Evidence for an alternate isoleucine biosynthesis pathway in G. sulfurreducens. 13
C metabolic flux analysis can be used as a tool for verifying genome annotation, optimizing metabolic models, and elucidating the physiological state of microorganisms (11
). In order to test the accuracy of the reconstructed central metabolic network of G. sulfurreducens
C labeling studies were initiated. An isotopomer balance model for G. sulfurreducens
, with the IMMs taken largely from an E. coli
), was developed.
In these studies, G. sulfurreducens
was cultured in fresh-water acetate-fumarate medium containing either 30% (mol/mol) [13
C]acetate (labeled at both carbons) or 13
C]fumarate (labeled at carbons 2 and 3). During growth on this medium, the tricarboxylic acid cycle (TCA) cycle functions as an open loop in which the succinate dehydrogenase reaction is bypassed (13
). Continual flux through the remaining reactions of the TCA cycle is maintained by coupling the secretion of succinate to the uptake of fumarate via the dicarboxylate exchanger, DcuB (5
). As a result, the TCA cycle intermediate, oxaloacetate, is derived primarily from exogenous fumarate. In fact, during growth on acetate-[13
C]fumarate medium, the mass isotopomer distribution of aspartate, which derives from oxaloacetate, matched that of the feed (30% doubly labeled/70% unlabeled). In contrast, during growth on [13
C]acetate-fumarate medium, aspartate was primarily unlabeled (Tables and ), confirming the presence of the open loop. Pyruvate is another common amino acid precursor. In G. sulfurreducens
, pyruvate biosynthesis occurs primarily via the condensation of acetyl-CoA and CO2
by the pyruvate-ferredoxin oxidoreductase (39
). In fact, leucine, which was predicted to be derived from acetyl-CoA and pyruvate, was labeled during growth on [13
C]acetate-fumarate, and essentially unlabeled during growth on acetate-[13
C]fumarate, confirming the central role of the pyruvate-ferredoxin oxidoreductase in pyruvate biosynthesis.
According to the annotated pathway (Fig. ), both oxaloacetate and pyruvate serve as precursors for isoleucine biosynthesis, and thus this amino acid should be labeled in the presence of both [13C]acetate and [13C]fumarate (Tables and , threonine-dependent pathway). However, the isotopomer mass distribution of isoleucine did not match the expected pattern: isoleucine was extensively labeled in the presence of [13C]acetate but poorly labeled in the presence of [13C]fumarate (Tables and ). This suggested that the annotated threonine-dependent pathway did not play a major role in isoleucine biosynthesis and that acetyl-CoA and/or pyruvate was the predominant precursor for this amino acid.
In the spirochete Leptospira interrogans
and in methanogenic Archaea
, the key isoleucine precursor, 2-oxobutanoate, is synthesized from acetyl-CoA and pyruvate via the citramalate pathway (17
) (Fig. ). The first dedicated step in this pathway is the condensation of pyruvate and acetyl-CoA by the enzyme citramalate synthase (CimA; EC 188.8.131.52). The introduction of this pathway into the G. sulfurreducens
isotopomer balance model significantly improved the agreement of experimental and predicted isotopomer mass distributions (Tables and ). The best fit was generated by allowing flux through both pathways, with the citramalate pathway serving as the primary route of isoleucine biosynthesis (Fig. ), accounting for 68 to 77% of the total flux to isoleucine.
FIG. 2. Predicted contributions of the threonine and citramalate pathways to isoleucine biosynthesis in wild-type G. sulfurreducens cultured in acetate-fumarate medium containing either 30% (mol/mol) [13C]acetate or [13C]fumarate. Model predictions are (more ...)
In order to determine whether the citramalate pathway was active in G. sulfurreducens
, crude soluble extracts were prepared from mid-log, freshwater acetate-fumarate cultures grown under the same conditions as those used for 13
C flux analysis studies and tested for the presence of citramalate synthase activity. These extracts contained 5.94 ± 0.49 nmol mg−1
of citramalate synthase activity, measured as the pyruvate-dependent release of CoA from acetyl-CoA (47
). The citramalate synthase appeared to have a high affinity for pyruvate, with 52.5% ± 4.9% of the activity remaining when the pyruvate concentration was reduced from 1 mM to 0.1 mM. Although these results were consistent with the presence of citramalate synthase, they were not conclusive due to the fact that isopropylmalate synthase, which catalyzes the first step in leucine biosynthesis, has residual citramalate synthase activity (20
). In addition, high levels of pyruvate-independent CoA release interfered with detection of the enzyme activity and accurate determination of the Km
Because both 13
C labeling studies and preliminary biochemical studies were consistent with the presence of citramalate synthase in G. sulfurreducens
, we examined the genome for candidate genes. Only two citramalate synthases had been characterized, those of L. interrogans
and Methanocaldococcus jannaschii
). Both citramalate synthases were homologous to isopropylmalate synthase (LeuA), which catalyzes the first step in the biosynthesis of leucine (47
). Examination of the G. sulfurreducens
genome revealed three LeuA family members: GSU1906, GSU1798, and GSU0937. Comparison to characterized enzymes suggested that GSU1906, which has 65% sequence similarity to LeuA of Salmonella enterica
serovar Typhimurium, encoded an isopropylmalate synthase, whereas GSU0937, which has 65% sequence similarity to NifV of Azotobacter vinelandii
, encoded a homocitrate synthase. The remaining candidate, GSU1798, which was annotated as a LeuA homolog (30
), is equally similar to characterized isopropylmalate and citramalate synthases; it is 46% similar to S. enterica
serovar Typhimurium LeuA and 45% similar to CimA from both L. interrogans
and M. jannaschii
. Thus, it was selected as the most likely candidate for a citramalate synthase in G. sulfurreducens
Because flux analysis indicated that the threonine-dependent pathway was a relatively minor contributor to isoleucine biosynthesis, we reexamined the genomic evidence for this pathway. Threonine ammonia-lyase (GSU0486) was the only enzyme unique to this pathway. Phylogenetic analysis of GSU0486, which was annotated as a biosynthetic threonine ammonia-lyase, IlvA (30
), revealed that it clustered with catabolic threonine ammonia-lyases (TdcB; EC 184.108.40.206), which are not inhibited by isoleucine and also catalyze the deamination of serine (37
) (Fig. ). Soluble extracts prepared from G. sulfurreducens
grown under the same conditions as the initial flux analysis experiment (log-phase, fresh-water acetate-fumarate medium) contained 227.9 ± 6.2 nmol mg−1
of isoleucine-insensitive threonine ammonia-lyase and 31.7 ± 2.3 nmol mg−1
of serine ammonia-lyase activity.
FIG. 3. Phylogenetic analysis of threonine ammonia-lyases. The phylogenetic tree was inferred from protein sequences by the neighbor-joining method using the BIONJ algorithm (14, 36) as previously described (8). Bootstrap values were calculated for 100 replicates. (more ...) Genetic evidence for two isoleucine biosynthetic pathways.
In order to corroborate the results of the preliminary biochemical analysis and evaluate the functions of the putative threonine ammonia-lyase (GSU0486; tdcB) and citramalate synthase (GSU1798; cimA) genes, three mutant strains were constructed: a threonine ammonia-lyase knockout mutant (DLCR5; tdcB::Knr), a citramalate synthase knockout mutant (DLCR6; cimA::Gmr), and a double knockout mutant (DLCR7; tdcB::Knr cimA::Gmr) (Fig. ). The single mutants grew on the standard plating medium, whereas the double mutant grew only on plates supplemented with 0.02% isoleucine. This indicated that there were no other pathways generating the key precursor 2-oxobutanoate and that both pathways contributed to the biosynthesis of isoleucine. Moreover, these genes could compensate for each other. During growth on acetate-fumarate medium (Fig. ), the growth rate and biomass yields of both single mutants were very similar to wild type, albeit there was a small increase in the doubling time of the citramalate synthase-deficient mutant relative to wild type (6 ± 0.13 h versus 5.25 ± 0.18 h). During growth on acetate-Fe(III) citrate medium, the rate of Fe(III) citrate reduction (Fig. ) and the final biomass yields of the two single mutants (data not shown) were essentially identical to the wild type.
FIG. 4. Construction and phenotypic characterization of citramalate synthase and threonine ammonia-lyase knockout mutants. (A) Scaled representation of the genotypes of mutants DLCR5, DLCR6, and DLCR7. White sections represent the deleted section of each gene. (more ...)
In order to confirm that GSU0486 and GSU1798 coded for threonine ammonia-lyase and citramalate synthase, respectively, soluble extracts of the wild-type and the three mutant strains were prepared from early-stationary-phase NBAF cultures, and enzymatic assays were performed (Table ). In the wild-type strain, the two activities were comparable to those obtained from extracts prepared from mid-log fresh-water medium cultures. As expected, threonine ammonia-lyase and serine ammonia-lyase activities were undetectable in DLCR5. Likewise, citramalate synthase activity was greatly reduced in DLCR6. Neither activity could be detected in the isoleucine auxotroph DLCR7. Isopropylmalate synthase activity was assayed as an internal control and was found to be identical or higher than wild type in the three mutant strains. These results indicate that the current annotation of GSU0486 as ilvA and GSU1798 as a leuA homolog does not reflect their actual enzymatic activities. We propose that they be reannotated as threonine/serine ammonia-lyase (tdcB) and citramalate synthase (cimA), respectively.
Enzymatic activities in wild-type and mutant strains
Unlike the closely related catabolic ammonia lyases of E. coli
and S. enterica
serovar Typhimurium, which have a strictly biodegradative role in these organisms (37
), the threonine ammonia-lyase of G. sulfurreducens
clearly participates in isoleucine biosynthesis. Despite the fact that the contribution of the threonine-dependent pathway to isoleucine biosynthesis in the wild-type strain was relatively minor (18 to 30%) (Fig. ), the amount of threonine ammonia-lyase activity in soluble extracts was about 25-fold higher than that of citramalate synthase activity. This discrepancy could be due to low intracellular concentrations of threonine and/or to inhibition of the enzyme by pyruvate, which occurs in E. coli
). A detailed biochemical characterization of these enzymes coupled with measurements of intracellular concentrations of amino acids and metabolites is therefore warranted.
In order to corroborate the roles of the citramalate synthase and the threonine ammonia-lyase in isoleucine biosynthesis, 13
C labeling studies were conducted in all three mutants and the wild-type strain. Isoleucine was 90 to 95% unlabeled in the isoleucine auxotroph DLCR7 (data not shown). A clear shift in the isoleucine flux ratio to primary use of the citramalate-dependent pathway in DLCR5 and the threonine-dependent pathway in DLCR6 was observed (Table and Fig. ). The residual fluxes in the deleted pathway in each case were likely due to imperfect model fit to the experimental data. Error in flux ratios can result from random variations in mass spectrometry data, loss of information in converting positional isotopomer distributions to mass isotopomer distributions of measurable fragments, and the nonlinearity of the optimization problem (45
Comparison of experimental and predicted mass isotopomer distributions during growth of wild-type (DL1) and mutant (DLCR5 and DLCR6) strains on acetate-fumarate medium containing 30% [13C]acetate and unlabeled fumarate
FIG. 5. Predicted contributions of threonine and citramalate pathways to isoleucine biosynthesis in the wild-type (DL1) and mutant strains during growth on acetate-fumarate (NBAF) medium. Model predictions are the best fit flux distributions, assuming both pathways (more ...) Distribution of the citramalate synthase.
The CimA protein from G. sulfurreducens constitutes the first characterized member of a phylogenetically distinct clade of citramalate synthases (Fig. , clade III). This clade contains representatives from a wide range of bacteria including Deltaproteobacteria, Alphaproteobacteria, Cyanobacteria, Deinococci, and Clostridia as well as members of the Archaea, such as Thermococcaceae. Clade III representatives were also found in the Actinobacteria, Sphingobacteria, Chlorobia, and the Chloroflexi. Inclusion of the LeuA and CimA sequences from these organisms did not affect the structure of the phylogenetic tree (data not shown). Clade III CimA homologs appear to be absent from the Beta-, Epsilon-, and Gammaproteobacteria. In some organisms that lack homologs of threonine ammonia-lyase, this class of citramalate synthases may constitute the only route for isoleucine biosynthesis; examples include Pelobacter propionicus, Pyrococcus furiosus, and all sequenced members of the Desulfovibrionaceae.
FIG. 6. Phylogenetic analysis of citramalate synthases. Phylogenetic tree was inferred from protein sequences by the neighbor-joining method using the BIONJ algorithm (14, 36) as previously described (8). Bootstrap values were calculated for 100 replicates. Biochemically (more ...)