A 2.0-kilobase DNA fragment carrying antibiotic resistance markers was inserted into the gltB gene of Escherichia coli previously cloned in a multicopy plasmid. Replacement of the chromosomal gltB+ gene by the gltB225::Qk mutation led to cells unable to synthesize glutamate synthase, utilize growth rate-limiting nitrogen sources, or derepress their glutamine synthetase. The existence of a gItBDF operon encoding the large (gltB) and small (gItD) subunits of glutamate synthase and a regulatory peptide (gltF) at 69 min of the E. coli linkage map was deduced from complementation analysis. A plasmid carrying the entire gltB+D+F+ operon complemented cells for all three of the mutant phenotypes associated with the polar g1tP22S::Q1 mutation in the chromosome. By contrast, plasmids carrying gltB+ only complemented cells for glutamate synthase activity. A major tricistronic mRNA molecule was detected from Northern (RNA blot) DNA-RNA hybridization experiments with DNA probes containing single genes of the operon. A 30,200-dalton polypeptide was identified as the gltF product, the lack of which was responsible for the inability of cells to use nitrogen-limiting sources associated with gltB225::Qk.

A procedure is described for the isolation of mutants of Azospirillum brasilense strains Sp7 (ATCC 29145) and Sp245 and Azospirillum lipoferum Br1702, which are unable to differentiate from vegetative vibrioids into encapsulated C forms. It is based on the characteristic of mutants, designated Enc−, to develop into white colonies in a background of wild-type red colonies (Enc+) on the surface of Congo Red plates. This behavior was ascribed to an inability of Enc− mutants to synthesize one or more as yet unidentified polysaccharides. The frequency of mutation from Enc+ to Enc− was a function of the particular strain and of the culture conditions employed. Enc− mutants from A. brasilense and A. lipoferum were at least as efficient as wild-type ancestors in adhering to and causing deformations of root hairs of germinated wheat seedlings.

The regulatory gene, glnF, of Escherichia coli was fused to the structural genes of the lac operon by use of the hybrid Mu phage derivative Mudl (Ap lac). Analysis of two of these fusions showed that the glnF gene is expressed constitutively, i.e., independent of either the nitrogen source in the growth medium or the availability of the glnA, glnL, glnG or glnF functional gene products. The orientation of the Mud1 (Ap lac) insertions was determined by chromosome mobilization in F-merogenotes carrying either of the two glnF::Mud1 chromosomal insertions isolated, and either one of a pair of F'lacZ::Mucts62 episomes; the two episomes differing in that their Mucts62 insertions are located in opposite orientations with regard to lacZ. The direction of chromosome mobilization by the Hfrs that were probably formed via Mu homology demonstrated that orientation of the glnF gene is clockwise relative to that of the chromosome.

Uptake of 14CH3NH+3 (methylammonium) was measured as a probe of NH+4 transport in intact Escherichia coli cells and derivatives impaired in the Ntr regulatory system. The results suggest that expression of the high affinity 14CH3NH+3 transport system (a) requires de novo polypeptide synthesis, (b) is activated by the glnG and glnF regulatory products under nitrogen limitation, and (c) is repressed under nitrogen excess by the glnL product. Cells deficient in glutamate synthase activity by virtue of their harbouring the gltB31 mutation were unable to activate synthesis of 14CH3NH+3 transport. This could explain the inability of cells carrying gltB mutations to grow on low concentrations of NH+4.

Two immunologically active polysaccharides were isolated from Nocardia asteroides (Poly I Na and Poly II Na) and N. brasiliensis (Poly I Nb and Poly II Nb). These polysaccharides were isolated from cell extracts and purified by methanol precipitation, chloroform extraction of extraneous material, and deproteinization with trichloroacetic acid. The crucial step used for separation of Poly I and Poly II from both nocardias was differential solubility. From dried preparations containing both polysaccharides, Poly I was solubilized at pH 10, whereas Poly II remained insoluble and was subsequently solubilized at pH 5. Poly I Na and Poly I Nb are apparently the same. Arabinose and galactose were the monosaccharide constituents of these polysaccharides, and their molar ratios were similar. Furthermore, PolW I Na and Poly I Nb crossreacted in agar diffusion precipitin tests with rabbit antisera prepared against either N. asteroides or N. brasilies. Either polysaccharide absorbed serum antibodies against the other. These polysaccharides can be regarded as groupspecific. Poly II Na and Poly II Nb are different and species-specific. They are composed of arabinose, galactose, and mannose but exhibit different molar ratios of these sugars according to species. They reacted only with homologous antisera.