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.     

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.     

Mycoplasma pneumoniae cytadherence: unravelling the tie that binds

Mycoplasma pneumoniae is the leading cause of pneumonia in older children and young adults. Mycoplasma adherence to the respiratory epithelium (cytadherence) is required for colonization and pathogenesis. Although considered to be among the smallest and simplest known prokaryotes, this cell-wall-less bacterium possesses a highly differentiated terminal structure that is thought to be functional in mycoplasma cell division, gliding motility, and cytadherence. Mutant analysis has identified mycoplasma proteins associated with cytadherence, and revealed novel regulatory features. Ultrastructural and biochemical studies have established the subcellular location and interaction of key components, several of which are phosphorylated by ATP-dependent kinase(s) in a manner that is responsive to changing nutritional conditions. This review summarizes recent progress in defining the composition, organization and regulation of the attachment organelle. What emerges is a picture of M. pneumoniae cytadherence as a multifactorial process that extends well beyond adhesin-receptor recognition.     

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.     

Mycoplasma genitalium mg200 and mg386 genes are involved in gliding motility but not in cytadherence

Isolation and characterization of transposon-generated Mycoplasma genitalium gliding-deficient mutants has implicated mg200 and mg386 genes in gliding motility. The proposed role of these genes was confirmed by restoration of the gliding phenotype in deficient mutants through gene complementation with their respective mg386 or mg200 wild-type copies. mg200 and mg386 are the first reported gliding-associated mycoplasma genes not directly involved in cytadherence. Orthologues of MG200 and MG386 proteins are also found in the slow gliding mycoplasmas, Mycoplasma pneumoniae and Mycoplasma gallisepticum, suggesting the existence of a unique set of proteins involved in slow gliding motility. MG200 and MG386 proteins share common features, such as the presence of enriched in aromatic and glycine residues boxes and an acidic and proline-rich domain, suggesting that these motifs could play a significant role in gliding motility.     

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.     

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.     

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.     

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.     

A 41-kDa variable surface protein of Mycoplasma gallisepticum has a counterpart in Mycoplasma imitans and Mycoplasma iowae

Abstract Mycoplasma gallisepticun, M. imitans and M. iowae are three morphologically similar avian Mycoplasma species, and M. gallisepticum and M. imitans have been shown to be antigenically related. Using a monoclonal antibody that binds to the previously described size- and phase-variant integral membrane surface protein PvpA of M. gallisepticum , we have identified in all three avian Mycoplasma species a 41-kDa surface antigen, which in M. gallisepticum and M. imitans was identified as peripheral membrane protein undergoing variation in expression among clonal isolates. Southern blot analysis using the pvpA gene as a probe demonstrated sequence homology with M. imitans and M. iowae genomic DNA and suggested that a pvpA -related gene that may encode the 41-kDa product exists in these two Mycoplasma species. These studies establish (i) that M. iowae is antigenically related to M. gallisepticum and M. imitans , (ii) that the three species share non-ribosomal gene sequences, and (iii) that peripheral membrane proteins contribute to Mycoplasma surface variation.     

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.     

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.     

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.     

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.     

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.     

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.     

Structural analysis of Mycoplasma pneumoniae by cryo-electron tomography

Bacteria of the genus Mycoplasma lack obvious homologs of prokaryotic or eukaryotic cytoskeletal, as well as motility-related genes (except FtsZ). Nevertheless, they maintain characteristic cell shapes and show adhesion and gliding abilities on both artificial surfaces and cells. Earlier genetic, biochemical, and electron microscopic analyses have shown that the tip structure, located at the tapered end of gliding mycoplasmas, is indispensable for this behavior. In this study, we have analyzed the fine structure of the Mycoplasma pneumoniae tip by cryo-electron tomography. We show that the central rod is surrounded by quasi-periodical electron-dense macromolecular complexes. Additional complexes are located at the distal end of the rod which connect the rod to the cytoplasmic membrane. Furthermore, we detect a structure at the proximal end of the rod that attaches the rod to the cell membrane. The surface protein complexes have been mapped in detail and their distribution on the cell surface has been visualized. Since the rod structures were detected at a close to native state of the cells, they allow us to build a hypothesis describing the motility mechanism of M. pneumoniae. Finally, we have evaluated the ribosome density of the organism by a template matching approach, whereby the reliability of the detection was supported by a comparative bioinformatics analysis.     

Identification of a novel nucleoside triphosphatase from Mycoplasma mobile: a prime candidate motor for gliding motility

A protein with a molecular mass of 42 kDa (P42) from Mycoplasma mobile, one of several mycoplasmas that exhibit gliding motility, was shown to be a novel NTPase (nucleoside triphosphatase). Although the P42 protein lacks a common ATP-binding sequence motif (Walker A), the recombinant proteins expressed in Escherichia coli certainly hydrolysed some nucleoside triphosphates, including ATP. The results of photoaffinity labelling by an ATP analogue supported that the P42 protein contains a specific binding site for ATP (or another nucleoside triphosphate). In the M. mobile genome, the P42 gene is located downstream of gli123, gli349 and gli521 genes, and they have been reported to be polycis-tronically transcribed. As the huge proteins encoded by gli123, gli349 and gli521 play a role in gliding motility of M. mobile, P42 might also have some kind of function in the gliding motility. The gliding motility of M. mobile is driven directly by ATP hydrolysis, but the key ATPase has not been identified. Our results showed that, among these four proteins, only P42 exhibited ATPase activity. Biochemical characteristics--optimal conditions for activity, substrate specificities, and inhibiting effects by ATP analogues--of the recombinant P42 proteins were very similar to those of a putative ATPase speculated from a previous analysis with a gliding 'ghost' whose cell membrane was permeabilized by Triton X-100. These results support the hypothesis that the P42 protein is the key ATPase in the gliding motility of M. mobile.     

Mutant analysis reveals a specific requirement for protein P30 in Mycoplasma pneumoniae gliding motility

The cell-wall-less prokaryote Mycoplasma pneumoniae, long considered among the smallest and simplest cells capable of self-replication, has a distinct cellular polarity characterized by the presence of a differentiated terminal organelle which functions in adherence to human respiratory epithelium, gliding motility, and cell division. Characterization of hemadsorption (HA)-negative mutants has resulted in identification of several terminal organelle proteins, including P30, the loss of which results in developmental defects and decreased adherence to host cells, but their impact on M. pneumoniae gliding has not been investigated. Here we examined the contribution of P30 to gliding motility on the basis of satellite growth and cell gliding velocity and frequency. M. pneumoniae HA mutant II-3 lacking P30 was nonmotile, but HA mutant II-7 producing a truncated P30 was motile, albeit at a velocity 50-fold less than that of the wild type. HA-positive revertant II-3R producing an altered P30 was unexpectedly not fully wild type with respect to gliding. Complementation of mutant II-3 with recombinant wild-type and mutant alleles confirmed the correlation between gliding defect and loss or alteration in P30. Surprisingly, fusion of yellow fluorescent protein to the C terminus of P30 had little impact on cell gliding velocity and significantly enhanced HA. Finally, while quantitative examination of HA revealed clear distinctions among these mutant strains, gliding defects did not correlate strictly with the HA phenotype, and all strains attached to glass at wild-type levels. Taken together, these findings suggest a role for P30 in gliding motility that is distinct from its requirement in adherence.     

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.