Title page for ETD etd-07122005-100946


Type of Document Dissertation
Author Schully, Kevin Lee
URN etd-07122005-100946
Title Regulatory Intricacies That Confer Temporal Control of Conjugation and Antibiotic Production Functions in Streptomyces: New Variations on Old Themes
Degree Doctor of Philosophy (Ph.D.)
Department Biological Sciences
Advisory Committee
Advisor Name Title
Gregg S. Pettis Committee Chair
David Donze Committee Member
Frederick A. Rainey Committee Member
John R. Battista Committee Member
Paul LaRock Dean's Representative
Keywords
  • operon
  • transcription termination
  • antitermination
  • streptomyces
  • conjugation
Date of Defense 2005-06-20
Availability unrestricted
Abstract
The complex life style of streptomycetes requires that the appropriate program of genes be expressed at the appropriate times. For example, conjugation occurs solely within the substrate mycelium stage of development while antibiotic production occurs in the aerial mycelium. Thus, complex regulatory mechanisms involving numerous modes of control have evolved that confer temporal regulation of functions governing these expression programs. Conjugation of the Streptomyces lividans plasmid pIJ101 utilizes only seven plasmid functions in its transmission. Two of these, tra and clt, are essential while three additional functions, spdA, spdB and kilB augment the process, with KorA and KorB regulating the transfer and spread functions. The transmission operon of pIJ101 is unique, in that multiple layers of transcriptional and posttranscriptional control converging to implement tight control of the transfer and spread functions, specifically the potentially lethal kilB gene product. When expressed unregulated, as a chromosomally integrated gene, kilB transcription decreases during the later stages of development, in sharp contrast to the temporally increasing pattern of KilB protein. However, when expressed on pIJ101, the kilB promoter is largely, if not completely repressed, and expression of kilB requires transcription readthrough from upstream. Furthermore, readthrough transcription terminates within a 105 base-pair intergenic region prior to kilB. Interestingly, kilB-operator-bound KorB repressor appears to act, at least in part, as an attenuator of operon transcription, perhaps by physically barring transcription elongation. Finally, the formation of a stem-and-loop in the readthrough transcript within the intercistronic region appears to promote antitermination of operon transcription to counteract the effects of the KorB roadblock. Regulation of antibiotic production in Streptomyces typically involves the streptomycete global regulatory mechanism bldA. The sweet potato pathogen Streptomyces ipomoeae strain 91-03 produces a bacteriocin-like antibiotic, ipomicin. In liquid culture, ipomicin is produced in low concentrations throughout exponential phase followed by a dramatic 10-fold increase as the culture enters stationary phase. In contrast, transcription of the ipomicin structural gene ipoA decreases as the culture ages. The contrasting patterns of ipoA transcription and ipomicin production, coupled to the fact that ipoA contains a TTA codon in its leader sequence, makes ipomicin a strong candidate for bldA regulation.
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