1,720,979 research outputs found
MYCOPLASMA PNEUMONIAE THYMIDINE PHOSPHORYLASE
No full textMycoplasma pneumoniae (Mpn) is a human pathogen causing acute respiratory diseases and accounts for approximately 30% cases of community-acquired pneumonia. Co-infection with Mycoplasmas compromises the efficacy of anticancer and antiviral nucleoside analog-based drugs due to the presence of Mycoplasma thymidine phosphorylase (TP). In this study, a TP-deficient strain of Mpn was generated in order to study the effect of Mpn TP in the metabolism of nucleoside analogs. Deficiency in TP activity led to increased uptake and incorporation of radiolabeled deoxyuridine and uracil but thymidine uptake was not affected. The activities of enzymes in the salvage of thymidine and deoxyuridine, e. g., thymidine kinase and uracil phosphoribosyltransferase were upregulated in the TP-deficient mutant, which may explain the increased uptake of deoxyuridine and uracil. Thirty FDA-approved anticancer and antiviral nucleoside and nucleobase analogs were used to screen their inhibitory activity toward the TP mutant and the wild type strain. Seven analogs were found to inhibit strongly the growth of both wild type and TP mutant. Differences in the inhibitory effect of several purine analogs between the two strains were observed. Further study is needed in order to understand the mechanism of inhibition caused by these analogs. Our results indicated that TP is not an essential gene for Mpn survival and TP deficiency affects other enzymes in Mpn nucleotide metabolism, and suggested that Mycoplasma nucleotide biosynthesis pathway enzymes are potential targets for future development of antibiotics
Expression of Mycoplasma proteins carrying an affinity tag in M. pneumoniae allows rapid purification and circumvents problems related to the aberrant genetic code
No full textIn Mycoplasma pneumoniae and several other mollicutes, the UGA opal codon specifies tryptophan rather than a translation stop. This often makes it difficult to express Mycoplasma proteins in heterologous hosts. In this work we demonstrate that mollicute proteins can be fused to an affinity tag and be expressed directly in M. pneumoniae. The protein can then be purified by affinity chromatography and be used for biochemical or any other desired analysis
Regulatory protein phosphorylation in Mycoplasma pneumoniae - A PP2C-type phosphatase serves to dephosphorylate HPR(Ser-P)
No full textAmong the few regulatory events in the minimal bacterium Mycoplasma pneumoniae is the phosphorylation of the HPr phosphocarrier protein of the phosphotransferase system. In the presence of glycerol, HPr is phosphorylated in an ATP-dependent manner by the HPr kinase/phosphorylase. The role of the latter enzyme was studied by constructing a M. pneumoniae hprK mutant defective in HPr kinase/phosphorylase. This mutant strain no longer exhibited HPr kinase activity but, surprisingly, still had phosphatase activity toward serine-phosphorylated HPr (HPr(Ser-P)). An inspection of the genome sequence revealed the presence of a gene (prpC) encoding a presumptive protein serine/threonine phosphatase of the PP2C family. The phosphatase PrpC was purified and its biochemical activity in HPr(Ser-P) dephosphorylation demonstrated. Moreover, a prpC mutant strain was isolated and found to be impaired in HPr(Ser-P) dephosphorylation. Homologues of PrpC are present in many bacteria possessing HPr(Ser-P), suggesting that PrpC may play an important role in adjusting the cellular HPr phosphorylation state and thus controlling the diverse regulatory functions exerted by the different forms of HPr
Interactions between Glycolytic Enzymes of <i>Mycoplasma pneumoniae</i>
No full textWith only 688 protein-coding genes, <i>Mycoplasma pneumoniae</i> is one of the smallest self-replicating organisms. These bacteria use glycolysis as the major pathway for ATP production by substrate-level phosphorylation, suggesting that this pathway must be optimized to high efficiency. In this study, we have investigated the interactions between glycolytic enzymes using the bacterial adenylate cyclase-based two-hybrid system. We demonstrate that most of the glycolytic enzymes perform self-interactions, suggesting that they form dimers or other oligomeric forms. In addition, enolase was identified as the central glycolytic enzyme of <i>M. pneumoniae</i> due to its ability to directly interact with all other glycolytic enzymes. Our results support the idea of the formation of a glycolytic complex in <i>M. pneumoniae</i> and we suggest that the formation of this complex might ensure higher fluxes through the glycolytic pathway than would be possible with isolated non-interacting enzymes.</jats:p
Upregulation of thymidine kinase activity compensates for loss of thymidylate synthase activity in Mycoplasma pneumoniae
No full textP>Thymidylate, an essential building block of DNA, is synthesized either from deoxyuridylate by thymidylate synthase (TS) or thymidine (dT) by thymidine kinase (TK). Thymidylate kinase (TMPK) phosphorylates dTMP to dTTP. Thymidine phosphorylase (TP) catalyses reversible phosphorolysis of dT. Using transposon mutagenesis M. pneumoniae TS gene (thyA/MPN320) was interrupted and requirement of these enzymes was studied. We found that TK activity and transcript levels and TP activity, but not TMPK or TS activity, are growth-phase-regulated, with induction at the exponential growth phase and a decline after the stationary phase. Inactivation of thyA results in upregulation of TK transcript and a 10-fold increase in TK activity, reduced TMPK level and it had no effect on TP activity. The level of [3H]-dT uptake and incorporation into DNA in the thyA mutant correlates with increases in TK activity, suggesting that dT uptake and metabolism is TK-dependent and that upregulation of TK activity in the thyA mutant compensates for the lack of ThyA activity. [3H]-dU uptake was low compared with dT, and incorporation of radioactivity into DNA in the thyA mutant indicates the presence of an alternative TS. Our results suggest that TK and TMPK are potential targets for the development of Mycoplasma-specific antibiotics
Implication of Glycerol and Phospholipid Transporters in Mycoplasma pneumoniae Growth and Virulence
No full textMycoplasma pneumoniae, the causative agent of atypical pneumonia, is one of the bacteria with the smallest genomes that are nonetheless capable of independent life. Because of their longstanding close association with their human host, the bacteria have undergone reductive evolution and lost most biosynthetic abilities. Therefore, they depend on nutrients provided by the host that have to be taken up by the cell. Indeed, M. pneumoniae has a large set of hitherto unexplored transporters and lipoproteins that may be implicated in transport processes. Together, these proteins account for about 17% of the protein complement of M. pneumoniae. In the natural habitat of M. pneumoniae, human lung epithelial surfaces, phospholipids are the major available carbon source. Thus, the uptake and utilization of glycerol and glycerophosphodiesters that are generated by the activity of lipases are important for the nutrition of M. pneumoniae in its common habitat. In this study, we have investigated the roles of several potential transport proteins and lipoproteins in the utilization of glycerol and glycerophosphodiesters. On the basis of experiments with the corresponding mutant strains, our results demonstrate that the newly identified GlpU transport protein (MPN421) is responsible for the uptake of the glycerophosphodiester glycerophosphocholine, which is then intracellularly cleaved to glycerol-3-phosphate and choline. In addition, the proteins MPN076 and MPN077 are accessory factors in glycerophosphocholine uptake. Moreover, the lipoproteins MPN133 and MPN284 are essential for the uptake of glycerol. Our data suggest that they may act as binding proteins for glycerol and deliver glycerol molecules to the glycerol facilitator GlpF.Fonds der Chemischen Industrie; Studienstiftung des Deutschen Volke
The Stability of Cytadherence Proteins in Mycoplasma pneumoniae Requires Activity of the Protein Kinase PrkC
No full textMycoplasma 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
The Phosphoproteome of the Minimal Bacterium Mycoplasma pneumoniae ANALYSIS OF THE COMPLETE KNOWN SER/THR KINOME SUGGESTS THE EXISTENCE OF NOVEL KINASES
No full textMycoplasma 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. Molecular & Cellular Proteomics 9:1228-1242, 2010
Going Beyond Counting First Authors in Author Co-citation Analysis
Full text linkThe present study examines one of the fundamental aspects of author co-citation analysis (ACA) - the way co-citation
counts are defined. Co-citation counting provides the data on which all subsequent statistical analyses and mappings
are based, and we compare ACA results based on two different types of co-citation counting - the traditional type that
only counts the first one among a cited work's authors on the one hand and a non-traditional type that takes into
account the first 5 authors of a cited work on the other hand. Results indicate that the picture produced through this non-traditional author co-citation counting contains more coherent author groups and is therefore considerably clearer. However, this picture represents fewer specialties in the research field being studied than that produced through the traditional first-author co-citation counting when the same number of top-ranked authors is selected and analyzed. Reasons for these effects are discussed
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