Metabolic reconstructions constitute a framework to organize genomic, proteomic, metabolomic, and other data sets and to assess the effects of perturbations on these elements at the network level. Accordingly, we present a metabolic reconstruction for YP CO92, a strain that is virulent to humans, and benchmark the reconstruction qualitatively against experimental growth data from eleven different carbon sources and quantitatively against growth rate and metabolite uptake and secretion rate data from two of the sources.
The two gaps in lysine and fatty acid biosynthesis highlighted in this work are significant because model simulations cannot occur unless both gaps are filled. The gap in lysine biosynthesis is noteworthy in that the disrupted gene, YPO0170, encodes a bifunctional enzyme, ArgD, that is involved in arginine biosynthesis as well : specifically, ArgD catalyzes the reversible conversion of N-acetyl-L-glutamate 5-semialdehyde and L-glutamate to N-acetyl-L-ornithine and 2-oxoglutarate, after which N-acetyl-L-ornithine is converted to L-ornithine. An absence of ArgD does not result in arginine auxotrophy, however, because YP CO92 contains an alternative, one-step route to L-ornithine from L-proline that is catalyzed by YPO4090. This reaction is included in the model. On the other hand, there is no clear paralog or other mechanism within YP CO92 that appears capable of replacing the missing fabI gene in fatty acid biosynthesis. At the amino acid level, there is only 28% identity between E. coli FabI and its best match in YP CO92, YPO3351. We therefore sought to identify possible candidate enzymes through an analysis of expression data for YP CO92 [Schrimpe-Rutledge AC and Adkins JN, unpublished data], reasoning that the unidentified gene will be located near other genes involved in fatty acid biosynthesis (specifically, the cluster from YPO1597 (fabH) through YPO1601 (fabF)), show correlated expression with the genes in this cluster, and be annotated as hypothetical. The best match based on these criteria is the hypothetical gene YPO1594. Consequently, we propose that YPO1594 might possess the ability to carry out the same catalytic function as FabI. Other genes showing correlated expression but located farther away from YPO1597-YPO1601 are YPO3732 and YPO2055.
Pathway analysis and model simulations led to the hypothesis that YP CO92 cannot utilize rhamnose and melibiose due to a missing sink reaction and perturbed gene regulation, respectively, but other possible mechanisms have been advanced. A disruption in gene regulation might contribute to the rhamnose-negative phenotype as well. Specifically, a recent study that compared the sequences of rhamnose fermentation genes in rhamnose-positive and rhamnose-negative strains suggested that a point mutation in the transcriptional activator RhaS might be responsible for the rhamnose-negative phenotype . Experimental data from a prior study indirectly support this claim . Rhamnose-negative strains can revert and gain the ability to metabolize the sugar at a low frequency, and an analysis of one such strain found that RhaB and RhaA had become active in the revertant . Since RhaS regulates the rhaBAD operon , these data imply that RhaS is the key regulatory protein controlling rhamnose utilization and that the mechanism involves rhaBAD. On the other hand, melibiose metabolism might be absent in YP CO92 because of one or more defects in inner membrane transport. We identified two possible melibiose symporters in YP CO92, YPO1582 and YPO0995 (melB). The former is the homolog of the putative melibiose symporter YP1470 from Y. pestis 91001, a strain that can utilize melibiose , but the 5' end of the coding sequence for YPO1582 has been disrupted by the IS element IS285. YPO1582 is therefore presumed to be a pseudogene. The latter is intact in YP CO92; however, the IS element IS1661 is located approximately 250 bp upstream from the 5' end of melB, raising the possibility that it has potentially disrupted the control of expression of melB. Proteomic data collected during mid-log growth on BCS medium support this hypothesis: no peptides for YPO0995 could be detected under these growth conditions [Schrimpe-Rutledge AC and Adkins JN, unpublished data].
Like rhamnose and melibiose, several hypotheses have been advanced to explain why YP CO92 cannot metabolize glycerol. Analysis of the YP CO92 genome sequence revealed the presence of a 93 bp in-frame deletion in glpD that might account for this phenotype: glpD encodes aerobic glycerol 3-phosphate dehydrogenase, an enzyme that is essential for glycerol utilization , and this deletion likely disrupts protein function. Intriguingly, this same deletion appeared in every glycerol-negative strain in one culture collection whereas all glycerol-positive strains from the same collection contained an intact glpD . There is a second annotated glycerol 3-phosphate dehydrogenase within the YP CO92 genome, gpsA, but the gpsA homolog in E. coli can only catalyze the transformation from dihydroxyacetone phosphate to glycerol 3-phosphate in an irreversible manner (Figure 3). A second defect occurs in the glpFKX operon and might contribute to the glycerol-negative phenotype as well: both the hypothetical protein glpX (YPO0089) and, more importantly, the glycerol kinase glpK (YPO0090) have also been disrupted by large deletions and are presumed to be pseudogenes. As with glpD, there is a second annotated glycerol kinase in the YP CO92 genome, YPO3312, but it is unknown whether YPO3312 can duplicate the function of YPO0090.
We saw slow growth for YP CO92 in both experimental measurements and model simulations in the absence of glycine and threonine, and the model predicts that this phenotype stems from insufficient supply of nitrogen. BCS medium does not contain an explicit source of nitrogen such as NH4Cl; therefore, YP CO92 most likely obtains elemental nitrogen through catabolism of one or more amino acids. Glycine, threonine, and serine can all interconvert. In turn, the breakdown of L-serine by L-serine dehydratase (YPO1771) or L-threonine by L-threonine dehydratase (YPO3896) leads to the direct formation of NH3. Simulations that exclude glycine and threonine from the in silico growth medium (serine is not a component of BCS medium) force nitrogen acquisition to occur through multi-step pathways that are less efficient, leading to slower growth. Providing supplemental nitrogen through sources such as NH4Cl, in contrast, leads to a faster in silico growth rate that varies with the uptake rate of the supplemental source.
It is well known that certain Y. pestis strains such as CO92 display methionine auxotrophy [25, 26], and the model highlights the importance of the methionine salvage pathway to this phenotype. Several reactions in this pathway are not currently associated with any genes in YP CO92; however, if this pathway is absent, the byproduct S-methyl-5'-thioadenosine (MTA) would be generated during reactions that consume S-adenosyl methionine, but MTA itself would never be consumed or degraded in any reaction. Such a situation is identical to the formation of L-lactaldehyde during rhamnose utilization and results in the same thermodynamically impossible outcome. Furthermore, this pathway is fully annotated in other Y. pestis strains such as Pestoides F and Angola, suggesting that it is present in YP CO92 as well but that the associated genes remain to be identified. For these reasons, the model includes a set of non-gene-associated reactions that recycles MTA back into methionine via the methionine salvage pathway, and predicts that these reactions are essential in YP CO92.