Abstract
Genomic DNA extracted from the symbiotically-competent, heterocyst-forming cyanobacterium Nostoc sp. strain 7801 was resistant to cleavage by a number of restriction endonucleases. A cosmid library of Nostoc DNA was prepared and maintained in the modification-limited Escherichia coli strain HB101. Analysis of cloned Nostoc DNA fragments indicated infrequent occurrence of restriction endonuclease recognition sites in the Nostoc genome.
The Nostoc genomic library was screened for sequences complementing mutations in the E. coli leucine and proline biosynthetic operons. Two cosmids complementing leuB were isolated but none for leuA, leuC, leuD, or proA were detected in 1000 cosmids. A 3.0 kb fragment subcloned from one of the cosmids complemented mutations in leuB when inserted into the HindIII site of pBR322 in either orientation, demonstrating that transcription of leuB originated within the cloned fragment. The cloned fragment also carries a second site capable of initiating transcription of fused antibiotic resistance genes. While transcription of Nostoc DNA sequences did occur in E. coli, unknown barriers must also exist that prevented additional biological complementation of specific E. coli mutations.
This is a preview of subscription content, log in via an institution to check access.
Similar content being viewed by others
Abbreviations
- Ap:
-
Ampicillin
- Cm:
-
chloramphenicol
- Km:
-
kanamycin
- kb:
-
kilobase pairs
- Ne:
-
neomycin
- R:
-
resistance
- S:
-
sensitivity
- Tc:
-
tetracycline
References
Allen MB, Arnon DI (1955) Studies on nitrogen-fixing blue-green algae. I. Growth and nitrogen fixation by Anabaena cylindrica Lemm. Plant Physiol 30:366–372
Berg DE (1977) Insertion and excision of the transposable kanamycin resistance determinant Tn5. In: Bukhari AI, Shapiro TA, Adhya SL (eds) DNA insertion elements, plasmids, and episomes. Cold Springs Harbor Laboratory, Cold Springs, NY, pp 205–212
Bolivar F, Backman K (1979) Plasmids of Escherichia coli as cloning vectors. In: Wu R (ed) Methods in enzymology, vol 68, Recombinant DNA. Academic Press, New York London, pp 245–267
Bolivar F, Rodriquez RL, Betlach MC, Boyer HW (1977) Construction and characterization of new cloning vehicles. I. Ampicillin-resistant derivatives of the plasmid pM89. Gene 2:75–93
Burns RO, Calvo J, Margolin P, Umbarger HE (1966) Expression of the leucine operon. J Bacteriol 91:1570–1567
Close T, Rodriquez R (1982) Construction and characterization of the chloramphenicol-resistance gene cartridge: A new approach to the transcriptional mapping of extrachromosomal elements. Gene 20:305–316
Close TJ, Zaitlin D, Kado CI (1984) Design and development of amplifiable broad-host-range cloning vectors: analysis of the vir region of Agrobacterium tumefaciens plasmid pTiC58. Plasmid 12:111–118
Ditta G, Stanfield S, Corbin D, Helinski D (1980) Broad host range DNA cloning system for gram-negative bacteria: construction of a gene bank of Rhizobium meliloti. Proc Natl Acad Sci USA 77:7347–7351
Enderlin CS, Meeks JC (1983) Pure culture and reconstitution of the Anthoceros-Nostoc symbiotic association. Planta 158:157–165
Fisher R, Tuli R, Haselkorn (1981) A cloned cyanobacterial gene for glutamine synthetase functions in Escherichia coli, but the enzyme is not adenylylated. Proc Natl Acad Sci USA 78:3393–3397
Gallie DR, Novak S, Kado CI (1985) Novel high- and low-copy stable cosmids for use in Agrobacterium and Rhizobium. Plasmid 14:171–175
Gupta M, Carr NG (1983) Isolation of polysomes from Nostoc sp. MAC and translation of messenger RNA in a heterologous cell-free system. J Gen Microbiol 129:2359–2366
Gurevitz M, Somerville CR, McIntosh L (1985) Pathway of assembly of ribulose bisphosphate carboxylase/oxygenase from Anabaena 7120 expressed in Escherichia coli. Proc Natl Acad Sci USA 82:6546–6550
Herrero A, Elhai J, Hohn B, Wolk CP (1984) Infrequent cleavage of cloned Anabaena variabilis DNA by restriction endonucleases from A. variabilis. J Bacteriol 160:781–784
Lambert GR, Scott JG, Carr NG (1984) Characterization and cloning of extrachromosomal DNA from filamentous cyanobacteria. FEMS Microbiol Letters 21:225–231
Mackey CJ, Zahler SA (1982) Insertion of bacteriophage SPB into the citF gene of Bacillus subtilis and specialized transduction of the iluBC-leu genes. J Bacteriol 151:1222–1229
Maniatis Y, Fritsch EF, Sambrook J (1982) Molecular cloning: A laboratory manual. Cold Springs Harbor Laboratory, Cold Springs, NY
Price CW, Fuson GB, Phaff HJ (1978) Genome comparison in yeast systematics: Delimitation of species within the genera Schwanniomyces, Saccharomyces, Debaryomyces, and Pichia. Microbiol Rev 42:161–193
Rodriquez RL, West RW, Heynecker HL, Bolivar F, Boyer HW (1979) Characterizing wild-type and mutant promoters of the tetracycline resistance gene in pBR313. Nucl Acids Res 6:3267–3287
Somers JM, Amzallag A, Middleton RB (1973) Genetic fine structure of the leucine operon of Escherichia coli K-12. J Bacteriol 113:1268–1272
Stewart WDP, Rowell P, Rai AN (1980) Symbiotic nitrogen fixing cyanobacteria. In: Stewart WDP, Gallon JR (eds) Nitrogen fixation. Academic Press, New York London, pp 239–277
Tumer NE, Robinson SJ, Haselkorn R (1983) Different promoters for the Anabaena glutamine synthetase gene during growth using molecular or fixed nitrogen. Nature 30:337–342
Van den Hondel CAMJJ, van Leen RW, van Arkel GA, Duyvesteyn M, de Waard A (1983) Sequence-specific nucleases from the cyanobacterium Fremyella diplosiphon, and a peculiar resistance of its chromosomal DNA towards cleavage by other restriction enzymes. FEMS Microbiol Letters 16:7–12
Author information
Authors and Affiliations
Rights and permissions
About this article
Cite this article
Cangelosi, G.A., Joseph, C.M., Rosen, J.J. et al. Cloning and expression of a Nostoc sp. leucine biosynthetic gene in Escherichia coli . Arch. Microbiol. 145, 315–321 (1986). https://doi.org/10.1007/BF00470864
Received:
Accepted:
Issue Date:
DOI: https://doi.org/10.1007/BF00470864