1.5.4
ROLE OF ExuR IN GALACTURONIC ACID TRANSPORT IN ERWINIA CHRYSANTHEMI EC16

V VALMEEKAM1, TL FREEMAN2 and MJD SAN FRANCISCO1

1Department of Biological Sciences, Texas Tech University, Lubbock, Texas 79409, USA; 2Department of Internal Medicine, Texas Tech University Health Sciences Center, Lubbock, Texas 79430, USA

Background and objectives
Polygalacturonate lyases and hydrolases are primary components of the virulence potential of phytopathogenic Erwinia chrysanthemi [1]. Degradation of the plant cell-wall pectate results in the formation of monomers and multimers of galacturonic acid. Transport of these degradation products is required for induction of the synthesis and secretion of pectate lyases and other cell wall-degrading enzymes by the bacterium. Thus uptake of pectate degradation products represents an important early event in the plant-bacterial interactions leading to disease development. Studies on these transport systems might be useful to understand the underlying mechanisms in sensing the host environment and moderation of virulence gene expression by this facultative pathogen. Uptake systems for galacturonic acid monomers and dimers have been described in E. chrysanthemi. Transport of the galacturonic acid monomer is mediated by the exuT gene product. We have cloned and sequenced the exuT gene from E. chrysanthemi EC16 and localized the polypeptide to the bacterial inner membrane. An exuT mutant was also constructed and was found to be reduced in pathogenicity [2]. The objective of this study was to understand the genetic regulation of exuT gene encoding the galacturonic acid permease in E. chrysanthemi EC16.

Materials and methods
The interrupted exuR gene was cloned into a low copy plasmid, and marker exchange recombinants were obtained after growing the plasmid-containing cells in a low-phosphate medium. Single copy exuT-lux gene fusions were mobilized into the Tn7 att site on the chromosome using a specialized vector containing Tn7 border sequences and a helper plasmid with the transposase functions. Galacturonic acid uptake was studied using a radio-labelled analogue, [14C]GA (0.58 mCi/mmol) at a final concentration of 0.1 mM. Assays were performed with cells grown in the presence or absence of inducer (galacturonic acid).

Results and conclusions
Galacturonic acid transport was inducible and its transcription negatively regulated by the repressor ExuR. ExuR belongs to the GntR family of transcriptional regulators with a highly conserved helix-turn-helix motif at the N-terminal region. Gene fusions with the 5' upstream sequence of exuT and luciferase reporter gene system indicated de-regulated constitutive expression in an exuR mutant. In planta expression of the exuT-luciferase gene fusion was distinctly higher in an exuR- background than the wild-type strain. The exuR mutant also revealed higher basal levels of exuT transcripts and galacturonic acid uptake. exuT gene expression was also subject to glucose-mediated catabolite repression as revealed by the [14C]GA uptake and reporter gene fusion studies. Analysis of the 5' upstream sequence of exuT indicated the presence of a strong binding site for CRP (catabolite repressor protein), the major activator of pectate lyase genes, and KdgR, the global regulator of pectinase synthesis, secretion and intracellular pectate catabolism. The possible involvement of the kdgR gene product in the regulation of exuT remains to be confirmed.

References
1. Collmer A, Keen NT, 1986. Annual Reviews of Phytopathology 24, 383-409.
2. Haseloff BJ, Freeman TL, Valmeekam V et al., 1998. Molecular Plant-Microbe Interactions, in press.