Maurizio Iaccarino

Requirement of sulfhydryl groups for the catalytic and tRNA recognition functions of isoleucyl-tRNA synthetase ☆

Abstract The reaction of isoleucyl-tRNA synthetase (IRS) with N -ethylmaleimide (NEM) and p -hydroxymercuribenzoate ( p MB) has been used to probe the role of sulfhydryl groups in the catalytic and tRNA recognition activities of the enzyme. The reaction of the protein with NEM has revealed one rapidly reacting sulfhydryl group and a class that reacts less than 10% as fast. Modification of the fast-reacting group with NEM markedly reduces the ATP-PP i exchange activity and the formation of Ile-tRNA but has no detectable effect on either the equilibrium constant or the rate of binding of tRNA Ile to the enzyme. In the presence of isoleucine and ATP, the reaction with NEM and the attendant loss of activities are prevented; other amino acids, other triphosphates, or tRNA, AMP and PP, do not protect the enzyme. The modification produced by reaction with NEM decreases the rate but not the extent of IRS(Ile-AMP) complex formation; thus, each of the altered molecules can react with isoleucine and ATP to form the enzyme-bound Ile-AMP but at less than 1% the rate. With native IRS, binding of isoleucine stimulates the rate of association and dissociation of tRNA Ile from the enzyme (Yarus & Berg, 1969); with NEM-modified IRS, this effect of isoleucine is reduced, indicating that the modification has interrupted the linkage between the isoleucine catalytic site and the tRNA binding site. Nine sulfhydryl groups per mole of IRS are revealed by titration with p MB. Inactivation of the ATP-PP i exchange reaches a limiting value of 90% when all of the sulfhydryl groups are titrated: Ile-tRNA synthesis, however, is inhibited more than 99% when five sulfhydryl groups are titrated. In contrast to the NEM-modification, binding of the mercurial to IRS markedly decreases the capacity of the protein to bind its cognate tRNA. The data suggest that there is one sulfhydryl group at or near the catalytic site, which must be free for maximal rate of Ile-AMP formation and utilization: this sulfhydryl group has no role in tRNA binding. Other sulfhydryl groups may contribute to the specific binding of the cognate tRNA either directly or by maintaining the needed protein conformation.

Organogenesis of legume root nodules

The N(2)-fixing nodules elicited by rhizobia on legume roots represent a useful model for studying plant development. Nodule formation implies a complex progression of temporally and spatially regulated events of cell differentiation/dedifferentiation involving several root tissues. In this review we describe the morphogenetic events leading to the development of these histologically well-structured organs. These events include (1) root hair deformation, (2) development and growth of infection threads, (3) induction of the nodule primordium, and (4) induction, activity, and persistence of the nodular meristem and/or of foci of meristematic activities. Particular attention is given to specific aspects of the symbiosis, such as the early stages of intracellular invasion and to differentiation of the intracellular form of rhizobia, called symbiosomes. These developmental aspects were correlated with (1) the regulatory signals exchanged, (2) the plant genes expressed in specific cell types, and (3) the staining procedures that allow the recognition of some cell types. When strictly linked with morphogenesis, the nodulation phenotypes of plant and bacterial mutants such as the developmental consequence of the treatment with metabolic inhibitors, metabolic intermediates, or the variation of physical parameters are described. Finally, some aspects of nodule senescence and of regulation of nodulation are discussed.

Auxotrophic mutant strains of Rhizobium etli reveal new nodule development phenotypes.

We report here the isolation and characterization of amino acid-requiring mutant strains of Rhizobium etli. We observe that the phenotype of most mutations, even when causing a strict auxotrophy, is overcome by cross-feeding from the host plant Phaseolus vulgaris, thereby allowing bacterial production of Nod factors and, consequently, nodule induction. Conversely, light and electron microscopy analysis reveals that the nodules induced by all mutants, including those with normal external morphology, are halted or strongly altered at intermediate or late stages of development. Moreover, some mutants induce nodules that display novel symbiotic phenotypes, such as specific alterations of the invaded cells or the presence of a reduced number of abnormally shaped uninvaded cells. Other mutants induce nodules showing an early and vast necrosis of the central tissue, a phenotype not previously observed in bean nodules, not even in nodules induced by a Fix- mutant. These observations indicate that amino acid auxotrophs represent a powerful tool to study the development of globose determinate-type nodules and emphasize the importance of establishing their histology and cytology before considerations of metabolic exchange are made.

Regulation of glutamine synthetase isoenzymes in Rhizobium leguminosarum biovar viceae

SUMMARY: Ammonia assimilation in Rhizobium leguminosarum biovar viceae strain RCR1001 (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 NH4CI. In contrast to reports for other Rhizobium species, R. leguminosarum can definitely utilize NH4C1 for growth. R. leguminosarum 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 post-translational 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 undetectabie 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.

Ectopic expression of the Rhizobium etli amtB gene affects the symbiosome differentiation process and nodule development

Under conditions of nitrogen limitation, soil bacteria of the genus Rhizobium are able to induce the development of symbiotic nodules on the roots of leguminous plants. During nodule organogenesis, bacteria are released endocytotically inside the invaded plant cells where they differentiate into their endosymbiotic form called bacteroids. Bacteroids surrounded by a plant-derived peribacteroid membrane are nondividing, organelle-like structures, called symbiosomes, that use nitrogenase to reduce N2 to ammonia. Experiments performed in vitro with isolated symbiosomes have previously led to the suggestion that the NH3 produced by the bacteroids is released as NH4+ into the plant cytosol. Furthermore, it was observed that the bacterial amtB (ammonium/methylammonium transport B) gene is switched off very early during symbiosis, just when bacteria are released into the host cells. We report here that the ectopic expression of amtB in bacteroids alters the ability of bacteria to invade the host cells and the sym...

Glutamine utilization by Rhizobium etli

We undertook the study of the use of glutamine (Gln) as the source of carbon and energy by Rhizobium etli. Tn5-induced mutagenesis allowed us to identify several genes required for Gln utilization, including those coding for two broad-range amino acid transporters and a glutamate dehydrogenase. The isolated mutants were characterized by the analysis of their capacity i) to grow on different media, ii) to transport Gln (uptake assays), and iii) to utilize Gln as the C energy source (CO2 production from Gln). We show that Gln is degraded through the citric acid cycle and that its utilization as the sole C source is related to a change in the bacterial cell shape (from bacillary to coccoid form) and a high susceptibility to a thiol oxidative insult. Both these data and the analysis of ntr-dependent promoters suggested that Gln-grown bacteria are under a condition of C starvation and N sufficiency, and as expected, the addition of glucose counteracted the morphological change and increased both the bacterial growth rate and their resistance to oxidative stress. Finally, a nodulation analysis indicates that the genes involved in Gln transport and degradation are dispensable for the bacterial ability to induce and invade developing nodules, whereas those involved in gluconeogenesis and nucleotide biosynthesis are strictly required.

Characterization and Cloning of Two Rhizobium leguminosarum Genes Coding for Glutamine Synthetase Activities

We have demonstrated that Rhizobium leguminosarum strain LPR1105 contains a heat stable and a heat labile glutamine synthetase (EC 6.3.1.2) activity similar to those described for other Rhizobiaceae. Most of the activity is heat stable when this strain is grown on glutamine as sole nitrogen source, but most is heat labile when grown on nitrate. Using a gene bank of R. leguminosarum DNA we have isolated two clones, which code for heat stable (p7D9) and heat labile (p4F7) glutamine synthetase activity, by complementing the glutamine auxotrophy of Klebsiella pneumoniae glnA mutants. Cross-hybridization of p7D9 with a fragment of the glnA gene of K. pneumoniae was observed, but no cross-hybridization between p7D9 and p4F7 was found. Since these two regions hybridize to genomic DNA of R. leguminosarum they are probably the structural genes for GSI and GSII, and the availability of these genes will make it possible to test this hypothesis. Clone p4F7 complements an ntrC+ but not an ntrC K. pneumoniae glnA mutant, suggesting that the ntrC gene is required for the complementation of the glutamine auxotrophy by this plasmid.

The Rhizobium GstI protein reduces the NH4+ assimilation capacity of Rhizobium leguminosarum.

We show that the protein encoded by the glutamine synthetase translational inhibitor (gstI) gene reduces the NH4+ assimilation capacity of Rhizobium leguminosarum. In this organism, gstI expression is regulated by the ntr system, including the PII protein, as a function of the nitrogen (N) status of the cells. The GstI protein, when expressed from an inducible promoter, inhibits glutamine synthetase II (glnII) expression under all N conditions tested. The induction of gstI affects the growth of a glutamine synthetase I (glnA-) strain and a single amino acid substitution (W48D) results in the complete loss of GstI function. During symbiosis, gstI is expressed in young differentiating symbiosomes (SBs) but not in differentiated N2-fixing SBs. In young SBs, the PII protein modulates the transcription of NtrC-regulated genes such as gstI and glnII. The evidence presented herein strengthens the idea that the endocytosis of bacteria inside the cytoplasm of the host cells is a key step in the regulation of NH4+ metabolism.