genes are found in certain Prosthecobacter
species including P. vanneervenii
, P. dejongeii
, and P. debontii
, but not P. fluviatilis 
. To begin, we verified that BtubA and BtubB proteins are in fact expressed in the species where the genes are present (Figures S1
). Western hybridization and PCR also confirmed the absence of BtubA and BtubB in P. fluviatilis
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 
, 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 (). 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 S3
, Movie S1
). Chemical fixatives were found to degrade the structures (Figure S4
), explaining why they were likely missed in previous conventional EM studies 
Cytoskeletal BtubA/B-candidate structures imaged in Prosthecobacter.
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 () with the same localization as had been reported for BtubA/B from immuno-fluorescence 
. 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 (). 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) (, , 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
). 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
Recombinant BtubA/B structures resemble the tube-like structures imaged in Prosthecobacter.
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 (). 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 
, 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 () were highly similar to those seen in experimental tomograms.
BtubA/B assembles into five-protofilament tubes.
To further confirm that the BtubA/B structures were in fact complete tubes and to obtain clearer cross-sectional views, btubAB
cells, recombinant E. coli
cells, and purified BtubA/B polymerized in vitro were all high-pressure-frozen, cryosectioned, and imaged (). Cryosections through BtubA/B tubes appeared pentagonal, suggesting five-protofilament tubes. Using the heterodimeric BtubA/B crystal structure 
, 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 (). 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 ). 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 
. 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 
, 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 
While the number of protofilaments in eukaryotic microtubules can vary, the lateral interactions between them are conserved 
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 
. Because the loops that are involved in these interactions are also present in BtubA and BtubB 
, 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 (). 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 ( and S10
), but did not clarify whether BtubA/B tubes have an “A-lattice” without seam or a “B-lattice” with seam 
. The latter seems more likely, however, since the B-lattice has been resolved in eukaryotic 13-protofilament microtubules, and is therefore depicted in . Based on our data, the BtubA/B crystal structure 
, 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 
and they form closely related structures differing mainly in the number of protofilaments, we suggest they be referred to as “bacterial microtubules” (bMTs).
Structural model of “bacterial microtubules.”
BtubA/B tubes have a helical, microtubule-like lattice.
It has been suggested that BtubA and BtubB evolved from modern eukaryotic α- and/or β-tubulins 
. If this were true, a phylogenetic association linking BtubA and BtubB to α- and/or β-tubulin would be expected. As shown previously 
, BtubA and BtubB are clearly members of the eukaryotic clade of tubulins (). A protein motif search (Table S1
), an identity matrix (Table S2
), and various treeing methods (, 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 () 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 
. 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) 
. 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.
BtubA and BtubB represent two novel tubulin subfamilies in the eukaryotic clade of tubulins.
Model for the evolution of BtubA/B.
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
, 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 
. 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
bacterium was involved in the evolution of eukaryotes 
, 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 
, recombinantly expressed in E. coli 
, 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.