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.     

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.     

Loss of co-chaperone TopJ impacts adhesin P1 presentation and terminal organelle maturation in Mycoplasma pneumoniae

Mycoplasma pneumoniae is a wall-less human respiratory tract pathogen that colonizes mucosal epithelium via a polar terminal organelle having a central electron-dense core and adhesin-related proteins clustered at a terminal button. A mutant lacking J-domain co-chaperone TopJ is non-cytadherent and non-motile, despite having a core and normal levels of the major cytadherence-associated proteins. J-domain co-chaperones work with DnaK to catalyse polypeptide binding and subsequent protein folding. Here we compared features of the topJ mutant with other cytadherence mutants to elucidate the contribution of TopJ to cytadherence function. The topJ mutant was similar ultrastructurally to a non-cytadherent mutant lacking terminal organelle proteins B/C, including aberrant core positioning and cell morphology in thin sections, but exhibited a hybrid satellite growth pattern with features of mutants both having and lacking a core. Time-lapse images of mycoplasmas expressing a YFP fusion with terminal organelle protein P41 suggested that terminal organelle formation/positioning was delayed or poorly co-ordinated with cell growth in the absence of TopJ. TopJ required a core for localization, perhaps involving HMW1. P1 trypsin accessibility on other non-cytadherent mutants was significantly enhanced over wild type but unexpectedly was reduced with topJ mutant cells, suggesting impaired processing, translocation and/or folding of this adhesin.     

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.     

Cellular engineering in a minimal microbe: structure and assembly of the terminal organelle of Mycoplasma pneumoniae

Mycoplasma pneumoniae is a minimal microbe with respect to cell envelope composition, biosynthetic and regulatory capabilities and genome size, yet it possesses a remarkably complex, multifunctional terminal organelle. This membrane-bound extension of the mycoplasma cell is defined by the presence of an electron-dense core that appears as paired, parallel bars oriented longitudinally and enlarging at the distal end to form a terminal button. Most non-cytadhering mutants of M. pneumoniae isolated to date exhibit defects in the architecture of the terminal organelle. Detailed characterization of those mutants has revealed the identities of many component proteins of the terminal organelle as well as the likely order in which some of those components are required. Additional questions regarding the composition of the electron-dense core, the means by which the terminal organelle is duplicated during cell division and the manner in which this process is regulated remain to be answered. Thus, it seems that there is much to be learned about cellular engineering and spatial regulation in these 'simple' cell wall-less bacteria.     

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.     

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.     

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.     

Molecular ruler of the attachment organelle in Mycoplasma pneumoniae

Length control is a fundamental requirement for molecular architecture. Even small wall-less bacteria have specially developed macro-molecular structures to support their survival. Mycoplasma pneumoniae, a human pathogen, forms a polar extension called an attachment organelle, which mediates cell division, cytadherence, and cell movement at host cell surface. This characteristic ultrastructure has a constant size of 250–300 nm, but its design principle remains unclear. In this study, we constructed several mutants by genetic manipulation to increase or decrease coiled-coil regions of HMW2, a major component protein of 200 kDa aligned in parallel along the cell axis. HMW2-engineered mutants produced both long and short attachment organelles, which we quantified by transmission electron microscopy and fluorescent microscopy with nano-meter precision. This simple design of HMW2 acting as a molecular ruler for the attachment organelle should provide an insight into bacterial cellular organization and its function for their parasitic lifestyles.     

Characterization of a major Mycoplasma penetrans lipoprotein and of its gene

Abstract A novel mycoplasmal species designated as Mycoplasma penetrans has been isolated recently from patients infected with human immunodeficiency virus. p35, a major antigen extracted from the membrane of this mycoplasma using Triton X-114 has been found to be a lipoprotein. After proteolytic treatment of p35, the sequence of one of the resulting peptides was determined and a corresponding oligonucleotide was deduced. Using this oligonucleotide as a probe the p35 gene was cloned and sequenced. Sequence analysis revealed an amino-terminal signal peptide with a potential acylation site which would result in a 35.3 kDa mature product. In addition, the p35 gene was followed by an open reading frame with a corresponding polypeptide partially homologous to p35, in particular to the N-terminus region.     

Genome sequence of Mycoplasma iowae strain 695, an unusual pathogen causing deaths in turkeys

Mycoplasma iowae is associated mainly with reduced hatchability in turkeys and is well known for the unusual ability of phenotypic variation in the Mycoplasma surface components as well as a relative resistance to heat, bile salts, and many antimicrobials. A subset of unique genes and a gene cluster responsible for these characteristics could be identified from the genome. Here, we report the first genome sequence of this species.     

Quantitative analysis of organelle abundance, morphology and dynamics

Recent data indicate that morphological characteristics of cell organelles are important for their function in the cell. These characteristics include not only their shape, number and size, but also their distribution in the cell. Moreover, the dynamics of processes that result in changes in these characteristics (e.g. organelle fission, fusion, autophagy, transport) influence the function of the cell. For a better understanding of these processes quantitative approaches are important. Here we give an overview of contemporary biochemical and microscopy methods that are used to quantify organelle abundance, morphology and the kinetics of the processes that cause changes in these properties.     

Sedimentation counting and morphology of Mycoplasma

Clark, Harold W. (The George Washington University School of Medicine, Washington, D.C.). Sedimentation counting and morphology of Mycoplasma. J. Bacteriol. 90:1373-1386. 1965.-The sedimentation technique for counting viral particles was applied to the quantitation and morphological identification of Mycoplasma in broth cultures. Mycoplasma, apparently in their native form, firmly adhered to the surface, when sedimented on glass cover slips or onto electron microscope grids. The sedimented cover slip preparations stained with crystal violet could be readily counted in the light microscope. The cultures sedimented onto electron microscope grids were readily counted at low magnification and provided excellent preparations for morphological examination at higher magnifications. It was found that air-dried Mycoplasma particles were enlarged considerably because of excessive flattening. Fixation of sedimented Mycoplasma particles in diluted OsO(4) prior to air drying yielded a more realistic morphology, with various sizes and shapes in the stages of the growth cycle exhibited. A new technique of differentially staining Mycoplasma colonies on agar plates was developed to facilitate the quantitation of viable colony-forming units for comparison with total counts. The use of plastic or Parafilm gaskets for dry mounting was developed to facilitate the handling and examination of the stained cover slip preparations. The results of this investigation indicated that the growth cycle of some Mycoplasma species includes a stage of hexadic fission with the cleavage of minimal reproductive units (less than 100 mmu) containing a limited deoxyribonucleic acid genetic coding molecule (approximately 4 x 10(6)).     

IgA肾病患者血液、尿液及咽拭子标本中穿通支原体的分离检出

目的探讨IgA肾病患者血液、尿液及咽拭子标本中穿通支原体（Mycoplasma penetrans,Mp）的分离检出率以及与病理型别相关性。方法采用分离培养法,共计从26例IgA肾病患者血液、尿液及咽拭子标本及38例正常对照相应标本中进行穿通支原体分离检测,对培养阳性标本用穿通支原体套式PCR进行证实。结果在11例（42．3％）患者血液与尿液或（和）咽拭子中同时分离到穿通支原体,单独尿液或咽拭子标本阳性分别为1例（3．8％）与7例（26．9％）。26例IgA肾病患者血液、尿液及咽拭子穿通支原体的分离检出率分别为42．3％、23．1％与57．7％;与38例正常对照组血液、尿液及咽拭子检出0例、2例（5．3％）与7例（18．4％）相比较,差异有非常显著性（P〈0．01）,在正常对照组中无2种以上标本同时检出穿通支原体。结论穿通支原体在ISA肾病患者的血液、尿液与咽拭子标本中均有较高的检出率且与病理型别有一定的相关性。     

The complete genomic sequence of Mycoplasma penetrans,an intracellular bacterial pathogen in humans

The complete genomic sequence of an intracellular bacterial pathogen, Mycoplasma penetrans HF-2 strain, was determined. The HF-2 genome consists of a 1 358 633 bp single circular chromosome containing 1038 predicted coding sequences (CDSs), one set of rRNA genes and 30 tRNA genes. Among the 1038 CDSs, 264 predicted proteins are common to the Mycoplasmataceae sequenced thus far and 463 are M.penetrans specific. The genome contains the two-component system but lacks the essential cellular gene, uridine kinase. The relatively large genome of M.penetrans HF-2 among mycoplasma species may be accounted for by both its rich core proteome and the presence of a number of paralog families corresponding to 25.4% of all CDSs. The largest paralog family is the p35 family, which encodes surface lipoproteins including the major antigen, P35. A total of 44 genes for p35 and p35 homologs were identified and 30 of them form one large cluster in the chromosome. The genetic tree of p35 paralogs suggests the occurrence of dynamic chromosomal rearrangement in paralog formation during evolution. Thus, M.penetrans HF-2 may have acquired diverse repertoires of antigenic variation-related genes to allow its persistent infection in humans.     

Purification and characterization of Mycoplasma penetrans Ca2+/Mg2+-dependent endonuclease.

The major nuclease from Mycoplasma penetrans has been purified to homogeneity. The enzyme seems to be present as a membrane-associated precursor of 50 kDa and as a peripheral membrane monomeric polypeptide of 40 kDa that is easily removed by washing of cells with isotonic buffers and in the aqueous phase upon Triton partitioning of Triton X-114-solubilized protein. The 40-kDa nuclease was extracted from M. penetrans cells by Triton X-114 and phase fractionation and was further purified by chromatography on Superdex 75 and chelating Sepharose (Zn2+ form) columns. By gel filtration, the apparent molecular mass was 40 kDa. The purified enzyme exhibits both a nicking activity on superhelical and linear double-stranded DNA and a nuclease activity on RNA and single-stranded DNA. No exonuclease activity was found for this enzyme. This nuclease required both Mg2+ (optimum, 5 mM) and Ca2+ (optimum, 2 mM) for activity and exhibited a pH optimum between pH 7 and 8 for DNase activity. It was inhibited by Zn2+, Mn2+, heparin, sodium dodecyl sulfate, and chelator agents such EDTA and EGTA, but no effect was observed with ATP, 2-mercaptoethanol, N-ethylmaleimide, dithiothreitol, nonionic detergents, phenylmethylsulfonyl fluoride, and iodoacetamide. Nuclease activity was inhibited by diethylpyrocarbonate at both pH 6 and 8 and by pepstatin, suggesting the involvement of a histidine and an aspartate in the active site. When added to human lymphoblast nuclei, the purified M. penetrans endonuclease induced internucleosomal fragmentation of the chomatin into oligonucleosomal fragments. On the basis of this result, and taking into account the fact that M. penetrans has the capacity to invade eucaryotic cells, one can suggest, but not assert, that produced Ca2+/Mg2+-dependent endonuclease may alter the nucleic acid metabolism of host cells by DNA and/or RNA degradation and may act as a potential pathogenic determinant.