The effect of 5-bromo-2'-deoxyuridine on growth and sporulation in Bacillus subtilis

Binnie, Craig (1982) The effect of 5-bromo-2'-deoxyuridine on growth and sporulation in Bacillus subtilis. PhD thesis, University of Glasgow.

Abstract

The aims of the work were to elucidate the manner in which the thymidine (TdR) analogue, 5-bromo-2'-deoxyuridine (BUdR) interferes with sporulation in Bacillus subtilis, to use BUdR as a density-label to analyse chromosome replication required for sporulation, and to assess the basis by which BUdR-tolerant strains can grow in medium containing the analogue. BUdR inhibited the growth of B. subtilis (thy A, thy B) when supplied in place of TdR, which is required for growth of this strain. An isogenic BUdR-tolerant derivative, but-32. grew normally with a BUdR to TdR ratio of between 7.5 : 1 and 15 : 1, but only between 1 and 5% of the cells, compared to control cells using TdR alone, were able to form spores. Three other BUdR-tolerant, TdR-requring strains were independently isolated, and the BUdR-tolerance mutations transferred by DNA-mediated transformation back into the thy A, thy B genetic background. Two mutations conferring tolerance to bromo-uracil (BU), the free base form of BUdR, were also separately transferred into the thy A, thy B background. Initial characterisation of these five strains, plus but-32, suggested that classes of BUdR-tolerance exist, and that the mutation conferring tolerance to BU also confers some resistance to BUdR, in medium containing TdR. The basis of the tolerance phenotype of strain but-32 seems to lie in the preferential uptake of TdR over BUdR into the cell, when the analogue partially replaces TdR in the medium. The results of incorporation experiments involving radiolabelled TdR and BUdR, CsCl density gradient analysis of BUdR-substituted DNA, and marker-frequency analysis of DNA replication indicated that BUdR had a markedly greater inhibitory effect on the rate of DNA replication in the BUdR-sensitive parent strain than in strain but-32, and that the level of substitution of BUdR for TdR in DNA was greater in the sensitive strain than in but-32. The use of 6-(p-hydroxyphenylazo)uracil (HPUra), a specific inhibitor of DNA synthesis, indicated that successful sporulation only occured on the completion of chromosome replication. Strain but-32 began to escape the inhibitory effect of HPUra on sporulation between 60 and 75 min after initiation of the process in starvation medium, the escape correlating with termination of chromosome replication, measured by marker-frequency analysis. Escape from BUdR inhibition of sporalation occured prior to, but in parallel with, escape from HPUra inhibition, indicating that BUdR inhibition was limited to the period of DNA replication which occurs at the onset of sporulation. Slowing down the rate of DNA synthesis with a subinhibitory concentration of HPUra delayed the escape of these cells from HPUra and BUdR inhibition of sporulation, and also inhibited sporulation, but to a lesser extent than BUdR. In addition, cells grown and resuspended in BUdR experienced delayed escape from HPUra inhibition of sporulation, suggesting that BUdR delayed completion of chromosome replication during sporulation. Prolonged DNA replication in sporulation medium with BUdR was confirmed by a) direct measurement of the rate of incorporation of [14c] - TdR into acid-insoluble material, b) density gradient analysis of BUdR-substituted DNA of strain but-32 during sporulation, and, c) by marker-frequency analysis of synchronised DNA replication during sporulation of Tsl34, a TdR-requiring mutant temperature-sensitive for initiation of DNA replication. (Abstract shortened by ProQuest.).

• Item Type: Thesis (PhD)
• Qualification Level: Doctoral
• Additional Information: Adviser: J G Coote
• Keywords: Microbiology
• Date of Award: 1982
• Depositing User: Enlighten Team
• Unique ID: glathesis:1982-72777
• Copyright: Copyright of this thesis is held by the author.
• Date Deposited: 11 Jun 2019 11:06
• Last Modified: 11 Jun 2019 11:06
• URI: https://theses.gla.ac.uk/id/eprint/72777