Pradel et al. 10.1073/pnas.0603760103. |
Fig. 5. Immunoblot analyses of MamK in AMB-1 and E. coli (A) and fluorescence images of TG1/p2020 (B). (A) Crude extracts (20 mg proteins each) were prepared from TG1/p6020 (lane 1), or from TG1/pBAD24 (empty plasmid) (lane 2), from M. magneticum AMB-1 wild type strain (lane 3), from a spontaneous mam mutant that loses the mamK gene (checked by PCR) (lane 4), and resolved on 12.5% SDS gels. The immunoblot was performed by the ECL method according to the manufacturer's instructions (Amersham Pharmacia Biosciences). Purification of MamK and generation of polyclonal antibody was carried out as described by Taoka et al. (1). The antibody was used at 1:10,000 dilution. (B) Fluorescence images of E. coli strain TG1 expressing MamK-GFP from p2020.
1. Taoka A, Asada R, Sasaki H, Anzawa K, Wu LF, Fukumori Y (2006) J Bacteriol 188:3805-3812.
Fig. 6. Multiple alignment of actin-like cytoskeletal proteins. The following sequences were downloaded from GenBank or SwissProt: eukaryotic actin proteins from yeast (ACT_YEAST, P60010), from rabbit 1(ATN_ActinRabi GI:229690); MreB proteins from Thermotoga maritima (MreB_THEMA1), Bacillus subtilis (MBL_BACSU and MREB_BACSU), E. coli (MREB_ECOLI), Caulobacter crescentus (MreB_CAUCR), Magnetospirillum magnetotacticum MS-1 (MreB_MS1 ZP_00055538), Magnetococcus sp. MC-1 (MreB_MC1, EAN29762.1), Magnetospirillum magneticum AMB-1 (MreB_AMB1, YP_422876.1); ParM proteins from Serratia marcescens (ParM_SERMA, Q6MY19_SERMA), E. coli (PARM_ECOLI), E. coli O157:H7, (ParM_0157 gi|13362102), Shigella dysenteriae Sd197 (ParM_Sdysen YP_406076.1), plasmid R-100 (ParM_R100, NP_052909.1); and MamK proteins from Magnetococcus sp. MC-1 (MamK_MC1, EAN27146.1) and Magnetospirillum magneticum AMB-1 (MamK_AMB1 YP_420328). The annotation of M. magnetotacticum MS-1 MamK (ZP_00054405.2) lacks the first 25 residues compared with its counterpart from Magnetospirillum magneticum AMB-1 (blue box). Because we showed that this segment encompassing part of the first phosphate-binding site is essential for the assembly of the M. magneticum AMB-1 MamK filament, we extended the MamK sequence of MS-1 (MamKc_MS1) to the first Met upstream of the annotated MamK according to the genomic sequence Contig3824 from 3928-4920 (http://genome.jgi-psf.org/cgi bin/blastOutput?db = magma&jobId = 290469&jobToken = 1452170032). We removed the first 13 residues from the annotated M. gryphiswaldense MSR-1 MamK (Q6NE59_9PROT), yielding MamKt_MRS-1, because they are absent from other MamK, ParM, or MreB proteins. Multiple sequence alignment and phylogenetic analyses were performed with CLC benchwork software.
Movie 1. Time-lapse experimental results of MamK filament assembly in individual cells. Shown are multiple sites of nucleation and assembly to a filament.
Movie 2. Time-lapse experimental results of MamK filament assembly in individual cells. Shown is asymmetric elongation of the filament.
Movie 3. Time-lapse experimental results of MamK filament assembly in individual cells. Shown is the MamK filament assembly in septating cells.