Studies on Growth and the Shikimate Pathway in Streptomyces

Moran, Alison Jean (1994) Studies on Growth and the Shikimate Pathway in Streptomyces. PhD thesis, University of Glasgow.

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The research presented in this thesis was carried out under the Antibiotics Club initiative and comprises of two main parts. The first part covers the determination of general conditions for the growth of S.coelicolor in a new liquid minimal medium and investigations into the improvement of the reproducibility of the onset of secondary metabolism of cultures grown in this medium in shake flasks. The second part covers the characterisation and investigation of some aromatic amino acid auxotrophs that had been isolated at the University of Manchester Institute of Science and Technology. These mutants were of interest particularly for cloning genes from the common pathway and also for flux studies, as hosts for the introduction and manipulation of specific pathway genes. This part also covers the complementation of one of the mutants with an S.coelicolor library and the sequence analysis of the DNA responsible. When investigated further, this clone was found to encode a putative transporter protein which also complemented a number of the other mutants. The growth of S.coelicolor and the onset of production of pigment in the new liquid minimal medium was very variable. Therefore, a number of observations were made on pregermination, storage and growth in of S.coelicolor in this medium, which may be used to improve the reproducibility of growth and the onset of secondary metabolite production in this medium. Firstly, pre-germinated spores may be stored frozen in glycerol. Therefore, cultures may be inoculated with a known number of viable spores and synchronous germination achieved. The mycelial density was shown to be proportional to inoculum size and to affect the growth profile and production of pigments of secondary metabolism. Thus, inoculation with a known number of spores would enable an optimum concentration of spores to be used consistantly. Secondly, the onset and range of production of pigments depended upon the constituents of the media, their concentrations and the pH. Glucose did not cause carbon catabolite repression of either the blue pigment or undecylprodigiosin although inhibition by phosphate of production of the blue pigment was noted. Apparent catabolite repression by nitrogen of production of undecylprodigiosin and the blue pigment by ammonium ions was also noted. The effect of pH, choice of buffer and nutrient source on the range of pigments produced was demonstrated by the sole production of yellow pigment under conditions of low pH. The yellow pigment was found to act as a pH indicator. Finally, mycelial pellets were still formed in the New Minimal Medium (NMM) despite the presence of the polyacrylate Junlon-110. Ten aromatic amino acid auxotrophs of S.lividans TK64 were investigated. These had been isolated at the University of Manchester Institute of Science and Technology (UMIST) using a strategy of random transposon mutagenesis and they were assigned numbers at that time. Cross-feeding experiments indicated that mutant 20 could act as a secretor for mutant 6, as a convertor. The mutants 3, 7, and 14 inhibited growth of the parent strain TK64. Mutant 6 appeared to have no detectable Shikimate Dehydrogenase (SDH) activity in a crude extract. The auxotrophy of the mutants was not relieved by quinic acid nor was protocatechuic acid produced. A library of S.coelicolor DNA in the vector pIJ916 was used to complement mutant 22. Fragments of the insert from the complementing plasmid pAW9162 were subcloned into the vector pIJ486/7, giving the smaller multicopy plasmids pAWl00 and pAW4865. The large insert in pAW9162, was able to complement the mutations in mutants 3, 7, 11, 14, 16, 20, 22 and 23 and the smaller insert, in pAWl00 and pAW4865 complemented mutants 22 and 16. Amino acid sequence comparison of the protein encoded by the insert in the recombinant plasmid pAWl00 (AW 100) indicated that it belonged to a known family (Family III) of membrane transport proteins many of which are driven by the proton-motive force. The amino-terminus of AW 100 was predicted to lie in the cytoplasm. However, sequence comparison indicated that the terminal transmembrane helix (TMH) had not been cloned. Therefore, although the insert contained in pAW100 only encoded a protein with thirteen TMHs, it would appear to be functional in vivo. Features which might be required for complex regulation were identified within the region upstream of a putative transcriptional start site of ORF-AWIOO. A repressor protein may be contained within the insert in pAW9162 as some other transporter proteins belonging to the same family as AW 100 are regulated by a divergently-transcribed DNA-binding repressor protein.

Item Type: Thesis (PhD)
Qualification Level: Doctoral
Keywords: Genetics
Date of Award: 1994
Depositing User: Enlighten Team
Unique ID: glathesis:1994-76079
Copyright: Copyright of this thesis is held by the author.
Date Deposited: 19 Nov 2019 16:51
Last Modified: 19 Nov 2019 16:51

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