On exposure to 1,2-propanediol (1,2-PD), Salmonella enterica serovar Typhimurium LT2 produces 1,2-PD utilization (Pdu) microcompartments (MCPs), nanoscale protein-bound shells that encapsulate metabolic enzymes. MCPs serve as a bioengineering platform to study reaction organization and enhance flux through specific pathways. However, a recently published assay of purified wild-type (WT) MCPs reported metabolic activity that differed markedly from that observed in vivo. Using kinetic modeling, we attribute these discrepancies to in vivo cell growth and to the cytosolic presence of MCP-associated enzymes and promiscuous alcohol dehydrogenases, which are not present in the purified MCPs. Assays of purified MCPs in E. coli lysate, together with an LT2 growth assay in which the native Pdu MCP-associated alcohol dehydrogenase, PduQ, was knocked out, support the conclusion that exogenous Pdu cytosolic enzyme activity can narrow the gap between in vitro and in vivo experiments. Our modeling further suggests that MCP-localized enzymes contribute little to in vivo metabolic flux downstream of PduCDE. We therefore propose a revised in vivo model of WT growth on 1,2-PD in which PduCDE is fully encapsulated, while much of the downstream Pdu activity occurs in the cytosol.
Salmonella enterica serovar Typhimurium LT2 produces bacterial microcompartments (MCPs) upon exposure to 1,2-propanediol (1,2-PD). 1,2-Propanediol utilization (Pdu) MCPs are 100-150 nanometers in diameter and are composed of eight shell proteins (PduABB'NJKTU) [1,2,3]. The protein shells encapsulate a catalytic core consisting of nine unique proteins (PduCDEGHLOPQSVW) [4]. Among these, the catalytic enzymes PduCDELPQW directly participate in 1,2-PD fermentation [1]. The Pdu core also encodes cobalamin adenylation [5,6,7] and diol dehydratase reactivation [8], reactions that are essential for optimal 1,2-PD fermentation. Biologically, MCPs are hypothesized to provide Salmonella with a growth advantage by enabling 1,2-PD metabolism while limiting cellular exposure to propionaldehyde, a toxic intermediate of 1,2-PD fermentation [9].
Pdu-mediated 1,2-PD fermentation consists of a five-reaction sequence (Figure 1). PduCDE first dehydrates 1,2-PD to propionaldehyde using adenosylcobalamin (AdoB 12 ) as a cofactor [10,11]. Propionaldehyde is then either converted to propionyl-coenyme A (propionyl-CoA) by PduP while converting nicotinamide adenine dinucleotide (NAD+) to nicotinamide adenine dinucleotide hydride (NADH) or to 1-propanol by PduQ while converting NADH to NAD+ [12,13]. Propionyl-CoA subsequently serves as a carbon source for central carbon metabolism via the 2-methylcitrate cycle (Figure 1) [14]. Reported metabolite dynamics indicate that 1-propanol is reassimilated under 1,2-PD-limited conditions and thus likely acts as a shunt for excess carbon flux through the Pdu pathway [2,3]. PduL and PduW convert propionyl-CoA to propionate while generating one adenosine triphosphate (ATP) [15,14]. Similar to 1-propanol, propionate is observed to act as a temporary sink for excess flux and is reconverted to propionyl-CoA under 1,2-PD-limited conditions [2,3].
A recently designed assay measured the dynamics of a functional 1,2-PD enzymatic pathway in purified Pdu MCPs [16]. However, 1,2-PD metabolite pathway dynamics in purified MCPs were substantially different from those observed upon growth of wild-type (WT) LT2 Salmonella on 1,2-PD as a sole carbon source [9,2,3]. First, in growth assays, WT LT2 consumed 1,2-PD more slowly than corresponding MCP isolates: WT depleted 1,2-PD within 18 hours, whereas MCP isolates were previously observed to consume it within the first hour (Figure 2). Second, in WT LT2 cells, the Pdu pathway was more efficient than MCP isolates, its kinetics unknown, including the cytosolic alcohol dehydrogenase reaction in the model resulted in fits with the majority of the WT 1-propanol dynamics attributed to the unconstrained enzyme. The temporal profile of metabolites during the ∆pduQ growth assay strongly suggest that ∆pduQ suffers from a redox imbalance due to the lack of PduQ. We show that fully encapsulated PduQ contributes little to the total alcohol dehydrogenase activity and, thus, conclude that cytosolic expression of PduQ is necessary to alleviate the redox imbalance.
Figure 1: Proposed reaction model of the Pdu MCPs with complete encapsulation of PduCDE and partial encapsulation of PduP, PduQ, PduL, and PduW. In this model, 1,2-propanediol is converted to propionyl-CoA, propionate, and 1-propanol. Propionate and 1-propanol act as shunts for excess carbon flux and are reconverted to propionyl-CoA. Propionyl-CoA is further metabolized to generate ATP via the 2-methylcitrate cycle, the tricarboxylic acid (TCA) cycle, and the electron transport chain [22,23,22,14,24,25,26]. The findings presented in this study support partial encapsulation of PduP, PduQ, PduL, and PduW.
Bacterial microcompartments are hypothesized to provide a growth advantage to Salmonella by sequestering toxic propionaldehyde and enhancing flux toward downstream metabolites. Recently, Palmero et al. 2025 and Archer et al. 2025 reported metabolite time courses of purified WT MCPs exposed to 1,2-PD and showed that MCP isolates rapidly consume 1,2-PD while producing only limited amounts of 1-propanol and propionyl-phosphate. This behavior contrasts with in vivo assays, in which 1,2-PD is metabolized or converted into extracellular metabolites which the cell subsequently uses for growth [3,2]. To confirm this previous observation, we conducted an in vivo assay of WT grown on 55 mM 1,2-PD and 150 nM Ado-B 12 (Figure 2B and SI Figure S1B). Consistent with prior observations, WT exposed to excess Ado-B 12 exhibited uninterrupted growth on 1,2-PD (Figure 2Bv). 1: Differences between in vitro and in vivo assays, proposed explanations for their origins, and methodology or evidence supporting these hypotheses.
Cultures consumed all available 1,2-PD within 18 h, during which WT grew at a mean rate of 0.203 optical density units per second and produced net propionaldehyde, propionate, and 1-propanol. Once the 1,2-PD pool was depleted, WT reassimilated the remaining extracellular metabolites. 1-propanol reached the highest p
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