The function of the PduJ microcompartment shell protein is determined by the genomic position of its encoding gene

Chowdhury, Chiranjit; Chun, Sunny; Sawaya, M.R.; Yeates, Todd O.; Bobik, Thomas A.

doi:10.1111/mmi.13423

Cited by 34 publications

(34 citation statements)

References 55 publications

(132 reference statements)

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“…TEM provided further support, as MCPs purified from two different mutants revealed structures that appeared to be large empty MCP shells. Putting this in context, prior studies showed that deletion of any individual shell protein of the Pdu MCP (other than PduB) did not prevent shell-core interactions, since MCPs purified from these mutants had the expected complement of core enzymes and shell proteins (32,50,52,59). Furthermore, we showed that an M38A mutation (which prevents translation of PduB=) also had relatively little effect on the association of the shell with the enzymatic core (Fig.…”

Section: Discussionmentioning

confidence: 67%

“…However, the question of specificity with regard to the binding of the enzymatic core to the shell is unresolved and somewhat paradoxical. Studies indicate that a single N-terminal enzyme-targeting sequence can bind multiple shell proteins (38,39) and vice versa, i.e., that a single shell protein can bind multiple core enzymes through targeting sequence associations (59,62,63). Recent findings have also shown that targeting sequences from diverse MCP systems (Eut and Grp) can direct the encapsulation of proteins into the Pdu MCP via a conserved hydrophobic motif (36).…”

Section: Discussionmentioning

confidence: 99%

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The N Terminus of the PduB Protein Binds the Protein Shell of the Pdu Microcompartment to Its Enzymatic Core

2017

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Bacterial microcompartments (MCPs) are extremely large proteinaceous organelles that consist of an enzymatic core encapsulated within a complex protein shell. A key question in MCP biology is the nature of the interactions that guide the assembly of thousands of protein subunits into a well-ordered metabolic compartment. In this report, we show that the N-terminal 37 amino acids of the PduB protein have a critical role in binding the shell of the 1,2-propanediol utilization (Pdu) microcompartment to its enzymatic core. Several mutations were constructed that deleted short regions of the N terminus of PduB. Growth tests indicated that three of these deletions were impaired MCP assembly. Attempts to purify MCPs from these mutants, followed by gel electrophoresis and enzyme assays, indicated that the protein complexes isolated consisted of MCP shells depleted of core enzymes. Electron microscopy substantiated these findings by identifying apparently empty MCP shells but not intact MCPs. Analyses of 13 site-directed mutants indicated that the key region of the N terminus of PduB required for MCP assembly is a putative helix spanning residues 6 to 18. Considering the findings presented here together with prior work, we propose a new model for MCP assembly.IMPORTANCE Bacterial microcompartments consist of metabolic enzymes encapsulated within a protein shell and are widely used to optimize metabolic process. Here, we show that the N-terminal 37 amino acids of the PduB shell protein are essential for assembly of the 1,2-propanediol utilization microcompartment. The results indicate that it plays a key role in binding the outer shell to the enzymatic core. We propose that this interaction might be used to define the relative orientation of the shell with respect to the core. This finding is of fundamental importance to our understanding of microcompartment assembly and may have application to engineering microcompartments as nanobioreactors for chemical production.

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Section: Discussionmentioning

confidence: 67%

Section: Discussionmentioning

confidence: 99%

The N Terminus of the PduB Protein Binds the Protein Shell of the Pdu Microcompartment to Its Enzymatic Core

2017

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“…3a, Table 1). We speculate that PduJ could compensate for the defects in the Pdu MCP structure caused by the lack of PduA, because PduA and PduJ proteins share high sequence (83%) and structural similarities 44 . In contrast, the PduBB' content per Pdu MCP that lacked PduA decreased by a factor of 4, and the ratio of PduB and PduB' in both complexes remained at 1:1 (Fig.…”

Section: Resultsmentioning

confidence: 99%

Decoding the stoichiometric composition and organisation of bacterial metabolosomes

et al. 2020

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Some enteric bacteria including Salmonella have evolved the propanediol-utilising microcompartment (Pdu MCP), a specialised proteinaceous organelle that is essential for 1,2propanediol 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.

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“…While pduA and pduK encode hexameric shell proteins, pduB and pduT encode pseudohexameric shell protein and pduN codes for the pentameric cap protein for formation of BMC shell (Figure 1). It is noteworthy that in Salmonella, both PduJ and PduM contributes to the formation of normal shaped BMC and are thought to be essential for structural integrity of BMCs (Sinha et al, 2012;Chowdhury et al, 2016). However, a standard pfam based (Pfam00936/Pfam03319) BMC domain search across the A. woodii genome resulted in the identification of four more clusters that could potentially encode BMC proteins ( Table 1).…”

Section: A Woodii Has Five Islands Potentially Encoding Several Diffmentioning

confidence: 99%

Exploring Bacterial Microcompartments in the Acetogenic Bacterium Acetobacterium woodii

et al. 2020

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The strictly anaerobic acetogenic bacterium Acetobacterium woodii is metabolically diverse and grows on variety of substrates which includes H 2 + CO 2 , sugars, alcohols and diols. It is unique in producing bacterial microcompartments (BMC) during growth on different substrates such as 1,2-propanediol, 2,3-butanediol, ethanol or fructose. In this study, we analyzed the genetic organization and expression of the BMC genes within the A. woodii genome, the previously described 18 gene pdu cluster as well as four other cluster potentially encoding one or two shell proteins. Expression analysis of respective gene clusters revealed that the pdu gene cluster is highly expressed during growth on 1,2-PD, 2,3-BD, ethanol and ethylene glycol. The promoter region upstream of the pduA gene was identified and used to establish a reporter gene assay based on chloramphenicol acetyl transferase as a reporter protein. The reporter gene assay confirmed the qPCR data and demonstrated that 1,2-PD is superior over ethanol and ethylene glycol as inducer. BMCs were enriched from cells grown on 2,3-BD and 1,2-PD and shown to have typical structure in electron micrographs. Biochemical analyses revealed several of the protein encoded by the pdu cluster to be part of the isolated BMCs. These data demonstrate a very unique situation in A. woodii in which apparently one BMC gene cluster in expressed during growth on different substrates.

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The function of the PduJ microcompartment shell protein is determined by the genomic position of its encoding gene

Cited by 34 publications

References 55 publications

The N Terminus of the PduB Protein Binds the Protein Shell of the Pdu Microcompartment to Its Enzymatic Core

The N Terminus of the PduB Protein Binds the Protein Shell of the Pdu Microcompartment to Its Enzymatic Core

Decoding the stoichiometric composition and organisation of bacterial metabolosomes

Exploring Bacterial Microcompartments in the Acetogenic Bacterium Acetobacterium woodii

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