To begin, we verified that BtubA and BtubB proteins are in fact expressed in the species where the genes are present Figures S1 and S2. Next, Prosthecobacter cells were grown under different conditions and plunge-frozen across EM grids. A total of cells were then imaged in 3-D by ECT. The spindle-shaped cells were polymorphic and exhibited prosthecae cellular stalks of different lengths. As seen in other bacterial phyla [25] , multiple classes of cytoskeletal structures were seen, but one class had a tube-like morphology and was frequently found in the harboring species, but never in the btubAB -lacking strain Figure 1.
The abundance of these tube-like structures was dependent on the species imaged as well as the growth conditions and growth stage, and was found to be highest in P. In sum, the tube-like structures were found in 48 of P. The tube-like structures were —1, nm long, always parallel to the cytoplasmic membrane, almost always localized in the stalk or in the transition zone between stalk and cell body, and occurred either individually or in bundles of two, three, or four Figure 1 , Figure S3 , Movie S1.
Chemical fixatives were found to degrade the structures Figure S4 , explaining why they were likely missed in previous conventional EM studies [11] , [22]. Shown are nm thick slices through cryotomograms. Arrows indicate cytoskeletal structures, which are also shown enlarged below. Asterisk in panel A identifies a sub-tomographic average.
Upper-left insets show low-magnification overviews of the cells; rectangles indicate areas imaged in 3-D. Bottom: 3-D segmentation of the bundle of panel B shown from two views four tubes are present.
Scale bars are nm. Tube-like structures were not seen in control E. The diameters and subunit repeat distances of all three structures in Prosthecobacter , recombinant E.
Finally, immunogold-staining using anti-BtubB antibodies localized the proteins to the same region of Prosthecobacter cells as the candidate structures seen by ECT Figures S5 and S6. Shown are nm thick slices through electron cryotomograms. Arrows indicate cytoskeletal structures. Black scale bars and white scale bar applies to enlarged images are nm. Because the opposing arcs observed here were always in this orientation facing each other and the beam path , it was clear that the structures must have been complete tubes distorted by the missing wedge rather than, for instance, parallel protofilaments, which would not be expected to always orient themselves in the same direction with respect to the electron beam.
These simulations recapitulated the experimental results well, since the density patterns Figure 3H were highly similar to those seen in experimental tomograms.
The black scale bar is 10 nm and applies to enlarged images and simulations in panels A—H; white scale bars are nm in panels E—G and 10 nm in panel I.
To maintain reasonable lateral interactions in such small tubes, protofilaments had to be spaced slightly closer 4. Thus only the five-protofilament model was consistent with the 7. Such an asymmetry can only arise from an uneven number of protofilaments, as demonstrated by simulated tomograms Figure S7 , further suggesting five rather than four or six protofilaments.
Because the left-right asymmetries in computational projections and in sub-tomographic averages at different positions along the tube axis remained consistent, the five protofilaments must be straight rather than twisting around the tube Figure S8. The alternative two protofilaments 7. Slight helical twists in the tubes in vitro may have caused the appearance of twisted pairs [17]. While the number of protofilaments in eukaryotic microtubules can vary, the lateral interactions between them are conserved [28] such that each protofilament is shifted 0.
The sum of five such shifts 4. The difference could be accommodated by a slightly different lateral interaction a stagger of 0. The latter seems more likely, however, since the B-lattice has been resolved in eukaryotic protofilament microtubules, and is therefore depicted in Figure 4.
A 2-D schematic of the proposed architecture of bacterial microtubules built from BtubA dark-blue and BtubB light-blue. Protofilaments are numbered 1—5. Seams and start-helices are indicated as in A. A Fourier transform of a simulated projection image 1. A prominent pair of elongated spots on the subunit-repeat layer line on either side of the meridian corresponds to the helical family J1.
Pairs of spots for the helical families J4 and J6 were very weak, likely because of destructive interference with the first minimum of the J1 Bessel-function. The asymmetry also shifted around the meridian depending on the rotation of the tube around its length axis Figure S The prominent pair of J1 spots on the subunit repeat layer line in all cases suggests a helical lattice, as all non-helical models lead to high-intensity spots on the meridian unpublished data.
Arrowheads indicate the subunit repeat layer line. Arrows mark the maxima of the J1, J4, J5, and J6 Bessel-functions, assuming outer rather than mass-weighted radii and therefore marking the expected meridional borders of spots.
As shown previously [11] , [12] , BtubA and BtubB are clearly members of the eukaryotic clade of tubulins Figure 6. A protein motif search Table S1 , an identity matrix Table S2 , and various treeing methods Figure 6 , Figure S11 , however, all failed to detect any stable associations between BtubA or BtubB with any eukaryotic tubulin subfamily. BtubA and BtubB should therefore be considered as two novel tubulin subfamilies, derived not from any particular modern subfamily but instead directly from ancient tubulins.
This hypothesis Figure 7 also seems more probable because, like FtsZ but unlike eukaryotic tubulins, BtubA and BtubB exhibit the presumably ancient properties of folding without chaperones and forming weak dimers [17] , [19] , [20]. It therefore appears that in tubulin evolution, heterodimer formation correlated with tube formation and the five-protofilament, one-start helix was the simplest and earliest microtubule architecture realized, which later evolved into the larger eukaryotic microtubule.
Here and in further phylogenetic analyses Figure S11 , Tables S1 and S2 , and Materials and Methods no stable associations between BtubA or BtubB and any tubulin subfamily were detected, in agreement with a previous less comprehensive study [11]. Numbers indicate how many sequences were included in a closed group. Tubulins, FtsZ, FtsZ-like, and TubZ all evolved from a common ancestor with the likely properties listed [5] , [9] , [58] — [61]. In contrast, BtubA and BtubB retained ancient features shared by FtsZ such as chaperone independence, weak dimerization, and both an activating T7 loop and short S9, S10 loop in both subunits [17] , [19] , [21].
The smaller, five-protofilament, one-start-helical architecture of the bacterial microtubule is therefore likely a primordial form. The appearance of the btubA , btubB , and bklc genes as a distinct bacterial operon inserted in the midst of functionally related genes, but in different places in the chromosomes in the three species concerned, still points to horizontal gene transfer [18].
Instead, it may have been from a yet-unidentified bacterial lineage that also carries the btubAB genes. It is presently debated whether an ancient Planctomycetes - Verrucomicrobia - Chlamydiae bacterium was involved in the evolution of eukaryotes [15] , [31] , [32] , but if so, such a relationship would be consistent with bMTs preceding modern eukaryotic MTs.
Because eukaryotic tubulins require chaperones and accessory proteins to fold and function properly, cell biological studies and anti-microtubule drug screenings typically require that tubulin be purified from tissue. A negative control was run without reverse transcriptase enzyme. PCR-reactions were analyzed by agarose gel electrophoresis. Primers [12] targeting conserved tubulin sequences were used to PCR-amplify potential tubulin genes from genomic P. After digestion, the PCR fragments were cloned into a digested vector derived from pHis Plasmid inserts were verified by sequencing.
For protein expression, plasmids were transformed into Escherichia coli C41 DE3 cells [34]. The proteins were expressed under control of the T7 promoter. C-terminally His-tagged P.
The column was washed with buffer A and the proteins were eluted using buffer A containing mM imidazole. Proteins were dialyzed into PBS 0. Erickson [19]. For negative staining, samples were applied to a Formvar-coated, carbon-coated, glow-discharged copper EM grid Electron Microscopy Sciences. Samples were aspirated and stained with 0. Tomographic tilt-series were acquired using the SerialEM [35] software package, then subsequently calculated and analyzed using IMOD [36].
Exponentially growing cells were prepared for immuno-EM by a modification of the method of Tokuyasu [37] , [38]. The cells were then pelleted and infiltrated with 2. Pellets were transferred to aluminum sectioning stubs Ted Pella, Inc. The samples were transferred to brass planchettes and rapidly frozen in a high-pressure freezing machine Bal-Tec HPM, Leica Microsystems. Cryosectioning of the vitrified samples was done as previously described [39] , [40].
Alternatively, EM grids were incubated in a static liquid culture, removed, blotted, and plunge-frozen. The grids were stored in liquid nitrogen. Pixels on the CCD represented 0. Three-dimensional reconstructions were calculated using the IMOD software package [36] or Raptor [45]. The averaging procedure described by Cope et al. The program addModPts was run to fill in model points every 4. Alternatively, model points were set manually at a distance of 42 nm or 21 nm for averaging unique sub-volumes.
The PEET software package [47] was used to align and average repeating sub-volumes. Isosurface rendering of the sub-volume averages was carried out using IMOD [36]. The protofilament was replicated and tubes were built using four, five, or six protofilaments, each shifted 0. The 5-nm protofilament spacing seen in eukaryotic microtubules seemed unreasonable in these much smaller-diameter tubes, since inter-protofilament interactions appeared impossible.
Protofilaments were therefore brought closer together 4. The tomogram simulation procedure described by Gan et al. All simulations were done with Bsoft [50] using imaging parameters close to the nominal experimental conditions. Briefly, a 3-D map was generated with a 0. Tomograms were reconstructed with IMOD [36] using the same settings as for the experimental data.
Tubes and other cell components in the tomogram were then segmented manually using Amira Visage Imaging GmbH. Subunit repeat distances in Prosthecobacter , E. Protein sequences were analyzed using the program PRINTS [51] in order to detect shared motifs and calculate a probability value for the likelihood that different BtubA or BtubB proteins belonged to a particular tubulin family.
To perform phylogenetic sequence analyses, two different databases were established using the ARB program package [52]. In both databases, the amino acid alignments were refined manually accounting for conserved tubulin domains. The identity matrix for a selection of representatives was generated using the ARB program package [52].
All programs are implemented in the ARB program package [52]. Tubulin and actin were apparently derived from bacterial precursors that had already evolved a wide range of cytoskeletal functions. The s and s saw extensive research on microtubules and actin. During this period, the consensus developed that these cytoskeletal elements were unique to eukaryotes and that nothing related to tubulin or actin existed in bacteria or archaeans. This consensus was overthrown in the s when a series of discoveries revealed that prokaryotes actually did have homologues of tubulin and actin and that these assembled into cytoskeletal filaments.
It is now generally accepted that eukaryotic microtubules and actin filaments originated from these prokaryotic homologues. The key discoveries of bacterial tubulin and actin were published in and are reviewed here on their 25th anniversary.
The discovery of FtsZ as a bacterial homologue of tubulin came first and was made independently by three groups de Boer et al. These independent studies each purified FtsZ protein from an Escherichia coli expression system and demonstrated that it bound and hydrolyzed GTP. That sequence, known as the tubulin signature sequence, was believed to be involved in the binding of GTP in the tubulins. The three groups all concluded that the GTP-binding site of FtsZ appeared to be related to that of tubulins.
A year earlier, before any link to tubulin was known, Bi and Lutkenhaus were the first to propose that FtsZ might be a cytoskeletal protein. They used immuno—electron microscopy to show that FtsZ localized to the invaginating septum in dividing E. In particular, de Boer et al. A subsequent study by Mukherjee and Lutkenhaus provided two major advances. First, they demonstrated that purified FtsZ could assemble in vitro into filamentous polymers.
This was strong support for the proposed role as cytoskeleton. Our laboratory took up the question of in vitro assembly and showed that FtsZ assembled in vitro into sheets of protofilaments and mini-rings that were similar to tubulin polymers Erickson et al. They had an identical complex fold, which is the ultimate test of homology. It turns out that FtsZ is not the only tubulin homologue in prokaryotes. Many archaeans have up to five FtsZ homologues, some with very divergent sequences that likely serve functions other than cell division.
The discovery of bacterial actins was complicated by the homology of actin to other protein families. Protein homology means shared ancestry. Sometimes, this is indicated by amino acid sequence identity, but often this sequence identity is too weak to recognize. Publication types Research Support, Non-U. Gov't Research Support, U.
Gov't, Non-P. A single microtubule contains 10 to 15 protofilaments 13 in mammalian cells that wind together to form a 24 nm wide hollow cylinder. Microtubules are structures that can rapidly grow via polymerization or shrink via depolymerization in size, depending on how many tubulin molecules they contain. This image is linked to the following Scitable pages:. Dynamic networks of protein filaments give shape to cells and power cell movement. Learn how microtubules, actin filaments, and intermediate filaments organize the cell.
Comments Close. The Comment you have entered exceeds the maximum length. Submit Cancel.
0コメント