Near-atomic resolution cryoelectron microscopy structure of the 30-fold homooligomeric SpoIIIAG channel essential to spore formation in
            <i>Bacillus subtilis</i>

Zeytuni, N.; Hong, Chuan; Flanagan, Kelly A.; Worrall, L.J.; Theiltges, Kate A.; Vuckovic, M.; Huang, Rick; Massoni, Shawn C.; Camp, Amy H.; Yu, Zhiheng; Strynadka, N.C.J.

doi:10.1073/pnas.1704310114

Cited by 35 publications

(28 citation statements)

References 56 publications

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“…During engulfment a complex composed of SpoIIQ synthesized in the forespore and the proteins in the SpoIIIA operon and GerM produced in the mother cell is assembled across the inner and outer forespore membranes [ 5 , 33 , 34 , 56 ]. Several proteins in this trans-envelope complex resemble components of specialized secretion systems found in Gram-negative bacteria [ 5 , 6 , 57 ] and three of them SpoIIQ, SpoIIIAH, and SpoIIIAG appear to form a channel in the space between the inner and outer forespore membranes [ 58 – 61 ]. It has thus been proposed that this complex functions as a feeding tube or secretion system to provide mother-cell-produced molecules to the forespore [ 4 – 6 ].…”

Section: Discussionmentioning

confidence: 99%

A two-step transport pathway allows the mother cell to nurture the developing spore in Bacillus subtilis

et al. 2017

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One of the hallmarks of bacterial endospore formation is the accumulation of high concentrations of pyridine-2,6-dicarboxylic acid (dipicolinic acid or DPA) in the developing spore. This small molecule comprises 5–15% of the dry weight of dormant spores and plays a central role in resistance to both wet heat and desiccation. DPA is synthesized in the mother cell at a late stage in sporulation and must be translocated across two membranes (the inner and outer forespore membranes) that separate the mother cell and forespore. The enzymes that synthesize DPA and the proteins required to translocate it across the inner forespore membrane were identified over two decades ago but the factors that transport DPA across the outer forespore membrane have remained mysterious. Here, we report that SpoVV (formerly YlbJ) is the missing DPA transporter. SpoVV is produced in the mother cell during the morphological process of engulfment and specifically localizes in the outer forespore membrane. Sporulating cells lacking SpoVV produce spores with low levels of DPA and cells engineered to express SpoVV and the DPA synthase during vegetative growth accumulate high levels of DPA in the culture medium. SpoVV resembles concentrative nucleoside transporters and mutagenesis of residues predicted to form the substrate-binding pocket supports the idea that SpoVV has a similar structure and could therefore function similarly. These findings provide a simple two-step transport mechanism by which the mother cell nurtures the developing spore. DPA produced in the mother cell is first translocated into the intermembrane space by SpoVV and is then imported into the forespore by the SpoVA complex. This pathway is likely to be broadly conserved as DPA synthase, SpoVV, and SpoVA proteins can be found in virtually all endospore forming bacteria.

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Section: Discussionmentioning

confidence: 99%

A two-step transport pathway allows the mother cell to nurture the developing spore in Bacillus subtilis

et al. 2017

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“…2. The S/N ratio ranges from 1.0 to 2.0 for smaller barrels, with values as low as 0.0 for larger barrels 30,31 . were split open, spread flat, and viewed from the inside of the barrel.…”

Section: Concentric β-Barrel Theorymentioning

confidence: 99%

Theory of concentric β-barrel structures: models of amyloid beta 42 oligomers, annular protofibrils, and transmembrane channels

Guy

Durell

Kayed

2018

Preprint

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Amyloid beta (Aβ) peptides are a major contributor to Alzheimer's disease. Previously, our group proposed molecular models of Aβ42 hexamers with two concentric antiparallel β-barrels that act as seeds from which dodecamers, octadecamers, both smooth and beaded annular protofibrils, and transmembrane channels form. Since then, numerous aspects of our models have been supported by experimental findings. Here we develop a more extensive range of models to be consistent with dimensions of assemblies observed in electron microscopy images of annular protofibrils and transmembrane assemblies. These models have the following features: Dodecamers with 2-concentric β-barrels are the major components of beaded annular protofibrils (bAPFs). These beads merge to form smooth annular protofibrils (sAPFs) that have three or four concentric β-barrels. Channels form from two to nine hexamers. Antiparallel C-terminus S3 segments form an outer transmembrane β-barrel. Half of the monomers of vertically asymmetric 12mer to 36mer channels form parallel transmembrane S2 β-barrels, and S1-S2 (N-terminus and middle) segments of the other half of the monomers form aqueous domains on the cis side of the membrane. Unit cells of 42-54mers have two more transmembrane S2 segments, with four concentric β-barrels in the transmembrane region and two concentric β-barrels on the cis side of the membrane.

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“…We also observed structures that appear to be channels crossing the septum (Fig. 1L) that may correspond to SpoIIQ-SpoIIIAH complexes 36–38 . Next, we observed a basement coat layer adjacent to the outer forespore membrane that is likely comprised of SpoVM and/or SpoIVA ( 39 , Fig.…”

Section: Resultsmentioning

confidence: 66%

The molecular architecture of engulfment during Bacillussubtilis sporulation

Khanna¹,

López‐Garrido²,

Zhao³

et al. 2018

Preprint

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18The study of cell biology is limited by the difficulty in visualizing cellular structures at high 19 spatial resolution within their native milieu. Here, we have visualized sporulation in 20 Bacillus subtilis using cryo-electron tomography coupled with cryo-focused ion beam 21 milling, a technique that allows the 3D reconstruction of cellular structures in near-native 22 state at molecular resolution. During sporulation, an asymmetrically-positioned septum 23 divides the cell into a larger mother cell and a smaller forespore. Subsequently, the 24 mother cell phagocytoses the forespore in a process called engulfment, which entails a 25 dramatic rearrangement of the peptidoglycan (PG) cell wall around the forespore. By 26 imaging wild-type sporangia, engulfment mutants, and sporangia treated with PG 27 synthesis inhibitors, we show that the initiation of engulfment does not entail the complete 28 dissolution of the septal PG by the mother cell SpoIIDMP complex, as was previously 29 thought. Instead, DMP is required to maintain a flexible septum that is uniformly and only 30 slightly thinned at the onset of engulfment. Then, the mother cell membrane migrates 31 around the forespore by forming tiny finger-like projections, the formation of which 32 requires both SpoIIDMP and new PG synthesized ahead of the leading edge of the 33 engulfing membrane. We propose a molecular model for engulfment membrane migration 34 in which a limited number of SpoIIDMP complexes tether the membrane to and degrade 35 the new PG ahead of the leading edge, thereby generating an irregular engulfing 36 membrane front. Our data also reveal other structures that will provide a valuable 37 resource for future mechanistic studies of endospore formation. 38 migration, cryo-electron tomography, cryo-focused ion beam milling.40 41 42 From an architectural point of view, bacterial cells are among the simplest forms of life on 43 the planet. Their cytoplasm is typically devoid of membrane bound organelles, and their 44 cellular morphology relies upon a semi-rigid peptidoglycan (PG) cell wall that imposes its 45 shape on the malleable cell membrane(s). Bacterial cells are inflated by their high internal 46turgor pressure, which pushes the membranes against the cell wall, causing the PG to 47 stretch and the cell to adopt its appropriate shape. Despite the apparent simplicity, studies 48 in the past few decades have demonstrated that bacterial cellular architecture is far more 49 complex than previously thought, in terms of both its ultrastructure and dynamic 50 capabilities 1-3 . 52Endospore formation in Bacillus subtilis represents a striking example of the dynamic 53 capabilities of bacterial cells, as it entails dramatic changes in cellular architecture. First, 54 the division sites shift to polar positions, generating a sporangium comprised of two cells: 55 a larger mother cell and a smaller forespore ( Fig. 1A; 4-6 ). The polar septum traps the 56 forespore chromosome, which is subsequently transported to the forespore by SpoIIIE, a 57 m...

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Near-atomic resolution cryoelectron microscopy structure of the 30-fold homooligomeric SpoIIIAG channel essential to spore formation in Bacillus subtilis

Cited by 35 publications

References 56 publications

A two-step transport pathway allows the mother cell to nurture the developing spore in Bacillus subtilis

A two-step transport pathway allows the mother cell to nurture the developing spore in Bacillus subtilis

Theory of concentric β-barrel structures: models of amyloid beta 42 oligomers, annular protofibrils, and transmembrane channels

The molecular architecture of engulfment during Bacillussubtilis sporulation

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