Skip to main content
NCBI home page
Journal of Bacteriology logoLink to Journal of Bacteriology
. 1990 Jun;172(6):2986–2995. doi: 10.1128/jb.172.6.2986-2995.1990

Multiple translational products from a Mycoplasma hyorhinis gene expressed in Escherichia coli.

S M Notarnicola 1, M A McIntosh 1, K S Wise 1
PMCID: PMC209098  PMID: 2188946

Abstract

We analyzed protein expression from a cloned Mycoplasma hyorhinis genomic fragment that produces in Escherichia coli a set of related polypeptides of 110, 100, 65, and 55 kilodaltons from a coding region of just over 3.0 kilobases. Expression of these multiple products resulted from a mechanism operating at the translational level but not from truncation at UGA termination codons, which are known to encode tryptophan in several mycoplasma species. The structural relatedness of the proteins was demonstrated by two-dimensional tryptic peptic mapping, but their generation by posttranslational processing was ruled out by pulse-chase labeling analysis. Examination of proteins expressed from plasmid constructs and tryptic peptide analysis of these polypeptides and the original set of proteins revealed that they share carboxy-terminal regions, an observation inconsistent with truncation at UGA codons. Expression of proteins from this cloned fragment was not dependent on vector sequences and was observed when the coding region was placed under control of a T7 promoter, suggesting that all products were translated from a single message. Expression of related products in mycoplasmas was examined by immunoblot analysis of M. hyorhinis proteins with antiserum against overexpressed recombinant proteins. A single 115-kilodalton mycoplasma protein was detected, which is larger than any of the related proteins expressed in E. coli. Our analysis indicated that translation initiation sites are used in E. coli that are not active in mycoplasmas, thereby defining differences between the translational regulatory signals of mycoplasmas and eubacteria.

Full text

PDF
2986

Images in this article

2988Image
on p.2988
2990Image
on p.2990
2991Image
on p.2991
2992Image
on p.2992
2993Image
on p.2993
2994Image
on p.2994

Selected References

These references are in PubMed. This may not be the complete list of references from this article.

  1. Andersen H., Christiansen G., Christiansen C. Electrophoretic analysis of proteins from Mycoplasma capricolum and related serotypes using extracts from intact cells and from minicells containing cloned mycoplasma DNA. J Gen Microbiol. 1984 Jun;130(6):1409–1418. doi: 10.1099/00221287-130-6-1409. [DOI] [PubMed] [Google Scholar]
  2. Birnboim H. C., Doly J. A rapid alkaline extraction procedure for screening recombinant plasmid DNA. Nucleic Acids Res. 1979 Nov 24;7(6):1513–1523. doi: 10.1093/nar/7.6.1513. [DOI] [PMC free article] [PubMed] [Google Scholar]
  3. Blanchard A., Wroblewski H., Barroso G. Localization of spiralin in Escherichia coli cells transformed with the recombinant plasmid pESI. Isr J Med Sci. 1987 May;23(5):414–417. [PubMed] [Google Scholar]
  4. Bonner W. M., Laskey R. A. A film detection method for tritium-labelled proteins and nucleic acids in polyacrylamide gels. Eur J Biochem. 1974 Jul 1;46(1):83–88. doi: 10.1111/j.1432-1033.1974.tb03599.x. [DOI] [PubMed] [Google Scholar]
  5. Boyer H. W., Roulland-Dussoix D. A complementation analysis of the restriction and modification of DNA in Escherichia coli. J Mol Biol. 1969 May 14;41(3):459–472. doi: 10.1016/0022-2836(69)90288-5. [DOI] [PubMed] [Google Scholar]
  6. Boyer M. J., Wise K. S. Lipid-modified surface protein antigens expressing size variation within the species Mycoplasma hyorhinis. Infect Immun. 1989 Jan;57(1):245–254. doi: 10.1128/iai.57.1.245-254.1989. [DOI] [PMC free article] [PubMed] [Google Scholar]
  7. Cohen S. N., Chang A. C., Hsu L. Nonchromosomal antibiotic resistance in bacteria: genetic transformation of Escherichia coli by R-factor DNA. Proc Natl Acad Sci U S A. 1972 Aug;69(8):2110–2114. doi: 10.1073/pnas.69.8.2110. [DOI] [PMC free article] [PubMed] [Google Scholar]
  8. Dudler R., Schmidhauser C., Parish R. W., Wettenhall R. E., Schmidt T. A mycoplasma high-affinity transport system and the in vitro invasiveness of mouse sarcoma cells. EMBO J. 1988 Dec 1;7(12):3963–3970. doi: 10.1002/j.1460-2075.1988.tb03283.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  9. Frazer A. C., Curtiss R., 3rd Production, properties and utility of bacterial minicells. Curr Top Microbiol Immunol. 1975;69:1–84. doi: 10.1007/978-3-642-50112-8_1. [DOI] [PubMed] [Google Scholar]
  10. Graves M. C., Rabinowitz J. C. In vivo and in vitro transcription of the Clostridium pasteurianum ferredoxin gene. Evidence for "extended" promoter elements in gram-positive organisms. J Biol Chem. 1986 Aug 25;261(24):11409–11415. [PubMed] [Google Scholar]
  11. Guindy Y. S., Samuelsson T., Johansen T. I. Unconventional codon reading by Mycoplasma mycoides tRNAs as revealed by partial sequence analysis. Biochem J. 1989 Mar 15;258(3):869–873. doi: 10.1042/bj2580869. [DOI] [PMC free article] [PubMed] [Google Scholar]
  12. Hager P. W., Rabinowitz J. C. Inefficient translation of T7 late mRNA by Bacillus subtilis ribosomes. Implications for species-specific translation. J Biol Chem. 1985 Dec 5;260(28):15163–15167. [PubMed] [Google Scholar]
  13. Inamine J. M., Ho K. C., Loechel S., Hu P. C. Evidence that UGA is read as a tryptophan codon rather than as a stop codon by Mycoplasma pneumoniae, Mycoplasma genitalium, and Mycoplasma gallisepticum. J Bacteriol. 1990 Jan;172(1):504–506. doi: 10.1128/jb.172.1.504-506.1990. [DOI] [PMC free article] [PubMed] [Google Scholar]
  14. Laemmli U. K. Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature. 1970 Aug 15;227(5259):680–685. doi: 10.1038/227680a0. [DOI] [PubMed] [Google Scholar]
  15. Mahairas G. G., Minion F. C. Transformation of Mycoplasma pulmonis: demonstration of homologous recombination, introduction of cloned genes, and preliminary description of an integrating shuttle system. J Bacteriol. 1989 Apr;171(4):1775–1780. doi: 10.1128/jb.171.4.1775-1780.1989. [DOI] [PMC free article] [PubMed] [Google Scholar]
  16. Messing J., Gronenborn B., Müller-Hill B., Hans Hopschneider P. Filamentous coliphage M13 as a cloning vehicle: insertion of a HindII fragment of the lac regulatory region in M13 replicative form in vitro. Proc Natl Acad Sci U S A. 1977 Sep;74(9):3642–3646. doi: 10.1073/pnas.74.9.3642. [DOI] [PMC free article] [PubMed] [Google Scholar]
  17. Renaudin J., Pascarel M. C., Bové J. M. Spiroplasma virus 4: nucleotide sequence of the viral DNA, regulatory signals, and proposed genome organization. J Bacteriol. 1987 Nov;169(11):4950–4961. doi: 10.1128/jb.169.11.4950-4961.1987. [DOI] [PMC free article] [PubMed] [Google Scholar]
  18. Rogers M. J., Simmons J., Walker R. T., Weisburg W. G., Woese C. R., Tanner R. S., Robinson I. M., Stahl D. A., Olsen G., Leach R. H. Construction of the mycoplasma evolutionary tree from 5S rRNA sequence data. Proc Natl Acad Sci U S A. 1985 Feb;82(4):1160–1164. doi: 10.1073/pnas.82.4.1160. [DOI] [PMC free article] [PubMed] [Google Scholar]
  19. SIMON E. H., TESSMAN I. THYMIDINE-REQUIRING MUTANTS OF PHAGE T4. Proc Natl Acad Sci U S A. 1963 Sep;50:526–532. doi: 10.1073/pnas.50.3.526. [DOI] [PMC free article] [PubMed] [Google Scholar]
  20. Samuelsson T., Guindy Y. S., Lustig F., Borén T., Lagerkvist U. Apparent lack of discrimination in the reading of certain codons in Mycoplasma mycoides. Proc Natl Acad Sci U S A. 1987 May;84(10):3166–3170. doi: 10.1073/pnas.84.10.3166. [DOI] [PMC free article] [PubMed] [Google Scholar]
  21. Su C. J., Tryon V. V., Baseman J. B. Cloning and sequence analysis of cytadhesin P1 gene from Mycoplasma pneumoniae. Infect Immun. 1987 Dec;55(12):3023–3029. doi: 10.1128/iai.55.12.3023-3029.1987. [DOI] [PMC free article] [PubMed] [Google Scholar]
  22. Tabor S., Richardson C. C. A bacteriophage T7 RNA polymerase/promoter system for controlled exclusive expression of specific genes. Proc Natl Acad Sci U S A. 1985 Feb;82(4):1074–1078. doi: 10.1073/pnas.82.4.1074. [DOI] [PMC free article] [PubMed] [Google Scholar]
  23. Taschke C., Klinkert M. Q., Wolters J., Herrmann R. Organization of the ribosomal RNA genes in Mycoplasma hyopneumoniae: the 5S rRNA gene is separated from the 16S and 23S rRNA genes. Mol Gen Genet. 1986 Dec;205(3):428–433. doi: 10.1007/BF00338078. [DOI] [PubMed] [Google Scholar]
  24. Taylor M. A., McIntosh M. A., Robbins J., Wise K. S. Cloned genomic DNA sequences from Mycoplasma hyorhinis encoding antigens expressed in Escherichia coli. Proc Natl Acad Sci U S A. 1983 Jul;80(13):4154–4158. doi: 10.1073/pnas.80.13.4154. [DOI] [PMC free article] [PubMed] [Google Scholar]
  25. Tegman V., Wallbrandt P., Nyström S., Johansson K. E., Jonsson B. H., Wieslander A. Cloning and expression of Acholeplasma laidlawii membrane acyl proteins in Escherichia coli. Isr J Med Sci. 1987 May;23(5):408–413. [PubMed] [Google Scholar]
  26. Trevino L. B., Haldenwang W. G., Baseman J. B. Expression of Mycoplasma pneumoniae antigens in Escherichia coli. Infect Immun. 1986 Jul;53(1):129–134. doi: 10.1128/iai.53.1.129-134.1986. [DOI] [PMC free article] [PubMed] [Google Scholar]
  27. Wise K. S., Watson R. K. Mycoplasma hyorhinis GDL surface protein antigen p120 defined by monoclonal antibody. Infect Immun. 1983 Sep;41(3):1332–1339. doi: 10.1128/iai.41.3.1332-1339.1983. [DOI] [PMC free article] [PubMed] [Google Scholar]
  28. Woese C. R. Bacterial evolution. Microbiol Rev. 1987 Jun;51(2):221–271. doi: 10.1128/mr.51.2.221-271.1987. [DOI] [PMC free article] [PubMed] [Google Scholar]
  29. Yamao F., Iwagami S., Azumi Y., Muto A., Osawa S., Fujita N., Ishihama A. Evolutionary dynamics of tryptophan tRNAs in Mycoplasma capricolum. Mol Gen Genet. 1988 May;212(2):364–369. doi: 10.1007/BF00334708. [DOI] [PubMed] [Google Scholar]
  30. Yamao F., Muto A., Kawauchi Y., Iwami M., Iwagami S., Azumi Y., Osawa S. UGA is read as tryptophan in Mycoplasma capricolum. Proc Natl Acad Sci U S A. 1985 Apr;82(8):2306–2309. doi: 10.1073/pnas.82.8.2306. [DOI] [PMC free article] [PubMed] [Google Scholar]
  31. Yanisch-Perron C., Vieira J., Messing J. Improved M13 phage cloning vectors and host strains: nucleotide sequences of the M13mp18 and pUC19 vectors. Gene. 1985;33(1):103–119. doi: 10.1016/0378-1119(85)90120-9. [DOI] [PubMed] [Google Scholar]

Articles from Journal of Bacteriology are provided here courtesy of American Society for Microbiology (ASM)

RESOURCES