ISHS Acta Horticulturae 273: V International Workshop on Fireblight
DETECTION AND CHARACTERIZATION OF AN IRON UPTAKE SYSTEM IN E. AMYLOVORA. |
| Authors: | J.L. Vanneste, D. Expert |
| Abstract:
Iron is an essential element required in numerous bacterial enzymatic reactions. In nature, iron is present in the ferric form (iron III) which is not soluble and thus not readily available to the bacteria. To circumvent this problem, microorganisms evolved specific iron uptake systems, that usually consist of i) the production and excretion of an iron ligand molecule called a siderophore and ii) a protein, located in the outer membrane of gram negative bacteria that serves as receptor for the iron-siderophore complex. In this study we show that the strain 1430 of E. amylovora possesses an inducible iron uptake mechanism. Furthermore, we present data that suggest that this mechanism could be involved in the virulence of E. amylovora. The comparison of growth curves of 1430 in complete medium and in iron depleted medium (figure 1) revealed that after a period of induction (part of the curve noted B) this strain of E. amylovora could grow normally under iron limiting conditions. This suggests that 1430 possesses an efficient iron uptake mechanism which is induced under iron limiting conditions. The part of the growth curve noted A could correspond to the growth due to residual amount of Fe in the medium. Production and excretion of a siderophore by 1430 was revealed by growing this strain on the "blue" medium devised by Schwyn and Neilands (1987). This medium contains a complex (Chrome Azurol S/iron (III)/ hexadecyltrimethylammonium bromide) that gives a blue color to the medium, but turns orange when the iron is removed from the complex. When grown on this medium colonies of 1430 were surrounded by an orange halo indicating that this strain produced and excreted a siderophore type molecule. Most siderophores isolated so far fall into two main groups; those based on catechol and those derived from the hydroxamic acid and called hydroxamate. These 2 types of siderophores can be identified by specific colorimetric assays, test of Arnow (1937) and test of Csàky (1948) respectively. A hydroxamate activity was detected in the supernatant of low iron cultures of 1430, whereas under the same conditions, no catechol activity could be detected. This indicates that under iron limiting conditions 1430 excretes an hydroxamate-type siderophore. Comparison of outer membrane proteins prepared from 1430 grown in iron depleted M63 minimal medium (EDDA M63) or in M63 medium, revealed that two proteins of 84 and 76 Kilodaltons were over-expressed under iron limiting conditions (figure. 2). Low iron inducible proteins migrating in the 70 – 80 Kilodaltons range are known to be | |
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