Abstract
Various bacteria localize metabolic pathways to proteinaceous organelles known as bacterial microcompartments (MCPs), enabling the metabolism of carbon sources to enhance survival and pathogenicity in the gut. There is considerable interest in exploiting bacterial MCPs for metabolic engineering applications, but little is known about the interactions between MCP signal sequences and the protein shells of different MCP systems. We found that the N-terminal sequences from the ethanolamine utilization (Eut) and glycyl radical-generating protein MCPs are able to target reporter proteins to the 1,2-propanediol utilization (Pdu) MCP, and that this localization is mediated by a conserved hydrophobic residue motif. Recapitulation of this motif by the addition of a single amino acid conferred targeting function on an N-terminal sequence from the ethanol utilization MCP system that previously did not act as a Pdu signal sequence. Moreover, the Pdu-localized signal sequences competed with native Pdu targeting sequences for encapsulation in the Pdu MCP. Salmonella enterica natively possesses both the Pdu and Eut operons, and our results suggest that Eut proteins might be localized to the Pdu MCP in vivo. We further demonstrate that S. enterica LT2 retained the ability to grow on 1,2-propanediol as the sole carbon source when a Pdu enzyme was replaced with its Eut homolog. Although the relevance of this finding to the native system remains to be explored, we show that the Pdu-localized signal sequences described herein allow control over the ratio of heterologous proteins encapsulated within Pdu MCPs.
Highlights
N-terminal signal sequences localize enzymes to bacterial microcompartments
We found that the N-terminal sequences from the ethanolamine utilization (Eut) and glycyl radical-generating protein MCPs are able to target reporter proteins to the 1,2-propanediol utilization (Pdu) MCP, and that this localization is mediated by a conserved hydrophobic residue motif
When expression of the encapsulation reporter construct pBADeutC1–20-gfp-ssrA was induced by the addition of 0.02% arabinose, fluorescent puncta were not observed in a ⌬eutR::FRT strain upon the addition of 30 mM ethanolamine and 150 nM vitamin B12, but puncta were observed in the ⌬pocR::FRT strain under the same conditions (Figs. 2 and 3)
Summary
N-terminal signal sequences localize enzymes to bacterial microcompartments. Results: Signal sequences from various microcompartments localize proteins to the 1,2-propanediol utilization (Pdu) microcompartment. Interactions between the N termini of encapsulated enzymes and the structural proteins of the associated MCP shell are a general mode of enzyme localization to diverse MCP systems, including the Pdu MCP [6, 7], the ethanolamine utilization (Eut) MCP [8], and the smaller MCP of Haliangium ochraceum [9] One of these interactions, between the N-terminal signal sequence of the Pdu enzyme PduP and the C terminus of the Pdu shell protein PduA, is modeled to be mediated by interactions between residues Glu, Ile, and Leu presented on the ␣-helical N terminus of PduP and His, Val, and Leu on the C terminus of PduA [6]. These results are useful as a guide to engineering the Pdu MCP to house biosynthetic pathways and raise questions as to whether the native Pdu and Eut systems maintain unique cargo protein content when natively co-expressed
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