The structure of the periplasmic FlaG–FlaF complex and its essential role for archaellar swimming motility

Tsai, Chi Lin; Tripp, Patrick; Sivabalasarma, Shamphavi; Zhang, Changyi; Rodriguez‐Franco, Marta; Wipfler, Rebecca L.; Chaudhury, Paushali; Banerjee, Ankan; Beeby, Morgan; Whitaker, Rachel J.; Tainer, John A.; Albers, Sonja

doi:10.1038/s41564-019-0622-3

Cited by 37 publications

(54 citation statements)

References 56 publications

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“…Also, as archaea do not have a cell wall, the S-layer may take over the function as a stator for the archaellar motor. The latter is supported by the fact that ArlF and ArlG (formerly referred to as FlaF and FlaG, respectively) were shown to interact with the S-layer glycoprotein (20,21). We thus subjected the ΔhvpssA and ΔhvpssD mutant strains to analysis for these physiological responses.…”

Section: Resultsmentioning

confidence: 99%

Lipid Anchoring of Archaeosortase Substrates and Midcell Growth in Haloarchaea

Halim

Schulze

DiLucido

et al. 2020

mBio

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The archaeal cytoplasmic membrane provides an anchor for many surface proteins. Recently, a novel membrane anchoring mechanism involving a peptidase, archaeosortase A (ArtA), and C-terminal lipid attachment of surface proteins was identified in the model archaeon Haloferax volcanii. ArtA is required for optimal cell growth and morphogenesis, and the S-layer glycoprotein (SLG), the sole component of the H. volcanii cell wall, is one of the targets for this anchoring mechanism. However, how exactly ArtA function and regulation control cell growth and morphogenesis is still elusive. Here, we report that archaeal homologs to the bacterial phosphatidylserine synthase (PssA) and phosphatidylserine decarboxylase (PssD) are involved in ArtA-dependent protein maturation. Haloferax volcanii strains lacking either HvPssA or HvPssD exhibited motility, growth, and morphological phenotypes similar to those of an ΔartA mutant. Moreover, we showed a loss of covalent lipid attachment to SLG in the ΔhvpssA mutant and that proteolytic cleavage of the ArtA substrate HVO_0405 was blocked in the ΔhvpssA and ΔhvpssD mutant strains. Strikingly, ArtA, HvPssA, and HvPssD green fluorescent protein (GFP) fusions colocalized to the midcell position of H. volcanii cells, strongly supporting that they are involved in the same pathway. Finally, we have shown that the SLG is also recruited to the midcell before being secreted and lipid anchored at the cell outer surface. Collectively, our data suggest that haloarchaea use the midcell as the main surface processing hot spot for cell elongation, division, and shape determination. IMPORTANCE The subcellular organization of biochemical processes in space and time is still one of the most mysterious topics in archaeal cell biology. Despite the fact that haloarchaea largely rely on covalent lipid anchoring to coat the cell envelope, little is known about how cells coordinate de novo synthesis and about the insertion of this proteinaceous layer throughout the cell cycle. Here, we report the identification of two novel contributors to ArtA-dependent lipid-mediated protein anchoring to the cell surface, HvPssA and HvPssD. ArtA, HvPssA, and HvPssD, as well as SLG, showed midcell localization during growth and cytokinesis, indicating that haloarchaeal cells confine phospholipid processing in order to promote midcell elongation. Our findings have important implications for the biogenesis of the cell surface.

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Section: Resultsmentioning

confidence: 99%

Lipid Anchoring of Archaeosortase Substrates and Midcell Growth in Haloarchaea

Halim

Schulze

DiLucido

et al. 2020

mBio

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“…Finally, though we have used the Sulfoscope to reveal fundamental novel aspects of cell division, we anticipate that this type of high-temperature live-imaging platform can be used to shed light on other exciting areas of Sulfolobus cell biology, including DNA remodeling [ 15 , 26 ], swimming [ 27 ], conjugation [ 28 , 29 ], viral infection [ 23 , 30 ], competition [ 31 , 32 ], and cell-cell fusion [ 33 ]. In addition, this system can now also be applied to the study of other thermophilic microbes, from eukaryotes to bacteria [ 34 , 35 ].…”

Section: Discussionmentioning

confidence: 99%

Live Imaging of a Hyperthermophilic Archaeon Reveals Distinct Roles for Two ESCRT-III Homologs in Ensuring a Robust and Symmetric Division

et al. 2020

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Summary Live-cell imaging has revolutionized our understanding of dynamic cellular processes in bacteria and eukaryotes. Although similar techniques have been applied to the study of halophilic archaea [ 1 , 2 , 3 , 4 , 5 ], our ability to explore the cell biology of thermophilic archaea has been limited by the technical challenges of imaging at high temperatures. Sulfolobus are the most intensively studied members of TACK archaea and have well-established molecular genetics [ 6 , 7 , 8 , 9 ]. Additionally, studies using Sulfolobus were among the first to reveal striking similarities between the cell biology of eukaryotes and archaea [ 10 , 11 , 12 , 13 , 14 , 15 ]. However, to date, it has not been possible to image Sulfolobus cells as they grow and divide. Here, we report the construction of the Sulfoscope , a heated chamber on an inverted fluorescent microscope that enables live-cell imaging of thermophiles. By using thermostable fluorescent probes together with this system, we were able to image Sulfolobus acidocaldarius cells live to reveal tight coupling between changes in DNA condensation, segregation, and cell division. Furthermore, by imaging deletion mutants, we observed functional differences between the two ESCRT-III proteins implicated in cytokinesis, CdvB1 and CdvB2. The deletion of cdvB1 compromised cell division, causing occasional division failures, whereas the ΔcdvB2 exhibited a profound loss of division symmetry, generating daughter cells that vary widely in size and eventually generating ghost cells. These data indicate that DNA separation and cytokinesis are coordinated in Sulfolobus , as is the case in eukaryotes, and that two contractile ESCRT-III polymers perform distinct roles to ensure that Sulfolobus cells undergo a robust and symmetrical division.

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“…Finally, while we have used the Sulfoscope to reveal fundamental novel aspects of cell division, we anticipate that this high-temperature imaging platform can be applied to shed light on other exciting areas of cell biology in Sulfolobus, including DNA remodelling [19,33], swimming [34], conjugation [35,36], viral infection [30,37], competition [38,39] and cell-cell fusion [40]. Live imaging of Sulfolobus will also uncover entirely new cell biology in these organisms, since so far, we have not been able to watch them alive.…”

Section: Discussionmentioning

confidence: 99%

Live cell imaging of the hyperthermophilic archaeonSulfolobus acidocaldariusidentifies complementary roles for two ESCRTIII homologues in ensuring a robust and symmetric cell division

et al. 2020

Preprint

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Live-cell imaging has revolutionized our understanding of dynamic cellular processes in bacteria and eukaryotes. While similar techniques have recently been applied to the study of halophilic archaea, our ability to explore the cell biology of thermophilic archaea is limited, due to the technical challenges of imaging at high temperatures. Here, we report the construction of the Sulfoscope, a heated chamber that enables live-cell imaging on an inverted fluorescent microscope. Using this system combined with thermostable fluorescent probes, we were able to image Sulfolobus cells as they divide, revealing a tight coupling between changes in DNA compaction, segregation and cytokinesis. By imaging deletion mutants, we observe important differences in the function of the two ESCRTIII proteins recently implicated in cytokinesis. The loss of CdvB1 compromises cell division, causing occasional division failures and fusion of the two daughter cells, whereas the deletion of cdvB2 leads to a profound loss of division symmetry, generating daughter cells that vary widely in size and eventually generating ghost cells. These data indicate that DNA separation and cytokinesis are coordinated in Sulfolobus, as is the case in eukaryotes, and that two contractile ESCRTIII polymers perform distinct roles to ensure that Sulfolobus cells undergo a robust and symmetrical division. Taken together, the Sulfoscope has shown to provide a controlled high temperature environment, in which cell biology of Sulfolobus can be studied in unprecedent details. AAP, DRM, GD, RH and BB conceived the study. Microscope design and construction and commercial heating stage testing was carried out by AAP, SC, GD, DRM and RH. Design of the heating cap and heating stage was carried out by AAP, JR, CR and MR. JR and MR constructed the heating cap and heating stage. AAP and DRM established the imaging methodology and dyes used. Live-imaging and imaging analysis were performed by AAP and DRM with help from SC. Genetics and strains were performed by AAP, MVW and KNS. Immunofluorescence and Western-Blots were performed by AAP. Flow Cytometry were performed by GTR. Chamber measurements were performed

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The structure of the periplasmic FlaG–FlaF complex and its essential role for archaellar swimming motility

Abstract: Reprints and permissions information is available at www.nature.com/reprints.

Cited by 37 publications

References 56 publications

Lipid Anchoring of Archaeosortase Substrates and Midcell Growth in Haloarchaea

Lipid Anchoring of Archaeosortase Substrates and Midcell Growth in Haloarchaea

Live Imaging of a Hyperthermophilic Archaeon Reveals Distinct Roles for Two ESCRT-III Homologs in Ensuring a Robust and Symmetric Division

Live cell imaging of the hyperthermophilic archaeonSulfolobus acidocaldariusidentifies complementary roles for two ESCRTIII homologues in ensuring a robust and symmetric cell division

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