Regulation of Glutamine Synthetase Isoenzymes in Rhizobium leguminosarum biovar viceae

Abstract

Ammonia assimilation in Rhizobium leguminosarum biovar viceae strain RCRlOO 1 (hereafter called R . leguminosarum) appears to take place only through the glutamine synthetase/glutamate synthase pathway since (a) no glutamate dehydrogenase was detected in crude extracts of bacteria grown in different nitrogen sources, and (b) the growth rate on glutamine as a nitrogen source was faster than that observed on NH4Cl. In contrast to reports for other Rhizobium species, R . leguminosarum can definitely utilize NH4C1 for growth. R. Ieguminosarum contains two glutamine synthetase isoenzymes, GSI and GSII, which can be detected in the presence of each other by differential heat stability, or separated by affinity chromatography or immunoabsorption with an antiserum raised against pure GSI. GSII does not cross-react with an anti-GSI antiserum. GSI was shown to be reversibly adenylylated and it was also shown that adenylylation inhibits the biosynthetic activity of this enzyme, in a similar way to that reported for Escherichia coli glutamine synthetase and in contrast to that observed for glutamine synthetase of Rhizobium sp. strain ANU289. The apparent adenylylation level in different growth conditions changes from 21% to 99%, indicating a physiological role of this posttranslational modification in the in vivo regulation of GSI activity. The intracellular concentration of GSI varies very little when R . leguminosarum is grown on different nitrogen sources (twofold when measured by the transferase assay, or fourfold when measured by ELISA). In addition, the concentration of mRNA specific for GSI in different nitrogen sources does not show appreciable differences. The intracellular concentration of GSII varies from a specific activity value higher than 1000 when R . leguminosarum is grown on glutamate or nitrate, to an undetectable level when grown on NH4C1. When NH4C1 is added to a culture growing in glutamate, GSII activity is rapidly diluted out, suggesting a post-translational mechanism of enzyme inhibition or inactivation. Chloramphenicol prevents the disappearance of GSII activity, thus suggesting that protein synthesis is required for this process.

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

“…1. viceae LPRllO5, a rifampicinresistant derivative of strain RCRlOOl (Hooykaas eta/., 1977). Growth conditions and preparations of cell extracts were as described previously (Rossi et al, 1989). Protein concentration was determined by the Bio-Rad protein assay using bovine serum albumin (BSA fraction V, Sigma) as a standard.…”

“…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). This phenomenon may be interpreted in several ways, as, for example, accumulation of an inhibitor or degradation of the enzyme; furthermore, a correlation with enzyme dilution following repression has not been reported.…”

“…GSI is similar to the single GS of enteric bacteria. It is an oligomeric, relatively heat stable enzyme, comprising 12 identical subunits of M , 59000, which can be adenylylated (Darrow & Knotts, 1977;Rossi et al, 1989). In contrast, GSII is similar to eukaryotic GS (Carlson & Chelm, 1986;De Vries et al, 1983;; it comprises 8 subunits of M , 42000 (De Vries et al, 1983) and is heat labile with respect to GSI.…”

“…1989), expression of GSII is controlled by the nitrogen source in the medium (Martin et al, 1988;Rossi et al, 1989). Although'very different in size and structure, GSI and GSII have similar K , values and similar turnover numbers for their substrates (Darrow, 1980).…”

“…GSI and GSII are products of different genes (Carlson et al, 1985;Colonna-Romano et al, 1987;Filser et al, 1986;Somerville & Kahn, 1983). While GSI is constitutively expressed (Darrow, 1980;Ludwig, 1980;Rossi et al, * Author for correspondence. Fax 81 7257202. t Present address : Istituto di Microbiologia, Naples, Italy.…”