Physiological factors affecting transformation of Azotobacter vinelandii

Abstract

A. vinelandii (ATCC 12837) can be transformed by exogenous DNA towards the end of exponential growth. Transformation occurs at very low frequencies when the DNA is purified or when the transformation is carried out in a liquid medium. Optimal transformation occurs on plates of BUrk N-free glucose medium containing either high phosphate (10 mM) or low Ca (0-0.29 mM) content. Higher levels of Ca are inhibitory, and Mg2+ is essential for transformation and growth. Extracellular polymer and capsule are increasingly inhibitory to transformation and are most abundant when the Ca content of the medium is high. Transformation is optimal at pH 7.0-7.1 and at 30.degree. C, conditions which also coincide with minimal extracellular polymer production. Nonencapsulated strains are excellent transformation recipients. Glycine-induced pleomorphism reduces the transformation frequency, and the degree of inhibition is dependent on the phosphate concentration of the medium. Rifampin resistance and shifts from adenine, hypoxanthine, uracil and nitrogenase auxotrophy to prototrophy can be achieved. Although single marker transfer is always greater than double marker transfer, rifampin resistance may be linked to hypoxanthine, adenine and uracil prototrophy at intervals of increasing distance. Rifampin resistance did not appear to be linked to nitrogenase.