title: Localization of Mycoplasma pneumoniae cytadherence-associated protein HMW2 by fusion with green fluorescent protein: implications for attachment organelle structure.
abstract: The terminal organelle of the cell wall-less pathogenic bacterium Mycoplasma pneumoniae is a complex structure involved in adherence, gliding motility and cell division. This membrane-bound extension of the mycoplasma cell possesses a characteristic electron-dense core. A number of proteins having direct or indirect roles in M. pneumoniae cytadherence have been previously localized to the terminal organelle. However, the cytadherence-accessory protein HMW2, which is required for the stabilization of several terminal organelle components, has been refractory to antibody-based approaches to subcellular localization. In the current study, we constructed a sandwich fusion of HMW2 and enhanced green fluorescent protein (EGFP) and expressed this fusion in wild-type M. pneumoniae and the hmw2- mutant I-2. The fusion protein was produced in both backgrounds at wild-type levels and supported stabilization of proteins HMW1, HMW3 and P65, and haemadsorption function in mutant I-2. Furthermore, the fusion protein was fluorescent and localized specifically to the terminal organelle. However, the EGFP moiety appeared to interfere partially with processes related to cell division, as transformant cells exhibited an increased incidence of bifurcated attachment organelles. These data together with structural predictions suggest that HMW2 is the defining component of the electron-dense core of the terminal organelle.
authors.affiliation: Department of Microbiology, University of Georgia, Athens, GA 30602, USA.
authors.forename: Mitchell F
authors.initials: MF
authors.lastname: Balish
authors.forename: Ryan T
authors.initials: RT
authors.lastname: Santurri
authors.forename: Alessondra M
authors.initials: AM
authors.lastname: Ricci
authors.forename: Kyungok K
authors.initials: KK
authors.lastname: Lee
authors.forename: Duncan C
authors.initials: DC
authors.lastname: Krause
mesh_terms: D001422:Bacterial Adhesion; D001426:Bacterial Proteins / Q000235:genetics / Q000378:metabolism / Q000502:physiology; D002135:Calcium-Binding Proteins; D015815:Cell Adhesion Molecules / Q000378:metabolism; D049452:Green Fluorescent Proteins; D008164:Luminescent Proteins / Q000378:metabolism; D008562:Membrane Glycoproteins / Q000378:metabolism; D008565:Membrane Proteins / Q000032:analysis / Q000378:metabolism; D008854:Microscopy, Electron; D008954:Models, Biological; D009154:Mutation; D009177:Mycoplasma pneumoniae / Q000378:metabolism* / Q000648:ultrastructure; D009419:Nerve Tissue Proteins / Q000378:metabolism; D015388:Organelles / Q000378:metabolism*; D011993:Recombinant Fusion Proteins / Q000378:metabolism; D050863:Synaptotagmin I; D050857:Synaptotagmins
chemical_list: D001426:Bacterial Proteins; D002135:Calcium-Binding Proteins; D015815:Cell Adhesion Molecules; C099979:HMW1 protein, Mycoplasma; C067481:HMW3 protein, Mycoplasma; D008164:Luminescent Proteins; D008562:Membrane Glycoproteins; D008565:Membrane Proteins; D009419:Nerve Tissue Proteins; D011993:Recombinant Fusion Proteins; D050863:Synaptotagmin I; D050857:Synaptotagmins; D049452:Green Fluorescent Proteins