Isolation and characterization of Pseudomonas putida mutants affected in arginine, ornithine and citrulline catabolism: function of the arginine oxidase and arginine succinyltransferase pathways.

Pseudomonas putida mutants impaired in the utilization of arginine are affected in either the arginine succinyltransferase pathway, the arginine oxidase route, or both. However, mutants affected in one of the pathways still grow on arginine as sole carbon source. Analysis of the products excreted by both wild-type and mutant strains suggests that arginine is mainly channelled by the oxidase route. Proline non-utilizing mutants are also affected in ornithine utilization, confirming the role of proline as an intermediate in ornithine catabolism. Mutants affected in ornithine cyclodeaminase activity still grow on proline and become unable to use ornithine. Both proline non-utilizing mutants and ornithine-cyclodeaminase-minus mutants are unable to use citrulline. These results, together with induction of ornithine cyclodeaminase when wild-type P. putida is grown on citrulline, indicate that utilization of citrulline as a carbon source proceeds via proline with ornithine as an intermediate. Thus in P. putida, the aerobic catabolism of arginine on the one hand and citrulline and ornithine on the other proceed by quite different metabolic segments.     

Control of the flux to arginine in Neurospora crassa: de-repression of the last three enzymes of the arginine pathway

The steady state concentrations of arginine and related intermediary metabolites of the arginine biosynthetic pathway in the eukaryote Neurospora crassa were varied and the concurrent de-repression of the enzymes ornithine transcarbamylase, argininosuccinate synthetase and argininosuccinase was measured. Pool variation was achieved endogenously by the introduction and combination of mutant enzymes with reduced specific activities. Measurements of activities of the mutationally unaltered enzymes showed various degrees of de-repression. The highest activity level for each of the three enzymes was about five times that found in the fully repressed wild-type strain. The variations observed in the pools were as follows: ornithine, 7-fold; citrulline, 700-fold; argininosuccinic acid, 400-fold; arginine, 300-fold.By this means a quantitative analysis of the process of repression is made possible. A strong correlation was found between the degree of de-repression of the three enzymes and the concentration of arginine. The de-repression follows a sigmoid curve with respect to arginine concentration. This is consistent with the interpretation that the pathway enzymes are subject to a repression system with arginine, or a simple derivative of it, acting as a co-repressor.     

Control of arginine metabolism in Neurospora: flux through the biosynthetic pathway.

The flux into the arginine biosynthetic pathway of Neurospora crassa was investigated using a mutant strain lacking the ornithine-degrading enzyme ornithine aminotransferase (EC 2.6.1.13). Flux was measured by the increase in the sum of the radioactivity (derived from [14C]glutamic acid) in the ornithine pool, the arginine pool, and arginine incorporated into proteins. Complete cessation of flux occurred immediately upon the addition of arginine to the growth medium. This response occurred prior to expansion of the arginine pool. After short-term exposure to arginine (80 min), flux resumed quickly upon exhaustion of arginine from the medium. This took place despite the presence of an expanded arginine pool. Initiation of flux required approximately 80 min when the mycelia were grown in arginine-supplemented medium for several generations before exhaustion of the exogenous arginine. The arginine pool of such mycelia was similar to that found in mycelia exposed to exogenous arginine for only 80 min. The results are consistent with rapid onset and release of feedback inhibiton of arginine biosynthesis in response to brief exposure to exogenous arginine. The insensitivity of flux to the size of the arginine pool is consistent with a role for compartmentation in this regulatory process. The lag in initiation of flux after long-term growth in the presence of exogenous arginine suggests the existence of an additional regulatory mechanism(s). Several possibilities are discussed.     

Arginine-specific carbamoyl phosphate metabolism in mitochondria of Neurospora crassa. Channeling and control by arginine

Citrulline is synthesized in mitochondria of Neurospora crassa from ornithine and carbamoyl phosphate. In mycelia grown in minimal medium, carbamoyl phosphate limits citrulline (and arginine) synthesis. Addition of arginine to such cultures reduces the availability of intramitochondrial ornithine, and ornithine then limits citrulline synthesis. We have found that for some time after addition of excess arginine, carbamoyl phosphate synthesis continued. Very little of this carbamoyl phosphate escaped the mitochondrion to be used in the pyrimidine pathway in the nucleus. Instead, mitochondrial carbamoyl phosphate accumulated over 40-fold and turned over rapidly. This was true in ornithine- or ornithine carbamoyltransferase-deficient mutants and in normal mycelia during feedback inhibition of ornithine synthesis. The data suggest that the rate of carbamoyl phosphate synthesis is dependent to a large extent upon the specific activity of the slowly and incompletely repressible synthetic enzyme, carbamoyl-phosphate synthetase A. In keeping with this conclusion, we found that when carbamoyl-phosphate synthetase A was repressed 2-10-fold by growth of mycelia in arginine, carbamoyl phosphate was still synthesized in excess of that used for residual citrulline synthesis. Again, only a small fraction of the excess carbamoyl phosphate could be accounted for by diversion to the pyrimidine pathway. The continued synthesis and turnover of carbamoyl phosphate in mitochondria of arginine-grown cells may allow rapid resumption of citrulline formation after external arginine disappears and no longer exerts negative control on ornithine biosynthesis.     

Catabolism of arginine, citrulline and ornithine by Pseudomonas and related bacteria

The distribution of the arginine succinyltransferase pathway was examined in representative strains of Pseudomonas and related bacteria able to use arginine as the sole carbon and nitrogen source for growth. The arginine succinyltransferase pathway was induced in arginine-grown cells. The accumulation of succinylornithine following in vivo inhibition of succinylornithine transaminase activity by aminooxyacetic acid showed that this pathway is responsible for the dissimilation of the carbon skeleton of arginine. Catabolism of citrulline as a carbon source was restricted to relatively few of the organisms tested. In P. putida, P. cepacia and P. indigofera, ornithine was the main product of citrulline degradation. In most strains which possessed the arginine succinyltransferase pathway, the first step of ornithine utilization as a carbon source was the conversion of ornithine into succinylornithine through an ornithine succinyltransferase. However P. cepacia and P. putida used ornithine by a pathway which proceeded via proline as an intermediate and involved an ornithine cyclase activity.     

Regulation of arginine-ornithine exchange and the arginine deiminase pathway in Streptococcus lactis.

Streptococcus lactis metabolizes arginine by the arginine deiminase (ADI) pathway. Resting cells of S. lactis grown in the presence of galactose and arginine maintain a high intracellular ornithine pool in the absence of arginine and other exogenous energy sources. Addition of arginine results in a rapid release of ornithine concomitant with the uptake of arginine. Subsequent arginine metabolism results intracellularly in high citrulline and low ornithine pools. Arginine-ornithine exchange was shown to occur in a 1-to-1 ratio and to be independent of a proton motive force. The driving force for arginine uptake in intact cells is supplied by the ornithine and arginine concentration gradients formed during arginine metabolism. These results confirm studies of arginine and ornithine transport in membrane vesicles of S. lactis (A. J. M. Driessen, B. Poolman, R. Kiewiet, and W. N. Konings, Proc. Natl. Acad. Sci. USA, 84:6093-6097). The activity of the ADI pathway appears to be affected by the internal concentration of (adenine) nucleotides. Conditions which lower ATP consumption (dicyclohexylcarbodiimide, high pH) decrease the ADI pathway activity, whereas uncouplers and ionophores which stimulate ATP consumption increase the activity. The arginine-ornithine exchange activity matches the ADI pathway most probably by adjusting the intracellular levels of ornithine and arginine. Regulation of the ADI pathway and the arginine-ornithine exchanger at the level of enzyme synthesis is exerted by glucose (repressor, antagonized by cyclic AMP) and arginine (inducer). An arginine/ornithine antiport was also found in Streptococcus faecalis DS5, Streptococcus sanguis 12, and Streptococcus milleri RH1 type 2.     

Analysis of ven3 and ven6 reticulate mutants reveals the importance of arginine biosynthesis in Arabidopsis leaf development

Arabidopsis thaliana reticulate mutants exhibit differential pigmentation of the veinal and interveinal leaf regions, a visible phenotype that often indicates impaired mesophyll development. We performed a metabolomic analysis of one ven6 (venosa6) and three ven3 reticulate mutants that revealed altered levels of arginine precursors, namely increased ornithine and reduced citrulline levels. In addition, the mutants were more sensitive than the wild-type to exogenous ornithine, and leaf reticulation and mesophyll defects of these mutants were completely rescued by exogenous citrulline. Taken together, these results indicate that ven3 and ven6 mutants experience a blockage of the conversion of ornithine into citrulline in the arginine pathway. Consistent with the participation of VEN3 and VEN6 in the same pathway, the morphological phenotype of ven3 ven6 double mutants was synergistic. Map-based cloning showed that the VEN3 and VEN6 genes encode subunits of Arabidopsis carbamoyl phosphate synthetase (CPS), which is assumed to be required for the conversion of ornithine into citrulline in arginine biosynthesis. Heterologous expression of the Arabidopsis VEN3 and VEN6 genes in a CPS-deficient Escherichia coli strain fully restored bacterial growth in minimal medium, demonstrating the enzymatic activity of the VEN3 and VEN6 proteins, and indicating a conserved role for CPS in these distinct and distant species. Detailed study of the reticulate leaf phenotype in the ven3 and ven6 mutants revealed that mesophyll development is highly sensitive to impaired arginine biosynthesis.     

Regulation of Arginase Activity by Intermediates of the Arginine Biosynthetic Pathway in Neurospora crassa

It has been found that, in Neurospora crassa, arginine synthesized from exogenous citrulline was not as effectively hydrolyzed as exogenous arginine. This was explained by the observed inhibition of arginase in vitro and in vivo by citrulline. The high arginine pool formed from exogenous citrulline feedback inhibits the arginine pathway. These two factors allow exogenous citrulline to be used adventitiously and efficiently as an arginine source. Finally, it was found that ornithine was a strong inhibitor of arginase. This suggests that the characteristically high ornithine pool of minimal cultures of Neurospora may act to control a potentially wasteful catabolism of endogenous arginine by arginase.     

Acetylhistidine as substrate for acetylornithinase: a new system for the selection of arginine regulation mutants in Escherichia coli

Summary The utilisation of acetylhistidine by histidine auxotrophs of E. coli K-12 was found to require a functioning acetylornithinase. The growth, on acetylhistidine-containing media, of his mutants possessing this enzyme was inhibited by arginine or its precursors acetylornithine, ornithine and citrulline. Mutants able to overcome this inhibition belonged to two classes: those (selected on acetylhistidine+acetylornithine or arginine) in which the arginine biosynthetic enzymes were repressible, as in the parent strains; and those (selected on acetylhistidine+acetylornithine, ornithine, citrulline or arginine) in which these enzymes were formed at high, non-repressible levels. The altered properties of the first class were shown genetically not to result from mutation in the argR or argECBH regions; the data are consistent with the second class carrying mutations at the argR locus.It is supposed that arginine, ornithine or citrulline, by repressing the formation of acetylornithinase, diminish the rate at which acetylhistidine can be utilised (although an acetylhistidine uptake system under arginine control would equally explain the results); non-repressible mutants would escape this effect. The kinetic properties, in crude extracts, of acetylornithinase from the parent strains and from members of each mutant class, with acetylornithine and acetylhistidine as substrates, were investigated. It was tentatively concluded that, in accord with the genetic results, the first class do not possess an acetylornithinase altered to make it function better with acetylhistidine as substrate. It is suggested that arginine may affect acetylhistidine utilisation by affecting its uptake in a manner not shared with ornithine or citrulline, as well as by repressing proteins of the arginine system, and that this arginine-specific effect is inoperative in the first class of mutants. The nature of the changes leading to ability to grow on acetylhistidine+acetylornithine remains unknown. Possible applications of these findings to the selection of hitherto undiscovered but potentially informative mutant types are discussed.     

Control of the ornithine cycle in Neurospora crassa by the mitochondrial membrane.

In Neurospora crassa, the mitochondrial membrane separates ornithine used in arginine biosynthesis from ornithine used in the arginine degradative pathway in the cytosol. Ornithine easily exchanges across the mitochondrial membrane under conditions appropriate for synthesis of the immediate biosynthetic product, citrulline. Neither of the two mitochondrial enzymes required for the ornithine-to-citrulline conversion is feedback inhibitable in vitro. Nevertheless, when arginine is added to cells and cytosolic ornithine increases as arginine degradation begins, the rate of citrulline synthesis drops immediately to about 20% of normal (B. J. Bowman and R. H. Davis, Bacteriol. 130:285-291, 1977). We have studied this phenomenon in citrulline-accumulating strains carrying the arg-1 mutation. Citrulline accumulation is blocked when arginine is added to an arg-1 strain but not to an arg-1 strain carrying a mutation conferring insensitivity of intramitochondrial ornithine synthesis to arginine. Thus, ornithine is evidently unable to enter mitochondria in normal (feedback-sensitive) cells. Other experiments show that cytosolic ornithine enters mitochondria readily except when arginine or other basic amino acids are present at high levels in the cells. We conclude that in N. crassa, the mitochondrial membrane has evolved as a secondary site of feedback inhibition in arginine synthesis and that this prevents a wasteful cycling of catabolic ornithine back through the anabolic pathway. This is compared to the quite different mechanism by which the yeast Saccharomyces cerevisiae prevents a futile ornithine cycle.     

Macrophage arginine metabolism and the inhibition or stimulation of cancer.

The potential of the immune system to inhibit or stimulate tumor growth is a vivid example of the "two-edged sword" nature of immune responses. Our results provide evidence that this dual capacity can be attributed, in part, to the dual pathways of arginine metabolism exhibited by intratumor macrophages. Specifically, i.p. tumor rejection in P815-preimmunized mice is accompanied by an upshift in intratumor macrophage arginine metabolism to the nitric oxide (NO) synthase pathway that yields citrulline and NO. A rapid and marked local increase in IFN-gamma (both mRNA and protein) in preimmunized mice during tumor rejection suggests that this cytokine plays a role in up-regulating nitric oxide production in vivo. Unlike tumor rejection, progressive i.p. P815 tumor growth in naive mice is associated with a marked decline in the production of citruline/NO by intratumor macrophages. Examination of macrophage arginine metabolism via arginase revealed a pattern opposite that of NO synthase. The local production of ornithine/urea markedly increases during progressive tumor growth whereas arginase activity decreases during tumor rejection. Inasmuch as nitric oxide inhibits tumor cell replication whereas ornithine is the precursor of polyamines required for cell replication, these results are consistent with the conclusion that the pathway macrophages use to metabolize arginine can influence the type of host immune responses against cancer and other conditions.     

Genetic and physiological characterization of Pseudomonas aeruginosa mutants affected in the catabolic ornithine carbamoyltransferase.

In Pseudomonas aeruginosa arginine can be degraded by the arginine "dihydrolase" system, consisting of arginine deiminase, catabolic ornithine carbamoyltransferase, and carbamate kinase. Mutants of P. aeruginosa strain PAO affected in the structural gene (arcB) of the catabolic ornithine carbamoyltransferase were isolated. Firt, and argF mutation (i.e., a block in the anabolic ornithine carbamoyltransferase) was suppressed specifically by a mutationally altered catabolic ornithine carbamoyltransferase capable of functioning in the anabolic direction. The suppressor locus arcB (Su) was mapped by transduction between hisII and argA. Second, mutants having lost suppressor activity were obtained. The Su- mutations were very closely linked to arcB (Su) and caused strongly reduced ornithine carbamoyltransferase activities in vitro. Under aerobic conditions, a mutant (PA0630) which had less than 1% of the wild-type catabolic ornithine carbamoyltransferase activity grew on arginine as the only carbon and nitrogen source, at the wild-type growth rate. When oxygen was limiting, strain PA0630 grown on arginine excreted citrulline in the stationary growth phase. These observations suggest that during aerobic growth arginine is not degraded exclusively via the dihydrolase pathway.     

The role of mitochondrially bound arginase in the regulation of urea synthesis: studies with [U-15N4]arginine, isolated mitochondria, and perfused rat liver

The main goal of the current study was to elucidate the role of mitochondrial arginine metabolism in the regulation of N-acetylglutamate and urea synthesis. We hypothesized that arginine catabolism via mitochondrially bound arginase augments ureagenesis by supplying ornithine for net synthesis of citrulline, glutamate, N-acetylglutamate, and aspartate. [U-(15)N(4)]arginine was used as precursor and isolated mitochondria or liver perfusion as a model system to monitor arginine catabolism and the incorporation of (15)N into various intermediate metabolites of the urea cycle. The results indicate that approximately 8% of total mitochondrial arginase activity is located in the matrix, and 90% is located in the outer membrane. Experiments with isolated mitochondria showed that approximately 60-70% of external [U-(15)N(4)]arginine catabolism was recovered as (15)N-labeled ornithine, glutamate, N-acetylglutamate, citrulline, and aspartate. The production of (15)N-labeled metabolites was time- and dose-dependent. During liver perfusion, urea containing one (U(m+1)) or two (U(m+2)) (15)N was generated from perfusate [U-(15)N(4)]arginine. The output of U(m+2) was between 3 and 8% of total urea, consistent with the percentage of activity of matrix arginase. U(m+1) was formed following mitochondrial production of [(15)N]glutamate from [alpha,delta-(15)N(2)]ornithine and transamination of [(15)N]glutamate to [(15)N]aspartate. The latter is transported to cytosol and incorporated into argininosuccinate. Approximately 70, 75, 7, and 5% of hepatic ornithine, citrulline, N-acetylglutamate, and aspartate, respectively, were derived from perfusate [U-(15)N(4)]arginine. The results substantiate the hypothesis that intramitochondrial arginase, presumably the arginase-II isozyme, may play an important role in the regulation of hepatic ureagenesis by furnishing ornithine for net synthesis of N-acetylglutamate, citrulline, and aspartate.     

Rat colon ornithine and arginine metabolism: coordinated effects after proliferative stimuli

Ornithine decarboxylase (ODC) catalyzes the first step in the polyamine biosynthetic pathway, a highly regulated pathway in which activity increases during rapid growth. Other enzymes also metabolize ornithine, and in hepatomas, rate of growth correlates with decreased activity of these other enzymes, which thus channels more ornithine to polyamine biosynthesis. Ornithine is produced from arginase cleavage of arginine, which also serves as the precursor for nitric oxide production. To study whether short-term coordination of ornithine and arginine metabolism exists in rat colon, ODC, ornithine aminotransferase (OAT), arginase, ornithine, arginine, and polyamine levels were measured after two stimuli (refeeding and/or deoxycholate exposure) known to synergistically induce ODC activity. Increased ODC activity was accompanied by increased putrescine levels, whereas OAT and arginase activity were reduced by either treatment, accompanied by an increase in both arginine and ornithine levels. These results indicate a rapid reciprocal change in ODC, OAT, and arginase activity in response to refeeding or deoxycholate. The accompanying increases in ornithine and arginine concentration are likely to contribute to increased flux through the polyamine and nitric oxide biosynthetic pathways in vivo.     

A multitracer stable isotope quantification of the effects of arginine intake on whole body arginine metabolism in neonatal piglets

We have previously shown that deficient arginine intake increased the rate of endogenous arginine synthesis from proline. In this paper, we report in vivo quantification of the effects of arginine intake on total endogenous arginine synthesis, on the rates of conversion between arginine, citrulline, ornithine, and proline, and on nitric oxide synthesis. Male piglets, with gastric catheters for diet and isotope infusion and femoral vein catheters for blood sampling, received a complete diet for 2 days and then either a generous (+Arg; 1.80 g x kg(-1) x day(-1); n = 5) or deficient (-Arg; 0.20 g.kg(-1).day(-1); n = 5) arginine diet for 5 days. On day 7, piglets received a primed, constant infusion of [guanido-(15)N(2)]arginine, [ureido-(13)C;5,5-(2)H(2)]citrulline, [U-(13)C(5)]ornithine, and [(15)N;U-(13)C(5)]proline in an integrated study of the metabolism of arginine and its precursors. Arginine synthesis (micromol x kg(-1) x h(-1)) from both proline (+Arg: 42, -Arg: 74, pooled SE: 5) and citrulline (+Arg: 67, -Arg: 120; pooled SE: 15) were higher in piglets receiving the -Arg diet (P < 0.05); and for both diets proline accounted for approximately 60% of total endogenous arginine synthesis. The conversion of proline to citrulline (+Arg: 39, -Arg: 67, pooled SE: 6) was similar to the proline-to-arginine conversion, confirming that citrulline formation limits arginine synthesis from proline in piglets. Nitric oxide synthesis (micromol x kg(-1) x h(-1)), measured by the rate conversion of [guanido-(15)N(2)]arginine to [ureido-(15)N]citrulline, was greater in piglets receiving the +Arg diet (105) than in those receiving the -Arg diet (46, pooled SE: 10; P < 0.05). This multi-isotope method successfully allowed many aspects of arginine metabolism to be quantified simultaneously in vivo.     

Arginine metabolism in wineLactobacillus plantarum: in vitro activities of the enzymes arginine deiminase (ADI) and ornithine transcarbamilase (OTCase)

This work was carried out to determine the activity of enzymes involved in arginine metabolism inLactobacillus plantarum isolated from wine and previously characterised at molecular level. The activity of the enzymes arginine deiminase and ornithine transcarbamylase was determined and citrulline and ornithine formed were analysed by HPLC analysis. Although the enzymatic activity was detected in all the strains analysed, a strong variability was observed between strains.Lactobacillus plantrum strain Lp60 is the strain with more possibilities to accumulate citrulline, precursor of the carcinogenic ethyl-carbamate, as showed by its high arginine deiminase activity and low ornithine transcarbamylase activity.     

pryB mutations as suppressors of arginine auxotrophy in Salmonella typhimurium.

Salmonella typhimurium strains which produce high constitutive levels of aspartate transcarbamoylase due to the pyrH700 mutation were found to grow more slowly in minimal medium than pyrH+ controls. The addition of arginine or citrulline but not ornithine restored normal growth rates. This requirement for arginine was completely suppressed by pyrB mutations and partially suppressed by pyrC and pyrD mutations. No suppression was observed with mutants at the pyrF locus. Introduction of leaky mutation argI2002 resulted in a more extreme arginine requirement and accentuated suppression by pyrB mutations. Suppression by the pyrC and pyrD mutations was reduced as a result of the incorporation of the leaky argI2002 allele. These results indicate that in pyrH700 strains carbamoyl phosphate is preferentially directed toward the formation of intermediates in the pyrimidine biosynthetic pathway. Arginine auxotrophy results from the reduced availability of carbamoyl phosphate for the biosynthesis of arginine. Suppression of this arginine dependence for growth is used as a convenient positive selection technique for pyrB mutations.     

Arginine catabolism in Lactobacillus sake isolated from meat.

Lactobacillus sake isolated from meat can hydrolyze arginine via the arginine deiminase pathway. Two enzymes, arginine deiminase and ornithine transcarbamylase, have been revealed by detection of their reaction products, citrulline and ornithine, respectively. The production of citrulline depends on the concentration of glucose in a synthetic medium; it does not occur when the concentration of glucose is 27.5 mM or higher.     

Effect of arginine,ornithine and citrulline supplementation upon performance and metabolism of trained rats

During intense exercise there is an augmented production of ammonia and IMP in the exercised muscle that could be related to the establishment of peripheral fatigue. In order to prevent this accumulation, the urea cycle in the liver eliminates ammonia in the form of urea and the skeletal muscle buffers the increase of ammonia via transamination reactions. In the present study we evaluated the effect of arginine, citrulline and ornithine supplementation, intermediates of the urea cycle, on the performance of sedentary and swimming-trained rats submitted to a single bout of exhaustive exercise. We also measured the glycogen content of the soleus and gastrocnemius muscles and of the liver, as well as the plasma concentrations of ammonia, urea, glutamine, glucose and lactate. The results indicate that arginine, citrulline and ornithine supplementation increased the flux of substrate through the reaction catalysed by glutamine synthetase, leading to increased glutamine production after an exhaustive bout of exercise, and of the mechanism involved in ammonia buffering.     

Bacillus subtilis 168 mutants resistant to arginine hydroxamate in the presence of ornithine or citrulline

Mutations in Bacillus subtilis 168 have been isolated that confer resistance to arginine hydroxamate in the presence, but not absence, of ornithine. Seven such Ahor mutants have been studied in detail. In common with certain classes of Ahr mutant (resistant to arginine hydroxamate in the absence of arginine precursors) described previously, these Ahor mutants showed little or no inducibility of enzymes of arginine catabolism. Mutants that showed no inducibility were unable to utilize arginine or ornithine as sole nitrogen source. The only biosynthetic enzyme to show any consistent differences in activity from the parent was ornithine carbamoyltransferase, whose level was slightly elevated in cells grown in the presence of ornithine or citrulline. PBS1 transduction crosses showed that two of the ahor mutations map at the ahrA locus, while a third (unique in its resistance to arginine hydroxamate in the presence of citrulline) mapped at a hitherto undescribed locus closely linked to metC, designated ahrD.