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CellSynthetic BiologyPeer-reviewed journal article

Genetic requirements for cell division in a genomically minimal cell

James F. PelletieriD, Lijie Sun, Kim S. Wise, Nacyra Assad-GarciaiD, Bogumil Jacek KarasiD, Thomas J. DeerinckiD, Mark H. EllismaniD, Andreas MershiniD, Neil GershenfeldiD, Ray-Yuan Chuang, John I. GlassiD, Elizabeth A. StrychalskiiD

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Abstract

Genomically minimal cells, such as JCVI-syn3.0, offer a platform to clarify genes underlying core physiological processes. While this minimal cell includes genes essential for population growth, the physiology of its single cells remained uncharacterized. To investigate striking morphological variation in JCVI-syn3.0 cells, we present an approach to characterize cell propagation and determine genes affecting cell morphology. Microfluidic chemostats allowed observation of intrinsic cell dynamics resulting in irregular morphologies. The addition of 19 genes not retained in JCVI-syn3.0 generated JCVI-syn3A, which presents significantly less morphological variation than JCVI-syn3.0. We further identified seven of these 19 genes, including two known cell division genes ftsZ and sepF and five genes of unknown function, required together to restore cell morphology and division similar to JCVI-syn1.0. This surprising result emphasizes the polygenic nature of cell morphology, as well as the importance of a Z-ring and membrane properties in the physiology of genomically minimal cells.

In-depth summary

Claude Sonnet 4.6 · max effort · 8 Nov 2026 · from the full text of version 1 (7 Oct 2020) · CC0 1.0

Seven genes, including ftsZ, sepF and five of unknown function, are jointly required to restore near-normal morphology and division in the minimal cell JCVI-syn3.0.

JCVI-syn3.0 has only 473 genes, 149 of them of unknown or generic function, and the physiology of its single cells was uncharacterized despite striking shape irregularity. This work uses a function-agnostic screen, from genome segment to gene cluster to single gene, to link specific genes to morphology and division.

Key results

  1. ShownSingle JCVI-syn3.0 cells and cellular forms are highly pleomorphic, unlike JCVI-syn1.0, and microfluidic chemostat imaging shows these shapes arise from dynamics intrinsic to cells rather than from shear flow.

    JCVI-syn1.0 formed round cells of ≈400 nm; JCVI-syn3.0 included large cellular forms with diameters ≥10 µm, and most JCVI-syn3.0 cells were large forms. Filaments showed branching or pearling, but no scission was observed. · Introduction; Dynamic propagation of JCVI-syn3.0; Morphological dynamics intrinsic to cells…

  2. ShownA segment-level screen localized the pleomorphic phenotype to minimized genomic segment 6. Replacing it with a version carrying 19 extra genes (JCVI-syn3A) greatly reduced morphological variation.

    RGD6 (minimized segment 6 in an otherwise JCVI-syn1.0 genome) mimicked JCVI-syn3.0 pleomorphism; the other minimized segments gave normal or nearly normal morphology. 76 genes were removed from segment 6; JCVI-syn3A doubling time was < 2 h. · Morphological diversity from a single minimized…; Genomic restoration of normal morphology in a…; Figure legends

  3. ShownAdding the ftsZ-containing cluster alone does not restore normal morphology. Seven genes (520, 521, 522, 527, 602, 604, 605) in clusters 1, 2 and 6 are all necessary and together sufficient in the JCVI-syn3.0 background.

    The 19 genes comprise a redundant rRNA operon (3 genes) plus 16 protein genes in eight clusters. JCVI-syn3.0+12345678 and +1267 were normal; cluster 7 was dispensable; deleting any of the seven genes from JCVI-syn3.0+126 reverted pleomorphism. · Genomic restoration of normal morphology in a…; Figure legends; Tables

  4. ShownThe genetic requirement is context dependent: JCVI-syn3A keeps nearly normal morphology after deleting cluster 1 (520-522), although that cluster is required in JCVI-syn3.0+126.

    Deletion of 520-522 from JCVI-syn3A retained nearly normal morphology (Fig S4C), whereas deleting any of the seven genes from JCVI-syn3.0+126 caused loss of normal morphology. · Genomic restoration of normal morphology in a…; Figure legends; Tables

  5. SuggestedFluorescence imaging of RGD6 suggests filaments carry cytoplasm and multiple nucleoids without scission, while vesicles are membrane-bound compartments lacking cytoplasmic marker. Bioinformatics link several unknown-function genes to membranes.

    Vesicles lacked mCherry from first appearance, stained with SP-DiOC18(3) and excluded fluorescent dextran. Genes 604 and 605 encode one transmembrane helix and a lipoprotein anchoring motif; FtsZ ≈640 vs FtsA ≈40 copies per cell in JCVI-syn3A. · Morphological diversity from a single minimized…; Systematic approach attributes morphology to…; JCVI-syn3A highlights aspects of Z-ring function; Figure legends

Methods, data and reproducibility

Reverse genetics on synthetic Mycoplasma genomes: gene additions and deletions at the segment, cluster and single-gene levels, with morphology scored by microscopy and cell-area distributions, plus time-lapse imaging in microfluidic chemostats.

  • Near-wild-type synthetic strain JCVI-syn1.0 (normal morphology reference) · Mycoplasma mycoides subspecies capri (synthetic genome) · CP002027
  • Minimal strain JCVI-syn3.0 (473 genes), pleomorphic · Mycoplasma mycoides-derived synthetic minimal cell · n = 473 genes · CP014940.1
  • JCVI-syn3A, JCVI-syn3.0 with 19 additional genes in segment 6 · Mycoplasma mycoides-derived synthetic minimal cell · n = 19 added genes · CP016816.2
  • RGD6, minimized segment 6 in an otherwise JCVI-syn1.0 genome; the other minimized segments were also tested individually in a JCVI-syn1.0 background · Mycoplasma mycoides-derived synthetic strain
  • Derivative strains with gene clusters 1 to 8 added to JCVI-syn3.0, and gene or cluster deletions from JCVI-syn3.0+126 and JCVI-syn3A (listed in Table S4) · Mycoplasma mycoides-derived synthetic strains · n = eight protein-coding clusters tested
Methods & tools
Microfluidic chemostat time-lapse imaging, Empirical gradient threshold (EGT) algorithm for cell-area distributions, DIC optical microscopy, Scanning electron microscopy, mCherry cytoplasmic marker, Hoechst, SP-DiOC18(3) and FITC-dextran fluorescent labels, CRISPR/Cas9 genome editing in yeast, Cre-lox cassette exchange, Genome transplantation, Zeiss Zen acquisition software
Reagents & environment
Synthetic strains JCVI-syn1.0, JCVI-syn3.0 (subclone 13-2) and JCVI-syn3A; yeast host VL6_48N_cas9; SP4 growth medium; Cre-lox plasmid GenBank MN982903.1; selection plasmid MN982904.
Experimental conditions
Zeiss Axio Imager 1 (63x, 1.4 objective); inverted microscope with 100x 1.46 NA objective and Hamamatsu Orca Flash 4.0 v2 sCMOS at 37.0 °C; time-lapse over ≈(10 to 16) h; Zeiss Merlin Fe-SEM.
Reproducibility gaps
No analysis code URL or public image repository is given; primer and strain tables are in supplementary files; strain or plasmid distribution terms are not stated.
Limitations (4)
  • The authors treat the extrusion-resolution model as suggested by their observations, and describe curvature-based FtsZ localization as speculation, so these mechanisms are not tested conclusions. · stated by the authors
  • The molecular functions of the five unknown-function genes remain unclear, and their association with the membrane is suggested only by bioinformatic analyses of sequence motifs and predicted helices. · stated by the authors
  • Morphology calls are largely categorical with area histograms; no statistical test or effect size supports the claim of significantly less variation, and the number of cells scored per strain is not stated. · noted in this summary
  • Cluster 1 is required in JCVI-syn3.0+126 but dispensable in JCVI-syn3A, so the seven-gene requirement is background dependent and may not generalize across genomic contexts. · noted in this summary

Read this if you study minimal cells, bacterial cell division, or genome-scale reverse genetics for assigning phenotypes to genes of unknown function.

Citations
142
FWCI
12.29
Field percentile
top 1%

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