ROLES OF SPOT AND FNR IN NH4-DEFICIENT MUTANTS OF ESCHERICHIA-COLI( ASSIMILATION AND OSMOREGULATION IN GOGAT (GLUTAMATE SYNTHASE))

An osmosensitive mutant of Escherichia coli was isolated and shown to harbor two mutations that were together necessary for osmosensitivity, One (ossB) was an insertion mutation in the gltBD operon, which encod es the enzyme glutamate synthase (GOGAT), involved in ammonia assimila tion and L-glutamate biosynthesis. The other (ossA) was in the fnr gen e, encoding the regulator protein FNR for anaerobic gene expression, S everal missense or deletion mutations in fnr and gltBD behaved like os sA and ossB, respectively, in conferring osmosensitivity. A mutation a ffecting the DNA-binding domain of FNR was recessive to fnr(+) with re spect to the osmotolerance phenotype but was dominant-negative for its effect on expression of genes in anaerobic respiration. Our results m ay most simply be interpreted as suggesting the requirement for monome ric FNR during aerobic growth of E. coli in high-osmolarity media, pre sumably for L-glutamate accumulation via the GOGAT-independent pathway (catalyzed by glutamate dehydrogenase [GDH]), but the mechanism of FN R action is not known. We also found that the spoT gene (encoding guan osine 3',5'-bispyrophosphate [ppGpp] synthetase II/ppGpp-3' pyrophosph ohydrolase), in multiple copies, overcomes the defect in NH4+ assimila tion associated with GOGAT deficiency and thereby suppresses osmosensi tivity in gltBD for strains. Enhancement of GDH activity in these deri vatives appears to be responsible for the observed suppression. Its li kely physiological relevance was established by the demonstration that growth of gltBD mutants (that are haploid for spoT(+)) on moderately low [NH4+] was restored with the use of C sources poorer than glucose in the medium, Our results raise the possibility that SpoT-mediated ac cumulation of ppGpp during C-limited growth leads to GDH activation an d that the latter enzyme plays an important role in N assimilation in situ hitherto unrecognized from studies on laboratory-grown cultures.