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Efficient models of polymerization applied to FtsZ ring assembly in Escherichia coli.

ABSTRACT: High protein concentrations complicate modeling of polymer assembly kinetics by introducing structural complexity and a large variety of protein forms. We present a modeling approach that achieves orders of magnitude speed-up by replacing distributions of lengths and widths with their average counterparts and by introducing a hierarchical classification of species and reactions into sets. We have used this model to study FtsZ ring assembly in Escherichia coli The model's prediction of key features of the ring formation, such as time to reach the steady state, total concentration of FtsZ species in the ring, total concentration of monomers, and average dimensions of filaments and bundles, are all in agreement with the experimentally observed values. Besides validating our model against the in vivo observations, this study fills some knowledge gaps by proposing a specific structure of the ring, describing the influence of the total concentration in short and long kinetics processes, determining some characteristic mechanisms in polymer assembly regulation, and providing insights about the role of ZapA proteins, critical components for both positioning and stability of the ring.

SUBMITTER: Ruiz-Martinez A

PROVIDER: S-EPMC5948971 | biostudies-literature | 2018 May

REPOSITORIES: biostudies-literature

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Efficient models of polymerization applied to FtsZ ring assembly in <i>Escherichia coli</i>.

Ruiz-Martínez Álvaro Á Bartol Thomas M TM Sejnowski Terrence J TJ Tartakovsky Daniel M DM

Proceedings of the National Academy of Sciences of the United States of America 20180423 19

High protein concentrations complicate modeling of polymer assembly kinetics by introducing structural complexity and a large variety of protein forms. We present a modeling approach that achieves orders of magnitude speed-up by replacing distributions of lengths and widths with their average counterparts and by introducing a hierarchical classification of species and reactions into sets. We have used this model to study FtsZ ring assembly in <i>Escherichia coli</i> The model's prediction of key ...[more]

PMID: 29686085

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Project description:BackgroundFtsZ is an essential cell division protein, which localizes at the middle of the bacterial cell to mediate cytokinesis. In vitro, FtsZ polymerizes and induces GTPase activity through longitudinal interactions to form the protofilaments, whilst lateral interactions result within formation of bundles. The interactions that participate in the protofilaments are similar to its eukaryotic homologue tubulin and are well characterized; however, lateral interactions between the inter protofilaments are less defined. FtsZ forms double protofilaments in vitro, though the key elements on the interface of the inter-protofilaments remain unclear as well as the structures involved in the lateral interactions in vivo and in vitro. In this study, we demonstrate that the highly conserved negative charge of glutamate 83 and the positive charge of arginine 85 located in the helix H3 bend of FtsZ are required for in vitro FtsZ lateral and longitudinal interactions, respectively and for in vivo cell division.ResultsThe effect of mutation on the widely conserved glutamate-83 and arginine-85 residues located in the helix H3 (present in most of the tubulin family) was evaluated by in vitro and in situ experiments. The morphology of the cells expressing Escherichia coli FtsZ (E83Q) mutant at 42°C formed filamented cells while those expressing FtsZ(R85Q) formed shorter filamented cells. In situ immunofluorescence experiments showed that the FtsZ(E83Q) mutant formed rings within the filamented cells whereas those formed by the FtsZ(R85Q) mutant were less defined. The expression of the mutant proteins diminished cell viability as follows: wild type > E83Q > R85Q. In vitro, both, R85Q and E83Q reduced the rate of FtsZ polymerization (WT > E83Q >> R85Q) and GTPase activity (WT > E83Q >> R85Q). R85Q protein polymerized into shorter filaments compared to WT and E83Q, with a GTPase lag period that was inversely proportional to the protein concentration. In the presence of ZipA, R85Q GTPase activity increased two fold, but no bundles were formed suggesting that lateral interactions were affected.ConclusionsWe found that glutamate 83 and arginine 85 located in the bend of helix H3 at the lateral face are required for the protofilament lateral interaction and also affects the inter-protofilament lateral interactions that ultimately play a role in the functional localization of the FtsZ ring at the cell division site.

Project description:BackgroundHeme proteins, such as hemoglobin, horseradish peroxidase and cytochrome P450 (CYP) enzyme, are highly versatile and have widespread applications in the fields of food, healthcare, medical and biological analysis. As a cofactor, heme availability plays a pivotal role in proper folding and function of heme proteins. However, the functional production of heme proteins is usually challenging mainly due to the insufficient supply of intracellular heme.ResultsHere, a versatile high-heme-producing Escherichia coli chassis was constructed for the efficient production of various high-value heme proteins. Initially, a heme-producing Komagataella phaffii strain was developed by reinforcing the C4 pathway-based heme synthetic route. Nevertheless, the analytical results revealed that most of the red compounds generated by the engineered K. phaffii strain were intermediates of heme synthesis which were unable to activate heme proteins. Subsequently, E. coli strain was selected as the host to develop heme-producing chassis. To fine-tune the C5 pathway-based heme synthetic route in E. coli, fifty-two recombinant strains harboring different combinations of heme synthesis genes were constructed. A high-heme-producing mutant Ec-M13 was obtained with negligible accumulation of intermediates. Then, the functional expression of three types of heme proteins including one dye-decolorizing peroxidase (Dyp), six oxygen-transport proteins (hemoglobin, myoglobin and leghemoglobin) and three CYP153A subfamily CYP enzymes was evaluated in Ec-M13. As expected, the assembly efficiencies of heme-bound Dyp and oxygen-transport proteins expressed in Ec-M13 were increased by 42.3-107.0% compared to those expressed in wild-type strain. The activities of Dyp and CYP enzymes were also significantly improved when expressed in Ec-M13. Finally, the whole-cell biocatalysts harboring three CYP enzymes were employed for nonanedioic acid production. High supply of intracellular heme could enhance the nonanedioic acid production by 1.8- to 6.5-fold.ConclusionHigh intracellular heme production was achieved in engineered E. coli without significant accumulation of heme synthesis intermediates. Functional expression of Dyp, hemoglobin, myoglobin, leghemoglobin and CYP enzymes was confirmed. Enhanced assembly efficiencies and activities of these heme proteins were observed. This work provides valuable guidance for constructing high-heme-producing cell factories. The developed mutant Ec-M13 could be employed as a versatile platform for the functional production of difficult-to-express heme proteins.

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Efficient models of polymerization applied to FtsZ ring assembly in Escherichia coli.

Publications

Efficient models of polymerization applied to FtsZ ring assembly in <i>Escherichia coli</i>.

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