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. 1989 Oct;171(10):5322–5324. doi: 10.1128/jb.171.10.5322-5324.1989

Bacillus subtilis mutant allele sup-3 causes lysine insertion at ochre codons: use of sup-3 in studies of translational attenuation.

W W Mulbry 1, N P Ambulos Jr 1, P S Lovett 1
PMCID: PMC210368  PMID: 2507520

Abstract

The mutation sup-3 in Bacillus subtilis suppresses ochre (TAA) mutations at each of three codons in the 5' end of the cat-86 coding sequence. The suppressor is shown to insert lysine at ochre codons. The efficiency of suppression by sup-3 is about 15%, as determined by changing a cat-86 Lys codon (codon 12) to an ochre codon and measuring the level of CAT in the suppressor-containing strain. The results obtained are discussed in light of previous observations that ochre mutations at cat leader codons 2 and 3 can be phenotypically suppressed by sup-3, whereas ochre mutations at leader codons 4 and 5 cannot. Translation of the cat leader is essential to inducible expression of cat. Our data support the interpretation that the nature of amino acids 2 through 5 of the leader peptide contributes to determining whether chloramphenicol can stall a ribosome in the leader, which in turn leads to induction of cat expression.

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Selected References

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  1. Alexieva Z., Duvall E. J., Ambulos N. P., Jr, Kim U. J., Lovett P. S. Chloramphenicol induction of cat-86 requires ribosome stalling at a specific site in the leader. Proc Natl Acad Sci U S A. 1988 May;85(9):3057–3061. doi: 10.1073/pnas.85.9.3057. [DOI] [PMC free article] [PubMed] [Google Scholar]
  2. Bradford M. M. A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal Biochem. 1976 May 7;72:248–254. doi: 10.1016/0003-2697(76)90527-3. [DOI] [PubMed] [Google Scholar]
  3. Costanzo M., Brzustowicz L., Hannett N., Pero J. Bacteriophage SPO1 genes 33 and 34. Location and primary structure of genes encoding regulatory subunits of Bacillus subtilis RNA polymerase. J Mol Biol. 1984 Dec 15;180(3):533–547. doi: 10.1016/0022-2836(84)90026-3. [DOI] [PubMed] [Google Scholar]
  4. Dubnau D. Induction of ermC requires translation of the leader peptide. EMBO J. 1985 Feb;4(2):533–537. doi: 10.1002/j.1460-2075.1985.tb03661.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  5. Dubnau D. Translational attenuation: the regulation of bacterial resistance to the macrolide-lincosamide-streptogramin B antibiotics. CRC Crit Rev Biochem. 1984;16(2):103–132. doi: 10.3109/10409238409102300. [DOI] [PubMed] [Google Scholar]
  6. Duvall E. J., Ambulos N. P., Jr, Lovett P. S. Drug-free induction of a chloramphenicol acetyltransferase gene in Bacillus subtilis by stalling ribosomes in a regulatory leader. J Bacteriol. 1987 Sep;169(9):4235–4241. doi: 10.1128/jb.169.9.4235-4241.1987. [DOI] [PMC free article] [PubMed] [Google Scholar]
  7. Duvall E. J., Lovett P. S. Chloramphenicol induces translation of the mRNA for a chloramphenicol-resistance gene in Bacillus subtilis. Proc Natl Acad Sci U S A. 1986 Jun;83(11):3939–3943. doi: 10.1073/pnas.83.11.3939. [DOI] [PMC free article] [PubMed] [Google Scholar]
  8. Duvall E. J., Williams D. M., Lovett P. S., Rudolph C., Vasantha N., Guyer M. Chloramphenicol-inducible gene expression in Bacillus subtilis. Gene. 1983 Oct;24(2-3):171–177. doi: 10.1016/0378-1119(83)90077-x. [DOI] [PubMed] [Google Scholar]
  9. Fox T. D. Identification of phage SP01 proteins coded by regulatory genes 33 and 34. Nature. 1976 Aug 26;262(5571):748–753. doi: 10.1038/262748a0. [DOI] [PubMed] [Google Scholar]
  10. Fox T. D., Losick R., Pero J. Regulatory gene 28 of bacteriophage SPO1 codes for a phage-induced subunit of RNA polymerase. J Mol Biol. 1976 Mar 5;101(3):427–433. doi: 10.1016/0022-2836(76)90157-1. [DOI] [PubMed] [Google Scholar]
  11. Georgopoulos C. P. Suppressor system in Bacillus subtilis 168. J Bacteriol. 1969 Mar;97(3):1397–1402. doi: 10.1128/jb.97.3.1397-1402.1969. [DOI] [PMC free article] [PubMed] [Google Scholar]
  12. Harwood C. R., Williams D. M., Lovett P. S. Nucleotide sequence of a Bacillus pumilus gene specifying chloramphenicol acetyltransferase. Gene. 1983 Oct;24(2-3):163–169. doi: 10.1016/0378-1119(83)90076-8. [DOI] [PubMed] [Google Scholar]
  13. Henner D. J., Hoch J. A. The Bacillus subtilis chromosome. Microbiol Rev. 1980 Mar;44(1):57–82. doi: 10.1128/mr.44.1.57-82.1980. [DOI] [PMC free article] [PubMed] [Google Scholar]
  14. Henner D. J., Steinberg W. Genetic location of the Bacillus subtilis sup-3 suppressor mutation. J Bacteriol. 1979 Aug;139(2):668–670. doi: 10.1128/jb.139.2.668-670.1979. [DOI] [PMC free article] [PubMed] [Google Scholar]
  15. Laredo J., Wolff V. L., Lovett P. S. Chloramphenicol acetyltransferase specified by cat-86: relationship between the gene and the protein. Gene. 1988 Dec 15;73(1):209–214. doi: 10.1016/0378-1119(88)90327-7. [DOI] [PubMed] [Google Scholar]
  16. Lipsky R. H., Rosenthal R., Zahler S. A. Defective specialized SP beta transducing bacteriophages of Bacillus subtilis that carry the sup-3 or sup-44 gene. J Bacteriol. 1981 Dec;148(3):1012–1015. doi: 10.1128/jb.148.3.1012-1015.1981. [DOI] [PMC free article] [PubMed] [Google Scholar]
  17. Mellado R. P., Vinuela E., Salas M. Isolation of a strong suppressor of nonsense mutations in Bacillus subtilis. Eur J Biochem. 1976 May 17;65(1):213–223. doi: 10.1111/j.1432-1033.1976.tb10408.x. [DOI] [PubMed] [Google Scholar]
  18. Mongkolsuk S., Chiang Y. W., Reynolds R. B., Lovett P. S. Restriction fragments that exert promoter activity during postexponential growth of Bacillus subtilis. J Bacteriol. 1983 Sep;155(3):1399–1406. doi: 10.1128/jb.155.3.1399-1406.1983. [DOI] [PMC free article] [PubMed] [Google Scholar]
  19. Shaw W. V. Chloramphenicol acetyltransferase from chloramphenicol-resistant bacteria. Methods Enzymol. 1975;43:737–755. doi: 10.1016/0076-6879(75)43141-x. [DOI] [PubMed] [Google Scholar]
  20. Taylor J. W., Ott J., Eckstein F. The rapid generation of oligonucleotide-directed mutations at high frequency using phosphorothioate-modified DNA. Nucleic Acids Res. 1985 Dec 20;13(24):8765–8785. doi: 10.1093/nar/13.24.8765. [DOI] [PMC free article] [PubMed] [Google Scholar]
  21. Zoller M. J., Smith M. Oligonucleotide-directed mutagenesis of DNA fragments cloned into M13 vectors. Methods Enzymol. 1983;100:468–500. doi: 10.1016/0076-6879(83)00074-9. [DOI] [PubMed] [Google Scholar]

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