gltBDF operon of Escherichia coli

Abstract

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.

Identification of phosphate starvation-inducible genes in Escherichia coli K-12 by DNA sequence analysis of psi::lacZ(Mu d1) transcriptional fusions

“…Also, glt mutants abolish derepression of the glnALG operon in a way that is overcome by mutations in the glnL (ntrB) gene. The gltF product could have a regulatory role in controlling the glnALG operon (9), which may or may not be associated with the control of glnALG expression by protein phosphorylation (31). A regulatory network that involves phosphorylation of protein regulators by heterologous sensory protein kinases could provide a mechanism for the overlapping controls on psi promoters (42).…”

“…A comparison of the psi:: lacZ(Mu dl) junction sequences with DNA data bases revealed that the psiQ32 and psiQ35: :lacZ(Mu dl)s lie in the gltB gene and the psiQ39::lacZ(Mu dl) lies in the gltD gene of the gltBDF operon for the large and small subunits of glutamate synthase (9). Unlike other psi fusions, the psiQ::lacZ(Mu dl)s were not previously tested for induction during nitrogen limitation because the psiQ mutants grew poorly on our low-ammonia medium (47), as expected for glt mutants.…”

Roles of SpoT and FNR in NH4+ assimilation and osmoregulation in GOGAT (glutamate synthase)-deficient mutants of Escherichia coli

“…These data implicate a defect in N assimilation as the basis for the osmosensitive phenotype of GJ193 and provide additional evidence that increased L-glutamate synthesis and accumulation are important in E. coli osmoregulation (2,13,37). Implied in this conclusion is the existence of a mechanism at high osmolarity for readjustment of the set point in feedback control of L-glutamate synthesis via both the GOGAT and GDH pathways (7,43), but the details of this mechanism are not known.…”

“…pHYD809 transformants of GJ193 were not complemented for growth on Ntr-regulated N sources such as L-arginine and L-ornithine ( Table 4), suggesting that (i) spoT-mediated suppression does not extend to functions that are regulated by gltF, the gene downstream of gltBD in the same operon (7,8), and (ii) osmosensitivity associated with the gltBD238::lac insertion is itself not due to a polar effect on the expression of gltF. Plasmid pHYD809, as well as the minimal spoT ϩ plasmid pHX41, was able to complement even a ⌬gltBDF fnr strain (GJ946) for both osmotolerance and growth on low [ GDH and GOGAT activity in pHYD809 derivatives of gltB mutants.…”

“…In low-[NH 4 ϩ ] medium, the latter is the only pathway that contributes to N assimilation (42,43). The two subunits of GOGAT are encoded by the gltB and gltD genes, which are organized (along with a third gene, gltF [7,8]) as an operon at 70 min on the linkage map.…”