PHENOTYPIC CHARACTERIZATION OF A TUNGSTEN-TOLERANT MUTANT OF AZOTOBACTER-VINELANDII

A tungsten-tolerant mutant strain (CA6) of Azotobacter vinelandii firs t described in 1980 (P. E. Bishop, D. M. L. Jarlenski, and D. R. Hethe rington, Proc. Natl. Acad. Sci. USA 77:7342-7346, 1980) has been furth er characterized. Results from growth experiments suggest that both ni trogenases 1 and 3 are utilized when CA6 grows in N-free medium contai ning Na2MoO4. Strain CA6.1.71, which lacks both nitrogenases 2 and 3, grew as well as strain CA in N-free medium containing Na2MoO4 after an initial lag. This indicates that nitrogenase 1 is fully functional in strain CA6. nifH-lacZ and anfH-lacZ transcriptional fusions were expr essed in CA6 in the presence of Na2MoO4. Thus, in contrast to wild-typ e strain CA, transcription of the anfHDGK gene cluster in strain CA6 i s not repressed by Mo. Expression of the vnfD-lacZ fusion was the same in both strains CA and CA6. In agreement with the results obtained wi th lac fusions, subunits of both nitrogenases 1 and 3 were found in pr otein extracts of CA6 cells grown in N-free medium containing Na2MoO4. How ever, CA6 cells, cultured in the presence of Na2WO4, accumulated nitrogenase 3 proteins without detectable amounts of nitrogenase 1 pro teins. This indicates that expression of Mo-independent nitrogenase 3 is the basis for the tungsten tolerance phenotype of strain CA6. A mea sure of Mo accumulation as a function of time showed that accumulation by strain CA6 was slower than that for strain CA. When Mo accumulatio n was studied as a function of Na2MoO4 concentration, the two strains accumulated similar amounts of Mo in the concentration range of 0 to 1 mu M Na2MoO4 during a 2-h period. Within the range of 1 to 5 mu M Na2 MoO4. Mo accumulation by strain CA increased linearly with increasing concentration whereas no further increases were observed for strain CA 6. These results are consistent with the possibility that the tungsten tolerance mutation carried by CA6 is in a Mo transport system.