1.6.7
MUTATION IN THE AVRBS1 AVIRULENCE GENE OF XANTHOMONAS CAMPESTRIS PV. VESICATORIA (Xcv) INFLUENCES CELLULAR PROTEIN SYNTHESIS DURING STARVATION

EN OKEY AND LW O'GARRO

Microbial Pathogenicity Group, Department of Biological and Chemical Science, University of The West Indies, PO Box 64, Bridgetown, Barbados

Background and objectives
Xanthomonas campestris pv. vesicatoria causes bacterial spot of pepper (Capsicum annuum) and tomato (Lycopersicum esculentum) in tropical areas. Control measures of this disease have met with marginal success mainly due to pathogenic variation in populations of the bacterium. Up to 15 known races have been detected. Recently an Xcv strain carrying mutation in the avrBs1 avirulence gene persisted longer and more abundantly than the wild-type parental strain during starvation in soil and detached pepper leaves [1]. It has also been demonstrated that bacterial cells adapt to stress conditions by inducing and/or repressing certain proteins [2]. The objective of this study was to determine the protein response to starvation of Xcv strains carrying mutant and wild-type avrBs1 avirulence genes.

Materials and methods
Two strains of Xcv (81-23 M13 and 81-23) were used in this study. Strain 81-23 M13 lacked avrBs1 avirulence activity and was derived from strain 81-23 by insertion of a transposon in the coding sequence of the gene. Each strain was grown in 25 ml nutrient broth (Difco) at room temperature with shaking (180 r.p.m.) for 18 h. Cells were harvested by centrifugation at 10,000 r.p.m. for 10 min. The harvested cells were suspended in 30 ml of each of the four media: (i) Dyes medium (per litre - 0.5 g KH2PO4; 0.5 g K2HPO4; 5.0 g NaCI; 0.2 g MgSO47H2O; 0.5 g yeast extract); (ii) asparagine medium (per litre - 0.5 g asparagine; 0.5 g KNO3; 0.2 g MgSO4; 0.1 g KH2PO4; 0.1 g CaCI2; 0.1 g NaCI; FeCI3 (trace amount); (iii) sterile distilled water; (iv) nutrient broth (8 g/l). Aliquots (3 ml) of each sample were obtained immediately, and at 30 min and 1, 2, 5, 24 and 48 h. An additional sample was taken after 14 days. Proteins were extracted and resolved on ID SDS-PAGE.

Results and conclusions
Significant differences in protein profiles were observed between the strains 81-23 and 81-23 M13 under starvation. In Dye's medium there were more pronounced protein bands of 95 and 42 kDa for strain 81-23. The 29-kDa band present in both strains (control) was absent from strain 81-23 M3 after 1 h. However, two new protein bands, 37.5 and 35 kDa, were observed in strain 81-23 M13 only after 14 days. In asparagine medium, the main difference between the strains were a 35-kDa band in strain 81-23 M13 after 30 min and a 37.5-kDa band after 24 h. There were more pronounced bands of 95 and 42 kDa in strain 81-23 in response to sterile distilled water. Also, a 29-kDa band was induced in strain 81-23 M13 after 2 h while a 72-kDa band was absent from strain 81-23 after 1 h.

These results indicate a greater shut down (global control) of protein synthesis in 81-23 NI 13 during starvation. Reduction in protein synthesis could be a result of reduced metabolic activity or cell size. This is an important strategy for long-term survival under stress [2]. The proteins (37.5, 35 and 29 kDa) which constantly increased could represent the core of the general starvation-inducible stimulon in Xcv. The sizes of these proteins are similar to those induced by heat shock in other bacteria, suggesting that there is some interconnecting regulation for these stresses in bacteria [2]. The high level of survival and abundance of strain 81-23 M13 in soil over its wild parental strain 81-23 reported [1] could be a result of more effective protein synthesis control as observed in this study.

References
1. O'Garro LW, Gibbs H, Newton A, 1997. Phytopathology 87, 960-966.
2. Holmquist L, Jouper-Jean A, Weichart D, 1993. FEMS Microbiology Ecology 12, 185-194.