Genes for the cytoskeletal protein tubulin in the bacterial genus Prosthecobacter

Abstract

Tubulins, the protein constituents of the microtubule cytoskeleton, are present in all known eukaryotes but have never been found in the Bacteria or Archaea. Here we report the presence of two tubulin-like genes [bacterial tubulin a (btuba) and bacterial tubulin b (btubb)] in bacteria of the genus Prosthecobacter (Division Verrucomicrobia). In this study, we investigated the organization and expression of these genes and conducted a comparative analysis of the bacterial and eukaryotic protein sequences, focusing on their phylogeny and 3D structures. The btuba and btubb genes are arranged as adjacent loci within the genome along with a kinesin light chain gene homolog. RT-PCR experiments indicate that these three genes are cotranscribed, and a probable promoter was identified upstream of btuba. On the basis of comparative modeling data, we predict that the Prosthecobacter tubulins are monomeric, unlike eukaryotic alpha and beta tubulins, which form dimers and are therefore unlikely to form microtubule-like structures. Phylogenetic analyses indicate that the Prosthecobacter tubulins are quite divergent and do not support recent horizontal transfer of the genes from a eukaryote. The discovery of genes for tubulin in a bacterial genus may offer new insights into the evolution of the cytoskeleton.

Fig 1.

Organization of the P. dejongeii tubulin genes in the genome. A probable promoter sequence upstream of btuba is marked. Consensus sequences and Shine–Dalgarno sequences are underscored.

Fig 2.

Agarose gel showing RT-PCR products with and without the addition of RT. Lane 1, 100-bp ladder (New England Biolabs); lane 2, BtubA primers − RT; lane 3, BtubA primers + RT; lane 4, BtubB primers − RT; lane 5, BtubB primers + RT.

Fig 3.

Comparison of the refined crystal structure of the bovine αβ tubulin dimer (A) with the modeled P. dejongeii BtubA/BtubB structures (B). Each structure indicates the position of GTP and a Mg²⁺ ion at the intradimer active site (N-site). The hydrophobic C-terminal loop and helices are marked in magenta. Rossmann folds are marked in green.

Fig 4.

Several notable changes are present at the P. dejongeii tubulin intradimer interface. Lysine residue 254, which neutralizes the N-site GTP phosphate groups (A) is highly conserved in eukaryotic β tubulin but is substituted for glutamic acid in BtubB (B). A key salt bridge at the interface of eukaryotic tubulins (C) is absent in the P. dejongeii tubulin dimer (D). BtubA and BtubB are >35% identical to bovine α and β tubulin. Models ranging from 1.5 to 2.5 Å rms deviation for this degree of sequence relationship are routinely produced using these methods (15, 16); thus, our models can be considered highly reliable. Figures were generated with MOLSCRIPT (21) and RASTER 3D (22).

Fig 5.

Phylogenetic trees showing the relationship of BtubA and BtubB relative to eukaryotic α and β tubulins (A) and other members of the tubulin family (B). Trees presented are parsimony trees rooted at the midpoint in A and outgroup rooted with ζ tubulin in B. Circles indicate bootstrap values. Nodes supported at >75% in the majority of analyses are indicated by the filled circles. Nodes supported at 50–74% in most analyses are indicated by the open circles. Unsupported nodes (<50%) have no circle. (Bar = 0.1 substitutions per site.)