Whole Surface Image of Mycoplasma mobile, Suggested by Protein Identification and Immunofluorescence Microscopy

Abstract

Mycoplasma mobile, a freshwater fish pathogen featured with robust gliding motility, binds to the surface of the gill, where it then colonizes. Here, to obtain a whole image of its cell surface, we identified the proteins exposed on the surface using the following methods. (i) The cell surface was labeled with sulfosuccinimidyl-6-(biotinamido) hexanoate and recovered by an avidin column. (ii) The cells were subjected to phase partitioning using Triton X-114, and the hydrophobic proteins were recovered. (iii) The membrane fraction was analyzed by two-dimensional gel electrophoresis. These recovered proteins were subjected to peptide mass fingerprinting, and a final list of 36 expressed surface proteins was established. The ratio of identified proteins to whole surface proteins was estimated through two-dimensional gel electrophoresis of the membrane fraction. The localization of three newly found proteins, Mvsps C, E, and F, has been clarified by immunofluorescence microscopy. Integrating all information, a whole image of the cell surface showed that the proteins for gliding that were localized at the base of the protrusion of flask-shaped M. mobile account for more than 12% of all surface proteins and that Mvsps, surface variants that were localized at both parts other than the neck, account for 49% of all surface proteins.

Localization of P42 and F 1 -ATPase α-Subunit Homolog of the Gliding Machinery in Mycoplasma mobile Revealed by Newly Developed Gene Manipulation and Fluorescent Protein Tagging

“…Gli349, MvspI, and DNA were localized at the neck, the head and body, and the body, respectively, in good agreement with previous studies (Fig. 1A) (12,13,55,58). P42 was colocalized with Gli349 but not with MvspI or with DNA localized in regions other than the neck, suggesting that P42 is involved in the gliding mechanism.…”

“…To date, the subcellular localizations have been clarified for 13 proteins in M. mobile (Fig. 1), and all proteins localizing at the neck part have been suggested to be involved in the gliding mechanism (3,22,55). In the present study, two other proteins were shown to be localized on the gliding machinery (Fig.…”

“…P42, which appears to have developed from an ancestor shared with FtsZ, has a special role in the gliding mechanism. This protein should be localized inside the cell at some structure other than the jellyfish tentacle or have an occluded position on the surface because a previous study in which surface proteins were biotinylated did not detect exposure of P42 (55), and it was not identified in the fraction of the jellyfish structure (22).…”

“…3B). To improve the fluorescence intensity, we tried using the promoters for the gene encoding an abundant M. mobile surface protein, MvspI (46,55,56), and an abundant Spiroplasma citri surface protein, spiralin (data not shown) (57). However, neither construct showed any fluorescence signal.…”

Novel mechanisms power bacterial gliding motility

“…The gliding machinery of M. mobile generates a force up to 27 pN, which enables cells to glide smoothly on a broad range of surfaces at a speed of 2.0-4.5 lm s 21 (Miyata et al, 2002). The M. mobile cell surface is covered by membrane-anchored proteins (Wu and Miyata, 2012). Examination of cells by electron microscopy revealed spike-like structures approximately 50 nm in length around the neck area of the bowling-pin-shaped cells (Miyata and Petersen, 2004).…”