Abstract
The recA gene of Mycobacterium tuberculosis has previously been cloned and sequenced (E. O. Davis, S. G. Sedgwick, and M. J. Colston, J. Bacteriol. 173:5653-5662, 1991). In this study, the expression of this gene was shown to be inducible in response to various DNA-damaging agents by using a transcriptional fusion to the reporter gene encoding chloramphenicol acetyltransferase. A segment of DNA around 300 bp upstream of the coding region was shown to be required for expression. However, primer extension analysis indicated that the transcriptional start sites were 47 and 93 bp upstream of the translation initiation codon. Sequence motifs with homology to two families of Escherichia coli promoters but also with significant differences were located near these proposed transcription start sites. The differences from the E. coli consensus patterns would explain the previously described lack of expression of the M. tuberculosis recA gene from its own promoter in E. coli. In addition, the M. tuberculosis LexA protein was shown to bind specifically to a sequence, GAAC-N4-GTTC, overlapping one of these putative promoters and homologous to the Bacillus subtilis Cheo box involved in the regulation of SOS genes. The region of DNA 300 bp upstream of the recA gene was shown not to contain a promoter, suggesting that it functions as an upstream activator sequence.
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- Bashyam M. D., Kaushal D., Dasgupta S. K., Tyagi A. K. A study of mycobacterial transcriptional apparatus: identification of novel features in promoter elements. J Bacteriol. 1996 Aug;178(16):4847–4853. doi: 10.1128/jb.178.16.4847-4853.1996. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Bayles K. W., Brunskill E. W., Iandolo J. J., Hruska L. L., Huang S., Pattee P. A., Smiley B. K., Yasbin R. E. A genetic and molecular characterization of the recA gene from Staphylococcus aureus. Gene. 1994 Sep 15;147(1):13–20. doi: 10.1016/0378-1119(94)90033-7. [DOI] [PubMed] [Google Scholar]
- Bertrand-Burggraf E., Hurstel S., Daune M., Schnarr M. Promoter properties and negative regulation of the uvrA gene by the LexA repressor and its amino-terminal DNA binding domain. J Mol Biol. 1987 Jan 20;193(2):293–302. doi: 10.1016/0022-2836(87)90220-8. [DOI] [PubMed] [Google Scholar]
- Brent R., Ptashne M. Mechanism of action of the lexA gene product. Proc Natl Acad Sci U S A. 1981 Jul;78(7):4204–4208. doi: 10.1073/pnas.78.7.4204. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Brosius J. Plasmid vectors for the selection of promoters. Gene. 1984 Feb;27(2):151–160. doi: 10.1016/0378-1119(84)90136-7. [DOI] [PubMed] [Google Scholar]
- Brosius J. Superpolylinkers in cloning and expression vectors. DNA. 1989 Dec;8(10):759–777. doi: 10.1089/dna.1989.8.759. [DOI] [PubMed] [Google Scholar]
- 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]
- Cheo D. L., Bayles K. W., Yasbin R. E. Elucidation of regulatory elements that control damage induction and competence induction of the Bacillus subtilis SOS system. J Bacteriol. 1993 Sep;175(18):5907–5915. doi: 10.1128/jb.175.18.5907-5915.1993. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Clark-Curtiss J. E., Jacobs W. R., Docherty M. A., Ritchie L. R., Curtiss R., 3rd Molecular analysis of DNA and construction of genomic libraries of Mycobacterium leprae. J Bacteriol. 1985 Mar;161(3):1093–1102. doi: 10.1128/jb.161.3.1093-1102.1985. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Colston M. J., Davis E. O. The ins and outs of protein splicing elements. Mol Microbiol. 1994 May;12(3):359–363. doi: 10.1111/j.1365-2958.1994.tb01025.x. [DOI] [PubMed] [Google Scholar]
- Das Gupta S. K., Bashyam M. D., Tyagi A. K. Cloning and assessment of mycobacterial promoters by using a plasmid shuttle vector. J Bacteriol. 1993 Aug;175(16):5186–5192. doi: 10.1128/jb.175.16.5186-5192.1993. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Davis E. O., Jenner P. J., Brooks P. C., Colston M. J., Sedgwick S. G. Protein splicing in the maturation of M. tuberculosis recA protein: a mechanism for tolerating a novel class of intervening sequence. Cell. 1992 Oct 16;71(2):201–210. doi: 10.1016/0092-8674(92)90349-h. [DOI] [PubMed] [Google Scholar]
- Davis E. O., Sedgwick S. G., Colston M. J. Novel structure of the recA locus of Mycobacterium tuberculosis implies processing of the gene product. J Bacteriol. 1991 Sep;173(18):5653–5662. doi: 10.1128/jb.173.18.5653-5662.1991. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Davis E. O., Thangaraj H. S., Brooks P. C., Colston M. J. Evidence of selection for protein introns in the recAs of pathogenic mycobacteria. EMBO J. 1994 Feb 1;13(3):699–703. doi: 10.1002/j.1460-2075.1994.tb06309.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Doukhan L., Predich M., Nair G., Dussurget O., Mandic-Mulec I., Cole S. T., Smith D. R., Smith I. Genomic organization of the mycobacterial sigma gene cluster. Gene. 1995 Nov 7;165(1):67–70. doi: 10.1016/0378-1119(95)00427-8. [DOI] [PubMed] [Google Scholar]
- Favre D., Viret J. F. Nucleotide sequence of the recA gene of Bordetella pertussis. Nucleic Acids Res. 1990 Jul 25;18(14):4243–4243. doi: 10.1093/nar/18.14.4243. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Fernandez de Henestrosa A. R., Calero S., Barbé J. Expression of the recA gene of Escherichia coli in several species of gram-negative bacteria. Mol Gen Genet. 1991 May;226(3):503–506. doi: 10.1007/BF00260664. [DOI] [PubMed] [Google Scholar]
- Gassel M., Alonso J. C. Expression of the recE gene during induction of the SOS response in Bacillus subtilis recombination-deficient strains. Mol Microbiol. 1989 Sep;3(9):1269–1276. doi: 10.1111/j.1365-2958.1989.tb00277.x. [DOI] [PubMed] [Google Scholar]
- Gonzalez-y-Merchand J. A., Colston M. J., Cox R. A. The rRNA operons of Mycobacterium smegmatis and Mycobacterium tuberculosis: comparison of promoter elements and of neighbouring upstream genes. Microbiology. 1996 Mar;142(Pt 3):667–674. doi: 10.1099/13500872-142-3-667. [DOI] [PubMed] [Google Scholar]
- Haijema B. J., van Sinderen D., Winterling K., Kooistra J., Venema G., Hamoen L. W. Regulated expression of the dinR and recA genes during competence development and SOS induction in Bacillus subtilis. Mol Microbiol. 1996 Oct;22(1):75–85. doi: 10.1111/j.1365-2958.1996.tb02657.x. [DOI] [PubMed] [Google Scholar]
- Haseltine W. A. Ultraviolet light repair and mutagenesis revisited. Cell. 1983 May;33(1):13–17. doi: 10.1016/0092-8674(83)90329-x. [DOI] [PubMed] [Google Scholar]
- 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]
- IYER V. N., SZYBALSKI W. A MOLECULAR MECHANISM OF MITOMYCIN ACTION: LINKING OF COMPLEMENTARY DNA STRANDS. Proc Natl Acad Sci U S A. 1963 Aug;50:355–362. doi: 10.1073/pnas.50.2.355. [DOI] [PMC free article] [PubMed] [Google Scholar]
- IYER V. N., SZYBALSKI W. MITOMYCINS AND PORFIROMYCIN: CHEMICAL MECHANISM OF ACTIVATION AND CROSS-LINKING OF DNA. Science. 1964 Jul 3;145(3627):55–58. doi: 10.1126/science.145.3627.55. [DOI] [PubMed] [Google Scholar]
- Jackson M., Berthet F. X., Otal I., Rauzier J., Martin C., Gicquel B., Guilhot C. The Mycobacterium tuberculosis purine biosynthetic pathway: isolation and characterization of the purC and purL genes. Microbiology. 1996 Sep;142(Pt 9):2439–2447. doi: 10.1099/00221287-142-9-2439. [DOI] [PubMed] [Google Scholar]
- Kenney T. J., Churchward G. Genetic analysis of the Mycobacterium smegmatis rpsL promoter. J Bacteriol. 1996 Jun;178(12):3564–3571. doi: 10.1128/jb.178.12.3564-3571.1996. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Lewis L. K., Jenkins M. E., Mount D. W. Isolation of DNA damage-inducible promoters in Escherichia coli: regulation of polB (dinA), dinG, and dinH by LexA repressor. J Bacteriol. 1992 May;174(10):3377–3385. doi: 10.1128/jb.174.10.3377-3385.1992. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Lin K. C., Shiuan D. A simple method for DNaseI footprinting analysis. J Biochem Biophys Methods. 1995 Feb;30(1):85–89. doi: 10.1016/0165-022x(94)00066-m. [DOI] [PubMed] [Google Scholar]
- Little J. W. Autodigestion of lexA and phage lambda repressors. Proc Natl Acad Sci U S A. 1984 Mar;81(5):1375–1379. doi: 10.1073/pnas.81.5.1375. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Little J. W. Mechanism of specific LexA cleavage: autodigestion and the role of RecA coprotease. Biochimie. 1991 Apr;73(4):411–421. doi: 10.1016/0300-9084(91)90108-d. [DOI] [PubMed] [Google Scholar]
- Little J. W., Mount D. W. The SOS regulatory system of Escherichia coli. Cell. 1982 May;29(1):11–22. doi: 10.1016/0092-8674(82)90085-x. [DOI] [PubMed] [Google Scholar]
- Little J. W., Mount D. W., Yanisch-Perron C. R. Purified lexA protein is a repressor of the recA and lexA genes. Proc Natl Acad Sci U S A. 1981 Jul;78(7):4199–4203. doi: 10.1073/pnas.78.7.4199. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Liu Y., Chatterjee A., Chatterjee A. K. Nucleotide sequence, organization and expression of rdgA and rdgB genes that regulate pectin lyase production in the plant pathogenic bacterium Erwinia carotovora subsp. carotovora in response to DNA-damaging agents. Mol Microbiol. 1994 Dec;14(5):999–1010. doi: 10.1111/j.1365-2958.1994.tb01334.x. [DOI] [PubMed] [Google Scholar]
- Lovett C. M., Jr, Cho K. C., O'Gara T. M. Purification of an SOS repressor from Bacillus subtilis. J Bacteriol. 1993 Nov;175(21):6842–6849. doi: 10.1128/jb.175.21.6842-6849.1993. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Matsui H., Sano Y., Ishihara H., Shinomiya T. Regulation of pyocin genes in Pseudomonas aeruginosa by positive (prtN) and negative (prtR) regulatory genes. J Bacteriol. 1993 Mar;175(5):1257–1263. doi: 10.1128/jb.175.5.1257-1263.1993. [DOI] [PMC free article] [PubMed] [Google Scholar]
- McEvoy J. L., Murata H., Chatterjee A. K. Genetic evidence for an activator required for induction of pectin lyase in Erwinia carotovora subsp. carotovora by DNA-damaging agents. J Bacteriol. 1992 Aug;174(16):5471–5474. doi: 10.1128/jb.174.16.5471-5474.1992. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 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]
- Movahedzadeh F., Colston M. J., Davis E. O. Characterization of Mycobacterium tuberculosis LexA: recognition of a Cheo (Bacillus-type SOS) box. Microbiology. 1997 Mar;143(Pt 3):929–936. doi: 10.1099/00221287-143-3-929. [DOI] [PubMed] [Google Scholar]
- Nakazawa T., Kimoto M., Abe M. Cloning, sequencing, and transcriptional analysis of the recA gene of Pseudomonas cepacia. Gene. 1990 Sep 28;94(1):83–88. doi: 10.1016/0378-1119(90)90471-3. [DOI] [PubMed] [Google Scholar]
- Perler F. B., Davis E. O., Dean G. E., Gimble F. S., Jack W. E., Neff N., Noren C. J., Thorner J., Belfort M. Protein splicing elements: inteins and exteins--a definition of terms and recommended nomenclature. Nucleic Acids Res. 1994 Apr 11;22(7):1125–1127. doi: 10.1093/nar/22.7.1125. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Pogson C. A., Simmons C. P., Strugnell R. A., Hodgson A. L. Cloning and manipulation of the Corynebacterium pseudotuberculosis recA gene for live vaccine vector development. FEMS Microbiol Lett. 1996 Sep 1;142(2-3):139–145. doi: 10.1111/j.1574-6968.1996.tb08421.x. [DOI] [PubMed] [Google Scholar]
- Predich M., Doukhan L., Nair G., Smith I. Characterization of RNA polymerase and two sigma-factor genes from Mycobacterium smegmatis. Mol Microbiol. 1995 Jan;15(2):355–366. doi: 10.1111/j.1365-2958.1995.tb02249.x. [DOI] [PubMed] [Google Scholar]
- Ranes M. G., Rauzier J., Lagranderie M., Gheorghiu M., Gicquel B. Functional analysis of pAL5000, a plasmid from Mycobacterium fortuitum: construction of a "mini" mycobacterium-Escherichia coli shuttle vector. J Bacteriol. 1990 May;172(5):2793–2797. doi: 10.1128/jb.172.5.2793-2797.1990. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Raymond-Denise A., Guillen N. Expression of the Bacillus subtilis dinR and recA genes after DNA damage and during competence. J Bacteriol. 1992 May;174(10):3171–3176. doi: 10.1128/jb.174.10.3171-3176.1992. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Revel-Viravau V., Truong Q. C., Moreau N., Jarlier V., Sougakoff W. Sequence analysis, purification, and study of inhibition by 4-quinolones of the DNA gyrase from Mycobacterium smegmatis. Antimicrob Agents Chemother. 1996 Sep;40(9):2054–2061. doi: 10.1128/aac.40.9.2054. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Rosenberg M., Court D. Regulatory sequences involved in the promotion and termination of RNA transcription. Annu Rev Genet. 1979;13:319–353. doi: 10.1146/annurev.ge.13.120179.001535. [DOI] [PubMed] [Google Scholar]
- Schnarr M., Oertel-Buchheit P., Kazmaier M., Granger-Schnarr M. DNA binding properties of the LexA repressor. Biochimie. 1991 Apr;73(4):423–431. doi: 10.1016/0300-9084(91)90109-e. [DOI] [PubMed] [Google Scholar]
- Snapper S. B., Lugosi L., Jekkel A., Melton R. E., Kieser T., Bloom B. R., Jacobs W. R., Jr Lysogeny and transformation in mycobacteria: stable expression of foreign genes. Proc Natl Acad Sci U S A. 1988 Sep;85(18):6987–6991. doi: 10.1073/pnas.85.18.6987. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Stolt P., Stoker N. G. Functional definition of regions necessary for replication and incompatibility in the Mycobacterium fortuitum plasmid pAL5000. Microbiology. 1996 Oct;142(Pt 10):2795–2802. doi: 10.1099/13500872-142-10-2795. [DOI] [PubMed] [Google Scholar]
- Stover C. K., de la Cruz V. F., Fuerst T. R., Burlein J. E., Benson L. A., Bennett L. T., Bansal G. P., Young J. F., Lee M. H., Hatfull G. F. New use of BCG for recombinant vaccines. Nature. 1991 Jun 6;351(6326):456–460. doi: 10.1038/351456a0. [DOI] [PubMed] [Google Scholar]
- Strohl W. R. Compilation and analysis of DNA sequences associated with apparent streptomycete promoters. Nucleic Acids Res. 1992 Mar 11;20(5):961–974. doi: 10.1093/nar/20.5.961. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Sugino A., Peebles C. L., Kreuzer K. N., Cozzarelli N. R. Mechanism of action of nalidixic acid: purification of Escherichia coli nalA gene product and its relationship to DNA gyrase and a novel nicking-closing enzyme. Proc Natl Acad Sci U S A. 1977 Nov;74(11):4767–4771. doi: 10.1073/pnas.74.11.4767. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Takiff H. E., Cimino M., Musso M. C., Weisbrod T., Martinez R., Delgado M. B., Salazar L., Bloom B. R., Jacobs W. R., Jr Efflux pump of the proton antiporter family confers low-level fluoroquinolone resistance in Mycobacterium smegmatis. Proc Natl Acad Sci U S A. 1996 Jan 9;93(1):362–366. doi: 10.1073/pnas.93.1.362. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 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]
- Weisemann J. M., Funk C., Weinstock G. M. Measurement of in vivo expression of the recA gene of Escherichia coli by using lacZ gene fusions. J Bacteriol. 1984 Oct;160(1):112–121. doi: 10.1128/jb.160.1.112-121.1984. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Weisemann J. M., Weinstock G. M. The promoter of the recA gene of Escherichia coli. Biochimie. 1991 Apr;73(4):457–470. doi: 10.1016/0300-9084(91)90113-f. [DOI] [PubMed] [Google Scholar]
- 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]
- Zhao X. J., McEntee K. DNA sequence analysis of the recA genes from Proteus vulgaris, Erwinia carotovora, Shigella flexneri and Escherichia coli B/r. Mol Gen Genet. 1990 Jul;222(2-3):369–376. doi: 10.1007/BF00633842. [DOI] [PubMed] [Google Scholar]
- van den Berg E. A., Geerse R. H., Pannekoek H., van de Putte P. In vivo transcription of the E. coli uvrB gene: both promoters are inducible by UV. Nucleic Acids Res. 1983 Jul 11;11(13):4355–4363. doi: 10.1093/nar/11.13.4355. [DOI] [PMC free article] [PubMed] [Google Scholar]