The sensitivity of 12 Frankia strains to heavy metals was determined by a growth inhibition assay. In general, all of the strains were sensitive to low concentrations (<0.5 mM) of Ag 1؉ , AsO 2 1؊ , Cd 2؉ , SbO 2 1؊ , and Ni 2؉ , but most of the strains were less sensitive to Pb 2؉ (6 to 8 mM), CrO 4 2؊ (1.0 to 1.75 mM), AsO 4 3؊ (>50 mM), and SeO 2 2؊ (1.5 to 3.5 mM). While most strains were sensitive to 0.1 mM Cu 2؉ , four strains were resistant to elevated levels of Cu 2؉ (2 to 5 mM and concentrations as high as 20 mM). The mechanism of SeO 2 2؊ resistance seems to involve reduction of the selenite oxyanion to insoluble elemental selenium, whereas Pb 2؉ resistance and Cu 2؉ resistance may involve sequestration or binding mechanisms. Indications of the resistance mechanisms for the other heavy metals were not as clear.Frankia, a member of the order Actinomycetales, forms a symbiotic nitrogen-fixing association with a variety of woody dicotyledonous plants (for reviews see references 3 and 28). The members of this bacterial genus are known to be associated with over 200 species of plants representing eight plant families. These bacteria fix N 2 from the atmosphere and produce a significant amount of the fixed nitrogen on the planet. Actinorhizal plants are ecologically important as pioneer community plants and have economic value in land reclamation, reforestation, and soil stabilization.The lack of a well-established genetic system is a major obstacle in the elucidation of the mechanism of actinorhizal nitrogen fixation and plant-microbe interactions (for reviews see references 22, 23, and 25). There is a paucity of genetic markers for Frankia. Some of the most useful genetic markers include resistance to antibiotics, resistance to antimetabolites, and resistance to heavy metals. These directly selectable traits provide a mechanism for positive selection in genetic studies and are also useful in the development of cloning vectors. For example, metal resistance has been useful in the development of cloning vectors for Rhodococcus, another member of the Actinomycetales (8). Previously, we developed a growth inhibition assay that was used to screen several Frankia strains for resistance to antibiotics (27). Although several important antibiotic resistance markers were described in that study, we were interested in identifying other selectable genetic markers. Since actinorhizal plants have been used for land reclamation in strip-mined areas, we reasoned that it is possible that the bacteria are resistant to heavy metals. The purpose of this study was to extend the use of our growth inhibition assay to identify new selectable markers, resistance to heavy metals.
Frankia strains ACN1AG (14), CcI3 (29), Cc1.17 (18), CN3 (20), CpI1 succinate variant (CpI1-S) (5, 26), CpI1 propionate variant (CpI1-P) (5, 26), DC12 (1), EI5c (17), EAN1pec (16), EuI1c (2), EUN1f (14), and QA3 (12) were grown and maintained in basal growth medium with NH 4 Cl as the nitrogen source, as described previously (26, 27). For the heavy metal s...
