Use of fluorescent-protein tagging to determine the subcellular localization of mycoplasma pneumoniae proteins encoded by the cytadherence regulatory locus.

Mycoplasma pneumoniae lacks a cell wall but has internal cytoskeleton-like structures that are assumed to support the attachment organelle and asymmetric cell shape of this bacterium. To explore the fine details of the attachment organelle and the cytoskeleton-like structures, a fluorescent-protein tagging technique was applied to visualize the protein components of these structures. The focus was on the four proteins--P65, HMW2, P41, and P24--that are encoded in the crl operon (for "cytadherence regulatory locus"), which is known to be essential for the adherence of M. pneumoniae to host cells. When the P65 and HMW2 proteins were fused to enhanced yellow fluorescent protein (EYFP), a variant of green fluorescent protein, the fused proteins became localized at the attachment organelle, enabling visualization of the organelles of living cells by fluorescence microscopy. The leading end of gliding M. pneumoniae cells, expressing the EYFP-P65 fusion, was observed as a focus of fluorescence. On the other hand, when the P41 and P24 proteins were labeled with EYFP, the fluorescence signals of these proteins were observed at the proximal end of the attachment organelle. Coexpression of the P65 protein labeled with enhanced cyan fluorescent protein clearly showed that the sites of localization of P41 and P24 did not overlap that of P65. The localization of P41 and P24 suggested that they are also cytoskeletal proteins that function in the formation of unknown structures at the proximal end of the attachment organelle. The fluorescent-protein fusion technique may serve as a powerful tool for identifying components of cytoskeleton-like structures and the attachment organelle. It can also be used to analyze their assembly.