1.5.10
THE REGULATION OF PATHOGENICITY GENE EXPRESSION IN XANTHOMONAS CAMPESTRIS MEDIATED BY A SMALL DIFFUSIBLE MOLECULE

H SLATER1, C BARBER1, A ALVAREZ-MORALES2, TJG WILSON1, JM DOW1 and MJ DANIELS1

1The Sainsbury Laboratory, Norwich Research Park, Colney, Norwich, NR4 7UH, UK; 2CINVESTAV, IPN Irapuato, Genetic Engineering Department, APDO POST. 629, Irapuato, GTO 36500, Mexico

Background and objectives
The rpf (regulation of pathogenicity factors) gene cluster is required for the synthesis of extracellular enzymes and polysaccharide (EPS), and hence pathogenicity, in the phytopathogen Xanthomonas campestris pv. campestris (Xcc). Two members of this gene cluster, rpfF and rpfB, are involved in a novel type of regulation mediated by a small diffusible molecule [1]. Production of extracellular endoglucanase and protease by rpfF (but not rpfB) mutants can be stimulated by growth in close proximity to rpfF+ strains, indicative of cross-feeding by a diffusible factor (DSF). The Xcc system is novel in that DSF is not an N-acyl homoserine lactone, and RpfF and RpfB are not related to Lux1/R proteins implicated in the synthesis of N-acyl homoserine lactone derivatives which are widely distributed in Gram-negafive bacteria. To identify further genes involved in the DSF system a molecular genetic approach has been taken.

Results and conclusions
A transcriptional fusion was constructed between the promoter region of the Xcc endoglucanase gene and the coding sequence of beta-glucuronidase (GUS). The rpfF mutant strain harbouring the endoglucanase:GUS reporter plasmid shows an increase in GUS activity when DSF is added exogenously or when in the presence of a DSF-producing strain. A mutant population of Xcc was generated by transposon mutagenesis, in which the transposon was introduced into the genome by a transmid system. This system allows the transposon and flanking DNA to be isolated as a plasmid that can replicate directly in E. coli. Screening these mutants with the GUS reporter strain as an assay for DSF production identified two DSF hyper-producing mutants. Paradoxically, both mutants (named 8557 and 8558) had reduced levels of extracellular enzymes and EPS and were reduced in pathogenicity. Although derived from independent transposon insertion events, the genes disrupted in these mutants in both cases were rpfC. RpfC belongs to a small sub-class of two-component regulators in which the sensor and response regulator domains are fused [2]. Immediately upstream from rpfC are rpfH and rpfG, which are also predicted to encode parts of a two-component regulatory system. It is possible that rpfH and rpfG are in an operon with rpfC. Further analysis revealed that an rpfH mutant also had elevated levels of DSF, although the levels were not as extreme as those measured for 8557 and 8558. Current work to investigate the transcriptional organization of these three genes will determine whether these phenotypes are the result of a polar effect. Further to this we are investigating if and how rpfC, rpfH and rpfG work together to couple the sensing of DSF to the production of enzymes and EPS, and also presumably to the negative regulation of DSF.

References
1. Barber CE, Tang JL, Feng JX et al., 1997. Molecular Microbiology 24, 555-566.
2. Tang JL, Liu YN, Barber CE et al., 1991. Molecular and General Genetics 226, 409-417.