DNA prepared from soil usually contains a brown-tinted inhibitor of the polymerase chain reaction (PCR) which limits the sensitivity of this technique for specific detection of microorganisms. To localize the inhibitor, soil fractions were tested for their inhibitory effect on the PCR reaction. A highly inhibitory activity, sufficient to account for the inhibition typically exhibited by soil DNA, was found to be tightly associated with the soil microorganism fraction. After cell breakage, the inhibitory material became soluble, and was not separable from DNA by standard purification procedures. A method was derived by which most of the inhibitory material could be selectively solubilized from the microorganism fraction without cell breakage, using successive washes with buffers differing in EDTA concentration. This technique was used to isolate a substance with characteristics suggesting that it is the major PCR inhibitor contaminating DNA purified from soil. It was found to be an organic, water-soluble compound of high molecular weight, and was present in a variety of soil types from different locations. It was found to be distinctly different in its solubility properties from humic and fulvic acids, and also in its FT-IR and NMR spectra. It forms a complex with protein and may inhibit the PCR reaction by an interaction with Taq DNA polymerase.

A mutant of Rhizobium melioti, 4R3, which is unable to grow on aspartate has been isolated. The defect is specific to aspartate utilization, since 4R3 is not an auxotroph and grows as well as its parent strain on other carbon and nitrogen sources. The defect was correlated with an inability to fix nitrogen within nodules formed on alfalfa. Transport of aspartate into the mutant cells was found to be normal. Analysis of enzymes involved in aspartate catabolism showed a significantly lower level of aspartate aminotransferase activity in cell extracts of 4R3 than in the wild type. Two unrelated regions identified from a genomic cosmid bank each complemented the aspartate catabolism and symbiotic defects in 4R3. One of the cosmids was found to encode an aspartate aminotransferase enzyme and resulted in restoration of aspartate aminotransferase activity in the mutant. Analysis of the region cloned in this cosmid by transposon mutagenesis showed that mutations within this region generate the original mutant phenotypes. The second type of cosmid was found to encode an aromatic aminotransferase enzyme and resulted in highly elevated levels of aromatic aminotransferase activity. This enzyme apparently compensated for the mutation by its ability to partially utilize aspartate as a substrate. These findings demonstrate that R. meliloti contains an aspartate aminotransferase activity required for symbiotic nitrogen fixation and implicate aspartate as an essential substrate for bacteria in the nodule.

Aspartate transport in Rhizubium meliluti was found to be mediated by at least two transport systems. High rates of aspartate uptake, necessary for growth on aspartate as a carbon source, required the dicarboxylate transport (Dct) system, which also transports succinate, fumarate and malate. The apparent K,,, for aspartate transport by this system was about 10 mM, compared to 15 p~ for succinate. This difference in affinity was also apparent in competitive inhibition studies, which showed that succinate effectively inhibits aspartate transport. Although aspartate was not a preferred substrate, it was a very efficient inducer of the Dct system. Both the Dct system and a second aspartate transport system were capable of supplying aspartate for use as a nitrogen source. The second system had a lower apparent K, for aspartate transport (1.5 mM), and was competitively inhibited by glutamate. This aspartateglutamate system was regulated independently from the Dct system, since it functioned in mutants lacking the Dct system regulatory genes dctB and dctD, and its induction did not coactivate the Dct system. Uptake kinetics in cultures growing on aspartate as nitrogen source showed rapid substrate exchange between extracellular and internal aspartate. R. meliloti was shown to be able to selectively activate the two uptake systems, and also regulated its metabolism as required to utilize aspartate as either carbon or nitrogen source.

A genetically marked Rhizobium meliloti strain, R692, was prepared by insertion of a 1.7-kb DNA segment from Tn903 between the nifHDK and fixABC genes in the nod megaplasmid. This DNA was used as a marker, detectable by polymerase chain reaction (PCR), for the specific identification of bacteria in soil samples and alfalfa nodules. This detection technique was tested by applying different titres of the marked strain to field plots seeded with alfalfa. Samples of soil and nodules were assayed for the presence of the marker DNA fragment by PCR using primers specific to the marker sequence. The experiments revealed that the bacteria could be detected directly in soil containing about 10(3)-10(4) bacteria/g, but greater sensitivity was prevented by potent PCR inhibitors present in the samples. The titre of the bacteria in the soil decreased rapidly after inoculation, dropping about 10-fold per week. Tests of vertical location of the bacteria in soil cores showed that the bacteria were initially dispersed to a depth of 18 cm, and subsequently retained viability in the top 2-8 cm. As few as 10 marked R. meliloti per gram of soil resulted in its establishment at detectable levels in nodules. Application of about 10(4)-10(5) bacteria/g soil was sufficient to give the maximum number of nodules per plant and resulted in 70-90% occupancy by the marked strain. Limited movement of the inoculant was detected by analysis of nodules from plants adjacent to the sites where the bacteria were applied, probably by movement in water. The experiments demonstrated the advantages of PCR for the monitoring of marked microorganisms in the environment.

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