An Investigation of the Molecular Genetics of Tetronasin Biosynthesis and Resistance in Streptomyces longisporoflavus

Linton, Kenneth James (1989) An Investigation of the Molecular Genetics of Tetronasin Biosynthesis and Resistance in Streptomyces longisporoflavus. PhD thesis, University of Glasgow.

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Streptomyces longisporoflavus makes tetronasin, a polyketide, polyether antibiotic. This thesis describes the isolation and preliminary characterisation of sequences from longisporoflavus which are probably involved in biosynthesis of, and/or resistance to tetronasin. A DNA library representative of the genome of a high-producing mutant, S. longisporoflavus strain 83E6, was constructed in the positive selection vector pIJ699 and contained in S. lividans TK64. The sensitivity of S. lividans to tetronasin was increased in the presence of 0.2M NaCl enabling tetronasin resistance determinants to be selected directly in the primary cloning recipient. Two non-identical recombinants (tetR1 and tetR5) containing 5.0 and 5.5kb of DNA respectively, were found to increase the tolerance of S. lividans to tetronasin. TetR5 also conferred resistance upon a tetronasin-super-sensitive strain of S. albus to a level comparable with that found in the wild-type S. longisporoflavus 4584. Although the functions of these sequences in the parental strain remain undetermined, they may by virtue of their activity in heterologous hosts, represent the first examples of the isolation of resistance determinants to an ionophore antibiotic. The 2.1kb actl fragment (Malpartida et al., 1987) which encodes components of the actinorhodin polyketide synthase, hybridized at low stringency to a 5kb BamHI fragment (AC36) from S. lonqisporoflavus. Paradoxically, a 4.3kb BamHI fragment from S. cinnamonensis (monl) with good sequence homology to actl (J. A. Robinson, pers. comm. ) failed to hybridize with the 5kb fragment in Southern analysis of total DNA. However monl did anneal to a 7kb BamHI fragment (MB74). Both fragments were isolated from partial libraries of S. longisporoflavus DNA constructed in pTZ18 R and contained in E. coli JM101. Cross hybridization of AC36 and MB74 with total DNA from several Streptomyces sp. which were known to produce polyketide antibiotics was investigated. At 65 C in 0.5xSSC both probes failed to hybridize with DNA fragments which correlated with restriction fragments known to be involved in polyketide biosynthesis. MB74 also had greater sequence homology with an 11kb BamHI fragment from S. cinnamonensis than the 4.3kb monl. Cross hybridization with isolated sequences involved in oxytetracycline (otcY). actinorhodin (actl) and putatively in monensin (monl) biosynthesis was tested. Not surprisingly, MB74 hybridized to a different restriction fragment from monl than actl and otcY. Interestingly, a fragment with a low level of homology to AC36 and otcY was discovered in MB74. It did not overlap with the sequences which hybridized to monl. A 2.8kb EcoRV/PvuII fragment from AC36 hybridized with actl and at 60 C, 2xSSC with monl. These data suggested that both AC36 and MB74 could be involved in polyketide biosynthesis but did not uniquely indicate sequences which might encode components of the tetronasin synthase. Recombinant bacteriophages which contained flanking DNA of each of the clones (AC36, MB74, tetR1 and tetR5) were identified by hybridization of radiolabelled probe to a representative library of S. longisporoflavus DNA contained in the lambda replacement vector EMBL3. Close linkage between any of the four clones was not established. However, sequences with homology to tetR1 were found, overlapping with, and in the DNA flanking, MB74. On its own MB74 was unable to confer resistance to tetronasin upon C. lividans or S. albus. Although S. longisporoflavus 4584 was shown to possess a photoreactivation system, conditions were defined for mutagenesis using short-wavelength UV light. Attempts to complement mutants which were deficient in the biosynthesis of tetronasin were inconclusive. Similarly, preliminary experiments designed to disrupt transcription of the endogenous copies of AC36 and MB74 in S. lonqisporoflavus 4584, require repitition. Therefore until otherwise established AC36, MB74 and sequences flanking the tetronasin resistance determinants must remain merely candidates for genes involved in tetronasin biosynthesis in longisporoflavus.

Item Type: Thesis (PhD)
Qualification Level: Doctoral
Keywords: Genetics
Date of Award: 1989
Depositing User: Enlighten Team
Unique ID: glathesis:1989-78001
Copyright: Copyright of this thesis is held by the author.
Date Deposited: 30 Jan 2020 15:44
Last Modified: 30 Jan 2020 15:44

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