Visualizing translation dynamics at atomic detail inside a bacterial cell.

Translation is the fundamental process of protein synthesis and is catalysed by the ribosome in all living cells1. Here we use advances in cryo-electron tomography and sub-tomogram analysis2,3 to visualize the structural dynamics of translation inside the bacterium Mycoplasma pneumoniae. To interpret the functional states in detail, we first obtain a high-resolution in-cell average map of all translating ribosomes and build an atomic model for the M. pneumoniae ribosome that reveals distinct extensions of ribosomal proteins. Classification then resolves 13 ribosome states that differ in their conformation and composition. These recapitulate major states that were previously resolved in vitro, and reflect intermediates during active translation. On the basis of these states, we animate translation elongation inside native cells and show how antibiotics reshape the cellular translation landscapes. During translation elongation, ribosomes often assemble in defined three-dimensional arrangements to form polysomes4. By mapping the intracellular organization of translating ribosomes, we show that their association into polysomes involves a local coordination mechanism that is mediated by the ribosomal protein L9. We propose that an extended conformation of L9 within polysomes mitigates collisions to facilitate translation fidelity. Our work thus demonstrates the feasibility of visualizing molecular processes at atomic detail inside cells.

Determination of metabolic activity in planktonic and biofilm cells of Mycoplasma fermentans and Mycoplasma pneumoniae by nuclear magnetic resonance.

Mycoplasmas are fastidious microorganisms, typically characterised by their restricted metabolism and minimalist genome. Although there is reported evidence that some mycoplasmas can develop biofilms little is known about any differences in metabolism in these organisms between the growth states. A systematic metabolomics approach may help clarify differences associated between planktonic and biofilm associated mycoplasmas. In the current study, the metabolomics of two different mycoplasmas of clinical importance (Mycoplasma pneumoniae and Mycoplasma fermentans) were examined using a novel approach involving nuclear magnetic resonance spectroscopy and principle component analysis. Characterisation of metabolic changes was facilitated through the generation of high-density metabolite data and diffusion-ordered spectroscopy that provided the size and structural information of the molecules under examination. This enabled the discrimination between biofilms and planktonic states for the metabolomic profiles of both organisms. This work identified clear biofilm/planktonic differences in metabolite composition for both clinical mycoplasmas and the outcomes serve to establish a baseline understanding of the changes in metabolism observed in these pathogens in their different growth states. This may offer insight into how these organisms are capable of exploiting and persisting in different niches and so facilitate their survival in the clinical setting.

Study of Two Separate Types of Macrolide-Resistant Mycoplasma pneumoniae Outbreaks.

To study the complete natural process of a Mycoplasma pneumoniae outbreak in a semiclosed room such as a primary school room, we investigated two separate M. pneumoniae outbreaks involving 81 students in total in two primary schools in Hangzhou, China. M. pneumoniae isolates from pharyngeal swabs were detected by fluorescence quantitative real-time PCR (RT-PCR) and culture. The class in school M had 39 students, with 12 (30.8%) with positive M. pneumoniae detection results. The class from school J had 42 students, with 13 (31.0%) positive. The strains from two classes were confirmed to represent two clones (3/4/5/7/2 and 5/4/5/7/2) and to be macrolide resistant (A2063G) according to P1 and multilocus variable-number tandem-repeat analysis (MLVA) genotyping, determination of MIC of antibiotics, and sequencing. Students with M. pneumoniae isolates detected were divided into three groups: those carrying the isolates, those with upper respiratory tract infection (URI), and those with pneumonia. Longitudinal sampling performed using pharyngeal swabs showed that the persistence of M. pneumoniae was longest in the group of students with pneumonia. M. pneumoniae causes pneumonia outbreaks in schools, and the incidence of pneumonia has a higher rate than that of URI. The persistence of M. pneumoniae, with a median duration of 79.50 days in the group of students with pneumonia, differs from that of the infection state.

Mycoplasma pneumoniae, an underutilized model for bacterial cell biology.

In recent decades, bacterial cell biology has seen great advances, and numerous model systems have been developed to study a wide variety of cellular processes, including cell division, motility, assembly of macromolecular structures, and biogenesis of cell polarity. Considerable attention has been given to these model organisms, which include Escherichia coli, Bacillus subtilis, Caulobacter crescentus, and Myxococcus xanthus. Studies of these processes in the pathogenic bacterium Mycoplasma pneumoniae and its close relatives have also been carried out on a smaller scale, but this work is often overlooked, in part due to this organism's reputation as minimalistic and simple. In this minireview, I discuss recent work on the role of the M. pneumoniae attachment organelle (AO), a structure required for adherence to host cells, in these processes. The AO is constructed from proteins that generally lack homology to those found in other organisms, and this construction occurs in coordination with cell cycle events. The proteins of the M. pneumoniae AO share compositional features with proteins with related roles in model organisms. Once constructed, the AO becomes activated for its role in a form of gliding motility whose underlying mechanism appears to be distinct from that of other gliding bacteria, including Mycoplasma mobile. Together with the FtsZ cytoskeletal protein, motility participates in the cell division process. My intention is to bring this deceptively complex organism into alignment with the better-known model systems.

Visual account of protein investment in cellular functions.

Proteomics techniques generate an avalanche of data and promise to satisfy biologists' long-held desire to measure absolute protein abundances on a genome-wide scale. However, can this knowledge be translated into a clearer picture of how cells invest their protein resources? This article aims to give a broad perspective on the composition of proteomes as gleaned from recent quantitative proteomics studies. We describe proteomaps, an approach for visualizing the composition of proteomes with a focus on protein abundances and functions. In proteomaps, each protein is shown as a polygon-shaped tile, with an area representing protein abundance. Functionally related proteins appear in adjacent regions. General trends in proteomes, such as the dominance of metabolism and protein production, become easily visible. We make interactive visualizations of published proteome datasets accessible at www.proteomaps.net. We suggest that evaluating the way protein resources are allocated by various organisms and cell types in different conditions will sharpen our understanding of how and why cells regulate the composition of their proteomes.

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.

Insights into the function of Mycoplasma pneumoniae protein P30 from orthologous gene replacement.

The attachment organelles of bacterial species belonging to the Mycoplasma pneumoniae phylogenetic cluster are required for host cytadherence, gliding motility and virulence. Despite being closely related, these bacteria possess distinct cellular morphologies and gliding characteristics. The molecular mechanisms for most attachment organelle phenotypes, including shape and ability to power motility, are obscure. The attachment organelle-associated P30 protein of M. pneumoniae is implicated in both adherence and motility, with mutations negatively impacting cell morphology, adherence, gliding and virulence. To test whether the P30 alleles of different mycoplasma species confer species-specific attachment organelle properties, we created an M. pneumoniae strain in which the Mycoplasma genitalium P30 orthologue, P32, was substituted for the native P30. Selected clones were visualized by scanning electron microscopy to assess morphology and by indirect immunofluorescence microscopy to localize P32. Cytadherence ability and gliding motility were assessed by haemadsorption assay and phase-contrast microcinematography, respectively. Cell and attachment organelle morphologies were indistinguishable from wild-type M. pneumoniae as well as M. pneumoniae II-3 expressing a C-terminally 6×His-tagged P30 construct. P32 was localized to the tip of the attachment organelle of transformant cells. Although a specific role for P30 in species-specific phenotypes was not identified, this first test of orthologous gene replacement in different mycoplasma species demonstrates that the differences in the M. pneumoniae and M. genitalium proteins contribute little if anything to the different attachment organelle phenotypes between these species.

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.

The phosphoproteome of the minimal bacterium Mycoplasma pneumoniae: analysis of the complete known Ser/Thr kinome suggests the existence of novel kinases.

Mycoplasma pneumoniae belongs to the Mollicutes, the group of organisms with the smallest genomes that are capable of host-independent life. These bacteria show little regulation in gene expression, suggesting an important role for the control of protein activities. We have studied protein phosphorylation in M. pneumoniae to identify phosphorylated proteins. Two-dimensional gel electrophoresis and mass spectrometry allowed the detection of 63 phosphorylated proteins, many of them enzymes of central carbon metabolism and proteins related to host cell adhesion. We identified 16 phosphorylation sites, among them 8 serine and 8 threonine residues, respectively. A phosphoproteome analysis with mutants affected in the two annotated protein kinase genes or in the single known protein phosphatase gene suggested that only one protein (HPr) is phosphorylated by the HPr kinase, HPrK, whereas four adhesion-related or surface proteins were targets of the protein kinase C, PrkC. A comparison with the phosphoproteomes of other bacteria revealed that protein phosphorylation is evolutionarily only poorly conserved. Only one single protein with an identified phosphorylation site, a phosphosugar mutase (ManB in M. pneumoniae), is phosphorylated on a conserved serine residue in all studied organisms from archaea and bacteria to man. We demonstrate that this protein undergoes autophosphorylation. This explains the strong conservation of this phosphorylation event. For most other proteins, even if they are phosphorylated in different species, the actual phosphorylation sites are different. This suggests that protein phosphorylation is a form of adaptation of the bacteria to the specific needs of their particular ecological niche.

The stability of cytadherence proteins in Mycoplasma pneumoniae requires activity of the protein kinase PrkC.

Mycoplasma pneumoniae belongs to the mollicutes, a group of bacteria that have strongly reduced genomes but that are nevertheless capable of independent life. With only three transcription factors, the regulatory features of these bacteria are very limited. Thus, posttranslational regulation might be important for M. pneumoniae. In addition to the highly specific HPr kinase, the M. pneumoniae prkC gene encodes the serine/threonine protein kinase C. In order to study the function(s) of this kinase, we isolated an M. pneumoniae mutant affected in PrkC. This mutation resulted in nonadherent growth and loss of cytotoxicity. Examination of the phosphorylation profile of the prkC mutant suggested that phosphorylation of cytadherence proteins was affected by the loss of this kinase. In contrast, inactivation of the prpC gene affecting the protein phosphatase that antagonizes PrkC-dependent phosphorylation resulted in more intensive phosphorylation of the cytadherence proteins HMW1 and HMW3 of the major adhesin P1 and of the surface protein MPN474. Moreover, loss of PrkC affects not only the phosphorylation state of the cytadherence proteins but also their intracellular accumulation. However, the expression of the corresponding genes was not affected by PrkC, suggesting that PrkC-dependent phosphorylation results in stabilization of the cytadherence proteins. The HMW proteins and P1 are part of the so-called terminal organelle of M. pneumoniae that is involved in gliding motility, cell division, and adhesion to host epithelial tissues. Our observations suggest that the posttranslational modification of cytadherence proteins by PrkC is essential for the development and function of the M. pneumoniae terminal organelle.

Pediatric respiratory infections by Mycoplasma pneumoniae.

Mycoplasma pneumoniae is one of the most common agents of community-acquired pneumonia in children and young adults. Although M. pneumoniae is a small bacterium that can reproduce in an artificial culture medium and is known to be sensitive to certain antibiotics in vitro as well as in vivo, the immunopathogenesis of M. pneumoniae in the human host is not fully understood. The epidemiologic characteristics, including periodic epidemics, and some clinical characteristics of M. pneumoniae are similar to those observed in systemic viral infections. Many experimental and clinical studies have suggested that the pathogenesis of lung injuries in M. pneumoniae infection is associated with a cell-mediated immune reaction, including high responsiveness to corticosteroid therapy. This paper presents an overview of M. pneumoniae infections, with emphasis on epidemiology, pathogenesis and treatment.

A processive lipid glycosyltransferase in the small human pathogen Mycoplasma pneumoniae: involvement in host immune response.

The human pathogen Mycoplasma pneumoniae has a very small genome but with many yet not identified gene functions, e.g. for membrane lipid biosynthesis. Extensive radioactive labelling in vivo and enzyme assays in vitro revealed a substantial capacity for membrane glycolipid biosynthesis, yielding three glycolipids, five phosphoglycolipids, in addition to six phospholipids. Most glycolipids were synthesized in a cell protein/lipid-detergent extract in vitro; galactose was incorporated into all species, whereas glucose only into a few. One (MPN483) of the three predicted glycosyltransferases (GTs; all essential) was both processive and promiscuous, synthesizing most of the identified glycolipids. These enzymes are of a GT-A fold, similar to an established structure, and belong to CAZy GT-family 2. The cloned MPN483 could use both diacylglycerol (DAG) and human ceramide acceptor substrates, and in particular UDP-galactose but also UDP-glucose as donors, making mono-, di- and trihexose variants. MPN483 output and processitivity was strongly influenced by the local lipid environment of anionic lipids. The structure of a major beta1,6GlcbetaGalDAG species was determined by NMR spectroscopy. This, as well as other purified M. pneumoniae glycolipid species, is important antigens in early infections, as revealed from ELISA screens with patient IgM sera, highlighting new aspects of glycolipid function.

Micoplasma en patología pulmonar pediátrica / Micoplasma in pediatric pulmonology patology

Mycoplasma pneumoniae es un germen patógeno frecuente del tracto respiratorio humano, especialmente en niños y adultos jóvenes. El desarrollo en los últimos años de nuevos métodos diagnósticos como el de reacción en cadena de la polimerasa (PCR), unido a métodos diagnósticos tradicionales, ha permitido ahondar en las características de la enfermedad por M. pneumoniae en pediatría.

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

Mycoplasmas are cell wall-less bacteria considered among the smallest and simplest prokaryotes known, and yet several species including Mycoplasma pneumoniae have a remarkably complex cellular organization highlighted by the presence of a differentiated terminal organelle, a membrane-bound cell extension distinguished by an electron-dense core. Adhesin proteins localize specifically to the terminal organelle, which is also the leading end in gliding motility. Duplication of the terminal organelle is thought to precede cell division, but neither the mechanism of its duplication nor its role in this process is understood. Here we used fluorescent protein fusions and time-lapse digital imaging to study terminal organelle formation in detail in growing cultures of M. pneumoniae. Individual cells ceased gliding as a new terminal organelle formed adjacent to an existing structure, which then migrated away from the transiently stationary nascent structure. Multiple terminal organelles often formed before cytokinesis was observed. The separation of terminal organelles was impaired in a nonmotile mutant, indicating a requirement for gliding in normal cell division. Examination of cells expressing two different fluorescent protein fusions concurrently established their relative order of appearance, and changes in the fluorescence pattern over time suggested that nascent terminal organelles originated de novo rather than from an existing structure. In summary, spatial and temporal analysis of terminal organelle formation has yielded insights into the nature of M. pneumoniae cell division and the role of gliding motility in that process.

Crystal structure of a protein associated with cell division from Mycoplasma pneumoniae (GI: 13508053): a novel fold with a conserved sequence motif.

UPF0040 is a family of proteins implicated in a cellular function of bacteria cell division. There is no structure information available on protein of this family. We have determined the crystal structure of a protein from Mycoplasma pneumoniae that belongs to this family using X-ray crystallography. Structural homology search reveals that this protein has a novel fold with no significant similarity to any proteins of known three-dimensional structure. The crystal structures of the protein in three different crystal forms reveal that the protein exists as a ring of octamer. The conserved protein residues, including a highly conserved DXXXR motif, are examined on the basis of crystal structure.

Transcription profiles of the bacterium Mycoplasma pneumoniae grown at different temperatures.

Applying microarray technology, we have investigated the transcriptome of the small bacterium Mycoplasma pneumoniae grown at three different temperature conditions: 32, 37 and 32 degrees C followed by a heat shock for 15 min at 43 degrees C, before isolating the RNA. From 688 proposed open-reading frames, 676 were investigated and 564 were found to be expressed (P < 0.001; 606 with P < 0.01) and at least 33 (P < 0.001; 77 at P < 0.01) regulated. By quantitative real-time PCR of selected mRNA species, the expression data could be linked to absolute molecule numbers. We found M.pneumoniae to be regulated at the transcriptional level. Forty-seven genes were found to be significantly up-regulated after heat shock (P < 0.01). Among those were the conserved heat shock genes like dnaK, lonA and clpB, but also several genes coding for ribosomal proteins and 10 genes of unassigned functions. In addition, 30 genes were found to be down-regulated under the applied heat shock conditions. Further more, we have compared different methods of cDNA synthesis (random hexamer versus gene-specific primers, different RNA concentrations) and various normalization strategies of the raw microarray data.

Characterization of a Mycoplasma pneumoniae hmw3 mutant: implications for attachment organelle assembly.

The proteins required for adherence of the pathogen Mycoplasma pneumoniae to host respiratory epithelial cells are localized to a polar structure, the attachment organelle. A number of these proteins have been characterized functionally by analysis of noncytadhering mutants, and many are components of the mycoplasma cytoskeleton. Mutations in some cytadherence-associated proteins have pleiotropic effects, including decreased stability of other proteins, loss of adherence and motility, and abnormal morphology. The function of protein HMW3, a component of the attachment organelle, has been difficult to discern due to lack of an appropriate mutant. In this paper, we report that loss of HMW3 resulted in decreased levels and more diffuse localization of cytoskeletal protein P65, subtle changes in morphology, inability to cluster the adhesin P1 consistently at the terminal organelle, reduced cytadherence, and, in some cells, an atypical electron-dense core in the attachment organelle. This phenotype suggests a role for HMW3 in the architecture and stability of the attachment organelle.

[Creation of a controlled model of respiratory mycoplasmosis]. / Sozdanie upravliaemoi modeli respiratornogo mikoplazmoza.

To create the controlled model of respiratory mecoplasmosis, laboratory animals with induced immunodefeciency were infected with M. pneumoniae strain having different degrees of virulence. Immunodeficient state was induced in susceptible animals (hamsters) by the injection of cyclophosphamide. The infection of immunodeficient animals with a virulent strain induced the development of severe manifest pneumonia with 50% mortality rate. The infection of the animals with induced immunodeficiency with the avirulent strain attenuated in the process of 10-year subculturing in acellular media led to the development of moderate pneumonia characterized by the proliferation of the infective agent in pulmonary tissues and the presence of pathomorphological changes. The simultaneous infection of control hamsters with the same strain did not induce the development of infection. The infection of the experimental animals with the avirulent strain in the presence of induced immunodeficiency resulted in the partial restoration of the virulent properties of the strain, which was manifested by the activation of the capacity of the infective agent for colonization and proliferation in body tissues of the animals.

Comparative analysis of the genomes of the bacteria Mycoplasma pneumoniae and Mycoplasma genitalium.

The sequenced genomes of the two closely related bacteria Mycoplasma genitalium and Mycoplasma pneumoniae were compared with emphasis on genome organization and coding capacity. All the 470 proposed open reading frames (ORFs) of the smaller M.genitalium genome (580 kb) were contained in the larger genome (816 kb) of M.pneumoniae. There were some discrepancies in annotation, but inspection of the DNA sequences showed that the corresponding DNA was always present in M. pneumoniae. The two genomes could be subdivided into six segments. The order of orthologous genes was well conserved within individual segments but the order of these segments in both bacteria was different. We explain the different organization of the segments by translocation via homologous recombination. The translocations did not disturb the continuous bidirectional course of transcription in both genomes, starting at the proposed origin of replication. The additional 236 kb in M.pneumoniae,compared with theM.genitalium genome, were coding for 209 proposed ORFs not identified in M.genitalium. Of these ORFs, 110 were specific to M.pneumoniae exhibiting no significant similarity to M.genitalium ORFs, while 76 ORFs were amplifications of ORFs existing mainly as single copies in M. genitalium. In addition, 23 ORFs containing a copy of either one of the three repetitive DNA sequences RepMP2/3, RepMP4 and RepMP5 were annotated in M.pneumoniae but not in M.genitalium,although similar DNA sequences were present. TheM.pneumoniae-specific genes included a restriction-modification system, two transport systems for carbohydrates, the complete set of three genes coding for the arginine dihydrolase pathway and 14 copies of the repetitive DNA sequence RepMP1 which were part of several different translated genes with unknown function.