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ABSTRACT Upon nutrient starvation,Chlamydia trachomatisserovar L2 (CTL) shifts from its normal growth to a non-replicating form, termed persistence. It is unclear if persistence reflects an adaptive response or a lack thereof. To understand this, transcriptomics data were collected for CTL grown under nutrient-replete and nutrient-starved conditions. Applying K-means clustering on transcriptomics data revealed a global transcriptomic rewiring of CTL under stress conditions in the absence of any canonical global stress regulator. This is consistent with previous data that suggested that CTL’s stress response is due to a lack of an adaptive response mechanism. To investigate the impact of this on CTL metabolism, we reconstructed a genome-scale metabolic model of CTL (iCTL278) and contextualized it with the collected transcriptomics data. Using the metabolic bottleneck analysis on contextualized iCTL278, we observed that phosphoglycerate mutase (pgm) regulates the entry of CTL to the persistence state. Our data indicate thatpgmhas the highest thermodynamics driving force and lowest enzymatic cost. Furthermore, CRISPRi-driven knockdown ofpgmin the presence or absence of tryptophan revealed the importance of this gene in modulating persistence. Hence, this work, for the first time, introduces thermodynamics and enzyme cost as tools to gain a deeper understanding on CTL persistence. IMPORTANCEThis study uses a metabolic model to investigate factors that contribute to the persistence ofChlamydia trachomatisserovar L2 (CTL) under tryptophan and iron starvation conditions. As CTL lacks many canonical transcriptional regulators, the model was used to assess two prevailing hypotheses on persistence—that the chlamydial response to nutrient starvation represents a passive response due to the lack of regulators or that it is an active response by the bacterium. K-means clustering of stress-induced transcriptomics data revealed striking evidence in favor of the lack of adaptive (i.e., a passive) response. To find the metabolic signature of this, metabolic modeling pin-pointed pgm as a potential regulator of persistence. Thermodynamic driving force, enzyme cost, and CRISPRi knockdown of pgm supported this finding. Overall, this work introduces thermodynamic driving force and enzyme cost as a tool to understand chlamydial persistence, demonstrating how systems biology-guided CRISPRi can unravel complex bacterial phenomena.