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Journal of Bacteriology, March 2001, p. 1621-1630, Vol. 183, No. 5
0021-9193/01/$04.00+0   DOI: 10.1128/JB.183.5.1621-1630.2001
Copyright © 2001, American Society for Microbiology. All rights reserved.

Visualization of the Attachment Organelle and Cytadherence Proteins of Mycoplasma pneumoniae by Immunofluorescence Microscopy

Shintaro Seto,1 Gerlinde Layh-Schmitt,2,dagger Tsuyoshi Kenri,3 and Makoto Miyata1,*

Department of Biology, Graduate School of Science, Osaka City University, Sumiyoshi-ku, Osaka 558-8585,1 and Department of Safety Research on Biologics, National Institute of Infectious Diseases, 4-7-1 Gakuen, Musashimurayama, Tokyo, 208-0011,3 Japan, and Hygiene-Institut, Abt. Hygiene und Medizinische Mikrobiologie, Universität Heidelberg, 69120 Heidelberg, Germany2

Received 28 September 2000/Accepted 14 November 2000

A method was developed for protein localization in Mycoplasma pneumoniae by immunofluorescence microscopy. The P1 adhesin protein was revealed to be located at least at one cell pole in all adhesive cells, as has been observed by immunoelectron microscopy. Cell images were classified according to P1 localization and assigned by DNA content. Cells with a single P1 focus at one cell pole had a lower DNA content than cells with two foci, at least one of which was positioned at a cell pole. Those with one focus at each cell pole had the highest DNA content, suggesting that the nascent attachment organelle is formed next to the old one and migrates to the opposite cell pole before cell division. Double staining revealed that the accessory proteins for cytadherence---HMW1, HMW3, P30, P90, P40, and P65---colocalized with the P1 adhesin in all cells. The localization of cytadherence proteins was also examined in cytadherence-deficient mutant cells with a branched morphology. In M5 mutant cells, which lack the P90 and P40 proteins, HMW1, HMW3, P1, and P30 were focused at the cell poles of short branches, and P65 showed no signal. In M7 mutant cells, which produce a truncated P30 protein, HMW1, HMW3, P1, P90, and P40 were focused, and P65 showed no signal. In M6 mutant cells, which express no HMW1 and a truncated P30 protein, the P1 adhesin was distributed throughout the entire cell body, and no signal was detected for the other proteins. These results suggest that the cytadherence proteins are sequentially assembled to the attachment organelle with HMW1 first, HMW3, P1, P30, P90, and P40 next, and P65 last.

* Corresponding author. Mailing address: Department of Biology, Graduate School of Science, Osaka City University, Sumiyoshi-ku, Osaka 558-8585, Japan. Phone 81(6)6605 3157. Fax: 81(6)6605 2522. E-mail: miyata{at}sci.osaka-cu.ac.jp.

dagger Present address: Procter & Gamble Pharmaceuticals, Health Care Research Center, Mason, OH 45040.

Journal of Bacteriology, March 2001, p. 1621-1630, Vol. 183, No. 5
0021-9193/01/$04.00+0   DOI: 10.1128/JB.183.5.1621-1630.2001
Copyright © 2001, American Society for Microbiology. All rights reserved.


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