Synthesis of empty bacterial microcompartments, directed organelle protein incorporation, and evidence of filament-associated organelle movement

Compartmentalization is an important process, since it allows the segregation of metabolic activities and, in the era of synthetic biology, represents an important tool by which defined microenvironments can be created for specific metabolic functions. Indeed, some bacteria make specialized proteinaceous metabolic compartments called bacterial microcompartments (BMCs) or metabolosomes. Here we demonstrate that the shell of the metabolosome (representing an empty BMC) can be produced within E. coil cells by the coordinated expression of genes encoding structural proteins. A plethora of diverse structures can be generated by changing the expression profile of these genes, including the formation of large axial filaments that interfere with septation. Fusing GFP to PduC, PduD, or PduV, none of which are shell proteins, allows regiospecific targeting of the reporter group to the empty BMC. Live cell imaging provides unexpected evidence of filament-associated BMC movement within the cell in the presence of Pdu

Solution structure of a bacterial microcompartment targeting peptide and its application in the construction of an ethanol bioreactor

Targeting of proteins to bacterial microcompartments (BMCs) is mediated by an 18-amino-acid peptide sequence. Herein, we report the solution structure of the N-terminal targeting peptide (P18) of PduP, the aldehyde dehydrogenase associated with the 1,2-propanediol utilization metabolosome from Citrobacter freundii. The solution structure reveals the peptide to have a well-defined helical conformation along its whole length. Saturation transfer difference and transferred NOE NMR has highlighted the observed interaction surface on the peptide with its main interacting shell protein, PduK. By tagging both a pyruvate decarboxylase and an alcohol dehydrogenase with targeting peptides, it has been possible to direct these enzymes to empty BMCs in vivo and to generate an ethanol bioreactor. Not only are the purified, redesigned BMCs able to transform pyruvate into ethanol efficiently, but the strains containing the modified BMCs produce elevated levels of alcohol

The Structural Basis of Coenzyme A Recycling in a Bacterial Organelle.

Bacterial Microcompartments (BMCs) are proteinaceous organelles that encapsulate critical segments of autotrophic and heterotrophic metabolic pathways; they are functionally diverse and are found across 23 different phyla. The majority of catabolic BMCs (metabolosomes) compartmentalize a common core of enzymes to metabolize compounds via a toxic and/or volatile aldehyde intermediate. The core enzyme phosphotransacylase (PTAC) recycles Coenzyme A and generates an acyl phosphate that can serve as an energy source. The PTAC predominantly associated with metabolosomes (PduL) has no sequence homology to the PTAC ubiquitous among fermentative bacteria (Pta). Here, we report two high-resolution PduL crystal structures with bound substrates. The PduL fold is unrelated to that of Pta; it contains a dimetal active site involved in a catalytic mechanism distinct from that of the housekeeping PTAC. Accordingly, PduL and Pta exemplify functional, but not structural, convergent evolution. The PduL structure, in the context of the catalytic core, completes our understanding of the structural basis of cofactor recycling in the metabolosome lumen

Recycling of vitamin B12 and NAD+ within the Pdu microcompartment of Salmonella enterica

Salmonella enterica is capable of utilizing 1,2-propanediol (1,2-PD) as a sole carbon and energy source in a coenzyme B12 (Adenosylcobalamin, AdoCbl) -dependent fashion that involves a bacterial microcompartment (MCP), the Pdu MCP. Pdu MCP is a polyhedral organelle composed entirely of protein subunits and its function is to sequester the intermediate propionaldehyde formed in the first step of 1,2-PD degradation in order to mitigate its toxicity and prevent DNA damage. Several sequentially-acting metabolic enzymes, including the diol dehydratase PduCDE and the propionaldehyde dehydrogenase PduP that respectively use AdoCbl and NAD+ as cofactors, are encapsulated within the solid protein shell of the MCP. During catalysis, the adenosyl-group of AdoCbl bound to PduCDE is periodically lost due to by-reactions and is usually replaced by a hydroxo-group resulting in the formation of an inactive OH-Cbl-enzyme complex, and the NAD+ is converted to NADH by PduP, which make it necessary to regenerate or/and replenish AdoCbl and NAD+ within the Pdu MCPs. Recent crystallography studies suggested that some Pdu MCP shell proteins, such as PduA, T and U, have pores that may mediate the transport of enzyme substrates/cofactors across the MCP shell. However, it\u27s possible that the cofactors might be regenerated in the local vicinity of the metabolic enzymes within the MCP, which could contribute to higher activity of the enzymes and thus to higher efficiency of 1,2-PD degradation. In this work, two proteins encoded by the pdu locus consisting of genes specifically involved in 1,2-PD utilization, PduS and PduQ, were overexpressed, purified and characterized in vitro, and their in vivo fuctions were investigated as well. Purified PduS enzyme exhibited the abilities to catalyze the two successive uni-electron reductions from cob(III)alamin to cob(I)alamin in vitro. The results indicated that PduS is a monomer and each monomer of PduS contains one non-covalently bound FMN and two [4Fe-4S] clusters which are oxygen-labile. Genetic studies showed that a pduS deletion decreased the growth rate of Salmonella on 1,2-PD supporting a role in cobalamin reduction in vivo. Further SDS-PAGE and Western blot of purified Pdu MCP and following MS-MS analysis demonstrated that the PduS protein is a component of the Pdu MCP. In addition, two-hybrid experiments indicated that PduS interacts with the PduO adenosyltransferase which is also located in the Pdu MCP and catalyzes the terminal step of AdoCbl synthesis. Purified PduQ enzyme was identified as an oxygen-sensitive iron-dependent alcohol dehydrogenase (ADH) that catalyzes the interconversion between propionaldehyde and 1-propanol in vitro. The propionaldehyde reduction activity of PduQ was about 45 times higher than that of the 1-propanol oxidation at pH 7.0, indicating that this enzyme is more efficient for catalyzing aldehyde reduction in cells where approximatedly neutral pH is maintained. Kinetic studies indicated PduQ has a high affinity for the substrate and cofactor involved in this reduction reaction, which also suggest that the physiological function of PduQ is the reduction of aldehyde with the conversion of NADH to NAD+. The pduQ deletion impaired growth on 1,2-PD and led to a lower cell density, indicating that PduQ was required to support the maximal rate of 1,2-PD degradation by Salmonella. SDS-PAGE and Western blot of purified Pdu MCP and subsequent MS-MS analysis demonstrated that the PduQ protein is also associated with the Pdu MCP. Co-affinity purification illustrated a specific strong interaction between PduQ and PduP. In conjunction with prior results, this work indicates that the Pdu MCP encapsulates a complete AdoCbl recycling system and that it is able to recycle the electron carrier NAD+ as well within the MCP lumen

PduL is an evolutionary distinct phosphotransacylase involved in B12-dependent 1,2-propanediol degradation by Salmonella enterica serovar Typhimurium LT2 and is associated with the propanediol utilization microcompartments

Salmonella enterica degrades 1,2-propanediol (1,2-PD) in a coenzyme B12-dependent manner. Prior enzymatic assays of crude cell extracts indicated that a phosphotransacylase (PTAC) was needed for this process, but the enzyme involved was not identified. Here we show that the pduL gene encodes an evolutionarily distinct PTAC used for 1,2-PD degradation. Growth tests showed that pduL mutants were unable to ferment 1,2-PD and were also impaired for aerobic growth on this compound. Enzyme assays showed that cell extracts from a pduL mutant lacked measurable PTAC activity in a background that also carried a pta mutation (the pta gene was previously shown to encode a PTAC enzyme). Ectopic expression of pduL corrected the growth defects of pta mutant. PduL fused to 8 C-terminal histidine residues (PduL-his8) was purified and its kinetic constants determined: Vmax = 51.7y7.6 ymol min-1 mg-1; and Km for propionyl-PO42- and acetyl-PO42- = 0.61 and 0.97 mM, respectively. Sequence analyses showed that PduL is unrelated in amino acid sequence to known PTAC enzymes and that PduL homologues are distributed among at least 49 bacterial species, but are absent from the Archaea and Eukarya. Immunoblots showed that PduL was a component of propanediol utilization microcompartment

Functional analysis of the propanediol utilization microcompartment shell proteins PduB and PduB\u27 in Salmonella enterica

Bacterial microcompartments (MCPs) are proteinaceous sub-cellular organelles that are widely distributed among bacteria and that function in a variety of processes ranging from global carbon fixation to enteric pathogenesis. MCPs consist of metabolic enzymes encapsulated with a protein shell. The role of the MCP is to harbor a specific metabolic pathway that produces a toxic or volatile intermediate and confine that intermediate to minimize cellular toxicity and carbon loss. To date, the protein shells of MCPs have been shown to play a functional role in transport of small metabolites through selective pores and in the encapsulation of lumen enzymes through short N-or C-terminal peptide extensions. Interestingly, homologs of the propanediol utilization (Pdu) MCP shell protein PduB’ have been crystallized in two forms, one that is closed and another that forms a large channel. This suggested that these proteins undergo conformational changes that allow the transport of larger enzymatic cofactor that the MCP needs to properly function. However, no mutational work has been done to examine residues that are responsible for such a large conformational change and assess its physiological significance. Charged residues (R78, K81) and Ramachandran outlier (D79), which are located at the center of the PduB’, are key structural components that when substituted with alanine cause MCP instability. Interestingly, substitutions of a channel residue A53 appears to cause central pore opening. In addition, results indicate that there is a functional difference between PduB and PduB’ despite the fact that they are identical in sequence except for a 37 amino acid N-terminal extension on PduB. The smaller protein, PduB’, is dispensable for MCP formation but the PduB protein which contains a 37 amino acid N-terminal extension is integral to MCP assembly and formation

Structure and Expression of Propanediol Utilization Microcompartments in Acetonema longum

Numerous bacteria assemble proteinaceous microcompartments to isolate certain biochemical reactions within the cytoplasm. The assembly, structure, contents, and functions of these microcompartments are active areas of research. Here we show that the Gram-negative sporulating bacterium Acetonema longum synthesizes propanediol utilization (PDU) microcompartments when starved or grown on 1,2-propanediol (1,2-PD) or rhamnose. Electron cryotomography of intact cells revealed that PDU microcompartments are highly irregular in shape and size, similar to purified PDU microcompartments from Salmonella enterica serovar Typhimurium LT2 that were imaged previously. Homology searches identified a 20-gene operon in A. longum that contains most of the structural, enzymatic, and regulatory genes thought to be involved in PDU microcompartment assembly and function. Transcriptional data on PduU and PduC, which are major structural and enzymatic proteins, respectively, as well as imaging, indicate that PDU microcompartment synthesis is induced within 24 h of growth on 1,2-PD and after 48 h of growth on rhamnose

Decoding the stoichiometric composition and organisation of bacterial metabolosomes

Some enteric bacteria including Salmonella have evolved the propanediol-utilising microcompartment (Pdu MCP), a specialised proteinaceous organelle that is essential for 1,2-propanediol degradation and enteric pathogenesis. Pdu MCPs are a family of bacterial microcompartments that are self-assembled from hundreds of proteins within the bacterial cytosol. Here, we seek a comprehensive understanding of the stoichiometric composition and organisation of Pdu MCPs. We obtain accurate stoichiometry of shell proteins and internal enzymes of the natural Pdu MCP by QconCAT-driven quantitative mass spectrometry. Genetic deletion of the major shell protein and absolute quantification reveal the stoichiometric and structural remodelling of metabolically functional Pdu MCPs. Decoding the precise protein stoichiometry allows us to develop an organisational model of the Pdu metabolosome. The structural insights into the Pdu MCP are critical for both delineating the general principles underlying bacterial organelle formation, structural robustness and function, and repurposing natural microcompartments using synthetic biology for biotechnological applications

Self-assembling, protein-based intracellular bacterial organelles: emerging vehicles for encapsulating, targeting and delivering therapeutical cargoes

Many bacterial species contain intracellular nano- and micro-compartments consisting of self-assembling proteins that form protein-only shells. These structures are built up by combinations of a reduced number of repeated elements, from 60 repeated copies of one unique structural element self-assembled in encapsulins of 24 nm to 10,000-20,000 copies of a few protein species assembled in a organelle of around 100-150 nm in cross-section. However, this apparent simplicity does not correspond to the structural and functional sophistication of some of these organelles. They package, by not yet definitely solved mechanisms, one or more enzymes involved in specific metabolic pathways, confining such reactions and sequestering or increasing the inner concentration of unstable, toxics or volatile intermediate metabolites. From a biotechnological point of view, we can use the self assembling properties of these particles for directing shell assembling and enzyme packaging, mimicking nature to design new applications in biotechnology. Upon appropriate engineering of the building blocks, they could act as a new family of self-assembled, protein-based vehicles in Nanomedicine to encapsulate, target and deliver therapeutic cargoes to specific cell types and/or tissues. This would provide a new, intriguing platform of microbial origin for drug delivery