A Central Regulatory Locus Controls Tetracycline Regulated Functions on a Bacteroides Tetracycline Resistant Conjugal Chromosomal Element
Stevens, Ann M.
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Permalink
https://hdl.handle.net/2142/72525
Description
Title
A Central Regulatory Locus Controls Tetracycline Regulated Functions on a Bacteroides Tetracycline Resistant Conjugal Chromosomal Element
Author(s)
Stevens, Ann M.
Issue Date
1993
Doctoral Committee Chair(s)
Salyers, Abigail A.
Department of Study
Microbiology
Discipline
Microbiology
Degree Granting Institution
University of Illinois at Urbana-Champaign
Degree Name
Ph.D.
Degree Level
Dissertation
Keyword(s)
Biology, Microbiology
Abstract
The genes necessary for regulated Plf activity on the Tc$\sp{\rm r}$ element were localized to an 8 kbp region. This region was known to contain the Tc$\sp{\rm r}$ gene, tetQ, which had already been sequenced. Sequencing of an additional 5 kbp downstream of tetQ has revealed two additional genes. Results support the hypothesis that these three genes form an operon with tetQ as the first gene. The two downstream genes exhibited amino acid homology with proteins from known two component regulatory systems. The protein corresponding to the environmental sensor has highest homology to BvgC, RcsC and PhoM. The protein corresponding to the effector had highest homology to HydG, NtrC and DctD, proteins known to interact with the $\sigma$54-holoenzyme of RNA polymerase. Other evidence confirmed that this operon was involved in regulation of transfer and mobilization as well as Plf activity. Accordingly, I have designated the two genes downstream of tetQ as rteA and rteB (regulation of Tc$\sp{\rm r}$ elements).
A $\beta$-glucuronidase (GUS) fusion containing tetQ and the 5$\sp\prime$ sequences of rteA, was sufficient for Tc-regulated expression from the tetQ promoter. Thus Tc-regulated transcription of the tetQ/rteA/rteB operon did not require RteA and RteB. I located a Tc-regulated promoter downstream of rteB. This promoter (P$\sb3$), unlike the tetQ promoter required the entire tetQ/rteA/rteB operon for expression. Thus, there appears to be at least two levels of regulation. The first is regulation of tetQ/rteA/rteB transcription, and the second is regulation of other promoters (e.g. of transfer genes or Plf genes on the NBUs) by the gene products of tetQ/rteA/rteB. The gene product transcribed from P$\sb3$ (ORF3) was analyzed both by sequencing it and disrupting it via an internal single crossover insertion into the chromosome. The disruption in ORF3 demonstrated the apparent involvement of this gene in the regulation of mobilization of co-resident plasmids.
A final objective was to determine what role, if any, the TetQ protein plays in regulation. My results indicate that an intact copy of tetQ is not necessary to obtain Tc-regulated Plf activity. Exogenous inducer, RteA and RteB are required. (Abstract shortened by UMI.)
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