Evidence that UGA is read as a tryptophan codon rather than as a stop codon by Mycoplasma pneumoniae, Mycoplasma genitalium, and Mycoplasma gallisepticum

Inamine, Julia M.; Ho, Kuo-Chieh; Loechel, Steve; Hu, Ping-Chuan

doi:10.1128/jb.172.1.504-506.1990

Cited by 120 publications

(81 citation statements)

References 12 publications

(4 reference statements)

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“…The RGD motif, which often plays a role in adhesion processes, was located near the C terminus (striped box). The two UGA opal termination codons which are used as Trp codons in M. pneumoniae (27) A single DPNAY pentapeptide appears not to be sufficient for binding these antibodies, since the anti-FP F10-2D antibodies show no cross-reactivity with HMW3 containing two DPNAY repeats separated by 33 amino acids. We searched the PROS-ITE database (1) for conserved peptide motifs and found the motif Arg-Gly-Asp (RGD) in domain III near the C terminus, confined to residues 399 to 401.…”

Section: Detection Of P65 In Triton X-100-insoluble Subfractionsmentioning

confidence: 99%

The proline-rich P65 protein of Mycoplasma pneumoniae is a component of the Triton X-100-insoluble fraction and exhibits size polymorphism in the strains M129 and FH

et al. 1995

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Previously, we described the identification of a novel Mycoplasma pneumoniae M129 protein, named P65 because of its apparent molecular mass of 65 kDa estimated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (T. Proft and R. Herrmann, Mol. Microbiol. 13:337-348, 1994). DNA sequence analysis of the P65 open reading frame (orfp65), however, revealed an ORF encoding a protein with a molecular weight of 47,034. This discrepancy can be explained by the unusual amino acid composition of this protein. According to the deduced amino acid sequence, the N-terminal half of P65 contains several penta-and hexapeptides (DPNAY and DPNQAY) forming a proline-rich acidic domain. Secondary-structure predictions indicated ␤-sheets and turns within that region, suggesting an extended and rigid conformation. Near the C terminus of P65 the tripeptide Arg-Gly-Asp (RGD) was found. This motif is known to play an important role in binding of extracellular matrix proteins to integrins. P65 could be located exclusively to the Triton X-100-insoluble cell fraction. The results of immunofluorescence microscopy and of immunoadsorption experiments indicated that P65 carries surface-exposed regions. Mild treatment of whole cells with proteases resulted in cleavage of a limited amount of P65 molecules, suggesting either that only a small percentage of P65 molecules are exposed on the surface or that protease cleavage is hampered by a compact protein conformation or by binding of an unknown component to P65. P65 exhibits size polymorphism in M. pneumoniae M129 and FH. This is caused by an intragenetic duplication of a 54-bp sequence within the FH orfp65. As a consequence, the number of DPNAY pentapeptides increased from 9 to 12 repeats in the FH strain.Mycoplasma pneumoniae is an extracellular pathogen of the human respiratory tract (26, 46) causing histopathological changes of lung epithelial cells, usually in older children and young adults (18). A critical step in bacterial colonization of the host cells is the specific adhesion to host cell receptors, mediated by bacterial adhesins. In M. pneumoniae, the P1 adhesin (3,17,25,26) and the adhesin-related 30-kDa protein (4, 10) have been identified. Both proteins are located mainly in a tip structure that functions as the attachment organelle of the bacterium. It could be demonstrated, by nearestneighbor analysis with a hydrophilic chemical cross-linker, that the product of open reading frame 6 (ORF6) of the P1 operon (28), a 40-kDa protein and 90-kDa protein (9, 34, 50) are located in close proximity to the P1 adhesin on the cell surface (35).Scanning and transmission electron microscope analyses of M. pneumoniae cells grown on grids and pretreated with Triton X-100 revealed a rodlike tip structure and a network of filamentous strands (22,41). Krause and coworkers identified a set of high-molecular-weight proteins (HMW1 to HMW5) which play an important role in cytadherence (30,31,(51)(52)(53) and appear to be involved in formation of a cytoskeletonlike structure (38). These proteins, ...

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Section: Detection Of P65 In Triton X-100-insoluble Subfractionsmentioning

confidence: 99%

The proline-rich P65 protein of Mycoplasma pneumoniae is a component of the Triton X-100-insoluble fraction and exhibits size polymorphism in the strains M129 and FH

et al. 1995

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“…In a first PCR, the genome regions surrounding the two genes of interest were amplified with the primers MpPdhBf1/r and MpEnof1/r (Table 1). These PCR products (1 : 10 diluted in HPLC water) were used as targets for the multiple mutation reaction (MMR; Hames et al, 2005) to amplify the complete genes and to mutate simultaneously the single TGA codon (encoding tryptophan in mycoplasmas; Inamine et al, 1990) occurring in both genes. Using pfu polymerase (Fermentas), the primer pairs MpPdhBVf/Vr and MpEnoVf/Vr create vector-specific overhangs, whereas the mutation primers MpPdhBM and MpEnoM change the TGA codons into TGG.…”

Section: Methodsmentioning

confidence: 99%

Characterization of pyruvate dehydrogenase subunit B and enolase as plasminogen-binding proteins in Mycoplasma pneumoniae

2013

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The obligate pathogenic mycoplasma species Mycoplasma pneumoniae uses a limited but effective repertoire of virulence factors to infect and colonize the human respiratory tract. Besides the development of a unique adhesion complex and the expression of tissue-damaging factors, surface-located glycolytic enzymes and their capacity to bind to components of the human extracellular matrix (ECM) support pathogen-host interactions. Here, we demonstrated that the glycolytic enzymes enolase (Mpn606) and pyruvate dehydrogenase subunit B (Mpn392; PDHB) of M. pneumoniae show concentration-dependent binding to human plasminogen. Monospecific polyclonal antisera against both recombinant proteins reduced the binding to plasminogen significantly. The surface location of PDHB but not of enolase was demonstrated using Triton X fractionation of M. pneumoniae total protein content, membrane fractionation, colony blotting, mild proteolysis of mycoplasma cells, and immunofluorescence tests. To characterize the binding site of plasminogen in surface-displaced PDHB, the mycoplasmal protein was separated into four recombinant proteins followed by investigation of the binding behaviour of peptides that overlap the protein part interacting with plasminogen. Spot analysis resulted in a novel region of 12 amino acids (FPAMFQIFTHAA, position 91 to 102 of PDHB), which is responsible exclusively for binding of human plasminogen and also interacts in a dose-dependent manner with this host protein. The data indicate that the plasminogen-binding enzymes enolase and especially the surface-associated PDHB may contribute to the pathogenesis of M. pneumoniae infections.

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“…Earlier minimal standards recommended that the base composition (G+C content, mol%) of chromosomal DNA be determined for each novel species, but there are frequent non-significant (homoplasic) overlaps in the values. The UGA codon usage appears to offer a sharp distinction between higher mollicute taxa (Yamao et al, 1985;Renaudin et al, 1986;Inamine et al, 1990;Citti et al, 1992;Navas-Castillo et al, 1992). Members of the Mycoplasmatales and Entomoplasmatales, so far as is known, utilize both UGA and UGG as tryptophan (W) codons, while only UGG encodes W in members of the Acholeplasmatales and Anaeroplasmatales and phytoplasmas.…”

Section: (Xii) Genomic and Genetic Analysesmentioning

confidence: 99%

Revised minimal standards for description of new species of the class Mollicutes (division Tenericutes)

Brown

Whitcomb

Bradbury

2007

International Journal of Systematic and Evolutionary Microbiology

138

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Minimal standards for novel species of the class Mollicutes (trivial term, mollicutes), last published in 1995, require revision. The International Committee on Systematics of Prokaryotes Subcommittee on the Taxonomy of Mollicutes proposes herein revised standards that reflect recent advances in molecular systematics and the species concept for prokaryotes. The mandatory requirements are: (i) deposition of the type strain into two recognized culture collections, preferably located in different countries; (ii) deposition of the 16S rRNA gene sequence into a public database, and a phylogenetic analysis of the relationships among the 16S rRNA gene sequences of the novel species and its neighbours; (iii) deposition of antiserum against the type strain into a recognized collection; (iv) demonstration, by using the combination of 16S rRNA gene sequence analyses, serological analyses and supplementary phenotypic data, that the type strain differs significantly from all previously named species; and (v) assignment to an order, a family and a genus in the class, with an appropriate specific epithet. The 16S rRNA gene sequence provides the primary basis for assignment to hierarchical rank, and may also constitute evidence of species novelty, but serological and supplementary phenotypic data must be presented to substantiate this. Serological methods have been documented to be congruent with DNA-DNA hybridization data and with 16S rRNA gene placements. The novel species must be tested serologically to the greatest extent that the investigators deem feasible against all neighbouring species whose 16S rRNA gene sequences show .0.94 similarity. The investigator is responsible for justifying which characters are most meaningful for assignment to the part of the mollicute phylogenetic tree in which a novel species is located, and for providing the means by which novel species can be identified by other investigators.

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Evidence that UGA is read as a tryptophan codon rather than as a stop codon by Mycoplasma pneumoniae, Mycoplasma genitalium, and Mycoplasma gallisepticum

Cited by 120 publications

References 12 publications

The proline-rich P65 protein of Mycoplasma pneumoniae is a component of the Triton X-100-insoluble fraction and exhibits size polymorphism in the strains M129 and FH

The proline-rich P65 protein of Mycoplasma pneumoniae is a component of the Triton X-100-insoluble fraction and exhibits size polymorphism in the strains M129 and FH

Characterization of pyruvate dehydrogenase subunit B and enolase as plasminogen-binding proteins in Mycoplasma pneumoniae

Revised minimal standards for description of new species of the class Mollicutes (division Tenericutes)

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