Microtubules in Bacteria: Ancient Tubulins Build a Five-Protofilament Homolog of the Eukaryotic Cytoskeleton

Microtubules play crucial roles in cytokinesis, transport, and motility, and are therefore superb targets for anti-cancer drugs. All tubulins evolved from a common ancestor they share with the distantly related bacterial cell division protein FtsZ, but while eukaryotic tubulins evolved into highly conserved microtubule-forming heterodimers, bacterial FtsZ presumably continued to function as single homopolymeric protofilaments as it does today

Abstract

Microtubules play crucial roles in cytokinesis, transport, and motility, and are therefore superb targets for anti-cancer drugs. All tubulins evolved from a common ancestor they share with the distantly related bacterial cell division protein FtsZ, but while eukaryotic tubulins evolved into highly conserved microtubule-forming heterodimers, bacterial FtsZ presumably continued to function as single homopolymeric protofilaments as it does today. Microtubules have not previously been found in bacteria, and we lack insight into their evolution from the tubulin/FtsZ ancestor. Using electron cryomicroscopy, here we show that the tubulin homologs BtubA and BtubB form microtubules in bacteria and suggest these be referred to as “bacterial microtubules” (bMTs). bMTs share important features with their eukaryotic counterparts, such as straight protofilaments and similar protofilament interactions. bMTs are composed of only five protofilaments, however, instead of the 13 typical in eukaryotes. These and other results suggest that rather than being derived from modern eukaryotic tubulin, BtubA and BtubB arose from early tubulin intermediates that formed small microtubules. Since we show that bacterial microtubules can be produced in abundance in vitro without chaperones, they should be useful tools for tubulin research and drug screening.

Author Summary

Bacteria are generally distinguished from the cells of fungi, plants, and animals (eukaryotes) not only by their much smaller size but also by the absence of certain subcellular structures such as nuclei, internal organelles, and microtubules. Using state-of-the-art microscopy, we demonstrate here that microtubules do exist in some bacteria. These bacterial microtubules are built from proteins that are closely related to the microtubule proteins in eukaryotes. Bacterial microtubules are smaller in diameter than their counterparts in eukaryotic cells but have the same basic architecture. We propose that bacterial microtubules represent primordial structures that preceded eukaryotic microtubules evolutionarily. Because bacterial microtubules can be produced and handled in the lab more easily than their eukaryotic counterparts, they may become useful tools for microtubule research and anti-cancer drug screening.

Introduction

Microtubules are among the most-studied eukaryotic subcellular structures [1]–[4]. Their crucial role in cell division, transport, and motility make them superb targets for anti-cancer drugs. All tubulins evolved from a common ancestor they share with the distantly related bacterial cell division protein FtsZ [5]–[9], but while eukaryotic α- and β-tubulins evolved into highly conserved tube-forming heterodimers [1],[4], bacterial FtsZ presumably continued to function as single homopolymeric protofilaments as it does today [10]. Although unidentified tubular structures have been seen in certain bacteria [3], tubulin genes have not been found in the genomes. The discovery of bacterial tubulin A (BtubA) and bacterial tubulin B (BtubB) in several Prosthecobacter strains was therefore exciting, since BtubA and BtubB are much more closely related to eukaryotic tubulins than to any other bacterial protein [11],[12]. Prosthecobacters belong to the Planctomycetes-Verrucomicrobia-Chlamydiae superphylum, whose members have been shown to possess various eukaryote-like features [13]–[15]. The function of BtubA/B in Prosthecobacter remains unclear, however, since they coexist with genuine FtsZ and are therefore unlikely to be the major cell division proteins [12],[16].

Since genomic organization and other evidence suggest prosthecobacters most probably acquired the btubAB genes by horizontal gene transfer [11],[12],[16]–[19], BtubA/B have been suggested to be descendants of modern eukaryotic α- and/or β-tubulins [6],[11],[17],[19],[20]. More recently, however, it was argued that they represent an ancient form, since (i) like FtsZ, BtubA/B assembles in diverse conditions and (ii) both BtubA and BtubB contain α- and β-tubulin-like features [21]. Just like α- and β-tubulins, BtubA/B form heterodimers which polymerize into protofilaments in vitro. Typically, 13 α/β-protofilaments align slightly staggered to form a hollow eukaryotic microtubule, but microtubule-like structures have not been described in BtubA/B preparations [17],[19],[21]. Cytoskeletal structures were also not observed in Prosthecobacter dejongeii cells by conventional thin-section electron microscopy (EM) [11],[22].

Reasoning that the structure of BtubA/B filaments might not have been preserved in vivo by conventional EM methods, here we sought to characterize BtubA/B structures using electron cryotomography (ECT) [23]. We show that BtubA/B form five-protofilament microtubules in vivo. Together with additional phylogenetic sequence analyses, these results support the notion that BtubA/B microtubules represent an ancient evolutionary form that led to modern eukaryotic 13-protofilament microtubules.

Results and Discussion

btubA and B genes are found in certain Prosthecobacter species including P. vanneervenii, P. dejongeii, and P. debontii, but not P. fluviatilis [11],[12],[24]. 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). Western hybridization and PCR also confirmed the absence of BtubA and BtubB in P. fluviatilis (Figure S2) [24].

Next, Prosthecobacter cells were grown under different conditions and plunge-frozen across EM grids. A total of 589 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. vanneervenii cells grown directly on EM grids (67% of cells imaged). In sum, the tube-like structures were found in 48 of 176 P. vanneervenii, 9 of 111 P. dejongeii, 15 of 151 P. debontii, and 0 of 151 P. fluviatilis cells. The tube-like structures were 200–1,200 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].

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Figure 1. Cytoskeletal BtubA/B-candidate structures imaged in Prosthecobacter.

Prosthecobacter vanneervenii cells showing tube-like BtubA/B-candidate structures occurring (A) individually or (B) in a bundle. Shown are 11-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 100 nm. See Figure S3 for further examples of BtubA/B structures.

Since genetic tools are not yet available for prosthecobacters, we applied labeling and heterologous expression approaches to test whether the candidate structures were in fact composed of BtubA/B as expected by their correlation with the presence of the genes. Recombinant Escherichia coli cells co-expressing BtubA and BtubB were imaged by ECT and exhibited strikingly similar tube-like structures running the length of the cells (Figure 2A) with the same localization as had been reported for BtubA/B from immuno-fluorescence [19]. Tube-like structures were not seen in control E. coli cells not expressing ButbA/B. Nearly identical tube-like structures were also seen when recombinant BtubA/B was polymerized in vitro and imaged by ECT (Figure 2B). The diameters and subunit repeat distances of all three structures (in Prosthecobacter, recombinant E. coli, and in vitro) were similar (7.6, 7.7, and 7.6 nm diameters, and 4.4, 4.4, and 4.2 nm repeat distances, respectively) (Figures 1, 2, and S3). 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). We conclude therefore that the tube-like structures are composed of BtubA/B, and the slight differences in repeat distance, straightness, and bundling in the three samples were due to differences in protein concentrations and/or the absence of other interacting proteins in vitro and in E. coli.

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Figure 2. Recombinant BtubA/B structures resemble the tube-like structures imaged in Prosthecobacter.

(A) E. coli cell co-expressing BtubA and BtubB (from P. dejongeii) and (B) recombinant BtubA/B polymerized in vitro exhibiting tube-like densities which are strikingly similar to those seen in Prosthecobacter. Shown are 11-nm thick slices through electron cryotomograms. Arrows indicate cytoskeletal structures. Black scale bars and white scale bar (applies to enlarged images) are 100 nm.

We have described the BtubA/B structures so far as “tube-like” because when acquiring a cryo-tomographic tilt-series, images of samples tilted beyond ∼65° cannot generally be included, so there is a missing “wedge” of data in reciprocal space that reduces the resolution in the direction of the electron beam. As a result, the “top” and “bottom” boundaries of cylindrical objects (considering the electron beam to be “vertical”) are smeared, leaving the sidewalls to appear like two arcs facing each other (Figure 3A–D). 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. Nevertheless different orientations of tubes with respect to the tilt axis aggravate the missing wedge artifact differently [26],[27], so to explore this effect tomograms of a known, tubular input structure consisting of BtubA/B crystal structures (see below) were simulated at different angles with respect to the tilt axis. These simulations recapitulated the experimental results well, since the density patterns (Figure 3H) were highly similar to those seen in experimental tomograms.

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Figure 3. BtubA/B assembles into five-protofilament tubes.

(A–D) Tomographic slices showing cross-sectional views of BtubA/B tubes in (A) prosthecobacters, (B) a sub-tomographic average from P. vanneervenii, (C) E. coli co-expressing BtubA/B (from P. vanneervenii), and (D) BtubA/B polymerized in vitro. (E, F) Images and (G) tomographic slices through cryosectioned, high-pressure-frozen (E) P. vanneervenii cells, (F) E. coli cells co-expressing BtubA/B, and (G) BtubA/B polymerized in vitro, showing that the BtubA/B structures are complete tubes. (H) Slices through simulated tomograms showing cross-sectional views of five-protofilament tube models lying in a plane perpendicular to the electron beam at different angles to the tilt-axis (from left to right 0°, 25°, 50°, 75°), showing how the well-known missing wedge effect recapitulates the apparent lack of density in the tops and bottoms of the tubes seen in the tomograms. (I) Pseudo-atomic model of a five-protofilament bacterial microtubule (blue; built from Protein Data Bank structure 2 btq) superimposed on the image of a cryo-sectioned BtubA/B tube (left). The tomographic slices are (A, C) 114 nm, (B, H) 11 nm, (D) 76 nm, and (G) 88 nm thick. The black scale bar is 10 nm and applies to enlarged images and simulations in panels A–H; white scale bars are 100 nm in panels E–G and 10 nm in panel I.

To further confirm that the BtubA/B structures were in fact complete tubes and to obtain clearer cross-sectional views, btubAB-harboring Prosthecobacter cells, recombinant E. coli cells, and purified BtubA/B polymerized in vitro were all high-pressure-frozen, cryosectioned, and imaged (Figure 3E–G). Cryosections through BtubA/B tubes appeared pentagonal, suggesting five-protofilament tubes. Using the heterodimeric BtubA/B crystal structure [17], we produced tube models with four, five, and six protofilaments for comparison. To maintain reasonable lateral interactions in such small tubes, protofilaments had to be spaced slightly closer (4.6 nm) than protofilaments in eukaryotic microtubules (5 nm), and this resulted in tube diameters of 6.7, 7.8, and 9.2 nm, respectively, for four-, five-, and six-protofilament tubes. Thus only the five-protofilament model was consistent with the 7.6-nm diameter measured in the tomograms, and the five-protofilament model fit the density of the BtubA/B tubes compellingly well (Figure 3I). Cross-sectional views of BtubA/B tubes in cryo-tomograms of whole cells and sub-tomogram averages often showed a left-right asymmetry (arrowheads in Figure 3A–C). 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).

Previous EM images of negatively stained, recombinant BtubA/B polymerized in vitro were not described as tubes, but as protofilament bundles or twisted pairs [17],[19],[21]. We obtained similar-looking images staining our own purified BtubA/B (Figure S9), but having observed clear tubes in vivo and noting the frequent pairing of parallel densities ∼7.6 nm apart in both our negatively stained images and the previously published images, we believe all these samples contained five-protofilament tubes as well. The alternative (two protofilaments 7.6 nm apart) seems unlikely since BtubA/B protofilaments are known to be only 4 nm in diameter [17], and would therefore have to be closer together to interact. 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 0.93 nm along the tube axis relative to its neighbors. In 13-protofilament microtubules, this shift results in a three-start helix around the microtubule and a seam where α- and β-subunits interact [29]. Because the loops that are involved in these interactions are also present in BtubA and BtubB [17], we expect BtubA/B protofilaments to be shifted similarly. The sum of five such shifts (4.65 nm) is similar to the subunit repeat distance measured in BtubA/B tubes (4.2 and 4.4 nm, respectively) and suggests that BtubA/B form one-start helical tubes (Figure 4). The difference could be accommodated by a slightly different lateral interaction (a stagger of 0.84–0.88 nm instead of 0.93 nm). In support of this model, the major features of Fourier transforms of BtubA/B tube images matched those of a one-start five-protofilament helix model (Figures 5 and S10), but did not clarify whether BtubA/B tubes have an “A-lattice” without seam or a “B-lattice” with seam [30]. The latter seems more likely, however, since the B-lattice has been resolved in eukaryotic 13-protofilament microtubules, and is therefore depicted in Figure 4. Based on our data, the BtubA/B crystal structure [17], and the known structural features of the eukaryotic microtubule, we conclude therefore that BtubA/B heterodimers form five-protofilament, one-start helical tubes in vivo with lateral and longitudinal interactions like their eukaryotic counterparts. Since BtubA/B are true homologs of eukaryotic tubulin [11],[12],[17] and they form closely related structures differing mainly in the number of protofilaments, we suggest they be referred to as “bacterial microtubules” (bMTs).

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Figure 4. Structural model of “bacterial microtubules.”

(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. (B) 3-D comparison of the architectures of a bacterial microtubule (left; BtubA in dark-blue; BtubB in light-blue) and a 13-protofilament eukaryotic microtubule (right; β-tubulin in black; α-tubulin in white). Seams and start-helices are indicated as in (A).

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Figure 5. BtubA/B tubes have a helical, microtubule-like lattice.

(A) Fourier transform of a simulated projection image (1.2 nm/pixel) of a five-protofilament BtubA/B-tube model (Figure 4) with a helical, microtubule-like lattice. 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 subunit-repeat layer line was surprisingly asymmetric probably because of the small number of protofilaments and the resulting lack of an extended “front” and ”back” side. The asymmetry also shifted around the meridian depending on the rotation of the tube around its length axis (Figure S10). (B–E) Fourier transforms of BtubA/B-tubes in (B) a 2-D slice through a subtomogram average (from within a P. vanneervenii cell), (C) a negatively stained projection image (of an in vitro assembled tube), (D) a cryo-EM projection image (of an in vitro assembled tube), and (E) a 2-D tomographic slice containing an in vitro assembled tube. 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).

It has been suggested that BtubA and BtubB evolved from modern eukaryotic α- and/or β-tubulins [11],[17],[19],[20]. If this were true, a phylogenetic association linking BtubA and BtubB to α- and/or β-tubulin would be expected. 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]. Furthermore, BtubA/B polymerizes in broader conditions and both proteins have mixtures of the structural characteristics found in α- and β-tubulin (activating T7 and short S9, S10 loops) [17],[21]. 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.

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Figure 6. BtubA and BtubB represent two novel tubulin subfamilies in the eukaryotic clade of tubulins.

In global phylogenetic analyses of the FtsZ/Tubulin superfamily, BtubA and BtubB stably clustered within the clade of eukaryotic tubulin subfamilies (i.e., the Tubulin family). A second stable group of sequences comprised bacterial and archaeal tubulin homologues (FtsZ, FtsZ-like, TubZ, RepX). The relationships between tubulin subfamilies were instable (except β-θ and α-κ). 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]. Shown is one representative maximum likelihood tree calculated using a 10% minimum similarity filter. A black circle indicates that the respective node/group was stable in different trees. Bar represents 1% estimated evolutionary distance. Numbers indicate how many sequences were included in a closed group.

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Figure 7. Model for the evolution of BtubA/B.

Tubulins, FtsZ, FtsZ-like, and TubZ all evolved from a common ancestor with the likely properties listed [5],[9],[58]–[61]. In contrast to the bacterial FtsZ, FtsZ-like, and TubZ proteins, the last common tubulin ancestor appears to have evolved to form heterodimers (consisting of “A”- and “B”-tubulins) with properties that enabled tube formation. Modern α- and β-tubulin further localized the activating T7 and short S9, S10 loop into different subunits, developed a need for chaperones, and began to form larger, ∼13-protofilament microtubules. 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 ancestry of the other supplemental tubulins γ through κ is unclear, except that θ- and κ-tubulins derived from β and α, respectively.

While BtubA/B likely represent an ancient form of tubulin, the origin of the genes found today in Prosthecobacter remains unclear. 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]. The lack of relatedness of BtubA/B to other tubulin families, however, makes clear that it was not a transfer from a modern eukaryote. Instead, it may have been from a yet-unidentified bacterial lineage that also carries the btubAB genes. The alternative, “vertical evolution” hypothesis is that btubAB was present in the last common ancestor of Verrucomicrobia, but the genes were simply lost by the other members of the phylum. 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. BtubA/B, however, is more stable, can be easily mutated [20],[21], recombinantly expressed in E. coli [17],[19]–[21], and as shown here, polymerized into microtubules in vitro. bMTs or eukaryotized derivatives could therefore complement eukaryotic microtubules as models and tools for tubulin research.

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Microtubules in Bacteria: Ancient Tubulins Build a Five-Protofilament Homolog of the Eukaryotic Cytoskeleton

Microtubules play crucial roles in cytokinesis, transport, and motility, and are therefore superb targets for anti-cancer drugs. All tubulins evolved from a common ancestor they share with the distantly related bacterial cell division protein FtsZ, but while eukaryotic tubulins evolved into highly conserved microtubule-forming heterodimers, bacterial FtsZ presumably continued to function as single homopolymeric protofilaments as it does today

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