Ammonium assimilation in Rhizobium phaseoli by the glutamine synthetase-glutamate synthase pathway

Abstract

Evidence from in vitro and in vivo studies showed that in Rhizobium phaseoli ammonium is assimilated by the glutamine synthetase (GS)-glutamate synthase NADPH pathway. No glutamate dehydrogenase activity was detected. R. phaseoli has two GS enzymes, as do other rhizobia. The two GS activities are regulated on the basis of the requirement for low (GSI) or high (GSII) ammonium assimilation. When the 2-oxoglutarate/glutamine ratio decreases, GSI is adenylylated. When GSI is inactivated, GSII is induced. However, induction of GSH activity varied depending on the rate of change of this ratio. GSII was inactivated after the addition of high ammonium concentrations, when the 2-oxoglutarate/glutamine ratio decreased rapidly. Ammonium inactivation resulted in alteration of the catalytic and physical properties of GSII. GSII inactivation was not relieved by shifting of the cultures to glutamate. After GSH inactivation, ammonium was excreted into the medium. Glutamate synthase activity was inhibited by some organic acids and repressed when cells were grown with glutamate as the nitrogen source.It is generally accepted that ammonium assimilation in rhizobia proceeds mainly through the glutamine synthetase (GS)-glutamate synthase (GOGAT) pathway (6,13,15,(19)(20)(21). However, in contrast with other bacteria, rhizobium contains two forms of GS, and this presents additional complexity in the study of ammonium assimilation. The GSI enzyme is similar to the enzymes found in other gramnegative bacteria in regard to its monomeric and oligomeric structure, postranslational regulation, and amino acid sequence (5, 6, 25). The GSII enzyme has a lower molecular weight and is heat labile (6, 7, 10, 16). Recently, in Bradyrhizobium japonicum, the gene coding for GSII was cloned, the enzyme was purified, and it was found that its amino acid sequence more closely resembles that of plant GS than that of bacterial GS (4). The function of the two GS forms from rhizobia in the free-living state and during symbiosis remains controversial, since there are several reports about the regulation of GS in rhizobia, without distinction between the two GS activities (13,15,19,20,27).Other studies by Darrow (6) and Ludwig (16) showed that ammonium has a strong negative modulation effect on GSII and a minor effect on GSI. GSI from free-living rhizobia (6, 16) and bacteroids (3) was mainly regulated by adenylylation. However, Howitt et al. (11) reported that, although GSII was severely repressed when rhizobia were grown on ammonium, GSI had the same biosynthetic activity, regardless of the adenylylation state of the enzyme (11).Glutamine auxothrophs have been isolated in Rhizobium cowpea 32H1 and R. meliloti (13, 16), but the number of mutations responsible for these phenotypes remains uncertain.Carlson et al. (5) and Somerville and Kahn (25) have isolated the GSI genes from B. japonicum and R. meliloti, respectively. The GSI gene of B. japonicum was constitutively transcribed in different nitrogen conditions (5). The R. meliloti GSI gene has been inte...

Dissociation by NH4Cl treatment of the enzymic activities of glutamine synthetase II from Rhizobium leguminosarum biovar viceae

“…These authors propose that a modified subunit with decreased transferase activity transmits to the neighbouring subunits a signal enhancing their biosynthetic activity. An apparent dissociation between transferase and biosynthetic activity may be present also in the data reported by Bravo & Mora (1988).…”

“…The experiments reported in this paper indicate that the reported rapid removal of GSII activity by NH4Cl treatment in crude extracts (Bravo & Mora, 1988;Fuchs & Keister, 1980;Howitt & Gresshoff, 1985;Ludwig, 1980;Rossi et al, 1989) may be explained as a post-translational modification, thus excluding other hypotheses such as accumulation of an inhibitor, change in the octamer/tetramer ratio or protein degradation. A post-transcriptional control was suggested by Adams & Chelm (1988) to explain the finding of the presence of GSII transcript in the absence of GSII transferase activity in N,-fixing bacteroids or free living microaerobic B. japonicum (Rao et al, 1978).…”

“…Although'very different in size and structure, GSI and GSII have similar K , values and similar turnover numbers for their substrates (Darrow, 1980). It has been reported that transferase activity of GSII assayed in crude extracts, is rapidly removed after addition of NH,Cl to a culture derepressed for GSII (Bravo & Mora, 1988;Fuchs & Keister, 1980;Howitt & Gresshoff, 1985 ;Ludwig, 1980). Addition of chloramphenicol prior to NH,Cl prevents disappearance of GSII transferase activity (Rossi et al, 1989).…”

Transcriptional activity of the symbiotic plasmid of Rhizobium etli is affected by different environmental conditions

“…The bacterial strains and plasmids used are listed in Table 1. RhiTobizun strains were grown at 30 "C in PY rich medium (Noel et al, 1984) or in Y minimal medium with different carbon and nitrogen sources, each at a concentration of 10 mM (Bravo & Mora, 1988). Carbon sources were sodium succinate, glucose or glycerol; nitrogen sources were ammonium chloride, potassium nitrate, sodium glutamate or glutamine.…”

Pathways for glutamate biosynthesis in the yeast Kluyveromyces lactis

“…In an alternative pathway demonstrated by Tempest et al (1970), glutamate is aminated to form glutamine by glutamine synthetase (GS ; EC 6;3;1;2), the amide group of which is then transferred reductively to 2-oxoglutarate by glutamate synthase (GOGAT ; EC 1;4;1;13), resulting in the net conversion of ammonium and 2-oxoglutarate to glutamate. The GS-GOGAT pathway has been found in several micro-organisms (Senior, 1975 ;Hummelt & Mora, 1980 ;Bravo & Mora, 1988 ;Marque! s et al, 1992) and in higher plants (Miflin et al, 1980).…”