Effect of Oxygen Concentration, Temperature and Combined Nitrogen on the Morphology and Nitrogenase Activity of Rhizobium sp. Strain 32H1 in Agar Culture

Abstract

207Cultures of Rhizobium sp. strain 3 2~1 grown in a layer of soft agar on top of a layer of harder agar developed maximum nitrogenase activity [80 to 90 nmol acetylene reduced h-l (mg protein)-l] after 12 d in air at 28 "C. Electron microscopy of sections of cores of the soft agar layer showed differences in the morphology of rhizobia in colonies growing at the surface, in the middle or at the bottom of the layer. The properties of rhizobia growing in the colonies in the middle of the soft agar layer suggested that only these contained nitrogenase. These rhizobia were present as a distinct band of cells passing through individual colonies at a constant depth in the agar. They were larger than normal vegetative Rhizobium, were pleiomorphic and were similar in morphology to the nitrogen-fixing bacteroids formed by strain 3 2~1 in cowpea root nodules. Their location within the soft agar layer changed with the O2 concentration in the atmosphere above the agar during growth, and they were not found in cultures showing little or no nitrogenase activity. Loss of nitrogenase activity occurred when cultures were grown at 33 "C, in a 02/Ar (1 :4, v/v) atmosphere, or when the concentration of combined nitrogen in the agar medium was increased from 2 to 20 mM-N.

Morphology and Nitrogenase Activity of Agar Cultures and Root Nodules Formed by D-Cycloserine-resistant Mutants of Rhizobium sp. Strain 32H1

“…The nitrogenase activities of agar cultures of strain 3 2~1 and of the D-cycloserine-resistant mutants of strain 3 2~1 were determined as described by Pankhurst & Craig (1978).…”

“…Air (1-5 ml) was withdrawn from the bottles with a syringe and replaced with acetylene to give an acetylene concentration of 0.1 atm. The bottles were then incubated at 25 "C. Gas samples (0.2 ml) were removed at various times after 30 min and analysed for acetylene and ethylene (C&) as described by Pankhurst & Craig (1978).…”

“…Root nodules were prepared for light and electron microscopy by the routine method of Craig & Williamson (1973). Agar cultures of strain 3 2~1 and of the D-cycloserine-resistant mutants of strain 3 2~1 were prepared for microscopy by the method of Pankhurst & Craig (1978). i The cultures were established with an inoculum containing 5 x 10' bacteria ml-l, and measurements were made after 14 d growth at 28 "C. Table 1 shows the minimum inhibitory concentrations of D-cycloserine for strain 3 2~1 and for the one-, two-and three-step D-cycloserine-resistant mutants.…”

“…Cultures of mutant 3 2~1 D-cycr-l/l contained a large zone of colonies with type 1 rhizobia (Pankhurst & Craig, 1978) (Fig. 1 b).…”

“…In view of the distinctive morphology of nitrogenase-active agar cultures of strain 3 2~1 (Pankhurst & Craig, 1978), it was of interest to examine the morphology of agar cultures of the three D-cycloserine-resistant mutants of strain 3 2~1 . Sections of 14-d-old agar cultures grown at 28 "C were therefore prepared and examined by light and electron microscopy.…”