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Effect of metabolosome encapsulation peptides on enzyme activity, coaggregation, incorporation, and bacterial microcompartment formation.

ABSTRACT: Metabolosomes, catabolic bacterial microcompartments (BMCs), are proteinaceous organelles that are associated with the breakdown of metabolites such as propanediol and ethanolamine. They are composed of an outer multicomponent protein shell that encases a specific metabolic pathway. Protein cargo found within BMCs is directed by the presence of an encapsulation peptide that appears to trigger aggregation before the formation of the outer shell. We investigated the effect of three distinct encapsulation peptides on foreign cargo in a recombinant BMC system. Our data demonstrate that these peptides cause variations in enzyme activity and protein aggregation. We observed that the level of protein aggregation generally correlates with the size of metabolosomes, while in the absence of cargo BMCs self-assemble into smaller compartments. The results agree with a flexible model for BMC formation based around the ability of the BMC shell to associate with an aggregate formed due to the interaction of encapsulation peptides.

SUBMITTER: Juodeikis R

PROVIDER: S-EPMC7221423 | biostudies-literature | 2020 May

REPOSITORIES: biostudies-literature

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Effect of metabolosome encapsulation peptides on enzyme activity, coaggregation, incorporation, and bacterial microcompartment formation.

Juodeikis Rokas R Lee Matthew J MJ Mayer Matthias M Mantell Judith J Brown Ian R IR Verkade Paul P Woolfson Derek N DN Prentice Michael B MB Frank Stefanie S Warren Martin J MJ

MicrobiologyOpen 20200213 5

Metabolosomes, catabolic bacterial microcompartments (BMCs), are proteinaceous organelles that are associated with the breakdown of metabolites such as propanediol and ethanolamine. They are composed of an outer multicomponent protein shell that encases a specific metabolic pathway. Protein cargo found within BMCs is directed by the presence of an encapsulation peptide that appears to trigger aggregation before the formation of the outer shell. We investigated the effect of three distinct encaps ...[more]

PMID: 32053746

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Project description:Bacterial microcompartments (BMCs) are large (∼100-nm) protein shells that encapsulate enzymes, their substrates, and cofactors for the purposes of increasing metabolic reaction efficiency and protecting cells from toxic intermediates. The best-studied microcompartment is the carbon-fixing carboxysome that encapsulates ribulose-1,5-bisphosphate carboxylase and carbonic anhydrase. Other well-known BMCs include the Pdu and Eut BMCs, which metabolize 1,2-propanediol and ethanolamine, respectively, with vitamin B12-dependent diol dehydratase enzymes. Recent bioinformatic analyses identified a new prevalent type of BMC, hypothesized to utilize vitamin B12-independent glycyl radical enzymes to metabolize substrates. Here we use genetic and metabolic analyses to undertake in vivo characterization of the newly identified glycyl radical enzyme microcompartment 3 (GRM3) class of microcompartment clusters. Transcriptome sequencing analyses showed that the microcompartment gene cluster in the genome of the purple photosynthetic bacterium Rhodobacter capsulatus was expressed under dark anaerobic respiratory conditions in the presence of 1,2-propanediol. High-performance liquid chromatography and gas chromatography-mass spectrometry analyses showed that enzymes coded by this cluster metabolized 1,2-propanediol into propionaldehyde, propanol, and propionate. Surprisingly, the microcompartment pathway did not protect these cells from toxic propionaldehyde under the conditions used in this study, with buildup of this intermediate contributing to arrest of cell growth. We further show that expression of microcompartment genes is regulated by a two-component system located downstream of the microcompartment cluster.IMPORTANCE BMCs are protein shells that are designed to compartmentalize enzymatic reactions that require either sequestration of a substrate or the sequestration of toxic intermediates. Due to their ability to compartmentalize reactions, BMCs have also become attractive targets for bioengineering novel enzymatic reactions. Despite these useful features, little is known about the biochemistry of newly identified classes of BMCs. In this study, we have undertaken genetic and in vivo metabolic analyses of the newly identified GRM3 gene cluster.

Project description:Ethanolamine (EA) is a compound prevalent in the gastrointestinal (GI) tract that can be used as a carbon, nitrogen, and/or energy source. Enterococcus faecalis, a GI commensal and opportunistic pathogen, contains approximately 20 ethanolamine utilization (eut) genes encoding the necessary regulatory, enzymatic, and structural proteins for this process. Here, using a chemically defined medium, two regulatory factors that affect EA utilization were examined. First, the functional consequences of loss of the small RNA (sRNA) EutX on the efficacy of EA utilization were investigated. One effect observed, as loss of this negative regulator causes an increase in eut gene expression, was a concomitant increase in the number of catabolic bacterial microcompartments (BMCs) formed. However, despite this increase, the growth of the strain was repressed, suggesting that the overall efficacy of EA utilization was negatively affected. Second, utilizing a deletion mutant and a complement, carbon catabolite control protein A (CcpA) was shown to be responsible for the repression of EA utilization in the presence of glucose. A predicted cre site in one of the three EA-inducible promoters, PeutS, was identified as the target of CcpA. However, CcpA was shown to affect the activation of all the promoters indirectly through the two-component system EutV and EutW, whose genes are under the control of the PeutS promoter. Moreover, a bioinformatics analysis of bacteria predicted to contain CcpA and cre sites revealed that a preponderance of BMC-containing operons are likely regulated by carbon catabolite repression (CCR).IMPORTANCE Ethanolamine (EA) is a compound commonly found in the gastrointestinal (GI) tract that can affect the behavior of human pathogens that can sense and utilize it, such as Enterococcus faecalis and Salmonella Therefore, it is important to understand how the genes that govern EA utilization are regulated. In this work, we investigated two regulatory factors that control this process. One factor, a small RNA (sRNA), is shown to be important for generating the right levels of gene expression for maximum efficiency. The second factor, a transcriptional repressor, is important for preventing expression when other preferred sources of energy are available. Furthermore, a global bioinformatics analysis revealed that this second mechanism of transcriptional regulation likely operates on similar genes in related bacteria.

Dataset Information

Effect of metabolosome encapsulation peptides on enzyme activity, coaggregation, incorporation, and bacterial microcompartment formation.

Publications

Effect of metabolosome encapsulation peptides on enzyme activity, coaggregation, incorporation, and bacterial microcompartment formation.

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