Cryo‐electron tomography analyses of terminal organelle mutants suggest the motility mechanism of Mycoplasma genitalium

The terminal organelle of Mycoplasma genitalium is responsible for bacterial adhesion, motility and pathogenicity. Localized at the cell tip, it comprises an electron‐dense core that is anchored to the cell membrane at its distal end and to the cytoplasm at its proximal end. The surface of the terminal organelle is also covered with adhesion proteins. We performed cellular cryoelectron tomography on deletion mutants of eleven proteins that are implicated in building the terminal organelle, to systematically analyze the ultrastructural effects. These data were correlated with microcinematographies, from which the motility patterns can be quantitatively assessed. We visualized diverse phenotypes, ranging from mild to severe cell adhesion, motility and segregation defects. Based on our observations, we propose a double‐spring ratchet model for the motility mechanism that explains our current and previous observations. Our model, which expands and integrates the previously suggested inchworm model, allocates specific functions to each of the essential components of this unique bacterial motility system.     

P65 truncation impacts P30 dynamics during Mycoplasma pneumoniae gliding

The cell wall-less prokaryote Mycoplasma pneumoniae is a major cause of community-acquired bronchitis and pneumonia in humans. Colonization is mediated largely by a differentiated terminal organelle, which is also the leading end in gliding motility. Cytadherence-associated proteins P30 and P65 appear to traffic concurrently to the distal end of developing terminal organelles. Here, truncation of P65 due to transposon insertion in the corresponding gene resulted in lower gliding velocity, reduced cytadherence, and decreased steady-state levels of several terminal organelle proteins, including P30. Utilizing fluorescent protein fusions, we followed terminal organelle development over time. New P30 foci appeared at nascent terminal organelles in P65 mutants, as in the wild type. However, with forward cell motility, P30 in the P65 mutants appeared to drag toward the trailing cell pole, where it was released, yielding a fluorescent trail to which truncated P65 colocalized. In contrast, P30 was only rarely observed at the trailing end of gliding wild-type cells. Complementation with the recombinant wild-type P65 allele by transposon delivery restored P65 levels and stabilized P30 localization to the terminal organelle.     

Functional domain analysis of the Mycoplasma pneumoniae co‐chaperone TopJ

Colonization of conducting airways of humans by the prokaryote Mycoplasma pneumoniae is mediated by a differentiated terminal organelle important in cytadherence, gliding motility and cell division. TopJ is a predicted J‐domain co‐chaperone also having domains unique to mycoplasma terminal organelle proteins and is essential for terminal organelle function, as well as stabilization of protein P24, which is required for normal initiation of terminal organelle formation. J‐domains activate the ATPase of DnaK chaperones, facilitating peptide binding and proper protein folding. We performed mutational analysis of the predicted J‐domain, central acidic and proline‐rich (APR) domain, and C‐terminal domain of TopJ and assessed the phenotypic consequences when introduced into an M. pneumoniae topJ mutant. A TopJ derivative with amino acid substitutions in the canonical J‐domain histidine–proline–aspartic acid motif restored P24 levels but not normal motility, morphology or cytadherence, consistent with a J‐domain co‐chaperone function. In contrast, TopJ derivatives having APR or C‐terminal domain deletions were less stable and failed to restore P24, but resulted in normal morphology, intermediate gliding motility and cytadherence levels exceeding that of wild‐type cells. Results from immunofluorescence microscopy suggest that both the APR and C‐terminal domains, but not the histidine–proline–aspartic acid motif, are critical for TopJ localization to the terminal organelle.     

Mycoplasma pneumoniae J-domain protein required for terminal organelle function

The cell wall-less prokaryote Mycoplasma pneumoniae causes tracheobronchitis and primary atypical pneumonia in humans. Colonization of the respiratory epithelium requires proper assembly of a complex, multifunctional, polar terminal organelle. Loss of a predicted J-domain protein also having domains unique to mycoplasma terminal organelle proteins (TopJ) resulted in a non-motile, adherence-deficient phenotype. J-domain proteins typically stimulate ATPase activity of Hsp70 chaperones to bind nascent peptides for proper folding, translocation or macromolecular assembly, or to resolve stress-induced protein aggregates. By Western immunoblotting all defined terminal organelle proteins examined except protein P24 remained at wild-type levels in the topJ mutant; previous studies established that P24 is required for normal initiation of terminal organelle formation. Nevertheless, terminal organelle proteins P1, P30, HMW1 and P41 failed to localize to a cell pole, and when evaluated quantitatively, P30 and HMW1 foci were undetectable in >40% of cells. Complementation of the topJ mutant with the recombinant wild-type topJ allele largely restored terminal organelle development, gliding motility and cytadherence. We propose that this J-domain protein, which localizes to the base of the terminal organelle in wild-type M. pneumoniae , functions in the late stages of assembly, positioning, or both, of nascent terminal organelles.     

Localization of Mycoplasma pneumoniae cytadherence-associated protein HMW2 by fusion with green fluorescent protein: implications for attachment organelle structure

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.     

Deletion of the Mycoplasma genitalium MG_217 gene modifies cell gliding behaviour by altering terminal organelle curvature

Motility is often a virulence factor of pathogenic bacteria. Although recent works have identified genes involved in gliding motility of mycoplasmas, little is known about the mechanisms governing the cell gliding behaviour. Here, we report that Mycoplasma genitalium MG217 is a novel protein involved in the gliding apparatus of this organism and it is, at least, one of the genes that are directing cells to move in narrow circles when they glide. In the absence of MG_217 gene, cells are still able to glide but they mainly move drawing erratic or wide circular paths. This change in the gliding behaviour correlates with a rearrangement in the terminal organelle disposition, suggesting that the terminal organelle operates as a guide to steer the mycoplasma cell in a specific direction. Immunogold labelling reveals that MG217 protein is located intracellular at the distal end of the terminal organelle, between the cell membrane and the terminal button. Such location is consistent with the idea that MG217 could act as a modulator of the terminal organelle curvature, allowing cells to move in specific directions.     

Cytoskeletal protein P41 is required to anchor the terminal organelle of the wall-less prokaryote Mycoplasma pneumoniae

The cell wall-less prokaryote Mycoplasma pneumoniae approaches the minimal requirements for a cell yet produces a complex terminal organelle that mediates cytadherence and gliding motility. Here we explored the molecular nature of the M. pneumoniae gliding machinery, utilizing fluorescent protein fusions and digital microcinematography to characterize gliding-altered mutants having transposon insertions in MPN311, encoding the cytoskeletal protein P41. Disruption of MPN311 resulted in loss of P41 and P24, the downstream gene product. Gliding ceases in wild-type M. pneumoniae during terminal organelle development, which occurs at the cell poles adjacent to an existing structure. In contrast, terminal organelle development in MPN311 mutants did not necessarily coincide with gliding cessation, and new terminal organelles frequently formed at lateral sites. Furthermore, new terminal organelles exhibited gliding capacity quickly, unlike wild-type M. pneumoniae. P41 and P24 localize at the base of the terminal organelle; in their absence this structure detached from the cell body of motile and dividing cells but retained gliding capacity and thus constitutes the gliding apparatus. Recombinant wild-type P41 restored cell integrity, establishing a role for this protein in anchoring the terminal organelle to the cell body.     

Triclosan: a widely used biocide and its link to antibiotics

Mycoplasmas are cell wall-less bacteria at the low extreme in genome size in the known prokaryote world, and the minimal nature of their genomes is clearly reflected in their metabolic and regulatory austerity. Despite this apparent simplicity, certain species such as Mycoplasma pneumoniae possess a complex terminal organelle that functions in cytadherence, gliding motility, and cell division. The attachment organelle is a membrane-bound extension of the cell and is characterized by an electron-dense core that is part of the mycoplasma cytoskeleton, defined here for working purposes as the protein fraction that remains after extraction with the detergent Triton X-100. This review focuses on the architecture and assembly of the terminal organelle of M. pneumoniae. Characterizing the downstream consequences of defects involving attachment organelle components has made it possible to begin to elucidate the probable sequence of certain events in the biogenesis of this structure.     

A minimized motile machinery for Mycoplasma genitalium

The cell wall‐less bacterium Mycoplasma genitalium uses specialized adhesins located at the terminal organelle to adhere to host cells and surfaces. The terminal organelle is a polar structure protruding from the cell body that is internally supported by a cytoskeleton and also has an important role in cell motility. We have engineered a M. genitalium null mutant for MG491 protein showing a massive downstream destabilization of proteins involved in the terminal organelle organization. This mutant strain exhibited striking similarities with the previously isolated MG_218 null mutant strain. Upon introduction of an extra copy of MG_318 gene in both strains, the amount of main adhesins P140 and P110 dramatically increased. These strains were characterized by microcinematography, epifluorescence microscopy and cryo‐electron microcopy, revealing the presence of motile cells and filaments in the absence of many proteins considered essential for cell adhesion and motility. These results indicate that adhesin complexes play a major role in the motile machinery of M. genitalium and demonstrate that the rod element of the cytoskeleton core is not the molecular motor propelling mycoplasma cells. These strains containing a minimized motile machinery also provide a valuable cell model to investigate the adhesion and gliding properties of this human pathogen.     

Domain analysis of protein P30 in Mycoplasma pneumoniae cytadherence and gliding motility

The cell wall-less prokaryote Mycoplasma pneumoniae causes bronchitis and atypical pneumonia in humans. Mycoplasma attachment and gliding motility are required for colonization of the respiratory epithelium and are mediated largely by a differentiated terminal organelle. P30 is a membrane protein at the distal end of the terminal organelle and is required for cytadherence and gliding motility, but little is known about the functional role of its specific domains. In the current study, domain deletion and substitution derivatives of P30 were engineered and introduced into a P30 null mutant by transposon delivery to assess their ability to rescue P30 function. Domain deletions involving the extracellular region of P30 severely impacted protein stability and adherence and gliding function, as well as the capacity to stabilize terminal organelle protein P65. Amino acid substitutions in the transmembrane domain revealed specific residues uniquely required for P30 stability and function, perhaps to establish correct topography in the membrane for effective alignment with binding partners. Deletions within the predicted cytoplasmic domain did not affect P30 localization or its capacity to stabilize P65 but markedly impaired gliding motility and cytadherence. The larger of two cytoplasmic domain deletions also appeared to remove the P30 signal peptide processing site, suggesting a larger leader peptide than expected. We propose that the P30 cytoplasmic domain may be required to link P30 to the terminal organelle core, to enable the P30 extracellular domain to achieve a functional conformation, or perhaps both.     

Mycoplasmas: a distinct cytoskeleton for wall-less bacteria

The bacterial genus Mycoplasma includes a large number of highly genomically-reduced species which in nature are associated with hosts either commensally or pathogenically. Several Mycoplasma species, including Mycoplasma pneumoniae, feature a multifunctional polar structure, the terminal organelle. Essential for colonization of the host and for gliding motility, the terminal organelle is associated with an internal cytoskeleton crucial to its assembly and function. This cytoskeleton is structurally and compositionally novel as compared with the cytoskeletons of other organisms, including other bacteria, is also involved in the cell division process. In this review we discuss the cytoskeletal structures and protein components of the attachment organelle and how they might interact and contribute to its various functions.     

Functional analysis of the Mycoplasma genitalium MG312 protein reveals a specific requirement of the MG312 N-terminal domain for gliding motility

The human pathogen Mycoplasma genitalium is known to mediate cell adhesion to target cells by the attachment organelle, a complex structure also implicated in gliding motility. The gliding mechanism of M. genitalium cells is completely unknown, but recent studies have begun to elucidate the components of the gliding machinery. We report the study of MG312, a cytadherence-related protein containing in the N terminus a box enriched in aromatic and glycine residues (EAGR), which is also exclusively found in MG200 and MG386 gliding motility proteins. Characterization of an MG_312 deletion mutant obtained by homologous recombination has revealed that the MG312 protein is required for the assembly of the M. genitalium terminal organelle. This finding is consistent with the intermediate-cytadherence phenotype and the complete absence of gliding motility exhibited by this mutant. Reintroduction of several MG_312 deletion derivatives into the MG_312 null mutant allowed us to identify two separate functional domains: an N-terminal domain implicated in gliding motility and a C-terminal domain involved in cytadherence and terminal organelle assembly functions. In addition, our results also provide evidence that the EAGR box has a specific contribution to mycoplasma cell motion. Finally, the presence of a conserved ATP binding site known as a Walker A box in the MG312 N-terminal region suggests that this structural protein could also play an active function in the gliding mechanism.     

The EAGR box structure: a motif involved in mycoplasma motility

Mycoplasma genitalium is an emerging human pathogen with the smallest genome found among self‐replicating organisms. M. genitalium presents a complex cytoskeleton with a differentiated protrusion known as the terminal organelle. This polar structure plays a central role in functions essential for the virulence of the microorganism, such as motility and cell‐host adhesion. A well‐conserved Enriched in Aromatic and Glycine Residues motif, the EAGR box, is present in many of the proteins found in the terminal organelle. We determined the crystal structure of the globular domain from M. genitalium MG200 that contains an EAGR box. This structural information is the first at near atomic resolution for the components of the terminal organelle. The structure revealed a dimer stabilized by a compact hydrophobic core that extends throughout the dimer interface. Monomers present a new fold that contains an accurate intra‐subunit symmetry relating two conspicuous hairpins. Some features, such as the domain plasticity and the presence and organization of the intra‐ and inter‐subunit symmetry axes, support a role for the EAGR box in protein–protein interactions. Genetic, biochemical and microcinematography analyses of MG200 variants lacking the EAGR box containing domain confirm the relevant and specific association of this domain with cell motility.     

Proteins P24 and P41 function in the regulation of terminal-organelle development and gliding motility in Mycoplasma pneumoniae

Mycoplasma pneumoniae is a major cause of bronchitis and atypical pneumonia in humans. This cell wall-less bacterium has a complex terminal organelle that functions in cytadherence and gliding motility. The gliding mechanism is unknown but is coordinated with terminal-organelle development during cell division. Disruption of M. pneumoniae open reading frame MPN311 results in loss of protein P41 and downstream gene product P24. P41 localizes to the base of the terminal organelle and is required to anchor the terminal organelle to the cell body, but during cell division, MPN311 insertion mutants also fail to properly regulate nascent terminal-organelle development spatially or gliding activity temporally. We measured gliding velocity and frequency and used fluorescent protein fusions and time-lapse imaging to assess the roles of P41 and P24 individually in terminal-organelle development and gliding function. P41 was necessary for normal gliding velocity and proper spatial positioning of new terminal organelles, while P24 was required for gliding frequency and new terminal-organelle formation at wild-type rates. However, P41 was essential for P24 function, and in the absence of P41, P24 exhibited a dynamic localization pattern. Finally, protein P28 requires P41 for stability, but analysis of a P28(-) mutant established that the MPN311 mutant phenotype was not a function of loss of P28.     

P110 and P140 Cytadherence-Related Proteins Are Negative Effectors of Terminal Organelle Duplication in Mycoplasma genitalium

Background

The terminal organelle is a complex structure involved in many aspects of the biology of mycoplasmas such as cell adherence, motility or cell division. Mycoplasma genitalium cells display a single terminal organelle and duplicate this structure prior to cytokinesis in a coordinated manner with the cell division process. Despite the significance of the terminal organelle in mycoplasma virulence, little is known about the mechanisms governing its duplication.

Methodology/Principal Findings

In this study we describe the isolation of a mutant, named T192, with a transposon insertion close to the 3′ end of the mg192 gene encoding for P110 adhesin. This mutant shows a truncated P110, low levels of P140 and P110 adhesins, a large number of non-motile cells and a high frequency of new terminal organelle formation. Further analyses revealed that the high rates of new terminal organelle formation in T192 cells are a direct consequence of the reduced levels of P110 and P140 rather than to the expression of a truncated P110. Consistently, the phenotype of the T192 mutant was successfully complemented by the reintroduction of the mg192 WT allele which restored the levels of P110 and P140 to those of the WT strain. Quantification of DAPI-stained DNA also showed that the increase in the number of terminal organelles in T192 cells is not accompanied by a higher DNA content, indicating that terminal organelle duplication does not trigger DNA replication in mycoplasmas.

Conclusions/Significance

Our results demonstrate the existence of a mechanism regulating terminal organelle duplication in M. genitalium and strongly suggest the implication of P110 and P140 adhesins in this mechanism.     

Transposon mutagenesis identifies genes associated with Mycoplasma pneumoniae gliding motility

The wall-less prokaryote Mycoplasma pneumoniae, a common cause of chronic respiratory tract infections in humans, is considered to be among the smallest and simplest known cells capable of self-replication, yet it has a complex architecture with a novel cytoskeleton and a differentiated terminal organelle that function in adherence, cell division, and gliding motility. Recent findings have begun to elucidate the hierarchy of protein interactions required for terminal organelle assembly, but the engineering of its gliding machinery is largely unknown. In the current study, we assessed gliding in cytadherence mutants lacking terminal organelle proteins B, C, P1, and HMW1. Furthermore, we screened over 3,500 M. pneumoniae transposon mutants individually to identify genes associated with gliding but dispensable for cytadherence. Forty-seven transformants having motility defects were characterized further, with transposon insertions mapping to 32 different open reading frames widely distributed throughout the M. pneumoniae genome; 30 of these were dispensable for cytadherence. We confirmed the clonality of selected transformants by Southern blot hybridization and PCR analysis and characterized satellite growth and gliding by microcinematography. For some mutants, satellite growth was absent or developed more slowly than that of the wild type. Others produced lawn-like growth largely devoid of typical microcolonies, while still others had a dull, asymmetrical leading edge or a filamentous appearance of colony spreading. All mutants exhibited substantially reduced gliding velocities and/or frequencies. These findings significantly expand our understanding of the complexity of M. pneumoniae gliding and the identity of possible elements of the gliding machinery, providing a foundation for a detailed analysis of the engineering and regulation of motility in this unusual prokaryote.     

Cell reproduction cycle of mycoplasma

The cell reproduction cycle of parasitic wall-free bacteria, mycoplasma, is reviewed. DNA replication of Mycoplasma capricolum starts at a fixed site neighboring the dnaA gene and proceeds to both directions after a short arrest in one direction. The initiation frequency fits to the slow speed of replication fork and DNA content is set constant. The replicated chromosomes migrate to one and three quarters of cell length before cell division to ensure delivery of the replicated DNA to daughter cells. The cell reproduction is based on binary fission but a branch is formed when DNA replication is inhibited. Mycoplasma pneumoniae has a terminal structure, designated as an attachment organelle, responsible for both host cell adhesion and gliding motility. Behavior of the organelle in a cell implies coupling of organelle formation to the cell reproduction cycle. Several proteins coded in three operons are delivered sequentially to a position neighboring the previous organelle and a nascent one is formed. One of the duplicated attachment organelles migrates to the opposite pole of the cell before cell division. It is becoming clear that mycoplasmas have specialized cell reproduction cycles adapted to the limited genome information and parasitic life.     

Electron cryotomography of Mycoplasma pneumoniae mutants correlates terminal organelle architectural features and function

The Mycoplasma pneumoniae terminal organelle functions in adherence and gliding motility and is comprised of at least eleven substructures. We used electron cryotomography to correlate impaired gliding and adherence function with changes in architecture in diverse terminal organelle mutants. All eleven substructures were accounted for in the prkC, prpC and P200 mutants, and variably so for the HMW3 mutant. Conversely, no terminal organelle substructures were evident in HMW1 and HMW2 mutants. The P41 mutant exhibits a terminal organelle detachment phenotype and lacked the bowl element normally present at the terminal organelle base. Complementation restored this substructure, establishing P41 as either a component of the bowl element or required for its assembly or stability, and that this bowl element is essential to anchor the terminal organelle but not for leverage in gliding. Mutants II‐3, III‐4 and topJ exhibited a visibly lower density of protein knobs on the terminal organelle surface. Mutants II‐3 and III‐4 lack accessory proteins required for a functional adhesin complex, while the topJ mutant lacks a DnaJ‐like co‐chaperone essential for its assembly. Taken together, these observations expand our understanding of the roles of certain terminal organelle proteins in the architecture and function of this complex structure.     

Mycoplasma pneumoniae Protein P30 Is Required for Cytadherence and Associated with Proper Cell Development

The attachment organelle of Mycoplasma pneumoniae is a polar, tapered cell extension containing an intracytoplasmic, electron-dense core. This terminal structure is the leading end in gliding motility, and its duplication is thought to precede cell division, raising the possibility that mutations affecting cytadherence also confer a defect in motility or cell development. Mycoplasma surface protein P30 is associated with the attachment organelle, and P30 mutants II-3 and II-7 do not cytadhere. In this study, the recombinant wild-type but not the mutant II-3 p30 allele restored cytadherence when transformed into P30 mutants by recombinant transposon delivery. The mutations associated with loss of P30 in mutant II-3 and reacquisition of P30 in cytadhering revertants thereof were identified by nucleotide sequencing of the p30 gene. Morphological abnormalities that included ovoid or multilobed cells having a poorly defined tip structure were associated with loss of P30. Digital image analysis confirmed quantitatively the morphological differences noted visually. Transformation of the P30 mutants with the wild-type p30 allele restored a normal morphology, as determined both visually and by digital image analysis, suggesting that P30 plays a role in mycoplasma cell development. Finally, the P30 mutants localized the adhesin protein P1 to the terminal organelle, indicating that P30 is not involved in P1 trafficking but may be required for its receptor-binding function.     

Truncated myosin XI tail fusions inhibit peroxisome, Golgi, and mitochondrial movement in tobacco leaf epidermal cells: a genetic tool for the next generation

Although organelle movement in higher plants is predominantly actin-based, potential roles for the 17 predicted Arabidopsis myosins in motility are only just emerging. It is shown here that two Arabidopsis myosins from class XI, XIE, and XIK, are involved in Golgi, peroxisome, and mitochondrial movement. Expression of dominant negative forms of the myosin lacking the actin binding domain at the amino terminus perturb organelle motility, but do not completely inhibit movement. Latrunculin B, an actin destabilizing drug, inhibits organelle movement to a greater extent compared to the effects of AtXIE-T/XIK-T expression. Amino terminal YFP fusions to XIE-T and XIK-T are dispersed throughout the cytosol and do not completely decorate the organelles whose motility they affect. XIE-T and XIK-T do not affect the global actin architecture, but their movement and location is actin-dependent. The potential role of these truncated myosins as genetically encoded inhibitors of organelle movement is discussed.