Skip to main page content
Access keys NCBI Homepage MyNCBI Homepage Main Content Main Navigation
, 199 (8)

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

Affiliations

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

Brent P Lehman et al. J Bacteriol.

Abstract

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.

Keywords: 1,2-propanediol; Salmonella; carboxysome; microcompartment; vitamin B12.

Figures

FIG 1
FIG 1
Multiple-sequence alignment of the first 53 amino acids of the PduB/PduB′ coding region, with their respective translation start sites indicated. The PduB protein has a 37-amino-acid N-terminal extension not present in PduB′ but is otherwise identical in amino acid sequence. Blue-highlighted residues have >80% amino acid sequence identity across the sequences shown, whose GI numbers are (top to bottom) 409994423, 545166260, 737633100, 490278627, 507082779, 506359047, 738157884, 696233319, 746121973, and 496088740.
FIG 2
FIG 2
Short N-terminal deletions in PduB result in faster growth of Salmonella on 1,2-propanediol minimal medium. The top panel is a schematic of N-terminal deletions that were tested. Residues highlighted in red indicate the deleted regions. Underlined residues correspond to a highly conserved putative α-helix. The pduBB′ deletion mutant is a control known to have a very high growth rate due to an MCP assembly defect. Other PduB deletion mutants are indicated in the legend. The M38A mutant changes the start codon of PduB′ and blocks its translation; thus, it is essentially a ΔpduB′ mutant. The table to the right shows the doubling time for each strain, and the error shown is one standard deviation determined from the results from three or more independent experiments. The wild type (WT) is Salmonella enterica LT2 serovar Typhimurium. OD600, optical density at 600 nm.
FIG 3
FIG 3
SDS-PAGE of MCPs purified from the PduB Δ27–32 mutant, the PduB M38A mutant, and the wild type (WT). Letters on the right indicate the Pdu protein represented by each band. Lysozyme (lys) is not an MCP component but was used to lyse cells. The percent yield of purified MCPs indicates grams of protein/gram of cells relative to the wild type. The specific activities of the major lumen enzymes, the PduCDE diol dehydratase and the PduP aldehyde dehydrogenase, are shown in micromoles per minute per milligram. Ten micrograms of protein was loaded in each lane. Gels were stained using a Coomassie-based protocol. The enzyme assay methods are described in Materials and Methods. The error shown is one standard deviation determined from the results from three or more independent experiments. M, molecular mass markers.
FIG 4
FIG 4
SDS-PAGE of MCPs purified from pduB deletion mutants with MCP assembly defects. WT, wild type. Letters on the right indicate the Pdu protein represented by each band. Lysozyme (lys) is not an MCP component but was used to lyse cells. The percent yield of purified MCPs indicates grams of protein/gram of cells relative to the wild type. The specific activities of the major lumen enzymes, the PduCDE diol dehydratase and the PduP aldehyde dehydrogenase, are shown in micromoles per minute per milligram. The error shown is one standard deviation determined from the results from three or more independent experiments. Ten micrograms of protein was loaded in each lane. Gels were stained using a Coomassie-based protocol. Enzyme assays are described in Materials and Methods.
FIG 5
FIG 5
Individual point mutations along the highly conserved putative α-helix on the N terminus of PduB result in fast growth on 1,2-propanediol minimal medium. WT, wild type. The PduB Δ6–12 mutant is a control strain known to have a very high growth rate due to an MCP assembly defect. Four pduB mutants with site-directed mutations (V7T, I19T, M11S, and V14T) were also tested. The table to the right shows the doubling time for each strain, and the error shown is one standard deviation determined from the results from three or more independent experiments. Growth curves were determined with a microplate reader, as described in Materials and Methods.
FIG 6
FIG 6
SDS-PAGE of MCPs purified from PduB mutants with site-directed mutations in the conserved N-terminal α-helix. WT, wild type. Letters on the right indicate the Pdu protein represented by each band. Lysozyme (lys) is not an MCP component but was used to lyse cells. The percent yield of purified MCPs indicates grams of protein/gram of cells relative to the wild type. The specific activities of the major lumen enzymes, the PduCDE diol dehydratase and the PduP aldehyde dehydrogenase, are given in micromoles per minute per milligram. The error shown is one standard deviation determined from the results from three or more independent experiments. Ten micrograms of protein was loaded in each lane. Gels were stained using a Coomassie-based protocol. Enzyme assays are described in Materials and Methods. On the right is a model that maps the residues of the PduB N terminus that were altered.
FIG 7
FIG 7
Transmission electron microscopy images of MCPs and MCP subcomplexes isolated from selected pduB deletion mutants. The wild-type strain is Salmonella enterica serovar Typhimurium LT2. MCP/MCP subcomplexes were negatively stained, as described previously (32, 43). Arrowheads indicate MCPs or putative empty MCP shells.

Similar articles

See all similar articles

Cited by 4 articles

Publication types

MeSH terms

Feedback