A system to investigate Pdu BMC biogenesis. (a) The chromosomal pdu operon of S. Typhimurium LT2 encodes structural genes (pduABB'JKMNTU), catalytic genes (pduCDELPQW for 1,2-propanediol degradation, and the pduGHOSX B12 recycling genes), and pduV (proposed to connect with filament-associated BMC movement 39 ). PduM is listed as a structural protein according to previous studies 28 . (b) The Pdu BMC shell encapsulates key enzymes that are required for 1,2-propanediol metabolism. (c) All Pdu proteins are visible by dual-labeling with fluorescence proteins (mCherry and sfGFP). Two series of vectors were constructed. First, PduA (a major shell protein) was labeled with sfGFP, and other structural components or PduE (a major enzymatic component) were labeled with mCherry. Second, PduE was labeled with sfGFP, and the structural components or other catalytic components were labeled with mCherry. (d) The presence of 1,2-propanediol (1,2-PD) is required to visualize Pdu BMCs. The fluorescence images show WT S. Typhimurium LT2 carrying pBAD-PduE-mCherry/PduA-sfGFP grown in microcompartment-inducing media (MIM), in the absence (-1,2-PD) or presence (+1,2-PD) of 1,2-propanediol.

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... genes encoding the proteins of the Pdu BMCs that mediate 1,2-PD degradation are located in the pdu operon (pduA-X) in the S. Typhimurium chromosome ( Fig. 1a and 1b). Transcription of the pdu operon occurs in the presence of 1,2-PD during anaerobic growth, and is activated by Crp and Arc 54,55 . To investigate the biogenesis pathway of Pdu BMCs in S. Typhimurium LT2, we designed a system based on the pBAD/MycHis vector with the arabinose-inducible ParaBAD promoter 56,57 and labeled individual Pdu ...

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... in the presence of 1,2-PD during anaerobic growth, and is activated by Crp and Arc 54,55 . To investigate the biogenesis pathway of Pdu BMCs in S. Typhimurium LT2, we designed a system based on the pBAD/MycHis vector with the arabinose-inducible ParaBAD promoter 56,57 and labeled individual Pdu proteins with mCherry and super-folder GFP (sfGFP) (Fig. 1c). We used the sfGFP tag to visualize the assembly of the PduA shell protein and the PduE cargo protein. In the PduA-sfGFP background, we fused mCherry to other structural components (PduB/B'/J/K/M/N/T/U) and PduE individually. In the PduE-sfGFP background, we fused mCherry to the structural proteins (PduB/B'/J/K/M/N/T/U) and other cargo ...

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... investigate the effect of increasing levels of protein expression, we detected the fluorescence of PduAsfGFP at various L-arabinose concentrations in microcompartment-inducing media (MIM) (Fig. S1). In the absence or a low concentration (0.00002%, g•mL -1 ) of L-arabinose, PduA-sfGFP appeared as discrete patches in cells, the typical subcellular distribution of Pdu BMCs as reported previously 24,58 . This expression of PduAsfGFP probably resulted from the low-level transcription of the ParaBAD promoter in the absence of ...

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... of S. Typhimurium LT2 in the absence of 1,2-PD does not stimulate Pdu BMC formation 15,24 . In our experimental model, growth in minimal media that lacked 1,2-PD (MIM-1,2-PD) allowed the visualization of expressed Pdu proteins diffused throughout the cytosol, confirming no formation of Pdu BMCs (Fig. 1d, Fig. S2). Growth in the presence of 1,2-PD induced the expression of endogenous Pdu proteins and the formation of Pdu BMCs with a typical clustered distribution, as indicated by in vivo colocalization of PduE-mCherry and PduA-sfGFP fluorescence (Fig. 1d, Fig. S3). This system and the generated S. Typhimurium LT2 mutant strains provided an ...

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... expressed Pdu proteins diffused throughout the cytosol, confirming no formation of Pdu BMCs (Fig. 1d, Fig. S2). Growth in the presence of 1,2-PD induced the expression of endogenous Pdu proteins and the formation of Pdu BMCs with a typical clustered distribution, as indicated by in vivo colocalization of PduE-mCherry and PduA-sfGFP fluorescence (Fig. 1d, Fig. S3). This system and the generated S. Typhimurium LT2 mutant strains provided an ideal platform to explore the assembly and roles of individual Pdu proteins during Pdu BMC ...

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... core. Moreover, PduB 1-37 serves as a linker peptide that acts as a bridge between the enzymatic core and shell assemblies, playing an essential role in the biogenesis of Pdu BMCs. Comparative genomic analysis revealed that PduB 1- 37 is conserved throughout the Klebsiella, Escherichia, Citrobacter, and Salmonella genera that carry the pdu operon (Fig. S10). In these four genera, the PduB 1-37 linker protein was conserved as highly as the other structural or essential Pdu proteins (PduA/B/D/J/N/U). In contrast, the PduX protein, which has an unknown function, was not highly conserved 38 . Because the predicted structures of PduB 1-37 from different genera were very similar (Fig. S10), ...

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... the pdu operon (Fig. S10). In these four genera, the PduB 1-37 linker protein was conserved as highly as the other structural or essential Pdu proteins (PduA/B/D/J/N/U). In contrast, the PduX protein, which has an unknown function, was not highly conserved 38 . Because the predicted structures of PduB 1-37 from different genera were very similar (Fig. S10), our findings suggest that PduB 1-37 may play a ubiquitous role in the assembly of the Pdu BMC in different bacterial ...

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... PduM and shell assemblies. To investigate the role of PduM in more detail, we studied the assembly of structural and catalytic Pdu proteins in the ΔpduM background ( Fig. 4a and 4b). We found that the catalytic enzymes PduG, PduO, PduP, and PduQ colocalized with PduE, whereas the shell proteins PduB, PduB', PduJ and PduK assembled with PduA ( Fig. 4b and 4c, Fig. S11), recapitulating the results observed in the ΔpduB 1-37 strain (Fig. 3). Moreover, confocal images of the ΔpduM strain expressing PduEmCherry and PduA-sfGFP revealed a relatively low level of physical correlation between shell assemblies and cargos ( Fig. 2b and ...

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... copyright holder for this this version posted December 9, 2021. ; https://doi.org/10. 1101 amino acid level) in all but one strain (Fig. S10). The high similarity of the predicted PduM structures from these genera (Fig. S10) suggests that PduM may play a critical role in Pdu BMC assembly. ...

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... copyright holder for this this version posted December 9, 2021. ; https://doi.org/10. 1101 amino acid level) in all but one strain (Fig. S10). The high similarity of the predicted PduM structures from these genera (Fig. S10) suggests that PduM may play a critical role in Pdu BMC assembly. ...

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... is another minor shell protein within the Pdu BMC 24 . The N-terminal region of PduK has a high sequence similarity to the hexameric shell protein PduA, and the C-terminal extension of PduK has an unknown function (Fig. S10). In the absence of PduK, Pdu BMCs were restricted to the cell poles (Fig. 2b) 27 , and SDS-PAGE and EM results revealed that the Pdu BMC structures are well-formed in the ΔpduK cells (Fig. S12), suggesting that PduK is essential for the spatial localization of Pdu ...

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... N-terminal region of PduK has a high sequence similarity to the hexameric shell protein PduA, and the C-terminal extension of PduK has an unknown function (Fig. S10). In the absence of PduK, Pdu BMCs were restricted to the cell poles (Fig. 2b) 27 , and SDS-PAGE and EM results revealed that the Pdu BMC structures are well-formed in the ΔpduK cells (Fig. S12), suggesting that PduK is essential for the spatial localization of Pdu ...

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... explore the role of PduK in the biogenesis of Pdu BMCs, we determined the distribution of individual Pdu proteins in the ΔpduB 1-37 /ΔpduK mutant (Fig. 4e). The major shell proteins PduB' and PduJ colocalized with PduA, forming assemblies at one cell pole (Fig. 4f and 4g, Fig. S11). Apart from those colocalized with PduA, some PduB' proteins were diffusely distributed throughout the cytosol of the ΔpduB 1-37 /ΔpduK cells, which was also seen in the WT background (Fig. S3). These findings are consistent with the comparison of Pdu protein abundance in cell extracts and isolated Pdu BMCs that indicated that a ...

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... of Pdu BMC shell proteins and cargos occur independently in vivo, and revealed that PduK is important for the assembly and subcellular distribution of both shell assemblies and entire Pdu BMCs. PduK orthologs were highly conserved and are predicted to have significant structural similarity between the bacterial genera that carry the pdu operon (Fig. S10), suggesting a universal role of PduK in Pdu BMC ...

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... tackle this question, we determined the in vivo diffusion dynamics of internal enzymes and shell proteins of Pdu BMCs, using fluorescence recovery after photobleaching (FRAP). We chose the elongated Pdu BMC structures in the ΔpduN strain (Fig. S13), which are suitable for photobleaching and quantitative analysis 64 . Growth assays suggest that the resulting Pdu BMCs in ΔpduN are metabolically functional (Fig. 2d). Fig. 6f and 6g show the representative FRAP image sequences of PduE-sfGFP (cargo) and PduA-sfGFP (shell) in the ΔpduN cells. The cargo enzymes exhibited a distinctly ...

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... comparison, FRAP of the PduGH-sfGFP proteins that are dispersedly distributed in the cytoplasm (Fig. 3d) in the absence of Pdu BMC formation revealed that the PduGH-sfGFP fluorescence recovered rapidly after only ~3 seconds (Fig. S14, Table S4), indicating that cargo enzymes exhibited highly dynamic mobility in the cytosolic solution than inside the Pdu BMC. While protein interactions and the internal arrangement of bacterial metabolosomes may differ between the canonical Pdu BMCs in the WT and the elongated structures of the Pdu BMCs in the ΔpduN strain, our ...

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... essential linker proteins to bind the shell and Rubisco matrix during β-carboxysome assembly 44 . The fact that orthologs of PduB 1-37 , PduK and PduM were identified in Klebsiella, Escherichia, Citrobacter, and Salmonella suggests that the general assembly principle of Pdu metabolosomes that we have identified may be pervasive among these genera (Fig. S10), and might extend to other metabolosomes that have multiple interior enzymes possessing structurally-similar encapsulation ...