe the integration of distinct metabolic processes with thiamine synthesis. Genetic analysis identified an allele of rpoD (rpoD1181) that allowed several HMP-requiring mutant strains to grow in the absence of thiamine. Our working model suggests that the rpoD1181 (S508Y) allele restores thiamine-independent growth by altering expression of an unidentified gene(s) whose product is required for efficient HMP-P synthesis.Thiamine synthesis in a purF iscA strain is restored by an rpoD allele. Salmonella enterica strain DM6176 (purF iscA) is unable to grow in the absence of thiamine (23). The iscA allele used is an insertion that is polar on hscAB (23). Mutations that suppressed the thiamine synthetic defect caused by the iscA lesion were identified. A 0.1-ml aliquot of an overnight culture of DM6176 was spread on a plate containing minimal medium plus gluconate and adenine. After overnight incubation at 37°C, more than 300 colonies that no longer required thiamine for growth were found. Ten random colonies were chosen for further analysis. In nine of the mutants, an insertion near the gltA locus, zbg-6391::Tn10d(Tc), was genetically linked to the causative lesion. On the basis of the proximity of gltA to the sdh genes and the knowledge that null mutations in sdh spared cellular thiamine pools (11), succinate dehydrogenase activity was measured in the nine mutants. Each of the nine mutants lacked detectable succinate dehydrogenase activity (data not shown), and they were not analyzed further.Multiple transposon insertions (M...

A connection between iron–sulfur cluster metabolism and the biosynthesis of 4-amino-5-hydroxymethyl-2-methylpyrimidine pyrophosphate in Salmonella enterica

“…The use of strain DM7060 (purG purE stm4068) and its derivatives to classify the target of defects in HMP synthesis has been described (Dougherty & Downs, 2004). Lesions in iscA, apbC, apbE, rseC and gshA were transduced into strain DM7060.…”

“…From these data it was concluded that the thiamine requirement caused by mutations affecting Fe-S cluster metabolism resulted from impaired conversion of AIR to HMP. Thus, disruption of Fe-S cluster metabolism joined reduced pantothenate synthesis (Allen et al, 2002;Dougherty & Downs, 2004) as a process that affects the conversion of AIR to HMP.…”

Analysis of ThiC Variants in the Context of the Metabolic Network of Salmonella enterica

“…PurK [5-(carboxyamino) imidazole ribonucleotide synthase, EC 6.3.4.18] and PurE [5-(carboxyamino) imidazole ribonucleotide mutase, EC 5.4.99.18] catalyze the enzymatic steps in the purine biosynthetic pathway that consume AIR. The purE3043 allele is expected to result in the accumulation of AIR in the presence of exogenous adenine (14,30). The purE3043 allele restored thiamine synthesis in strains with ThiC E281K and ThiC V267M variants, as judged by the growth rate of ϳ0.20 h Ϫ1 compared to one of ϳ0.07 h Ϫ1 in adenine medium (Table 2).…”

Mutations in the Primary Sigma Factor σ A and Termination Factor Rho That Reduce Susceptibility to Cell Wall Antibiotics

“…Indeed, there are relatively few examples where altered-function mutations in a primary (essential) factor have emerged in random genetic selections (as opposed to experiments explicitly designed to recover such mutations). We are aware of three examples that include mutations leading to (i) an A-signaling defect in Myxococcus xanthus (73), (ii) suppression of defects associated with an inability to synthesize ppGpp in E. coli (74), and (iii) increased conversion of aminoimidazole ribotide to hydroxymethyl pyrimidine in Salmonella enterica (75). Here, we extend this list to include changes in antibiotic susceptibility.…”