Genetic requirements for cell division in a genomically minimal cell

Pelletier, James F.; Sun, Lijie; Wise, Kim S.; Assad-García, Nacyra; Karas, Bogumil J.; Deerinck, Thomas J.; Ellisman, Mark H.; Mershin, Andreas; Chuang, Ronald Y.; Glass, John I.; Strychalski, Elizabeth A.

doi:10.1101/2020.10.07.326892

Cited by 10 publications

(16 citation statements)

References 59 publications

(26 reference statements)

Supporting

Mentioning

Contrasting

Order By: Relevance

“…Finally, the robust initiation of the Δ4 cells and its mechanistic implications was a major surprise for us. At the same time, we also find them gratifying from an evolutionary perspective [33,34]. For example, the First Cell would only need one master regulator protein with intrinsic ATPase activity for robust initiation, and different organisms may have acquired organism-specific additional control mechanisms to improve the precision in initiation during evolution (Figure 2).…”

Section: Discussionmentioning

confidence: 99%

Robust control of replication initiation in the absence of DnaA-ATP ↔ DnaA-ADP regulatory elements inEscherichia coli

Boesen

Charbon

et al. 2022

Preprint

View full text Add to dashboard Cite

Replication initiation, especially the widely conserved master initiator protein DnaA, is one of the most well-studied biological problems in bacteria. Specifically, conversion between the ATP and ADP form of DnaA during growth is critical for initiation control. In Escherichia coli, the regulatory elements for conversion have been discovered and extensively characterized over decades. However, they are not widely conserved in bacteria, raising questions on how to generalize the findings in E. coli to other organisms. In this work, we show that the intrinsic ATPase activity of DnaA itself is sufficient for robust and precise initiation control. We constructed and studied E. coli mutants lacking the extrinsic control of either DnaA-ATP → DnaA-ADP (by hda and datA) or DnaA-ADP → DnaA-ATP (by DARS1 and DARS2) at the single-cell and population levels. These cells showed distinct and opposing characteristics in initiation timing, the degree of initiation asynchrony, and cell-to-cell variability. Strikingly, when all four regulatory elements were deleted, E. coli exhibited a near wild-type phenotype, with only mildly increased intrinsic and extrinsic initiation noise. By further characterizing the DnaA variants with increased and decreased ATPase activity, we conclude that DnaA is the only requirement for robust initiation, shedding new evolutionary light on cell-cycle control in bacteria.

show abstract

Section: Discussionmentioning

confidence: 99%

Robust control of replication initiation in the absence of DnaA-ATP ↔ DnaA-ADP regulatory elements inEscherichia coli

Boesen

Charbon

et al. 2022

Preprint

View full text Add to dashboard Cite

show abstract

“…Considering that the genome of JCVI-syn3.0 is chemically synthesised, its genome can be altered in any way that can yield a viable cell. JCVI-syn3A (CPO16816) and JCVI-syn3B (Pelletier et al, 2020) are the variants of the minimal cell JCVI-syn3.0 that contain an additional 19 genes that make the cell morphology more stable and working with bacteria easier. JCVI-syn3B contains an additional dual loxP landing pad that enables direct insertion of a new DNA between the loxP sites in a Cre recombinase-mediated reaction.…”

Section: Discussionmentioning

confidence: 99%

“…We utilised near minimal bacterium JCVI-syn3A (GenBank CP016816.2) harbouring the minimal synthetic Mycoplasma mycoides genome and JCVI-syn3B harbouring dual loxP landing pad in the JCVI-syn3A genome (Hossain, Deter, Peters, & Butzin, 2021;Pelletier et al, 2020) as a mycoplasma vehicle to express U. parvum surface membrane lipoprotein multiple banded antigen (MBA) and UpVF.…”

Section: Syn-mba-upvf Induced Cell Membrane Permeabilitymentioning

confidence: 99%

Blockade of endoplasmic reticulum stress‐induced cell death by Ureaplasma parvum vacuolating factor

Nishiumi

Kawai

Nakura

et al. 2021

Cellular Microbiology

Self Cite

View full text Add to dashboard Cite

Previously, we found that Ureaplasma parvum internalised into HeLa cells and cytosolic accumulation of galectin-3. U. parvum induced the host cellular membrane damage and survived there. Here, we conducted vesicular trafficking inhibitory screening in yeast to identify U. parvum vacuolating factor (UpVF). U. parvum triggered endoplasmic reticulum (ER) stress and upregulated the unfolded protein response-related factors, including BiP, P-eIF2 and IRE1 in the host cells, but it blocked the induction of the downstream apoptotic factors. MicroRNA library screening of U. parvuminfected cells and UpVF-transfected cells identified miR-211 and miR-214 as the negative regulators of the apoptotic cascade under ER stress. Transient expression of UpVF induced HeLa cell death with intracellular vacuolization; however, some stable UpVF transformant survived. U. parvum-infected cervical cell lines showed resistance to actinomycin D, and UpVF stable transformant cell lines exhibited resistance to Xray irradiation, as well as cisplatin and paclitaxel. UpVF expressing cervical cancer xenografts in nude mice also acquired resistance to cisplatin and paclitaxel. A mycoplasma expression vector based on Mycoplasma mycoides, Syn-MBA (multiple banded antigen)-UpVF, reduced HeLa cell survival compared with that of Syn-MBA after 72 hr of infection. These findings together suggest novel mechanisms for Ureaplasma infection and the possible implications for cervical cancer malignancy. Take Aways• Ureaplasmal novel virulence factor, UpVF, was identified.• UpVF triggered ER stress but suppressed apoptotic cascade via miR-211 and -214.• UpVF conferred resistance to anticancer treatments both in vivo and in vitro.• Dual expression of MBA and UpVF in JCVI-syn3B showed host cell damage.

show abstract

“…With the exception of JCVI-syn3B (GenBank accession number acquisition in progress), all the synthetic mycoplasma strains used were previously described (Gibson et al, 2010;Hutchison et al, 2016;Pelletier et al, 2021). Similar to JCVI-syn3A, JVCI-syn3B possesses a second rRNA operon copy, lacks a gene (MMSYN1_0531) coding for an efflux protein, and has 16 protein coding genes that were added back into the JCVI-syn3.0 genome to render the cell with a more wild type Mmc morphology and growth rate (Breuer et al, 2019;Pelletier et al, 2021). The strain also contains a landing pad system (dual LoxP sites) that facilitates genetic manipulation.…”

Section: Mycoplasma Strains and Mammalian Cells Culturesmentioning

confidence: 99%

“…The JCVI-syn3.0 has the smallest genome of any known organism that can be grown in axenic culture. We have also constructed variants of minimal cell JCVI-syn3.0 that have a small number of added non-essential genes that result in a cell with properties more like wild type Mmc (Breuer et al, 2019;Hossain et al, 2021;Pelletier et al, 2021).…”

Section: Introductionmentioning

confidence: 99%

Minimal cell JCVI-syn3B as a chassis to reveal the mechanisms behind Mycoplasma host–pathogen interactions

Bittencourt¹,

Brown

Assad-García

et al. 2022

Preprint

Self Cite

View full text Add to dashboard Cite

Mycoplasmas are atypical bacteria that are obligate parasites of animals and plants. They are known for their capacity to contaminate and parasitize mammalian cell cultures, thereby offering fortuitous models to examine the host-microbe interaction. We used a near wildtype strain of the goat pathogen Mycoplasma mycoides (Mmc) and several minimized Mmc strains lacking genes not essential for growth in laboratory media to investigate host-mycoplasma interactions. Stains with near minimal genomes were incapable of surviving in co-culture with mammalian HEK-293T and HeLa cells. We identified a cluster of eight non-essential genes that when added back to the minimized strains enabled mycoplasma attachment to mammalian cells. Those genes did not restore the capacity of the minimal cell to grow when co-cultured with mammalian cells indicating processes of attaching to mammalian cells and parasitism involve different genes. Furthermore, minimized Mmc strains were not phagocytized by human myeloid cells unlike the near wildtype Mmc.

show abstract

Genetic requirements for cell division in a genomically minimal cell

Cited by 10 publications

References 59 publications

Robust control of replication initiation in the absence of DnaA-ATP ↔ DnaA-ADP regulatory elements inEscherichia coli

Robust control of replication initiation in the absence of DnaA-ATP ↔ DnaA-ADP regulatory elements inEscherichia coli

Blockade of endoplasmic reticulum stress‐induced cell death by Ureaplasma parvum vacuolating factor

Minimal cell JCVI-syn3B as a chassis to reveal the mechanisms behind Mycoplasma host–pathogen interactions

Contact Info

Product

Resources

About