Title page for ETD etd-10112004-092507


Type of Document Dissertation
Author Col, Bekir
Author's Email Address bcol@vt.edu
URN etd-10112004-092507
Title Regulation of Fructose 1,6-bisphosphatase II (GlpX) Gene Expression in Escherichia coli
Degree PhD
Department Biochemistry
Advisory Committee
Advisor Name Title
Larson, Timothy J. Committee Chair
Dean, Dennis R. Committee Member
Gillaspy, Glenda E. Committee Member
Popham, David L. Committee Member
Stevens, Ann M. Committee Member
Keywords
  • glp regulon
  • gluconeogenesis
  • carbon metabolism
  • fructose 1 6-bisphosphatase
  • glpFKX operon
Date of Defense 2004-09-29
Availability unrestricted
Abstract
The glpX gene of Escherichia coli encodes fructose 1,6-bisphosphatase II (FBPase II), an enzyme that would appear to be redundant with FBPase I, encoded by fbp. However, glpX mutants have no apparent phenotype, while fbp mutants are unable to grow on gluconeogenic substrates as sole carbon sources, suggesting that GlpX function is insufficient for growth of fbp mutants under these conditions. To gain insight into the physiological functions of the FBPases, regulation of glpX expression was investigated. It was found that glpX is transcribed as part of a complex glpFKX operon containing promoters upstream of glpF, glpK and glpX (PglpF, PglpK, PglpX, respectively). Transcription start sites of PglpX were found at -24 and -41 relative to the ATG translation initiation site using primer extension analysis. Unlike PglpF, these newly found promoters were not subject to regulation by GlpR or cAMP-CRP. Cra (Catabolite Repressor/Activator) positively regulated expression from PglpK and PglpX by increasing transcription approximately 2 fold. Western analysis using GlpX polyclonal antibodies revealed that GlpX levels were higher in cultures grown on glycerol compared with levels in maltose- or glucose-grown cultures (glycerol>maltose>glucose). Various strains and growth conditions were used to show that GlpX levels are regulated by GlpR, suggesting that PglpF can give rise to expression of glpX. GlpX protein was present in a strain containing a polar insertion in glpK, indicating that PglpX can also give rise to expression of glpX. Strains deficient in FBPase I or CsrA (carbon starvation regulator) did not reveal any difference in GlpX levels with respect to the wild type. All of these data indicate that glpX expression is achieved by its own promoter as well as the operon promoter, PglpF. Finally, the results show that the delta-fbp phenotype is not due to the absence of GlpX.
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