Use of Fluorescent-Protein Tagging To Determine the Subcellular Localization of Mycoplasma pneumoniae Proteins Encoded by the Cytadherence Regulatory Locus

Abstract

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 Mycoplasma pneumoniae, one of the smallest self-replicating bacteria known, is a causative agent of bronchitis and primary atypical pneumonia in humans (43,44). M. pneumoniae lacks a cell wall and hence has a pleomorphic cell shape. However, a majority of M. pneumoniae cells in cultures are filamentous and have a differentiated terminal structure at one pole. This terminal structure, the attachment organelle, is a tapered membrane protrusion responsible for the adherence of M. pneumoniae to host respiratory epithelium (cytadherence) (23, 24). The attachment organelle renders M. pneumoniae cells asymmetric and functions as a leading end for gliding motility. This organelle also may have a role in initiating cell division in M. pneumoniae, because the bifurcation of the attachment organelle seems to occur prior to the binary fission of M. pneumoniae (2,6,7,25,26,34,36,48).The attachment organelle and polar filamentous cell shape of M. pneumoniae are thought to be stabilized by intracellular cytoskeleton-like structures, which have been observed in electron micrographs of M. pneumoniae (5,25,33). The most remarkable architectural feature of the cytoskeleton-like structures is the electron-dense core, a rod-like structure that exists longitudinally at the center of the attachment organelle (33). This rod-like structure, measuring about 300 nm long and 80 nm thick, has a knob at the distal end (terminal button) (33,45). A network of fibrous structures is also observed in the cytoplasm of M. pneumoniae (33). These cytoskeleton-like structures are major components of the Triton X-100-insoluble fraction of M. pneumoniae cells (Triton shell) and are thought to have a scaffold-like function upon which other cell components construct M. pneumoniae cells (45,51).A recent report indicated that the Triton X-100-insoluble fraction contains about 100 proteins, including most of the known proteins required for cytadherence (P1, B, C, HMW1, HMW2, and HMW3) (45). These cytadherence-related proteins are believed to be the main components of the attachment organelle and are encoded in three operons, designated p1, hmw, and crl, in the genome (24,25). Protein P1 (encoded in the p1 operon) is a major adhesin molecule responsible for cytadherence and is densely clustered at the surface of the attachment organelle (9,18,26,48). Proteins B, C, HMW1, HMW2, and HMW3, called cytadherence accessory proteins,...

Terminal organelle development in the cell wall-less bacterium Mycoplasma pneumoniae

“…To begin to test that model, we examined fluorescence patterns in growing mycoplasma cultures producing either P41-YFP or P65-YFP and P30-cyan fluorescent protein (CFP) for their relative timing of appearance. Proteins P65 and P41 are novel cytoskeletal elements of unknown function expressed from the same transcriptional unit in M. pneumoniae and localize to the distal end and base, respectively, of the terminal organelle (24,25). Transformants producing P65-YFP alone exhibited a pattern of new terminal organelle development comparable to that of P30-YFP, and in cells producing both fusions, P30-CFP and P65-YFP were observed in pairs in Ͼ95% of the cells examined, suggesting near-concurrent incorporation into the terminal organelle (data not shown).…”

Protein Kinase/Phosphatase Function Correlates with Gliding Motility in Mycoplasma pneumoniae

“…Accordingly, their distinct functions in gliding motility are difficult to define by mutagenesis alone. Given that the M. pneumoniae genome exhibits no homology to elements of defined gliding mechanisms (2,3), including those of other gliding mycoplasmas (11)(12)(13)(14), it was necessary to perform saturating transposon mutagenesis in order to identify the components specific to gliding (15). Transposon insertions in the genes encoding the cytoskeletal proteins P41 and P65 (MPN311 and MPN309, respectively) (16), which are known to localize to the terminal structure of M. pneumoniae (7), produce gliding-deficient phenotypes (15,17), but the defect in each of them is not clearly associated with an actual gliding motor and suggests little about structure and function of the motor.…”

Processing Is Required for a Fully Functional Protein P30 in Mycoplasma pneumoniae Gliding and Cytadherence

“…To assess if the reduced function was due to the lower steady-state levels of pre-P30, we engineered this derivative under the control of the tuf promoter (tuf-pre-P30) for enhanced expression (18). This resulted in pre-P30 levels more similar to that of wild-type P30, with no mature P30 detected by immunoblotting (Fig.…”