We developed a protocol which yields purified bacterial DNA from the soil bacterial community. The bacteria were first dispersed and separated from soil particles in the presence of polyvinylpolypyrrolidone, which removes humic acid contaminants by adsorption to this insoluble polymer. The soil bacteria were then collected by centrifugation and lysed by using a comprehensive protocol designed to maximize disruption of the various types of bacteria present. Total bacterial DNA was purified from the cell lysate and remaining soil contaminants by using equilibrium density gradients. The isolated DNA was essentially pure as determined by UV spectral analysis, was at least 48 kilobases long, and was not subject to degradation, which indicated that there was no contaminating nuclease activity. The isolated DNA was readily digested by exogenously added restriction endonucleases and successfully analyzed by slot blot and Southern blot hybridizations. Using single-stranded, 32P-labeled DNA probes, we could detect and quantitate the presence of a specific microbial population in the natural soil community on the basis of the presence of a DNA sequence unique to that organism. The sensitivity of our methodology was sufficient to detect Bradyrhizobium japonicum at densities as low as 4.3 x 10 cells per g (dry weight) of soil, which corresponds to about 0.2 pg of hybridizable DNA in a l-,g DNA sample.

Aurintricarboxylic acid (ATA) is a general inhibitor of nucleases. ATA has been shown to inhibit the following enzymes in vitro: DNAse I, RNAse A, S1 nuclease, exonuclease III, and restriction endonucleases Sal I, Bam HI, Pst I and Sma I. The observed inhibition is consistent with the proposal by Blumenthal and Landers (BBRC 55, 680, 1973) that most nucleic acid binding proteins will be sensitive to ATA. The action of ATA as a nuclease inhibitor can be used to advantage in the isolation of cellular nucleic acids.

Bradyrhizobium japonicum induces the formation of nitrogen-fixing symbiotic root nodules on soybean plants. The B. japonicum genome encodes two isoforms of glutamine synthetase (GS). One form, GSI, encoded by the gene glnA, is similar in structure and activity to the enzyme found in all other bacteria. The second form, GSII, encoded by glnII, is structurally related to the eucaryotic enzyme. Genetic analyses indicate that glnA or glnII alone is sufficient to provide glutamine prototrophy, whereas the double mutation glnA glnII produces glutamine auxotrophy. The glnA gene is transcribed from a single promoter that has a structure most similar to that of the bacterial consensus promoter. The level of transcription of glnA is not specifically affected by nitrogen limitation of growth. The glnII gene is also transcribed from a single promoter; however, this promoter has structural features characteristic of promoters controlled by the nitrogen regulation system. In contrast to glnA, physiological studies indicate that glnII transcription is regulated in response to nitrogen source availability. Under aerobic growth conditions, expression of glnII is induced when growth is limited by nitrogen source depletion as expected for regulation by the nitrogen regulation system.

We isolated the ntrC gene from Bradyrhizobiumjaponicum, the endosymbiont of soybean (Glycine max), and examined its role in regulating nitrogen assimilation. Two independent ntrC mutants were constructed by gene replacement techniques. One mutant was unable to produce NtrC protein, while the other constitutively produced a stable, truncated NtrC protein. Both ntrC mutants were unable to utilize potassium nitrate as a sole nitrogen source. In contrast to wild-type B. japonicum, the NtrC null mutant lacked ginII transcripts in aerobic, nitrogen-starved cultures. However, the truncated-NtrC mutant expressed glnIl in both nitrogenstarved and nitrogen-excess cultures. Both mutants expressed glnll under oxygen-limited culture condiftions and in symbiotic cells. These results suggest that nitrogen assimilation in B. japonicum is regulated in response to both nitrogen limitation and oxygen limitation and that separate regulatory networks exist in free-living and symbiotic cells.

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