9419403 Chatterjee Erwinia carotovora subsp. carotovora (hereafter Ecc) and most other soft-rotting Erwinia species produce an assortment of extracellular degradative enzymes such as pectin Iyase (Pnl), pectate Iyase (Pel), polygalacturonase (Peh), cellulase (Cel), protease (Prt) and phospholipase. A long-term goal of this research is to clarify the genetic and physiological aspects of extracellular enzyme production in soft-rotting Erwinia and to elucidate the roles of these enzymes in bacterial plant pathogenicity. Purified Pnls, like Pel and Peh, can macerate plant tissues. Genetic data also indicate that Pnl contributes to virulence of Ecc. However, our research, supported by NSF, has revealed that Pnl is regulated differently from Pel, Peh, Cel and Prt. In Ecc71, the model Ecc strain used in my laboratory, as well as in many other Erwinia, Pnl production is activated in response to stress imposed by DNA-damaging agents. This unusual response, which links the production of a virulence factor with DNA damage, is controlled by a unique regulatory system involving, in addition to RecA, the newly discovered rdg genes (rdg = regulator of damage-inducible gene). The 2.1 kb rdg region contains two regulatory genes, rdgA and rdgB, organized as separate transcriptional units. The presence of helix-turn-helix motifs in both RdgA and RdgB indicates that they probably can bind DNA. There is high homology between RdgA and bacteriophage repressors and between RdgB and transcriptional activators. These features and structural similarity between the putative operators in rdgA and rdgB prompted the hypothesis that RdgA, as a repressor, may regulate its own expression and possibly also rdgB expression. By promoter switching, it has been determined that rdgB product activated Pnl production in E. coli, yet deletion analysis of rdg+ DNA indicated that when rdgB was driven by its own promoter, functions of both rdgA and rdgB were necessary for the induction of Pnl production by mitomycin C (MC). The activation of rdgB transcription was subsequently found to require MC and functional recA and rdgA, implicating a role of a RecA processed RdgA derivative in rdgB expression. Thus, RdgA is postulated to play two key roles: in the native state RdgA functions as a repressor whereas the RecA processed RdgA serves as a transcriptional activator. The major goals of this project are to test this hypothesis and to elucidate the mechanism by which RdgB activates Pnl production in Ecc71. The specific objectives are (a) to fractionate RdgA, (b) to characterize RdgA derivatives produced by RecA* (i.e., activated RecA with protease activity), (c) to elucidate the regulation of rdgA by the use of reporter genes and DNA binding assays, (d) to identify the RecA* derived RdgA product required for the activation of rdgB transcription, and (e) to examine binding of RdgB to the sequences upstream of PnlA, the structural gene for pectin Iyase. %%% This research should elucidate the molecular mechanisms by which a novel regulatory circuit controls the production of a bacterial plant virulence factor. It could lead to better ways to combat bacterial diseases of crop plants. ***

Agency
National Science Foundation (NSF)
Institute
Division of Molecular and Cellular Biosciences (MCB)
Application #
9419403
Program Officer
Philip Harriman
Project Start
Project End
Budget Start
1995-03-15
Budget End
1999-02-28
Support Year
Fiscal Year
1994
Total Cost
$315,000
Indirect Cost
Name
University of Missouri-Columbia
Department
Type
DUNS #
City
Columbia
State
MO
Country
United States
Zip Code
65211