• We are sorry, but NCBI web applications do not support your browser and may not function properly. More information
Logo of jbacterPermissionsJournals.ASM.orgJournalJB ArticleJournal InfoAuthorsReviewers
J Bacteriol. Jul 1996; 178(14): 4122–4130.
PMCID: PMC178169

Cloning, sequencing, and characterization of the Bacillus subtilis biotin biosynthetic operon.

Abstract

A 10-kb region of the Bacillus subtilis genome that contains genes involved in biotin-biosynthesis was cloned and sequenced. DNA sequence analysis indicated that B. subtilis contains homologs of the Escherichia coli and Bacillus sphaericus bioA, bioB, bioD, and bioF genes. These four genes and a homolog of the B. sphaericus bioW gene are arranged in a single operon in the order bioWAFDR and are followed by two additional genes, bioI and orf2. bioI and orf2 show no similarity to any other known biotin biosynthetic genes. The bioI gene encodes a protein with similarity to cytochrome P-450s and was able to complement mutations in either bioC or bioH of E. coli. Mutations in bioI caused B. subtilis to grow poorly in the absence of biotin. The bradytroph phenotype of bioI mutants was overcome by pimelic acid, suggesting that the product of bioI functions at a step prior to pimelic acid synthesis. The B. subtilis bio operon is preceded by a putative vegetative promoter sequence and contains just downstream a region of dyad symmetry with homology to the bio regulatory region of B. sphaericus. Analysis of a bioW-lacZ translational fusion indicated that expression of the biotin operon is regulated by biotin and the B. subtilis birA gene.

Full Text

The Full Text of this article is available as a PDF (363K).

Selected References

These references are in PubMed. This may not be the complete list of references from this article.
  • Abbott J, Beckett D. Cooperative binding of the Escherichia coli repressor of biotin biosynthesis to the biotin operator sequence. Biochemistry. 1993 Sep 21;32(37):9649–9656. [PubMed]
  • Alexeev D, Bury SM, Boys CW, Turner MA, Sawyer L, Ramsey AJ, Baxter HC, Baxter RL. Sequence and crystallization of Escherichia coli dethiobiotin synthetase, the penultimate enzyme of biotin biosynthesis. J Mol Biol. 1994 Jan 14;235(2):774–776. [PubMed]
  • Asturias JA, Eltis LD, Prucha M, Timmis KN. Analysis of three 2,3-dihydroxybiphenyl 1,2-dioxygenases found in Rhodococcus globerulus P6. Identification of a new family of extradiol dioxygenases. J Biol Chem. 1994 Mar 11;269(10):7807–7815. [PubMed]
  • Backman K, Chen YM, Magasanik B. Physical and genetic characterization of the glnA--glnG region of the Escherichia coli chromosome. Proc Natl Acad Sci U S A. 1981 Jun;78(6):3743–3747. [PMC free article] [PubMed]
  • Barker DF, Campbell AM. Genetic and biochemical characterization of the birA gene and its product: evidence for a direct role of biotin holoenzyme synthetase in repression of the biotin operon in Escherichia coli. J Mol Biol. 1981 Mar 15;146(4):469–492. [PubMed]
  • Birch OM, Fuhrmann M, Shaw NM. Biotin synthase from Escherichia coli, an investigation of the low molecular weight and protein components required for activity in vitro. J Biol Chem. 1995 Aug 11;270(32):19158–19165. [PubMed]
  • Bower S, Perkins J, Yocum RR, Serror P, Sorokin A, Rahaim P, Howitt CL, Prasad N, Ehrlich SD, Pero J. Cloning and characterization of the Bacillus subtilis birA gene encoding a repressor of the biotin operon. J Bacteriol. 1995 May;177(9):2572–2575. [PMC free article] [PubMed]
  • Cleary PP, Campbell A. Deletion and complementation analysis of biotin gene cluster of Escherichia coli. J Bacteriol. 1972 Nov;112(2):830–839. [PMC free article] [PubMed]
  • Cronan JE., Jr The E. coli bio operon: transcriptional repression by an essential protein modification enzyme. Cell. 1989 Aug 11;58(3):427–429. [PubMed]
  • Dassa E, Hofnung M. Sequence of gene malG in E. coli K12: homologies between integral membrane components from binding protein-dependent transport systems. EMBO J. 1985 Sep;4(9):2287–2293. [PMC free article] [PubMed]
  • Donadio S, Katz L. Organization of the enzymatic domains in the multifunctional polyketide synthase involved in erythromycin formation in Saccharopolyspora erythraea. Gene. 1992 Feb 1;111(1):51–60. [PubMed]
  • Dower WJ, Miller JF, Ragsdale CW. High efficiency transformation of E. coli by high voltage electroporation. Nucleic Acids Res. 1988 Jul 11;16(13):6127–6145. [PMC free article] [PubMed]
  • Dubnau D, Davidoff-Abelson R. Fate of transforming DNA following uptake by competent Bacillus subtilis. I. Formation and properties of the donor-recipient complex. J Mol Biol. 1971 Mar 14;56(2):209–221. [PubMed]
  • Florentin D, Bui BT, Marquet A, Ohshiro T, Izumi Y. On the mechanism of biotin synthase of Bacillus sphaericus. C R Acad Sci III. 1994 Jun;317(6):485–488. [PubMed]
  • Gibson KJ, Lorimer GH, Rendina AR, Taylor WS, Cohen G, Gatenby AA, Payne WG, Roe DC, Lockett BA, Nudelman A, et al. Dethiobiotin synthetase: the carbonylation of 7,8-diaminonanoic acid proceeds regiospecifically via the N7-carbamate. Biochemistry. 1995 Sep 5;34(35):10976–10984. [PubMed]
  • Gloeckler R, Ohsawa I, Speck D, Ledoux C, Bernard S, Zinsius M, Villeval D, Kisou T, Kamogawa K, Lemoine Y. Cloning and characterization of the Bacillus sphaericus genes controlling the bioconversion of pimelate into dethiobiotin. Gene. 1990 Mar 1;87(1):63–70. [PubMed]
  • Hatfield D, Hofnung M, Schwartz M. Genetic analysis of the maltose A region in Escherichia coli. J Bacteriol. 1969 May;98(2):559–567. [PMC free article] [PubMed]
  • Haydock SF, Dowson JA, Dhillon N, Roberts GA, Cortes J, Leadlay PF. Cloning and sequence analysis of genes involved in erythromycin biosynthesis in Saccharopolyspora erythraea: sequence similarities between EryG and a family of S-adenosylmethionine-dependent methyltransferases. Mol Gen Genet. 1991 Nov;230(1-2):120–128. [PubMed]
  • He JS, Ruettinger RT, Liu HM, Fulco AJ. Molecular cloning, coding nucleotides and the deduced amino acid sequence of P-450BM-1 from Bacillus megaterium. Biochim Biophys Acta. 1989 Dec 22;1009(3):301–303. [PubMed]
  • Horinouchi S, Weisblum B. Nucleotide sequence and functional map of pC194, a plasmid that specifies inducible chloramphenicol resistance. J Bacteriol. 1982 May;150(2):815–825. [PMC free article] [PubMed]
  • Huang W, Jia J, Gibson KJ, Taylor WS, Rendina AR, Schneider G, Lindqvist Y. Mechanism of an ATP-dependent carboxylase, dethiobiotin synthetase, based on crystallographic studies of complexes with substrates and a reaction intermediate. Biochemistry. 1995 Sep 5;34(35):10985–10995. [PubMed]
  • Hueck CJ, Hillen W. Catabolite repression in Bacillus subtilis: a global regulatory mechanism for the gram-positive bacteria? Mol Microbiol. 1995 Feb;15(3):395–401. [PubMed]
  • Ifuku O, Kishimoto J, Haze S, Yanagi M, Fukushima S. Conversion of dethiobiotin to biotin in cell-free extracts of Escherichia coli. Biosci Biotechnol Biochem. 1992 Nov;56(11):1780–1785. [PubMed]
  • Ifuku O, Koga N, Haze S, Kishimoto J, Wachi Y. Flavodoxin is required for conversion of dethiobiotin to biotin in Escherichia coli. Eur J Biochem. 1994 Aug 15;224(1):173–178. [PubMed]
  • Ifuku O, Miyaoka H, Koga N, Kishimoto J, Haze S, Wachi Y, Kajiwara M. Origin of carbon atoms of biotin. 13C-NMR studies on biotin biosynthesis in Escherichia coli. Eur J Biochem. 1994 Mar 1;220(2):585–591. [PubMed]
  • Inoue H, Nojima H, Okayama H. High efficiency transformation of Escherichia coli with plasmids. Gene. 1990 Nov 30;96(1):23–28. [PubMed]
  • Jiang XM, Neal B, Santiago F, Lee SJ, Romana LK, Reeves PR. Structure and sequence of the rfb (O antigen) gene cluster of Salmonella serovar typhimurium (strain LT2). Mol Microbiol. 1991 Mar;5(3):695–713. [PubMed]
  • Lauer G, Rudd EA, McKay DL, Ally A, Ally D, Backman KC. Cloning, nucleotide sequence, and engineered expression of Thermus thermophilus DNA ligase, a homolog of Escherichia coli DNA ligase. J Bacteriol. 1991 Aug;173(16):5047–5053. [PMC free article] [PubMed]
  • Lemoine Y, Wach A, Jeltsch JM. To be free or not: the fate of pimelate in Bacillus sphaericus and in Escherichia coli. Mol Microbiol. 1996 Feb;19(3):645–647. [PubMed]
  • Lerner CG, 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. [PMC free article] [PubMed]
  • Lopilato J, Bortner S, Beckwith J. Mutations in a new chromosomal gene of Escherichia coli K-12, pcnB, reduce plasmid copy number of pBR322 and its derivatives. Mol Gen Genet. 1986 Nov;205(2):285–290. [PubMed]
  • Moran CP, Jr, Lang N, LeGrice SF, Lee G, Stephens M, Sonenshein AL, Pero J, Losick R. Nucleotide sequences that signal the initiation of transcription and translation in Bacillus subtilis. Mol Gen Genet. 1982;186(3):339–346. [PubMed]
  • Ohshiro T, Yamamoto M, Izumi Y, Bui BT, Florentin D, Marquet A. Enzymatic conversion of dethiobiotin to biotin in cell-free extracts of a Bacillus sphaericus bioB transformant. Biosci Biotechnol Biochem. 1994 Sep;58(9):1738–1741. [PubMed]
  • Otsuka AJ, Buoncristiani MR, Howard PK, Flamm J, Johnson C, Yamamoto R, Uchida K, Cook C, Ruppert J, Matsuzaki J. The Escherichia coli biotin biosynthetic enzyme sequences predicted from the nucleotide sequence of the bio operon. J Biol Chem. 1988 Dec 25;263(36):19577–19585. [PubMed]
  • Overduin P, Boos W, Tommassen J. Nucleotide sequence of the ugp genes of Escherichia coli K-12: homology to the maltose system. Mol Microbiol. 1988 Nov;2(6):767–775. [PubMed]
  • Pai CH. Genetics of biotin biosynthesis in Bacillus subtilis. J Bacteriol. 1975 Jan;121(1):1–8. [PMC free article] [PubMed]
  • Ploux O, Marquet A. The 8-amino-7-oxopelargonate synthase from Bacillus sphaericus. Purification and preliminary characterization of the cloned enzyme overproduced in Escherichia coli. Biochem J. 1992 Apr 15;283(Pt 2):327–331. [PMC free article] [PubMed]
  • Ploux O, Soularue P, Marquet A, Gloeckler R, Lemoine Y. Investigation of the first step of biotin biosynthesis in Bacillus sphaericus. Purification and characterization of the pimeloyl-CoA synthase, and uptake of pimelate. Biochem J. 1992 Nov 1;287(Pt 3):685–690. [PMC free article] [PubMed]
  • Reed KE, Cronan JE., Jr Lipoic acid metabolism in Escherichia coli: sequencing and functional characterization of the lipA and lipB genes. J Bacteriol. 1993 Mar;175(5):1325–1336. [PMC free article] [PubMed]
  • Rolfes RJ, Zalkin H. Escherichia coli gene purR encoding a repressor protein for purine nucleotide synthesis. Cloning, nucleotide sequence, and interaction with the purF operator. J Biol Chem. 1988 Dec 25;263(36):19653–19661. [PubMed]
  • Sanyal I, Cohen G, Flint DH. Biotin synthase: purification, characterization as a [2Fe-2S]cluster protein, and in vitro activity of the Escherichia coli bioB gene product. Biochemistry. 1994 Mar 29;33(12):3625–3631. [PubMed]
  • Sanyal I, Gibson KJ, Flint DH. Escherichia coli biotin synthase: an investigation into the factors required for its activity and its sulfur donor. Arch Biochem Biophys. 1996 Feb 1;326(1):48–56. [PubMed]
  • Sloma A, Ally A, Ally D, Pero J. Gene encoding a minor extracellular protease in Bacillus subtilis. J Bacteriol. 1988 Dec;170(12):5557–5563. [PMC free article] [PubMed]
  • Sloma A, Rufo GA, Jr, Theriault KA, Dwyer M, Wilson SW, Pero J. Cloning and characterization of the gene for an additional extracellular serine protease of Bacillus subtilis. J Bacteriol. 1991 Nov;173(21):6889–6895. [PMC free article] [PubMed]
  • Speck D, Ohsawa I, Gloeckler R, Zinsius M, Bernard S, Ledoux C, Kisou T, Kamogawa K, Lemoine Y. Isolation of Bacillus sphaericus biotin synthesis control mutants: evidence for transcriptional regulation of bio genes. Gene. 1991 Dec 1;108(1):39–45. [PubMed]
  • Spizizen J. TRANSFORMATION OF BIOCHEMICALLY DEFICIENT STRAINS OF BACILLUS SUBTILIS BY DEOXYRIBONUCLEATE. Proc Natl Acad Sci U S A. 1958 Oct 15;44(10):1072–1078. [PMC free article] [PubMed]
  • Stassi D, Donadio S, Staver MJ, Katz L. Identification of a Saccharopolyspora erythraea gene required for the final hydroxylation step in erythromycin biosynthesis. J Bacteriol. 1993 Jan;175(1):182–189. [PMC free article] [PubMed]
  • Stokes HW, Hall BG. Sequence of the ebgR gene of Escherichia coli: evidence that the EBG and LAC operons are descended from a common ancestor. Mol Biol Evol. 1985 Nov;2(6):478–483. [PubMed]
  • Taira K, Hirose J, Hayashida S, Furukawa K. Analysis of bph operon from the polychlorinated biphenyl-degrading strain of Pseudomonas pseudoalcaligenes KF707. J Biol Chem. 1992 Mar 5;267(7):4844–4853. [PubMed]
  • Tinoco I, Jr, Borer PN, Dengler B, Levin MD, Uhlenbeck OC, Crothers DM, Bralla J. Improved estimation of secondary structure in ribonucleic acids. Nat New Biol. 1973 Nov 14;246(150):40–41. [PubMed]
  • Vieira J, Messing J. The pUC plasmids, an M13mp7-derived system for insertion mutagenesis and sequencing with synthetic universal primers. Gene. 1982 Oct;19(3):259–268. [PubMed]
  • Weickert MJ, Chambliss GH. Site-directed mutagenesis of a catabolite repression operator sequence in Bacillus subtilis. Proc Natl Acad Sci U S A. 1990 Aug;87(16):6238–6242. [PMC free article] [PubMed]
  • Wen LP, Fulco AJ. Cloning of the gene encoding a catalytically self-sufficient cytochrome P-450 fatty acid monooxygenase induced by barbiturates in Bacillus megaterium and its functional expression and regulation in heterologous (Escherichia coli) and homologous (Bacillus megaterium) hosts. J Biol Chem. 1987 May 15;262(14):6676–6682. [PubMed]
  • Wierenga RK, Terpstra P, Hol WG. Prediction of the occurrence of the ADP-binding beta alpha beta-fold in proteins, using an amino acid sequence fingerprint. J Mol Biol. 1986 Jan 5;187(1):101–107. [PubMed]
  • 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. [PubMed]
  • Youngman P, Perkins JB, Losick R. Construction of a cloning site near one end of Tn917 into which foreign DNA may be inserted without affecting transposition in Bacillus subtilis or expression of the transposon-borne erm gene. Plasmid. 1984 Jul;12(1):1–9. [PubMed]
  • Zuber P, Losick R. Role of AbrB in Spo0A- and Spo0B-dependent utilization of a sporulation promoter in Bacillus subtilis. J Bacteriol. 1987 May;169(5):2223–2230. [PMC free article] [PubMed]

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

Formats:

Related citations in PubMed

See reviews...See all...

Cited by other articles in PMC

See all...

Links