Dataset Information

Genetic analysis of the protein shell of the microcompartments involved in coenzyme B12-dependent 1,2-propanediol degradation by Salmonella.

ABSTRACT: Hundreds of bacterial species use microcompartments (MCPs) to optimize metabolic pathways that have toxic or volatile intermediates. MCPs consist of a protein shell encapsulating specific metabolic enzymes. In Salmonella, an MCP is used for 1,2-propanediol utilization (Pdu MCP). The shell of this MCP is composed of eight different types of polypeptides, but their specific functions are uncertain. Here, we individually deleted the eight genes encoding the shell proteins of the Pdu MCP. The effects of each mutation on 1,2-PD degradation and MCP structure were determined by electron microscopy and growth studies. Deletion of the pduBB', pduJ, or pduN gene severely impaired MCP formation, and the observed defects were consistent with roles as facet, edge, or vertex protein, respectively. Metabolite measurements showed that pduA, pduBB', pduJ, or pduN deletion mutants accumulated propionaldehyde to toxic levels during 1,2-PD catabolism, indicating that the integrity of the shell was disrupted. Deletion of the pduK, pduT, or pduU gene did not substantially affect MCP structure or propionaldehyde accumulation, suggesting they are nonessential to MCP formation. However, the pduU or pduT deletion mutants grew more slowly than the wild type on 1,2-PD at saturating B(12), indicating that they are needed for maximal activity of the 1,2-PD degradative enzymes encased within the MCP shell. Considering recent crystallography studies, this suggests that PduT and PduU may mediate the transport of enzyme substrates/cofactors across the MCP shell. Interestingly, a pduK deletion caused MCP aggregation, suggesting a role in the spatial organization of MCP within the cytoplasm or perhaps in segregation at cell division.

SUBMITTER: Cheng S

PROVIDER: S-EPMC3067621 | biostudies-literature | 2011 Mar

REPOSITORIES: biostudies-literature

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Genetic analysis of the protein shell of the microcompartments involved in coenzyme B12-dependent 1,2-propanediol degradation by Salmonella.

Cheng Shouqiang S Sinha Sharmistha S Fan Chenguang C Liu Yu Y Bobik Thomas A TA

Journal of bacteriology 20110114 6

Hundreds of bacterial species use microcompartments (MCPs) to optimize metabolic pathways that have toxic or volatile intermediates. MCPs consist of a protein shell encapsulating specific metabolic enzymes. In Salmonella, an MCP is used for 1,2-propanediol utilization (Pdu MCP). The shell of this MCP is composed of eight different types of polypeptides, but their specific functions are uncertain. Here, we individually deleted the eight genes encoding the shell proteins of the Pdu MCP. The effect ...[more]

PMID: 21239588

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Project description:The propanediol utilization (pdu) operon of Salmonella enterica serovar Typhimurium LT2 contains genes needed for the coenzyme B(12)-dependent catabolism of 1,2-propanediol. Here the completed DNA sequence of the pdu operon is presented. Analyses of previously unpublished pdu DNA sequence substantiated previous studies indicating that the pdu operon was acquired by horizontal gene transfer and allowed the identification of 16 hypothetical genes. This brings the total number of genes in the pdu operon to 21 and the total number of genes at the pdu locus to 23. Of these, six encode proteins of unknown function and are not closely related to sequences of known function found in GenBank. Two encode proteins involved in transport and regulation. Six probably encode enzymes needed for the pathway of 1,2-propanediol degradation. Two encode proteins related to those used for the reactivation of adenosylcobalamin (AdoCbl)-dependent diol dehydratase. Five encode proteins related to those involved in the formation of polyhedral organelles known as carboxysomes, and two encode proteins that appear distantly related to those involved in carboxysome formation. In addition, it is shown that S. enterica forms polyhedral bodies that are involved in the degradation of 1,2-propanediol. Polyhedra are formed during either aerobic or anaerobic growth on propanediol, but not during growth on other carbon sources. Genetic tests demonstrate that genes of the pdu operon are required for polyhedral body formation, and immunoelectron microscopy shows that AdoCbl-dependent diol dehydratase is associated with these polyhedra. This is the first evidence for a B(12)-dependent enzyme associated with a polyhedral body. It is proposed that the polyhedra consist of AdoCbl-dependent diol dehydratase (and perhaps other proteins) encased within a protein shell that is related to the shell of carboxysomes. The specific function of these unusual polyhedral bodies was not determined, but some possibilities are discussed.

Project description:UnlabelledBacterial microcompartments (MCPs) are a diverse family of protein-based organelles composed of metabolic enzymes encapsulated within a protein shell. The function of bacterial MCPs is to optimize metabolic pathways by confining toxic and/or volatile metabolic intermediates. About 20% of bacteria produce MCPs, and there are at least seven different types. Different MCPs vary in their encapsulated enzymes, but all have outer shells composed of highly conserved proteins containing bacterial microcompartment domains. Many organisms have genes encoding more than one type of MCP, but given the high homology among shell proteins, it is uncertain whether multiple MCPs can be functionally expressed in the same cell at the same time. In these studies, we examine the regulation of the 1,2-propanediol (1,2-PD) utilization (Pdu) and ethanolamine utilization (Eut) MCPs in Salmonella. Studies showed that 1,2-PD (shown to induce the Pdu MCP) represses transcription of the Eut MCP and that the PocR regulatory protein is required. The results indicate that repression of the Eut MCP by 1,2-PD is needed to prevent detrimental mixing of shell proteins from the Eut and Pdu MCPs. Coexpression of both MCPs impaired the function of the Pdu MCP and resulted in the formation of hybrid MCPs composed of Eut and Pdu MCP components. We also show that plasmid-based expression of individual shell proteins from the Eut MCP or the β-carboxysome impaired the function of Pdu MCP. Thus, the high conservation among bacterial microcompartment (BMC) domain shell proteins is problematic for coexpression of the Eut and Pdu MCPs and perhaps other MCPs as well.ImportanceBacterial MCPs are encoded by nearly 20% of bacterial genomes, and almost 40% of those genomes contain multiple MCP gene clusters. In this study, we examine how the regulation of two different MCP systems (Eut and Pdu) is integrated in Salmonella. Our findings indicate that 1,2-PD (shown to induce the Pdu MCP) represses the Eut MCP to prevent detrimental mixing of Eut and Pdu shell proteins. These findings suggest that numerous organisms which produce more than one type of MCP likely need some mechanism to prevent aberrant shell protein interactions.

Project description:The spatial organization of metabolism is common to all domains of life. Enteric and other bacteria use subcellular organelles known as bacterial microcompartments to spatially organize the metabolism of pathogenicity-relevant carbon sources, such as 1,2-propanediol. The organelles are thought to sequester a private cofactor pool, minimize the effects of toxic intermediates, and enhance flux through the encapsulated metabolic pathways. We develop a mathematical model of the function of the 1,2-propanediol utilization microcompartment of Salmonella enterica and use it to analyze the function of the microcompartment organelles in detail. Our model makes accurate estimates of doubling times based on an optimized compartment shell permeability determined by maximizing metabolic flux in the model. The compartments function primarily to decouple cytosolic intermediate concentrations from the concentrations in the microcompartment, allowing significant enhancement in pathway flux by the generation of large concentration gradients across the microcompartment shell. We find that selective permeability of the microcompartment shell is not absolutely necessary, but is often beneficial in establishing this intermediate-trapping function. Our findings also implicate active transport of the 1,2-propanediol substrate under conditions of low external substrate concentration, and we present a mathematical bound, in terms of external 1,2-propanediol substrate concentration and diffusive rates, on when active transport of the substrate is advantageous. By allowing us to predict experimentally inaccessible aspects of microcompartment function, such as intra-microcompartment metabolite concentrations, our model presents avenues for future research and underscores the importance of carefully considering changes in external metabolite concentrations and other conditions during batch cultures. Our results also suggest that the encapsulation of heterologous pathways in bacterial microcompartments might yield significant benefits for pathway flux, as well as for toxicity mitigation.

Dataset Information

Genetic analysis of the protein shell of the microcompartments involved in coenzyme B12-dependent 1,2-propanediol degradation by Salmonella.

Publications

Genetic analysis of the protein shell of the microcompartments involved in coenzyme B12-dependent 1,2-propanediol degradation by Salmonella.

Similar Datasets

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