Visualization of the attachment organelle and cytadherence proteins of Mycoplasma pneumoniae by immunofluorescence microscopy

A method was developed for protein localization in Mycoplasma pneumoniae by immunofluorescence microscopy. The P1 adhesin protein was revealed to be l ocated at least at one cell pole in all adhesive cells, as has been observe d 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 on e 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 o rganelle is formed next to the old one and migrates to the opposite cell po le before cell division. Double staining revealed that the accessory protei ns for cytadherence-HMW1, HMW3, P30, P90, P40, and P65-colocalized with the P1 adhesin in all cells. The localization of cytadherence proteins was als o examined in cytadherence-deficient mutant cells with a branched morpholog y. In M5 mutant cells, which lack the P90 and P40 proteins, HMW1, HMW3, pi, and P30 were focused at the cell poles of short branches, and P65 showed n o 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 w as distributed throughout the entire cell body, and no signal was detected for the other proteins. These results suggest that the cytadherence protein s are sequentially assembled to the attachment organelle with HMW1 first, H MW3, pi, P30, P90, and P40 next, and P65 last.