Genetic evidence for 5-aminoimidazole-4-carboxamide ribonucleotide (AICAR) as a negative effector of cytochrome terminal oxidase cbb 3 production in Rhizobium etli

Abstract

A Rhizobium etli Tn5mob-induced mutant (CFN035) exhibits an enhanced capacity to oxidize N,N,N',N', tetramethyl-p -phenylenediamine (TMPD), a presumptive indicator of elevated cytochrome c terminal oxidase activity. Sequencing of the mutated gene in CFN035 revealed that it codes for the amidophosphoribosyl transferase enzyme (PurF) that catalyzes the first step in the purine biosynthetic pathway. Two c-type cytochromes with molecular weights of 32 and 27 kDa were produced in strain CFN035, which also produced a novel CO-reactive cytochrome (absorbance trough at 553 nm), in contrast to strain CE3 which produced a single 32 kDa c-type protein and did not produce the 553 nm CO-reactive cytochrome. A wild-type R. etli strain that expresses the Bradyrhizobium japonicum fixNOQP genes, which code for the symbiotic cytochrome terminal oxidase cbb3, produced similar absorbance spectra (a trough at 553 nm in CO-difference spectra) and two c-type proteins similar in size to those of strain CFN035, suggesting that CFN035 also produces the cbb3 terminal oxidase. The expression of a R. etli fixN-lacZ gene fusion was measured in several R. etli mutants affected in different steps of the purine biosynthetic pathway. Our analysis showed that purF, purD, purQ, purL, purY, purK and purE mutants expressed three-fold higher levels of the fixNOQP operon than the wild-type strain. The derepressed expression of fixN was not observed in a purH mutant. The purH gene product catalyzes the conversion of 5-aminoimidazole-4-carboxamide ribonucleotide (AICAR) to 5-form-aminoimidazole-4-carboxamide ribonucleotide (FAICAR) and inosine. Supplementation with AICA riboside lowered the levels of fixN expression in the purF mutants. These data are consistent with the possibility that AICAR, or a closely related metabolite, is a negative effector of the production of the symbiotic terminal oxidase cbb3 in R. etli.

A purF mutant of Mycobacterium smegmatis has impaired survival during oxygen-starved stationary phase The GenBank accession number for the sequence reported in this paper is AJ278609.

“…a-Horsman et al, 1994). Analysis of the transcription of the fixNOQP operon, encoding cytochrome cbb $, in different R. etli purine biosynthetic mutants, suggests that 5 aminoimidazole-4-carboxamide ribonucleotide (AICAR) or a related metabolite is a negative regulator of this oxidase (Soberon et al, 1997). It would be interesting to investigate the survival phenotypes of other M. smegmatis purine biosynthesis mutants.…”

“…Alternatively, the O # -starvation phenotype may not be a consequence of starvation for purines directly but results from a shortage of one or more intermediates in the purine biosynthesis pathway. Important in this context is the finding that a Rhizobium etli purF mutant and certain other purine biosynthesis mutants overexpress cytochrome cbb $, a cytochrome c oxidase (Soberon et al, 1997 ;Garcı! a-Horsman et al, 1994).…”

Metabolic Flux in Both the Purine Mononucleotide and Histidine Biosynthetic Pathways Can Influence Synthesis of the Hydroxymethyl Pyrimidine Moiety of Thiamine in Salmonella enterica

“…Neither the biochemistry of this conversion nor the enzyme(s) involved has been rigorously defined, and thus it is difficult to suggest a mechanism or more precise site for the target or the AICAR effect. However, AICAR has been implicated previously in regulation, both as a negative effector of cytochrome terminal oxidase production in Rhizobium etli (50) and as a proposed alarmone for C-1-folate deficiency (8). The work presented here has expanded our model system for metabolic integration by defining additional parameters of the interaction between thiamine and purine mononucleotide synthesis, identifying a new connection between histidine and thiamine biosynthesis, and defining the part of the thiamine pathway that is affected by coenzyme A levels in the cell.…”

A purL mutant of Sinorhizobium fredii HH103 is symbiotically defective and altered in its lipopolysaccharide

“…§Fåhraeus nutritive solution was supplemented with 0?8 mg adenine ml 21 . conditions, higher levels of the fixNOQP operon, which codes for the symbiotic cytochrome terminal oxidase ccb 3 (Soberon et al, 1997). Moreover, Rhizobium sp.…”