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Volume 142, Issue 11

Organization of the 168 chromosome between and the attachment site of the SP prophage: use of Long Accurate PCR and yeast artificial chromosomes for sequencing Free

Abstract

Within the genome sequencing project, the region between and was assigned to our laboratory. In this report we present the sequence of the last 36 kb of this region, between the operon and the attachment site of the prophage. A two-step strategy was used for the sequencing. In the first step, total chromosomal DNA was cloned in phage M13-based vectors and the clones carrying inserts from the target region were identified by hybridization with a cognate yeast artificial chromosome (YAC) from our collection. Sequencing of the clones allowed us to establish a number of contigs. In the second step the contigs were mapped by Long Accurate (LA) PCR and the remaining gaps closed by sequencing of the PCR products. The level of sequence inaccuracy due to LA PCR errors appeared to be about 1 in 10000, which does not affect significantly the final sequence quality. This two-step strategy is efficient and we suggest that it can be applied to sequencing of longer chromosomal regions. The 36 kb sequence contains 38 coding sequences (CDSs), 19 of which encode unknown proteins. Seven genetic loci already mapped in this region, and were identified. Eleven CDSs were found to display significant similarities to known proteins from the data banks, suggesting possible functions for some of the novel genes: may encode a cold shock protein; the first bacterial homologue of chalcone synthase; a 5′ to 3′ exonuclease, similar to that of DNA polymerase I of and a stress-response-associated protein. The protein encoded by has homology with the transcriptional NifA-like regulators. The arrangement of the genes relative to possible promoters and terminators suggests 19 potential transcription units.

  • Received:
  • Accepted:
  • Revised:
  • Published Online:
Keyword(s): Bacillus subtilis , genome sequencing , Long Accurate PCR and yeast artificial chromosome
© Society for General Microbiology 1996
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1996-11-01
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References

  1. Albano M., Breitling R., Dubnau D. 1989; Nucleotide sequence and genetic organization of theBacillus subtilis comGoperon. J Bacteriol 171:5386–5404
     [Google Scholar]
  2. Altschul S. F., Gish W., Miller W., Myers E. W., Lipman D. J. 1990; Basic Local Alignment Search Tool. J Mol Biol 215:403–410
     [Google Scholar]
  3. Anagnostopoulos C., Piggot P. J., Hoch J. A. 1993; The genetic map of Bacillus subtilis.. In Bacillus subtilis and Other Grampositive Bacteria : Biochemistry, Physiology and Molecular Genetics pp. 425–461 Sonenshein A. L., Hoch J. A., Losick R. Edited by Washington, DC: American Society for Microbiology;
     [Google Scholar]
  4. Azevedo V., Alvarez E., Zumstein E., Damiani G., Sgaramella V., Ehrlich S. D., Serror P. 1993; An ordered collection of Bacillus subtilisDNA segments cloned in yeast artificial chromosomes. Proc Natl Acad Sci USA 906047–6051
     [Google Scholar]
  5. Babitzke P., Gollnick P., Yanofsky C. 1992; The mtrABoperon of Bacillus subtilisencodes GTP cyclohydrolase I (MtrA), an enzyme involved in folic acid biosynthesis, and MtrB, a regulator of tryptophan biosynthesis. J Bacteriol 174:2059–2064
     [Google Scholar]
  6. Barat M., Anagnostopoulos C., Schneider A. -M. 1965; Linkage relationships of genes controlling isoleucine, valine, and leucine biosynthesis in Bacillus subtilis.. J Bacteriol 90:357–369
     [Google Scholar]
  7. Barnes W. M. 1994; PCR amplification of up to 35-kb DNA with high fidelity and yield from λ bacteriophage templates. Proc Natl Acad Sci USA 912216–2220
     [Google Scholar]
  8. Bensing B. A., Dunny G. M. 1993; Cloning and molecular analysis of gene affecting expression of binding substance, the recipient-encoded receptor(s) mediated mating aggregate formation in Enterococcus faecalis.. J Bacteriol 175:7421–7429
     [Google Scholar]
  9. Bower S., Perkins J., Yocum R. R., Serror P., Sorokin A., Rahaim P., Howitt C. L., Prasad N., Ehrlich S. D., Pero J. 1995; Cloning and characterization of the Bacillus subtilis birAgene encoding a repressor of the biotin operon. J Bacteriol 171:2572–2575
     [Google Scholar]
  10. Bruand G., Sorokin A., Serror P., Ehrlich S. D. 1995; Nucleotide sequence of the Bacillus subtilis dnaDgene. Microbiology 141:321–322
     [Google Scholar]
  11. Buchanan C. E., Ling M. -L. 1992; Isolation and sequence analysis of dacB,which encodes a sporulation-specific penicillinbinding protein in Bacillus subtilis.. J Bacteriol 174:1717–1725
     [Google Scholar]
  12. Calogero S., Gardan R., Glaser P., Schweizer J., Rapoport G., Débarbouillé M. 1994; RocR, a novel regulator controlling arginine utilization in Bacillus subtilis,belongs to NtrC/NifA family of transcription activators. J Bacteriol 176:1234–1241
     [Google Scholar]
  13. Cheng S., Fockler C., Barnes W. M., Higuchi R. 1994; Effective amplification of long targets from cloned inserts and human genomic DNA. Proc Natl Acad Sci USA 915695–5699
     [Google Scholar]
  14. Dear S., Staden R. 1991; A sequence assembly and editing program for efficient management of large projects. Nucleic Acids Res 19:3907–3911
     [Google Scholar]
  15. Del Castillo I., Gonz-les-Pastor J. E., San Millàn amp;Moreno. 1991; Nucleotide sequence of the Escherichia coliregulatory gene mprAand construction and characterisation of mprAdeficient mutants. J Bacterial 173:3924–3929
     [Google Scholar]
  16. van Dijl J. M., de Jong A., Vehmaanperä J., Venema G., Bron S. 1992; Signal peptidase I of Bacillus sub tills: patterns of conserved amino acids in prokaryotic and eukaryotic type I signal peptidases. EMBO J 11:2819–2828
     [Google Scholar]
  17. Donovan W., Zheng L., Sandman K., Losick R. 1987; Genes encoding spore coat polypeptides from Bacillus subtilis.. J Mol Biol 196:1–10
     [Google Scholar]
  18. Driks A., Roels S., Beall B., Moran C. P.JR Losick R. 1994; Subcellular localization of proteins involved in the assembly of the spore coat of Bacillus subtilis.. Genes Dev 8:234–244
     [Google Scholar]
  19. Driver R. P., Lawther R. P. 1985; Restriction endonuclease analysis of ilvGEDAoperon of members of the family Enterobacteriaceae.. J Bacteriol 162:1317–1319
     [Google Scholar]
  20. Drummond A. M., Whitty P., Wootton J. 1986; Sequence and domain relationships of ntrCand nifAfrom Klebsiella pneumoniae: homologies to other regulatory proteins. EMBO J 5:441–447
     [Google Scholar]
  21. Dujon B.others 1994; Complete DNA sequence of yeast chromosome XI. Nature 369:371–378
     [Google Scholar]
  22. Errington J. 1986; A general method for fusion of the Escherichia coli lacZgene to chromosomal genes in Bacillus subtilis. J Gen Microbiol 132:2953–2961
     [Google Scholar]
  23. Fleischmann R. D.others 1995; Whole-genome random sequencing and assembly of Haemophilus influenzaeRd. Science 269:496–512
     [Google Scholar]
  24. Fraser C. M.others 1995; The minimal gene complement of Mycoplasma genitalium.. Science 270:397–403
     [Google Scholar]
  25. Godon J. J., Chopin M. C., Ehrlich S. D. 1992; Branched-chain amino acid biosynthesis genes in Lactococcus lactis.. J Bacteriol 174:6580–6589
     [Google Scholar]
  26. Gray J. V., Golinelli-Pimpaneau G., Knowles J. R. 1990; Monofunctional chorismate mutase from Bacillus subtilis: purification of the protein, molecular cloning of the gene, and overexpression of the gene product in Escherichia coli. . Biochemistry 29:376–383
     [Google Scholar]
  27. Henner D. J., Band L., Shimotsu H. 1984; Nucleotide sequence of the Bacillus subtilistryptophan operon. Gene 34:169–177
     [Google Scholar]
  28. Henner D. J., Band L., Flaggs G., Chen E. 1986; The organization and nucleotide sequence of the Bacillus subtilis hisH, tyrAand aroEgenes. Gene 49:147–152
     [Google Scholar]
  29. Herrler M., Bang H., Marhiel M. A. 1994; Cloning and characterization of ppiB,a Bacillus subtilisgene which encodes a cyclosporin A-sensitive peptidyl-prolyl cis-transisomerase. Mol Microbiol 11:1073–1083
     [Google Scholar]
  30. Hoch J., Mathews J. 1972; Genetic studies in Bacillus subtilis.. In Spores V pp. 113–116 Halvorson H. O., Hanson R., Campbell L. L. Edited by Washington, DC: American Society for Microbiology;
     [Google Scholar]
  31. Holland D., Ben-Hayyim G., Foltin Z., Camoin L., Strosberg A., Eshdat Y. 1993; Molecular organisation of salt-stress associated protein in citrons: protein and cDNA peroxidase. Plant Mol Biol 21:923–927
     [Google Scholar]
  32. Itaya M., Kondo K. 1991; Molecular cloning of a ribonuclase H (RNase HI) gene from an extreme thermophile Thermus thermophilusHB8 : a thermostable RNase HI can functionally replace the Escherichia colienzyme in vivo.. Nucleic Acids Res 16:4443–4449
     [Google Scholar]
  33. Iwakura M., Kawata M., Tsuda K., Tanaka T. 1988; Nucleotide sequences of the thymidilate synthase B and dihydrofolate reductase genes contained in one Bacillus subtilisoperon. Gene 64:9–20
     [Google Scholar]
  34. Leavitt M. C., Ito J. 1989; T5 DNA polymerase: structural- functional relationships to other DNA polymerases. Proc Natl Acad Sci USA 864465–4469
     [Google Scholar]
  35. Ludwig H. G., Lottspeich F., Henschen A., Bacher A. 1987; Heavy riboflavin synthase of Bacillus subtilis: primary structure of the β subunit. J Biol Chem 262:1016–1021
     [Google Scholar]
  36. McLaughlin J. R., Murray GL., Rabinowitz J. C. 1981; Unique features in the ribosome-binding site sequence of the Grampositive Staphylococcus aureusβ-lactamase gene. J Biol Chem 256:11183–11291
     [Google Scholar]
  37. Mares R., Urbanowski M. L., Stauffer G. V. 1992; Regulation of the Salmonella typhimurium met Agene by the MetR protein and homocysteine. J Bacteriol 174:390–397
     [Google Scholar]
  38. Marklund B. I., Tennent J. M., Garcia E., Hamers A., Baga M., Lindberg F., Gaastra W., Normark S. 1992; Horizontal gene transfer of the Escherichia coli papand prspili operon as a mechanism for the development of tissue-specific adhesive properties. Mol Microbiol 6:2225–2245
     [Google Scholar]
  39. Martin-Verstaete I., Débarbouillé M., Klier A., Rapoport G. 1994; Interactions of wild-type and truncated LevR of Bacillus subtiliswith the upstream activating sequence of the levanase operon. J Mol Biol 241:178–192
     [Google Scholar]
  40. Messing J., Vieira J. 1982; A new pair of M13 vectors for selecting either DNA strand of double-digest restriction fragments. Gene 19:269–276
     [Google Scholar]
  41. Micka B., Marahiel M. A. 1992; The DNA-binding protein HBsu is essential for normal growth and development in Bacillus subtilis.. Biochimie 74:641–650
     [Google Scholar]
  42. Mironov V. N., Kraev A. S., Chikindas M. L., Chernov B. K., Stepanov A. I., Skryabin K. G. 1994; Functional organization of the riboflavin biosynthesis operon from Bacillus subtilisSHgw. Mol Gen Genet 242:201–208
     [Google Scholar]
  43. Morbidoni H. R., de Mendoza D., Cronan J. E.Jr 1995; Synthesis of sn-glycerol 3-phosphate, a key precursor of membrane lipids in Bacillus subtilis. . J Bacteriol 177:5899–5905
     [Google Scholar]
  44. Okasaki R., Kornberg A. 1964; Enzymatic synthesis of deoxyribonucleic acid. XV. Purification and properties of a polymerase from Bacillus subtilis.. J Biol Chem 239:259–268
     [Google Scholar]
  45. Oliver S. G.others 1992; The complete DNA sequence of yeast chromosome III. Nature 357:38–46
     [Google Scholar]
  46. Ordal G. W., Parker H. M., Kirby J. R. 1985; Complementation and characterization of chemotaxis mutants of Bacillus subtilis.. J Bacteriol 164:802–810
     [Google Scholar]
  47. Pearson W. R., Lipman D. J. 1988; Improved tools for biological sequence comparison. Proc Natl Acad Sci USA 852444–2448
     [Google Scholar]
  48. Phillips D. A., Kapulnik Y. 1995; Plant isoflavonoid, pathogens and symbionts. Trends Microbiol 2:58–64
     [Google Scholar]
  49. Popham D. L., Setlow P. 1995; Cloning, nucleotide sequence and mutagenesis of the Bacillus subtilis ponAoperon, which codes for penicillin-binding protein (PBP) 1 and a PBP-related factor. J Bacteriol 177:326–335
     [Google Scholar]
  50. Popham D. L., Szeto D., Keener J., Kustu S. 1989; Function of a bacterial activator protein that binds to the transcriptional enhancers. Science 243:629–635
     [Google Scholar]
  51. Popham D. L., lllades-Aniar B., Setlow P. 1995; The Bacillus subtilis dacBgene, encoding penicillin-binding protein 5, is part of a three-gene operon required for proper spore cortex synthesis and spore core dehydration. J Bacteriol 177:4721–4729
     [Google Scholar]
  52. Reich G., Gardiner K. J., Olsen G. J., Pace B., Marsh T. L., Pace N. R. 1986; The RNA component of the Bacillus subtilisRNase P. Sequence, activity and partial secondary structure. J Biol Chem 261:7888–7893
     [Google Scholar]
  53. Renault P., Godon J. J., Goupil N., Delorme C., Ehrlich S. D. 1995; Metabolic operon in lactococci. Dev Biol Stand 85:431–441
     [Google Scholar]
  54. Ryder T. B., Hedrick S. A., Belt J. N., Liang X., Clouse S. D., Lamb C. J. 1987; Organization and differential activation of a gene family encoding the plant defense enzyme chalcone synthase in Phaseolus vulgaris.. Mol Gen Genet 210:219–233
     [Google Scholar]
  55. Saxild H. H., Nygaard P. 1987; Genetic and physiological characterization of Bacillus subtilismutants resistant to purine analogs. J Bacteriol 169:2977–2983
     [Google Scholar]
  56. Sayers J. R., Eckstein F. 1991; A single-strand specific endonuclease activity copurifies with overexpressed T5 D15 exonuclease. Nucleic Acids Res 19:4127–4132
     [Google Scholar]
  57. Schindelin H., Marahiel M. A., Heinemann U. 1993; Universal nucleic acid binding domain revealed by crystal structure of the Bacillus subtilismajor cold shock protein. Nature 364:164–168
     [Google Scholar]
  58. Schnuchel A., Wiltscheck R., Czisch M., Herrier M., Willinsky G., Graumann P., Marahiel M. A., Holak T. A. 1993; Structure in solution of the major cold shock protein from Bacillus subtilis.. Nature 364:169–171
     [Google Scholar]
  59. Schott K., Kellermann J., Lottspeich F., Bacher A. 1990; Riboflavin synthases of Bacillus subtilis'.purification and amino acid sequence of the α subunit. J Biol Chem 265:4204–4209
     [Google Scholar]
  60. Schröder K., Graumann P., Schnuchel A., Holak T. A., Marahiel M. A. 1995; Mutational analysis of the putative nucleic acid-binding surface of the cold-shock domain, CspB, revealed an essential role of aromatic and basic residues in binding of the singlestranded DNA containing the Y-box motif. Mol Microbiol 16:699–708
     [Google Scholar]
  61. Sorokin A., Zumstein E., Azevedo V., Ehrlich S. D., Serror P. 1993; The organization of the Bacillus subtilis168 chromosome region between the spoVAand serAgenetic loci, based on sequence data. Mol Microbiol 10:385–395
     [Google Scholar]
  62. Sorokin A., Serror P., Pujic P., Azevedo V., Ehrlich S. D. 1995; The Bacillus subtilischromosome region encoding homologues of the Escherichia coli mss Aand rpsAgene products. Microbiology 141:311–319
     [Google Scholar]
  63. Sorokin A., Azevedo V., Zumstein E., Galleron N., Ehrlich S. D., Serror P. 1996a; Sequence analysis of the Bacillus subtilischromosome region between the serAand kdgloci cloned in a yeast artificial chromosome. Microbiology 142:2005–2016
     [Google Scholar]
  64. Sorokin A., Lapidus L., Capuano V., Galleron N., Pujic P., Ehrlich S. D. 1996b; A new approach using Multiplex Long Accurate PCR and yeast artificial chromosomes for bacterial chromosome mapping and sequencing. Genome Res 6:448–453
     [Google Scholar]
  65. Sun G., Sharkova E., Chesnut R., Birkey S., Duggan M. F., Sorokin A., Pujic P., Ehrlich S. D., Hulett M. F. 1996; Regulators of aerobic and anaerobic respiration in Bacillus subtilis. . J Bacteriol 178:1374–1385
     [Google Scholar]
  66. Taha M. K., So M., Seifert H. S., Billyard E., Marchal C. 1988; Pilin expression in Neisseria gonorrhoeaeis under both positive and negative transcriptional control. EMBO J 7:4367–4378
     [Google Scholar]
  67. Tanaka T., Kawata M., Nagami Y., Uchiyama H. 1987; prtRenhances the mRNA level of the Bacillus subtilisextracellular proteases. J Bacteriol 169:3044–3050
     [Google Scholar]
  68. Willinsky G., Bang H., Fisher G., Marahiel M. A. 1992; Characterization of cspB,a Bacillus subtilisinducible cold shock gene affecting cell viability at low temperature. J Bacteriol 174:6326–6335
     [Google Scholar]
  69. Yamamoto J., Shimizu M., Yamane K. 1991; Molecular cloning and analysis of nucleotide sequence of the Bacillus subtilis lysAgene region using B. subtilisphage vectors and a multi-copy plasmid, pUBllO. Agric Biol Chem 55:1615–1626
     [Google Scholar]
  70. Yang M., Shimotsu H., Ferrari E., Henner D. J. 1987; Characterization and mapping of Bacillus subtilis ptrRgene. J Bacteriol 169:434–437
     [Google Scholar]
  71. Yazdi M. A., Moir A. 1990; Characterization and cloning of the gerClocus of Bacillus subtilis.. J Gen Microbiol 136:1335–1342
     [Google Scholar]
  72. Yu J., Hederstedt L., Piggot P. 1995; The cytochrome becomplex (menaquinone: cytochrome creductase) in Bacillus subtilishas a non-traditional subunit organisation. J Bacteriol 177:6751–6760
     [Google Scholar]
  73. Zeigler D. R., Dean D. H. 1990; Orientation of the genes in the Bacillus subtilischromosome. Genetics 125:703–708
     [Google Scholar]
  74. Zimmermann J., Voss H., Kristensen T., Schwager G., Stegemann J., Erfle H., Ansorge W. 1989; Automated preparation and purification of Ml 3 templates for DNA sequencing. Methods Mol Cell Biol 1:29–34
     [Google Scholar]
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Organization of the Bacillus subtilis 168 chromosome between kdg and the attachment site of the SPβ prophage: use of Long Accurate PCR and yeast artificial chromosomes for sequencing
Microbiology 142, 3005 (1996); https://doi.org/10.1099/13500872-142-11-3005
/content/journal/micro/10.1099/13500872-142-11-3005
/content/journal/micro/10.1099/13500872-142-11-3005
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