Phylogenetic analysis identifies many uncharacterized actin-like proteins (Alps) in bacteria: regulated polymerization, dynamic instability and treadmilling in Alp7A

Abstract

Actin, one of the most abundant proteins in the eukaryotic cell, also has an abundance of relatives in the eukaryotic proteome. To date though, only five families of actins have been characterized in bacteria. We have conducted a phylogenetic search and uncovered more than 35 highly divergent families of actin-like proteins (Alps) in bacteria. Their genes are found primarily on phage genomes, on plasmids and on integrating conjugative elements, and are likely to be involved in a variety of functions. We characterize three Alps and find that all form filaments in the cell. The filaments of Alp7A, a plasmid partitioning protein and one of the most divergent of the Alps, display dynamic instability and also treadmill. Alp7A requires other elements from the plasmid to assemble into dynamic polymers in the cell. Our findings suggest that most if not all of the Alps are indeed actin relatives, and that actin is very well represented in bacteria.

Fig. 1

Phylogenetic analysis identifies more than 35 families of bacterial actins. (A) Phlyogenetic tree of the bacterial actins-like proteins (Alps). Protein sequences were derived from the BLAST search series as described in the text and in the Experimental procedures. The tree was generated by the neighbor-joining method, and bootstrap values corresponding to confidence levels are indicated for selected branches. Color and number assignments for each family are arbitrary and do not signify relatedness. The five previously characterized families are indicated, as are representatives of three new families: Alp6A, previously designated as GP207 of Bacillus thuringiensis phage 0305ϕ8-36 (Thomas et al., 2007); Alp7A, previously designated as OrfB of Bacillus subtilis natto plasmid pLS20 (Meijer et al., 1995); Alp8A, previously designated as Orf250 of Proteus vulgaris plasmid Rts1 (Murata et al., 2002). See Table S1 in the Supporting Information for accession numbers and sources. (B) Alignment of the PHOSPHATE 1, CONNECT 1 and PHOSPHATE 2 regions (as per Bork et al., 1992) of human beta-actin and representatives of the eight families: B. subtilis MreB, B. subtilis FtsA, E. coli plasmid R1 ParM, M. magnetospirullum MamK, B. subtilis natto plasmid pLS32 AlfA, Alp 6A, Alp7A, Alp8A. Conserved residues corresponding to actin D11 (red), G13 (red), Q137 (blue), D154 (red), and G156 (red) are highlighted. (C-F) Fluorescence microscopy images of (C) pP_xylalp6A-gfp/DH5α, (D) pP_xylalp7A-gfp/MG1655, (E) pP_xylalp7A/MG1655, and (F) pP_trcalp8A-gfp/TOP10; the promoter is not the true P_trc promoter but the variant that is present in plasmid pDSW210 (Weiss et al., 1999). Scale bar (F) equals 1 μm; all images are at the same scale.

Fig. 2

Alp7A is required for plasmid stability; Alp7A-GFP can function in its place. (A) Plasmid derivatives of the alp7AR region of B. subtilis natto plasmid pLS20. The uppermost schematic depicts the alp7AR operon, the divergently transcribed orfA gene, and the intervening origin of replication. The insert in (1) mini-pLS20: the entire alp7AR operon is included as is a portion of orfA containing a putative replication terminator and decatenation site (Meijer et al., 1995); (2) mini-pLS20Δ(alp7AR), containing only the origin of replication; (3) mini-pLS20Δ(alp7A): as mini-pLS20, but alp7A is replaced by an in-frame deletion of the gene; (4) mini-pLS20alp7A-gfp: as mini-pLS20, but alp7A is replaced by alp7A-gfp; the sequence that is immediately upstream of alp7R in pLS20 is included so as to reproduce its native translational context. (B and C) Plasmid retention in logarithmic phase cultures in the absence of antibiotic selection: (B) mini-pLS20 (black), mini-pLS20Δ(alp7AR) (red), mini-pLS20Δ(alp7A) (blue); (C) mini-pLS20alp7A-gfp (green), mini-pLS20alp7A(D212A) (blue), mini-pLS20alp7A(E180A) (red). (D) Chromosomal constructs for plasmid complementation experiment in Panel E: P_xylalp7A-gfp or P_xylalp7A were integrated into the chromosome of B. subtilis strain PY79 at thrC. (E) Restoration of plasmid stability to mini-pLS20Δ(alp7A) by expression of Alp7A or Alp7A-GFP from inducible chromosomal constructs (Panel D). Strains were grown in the presence or absence of 0.25% xylose for 21 generations. (F) Immunoblot of PY79 transformants containing (lane 1) pLS20cat alp7A∷pMUTINalp7A-gfp; (lane 2) mini-pLS20alp7A-gfp; (lanes 3-7) xylose induction profile of Alp7A-GFP produced from the chromosome in mini-pLS20Δ(alp7A)/PY79 thrC∷xylR⁺P_xylAalp7A-gfp. The two panels are derived from a single filter that was probed with anti-Alp7A antisera.

Fig. 3

Alp7A forms filaments in vivo. (A) Immunoblot of B. subtilis strain PY79 or transformants of PY79 carrying plasmids containing alp7A or alp7A-gfp: (lane 1) no plasmid; (lane 2) pLS20cat; (lane 3) mini-pLS20; (lane 4) pLS20cat alp7A∷pMUTINalp7A-gfp; (lane 5) mini-pLS20alp7A-gfp. The filter was probed with anti-Alp7A antisera. (B-I) Fluorescence microscopy images of (B, C) mini-pLS20alp7A-gfp/PY79; (D, E) pLS20cat alp7A∷pMUTINalp7A-gfp/PY79 (not deconvolved); (F, G) mini-pLS20alp7A(D212A)-gfp/PY79; (H and I) mini-pLS20alp7A(E180A)-gfp/PY79. (B, D, F, H) Membranes stained with FM4-64. (J) FRAP analysis of Alp7A(E180A)-GFP. Left panel, fluorescence microscopy images pre-bleach, post-bleach, 30 s post-bleach, 60 s post-bleach; right panel, corresponding fluorescence intensity plot (linear scale, arbitrary units). Scale bar (I, J) equals 1 μm; all images are at the same scale.

Fig. 4

Alp7A filaments show dynamic instability in vivo. (A) Images from time-lapse fluorescence microscopy of mini-pLS20alp7A-gfp/PY79. Scale bar equals 1 μm; all images are at the same scale. (B-D) Growth and shrinkage of individual filaments. The filaments in (A) are tracked in (B); the white circles correspond to the filament on the left, the blue circles to the filaments on the right. The filament on the left corresponds to that of Movie S2. (E) Images from time-lapse fluorescence microscopy of pLS20cat alp7A∷pMUTINalp7A-gfp/PY79. Scale bar equals 1 micron; all images are to the same scale. (F-H) Growth and shrinkage of individual filaments. The filament in (E) corresponds to that of Movie S3 and is tracked in (F).

Fig. 5

Production of dynamic filaments requires additional elements of pLS20. (A-D) Filament length (microns) as a function of time (seconds) of two representative filaments in strain PY79 thrC∷xylR⁺P_xylAalp7A-gfp containing (A) no plasmid (see Movie S7); (B) mini-pLS20 (see Movie S8); (C) mini-pLS20Δ(alp7A) (see Movie S9); (D) mini-pLS20Δ(alp7AR).

Fig. 6

DNA containing alp7R and the DNA directly upstream of alp7A lowers the critical concentration for Alp7A filament formation. B. subtilis strain PY79 thrC∷xylR⁺P_xylAalp7A-gfp has a chromosomal copy of alp7A-gfp expressed from the xylose promoter (Fig. 2D). This strain or a transformant containing the mini-pLS20Δ(alp7A) plasmid were grown in various concentrations of xylose, and alp7A-gfp expression was monitored by immunoblot with anti-Alp7A antisera. (A-B) Xylose-induction profile of the strain lacking the mini-pLS20Δ(alp7A) plasmid (A) or containing the plasmid (B); (A, first lane) Alp7A-GFP produced from mini-pLS20alp7A-GFP. (C-L) Fluorescence microscopy images of glutaraldehyde-fixed cells of the strain lacking the plasmid (C-G), or containing the plasmid (H-L) after induction with xylose for 1 h at (C and H) 0.01%; (D and I) 0.025%; (E and J) 0.05%; (F and K) 0.10 %; (G and L) 0.25%. Scale bar (G) equals 1 μm; all images are at the same scale. (M) Percentage of cells containing at least one filament in strains containing the plasmid (green circles) or lacking the plasmid (black circles) after xylose induction. At least 100 cells were scored for each xylose concentration. (N) Quantitation of immunoblots in (A) black circles, and (B) green circles.

Fig. 7

Alp7A filaments colocalize with mini-pLS20, push plasmids apart, and treadmill. (A-F) Fluorescence microscopy images of fixed cells containing LacI-CFP tagged mini-pLS20alp7A-gfp. (A and D) Membranes (FM 4-64) and filaments (Alp7A-GFP); (B and E) Plasmid foci (LacI-CFP) and filaments (Alp7A-GFP); (C and F) Plasmid foci (LacI-CFP). Scale bar equals 1 μm; all images are at the same scale. (G) Plasmids per cell vs. cell length. (H) Filaments per cell vs. cell length. (I) Plasmids per cell vs. filaments per cell. The area of the spheres corresponds to the number of occurrences. For (G-I), 91 cells containing 173 filaments and 546 plasmid foci were examined. There was an average of 5.9 plasmid foci per cell, which is consistent with the reported plasmid copy number (Meijer et al., 1995), and there was an average of 1.9 filaments per cell. (J and K) Time-lapse of growing cells containing LacI-CFP tagged mini-pLS20alp7A-gfp, showing plasmids (blue) pushed apart by a filament (green). Images were collected at the indicated time intervals (seconds). (L) Photobleaching analysis reveals treadmilling behavior. A pre-bleach image (-4 seconds) and post-bleach images that were collected at 4 second intervals are shown. The distance between the left end of the filament (line a) and the bleached zone boundary (line b) increases with time, as the right end undergoes depolymerization (line c). (M) A schematic illustrating how fluxing can occur. If a filament containing a plus and minus end is treadmilling in place, then after photobleaching a small region (red circle), the bleached subunits (black circles) will “flux” in one direction as new subunits add to the plus end. (N) Photobleaching of filaments containing plasmids (blue) at each end. A pre-bleach image (-7 s) and 9 post-bleach images taken at the indicated times (seconds) are shown (left panel) beside three dimensional GFP fluorescence intensity plots corresponding to selected time points (right panel). Over time, the bleached zone (red bracket in plots) moves to the left as a region of lower fluorescence intensity (white bracket) increases in length.