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. 1991 Sep;173(18):5653–5662. doi: 10.1128/jb.173.18.5653-5662.1991

Novel structure of the recA locus of Mycobacterium tuberculosis implies processing of the gene product.

E O Davis 1, S G Sedgwick 1, M J Colston 1
PMCID: PMC208294  PMID: 1909321

Abstract

A fragment of Mycobacterium tuberculosis DNA containing recA-like sequences was identified by hybridization with the Escherichia coli recA gene and cloned. Although no expression was detected from its own promoter in E. coli, expression from a vector promoter partially complemented E. coli recA mutants for recombination, DNA repair, and mutagenesis, but not for induction of phage lambda. This clone produced a protein which cross-reacts with antisera raised against the E. coli RecA protein and was approximately the same size. However, the nucleotide sequence of the cloned fragment revealed the presence of an open reading frame for a protein about twice the size of other RecA proteins and the cloned product detected by Western blotting (immunoblotting). The predicted M. tuberculosis RecA protein sequence was homologous with RecA sequences from other bacteria, but this homology was not dispersed; rather it was localized to the first 254 and the last 96 amino acids, with the intervening 440 amino acids being unrelated. Furthermore, the junctions of homology were in register with the uninterrupted sequence of the E. coli RecA protein. Identical restriction fragments were found in the genomic DNAs of M. tuberculosis H37Rv and H37Ra and of M. bovis BCG. It is concluded that the ancestral recA gene of these species diversified via an insertional mutation of at least 1,320 bp of DNA. Possible processing mechanisms for synthesizing a normal-size RecA protein from this elongated sequence are discussed.

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Selected References

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  1. Allen B. W. Mycobacterium tuberculosis strain H37Rv. J Med Lab Technol. 1969 Oct;26(4):389–390. [PubMed] [Google Scholar]
  2. Bagg A., Kenyon C. J., Walker G. C. Inducibility of a gene product required for UV and chemical mutagenesis in Escherichia coli. Proc Natl Acad Sci U S A. 1981 Sep;78(9):5749–5753. doi: 10.1073/pnas.78.9.5749. [DOI] [PMC free article] [PubMed] [Google Scholar]
  3. Belfort M. Self-splicing introns in prokaryotes: migrant fossils? Cell. 1991 Jan 11;64(1):9–11. doi: 10.1016/0092-8674(91)90201-9. [DOI] [PubMed] [Google Scholar]
  4. Bolivar F., Rodriguez R. L., Greene P. J., Betlach M. C., Heyneker H. L., Boyer H. W., Crosa J. H., Falkow S. Construction and characterization of new cloning vehicles. II. A multipurpose cloning system. Gene. 1977;2(2):95–113. [PubMed] [Google Scholar]
  5. Bowles D. J., Marcus S. E., Pappin D. J., Findlay J. B., Eliopoulos E., Maycox P. R., Burgess J. Posttranslational processing of concanavalin A precursors in jackbean cotyledons. J Cell Biol. 1986 Apr;102(4):1284–1297. doi: 10.1083/jcb.102.4.1284. [DOI] [PMC free article] [PubMed] [Google Scholar]
  6. 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]
  7. Burckhardt S. E., Woodgate R., Scheuermann R. H., Echols H. UmuD mutagenesis protein of Escherichia coli: overproduction, purification, and cleavage by RecA. Proc Natl Acad Sci U S A. 1988 Mar;85(6):1811–1815. doi: 10.1073/pnas.85.6.1811. [DOI] [PMC free article] [PubMed] [Google Scholar]
  8. Carlsson J., Carpenter V. S. The recA+ gene product is more important than catalase and superoxide dismutase in protecting Escherichia coli against hydrogen peroxide toxicity. J Bacteriol. 1980 Apr;142(1):319–321. doi: 10.1128/jb.142.1.319-321.1980. [DOI] [PMC free article] [PubMed] [Google Scholar]
  9. Carrington D. M., Auffret A., Hanke D. E. Polypeptide ligation occurs during post-translational modification of concanavalin A. Nature. 1985 Jan 3;313(5997):64–67. doi: 10.1038/313064a0. [DOI] [PubMed] [Google Scholar]
  10. Cheo D. L., Bayles K. W., Yasbin R. E. Cloning and characterization of DNA damage-inducible promoter regions from Bacillus subtilis. J Bacteriol. 1991 Mar;173(5):1696–1703. doi: 10.1128/jb.173.5.1696-1703.1991. [DOI] [PMC free article] [PubMed] [Google Scholar]
  11. 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]
  12. Cowing D. W., Bardwell J. C., Craig E. A., Woolford C., Hendrix R. W., Gross C. A. Consensus sequence for Escherichia coli heat shock gene promoters. Proc Natl Acad Sci U S A. 1985 May;82(9):2679–2683. doi: 10.1073/pnas.82.9.2679. [DOI] [PMC free article] [PubMed] [Google Scholar]
  13. DAVIS B. D., MINGIOLI E. S. Mutants of Escherichia coli requiring methionine or vitamin B12. J Bacteriol. 1950 Jul;60(1):17–28. doi: 10.1128/jb.60.1.17-28.1950. [DOI] [PMC free article] [PubMed] [Google Scholar]
  14. Devereux J., Haeberli P., Smithies O. A comprehensive set of sequence analysis programs for the VAX. Nucleic Acids Res. 1984 Jan 11;12(1 Pt 1):387–395. doi: 10.1093/nar/12.1part1.387. [DOI] [PMC free article] [PubMed] [Google Scholar]
  15. Dreyfus L. A. Molecular cloning and expression in Escherichia coli of the recA gene of Legionella pneumophila. J Gen Microbiol. 1989 Nov;135(11):3097–3107. doi: 10.1099/00221287-135-11-3097. [DOI] [PubMed] [Google Scholar]
  16. Dutreix M., Moreau P. L., Bailone A., Galibert F., Battista J. R., Walker G. C., Devoret R. New recA mutations that dissociate the various RecA protein activities in Escherichia coli provide evidence for an additional role for RecA protein in UV mutagenesis. J Bacteriol. 1989 May;171(5):2415–2423. doi: 10.1128/jb.171.5.2415-2423.1989. [DOI] [PMC free article] [PubMed] [Google Scholar]
  17. Ennis D. G., Ossanna N., Mount D. W. Genetic separation of Escherichia coli recA functions for SOS mutagenesis and repressor cleavage. J Bacteriol. 1989 May;171(5):2533–2541. doi: 10.1128/jb.171.5.2533-2541.1989. [DOI] [PMC free article] [PubMed] [Google Scholar]
  18. Fine P. E. BCG vaccination against tuberculosis and leprosy. Br Med Bull. 1988 Jul;44(3):691–703. doi: 10.1093/oxfordjournals.bmb.a072277. [DOI] [PubMed] [Google Scholar]
  19. Fine P. E., Rodrigues L. C. Modern vaccines. Mycobacterial diseases. Lancet. 1990 Apr 28;335(8696):1016–1020. doi: 10.1016/0140-6736(90)91074-k. [DOI] [PubMed] [Google Scholar]
  20. Gough J. A., Murray N. E. Sequence diversity among related genes for recognition of specific targets in DNA molecules. J Mol Biol. 1983 May 5;166(1):1–19. doi: 10.1016/s0022-2836(83)80047-3. [DOI] [PubMed] [Google Scholar]
  21. HOWARD-FLANDERS P., SIMSON E., THERIOT L. A LOCUS THAT CONTROLS FILAMENT FORMATION AND SENSITIVITY TO RADIATION IN ESCHERICHIA COLI K-12. Genetics. 1964 Feb;49:237–246. doi: 10.1093/genetics/49.2.237. [DOI] [PMC free article] [PubMed] [Google Scholar]
  22. Hassett D. J., Charniga L., Cohen M. S. recA and catalase in H2O2-mediated toxicity in Neisseria gonorrhoeae. J Bacteriol. 1990 Dec;172(12):7293–7296. doi: 10.1128/jb.172.12.7293-7296.1990. [DOI] [PMC free article] [PubMed] [Google Scholar]
  23. Hirata R., Ohsumk Y., Nakano A., Kawasaki H., Suzuki K., Anraku Y. Molecular structure of a gene, VMA1, encoding the catalytic subunit of H(+)-translocating adenosine triphosphatase from vacuolar membranes of Saccharomyces cerevisiae. J Biol Chem. 1990 Apr 25;265(12):6726–6733. [PubMed] [Google Scholar]
  24. Holmes D. S., Quigley M. A rapid boiling method for the preparation of bacterial plasmids. Anal Biochem. 1981 Jun;114(1):193–197. doi: 10.1016/0003-2697(81)90473-5. [DOI] [PubMed] [Google Scholar]
  25. Howard-Flanders P., West S. C., Stasiak A. Role of RecA protein spiral filaments in genetic recombination. Nature. 1984 May 17;309(5965):215–219. doi: 10.1038/309215a0. [DOI] [PubMed] [Google Scholar]
  26. Jacobs W. R., Docherty M. A., Curtiss R., 3rd, Clark-Curtiss J. E. Expression of Mycobacterium leprae genes from a Streptococcus mutans promoter in Escherichia coli K-12. Proc Natl Acad Sci U S A. 1986 Mar;83(6):1926–1930. doi: 10.1073/pnas.83.6.1926. [DOI] [PMC free article] [PubMed] [Google Scholar]
  27. Kane P. M., Yamashiro C. T., Wolczyk D. F., Neff N., Goebl M., Stevens T. H. Protein splicing converts the yeast TFP1 gene product to the 69-kD subunit of the vacuolar H(+)-adenosine triphosphatase. Science. 1990 Nov 2;250(4981):651–657. doi: 10.1126/science.2146742. [DOI] [PubMed] [Google Scholar]
  28. Kjems J., Garrett R. A. Ribosomal RNA introns in archaea and evidence for RNA conformational changes associated with splicing. Proc Natl Acad Sci U S A. 1991 Jan 15;88(2):439–443. doi: 10.1073/pnas.88.2.439. [DOI] [PMC free article] [PubMed] [Google Scholar]
  29. Lerner C. G., Inouye M. Low copy number plasmids for regulated low-level expression of cloned genes in Escherichia coli with blue/white insert screening capability. Nucleic Acids Res. 1990 Aug 11;18(15):4631–4631. doi: 10.1093/nar/18.15.4631. [DOI] [PMC free article] [PubMed] [Google Scholar]
  30. Lipman D. J., Pearson W. R. Rapid and sensitive protein similarity searches. Science. 1985 Mar 22;227(4693):1435–1441. doi: 10.1126/science.2983426. [DOI] [PubMed] [Google Scholar]
  31. Lloyd R. G., Buckman C. Genetic analysis of the recG locus of Escherichia coli K-12 and of its role in recombination and DNA repair. J Bacteriol. 1991 Feb;173(3):1004–1011. doi: 10.1128/jb.173.3.1004-1011.1991. [DOI] [PMC free article] [PubMed] [Google Scholar]
  32. Love P. E., Yasbin R. E. Induction of the Bacillus subtilis SOS-like response by Escherichia coli RecA protein. Proc Natl Acad Sci U S A. 1986 Jul;83(14):5204–5208. doi: 10.1073/pnas.83.14.5204. [DOI] [PMC free article] [PubMed] [Google Scholar]
  33. Lovett C. M., Jr, Roberts J. W. Purification of a RecA protein analogue from Bacillus subtilis. J Biol Chem. 1985 Mar 25;260(6):3305–3313. [PubMed] [Google Scholar]
  34. Lowrie D. B. How macrophages kill tubercle bacilli. J Med Microbiol. 1983 Feb;16(1):1–12. doi: 10.1099/00222615-16-1-1. [DOI] [PubMed] [Google Scholar]
  35. Marrero R., Yasbin R. E. Cloning of the Bacillus subtilis recE+ gene and functional expression of recE+ in B. subtilis. J Bacteriol. 1988 Jan;170(1):335–344. doi: 10.1128/jb.170.1.335-344.1988. [DOI] [PMC free article] [PubMed] [Google Scholar]
  36. McEntee K. Genetic analysis of the Escherichia coli K-12 srl region. J Bacteriol. 1977 Dec;132(3):904–911. doi: 10.1128/jb.132.3.904-911.1977. [DOI] [PMC free article] [PubMed] [Google Scholar]
  37. McEvoy J. L., Murata H., Chatterjee A. K. Molecular cloning and characterization of an Erwinia carotovora subsp. carotovora pectin lyase gene that responds to DNA-damaging agents. J Bacteriol. 1990 Jun;172(6):3284–3289. doi: 10.1128/jb.172.6.3284-3289.1990. [DOI] [PMC free article] [PubMed] [Google Scholar]
  38. Mead D. A., Szczesna-Skorupa E., Kemper B. Single-stranded DNA 'blue' T7 promoter plasmids: a versatile tandem promoter system for cloning and protein engineering. Protein Eng. 1986 Oct-Nov;1(1):67–74. doi: 10.1093/protein/1.1.67. [DOI] [PubMed] [Google Scholar]
  39. Miller R. V., Kokjohn T. A. General microbiology of recA: environmental and evolutionary significance. Annu Rev Microbiol. 1990;44:365–394. doi: 10.1146/annurev.mi.44.100190.002053. [DOI] [PubMed] [Google Scholar]
  40. Nohmi T., Battista J. R., Dodson L. A., Walker G. C. RecA-mediated cleavage activates UmuD for mutagenesis: mechanistic relationship between transcriptional derepression and posttranslational activation. Proc Natl Acad Sci U S A. 1988 Mar;85(6):1816–1820. doi: 10.1073/pnas.85.6.1816. [DOI] [PMC free article] [PubMed] [Google Scholar]
  41. Owttrim G. W., Coleman J. R. Regulation of expression and nucleotide sequence of the Anabaena variabilis recA gene. J Bacteriol. 1989 Oct;171(10):5713–5719. doi: 10.1128/jb.171.10.5713-5719.1989. [DOI] [PMC free article] [PubMed] [Google Scholar]
  42. Ramesar R. S., Abratt V., Woods D. R., Rawlings D. E. Nucleotide sequence and expression of a cloned Thiobacillus ferrooxidans recA gene in Escherichia coli. Gene. 1989 May 15;78(1):1–8. doi: 10.1016/0378-1119(89)90308-9. [DOI] [PubMed] [Google Scholar]
  43. Roca A. I., Cox M. M. The RecA protein: structure and function. Crit Rev Biochem Mol Biol. 1990;25(6):415–456. doi: 10.3109/10409239009090617. [DOI] [PubMed] [Google Scholar]
  44. Sancar A., Rupp W. D. Physical map of the recA gene. Proc Natl Acad Sci U S A. 1979 Jul;76(7):3144–3148. doi: 10.1073/pnas.76.7.3144. [DOI] [PMC free article] [PubMed] [Google Scholar]
  45. Sanger F., Nicklen S., Coulson A. R. DNA sequencing with chain-terminating inhibitors. Proc Natl Acad Sci U S A. 1977 Dec;74(12):5463–5467. doi: 10.1073/pnas.74.12.5463. [DOI] [PMC free article] [PubMed] [Google Scholar]
  46. Sedgwick S. G., Goodwin P. A. Differences in mutagenic and recombinational DNA repair in enterobacteria. Proc Natl Acad Sci U S A. 1985 Jun;82(12):4172–4176. doi: 10.1073/pnas.82.12.4172. [DOI] [PMC free article] [PubMed] [Google Scholar]
  47. Shih C. K., Wagner R., Feinstein S., Kanik-Ennulat C., Neff N. A dominant trifluoperazine resistance gene from Saccharomyces cerevisiae has homology with F0F1 ATP synthase and confers calcium-sensitive growth. Mol Cell Biol. 1988 Aug;8(8):3094–3103. doi: 10.1128/mcb.8.8.3094. [DOI] [PMC free article] [PubMed] [Google Scholar]
  48. Shinagawa H., Iwasaki H., Kato T., Nakata A. RecA protein-dependent cleavage of UmuD protein and SOS mutagenesis. Proc Natl Acad Sci U S A. 1988 Mar;85(6):1806–1810. doi: 10.1073/pnas.85.6.1806. [DOI] [PMC free article] [PubMed] [Google Scholar]
  49. Slilaty S. N., Little J. W. Lysine-156 and serine-119 are required for LexA repressor cleavage: a possible mechanism. Proc Natl Acad Sci U S A. 1987 Jun;84(12):3987–3991. doi: 10.1073/pnas.84.12.3987. [DOI] [PMC free article] [PubMed] [Google Scholar]
  50. Slilaty S. N., Rupley J. A., Little J. W. Intramolecular cleavage of LexA and phage lambda repressors: dependence of kinetics on repressor concentration, pH, temperature, and solvent. Biochemistry. 1986 Nov 4;25(22):6866–6875. doi: 10.1021/bi00370a020. [DOI] [PubMed] [Google Scholar]
  51. Staden R. Automation of the computer handling of gel reading data produced by the shotgun method of DNA sequencing. Nucleic Acids Res. 1982 Aug 11;10(15):4731–4751. doi: 10.1093/nar/10.15.4731. [DOI] [PMC free article] [PubMed] [Google Scholar]
  52. Thole J. E., Dauwerse H. G., Das P. K., Groothuis D. G., Schouls L. M., van Embden J. D. Cloning of Mycobacterium bovis BCG DNA and expression of antigens in Escherichia coli. Infect Immun. 1985 Dec;50(3):800–806. doi: 10.1128/iai.50.3.800-806.1985. [DOI] [PMC free article] [PubMed] [Google Scholar]
  53. Walker G. C. Mutagenesis and inducible responses to deoxyribonucleic acid damage in Escherichia coli. Microbiol Rev. 1984 Mar;48(1):60–93. doi: 10.1128/mr.48.1.60-93.1984. [DOI] [PMC free article] [PubMed] [Google Scholar]
  54. 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]
  55. Yarranton G. T., Sedgwick S. G. Cloned truncated recA genes in E. coli II. Effects of truncated gene products on in vivo recA+ protein activity. Mol Gen Genet. 1982;185(1):99–104. doi: 10.1007/BF00333797. [DOI] [PubMed] [Google Scholar]
  56. Young D. B. Structure of mycobacterial antigens. Br Med Bull. 1988 Jul;44(3):562–583. doi: 10.1093/oxfordjournals.bmb.a072268. [DOI] [PubMed] [Google Scholar]

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